Effects of Bacillus subtilis supplementation in soybean meal-based diets on growth performance, diet digestibility and gut health in bullfrog Lithobates catesbeianus

Aquaculture Reports 16 (2020) 100273

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Effects of Bacillus subtilis supplementation in soybean meal-based diets on growth performance, diet digestibility and gut health in bullfrog Lithobates catesbeianus

T

Jibin Lina, Qiuhui Zenga, Chunxiao Zhanga, Kai Songa, Kangle Lua, Ling Wanga,*, Samad Rahimnejadb a

Xiamen Key Laboratory for Feed Quality Testing and Safety Evaluation, FisheriesCollege, Jimei University, Xiamen, 361021, China University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zatisi 728/ II, 389 25, Vodnany, Czech Republic

b

A R T I C LE I N FO

A B S T R A C T

Keywords: Bullfrog Soybean meal-based diet Bacillus subtilis Diet digestibility Gut health

An 8-week feeding experiment was carried out to evaluate the supplemental effects of Bacillus subtilis in all-plant protein diets on growth, nutrients digestibility, and intestinal histomorphology and microbial composition in bullfrog Lithobates catesbeianus. Totally four diets were prepared: a non-supplemented control diet and three probiotic diets containing 1 × 105, 1 × 107 or 1 × 109 CFU g−1 of B. subtilis (Con, BS5, BS7 and BS9 diets). The experiment was done in triplicates and bullfrogs averaging 42.5 ± 0.2 g were fed to visual satiety twice daily. No significant changes in growth, feed utilization, body composition and survival rate were found by probiotic application (P > 0.05). However, significant improvements in apparent digestibility coefficients of protein, calcium and phosphorus were found by probiotic supplementation. Moreover, whole-body phosphorous and calcium contents were enhanced by probiotic administration. The highest villus height and thickness were recorded in the BS5 group. Operational taxonomic unit (OTU) diversity and richness significantly increased by application of 1 × 107 CFU g-1 of B. subtilis. Furthermore, B. subtilis abundance in the gut was enhanced with increasing the probiotic inclusion level indicating the successful establishment of the probiotic. The findings in this research showed that supplementing 1 × 107 CFU g−1 of B. subtilis in soybean meal-based diets for bullfrog beneficially influences nutrients digestibility and uptake, and intestinal structure and microbial composition.

1. Introduction Fish meal has been traditionally used as a high-quality protein source in aquafeed. However, the increased price of fish meal which is rooted in its stagnant supply and increased demand has urged the nutritionist to search for potential candidates to replace fish meal (Zhang et al., 2018). Soybean meal has been recognized as a promising alternative because of its competitive nutrient composition, stable supply and reasonable price (Trushenski et al., 2006; Wang et al., 2006; Barrows et al., 2007). However, high dietary inclusion levels of soybean meal has led to reduced weight gain, nutrients digestibility, palatability and feed utilization in several aquatic animals such as hybrid striped bass (Morone saxatilis×M. chrysops) (Brown et al., 1997), cobia (Rachycentron canadum) (Zhou et al., 2005) and bullfrog (Lithobates catesbeianus) (Zhang et al., 2015; Yang et al., 2019). Probiotics beneficially impact the host health through improving

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feed utilization, contribution to the enzymatic digestion, preventing pathogenic microorganisms, and exerting growth-promoting effects (Verschuere et al., 2000; Irianto and Austin, 2002; Merrifield et al., 2010a; Nayak, 2010). Potential of probiotics in improving utilization of high plant protein diets has been demonstrated in rainbow trout (Oncorhynchus mykiss) (Sealey et al., 2009) and Pacific white shrimp (Litopenaeus vannamei) (Olmos et al., 2011). This could be mediated through altering the gut structure and/or microbiota which may subsequently influence the sensitivity of aquatic animals to high plant protein diets (Sealey et al., 2009). Bacillus spp. are one of the widely studied probiotic genera in aquaculture (Wang et al., 2008; Cha et al., 2013; Rahimnejad et al., 2019). The spore-forming ability of the Bacillus species increases their resistance to gastric conditions and their survivability (Podolsky, 1998; Shah, 2000; Cha et al., 2013). Bacillus subtilis exhibited a multitude of beneficial properties when supplemented in the diets for grouper

Corresponding author at: Fisheries College, Jimei University, No. 43 Yindou Road, Xiamen, 361021, China. E-mail address: [email protected] (L. Wang).

https://doi.org/10.1016/j.aqrep.2020.100273 Received 21 October 2019; Received in revised form 3 December 2019; Accepted 1 January 2020 2352-5134/ © 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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(Epinephelus coioides) (Sun et al., 2010), among which is its ability in producing a wide range of extracellular substances including phytase (Kim et al., 1998). The phytase producing property of B. subtilis could be used as means to improve phosphate bioavailability from plant feedstuffs in aquafeed (Cheng and Hardy, 2002; Tsuji et al., 2015). The extracellular phytase activity of B. subtilis has been reported to be 15.97 U ml−1, which is equivalent to 600 U kg−1 of commercial phytase (Kalsi et al., 2016). Bullfrog (Lithobates catesbeianus) has become a promising candidate amphibian for aquaculture due to its increased meat popularity worldwide (Pasteris et al., 2006; Huang et al., 2014; Zhang et al., 2015, 2016a; Zhang et al., 2016b; Li et al., 2019). The objectives of the present study were to examine the effects of supplementing B. subtilis in a soybean meal-based diet on growth performance, nutrients digestibility and utilization, gut histomorphology and microbial composition in bullfrog.

Table 1 Formulation and proximate composition of the basal diet (% dry matter). Ingredients a

Soybean meal Wheat flour Lecithin Soybean oil Fish oil Choline Vitamin premixb Mineral premixc L-Ascorbate-2-phosphate Mold inhibitor Antioxidant CaCO3 L-lysine HCL DL-methionine Y2O3

%

Proximate composition

%

60 28.69 1 2 3 0.5 0.1 0.5 0.1 0.05 0.05 3.15 0.28 0.48 0.1

Dry matter (DM) Protein Lipid Ash Calcium Total phosphorus

86.8 39.94 7.07 7.90 0.7 0.55

a

2. Materials and methods

Jiakang Feed Co., Ltd., Xiamen, China. Vitamin premix (mg or g/kg diet): thiamin, 10 mg; riboflavin, 8 mg; pyridoxine HCl, 10 mg; vitamin B12, 0.2 mg, vitamin K3, 10 mg; inositol, 100 mg; pantothenic acid, 20 mg; niacin acid, 50 mg; folic acid, 2 mg; biotin, 2 mg; retinol acetate, 400 mg; cholecalciferol, 5 mg; alpha-tocopherol, 100 mg; ethoxyquin, 150 mg; wheat middling 0.1328 g. c Mineral premix (mg or g/kg): KCI, 200 mg; KI, 60 mg; CoSO4, 100 mg CuSO4·5H2O, 24 mg; FeSO4.H2O, 400 mg; ZnSO4·H2O, 174 mg; MnSO4·H2O, 78 mg; MgSO4·7H2O, 800 mg; Na2SeO3, 50 mg; Zeolite, 3.114 g. b

2.1. Preparation of the probiotic strain The tested probiotic species was isolated from intestinal microflora of healthy bullfrog and identified as B. subtilis by 16S ribosomal RNA gene sequencing (Majorbio Bio-pharm Technology Co., Ltd. Shanghai, China). The sample was enriched in 100 ml Luria–Bertani (LB) (pH = 5.5) supplemented with 2 % (w/v) sodium phytate and incubated at 37 °C for 42 h at 150 rpm. A phytase screening medium (PSM) was used for isolation of phytase-producing bacterial strains (Jorquera et al., 2011). Halo zone-forming bacterial colonies were purified on LB agar medium, further point inoculated on phytase screening agar plates. Prior to use the strain was preserved at 4 °C in Luria–Bertani (LB) with 15 % sterile glycerol.

meal was used as the dietary protein source, and a mixture of fish oil and soybean oil was used as dietary lipid source. Three additional diets were prepared by supplementing the basal diet with spore of B. subtilis at 1 × 105 (BS5), 1 × 107 (BS7) and 1 × 109 (BS9) CFU g−1. All the feed ingredients were finely ground and passed through a 250-μm sieve. All dry ingredients were mixed thoroughly in a mixer before adding oil and appropriate amount of water. Then the mixture was transferred into an MY45 single screw extrusion machine (Xiamen Fishing Machinery Feed Machinery Co., Xiamen, China) to produce neutrally buoyant 4.0 × 5.5 mm pellets. The pellets were dried to a moisture content of approximately 10 % in an air convection oven at 45 °C. After drying, spore of B. subtilis was supplemented to the basal diet by spraying. Sterilized saline (0.85 %) was sprayed at the same dose to the basal diet. Then, the pellets were dried in a forced-air environment at 20 °C for about 36 h and stored at -20 °C until used. B. subtilis concentrations in the experimental diets were determined using the spread-plate technique. Briefly, 1 g of each test diet was randomly sampled and serially 10-fold diluted in phosphate-buffered saline solution (PBS; pH 7.2) and 100 ml of each dilution was then spread on mannitol-egg yolk-polymyxin agar (MYP agar, Difco, USA) in order to estimate the probiotic concentration (CFU g−1) (Zokaeifar et al., 2012). The analyzed concentration of B. subtilis in the experimental diets are shown in Table 2.

2.2. Cultivation of the bacterial strain B. Subtilis was cultivated in 100 ml spore medium [soybean meal 1 %, glucose 1 %, NaCl 0.5 %, MnSO4·H2O 0.06 %, pH = 7.0] in a 250-ml baffled flask at 31 °C for 96 h with shaking at 150 rpm. The proportion of spores was more than 95 %. The culture was then centrifuged at 5000 g for 10 min at 4 °C. After discarding the supernatant, the pelleted culture was re-suspended and washed three times in sterile Normal Saline Solution (NSS, 0.85 % NaCl). The cell density of the suspension was estimated using spectrophotometer at 600 nm and correlated to the colony-forming unit (CFU) using the spread-plate technique. The cell concentration was approximately 1 × 109 CFU ml−1. The prepared suspension was kept at 4 °C until used. 2.3. Phytase assay The B. subtilis was inoculated in phytase liquid medium (wheat bran extract, WBE) [containing, per liter: 100 ml wheat bran extract (10 g wheat bran was added to 90 ml water, then the filtrate constant volume to 100 ml), 1 g tryptone, 0.04 g (NH4)2SO4, 0.02 g MgSO4·7H2O, 0.005 g KH2PO4, 0.004 g K2HPO4, 0.2 g CaCl2, pH = 7.0] at 30 °C for 72 h at 150 rpm. Cells were removed by centrifugation at 12 000 g for 5 min at 4 °C. The supernatant served as crude enzyme. Phytase activity was assayed according to the method of vanadium ammonium molybdate in accordance with China standard GB/T 18634-2009. One unit of phytase activity was defined as the amount of enzyme that releases 1 u M of inorganic phosphate per min under standard assay conditions. The phytase activity of B. subtilis was 26.31 U ml−1.

2.5. Feeding trial This study was approved by the ethics committee on animal experimentation of Jimei University. Bullfrogs, produced from the same batch, were purchased from a commercial farm in Xiamen (Fujian Table 2 Analyzed concentrations of B. subtilis in the experimental diets.

Control BS5 BS7 BS9

2.4. Experimental diets A basal diet was formulated to contain 39.9 % protein and 7.0 % lipid (Table 1) to meet the nutrient requirements of bullfrog. Soybean

Expected (CFU g−1)

Analyzed (CFU g−1)

0 1 × 105 1 × 107 1 × 109

0 1.07 × 105 1.15 × 107 0.91 × 109

Values are means of triplicate groups. 2

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2.7.2. Diet digestibility Yttrium oxide in the diets and feces samples were determined as described by Furukawa and Tsukahara (1966). Calcium and phosphorus in the diets and feces were analyzed by inductively coupled plasma atomic emission spectrophotometer (ICPOES, Prodigy7, Leeman Labs, USA). The apparent digestibility coefficients (ADCs) of dry matter, protein, lipid, calcium and phosphorus were calculated using the following formula (NRC, 2011):

province, China) and transported to aquaculture facility of Jimei University. They were first stocked into an indoor aquarium (150 × 70 × 60 cm) and fed the basal diet twice daily (08:30 and 18:00) for two weeks to acclimatize them to the test feed and experimental conditions. At the end of the acclimation period, bullfrogs of similar size (42.5 ± 0.2 g) were randomly distributed into 12 indoor aquaria (68 × 38 × 36 cm) with average water depth of 4 cm at a density of 12 bullfrogs per aquarium. The experiment was performed in three replicates and bullfrogs were hand-fed the test diets to apparent satiety twice daily (08:00 and 18:00) for 8 weeks. Uneaten feed, if any, was taken out with net 30 min after feeding, dried and feed intake was calculated by subtracting the weight of uneaten feed from the amount of total supplied feed. All the water in each aquarium was changed after each meal. During the experimental period, water temperature ranged from 24 to 28 °C and photoperiod was maintained on a 12:12 (light: dark) schedule.

ADC of dry matter (%) = (1-Y2O3 in diet/Y2O3 in feces) × 100 %. ADC of nutrient (%) = [1-(Y2O3 in diet/Y2O3 in feces) × (nutrient in feces/nutrient in diet)] × 100 %. 2.7.3. Intestinal morphology The fixed jejunum samples were wrapped in gauze and rinsed in running water for 24 h. Subsequently, the samples were dehydrated in ethanol, infiltrated in xylene and embedded in paraffin, according to the standard histological procedures. The samples were cut serially (6 μm thick) using a rotary microtome, stained with hematoxylin and eosin, and alterations in intestinal epithelia photographed using Upright automatic biological microscope (Leica DM5500B, Germany).

2.6. Sample collection At the end of the feeding trial, bullfrogs were fasted for 24 h prior to sample collection. All the bullfrogs in each aquarium were counted and individually weighed to determine survival rate and growth parameters. Eight bullfrogs per aquarium were randomly collected. Two bullfrogs were randomly sampled for analysis of whole-body composition and the remaining six bullfrogs were collected for tissue sampling. The liver samples were collected from the same six bullfrogs per tank, transferred into 1.5-ml tubes (RNase-Free; Axygen), flash frozen in liquid nitrogen and then stored at -80 °C until subsequent analysis of antioxidant capacity parameters. The jejunum samples were collected from the same six bullfrogs, flushed with ice-cold phosphate-buffered saline (PBS saline, pH 7.4), and then fixed in 10 % formalin for morphological analyses. In order to evaluate the effects on gut microbiota, the jejunum of three bullfrogs per tank were sampled under the sterile condition. The abdomen of the bullfrog was carefully wiped with ethanol and the abdominal cavity was opened with sterile instruments. The jejunum of bullfrog was cut with sterile scalpel and rinsed carefully with physiological saline solution (0.85 %) to remove all intestine contents and emptied into sterile eppendorf tubes and frozen, until for microbiota analysis (Majorbio Bio-pharm Technology Co., Ltd. Shanghai, China). At the end of the feeding trial, the remaining bullfrogs were fed with their corresponding diets and the feces samples were collected using fecal collection apparatus as described by Zhang et al. (2015). After feeding the bullfrogs at 08:30, the apparatus was placed on the bottom of each aquarium with 3 cm of water covering the bottom. This apparatus occupied 60 % of the bottom of each aquarium (150 × 70 × 60 cm), which allowed bullfrog to move freely, rest and defecate without apparent stress. After 23 h, the apparatus was removed from the aquarium, and the sieve plate was pulled out, and feces were collected. The feces were then freeze-dried, ground using a 1 mm screen and stored at −20 °C for chemical analyses. Fecal samples from each tank were pooled at the end of experiment.

2.7.4. Gut microbiota For jejunum microbiota analysis, total genomic DNA was extracted from the intestinal contents of Control, BS5, BS7 and BS9 groups with the TIANamp Stool DNA Kit (Tiangen Biotech (Beijing) Co., Ltd), following the manufacturer's recommendations and sent for 16S rRNA gene sequence analysis with high-throughput sequencing [(Shanghai) Majorbio Co., Ltd]. 2.8. Statistical analysis All data were pooled within each replicate and analyzed using oneway analysis of variance (one-way ANOVA) and Tukey's multiple range tests. Analyses were performed using SPSS version 17.0 (SPSS Inc, Chicago, IL, USA). Data are presented as mean ± standard error of the mean (SE). The level of significance was set at P < 0.05. 3. Results 3.1. Growth performance and diet digestibility Growth performance, feed utilization and survival rate of bullfrog were not significantly influenced by dietary treatments (P > 0.05) (Table 3). FE, PER and HSI showed an increasing tendency by increasing probiotic supplementation level, however, the differences were not significant. ADCs of protein and calcium were significantly improved in the group received BS7 diet (Table 4). Moreover, significantly higher ADC of phosphorous was detected in BS7 and BS9 groups. 3.2. Whole-body composition

2.7. Analytical methods Probiotic application did not affect whole-body proximate composition (Table 5). However, a significant enhancement in whole-body phosphorus content was found in BS7 group in comparison to the control group (Fig. 1). Moreover, whole-body calcium content was significantly enhanced in all the probiotic treated groups (Fig. 1).

2.7.1. Chemical composition Proximate composition of diets and whole-body samples was analyzed according to the standard methods of the Association of Official Chemists (Association of Official Analytical Chemists (AOAC, 1995). Moisture was determined by oven drying at 105 °C until a constant weight was reached. Crude protein content (nitrogen×6.25) was measured using Auto Dumas burning nitrogen fixation equipment (Rapid N III, Germany). Crude lipid was determined by ether-extraction and ash by incineration at 550 °C for 8 h, and ash content was measured by the combustion method in a muffle furnace at 550 °C for 8 h.

3.3. Gut morphology and microbiota The highest values of villus height and thickness were detected in BS7 group which were significantly different from those of the BS9 group (Table 6, Fig. 2). However, gut muscular thickness did not 3

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Table 3 Growth performance, feed utilization and survival (42.5 ± 0.2 g) fed the experimental diets for 8 weeks.

rate

of

Table 5 Whole-body composition of bullfrog fed the experimental diets for 8 weeks (% wet weight).

bullfrog

Diets

FBWa (g) WGb (%) FRc (%/d) SGRd (%/d) FEe PERf HSIg (%) Survival (%)

Moisture

Control

BS5

BS7

BS9

91.3 ± 0.49 115 ± 1.46 1.10 ± 0.03 1.37 ± 0.01 1.10 ± 0.03 3.27 ± 0.10 4.71 ± 0.37 94.47 ± 2.77

87.2 ± 1.83 106 ± 4.21 1.11 ± 0.01 1.29 ± 0.04 1.07 ± 0.03 3.16 ± 0.09 4.76 ± 0.11 100 ± 0.00

82.8 ± 3.33 94.84 ± 7.94 1.08 ± 0.05 1.19 ± 0.07 1.10 ± 0.08 3.27 ± 0.24 4.69 ± 0.35 100 ± 0.00

91.9 ± 6.62 116 ± 15.3 1.01 ± 0.04 1.37 ± 0.12 1.25 ± 0.01 3.44 ± 0.27 5.23 ± 0.58 94.5 ± 2.77

Control BS5 BS7 BS9

77.15 79.11 77.66 77.59

± ± ± ±

Protein 0.63 1.16 0.84 1.45

15.89 14.90 15.89 16.04

Lipid

± ± ± ±

0.57 0.66 0.57 0.85

5.44 4.85 5.58 5.23

Ash ± ± ± ±

0.09 0.14 0.24 0.29

2.24 2.20 2.32 2.33

± ± ± ±

0.12 0.14 0.04 0.12

Values are presented as mean ± SE. The lack of superscript letters indicates no significant difference among treatments (P > 0.05).

Values are means of triplicate groups and presented as mean ± SE. Values in the same column having different. The lack of superscript letters indicates no significant different difference among treatments (P > 0.05). a Final body weight. b Feeding rate = [dry feed fed/ (total final weight(g)/2 + total initial weight(g)/2)/days] ×100. c Weight gain = [(final body weight-initial body weight)/initial body weight] ×100. d pecific growth rate= [Ln (final body weight)-Ln (initial body weight)]/ days ×100. e Feed efficiency = weight gain/dry feed fed. f Protein efficiency ratio= (total final weight(g)-total initial weight(g))/total dry protein consumed (g). g Hepatosomatic index= (liver weight/body weight) ×100.

Fig. 1. Whole-body calcium (Ca) and phosphorus (P) content of bullfrog fed the experimental diets for 8 weeks (% dry weight). Values are means of triplicate groups. Bars with different letters are significantly different (P < 0.05). Table 6 Jejunum morphology of bullfrog fed the experimental diets for 8 weeks.

significantly differ among the experimental groups. The operational taxonomic units (OTUs) are shown in Fig. 3. A total number of 2413 OTUs were observed, of which 269 OTUs (11.1 %) were shared among the four groups. The unique OTUs number for control, BS5, BS7 and BS9 groups were 424, 179, 452 and 338, respectively (Fig. 3). The BS7 group exhibited significantly higher community richness (ACE and Chao 1) and diversity (Shannon) compared to control (Table 7). The most abundant phylum was Proteobacteria, followed by Tenericutes, Chloroflexi and Firmicutes, respectively (Fig. 4). Abundance of Proteobacteia was enhanced by B. subtilis supplementation in a dose dependent manner. Moreover, the highest abundance of Firmicutes was recorded in the BS7 group. Mycoplasma was the most abundant genus, and their number decreased by increasing the probiotic supplementation level (Table 8). An increasing trend in abundance of Lactococcus bacteria was observed by increment of the probiotic inclusion level, although the differences were not significant (P > 0.05). Number of Bacillus spp. in bullfrog gut increased by B. subtilis supplementation in a dose dependent manner.

VHa (μm) Control BS5 BS7 BS9

515.1 630.0 621.2 453.1

± ± ± ±

VTb (μm) 15.12ab 20.00a 18.19a 46.96b

106.7 138.4 113.8 107.3

± ± ± ±

MTc (μm) 6.74a 6.14b 3.25ab 3.40a

84.40 79.60 75.77 73.88

± ± ± ±

9.40 9.60 4.23 6.13

Values are means of triplicate groups and presented as mean ± SE. Values in the same column having different superscript letters are significantly different (P < 0.05). The lack of superscript letters indicates no significant difference among treatments (P > 0.05). a Villus height. b Villus thickness. c Muscular thickness.

(Carvalho et al., 2011) and tambaqui (Colossoma macropomum) (Da Paixao et al., 2017). However, Ai et al. (2011) reported improved SGR and FE in juvenile large yellow croaker (Larimichthys crocea) fed B. subtilis containing diets. This inconsistency could stem from the differences in the species, probiotic dose and experimental conditions (Das et al., 2008). A rohu carp (Labeorohita Hamilton) study showed the enhancement of FE and PER when B. subtilis was included in the diet (Bairagi et al., 2004). Likewise, in the current study slight improvements in FE and PER were found by probiotic administration. Additionally, in this study significant improvements in ADCs of protein, calcium and phosphorous were achieved by B. subtilis application. It has been demonstrated that gastrointestinal bacteria play role in digestion of nutrients and contribute to the microorganisms in rohu (Labeo rohita)

4. Discussion B. subtilis has been recognized as a beneficial probiotic in aquatic animals owing to its enzyme producing ability which contributes to digestion of nutrients leading to growth promoting effects (Sogarrd and Suhr-Jessen, 1990; Gatesoupe, 1991). In the current study, WG, SGR and FR of bullfrog were not affected by the probiotic application which is consistent with the findings in Nile tilapia (Oreochromis niloticus)

Table 4 Apparent digestibility coefficients of nutrients (%) in bullfrog fed the experimental diets for 8 weeks.

Control BS5 BS7 BS9

Dry matter

Protein

83.04 76.46 83.60 79.05

86.58 87.12 91.03 88.18

± ± ± ±

0.40 3.64 2.90 1.78

± ± ± ±

0.56a 1.65ab 1.41b 0.97ab

Lipid

Calcium

8569 ± 0.41 80.34 ± 3.01 86.47 ± 2.40 82.50 ± 1.44

56.61 58.79 69.03 66.12

± ± ± ±

Phosphorus 3.05a 4.03ab 0.62b 6.51ab

41.40 42.09 52.43 50.69

± ± ± ±

4.22a 4.55a 0.38b 5.07b

Values are means of triplicate groups and presented as mean ± SE. Values in the same column having different superscript letters are significantly different (P < 0.05). The lack of superscript letters indicates no significant difference among treatments (P > 0.05). 4

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Fig. 2. Jejunum structure of bullfrog fed the experimental diets for 8 weeks (A: Control, B: BS5, C: BS7, D: BS9, scale bar-100 μm).

(Bairagi et al., 2004), common carp (Cyprinus carpio) (Wang and Xu, 2006) and shrimp (Wang, 2007) by providing bioactive compounds including enzymes, amino acids and vitamins (Bairagi et al., 2002). The positive effects observed on nutrients digestibility in this study could be associated with production of secondary metabolites such as protease or phytase by B. subtilis which may aid in the digestion and uptake of nutrients from soybean meal-based diets (Kalsi et al., 2016). This agrees with the findings in rockfish (Sebastes schlegeli) fed soy bean meal containing diets (Yoo et al., 2005). The lack of significant difference in whole-body proximate composition among experimental groups in this study is consistent with previous findings in rainbow trout (Oncorhynchus mykiss) (Merrifield et al., 2010b; Ramos et al., 2015), Japanese eel (Anguilla japonica) (Lee et al., 2018) and Tambaqui (Colossoma macropomum) (Da Paixao et al., 2017). However, in this study B. subtilis administration beneficially influenced body P and Ca contents which agrees with the results of studies on rainbow trout (Oncorhynchus mykiss) (Sugiura et al., 2001) and common carp, (Cyprinus carpio) (Schafer et al., 1995). The facilitated P and Ca retention in this study could be ascribed to the phytase-producing capability of B. subtilis which contributes to the release of protein, P and

Fig. 3. Venn diagram showing the unique and shared OTUs in the experimental groups.

Table 7 Operational taxonomic unit richness (Chao 1 & Ace) and diversity (Shannon & Simpson) indices of the intestinal bacterial in bullfrog fed the experimental diets for 8 weeks. Sampling depth

Mean sequence Richness estimate ACE Chao 1 Diversity estimators Shannon Simpson Coverage

Diets Control

BS5

26304

BS7

26304 ab

BS9

26304 a

26304 c

450.0 ± 31.00 462.5 ± 32.50ab

419.0 ± 25.06 430.0 ± 37.02a

567.7 ± 18.89 574.0 ± 15.28c

471.0 ± 21.50ab 487.0 ± 17.09abc

3.71 ± 0.29b 0.13 ± 0.01cd 0.9983

2.50 ± 0.11a 0.21 ± 0.02d 0.9979

4.61 ± 0.20c 0.06 ± 0.01ab 0.9988

4.01 ± 0.17bc 0.08 ± 0.01bc 0.9987

Values are means of triplicate groups and presented as mean ± SE. Values in the same column row having different superscript letters are significantly different (P < 0.05). The lack of superscript letters indicates no significant difference among treatments (P > 0.05). 5

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Moreover, a striped bass (Morone saxatilis) study showed that microbial phytase supplementation level significantly influences the protein utilization (Papatryphon et al., 1999). Microbial phytase promotes proteins and amino acids utilization through the breakdown of phytate–protein complexes (Kornegay and Qian, 1996). In addition, it neutralizes the adverse impacts caused by phytate on utilization of protein and other dietary components in monogastric animals’ feed (Mitchell et al., 1997). In the present study, ADC of protein was also improved by probiotic application which agrees with the results of a Korean rockfish (Sebastes schlegeli) study (Yoo et al., 2005). Probiotics are applied to achieve a balanced intestinal microflora that favorably influences the animal’s health (Tannock, 1998). In the current study, abundance of Bacillus spp. was enhanced by increasing dietary inclusion level of B. subtilis showing that the bacterium could dominate the bullfrog’s gut and exerts its beneficial impacts on nutrients digestibility and uptake. Gut morphological parameters such as villi length and muscular thickness are used as common indicators of gut condition and function in aquatic animals. The length of intestinal villi is one of the key factors that determines the degree of nutrients uptake (Klurfeld, 1999), indicating the correlation between intestinal morphology and digestive function (Aly et al., 2008). In the current study, supplementation of B. subtilis at 1 × 105 CFU g−1 led to the enhanced villi length and thickness. A similar observation was reported by Pirarat et al. (2011) in Nile tilapia (Oreochromis niloticus). The increased villi length indicates enhanced surface area and facilitated absorption of nutrients (Caspary, 1992). Decreased villus height and thickness, and muscular thickness in jejunum at higher inclusion levels of B. subtilis in this study is in contrast with earlier findings in a gibel carp (Carassius auratus gibelio) study (Chen et al., 2014). This may indicate that B. subtilis differently affects the various intestinal segments (Pirarat et al., 2011). Based on this result, more research is required on the mechanism through which B.

Fig. 4. The distribution bar plot of different bacterial phyla in the experimental groups. Values are means of triplicate groups.

Ca bound to phytic acid in soybean meal (Zhang et al., 2009; Kalsi et al., 2016; Deepa et al., 2011; Greiling et al., 2019). In agreement to our results, Vandenberg et al. (2012) found a similar trend for ADCs of protein in rainbow trout fed microbial phytase containing diets.

Table 8 Composition of bacterial genera in the intestine of bullfrog fed the experimental diets for 8 weeks.

Mycoplasma Ralstonia Sphingomonas Burkholderia Rhizobium Beijerinckiaceae_uncultured Novosphingobium Cetobacterium Acidobacteriaceae_Subgroup_1_uncultured Pleomorphomonas Pseudomonas Lysobacter Subgroup_2_norank Lactococcus Anaerolineaceae_uncultured Plesiomonas Enterococcus JG37-AG-4_norank Citrobacter Hydrogenophaga JG30a-KF-32_norank Aeromonas Candidatus_Competibacter Bradyrhizobiaceae_unclassified Enterobacteriaceae_unclassified Bacteria_unclassified TK10_norank HSB_OF53-F07_norank Bacillus MSB-5B2_norank Prevotella_9 Bosea

Control

BS5

BS7

BS9

29.70 ± 10.50 10.22 ± 7.11 3.07 ± 1.06 2.43 ± 1.33 0.40 ± 0.13 0.06 ± 0.04 0.87 ± 0.27 0.51 ± 0.47 2.68 ± 2.29 0.00 ± 0.00 0.55 ± 0.29 3.68 ± 3.68 1.45 ± 1.04 0.41 ± 0.32 1.10 ± 0.27 1.41 ± 1.39 0.67 ± 0.15 1.10 ± 0.93 0.28 ± 0.23 0.02 ± 0.01 0.32 ± 0.17 0.18 ± 0.06 0.00 ± 0.00 0.47 ± 0.17 0.33 ± 0.24 0.46 ± 0.08 0.63 ± 0.42 0.45 ± 0.21 0.10 ± 0.03a 0.67 ± 0.26 0.39 ± 0.16 0.14 ± 0.14

19.77 ± 2.27 16.89 ± 9.10 3.43 ± 1.31 3.43 ± 1.64 2.27 ± 1.78 0.57 ± 0.56 0.77 ± 0.30 0.39 ± 0.23 0.59 ± 0.48 2.51 ± 2.49 1.58 ± 1.29 0.01 ± 0.01 0.45 ± 0.36 1.12 ± 1.07 0.43 ± 0.20 0.43 ± 0.23 0.40 ± 0.12 0.35 ± 0.31 1.33 ± 0.90 0.02 ± 0.01 0.07 ± 0.01 0.05 ± 0.03 0.00 ± 0.00 0.22 ± 0.08 0.51 ± 0.48 0.06 ± 0.02 0.19 ± 0.11 0.02 ± 0.02 0.20 ± 0.03a 0.04 ± 0.04 0.12 ± 0.02 0.58 ± 0.54

13.74 ± 2.10 15.99 ± 8.76 4.38 ± 0.08 3.84 ± 1.58 1.44 ± 0.73 0.03 ± 0.02 1.31 ± 0.25 0.23 ± 0.39 0.54 ± 0.36 0.23 ± 0.17 1.12 ± 0.79 0.00 ± 0.00 0.84 ± 0.34 0.49 ± 0.24 0.80 ± 0.11 0.35 ± 0.17 0.88 ± 0.17 0.72 ± 0.43 0.10 ± 0.03 2.22 ± 2.19 0.87 ± 0.61 0.07 ± 0.00 1.83 ± 1.79 0.59 ± 0.27 0.66 ± 0.49 0.53 ± 0.19 0.47 ± 0.11 0.72 ± 0.35 0.34 ± 0.14ab 0.36 ± 0.12 0.75 ± 0.32 0.16 ± 0.12

8.88 ± 2.85 18.66 ± 9.20 6.64 ± 1.67 3.72 ± 1.53 6.57 ± 6.02 7.58 ± 7.58 1.49 ± 0.30 3.23 ± 1.86 0.51 ± 0.31 1.37 ± 1.24 0.52 ± 0.08 0.05 ± 0.05 0.80 ± 0.42 1.29 ± 0.97 0.82 ± 0.10 0.87 ± 0.31 0.94 ± 0.10 0.53 ± 0.29 0.74 ± 0.50 0.04 ± 0.02 0.93 ± 0.84 1.60 ± 1.54 0.02 ± 0.01 0.52 ± 0.22 0.21 ± 0.10 0.66 ± 0.30 0.33 ± 0.10 0.40 ± 0.31 0.92 ± 0.37b 0.47 ± 0.23 0.23 ± 0.10 0.53 ± 0.51

Values are means of triplicate groups and presented as mean ± SE. Values in the same row having different superscript letters are significantly different (P < 0.05). The lack of superscript letters indicates no significant difference among treatments (P > 0.05). 6

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subtilis influences the intestinal morphology. Gut microbiota is a highly complex ecosystem that plays crucial roles in immune function, digestion and absorption of nutrients and metabolic status of the host (Flint et al., 2008; Ma et al., 2017). Our results revealed the increased gut microbiota richness and diversity by increasing the B. subtilis inclusion level up to 1 × 107 CFU g−1. The most OTU sequences were related to Proteobacteria, Tenericutes, Chloroflexi and Firmicutes which agrees with results of a common carp (Cyprinus carpio L.) study (Li et al., 2013). Hassaan et al. (2016) showed that dietary supplementation of B. subtilis increases the total bacterial counts of gut microflora in Nile tilapia. Diversity of gut microflora is deemed to influence the growth and intrusion of harmful bacteria (Turnbaugh et al., 2006; Ley et al., 2008). However, a negative impact of B. subtilis was found on diversity of gut microbiota at higher inclusion levels which could be as a result of an imbalance in intestinal bacteria caused by excessive level of exogenous bacteria. At the phyla level, Proteobacteria and Tenericutes dominated the intestinal microbial community which is in agreement with the findings in largemouth bronze gudgeon (Coreius guichenoti) (Li et al., 2016). Uchii et al. (2006) suggested that the taxonomic composition of the fish intestinal microbiota is highly responsive to the feeding habit, whereas several authors claimed that intestinal microbiota of grass carp is more resemble to the water and sediment microbiota than the food microbiota (Wu et al., 2012). Dietary application of B. subtilis led to the increased proportion of Firmicutes in the gut, which includes Bacillus and Lactobacillu species with beneficial impacts on gut health (Russell and Diez, 1998). At the genus level, with increasing B. subtilis dose the proportion of Bacillus increased significantly. Maruta et al. (1996) also demonstrated that the ingestion of Bacillus species contributes to restoration of the normal microbial flora following extensive antibiotic usage or diseases. Hence, B. subtilis may play a role in improving proportion of beneficial bacteria and maintaining gut microflora balance.

Chemists, Arlington, VA. Bairagi, A., Ghosh, K.S., Sen, S.K., Ray, A.K., 2002. Enzyme producing bacterial flora isolated from fish digestive tracts. Aquac. Int. 10 (2), 109–121. Bairagi, A., Ghosh, K.S., Sen, S.K., Ray, A.K., 2004. Evaluation of the nutritive value of Leucaena leucocephala leaf meal, inoculated with fish intestinal bacteria Bacillus subtilis and Bacillus circulans in formulated diets for rohu, Labeo rohita (Hamilton) fingerlings. Aquac. Res. 35 (5), 436–446. Brown, P.B., Twibell, R., Jonker, Y., Wilson, K.A., 1997. Evaluation of three soybean products in diets fed to juvenile hybrid striped bass Morone saxatilis×M. chrysops. J. World Aquacult. Soc 28 (3), 215–223. Carvalho, J.V., Lira, A.D., Costa, D.S.P., Moreira, E.L.T., Pinto, L.F.B., Abreu, R.D., Albinati, R.C.B., 2011. Desempenho zootécnico e morfometria intestinal de alevinos de tilápia-do-Nilo alimentados com Bacillus subtilis ou mananoligossacarídeo. Rev. Bras. Saude. Prod. Anim. 12, 176–187. Caspary, W.F., 1992. Physiology and pathophysiology of intestinal absorption. Am. J. Clin. Nutr. 55 (1), 299–308. Cha, J.H., Rahimnejad, S., Yang, S.Y., Kim, K.W., Lee, K.J., 2013. Evaluations of Bacillus spp. as dietary additives on growth performance, innate immunity and disease resistance of olive flounder (Paralichthys olivaceus) against Streptococcus iniae and as water additives. Aquaculture 402-403, 50–57. Chen, Y., Zhu, X., Yang, Y., Han, D., Jin, J., Xie, S., 2014. Effect of dietary chitosan on growth performance, haematology, immune response, intestine morphology, intestine microbiota and disease resistance in gibel carp (Carassius auratus gibelio). Aquacult. Nutr. 20, 532–546. Cheng, Z.J., Hardy, R.W., 2002. Effect of microbial phytase on apparent nutrient digestibility of barley, canola meal, wheat and wheat middlings, measured in vivo using rainbow trout (Oncorhynchus mykiss). Aquacul. Nutr. 8, 271–277. Da Paixao, A.E.M., dos Santos, J.C., Pinto, M.S., Pereira, D.S.P., de Oliveira Ramos, C.E.C., Cerqueira, R.B., da Silva, R.F., 2017. Effect of commercial probiotics (Bacillus subtilis and Saccharomyces cerevisiae) on growth performance, body composition, hematology parameters, and disease resistance against Streptococcus agalactiae in tambaqui (Colossoma macropomum). Aquac. Int. 25 (6), 2035–2045. Das, S., Ward, L.R., Burke, C., 2008. Prospects of using marine Actinobacteria as probiotics in aquaculture. Appl. Microbiol. Biotechnol. 81 (3), 419–429. Deepa, C., Jeyanthi, G.P., Chandrasekaran, D., 2011. Effect of phytase and citric acid supplementation on the growth performance, phosphorus, calcium and nitrogen retention on broiler chicks fed with low level ofavailable phosphorus. Asian J. Poult. Sci. 5 (1), 28–34. Flint, H.J., Bayer, E.A., Rincon, M.T., Lamed, R., White, B.A., 2008. Polysaccharide utilization by gut bacteria: potential for new insights from genomic analysis. Nat. Rev. Microbiol. 6, 121–131. Furukawa, A., Tsukahara, H., 1966. On the acid digestion method for determination of chromic oxide as an index substance in the study of digestibility of fish fed. Bull. Jpn. Soc. Sci. Fish. 32, 502–506. Gatesoupe, F.J., 1993. Bacillus sp. spores as food additive for the rotifer Brachiomus plicatilis: improvement of their bacte’rial environment and their dietary value for larval turbot, Scophthalmus maximus. In: In: Kaushik, L., S.J, Luquet (Eds.), P., Fish Nutrition in Practice 1991. INRA Editions, Paris, pp. 561–568. Greiling, A.M., Tschesche, C., Baardsen, G., Krockel, S., Koppe, W., Rodehutscord, M., 2019. Effects of phosphate and phytase supplementation on phytate degradation in rainbow trout (Oncorhynchus mykiss W.) and Atlantic salmon (Salmo salar L.). Aquaculture 503, 467–474. Hassaan, M.S., Soltan, M.A., Jarmołowicz, S., Abdo, H.S., 2016. Combined effects of dietary malic acid and Bacillus subtilis on growth, gut microbiota and blood parameters of Nile tilapia (Oreochromis niloticus). Aquac. Nutr. 24 (1), 83–93. Huang, K.K., Zhang, C.X., Wang, L., Song, K., Huang, F., 2014. Effects of dietary protein and lipid levels on growth of bullfrog, Rana catesbeiana. J. Fish. China 38, 877–887 in Chinese with English abstract. Irianto, A., Austin, B., 2002. Probiotics in aquaculture. J. Fish Dis. 25, 633–642. Jorquera, M.A., Crowley, D.E., Maschener, P., Greiner, R., Fernández, M.T., Romero, D., Menezes-Blackburn, D., De La, Luz, Mora, M., 2011. Identification of β-propeller phytase-encoding genes in culturable Paenibacillus and Bacillus spp. from the rhizosphere of pasture plants on volcanic soils. FEMS Microbiol. Ecol. 75 (1), 163–172. Kalsi, H.K., Singh, R., Dhaliwal, H.S., Kumar, V., 2016. Phytases from Enterobacter and Serratia species with desirable characteristics for food and feed applications. 3 Biotech 6, 64. Kim, Y.O., Kim, H.K., Bae, K.S., Yu, J.H., Oh, T.K., 1998. Purification and properties of a thermostable phytase from Bacillus sp. DS11. Enzyme. Microb. Tech. 22 (1), 2–7. Klurfeld, D.M., 1999. Nutritional regulation of gastrointestinal growth. Front. Biosci. 4, 299–302. Kornegay, E.T., Qian, H., 1996. Replacement of inorganic phosphorus by microbial phytase for young pigs fed on a maize–soyabean-meal diet. Brit. J. Nutr. 76, 563–578. Lee, S., Katya, K., Hamidoghli, A., Hong, J., Kim, D.J., Bai, S.C., 2018. Synergistic effects of dietary supplementation of Bacillus subtilis WB60 and mannanoligosaccharide (MOS) on growth performance, immunity and disease resistance in Japanese eel, Anguilla japonica. Fish Shellfish Immun. 83, 283–291. Ley, R.E., Lozupone, C.A., Hamady, M., Knight, R., Gordon, J.I., 2008. Worlds within worlds: evolution of the vertebrate gut microbiota. Nat. Rev. Microbiol. 6 (10), 776–788. Li, X.M., Yan, Q.Y., Ringø, E., Wu, X.B., He, Y.F., Yang, D.G., 2016. The influence of weight and gender on intestinal bacterial community of wild largemouth bronze gudgeon (Coreius guichenoti, 1874). BMC Microbiol. 16 (1), 191–198. Li, X.M., Yan, Q.Y., Xie, S.Q., Hu, W., Yu, Y.H., Hu, Z.H., 2013. Gut microbiota contributes to the growth of fast growing transgenic common carp (Cyprinus carpio L.). PLoS One 8, 1–11. Li, X., Rahimnejad, S., Wang, L., Lu, K.L., Song, K., Zhang, C.X., 2019. Substituting fish

5. Conclusion These findings show that B. subtilis supplementation at 1 × 107 CFU g leads to significant improvement of protein, calcium and phosphorous digestibility in bullfrog fed a soybean meal-based diet. Furthermore, gut morphology and microbial composition were beneficially influenced by the probiotic application. −1

Data sharing statement No additional unpublished data are available. Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgments This work was funded by the National Natural Science Foundation of China (grant no.31602172), and the Science and Technology Leading Project of Fujian Province (grant no. 2017N0021). We thank Xiamen Jiakang Feed Co., Ltd. for donating the feed ingredients. References Ai, Q.H., Xu, H.G., Mai, K.S., Xu, W., Wang, J., Zhang, W.B., 2011. Effects of dietary supplementation of Bacillus subtilis and fructooligosaccharide on growth performance, survival, non-specific immune response and disease resistance of juvenile large yellow croaker, Larimichthys crocea. Aquaculture 317, 155–161. Aly, S.M., Ahmed, Y.A., Ghareeb, A.A., Mohamed, M.F., 2008. Studies on Bacillus subtilis and Lactobacillus acidophilus, as potential probiotics, on the immune response and resistance of Tilapia nilotica (Oreochromis niloticus) to challenge infections. Fish Shellfish Immun. 25, 128–136. Association of Official Analytical Chemists (AOAC), 1995. Official Methods of Analysis of Official Analytical Chemists International, 16th ed. Association of Official Analytical

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J. Lin, et al.

Sun, Y.Z., Yang, H.L., Ma, R.L., Lin, W.Y., 2010. Probiotic applications of two dominant gut Bacillus strains with antagonistic activity improved the growth performance and immune responses of grouper Epinephelus coioides. Fish Shellfish Immun. 29, 803–809. Tannock, G.W., 1998. Studies of the intestinal microflora: a prerequisite for the development of probiotics. Int. Dairy J. 8, 527–533. Trushenski, J.T., Kasper, C.S., Kohler, C.C., 2006. Challenges and opportunities in finfish nutrition. N. Am. J. Aquaculture 68 (2), 122–140. Tsuji, S., Tanaka, K., Takenaka, S., Yoshida, K., 2015. Enhanced secretion of natto phytase by Bacillus subtilis. Biosci. Biotech. Bioch. 79 (11), 1906–1914. Turnbaugh, P.J., Ley, R.E., Mahowald, M.A., Magrini, V., Mardis, E.R., Gordon, J.I., 2006. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444 (7122), 1027–1031. Uchii, K., Matsui, K., Yonekura, R., Tani, K., Kenzaka, T., Nasu, M., Kawabata, Z., 2006. Genetic and physiological characterization of the intestinal bacterial microbiota of Bluegill (Lepomis macrochirus) with three different feeding habits. Microb. Ecol. 51, 277–283. Vandenberg, G.W., Scott, S.L., De La Noüe, J., 2012. Factors affecting nutrient digestibility in rainbow trout (Oncorhynchus mykiss) fed a plant protein-based diet supplemented with microbial phytase. Aquacult. Nutr. 18, 369–379. Verschuere, L., Rombaut, G., Sorgeloos, P., Verstraete, W., 2000. Probiotic bacteria as biological control agents in aquaculture. Microbiol. Mol. Biol. Rev. 64, 655–671. Wang, Y.B., 2007. Effect of probiotics on growth performance and digestive enzyme activity of the shrimp Penaeus vannamei. Aquaculture 269, 259–264. Wang, Y.B., Li, J.R., Lin, J., 2008. Probiotics in aquaculture: challenges and outlook. Aquaculture 281, 1–4. Wang, Y.B., Xu, Z.R., 2006. Effect of probiotics for common carp (Cyprinus carpio) based on growth performance and digestive enzyme activities. Animal Feed Sci. Tech 127, 283–292. Wang, Y., Kong, L.J., Li, C., Bureau, D.P., 2006. Effect of replacing fish meal with soybean meal on growth, feed utilization and carcass composition of cuneate drum (Nibea miichthioides). Aquaculture 261 (4), 1307–1313. Wu, S., Wang, G., Angert, E.R., Wang, W., Li, W., Zou, H., 2012. Composition, diversity, and origin of the bacterial community in grass carp intestine. PLoS One 7, 1–11. Yang, M.Q., Rahimnejad, S., Zhang, C.X., Song, K., Lu, K.L., Wang, L., 2019. Histomorphology of gastrointestinal tract in bullfrog Rana (Lithobates) catesbeianaandevaluation of thechangesinducedby a soybean meal‐based diet. Aquacult. Res. https://doi.org/10.1111/are.14361. Yoo, G.Y., Wang, X., Choi, S., Han, K., Kang, J.C., Bai, S.C., 2005. Dietary microbial phytase increased the phosphorus digestibility in juvenile Korean rockfish Sebastes schlegeli fed diets containing soybean meal. Aquaculture 243, 315–322. Zhang, C.X., Feng, W., Wang, L., Song, K., Lu, K.L., Li, P., 2016b. Optimal dietary methionine requirement of bullfrog Rana (Lithobates) catesbeiana. Aquaculture 464, 576–581. Zhang, C.X., Huang, K.K., Wang, L., Song, K., Lu, K.L., Zhang, L., Li, P., 2016a. Optimal dietary carbohydrate to lipid ratio for bullfrog Rana (Lithobates) catesbeiana. Aquacult. Res. 47, 3332–3340. Zhang, C.X., Huang, K.K., Wang, L., Song, K., Zhang, L., Li, P., 2015. Apparent digestibility coefficients and amino acid availability of common protein ingredients in the diets of bullfrog, Rana (Lithobates) catesbeiana. Aquaculture 437, 38–45. Zhang, C.X., Rahimnejad, S., Wang, Y.R., Lu, K.L., Song, K., Wang, L., Mai, K.S., 2018. Substituting fish meal with soybean meal in diets for Japanese seabass (Lateolabrax japonicus): effects on growth, digestive enzymes activity, gut histology, and expression of gut inflammatory and transporter genes. Aquaculture 483, 173–182. Zhang, M.L., Zhang, M.H., Zhang, C.H., Du, H.M., Wei, G.F., Pang, X.Y., Zhou, H.K., Liu, B.B., Zhao, L.P., 2009. Pattern extraction of structural responses of gut microbiota to rotavirus infection via multivariate statistical analysis of clone library data. FEMS Microbiol. Ecol. 70 (2), 177–185. Zhou, Q.C., Mai, K.S., Tan, B.P., Liu, Y.J., 2005. Partial replacement of fishmeal by soybean meal in diets for juvenile cobia (Rachycentron canadum). Aquacul. Nutr. 11, 175–182. Zokaeifar, H., Balcazar, J.L., Saad, C.R., Kamarudin, M.S., Sijam, K., Arshad, A., Nejat, N., 2012. Effects of Bacillus subtilis on the growth performance, digestive enzymes, immune gene expression and disease resistance of white shrimp, Litopenaeus vannamei. Fish Shellfish Immun. 33 (4), 683–689.

meal with housefly (Musca domestica) maggot meal in diets for bullfrog Rana (Lithobates) catesbeiana: Effects on growth, digestive enzymes activity, antioxidant capacity and gut health. Aquaculture 499, 295–305. Ma, S., Sun, Y., Wang, F., Mi, R., Wen, Z., Li, X., et al., 2017. Effects of tussah immunoreactive substances on growth, immunity, disease resistance against Vibrio splendidus and gut microbiota profile of Apostichopus japonicus. Fish Shellfish Immun. 63, 471–479. Maruta, K.H., Miyazaki, S., Masuda, M., Takahashi, T., Marubashi, Y., Tadano, Y., Takahashi, H., 1996. Exclusion of intestinal pathogens by continuous feeding with Bacillus subtilis C-3102 and its influence on the intestinal microflora in broilers. Animal Sci. Tech 67 (3), 273–280. Merrifield, D.L., Bradley, G., Baker, R.T.M., Davies, S.J., 2010b. Probiotic applications for rainbow trout (Oncorhynchus mykiss, Walbaum) II. Effects on growth performance, feed utilization, intestinal microbiota and related health criteria postantibiotic treatment. Aquacul. Nutr. 16 (5), 496–503. Merrifield, D.L., Dimitroglou, A., Foey, A., Davies, S.J., Baker, R.T.M., Bøgwald, J., Castex, M., Ringø, E., 2010a. The current status and future focus of probiotic and prebiotic applications for salmonids. Aquaculture 302, 1–18. Mitchell, D.B., Vogel, K., Weimann, B.J., Pasamontes, L., van Loon, A.P., 1997. The phytase subfamily of histidine acid phosphatases: isolation of genes for two novel phytases from the fungi Aspergillus terreus and Myceliophthora thermophila. Microbiology 143, 245–252. Nayak, S.K., 2010. Probiotics and immunity: a fish perspective. Fish Shellfish Immun. 29, 2–14. NRC, 2011. Nutrient Requirements of Fish and Shrimp. The National Academies Press, Washington, DC, USA. Olmos, J., Ochoa, L., Paniagua-Michel, J., Contreras, R., 2011. Functional feed assessment on Litopenaeus vannamei using 100% fish meal replacement by soybean meal, high levels of complex carbohydrates and Bacillus probiotic strains. Mar. Drugs 9, 1119–1132. Papatryphon, E., Howell, R.A., Soares, J.H., 1999. Growth and mineral absorption by striped bass Morone saxatilis fed a plant feedstuff based diet supplemented with phytase. J. World Aquacult. Soc 30, 161–173. Pasteris, S.E., Bühler, M.I., Nader-Macías, M.E., 2006. Microbiological and histological studies of farmed-bullfrog (Rana catesbeiana) tissues displaying red-leg syndrome. Aquaculture 251, 11–18. Pirarat, N., Pinpimai, K., Endo, M., Katagiri, T., Ponpornpisit, A., Chansue, N., Maita, M., 2011. Modulation of intestinal morphology and immunity in Nile tilapia (Oreochromis niloticus) by Lactobacillus rhamnosus GG. Res. Vet. Sci. 91 (3), 92–97. Podolsky, S., 1998. Cultural divergence: Elie Metchnikoff’s Bacillus bulgaricus therapy and his underlying concept of health. B. Hist. Med. 72 (1), 1–27. Rahimnejad, S., Lu, K.L., Wang, L., Song, K., Mai, K.S., Davis, D.A., Zhang, C.X., 2019. Replacement of fish meal with Bacillus pumillus SE5 and Pseudozyma aphidis ZR1 fermented soybean meal in diets for Japanese seabass (Lateolabrax japonicus). Fish Shellfish Immun. 84, 987–997. Ramos, M.A., Gonçalves, J.F.M., Batista, S., Costas, B., Pires, M.A., Rema, P., Ozorio, R.O.A., 2015. Growth, immune responses and intestinal morphology of rainbow trout (Oncorhynchus mykiss) supplemented with commercial probiotics. Fish Shellfish Immun. 45 (1), 19–26. Russell, J.B., Diez, C.F., 1998. The effects of fermentation acids on bacterial growth. Adv. Microb. Physiol. 39, 205–234. Schafer, A., Koppe, W.M., Meyer-Burgdorff, K.H., Gunther, K.D., 1995. Effects of microbial phytase on utilization of native phosphorus by carp in diet based on soybeanmeal. Water Sci. Technol. 31 (10), 149–155. Sealey, W.M., Barrows, F.T., Smith, C.E., Overturf, K., LaPatra, S.E., 2009. Soybean meal level and probiotics in first feeding fry diets alter the ability of rainbow trout Oncorhynchus mykiss to utilize high levels of soybean meal during grow-out. Aquaculture 293, 195–203. Shah, N.P., 2000. Probiotic bacteria: selective enumeration and survival in dairy foods. J. Dairy Sci. 83 (4), 894–907. Sogarrd, D.H., Suhr-Jessen, T., 1990. Microbialsfor feed beyond lacticacid bacteria. Feed Int. 11 (1), 36–38 32-34. Sugiura, S.H., Gabaudan, J., Dong, F.M., Hardy, R.W., 2001. Dietary microbial phytase supplementation and the utilization of phosphorus, trace minerals and protein by rainbow trout [Oncorhynchus mykiss (Walbaum)] fed soybean meal-based diets. Aquac. Res. 32 (7), 583–592.

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