Effects of Bacillus velezensis Supplementation on the Growth Performance, Immune Responses, and Intestine Microbiota of Litopenaeus vannamei

In the present study, Bacillus velezensis (BV007, CGMCC No. 20039) was isolated from the gut of Litopenaeus vannamei, and the effects of BV007 on the growth performance, immune responses, and intestine microbiota of the shrimp were investigated. A total of 1,200 healthy shrimp (3.0 ± 0.3 cm, 0.32 ± 0.8 g) were randomly divided into four groups, and fed diets supplemented with different levels of BV007 (C: 0; BV1: 1 × 105 CFU/g; BV2: 1 × 107 CFU/g; and BV3: 1 × 109 CFU/g) for 8 weeks. The results showed a significantly increased final body length (FBL), length gain rate (LGR), final body weight (FBW), weight gain rate (WGR), plumpness index (PI), and specific growth rate (SGR) in shrimp fed with BV007 for 42 days compared with shrimp fed with control diet. The activity of α-amylase in hepatopancreas was also significantly increased in the BV007-administered groups. After 42 days of growth trial, the challenge test with Vibrio parahaemolyticus was conducted for 2 weeks. The enhanced immune responses were exhibited by shrimp fed with BV007 after V. parahaemolyticus challenge, particularly in respiratory bursts and superoxide dismutase, catalase, and alkaline phosphatase activities. Moreover, the administration of BV007 could considerably increase the abundance of potential probiotics (Bacillus) and reduced the abundances of potential pathogenic bacteria (Vibrio) in shrimp intestines. In conclusion, the dietary supplementation with B. velezensis BV007 could promote the growth performance, enhance the immune responses, and modulate the intestine microbiota of shrimp, and 107 CFU/g feed was recommended to be used as a feed additive to enhance the growth and health status of shrimp.


INTRODUCTION
The Pacific white shrimp Litopenaeus vannamei is one of the major cultivated aquaculture crustacean species worldwide. In recent years, shrimp aquaculture has developed rapidly and intensively, but the low growth rates and disease have occurred more frequently, causing considerable economic losses (Neiland et al., 2001). The use of hormones and antibiotics as a traditional way to prevent and treat infectious diseases has led to the evolution of pathogenic bacteria and environmental pollution. Great effort has been invested to develop the potential alternatives to antibiotics to avoid the creation of bacterial resistance and high antibiotic residues. Currently, probiotics, prebiotics, and medicinal herbs have shown to be a vital alternative additive for the improvement of host health (Raman et al., 2019;Dawood et al., 2020;Zhang et al., 2021).
Probiotics are valuable in animal production due to their improvement of feed value, enzymatic contribution to digestion, and activation of the immune responses (Gao et al., 2018;Qin et al., 2020). For example, oral administration of Bacillus PC465 enhanced the growth performance and survival rate of L. vannamei (Chai et al., 2016). Dietary supplementation with B. licheniformis at 10 5 CFU/ml for 8 weeks enhanced abalones growth and enhanced disease resistance to Vibrio parahemolyticus (Gao et al., 2018). Moreover, the activities of glutamic-pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT) were significantly lower in shrimp fed with Enterococcus faecalis supplemented groups as compared with control shrimp . In addition, dietary Bacillus amyloliquefaciens A23 at 10 8 CFU/g significantly enhanced the intestinal microbial diversity of Procambarus clarkii (Xu et al., 2021). Besides the promotion of Lac. pentosus, BD6 feeding can increase the relative abundance of beneficial bacteria and reduce the abundance of harmful pathogenic bacteria in the gut flora of shrimp, thus regulating the host immune system (Lin et al., 2017).
Although many studies suggest that B. subtilis possesses a number of beneficial traits when used as a supplement in the shrimp diets (Liu et al., 2010;Fu et al., 2011;Zokaeifar et al., 2012Zokaeifar et al., , 2014Interaminense et al., 2018Interaminense et al., , 2019, few studies have investigated the appropriate dose of B. velezensis in L. vannamei farming. Therefore, the current study aimed to find the optimum supplementation level of B. velezensis and to investigate the potential effect on the growth performance, feed utilization, immune responses, and gut microbiota of L. vannamei.

Bacterial Strain and Diet Preparation
Bacillus velezensis (named as BV007) was previously isolated from healthy L. vannamei intestines. The intestines were immersed in sterile phosphate buffered saline (PBS) (0.05 M, pH 7.4), shook, and eluted at 30 • C for 30 min. Spread the eluate on LB nutrient agar (Beijing Land Bridge Tech. Co. Ltd.) plates with gradient dilution and incubated at 30 • C for 24 h. After picking a single colony and streaking for purification, the strain 007 with a wide range of enzyme-producing abilities was obtained.
The genetic characterization of B. velezensis was accomplished by using 16S rDNA sequence analysis, and the strain was deposited by the China General Microbiological Culture Center (CGMCC no. 20039).
In the diet preparation, the bacterial strains were inoculated in nutrient broth, collected in sterilized water, and adjusted to 10 9 CFU/ml before feed production. The ingredients of the basal diet were measured to contain 41.35% crude protein and 7.67% crude lipid with fish meal, corn gluten meal, and soybean meal as the fundamental protein source whereas the main lipid sources were soybean oil and soy lecithin oil. Three experimental diets were supplemented with different levels of BV007: 10 5 CFU/g (BV1), 10 7 CFU/g (BV2), and 10 9 CFU/g (BV3). The group without BV007 was served as a control (C). The bacterial amounts of experimental diets were also confirmed by the plate count method, and the colonies that emerged on the plates were counted. The four formulated experimental diets were extruded through a 1.2-mm die. The resulting pellets were dried at 25 • C with the aid of an air conditioner. After drying, all diets were stored at 4 • C until usage.

Experimental Design and Daily Management
Healthy L. vannamei (body length: 3.0 ± 0.3 cm, body weight: 0.32 ± 0.8 g) were purchased from a local farm (Yantai, Shandong Province, China) and acclimated at 25-27 • C and 30% of salinity for 2 weeks before processing. The shrimp were randomly dispersed into 12 fiberglass tanks (60 L), each containing 100 shrimp. The shrimp were fed four times daily (06:00, 12:00, 17:00, and 22:00) at 6% of body weight. The cumulative mortality of the shrimp was recorded daily. After 42 days of feeding, the total number of shrimp and the weight in each tank was quantified to calculate the survival rate (SR, in percentage), final body weight (FBW), final body length (FBL), plumpness index (PI), specific growth rate (SGR, %/d) according to the methods described previously (Velmurugan et al., 2015;Amoah et al., 2019b).

Sample Collection
Hemolymph samples (eight replicates were tested in each treatment, and three shrimps were tested in each replicate [n = 24]) were individually withdrawn from the pericardial cavity of the shrimp using a 1-ml sterile syringe containing an anticoagulant (30 mM trisodium citrate, 0.34 M sodium chloride, and 10 mM EDTA at pH 7.55 with an osmolality adjusted to 780 mOsm/kg with glucose). The volume ratio of anticoagulant to hemolymph was 1:1. The harvested hemocytes were centrifuged at 4 • C, 4,000 rpm/min for 10 min, collected, and adjusted to 10 6 cells/ml with PBS (0.05 M, pH 7.4) for the phagocytic activity and respiratory burst assays. In addition, the hepatopancreas was collected for the assay of digestive, antioxidant, and immunerelated enzyme activities, eight replicates were tested in each treatment, and three shrimps were tested in each replicate (n = 24). The middle intestines and hepatopancreas of each group were collected sterile into 4% paraformaldehyde and stored at 4 • C for the analysis of microorganisms (three parallel assays).

Phagocytosis Assay
The phagocytosis assay was adapted from the method of Delaporte (Delaporte et al., 2003) with minor modifications. Briefly, the hemolymph was mixed with 2.3% yellow-green FluoSpheres (diameter 2.0 µm, Polyscience, Eppelheilm, Germany), and incubated in the dark at 18 • C for 1 h with rotation. After incubation, the hemocytes were analyzed by flow cytometry (BD Accuri C6 Plus, BD Biosciences, America) using the FL-1 tunnel to detect hemocytes containing fluorescent beads. The phagocytosis rates were expressed as the percentage of hemocytes that engulfed three or more beads.

Respiratory Burst Activity
Respiratory burst activity was analyzed according to the protocols described by Kalgraff et al., 2011. Briefly, hemocytes (1 × 10 6 cells/ml) were challenged by phorbol 12-myristate 13acetate (PMA; final concentration of 0.1 µg/ml) for 10 min. Subsequently, dihydrorhodamine 123 (DHR 123) was added to a final concentration of 2 µg/ml and then incubated for 30 min prior to flow cytometry analysis. The flow cytometry analyses were done on a BD FACSCalibur flow cytometer equipped with a 15 mW 488 nm argon ion laser. Further data analyses were done using FCS Express 3 software (De Novo Software, CA, USA).

Real-Time PCR Analysis
Total RNA was isolated from hepatopancreas of different treatments using RNAiso (TaKaRa, Japan) reagent. cDNA synthesis was performed according to Promega M-MLV RT Usage information. The temporal expression profiles were carried out with an Applied Biosystem 7500 fast real-time PCR system (Applied Biosystems, MA, USA) using SYBR Green I (Biotek, VT, USA). The PCR amplification program was 95 • C for 10 min, then 40 cycles of 95 • C for 15 s, 60 • C for 1 min, followed by 95 • C for 15 s, 60 • C for 1 min, and 95 • C for 15 s. At the end of each PCR, a dissociation curve analysis of amplification products was performed to confirm the purity of the PCR product. The 2 − CT method was used to analyze the expression levels (Livak and Schmittgen, 2001). All data were given in terms of relative mRNA expressed as mean ± SD (N = 6). The primers for qRT-PCR of genes for antioxidant-related genes (cat and sod), antibacterial peptides genes (crustin and penaiedin 3a), pattern recognition receptors (lgbp and lec), along with the housekeeping gene β-actin, are shown in Table 1.

Histopathological Examination of the Intestine and Hepatopancreas
Three similar-sized shrimp from each tank were sampled and dissected to obtain the midgut and the hepatopancreas (5 mm 3 ). The fixed intestinal and hepatopancreas samples were dehydrated in a graded series of ethyl alcohol and embedded in paraffin. Then, the paraffin blocks were sliced transversely into 5-µm thick sections and stained with H&E. The villus length of intestinal slice and the cell type of hepatopancreas slice were measured by a light microscope with a computerized image system (Olympus, DP73, Tokyo, Japan).

Intestinal Microbial Composition
The intestinal community of the micro-organisms was performed according to Suo (Suo et al., 2017) with slight changes. The total DNA of microbes in the intestine was extracted directly with the E.Z.N.A. Stool DNA Kit (Omega Bio-tek, Inc., GA, USA) (Xin et al., 2015). The amplification and sequencing of the hypervariable region (V4 + V5) of the bacterial 16S DNA gene was performed for the sequencing analysis and species identification. High-throughput sequencing was performed on the IonS5TMXL platform (Novogene, China). The sequencing reads were assigned to each sample according to the individual unique barcode. The sequences were analyzed with the QIIME software package (Quantitative Insights Into Microbial Ecology) and the UPARSE pipeline (Caporaso et al., 2011). The reads were first filtered and clustered into operational taxonomic units (OTUs) at an identity threshold of 97%. The alpha and beta diversity analyses were then performed as described by Amoah et al. (2019b).

Vibrio Parahaemolyticus Challenge
Vibrio parahaemolyticus was used for the challenge experiment (Amoah et al., 2019b). At the end of the culture experiment, 30 shrimps from each tank were injected with V. parahaemolyticus (final concentration 2×10 7 CFU). In each tank, 20 shrimps were used for cumulative mortality assay, which was recorded daily for 14 days. The other 10 shrimps were cultured for immunoassays and 1 week after bacterial challenge, both the hemocytes and hepatopancreas were sampled and analyzed as described above. In addition, each treatment group was continuously fed with the experimental diets. The cumulative mortality rate was calculated following the formula of Liu et al. (2017).

Statistical Analysis
The experimental data are presented as the mean ± SE. The data were statistically analyzed for significance using a one-way ANOVA, and then the differences among the means at P < 0.05 were tested with Duncan's multiple range test using SPSS 23.0 (SPSS Inc., 2005, IL, USA).

RESULT Growth Performance and Survival
The growth performance and survival of L. vannamei fed experimental diets are shown in Table 2. The results showed that FBL, FBW, PI, and SGR were significantly improved by the different levels of probiotics supplementation, whereas no significant differences were observed in SR among the control and probiotic diets.

Phagocytic and Respiratory Burst Activity
After 42 days of feeding, there was a significant increase in the phagocytic activity of shrimp fed diets supplemented with B. velezensis as compared to the control group ( Figure 1A). However, no significant increase in the rates of phagocytosis was found in either the probiotics-supplemented or nonsupplemented shrimps after the V. parahaemolyticus challenge ( Figure 1B). Compared with the control group, significantly reduced reactive oxygen species (ROS) production was observed in the BV3 group, but not in the BV1 or BV2 groups ( Figure 1C).
In contrast, the production of ROS by hemocytes decreased statistically in the BV1, BV2, and BV3 groups when shrimp were challenged with V. parahaemolyticus (P < 0.01; Figure 1D).

Digestive Enzyme Activities
As shown in Table 3, the specific activity of the α-amylase enzyme was enhanced in B. velezensis-supplemented groups.
As compared with the control group, the AMS activity was enhanced significantly in shrimp fed BV1, BV2, and BV3 diets with the highest activity observed in the BV2 group (P < 0.01). However, no significant differences were observed in TRP or LPS activities among any of the groups. In addition, the GOT and GPT activities were significantly reduced in the BV3 and BV1 groups as compared with the control group, respectively (P < 0.05).

Immune-Related Enzyme Activities
The activities of ACP showed no differences among the tested groups after being challenged with V. parahaemolyticus (Table 4). Similarly, no significant difference in AKP activity  was detected among the BV1, BV2, and BV3 groups. However, the AKP activity was enhanced significantly after V. parahaemolyticus challenge among the tested groups (P < 0.01; Table 4). Both the CAT and SOD activities were enhanced in B. velezensis-supplemented groups and in the groups challenged with pathogenic bacteria ( Table 4). The highest SOD and CAT activities were observed in the BV2 (P < 0.01; Table 4) and BV3 (P < 0.01; Table 4) groups.

Expression of Immune-Related Genes
As revealed in Figure 2, the feeding dosage of B. velezensis had a significant effect on the expression levels of immune-related genes. The expression levels of cat were significantly induced in BVs groups (P < 0.05, Figure 2A). After V. parahaemolyticus challenge, the cat transcripts in BV1 group were significantly higher than other groups (P < 0.05, Figure 2A). As concerned to sod expression, the higher expression levels were observed in BV2 and BV3 groups compared with C and BV1 groups, while 4 | Immune-related enzyme activities in the hepatopancreas of L. vannamei fed diets containing 0 CFU/g (C), 10 5 CFU/g (BV1), 10 7 CFU/g (BV2), or 10 9 CFU/g (BV3) B. velezensis for 6 weeks and then, infected with 10 7 CFU/ml V. parahaemolyticus for one week (use words with a superscript comma). the levels were significantly upregulated in BV1 and BV2 groups after the bacterial challenge (P < 0.05, Figure 2B). No significant changes in the expression levels of antibacterial peptides (crustin and penaeidin 3α) were observed among C, BV1, BV2, and BV3 groups. After the bacterial challenge, the expression levels of crustin and penaeidin 3α in BV2 were significantly higher than the other groups (P < 0.05, Figures 2C,D). In addition, the expression levels of lgbp and lec were highly enhanced regardless of whether the shrimp were bacterial challenged, especially in BV1 and BV2 groups (P < 0.05, Figures 2E,F).

Intestinal and Hepatopancreas Morphology
The intestinal and hepatopancreas morphometric parameters were presented in Figure 3 and Table 5. The liver tubule of hepatopancreas in the BV addition groups are arranged relatively tightly and significantly generally star-shaped or polygonal (P < 0.05), while the lumen of the control group is mostly circular. Besides, the control group distributed many differentiated E cells (embryonic cells), while the BV addition group distributed more B cells (secreting cells), F cells (fibrocyte), and R cells (absorbing cells). The shrimps from all dietary treatments had an intact epithelial barrier with extensive mucosal folds and abundant Microvilli. The mucosal structure of the control group was slightly loose, the length of intestinal villi was significantly shorter (P < 0.05), and the number of mucosal epithelial cells was less. The intestinal tissue structures of the BV addition groups were relatively complete, and the length of the intestinal villi was increased and closely connected with the intestinal wall.

Richness and Diversity of Intestinal Microflora
The alpha diversity was calculated using the Shannon and Simpson indices and the Chao1, ACE, and PD whole tree estimators ( Table 6). The nonparametric species-richness estimator Chao1 showed a minimum OTUs count of 287 in the BV1 group and a maximum count of 730 in the BV2 group (P < 0.05). The highest level of supplemented probiotics (10 9 CFU/g) also had a higher OTU count than the control group (P < 0.05). The number of observed species, Shannon index, and ACE showed similar trends with BV3 exhibiting significantly higher values than the other groups. Phylogenetic diversity was measured using the PD whole tree estimator, which ranged from 26.50 to 52.64. All these indices suggested that the BV2 and BV3 groups had a higher microbial diversity in their intestines, whether measured via richness or evenness. A Venn diagram was constructed to identify the OTUs held in common or unique to shrimp under different diets. In this regard, 961 OTUs were shared among all the shrimp gut samples (Figure 4). In contrast,43,49,197,and 245 OTUs were unique to the C, BV1, BV2, and BV3 diets, respectively. This observation suggests that the exposure of shrimp to different graded probiotics led to the selection of unique microbial populations.

Bacterial Composition of Intestinal Microflora
The bacterial composition at the genus level in the intestine of L. vannamei is represented in Figure 5. The most abundant genera in the shrimp intestine were Candidatus Bacilloplasma, Vibrio, Pseudoalteromonas, Bacillus, and Tenacibaculum. After 42 days of feeding, the relative abundance of the predominant genera differed between the control and the groups fed with the BV2 and BV3 diets. In particular, the proportions of Vibrio and Pseudoalteromonas in the BV2 group (11.7 and 5.7%) were decreased compared with the C group (30.2 and 10.9%), BV1 group (15.2 and 10.7%), and BV3 group (17.9 and 8.8%). In addition, the proportion of Bacillus in the BV2 group (7.7%) was significantly higher than that in the C group (0.2%, P < 0.05).

Challenge Test
After the 2-weeks challenge with V. parahaemolyticus, the cumulative mortality rates of L. vannamei were shown in Figure 6. It was observed that the cumulative mortality was significantly lower in the treated groups than in the untreated ones, that is, 97.2, 61.1, 30.6, and 51.4% for shrimps fed with the C, BV1, BV2, and BV3, respectively. The results were shown as mean ± SE (N = 6).

DISCUSSION
Many strains of some Bacillus sp. are currently used as probiotic dietary supplements in aquatic animal feeds (Foysal and Lisa, 2018;Kewcharoen and Srisapoome, 2019;Kuebutornye et al., 2019;Zhai et al., 2019;Zhou et al., 2019;Liu et al., 2020). These probiotics not only produce certain essential micronutrients that promote better growth and feed utilization of the hosts (Xie et al., 2019) but also participate in the digestion processes that break down nutrients, such as carbohydrates, proteins, and lipids by producing extracellular enzymes (e.g., amylase, trypsin, lipase) Zhang et al., 2020). In this study, a basal diet with continuous supplementation of B. velezensis was shown to increase the growth performance of shrimp, which is consistent with the results of previous studies in tilapia (Galagarza et al., 2018;Hassaan et al., 2018), carp (Fan et al., 2018;Jiang et al., 2019), and grouper (Li et al., 2019b). The observed improvement in growth performance could be ascribed to the enhanced intestinal digestive enzyme activities (Bokkenheuser et al., 1988;Xu et al., 2003;Fu et al., 2018). In the present study, the digestive enzymes, such as α-amylase (known to catalyze the hydrolysis of starch into sugars), lipase (known to catalyze the hydrolysis of fats and lipids), and trypsin (catalyzing the hydrolysis of proteins into smaller peptides) significantly increased in the treated groups compared with the untreated shrimp (Rawlings and Barrett, 1994;Svendsen, 2000). Similar observations were recorded in Apostichopus japonicus and Salmo salar L. that were fed diets supplemented with B. velezensis Wang J. et al., 2019). It was also noted that Bacillus genus might secrete a wide range of exoenzymes that aid in the nutritional enhancement of the host. As a result, the increase in digestive enzyme activities can be linked to the elevated beneficial bacteria in the Bacillustreated groups since they secrete chemical compounds, such as protease, amylases, and β-galactosidases. Notably, the GPT and GOT activities were reduced in the B. velezensis-supplemented groups, indicating that no hepatopancreas tissue damage or dysfunction was induced by the addition of probiotics (Cao et al., 2012). The modulation of immune responses and disease resistance is a major function induced by probiotics. As a result, both cellular and humoral immunity are stimulated in response to a pathogenic challenge (Goulart et al., 2019;Ng et al., 2019;Wang G. et al., 2019). In this study, the phagocytosis of L. vannamei hemocytes was increased with the addition of B. velezensis. Similarly, Rengpipat et al. (2000) found that the use of Bacillus S11 could activate cellular immune defenses of Penaeus monodon. During phagocytosis, respiratory bursts were used to kill bacterial pathogens, relying in particular on ROS, but their overproduction can cause damage to biomolecules, such as lipids, proteins, and nucleic acids, and generate oxidative stress (Spencer et al., 2019). After infection, BVs groups significantly reduced the damage of ROS to the body, possibly by modulation of the internal antioxidants and antioxidative systems (Chen et al., 2015). The SOD and CAT are considered to be molecular biomarkers for evaluating the oxidative stress status of aquatic organisms (Valavanidis et al., 2006;Yang et al., 2015). After the bacterial challenge, the activities of SOD and CAT showed similar trends, increasing slightly at first and then decreasing, which suggest that dietary supplemented probiotics improved the hepatopancreas antioxidant capacity of shrimp. ACP and AKP are necessary for phosphorylation and dephosphorylation, which are very important to the crustacean immune system (Yang et al., 2007). In the present study, the AKP activities in B. velezensis-supplemented groups were significantly higher than that in the control group. Similar results have also been found by Amoah and Cai (Amoah et al., 2019b;Cai et al., TABLE 5 | Statistics of the morphology and structure of hepatopancreas and midgut of L. vannamei fed diets containing 0 CFU/g (C), 10 5 CFU/g (BV1), 10 7 CFU/g (BV2), and 10 9 CFU/g (BV3) B. velezensis for 6 weeks. The values (mean of 8 replicates ± SD) with different letters (a, b, c) represent statistically significant differences based on the LSD method (P < 0.05, one-way ANOVA).

2019). The results suggested that dietary supplementation of
Paenibacillus polymyxa, B. licheniformis, and Bacillus flexus enhanced the growth, hepatopancreas immune, and antioxidant activities of shrimps. In addition, the expressions of immunerelated genes were highly induced in BVs groups. Among them, crustin and penaeidin 3α are vital antibacterial peptides in shrimps, powerful for the elimination of Vibrio (Cuthbertson et al., 2002(Cuthbertson et al., , 2005Muñoz et al., 2002;Arockiaraj et al., 2013). The enhanced expression of these genes contributes to resist invading bacteria and improve non-specific immunity (Muñoz et al., 2002;Arockiaraj et al., 2013;Cuthbertson et al., 2005). Overall, the dietary supplemented B. velezensis BV007 could boost the innate immunity of shrimp at an appropriate level. The intestinal microbiota of shrimp plays an essential role in mediating immunity, nutrient metabolism, and energy homeostasis (Zhang et al., 2020). The previous studies have shown that probiotics in the shrimp diet could effectively modulate the intestinal microbiota, improve growth performance, and enhance disease resistance (Yang et al., 2015;Suo et al., 2017;Amoah et al., 2019a;Xie et al., 2019). In the present study, a greater diversity of bacterial species was found in the BV007 supplemented groups than that in the control group. The reason may lie in the changed metabolic activities or beneficial effects on gut microbiota regulated by B. velezensis (He et al., 2019;Li et al., 2019a). Notably, the Vibrio species are severe pathogens in aquatic organisms (Hsu and Chen, 2007). In 2013, severe acute hepatopancreatic necrosis disease (AHPND) was induced by V. parahaemolyticus (Tran et al., 2013) causing mass mortality of shrimps specifically in the affected Southeast Asian countries (Hsu and Chen, 2007). In this study, cumulative mortality of the probiotics treated groups was significantly lower FIGURE 4 | A Venn diagram showing the unique and shared operational taxonomic units (OTUs) among groups of L. vannamei fed diets containing 0 CFU/g (C), 10 5 CFU/g (BV1), 10 7 CFU/g (BV2), or 10 9 CFU/g (BV3) B. velezensis.
than the untreated group after V. parahaemolyticus challenge. It suggested that B. velezensis 007 could improve the disease resistance of L. vannamei. Similar results were also proved by Zokaeifar et al. (2012) and Amoah et al. (2019b). They suggested that Bacillus subtilis and Bacillus coagulans ATCC 7050 enhanced the anti-disease ability in shrimp. The results might be attributed to the increased abundances of beneficial genus bacteria Bacillus FIGURE 5 | Relative abundance of the top 10 classes at the genus level of intestinal microflora of L. vannamei fed diets containing 0 CFU/g (C), 10 5 CFU/g (BV1), 10 7 CFU/g (BV2), or 10 9 CFU/g (BV3) B. velezensis. Error bars with different letters (a, b, c) represent statistically significant differences based on the LSD method (P < 0.05, one-way ANOVA). The results were shown as mean ± SE (N = 3). and enhanced immune responses in the treated groups (Amoah et al., 2019b).
The present study showed that diet supplementation with the potential probiotic B. velezensis BV007 could significantly promote growth performance, enhance immune response, and disease resistance in L. vannamei. Based on these findings, we conclude that a dose of 10 7 CFU/g feed provides health and growth benefits by improving the digestive enzyme activities, immune responses, intestinal development, and gut microflora.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.

AUTHOR CONTRIBUTIONS
SH conceived and designed the experiments. LC and CL conducted the experiments. BL, HZ, and QZ analyzed the data. LR, XZ, CS, and JG contributed reagents, materials, and analytical tools. LC, CL, and SH wrote the manuscript. All authors contributed to the article and approved the submitted version.