effects: To white shrimp (Litopenaeus vannamei) postlarvae and to the rearing water
Yan Cai, Wei Yuan, Shifeng Wang, Weiliang Guo, An Li, Yue Wu, Xin Chen, Zhuling Ren, Yongcan Zhou
PII: S0044-8486(18)31105-0
DOI: doi:10.1016/j.aquaculture.2018.08.024
Reference: AQUA 633466
To appear in: aquaculture Received date: 25 May 2018 Revised date: 3 August 2018 Accepted date: 13 August 2018
Please cite this article as: Yan Cai, Wei Yuan, Shifeng Wang, Weiliang Guo, An Li, Yue Wu, Xin Chen, Zhuling Ren, Yongcan Zhou , In vitro screening of putative probiotics and their dual beneficial effects: To white shrimp (Litopenaeus vannamei) postlarvae and to the rearing water. Aqua (2018), doi:10.1016/j.aquaculture.2018.08.024
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In vitro screening of putative probiotics and their dual beneficial
effects: to white shrimp (Litopenaeus vannamei) postlarvae and to the rearing water
Yan Caia,b,c, Wei Yuana,c, Shifeng Wanga,b*, Weiliang Guoa,c, An Lia,c, Yue Wua,c, Xin Chen a,c, Zhuling Rena,c, Yongcan Zhoua,b,c*
aState Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, Haikou, Hainan, 570228, PR China
bKey Laboratory of Tropical Biological Resources of Ministry of Education, Hainan University, Haikou, Hainan, 570228, PR China
cHainan Provincial Key Laboratory for Tropical Hydrobiology and Biotechnology, College of Marine Science, Hainan University, Haikou, Hainan, 570228, PR China
* Corresponding authors:
Shifeng Wang, Email: [email protected], Tel: +86-13519805769, Address:
College of Marine Science, 58 Renmin Road, Haikou, Hainan, 570228, PR China.
Yongcan Zhou, Email: [email protected], Tel: +86-13078932128, Address: College of Marine Science, 58 Renmin Road, Haikou, Hainan, 570228, PR China.
The first author is Yan Cai, the co-first author is Wei Yuan. These authors contributed equally to this work.
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Abstract
Shrimp aquaculture has been severely threatened by vibriosis. Traditional methods to combat Vibrio diseases with antibiotics and chemotherapeutics have been discouraged due to their potential negative consequences. The aim of this study was: firstly screen potential probiotics using in vitro assays; then evaluate the beneficial effects of the screened probiotics in Litopenamei vannamei postlarvae by in vivo feeding experiments. Pre-screening of 12 candidate probiotics based on criteria such as extracellular enzyme production, haemolytic activity, tolerance to bile salt, tolerance to gastrointestinal stress, inhibitory activity against Vibrio pathogens, cell surface hydrophobicity and autoaggregation resulted in two potential probiotic isolates: LS-1 and LD-1. Both isolates showed good probiotic characteristics on all criteria above.
Biochemical and 16S rRNA sequencing analysis identified LS-1 and LD-1 as Bacillus licheniformis and Bacillus flexus, respectively. In vivo feeding experiments with LS-1, or LD-1, or LS-1+LD-1 as dietary supplementation demonstrated that both strains could effectively enhance the growth, innate immune enzyme activities, digestive enzyme activities, stress tolerance and disease resistance of L. vannamei. In addition, both strains also illustrated water quality improving effects in the probiotic supplement feeding experiments. This is one of the few reports of Bacillus probiotics that display dual beneficial effects: both to the host and to the rearing water. Results from this study demonstrated that Bacillus licheniformis strain LS-1 and Bacillus flexus strain LD-1 are promising probiotic candidates to provide multiple benefits for shrimp rearing.
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Keywords:
Litopenamei vannamei; Probiotics; In vitro screening; Growth; Disease resistance; Water qualityACCEPTED MANUSCRIPT
1. Introduction
White shrimp (Litopenamei vannamei) is one of the most important cultivated shrimp species worldwide. With a production of 3,314,447 metric ton (mt) in 2013, white shrimp represented 74.4 % of total shrimp and prawn production (4,454,602 mt) globally (FAO, 2015). However, in recent years vibriosis has become the most serious threat to Penaeid shrimp production (Joseph, et al., 2015; Joshi et al., 2014;
Thitamadee et al., 2016; Zorriehzahra and Banaederakhshan, 2015). Mass mortality of larval shrimp in hatcheries has been associated with vibriosis. In the first quarter of 2013, Thailand suffered a 33% drop of shrimp production due to vibriosis (Joshi et al., 2014). In 2011, some farms in southeastern China even lost 80% of their shrimp stock to vibriosis breakouts (FAO, 2013; Zorriehzahra and Banaederakhshan, 2015).
Traditional methods to combat Vibrio diseases with antibiotics and chemotherapeutics have been discouraged due to their potential negative consequences, such as drug resistance, drug residue and environment pollution （Alcaide et al., 2005; Sarter et al., 2007）In the search for more effective and environmentally friendly treatments, probiotics have emerged as a promising alternative (Balcázar et al., 2006; Verschuere et al., 2000).
Probiotics are live microorganisms that confer health benefits on the host when administered in adequate doses (Reid et al., 2003). The mechanisms of probiotic bacteria’s beneficial actions include antagonism to pathogens, increasing immune response, enzymatic contribution to digestion, ability of cells to produce metabolites
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(like vitamins), colonization or adhesion properties, etc (Akhter et al., 2015). Based on their different administration routes and function channels, probiotics in aquaculture could be roughly divided into two large groups: a) gut probiotics, which are administered orally and produce beneficial effects through the gut and b) aquatic probiotics, which are administered into the rearing water and can improve water quality or reduce the number of pathogenic bacteria in the water (Qi et al., 2009). For screening of gut probiotics, certain characteristics were essential: the organism must be non-toxic to the host; they must be able to survive the long feed storage time and unfavorable conditions in the gastrointestinal tract of the host. In addition to these essential characteristics, the organisms should preferably possess some other beneficial functional properties, such as exclusion of pathogens and digestive enzyme activities. For aquatic probiotics, they usually possess physiological functions such as consuming extra nutrition in the rearing water, competing with potential pathogenic bacteria for nutrition or directly killing harmful bacteria. However, the most important criterion for the selection of probiotic strains, gut probiotics or aquatic probiotics, is the ability to bring benefits to the host. The benefits include: a) enhancing growth performance, b) boosting immunity, c) promoting disease resistance (Balcázar et al., 2006; Begley et al., 2006; Mishra and Prasad, 2005; Tsai et al., 2005). To date, various probiotics have been reported in aquaculture researches; however, gut probiotics account for the majority of them. There were few reported probiotics that possess the merits of both gut probiotics and aquatic probiotics (Faramarzi et al., 2011;
Newaj-Fyzul et al., 2014; Sorroza et al., 2012; Wang et al., 2008).
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In the present study, potential probiotics were firstly pre-screened from 12 candidate probiotics based on criteria such as extracellular enzyme production, haemolytic activity, tolerance to bile salt, tolerance to gastrointestinal stress, inhibitory activity against Vibrio pathogens, cell surface hydrophobicity and autoaggregation. Then the resulting 2 pre-selected putative probiotics were identified.
Finally, the effects of dietary supplementation of these putative probiotics on the growth, immune/digestive enzyme activities, stress tolerance and disease resistance of L. vannamei were evaluated. At the same time, the water quality improving effects of the 2 putative probiotics were also examined during the probiotic supplement feeding trial.
2. Materials and methods
2.1. Candidate probiotic bacteria
12 Bacillus spp. isolates (designated SY-1, SY-2, DF-1, LS-1, LS-2, LS-3, LS-4, LS-5, LD-1, LD-2, LD-3, LD-4) originated from Hainan aquaculture pond waters/sediments were found to display water quality improving properties in a previous study (Wu, 2014). These isolates were stored in 25% glycerol at − 80°C until this study was performed. Prior to experiments, the bacteria were cultured in Luriae Bertani (LB) medium at 30 °C for 18 h. The cell pellets were washed in phosphate-buffered saline (PBS, pH 7.4) twice and re-suspended in the same PBS to a final concentration of 108 CFU/mL. Before cell surface hydrophobicity and autoaggregation assays, the bacteria suspension concentration might be adjusted to
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reach an absorbance of 1.0 at 600 nm.
2.2. Evaluation of probiotic properties
Extracellular enzyme production, haemolytic activity, tolerance to bile salt, tolerance to gastrointestinal stress, inhibitory activity against Vibrio pathogens, cell surface hydrophobicity and autoaggregation were studied as selection criteria for potential probiotics. Afterwards, the selected isolates were characterized using Gram stain, cellular morphology, and DNA sequence analysis.
2.2.1. Extracellular enzyme production
The production of extra-cellular amylase, protease and lipase were studied using the agar well diffusion method (Leyva-Madrigal et al., 2011). Briefly, wells (6 mm diameter) were made on every plate with sterile borer. 60 μL of bacterial suspension was introduced into each well and plates were incubated at 30 °C for 24 h. For extra-cellular amylase production, bacteria isolates were inoculated on starch (1%) supplemented agar plates and amylase activity were observed by the clear zones around the wells after evenly spraying the culture plates with 1% Lugol’s iodine solution (Jacobs and Gerstein, 1960). For extra-cellular protease production, the isolates were inoculated on skimmed milk enriched agar plates and the clear zones on the plates indicated protease activity (Jacobs and Gerstein, 1960). For extra-cellular lipase production, the isolates were inoculated onto trioleoylglycerol and rhodamine B supplemented agar plates and lipase activity was identified by the appearance of orange fluorescent halos around bacterial colonies visible upon UV irradiation (Kouker and Jaeger, 1987).
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2.2.2. Haemolytic activity
Haemolysis activities of the bacterial isolates were analyzed on blood agar following the protocol of Luis-Villaseñor et al. (2011) with some modifications.
Briefly, the isolates were spot inoculated on haemolytic plates and incubated at 30°C for 24 h. Then hemolysis activities were determined by observing the plates: isolates that caused no change on the agar around the colonies were considered to be non haemolytic (γ-hemolysis) and isolates showing a clean zone around colonies were considered to be haemolytic (β haemolysis).
2.2.3. Tolerance to bile salt
The bile salt tolerance of isolates was determined using the method described by Swain et al. (2009) with some modifications. Each isolate was incubated (1%, v/v) into LB broth (pH 7.0) containing 0%, 0.15%, 0.30% and 0.60% (w/v) of oxgall (Sigma Chemical Co., USA), respectively. The mixtures were incubated at 30 ºC for 24 h. The total viable counts and survival rates were determined using the plate method with LB medium. LB broth with 0% oxgall served as control and survival rates of the isolates were represented in percentage.
2.2.4. Tolerance to gastrointestinal stress
Tolerance to gastrointestinal stress was assessed using the method described by Manhar et al. (2015) with some modifications. Simulated gastric fluid (SGF) was prepared by mixing pepsin (0.3 mg/mL, HiMedia) and NaCl (0.5%, w/v) in PBS (pH adjusted to 3) before sterilizing the PBS mixture by filtration (Charteris et al., 1998).
Simulated intestinal fluid (SIF) was prepared by adding pancreatin (0.1 mg/mL,
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Sigma) to PBS (pH adjusted to 6.8) before sterilization through filtration (Maragkoudakis et al. 2006). Re-suspended bacteria isolates were incubated in SGF or SIF at 30 °C. After 0, 30, 60 and 180 min of incubation, the survival rates were determined by the plate method using LB medium incubated at 30 ºC for 48 h.
2.2.5. Antibacterial activity
The antimicrobial activities of the four potential probiotic isolates were detected using Vibrio harveyi (ATCC 33843), V. vulnificus (ATCC 43382), V.
parahaemolyticus (ATCC 43996) and V. alginolyticus (ATCC 17750) as indicator strains. Antibacterial activity was determined using the agar spot method according to Schillinger and Lücke (1989). Briefly, overnight bacteria cultures were spotted onto the surface of LB agar plates, incubated at 30 °C for 24h to allow colonies to develop.
Then a layer of soft agar medium (nutrient broth containing 0.5% w/v agar) containing proximately 107 CFU/mL live indicator strains was gently poured onto the previously prepared LB agar plates. The double layered plates were then incubated at 30 °C for another 24h and inhibition halos were measured. All experiments were performed in triplicate.
2.2.6. Cell surface hydrophobicity
The cell surface hydrophobicity of the four potential probiotics was determined according to the method described by Rosenberg et al. (1980) with slight modifications. Briefly, overnight bacteria cultures were washed twice, re-suspended and the absorbance of the cell suspension was measured at 600 nm (A0). Then the cell suspension was added to the equal volume of hydrocarbon (xylene) and vortexed for 2
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min. The two phases were allowed to separate for 1 h and the aqueous phase was gently taken out to measure its absorbance at 600 nm (A1). The cell surface hydrophobicity was calculated as:
Hydrophobicity (%)= [(A0 – A1)/ A0]×100
Where A0 represents the absorbance before mixing with xylene and A1 represents the absorbance after mixing with xylene for 1 h.
2.2.7. Autoaggregation
Autoaggregation assay was performed following Del Re’s method (Del Re et al., 2000) with some modifications. The bacterial suspension (4 mL) was vortexed for 20 s and incubated at room temperature. The absorbance of the upper zone aqueous phase was measured at 600nm before and after incubation. Autoaggregation percentage was calculated as:
Autoaggregation (%) = [1- (At/A0)]×100
where At represents the absorbance at different time intervals of 1 , 2 , 3, 4 and 5 h, A0 represents the absorbance at 0 h.
2.3. Identification of selected probiotic isolates
The morphological and biochemical characteristics of selected probiotic isolates were tested based on Bergey's Manual of Systematic Bacteriology. The genetic identification was conducted using 16S rRNA sequencing. Obtained sequences were subjected to BLAST searches in the NCBI GenBank database (Altschul et al., 1990) and the phylogenetic tree was constructed by neighborjoining (NJ) method using MEGA software (Version 5.05) (Tamura et al., 2011).
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2.4. In vivo shrimp feeding trial 2.4.1. Experimental diets
A commercial shrimp feed (41% protein, 4.5% fat, 4.5% fiber and 16% ash, Evergreen Feed, Guangdong, China) was used as the basal diet. The experimental diet was formulated by supplementing the basal diet with selected putative probiotic LS-1, or LD-1, or both (LS-1+LD-1). Briefly, the bacteria were cultured in LB medium at 30 °C for 18 h. The cell pellets were washed in PBS (pH 7.4) twice and re-suspended in the same PBS to a final concentration of 2.0×1010 CFU/mL. Then bacteria suspension were thoroughly homogenized and sprayed on the basal feed pellets (100 mL bacteria suspension/kg feed) to obtain three experimental diets: LS-1(2.0×109 CFU/g diet), LD-1(2.0×109 CFU/g diet) and LS-1+LD-1(1.0×109 CFU/g diet LS-1 + 1.0×109 CFU/g diet LD-1). The control diet was prepared by adding the same volume of PBS (without probiotic bacteria) to the basal diet. The prepared diets were stored at 4ºC before use. Because preliminary experiments showed that the final concentrations of probiotic bacteria in the experimental diets could remain steady within 7 days, in our formal experiments, experimental diets were prepared every 5 days in order to guarantee the vitality of the probiotics.
2.4.2. Shrimp rearing and experimental design
Healthy L. vannamei postlarvae aged of 5 days (PL5) were obtained from a local hatchery (Dingda Mari-culture Co. Ltd., Hainan, China) and acclimated in filtered aerated seawater (salinity 25‰, 28 ± 1ºC) for a week prior to feeding experiments.
During the acclimation period, shrimp postlarvae were fed thrice daily (8:00, 16:00
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and 22:00) with commercial shrimp diet (41% protein, 4.5% fat, 4.5% fiber and 16%
ash, Evergreen Feed, Guangdong, China) at a ratio of 3% of body weight. At the beginning of the feeding trial, shrimp postlarvae (averaging weight 0.063 ± 0.001g per 20 individuals) were starved for 24h before being divided into twelve 60L-fiberglass tanks (500 shrimp postlarvae per tank) filled with aerated seawater.
The feeding trial continued for 21 days and included four treatments: control (basal diet without probiotics), LS-1(basal diet supplemented with LS-1), LD-1 (basal diet supplemented with LD-1), and LS-1+LD-1 (basal diet supplemented with LS-1 and LD-1). Each treatment consisted of three replicates. Rearing water in all tanks was not changed throughout the study period. Water temperature, dissolved oxygen, pH were maintained in the range of 28 ± 1 °C, 6.9 ± 0.5 mg/L and 7.5 ± 0.7, respectively. At the end of the 21-day feeding trial, the shrimp postlarvae grew into juvenile stage and were called juvenile shrimps accordingly.
2.5. Growth performance
At the end of the feeding trial, juvenile shrimps were fasted for 24h and 20 juvenile shrimps from each tank were randomly chosen to measure growth performance. The growth performance and survival of juvenile shrimps for all groups were calculated using the following equations:
Weight gain rate (WGR, %) = (Wt – W0) / Wt ×100
Specific growth rate (SGR, %day-1) = [lnWt – lnW0] / t ×100 Survival rate (%) = (Nt / N0) × 100
where, W0 and Wt are the initial weight (g) and final weight (g) of 20 shrimps, N0
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and Nt are initial and final number of shrimp, and t represents culture time in days.
2.6. Determination of water quality
2.6.1. TAN, NO2-N and COD concentrations
During the feeding trial, total ammonia nitrogen (TAN), nitrite nitrogen (NO2-N) and chemical oxygen demand (COD) of the rearing sea water were measured every other day before feeding time. TAN was measured by hypo-bromate oxidimetry method (Bower and Bidwell, 1978); NO2-N was measured by colorimetry method (ISO 13395, 1996); COD was measured with a ET99722 COD meter (Lovibond®,Genman).
2.6.2. Total bacteria and Vibrio spp. bacteria count in water
During the feeding trial, rearing water samples were taken every other day and the total bacteria and Vibrio spp. bacteria count in the respective water samples were determined. Water samples were serially diluted ten-fold. Then each dilution was spread onto triplicate Marine Agar 2216 (2% NaCl; Oxoid, Basingstoke, UK) and thiosulfate citrate bile salt sucrose agar (TCBS; Difco, Detroit, MI, USA). Marine Agar 2216 was used for total bacterial count and TCBS media for viable Vibrio spp.
bacteria count. After the agar plates inoculated with each dilution were incubated at 30ºC for 18h, CFU mL-1 were determined for viable bacterial populations (Dong et al., 2017).
2.7. Biochemical analyses
At the end of the feeding trial, after starving for 24 h, 20 juvenile shrimps from each tank (i.e., 20 × 3 = 60 shrimps per group) were randomly collected, rinsed with
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distilled water and homogenized in PBS (pH 7.5) at a ratio of 1:9 (w/v). Then the homogenate was centrifuged (5000×g for 15 min at 4°C) and the supernatant were stored at –80 °C for further enzymatic analysis. The lysozyme，peroxidase， alkaline phosphatase and digestive enzyme (including protease, amylase and lipase) activities were determined using commercial assay kits (Nanjing Jiancheng Institute, Nanjing, China) according to the manufacturer’s instructions. Lysozyme activity was determined by the decrease in transmittance which was caused by the lysis of bacteria (Sun et al., 2015). Peroxidase (POD) activity was estimated by detecting a colorimetric oxidation product (570 nm) from the reaction between H2O2 and a probe under the presence of peroxidase (Li et al., 2006). Alkaline phosphatase (AKP) activity was determined with p-nitrophenyl phosphate as the substrate and p-nitrophenol as the standard (Sanchooli et al., 2012). The soluble protein concentration was measured using bovine serum albumin as a standard (Bradford, 1976). Protease activity was assayed with Folin-phenol reagent using the manufacturer’s method (Biological department of Sun Yat-sen University, 1979).
Amylase activity was measured using soluble starch as the substrate and iodine solution to reveal non-hydrolyzed starch (Robyt and Whelan, 1968). Lipase activity was determined through detecting the amount of p-nitrophenyl palmitate released from the substrate (Winkler and Stuckman, 1979). All enzyme activity assays were performed in triplicate.
2.8. Low salinity stress test
At the end of the feeding trial, a low salinity stress test was conducted in 1000 mL
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beakers. Briefly, 90 juvenile shrimps from each diet group (30 shrimps from each tank) were transferred from regular seawater (25‰) to 500 mL fresh water (0‰). The stress test was performed in triplicates. All beakers were constantly aerated and no feed was provided during the test. Mortalities of juvenile shrimps were recorded every hour until shrimps in one group died out (Zheng et al., 2017).
2.9. Challenge test
Pathogen Vibrio harveyi strain GDH11385 was previously isolated from moribund Epinephelus coioides (Xu et al., 2017) and maintained as a frozen stock in tryptic soy broth with 25% (v/v) glycerol at -80 °C until needed. Prior to the challenge trial, frozen stock cultures were revived by first streaking the culture on thiosulfate citrate bile salt sucrose agar (TCBS; Difco, Detroit, MI, USA) plates and cultured at 28 °C for 16 hours, then well isolated single colonies were inoculated in Marine Broth 2216E (2% NaCl; Oxoid, Basingstoke, UK) at 28 °C for 12 hours. The broth culture was then centrifuged at 1500 ×g for 10 min at 4 °C. The bacterial pellet was harvested, washed and re-suspended in phosphate-buffered saline (PBS, pH 7.0).
Prior to challenge test, the 7-day LD50 was determined by immersing juvenile shrimps (30 shrimps per treatment) into four serial dilution (105, 106, 107 and 108 CFU mL-1) of V. harveyi and the result showed that the 7-day LD50 by immersion was 2×107 CFU mL-1. At the end of the feeding trial, 30 juvenile shrimps from each tank (each treatment consisting of three tanks as replicates, i.e. 30 shrimps/tank ×3 tanks/treatment = 90 shrimps/treatment) were immersed with live V. harveyi at the final concentration of 2×107 CFU mL-1. No feed was given to the shrimp during the
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challenge test. Mortality was monitored for 7 days after the challenge. Relative percentage survival (RPS) was calculated as: RPS = Number of surviving shrimps after challenge/Number of shrimps immersed with pathogen bacteria×100 (Lin et al., 2013).
2.10. Statistical analysis
Statistical analysis was carried out using SPSS for Windows (SPSS Inc., 22.0, Chicago, IL, USA). Normality and equality of variance of data were assessed using the Shapiro-Wilk and Levene's test, respectively. Data were arcsine or Log transformed when necessary to meet the normality and homoscedastic assumptions (Bansemer et al., 2016). Where normality and homoscedastic assumptions were met, data were analyzed using one-way analysis of variance (ANOVA) followed by a post-hoc Duncan test to determine significant differences. Where data violated the normality and homoscedastic assumptions even after transformation, the Kruskal-Wallis test was performed. Significance for all analyses was set at P< 0.05.
All data are presented as mean ± standard deviation.
3. Results
3.1. Evaluation of probiotic properties
Extracellular enzyme production tests showed that all the 12 candidate probiotic isolates displayed various degree of extracellular protease activity, with higher protease activity observed in LD-1, LS-1 and SY-1. LD-1, LS-1 and SY-1 also exhibited higher amylase activities. Besides, LD-1 also exhibited highest lipase
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activity among the 12 isolates (Table 1).
Hemolysis test showed that of the 12 isolates, only LD-1, LD-2, SY-1 and LS-1 exhibit no haemolytic activity. All the other 8 isolates were β (Beta) hemolytic.
Therefore, LD-1, LD-2, SY-1 and LS-1 were selected as potential probiotics for the following tests.
Tolerance to simulated gastric fluid tests showed that at the presence of gastric fluid, the viable counts for all 4 strains were reduced with the extension of exposure time. Among the 4 potential probiotic isolates, LD-2 were most sensitive to SGF, its viability reduced almost 25% after 180 min of exposure to SGF (Fig. A. 1A). As for tolerance to simulated intestinal fluid, all isolates showed relatively high tolerance to SIF (Fig. A. 1B).
As shown in Table 2, viable counts for all isolates were significantly reduced in bile salt. Especially for SY-1, even in the lowest bile salt concentration, no viable bacteria could be counted. However, LS-1 exhibited high bile salt tolerance. At highest bile salt concentration (0.60%), it still remained 67-68% viability.
The antagonistic properties of the 4 putative probiotic isolates against several Vibrio pathogens were presented in Table 3. LS-1, LD-1 and SY-1 demonstrated medium to high antagonism activities against 2 of the 4 Vibrio pathogens. LD-2 showed no inhibitory activity against any of the 4 Vibrio pathogens.
As reported in Table A.1, for all the four potential probiotics, the hydrophobicity values were above 50%, ranging from 55.3 to 73.2%. The autoaggregation percentages of all four isolates increased as the culture time increased, with LS-1 and
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LD-1 showing the highest values (58.5% and 52%, respectively) at 5h.
In summary, the results from above tests showed that among the 4 potential probiotic isolates, LD-2 exhibited the lowest extracellular enzyme activity levels, lowest tolerance to SGF, and no antagonism activity against pathogen bacteria; SY-1 were highly sensitive to bile salt; both LD-2 and SY-1 displayed the lowest hydrophobicity and autoaggregation ability. Therefore, based on the above results, LD-2 and SY-1 were disregarded and LS-1 and LD-1 were chosen as the finally selected putative probiotic isolates for the following identification and in vivo feeding experiments.
3.2. Characterization of the finally selected putative probiotic isolates
Gram staining indicated that both LS-1 and LD-1 were gram-positive. BLASTN search analysis of the 16S rRNA sequences was performed and results showed LS-1 had the maximum identity with Bacillus licheniformis (identity value: 99%; E value:0.0) and LD-1 had the maximum identity with Bacillus fluxus (identity value:
99%; E value:0.0). Both physiological and biochemical analyses also identified LS-1 and LD-1 as Bacillus licheniformis and Bacillus flexus, respectively (Table A.2, Fig.
1). The sequences were submitted to the GenBank and the accession numbers of the strains LS-1 and LD-1 were MH362706 and MH362707, respectively.
3.3. Growth performance
Growth performance and survival of L. vanmamei fed different experimental diets were shown in Table 4. At the end of 21 days of feeding trial, final length, final weight and specific growth rate (SGR) of three probiotic supplemented groups were
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all improved comparing to those of control group (P < 0.05), however, the increases were only significant in shrimps fed LS-1 and LS-1+LD-1; they were not significant in shrimps fed LD-1 diet. In addition, the survival rates of all three probiotic supplemented groups were also significantly enhanced (P < 0.05) and the highest significant value (69.31±0.04%) was obtained in shrimp group fed diet LS-1.
3.4. Water quality of the rearing water 3.4.1. TAN, NO2-N and COD concentrations
For most of the feeding trial period, TAN and COD concentrations were significantly lower in probiotic supplemented groups compared to the control group.
While in the same time, NO2-N concentrations did not show significant differences between control and probiotic treated groups (Fig. 2A-C).
3.4.2. Total bacteria and Vibrio spp. bacteria count
Results showed that during the whole feeding trial period, the total bacteria counts in the rearing water were not significantly different between the probiotic supplemented groups and control (Fig. A.2). However，Vibrio spp. bacteria counts were significantly reduced in the probiotic supplemented groups in comparison with control since the first day on (Fig. 2D).
3.5. Biochemical analyses
3.5.1. Immune-related enzyme activities
AKP activities in the three probiotic supplemented groups were all significantly increased when compared with control. Within the three probiotic supplemented groups, LS-1 showed the highest AKP activity level. For POD activity, only LD-1
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supplemented group was significantly higher than control. LS-1 and LS-1+LD-1 groups showed no significantly differences. Similarly, the only significantly higher LSZ activity was observed in LD-1 supplemented group. The LSZ activities in the other two probiotic supplemented groups were not significantly different from control (Table 5).
3.5.2. Digestive enzyme activities
Protease and lipase activities of juvenile shrimps in LS-1 group increased more than three times that of control group. Protease and lipase activities of juvenile shrimps in the other two probiotic supplemented groups (LD-1 group and LS-1+
LD-1 group) were not significantly different from control. Amylase activities of juvenile shrimps in all three probiotic supplemented groups showed no significant differences when compared with control (Table 5).
3.6. Stress Tolerance
Fresh water stress test showed that diet supplement of LS-1, LD-1 or LS-1+LD-1 could improve the freshwater stress tolerance of L. vanmamei. The cumulative mortalities of all three probiotic added groups were significantly lower than the control group on the last hour of the stress test (P < 0.05) (Fig. 3).
3.7. Challenge test
The challenge test showed that the survival rates of juvenile shrimps in all three probiotic supplemented groups were significantly higher than that of shrimps in control group (P < 0.05). However, survival rates showed no differences among LS-1, LD-1 and LS-1+LD-1 groups (P > 0.05) (Fig. 4).
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4. Discussion
4.1. Antagonism of Bacillus spp. strains
The isolated strain LD-1 and LS-1 were identified as B. licheniforms and B. flexus.
Antagonism tests showed that LD-1 and LS-1 displayed marked antagonistic activity against pathogenic Vibrio strains such as, Vibrio harveyi, Vibrio vulnificus, Vibrio parahaemolyticus and Vibrio alginolyticus. The antagonistic activity of various Bacillus spp. has been reported in many previous studies. For example, Liu et al.
(2014) found that Bacillus subtilis could effectively inhibit the growth of V.
anguillarum, V. vulnificus, V. alginolyticus, V. harveyi and V. parahaemolyticus.
Irasema et al. (2011) reported inhibitory activities of four Bacillus strains against V.
harveyi, V. parahaemolyticus, V. campbelli, V. alginolyticus, and V. vulnificus. Similar results were also obtained by Decamp et al. (2008), Balcázar et al. (2007) and Chandran et al.(2014), all of whom reported that species of the genus Bacillus showed inhibitory activity against various shrimp Vibrio pathogens. Various causes might attribute to the inhibitory effects of Bacillus to pathogenic Vibrio, including:
production of bacitracin, gramicidin S, polymyxin, or tyrothricin; competition of essential nutrients; alteration of the pH in the growth medium; and production of volatile compounds (Chaurasia et al., 2005; Drablos et al., 1999; Gullian et al., 2004;
Morikawa et al., 1992; Perez et al., 1993; Yilmaz et al., 2006). The exact causes of the antagonistic activities of LD-1 and LS-1 might include one or a few of the above mechanisms and it should be further explored.
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4.2. Water quality controlling effect
Inappropriate water quality is a major factor that contributes to the outbreaks of shrimp diseases. Not only the high abundance of pathogenic bacteria in the rearing water could directly infect shrimps (Thakura and Lin, 2003), the increasing concentrations of toxic ions such as ammonium and nitrite, resulting from the accumulation of residual food and fecal matter, would adversely affect the health status of cultivated shrimp, weakens its immune system and render the cultivated shrimp vulnerable to pathogenic infections and consequently high mortality (Chumpol et al., 2017; Lightner et al., 2006; Thakura and Lin, 2003). Better water quality control is recommended in shrimp aquaculture and probiotics has showed a great potential in eliminating harmful bacteria as well as decreasing the concentrations of toxic ions in shrimp rearing ponds (Boyd and Gross, 1998; Gomez-Gil et al., 2000;
Nimrat et al., 2012). For instance, a mixed purple nonsulfur bacteria were found to be a good candidate for applying in white shrimp cultivation to maintain water quality since they significantly decreased the levels of NH4+
, NO2-
, NO3-
and COD compared to control (Chumpol et al., 2017). Dalmin et al. (2001) declared that some gram-positive Bacillus spp. were able to transform organic matter to CO2, thus removing organic matter from culture systems (Dalmin et al., 2001). Aerobic denitrification performances were also reported in certain Bacillus spp., such as B.
cereus, B. subtilis, B. coagulans and B. licheniform (Joong et al., 2005; Song et al., 2011). In this study, though B. licheniform and B. flexus were not directly added into the water, they unavoidably entered the rearing water when shrimp diet pellets mixed
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with probiotic bacteria were spread into the rearing water to feed the shrimp postlarvae. Because the only differences between control group and experimental group water treatments were: for experimental groups, candidate probiotics entered the rearing water along with basal feed pellets during the feeding process while for control groups, only basal feed pellets were added, we inferred that the differences in water quality between control and experimental groups were induced by the action of candidate probiotics.
4.3. Growth and survival
In the present study, substantial enhancement of growth and survival rates was observed in LS-1 and LD-1 supplemented groups over controls. This was in accordance with many other researches investigating the effects of Bacillus as probiotics (Boonthai et al., 2011; Far et al., 2009; Nimrat et al., 2011; Utiswannakul et al., 2011). The growth enhancing effects of probiotics may be attributed to its ability to release digestive enzymes, production of vitamins, breakdown of indigestible components, prevention of intestinal disorders, up-regulation of growth-related gene expressions and reducing the severity of cellular stress (Avella et al., 2010; Doan et al., 2018; Hoseinifar et al., 2015; Lara-Flores, 2011; Liu et al., 2017; Liu et al., 2012;
Shaheen et al., 2014). In the present study, the consistency of significantly enhanced digestive enzyme activities in shrimps and significantly increased shrimp growth indicated that digestive enzymes play an important role in shrimp growth. However, though both LS-1 and LD-1 could produce exo-enzymes, the exact mechanism of LS-1 or LD-1’s growth promoting effects were not clear.
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4.4. Digestive enzyme activity
Since digestive enzymes are very efficient at metabolizing a large variety of carbohydrate, lipids, and proteins (Liu et al., 2009; Zokaeifar et al., 2012), it is widely believed that higher digestive enzyme activities in the digestive tract can enhance the digestive capability and growth performance of the host and that the level of digestive enzyme activity is a useful comparative indicator of food utilization rate, digestive capacity, and growth performance of the host (Suzer et al., 2008; ten Doeschate and Coyne, 2008; Ueberschar, 1991). Our results agreed with above opinions. Our experiment showed that shrimps with significant higher digestive enzyme activities (LS-1 group) also exhibited significant higher growth rate comparing to control. In contrast, in LD-1 group non-significant higher digestive enzyme was accompanied with non-significant higher growth rate. However, it is not clear how LS-1 or LD-1 enhanced the digestive enzyme activities in the shrimps. One explanation is: since exo-enzymes (such as proteases, lipase and amylases) are very efficient at metabolizing a large variety of carbohydrate, lipids, and proteins (Liu et al., 2009), it was the exo-enzymes produced by the probiotics that enhanced the digestive capacities of the host (ten Doeschate and Coyne, 2008; Zokaeifar et al., 2012). This theory was partially supported by a number of Bacillus studies which have shown that numerous Bacillus spp. with exo-enzyme activities, like LS-1 or LD-1 in the present study, can effectively improve the host's growth performance. And it was believed that the exo-enzymatic activity of Bacillus spp. is a main reason for its ability to improve digestive enzyme activities of the host (Han et al., 2015). The second explanation for
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action of probiotics in promoting the inner-gut digestive enzyme activities is: the dietary supplementation of probiotics stimulates the endogenous digestive enzyme secretion in the host, thus enhancing the digestive capacity, and growth performance of the host (Dawood et al., 2016; Suzer et al., 2008; Wu et al., 2012; Ziaei-Nejad et al., 2006). In the present study, both LS-1 and LD-1 produce exo-enzymes, and both strains could enhance the digestive enzyme activities in the shrimps. Seen from this respect, our results were in accordance with the first explanation. However, we also noticed that the exo-enzyme production capacities of the two stains were not proportional to the digestive enzyme activity level enhancement they induced in shrimp. Though LD-1 possess high enzyme activities (protease+++, Lipase+++, amylase+++), shrimps in the LD-1 group only exhibit non-significant digestive enzyme increase. On the other hand, LS-1, with minor lipase activity and high amylase activity (protease++++, lipase+, amylase+++), induced significant increases in the lipase activities and non-significant change in amylase activity in shrimp.
Therefore, the first theory (exo-enzyme production) alone couldn’t explain our results.
However, it might not be merely the second theory either. Several other factors, such as differentially reduced survivals of probiotics in the host gut, and the change of the exo-enzyme production capacity of probiotics in the inner gut environment of the host might have also played a role in deciding the inner-gut digestive enzyme activity levels of the host. To fully explain the mechanism of exo-enzyme producing probiotics’ actions on host, more specifically designed in vivo experiments should be conducted in future.
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4.5. Stress tolerance and disease resistance
Fresh water stress and Vibrio pathogen challenge test demonstrated that diet supplement of LS-1, LD-1 or LS-1+LD-1 could significantly improve the stress tolerance and disease resistance of L. vanmamei. This could be partially explained by the significantly enhanced immune-related enzyme activities observed in LS-1, LD-1 and LS-1+LD-1 supplemented groups. Mix of LS-1 and LD-1 didn’t seem to bring any synergic effect since the best survival, stress tolerance and disease resistance performances were not observed in mix probiotic group. Similarly, numerous previous studies have reported that supplementation of Bacillus probiotics promoted the stress tolerance, survival rate, immune responses and disease resistance of aquatic animals.
Rengpipat et al. (2000) determined that the use of Bacillus spp. provided disease protection for tiger shrimp (P. monodon) by activating both cellular and humoral immune defenses. B. coagulans supplemented at a certain concentration could significantly increase survival rate of L. vannamei larvae (Zhou et al., 2009). Dietary supplement of B. subtilis HAINUP40 can effectively enhances the innate immune responses and the protection against Streptococcus agalactiae infection in Nile tilapia (Liu et al., 2017). Administration of B. velezensis also significantly enhanced various Nile tilapia innate immune parameters as well as its resistance against S. agalactiae infection (Doan et al., 2018). B. cereus could enhance various immunological parameters in P. monodon (Chandran et al., 2014). Dietary supplementation of B.
licheniformis promoted abalones’s resistance to V. parahaemolyticus infection (Gao et al., 2018).
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5. Conclusions
In summary, probiotic properties of 12 Bacillus spp. isolates (including extracellular enzyme production, haemolytic activity, tolerance to bile salt, tolerance to gastrointestinal stress, inhibitory activity against Vibrio pathogens, cell surface hydrophobicity and autoaggregation) were evaluated using in vitro experiments and two isolates (LS-1 and LD-1) with good probiotic potentials were finally selected and subsequently identified as B. licheniformis and B. flexus. The following in vivo feeding experiments demonstrated that these two stains not only improved the water quality of the rearing water, they also enhanced the growth, survival, digestive enzyme activities, immune response, stress tolerance and disease resistance of L.
vannamei postlarvae.
Appendices
Table A.1 Hydrophobicity and Autoaggregation ability of the 4 potential probiotic isolates.
Table A.2 Physiological and biochemical characteristics of LS-1 and LD-1.
Fig. A.1 Tolerance to gastrointestinal stress.
Fig.A.2 Variation of total bacteria count in the rearing water.
Acknowledgments
This study was funded by the National Natural Science Foundation of China (No.
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31560725, No. 41666006); the Special Program for Marine Science and Technology o f Hainan (No. 2015XH02).
Appendix Figures
Fig. A.1 Tolerance to gastrointestinal stress. (A) in simulated gastric fluid;
(B) in simulated intestinal fluid.
a
a a
a
b a a
b b
a
b b
b
b b b
0 2 4 6 8 10
LS-1 LD-1 LD-2 SY-1
Viable Counts (Lg CFU/mL)
Strains
Simulated gastric fluid
0 min 30 min 60 min 180 min
a
a
a a
a a
b b
a
b
c
c a
c
d d
0 2 4 6 8 10
LS-1 LD-1 LD-2 SY-1
Viable Counts (Lg CFU/mL)
Strains
Simulated intestinal fluid
0 min 30 min 60 min 180 min
A
B
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Fig.A.2 Variation of total bacteria count in the rearing water.
Appendix Tables
Table A.1 Hydrophobicity and Autoaggregation ability of the 4 potential probiotic isolates.
Isolates Hydrophobicity (%) Autoaggregation (%)
1h 2h 3h 4h 5h
LD-1 73.2±8.02 11.2±2.41a 34.7±6.48b 36.1±3.96b 49.5±4.4c 52±3.50c LS-1 64±8.25 26.2±0.47a 31.0±1.53ab 35.1±4.50b 51.6±3.67c 58.5±3.23d LD-2 55.3±7.97 12.3±1.29a 16.6±1.89b 21.2±1.72c 27.4±0.58d 25.8±1.99d SY-1 55.8±17.86 19.9±0.15a 27.3±0.78b 34.7±0.32c 37.0±1.68d 46.3±0.17e Values (means ± SD, n=3) with different superscript letters in a row show significant differences (P < 0.05). The same as below.
Table A.2 Physiological and biochemical characteristics of LS-1 and LD-1.
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and biochemical tests
Bacillus licheniformis Bacillus flexus LS-1 B. licheniformisa LD-1 B. flexusa
Growth at 50 ºC + + + +
Tolerance to
7%NaCl + + + +
Motility + + + +
Starch
Hydrolysis + + + +
Gelatin
Liquefaction + + + +
Voges-proskauer + + + +
Citrate + + + +
Nitrate + + - -
Catalase + + + +
Propionate + + + +
D-glucose + + ND ND
L-arabinose + + + +
D-mannitol + + + +
D-xylose + + + +
“+”: positive; “-”: negative; a : phenotypic characteristics as described in Bergeys' manual of systemic bacteriology.
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