Aquaculture Reports 21 (2021) 100822

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Aquaculture Reports
journal homepage: www.elsevier.com/locate/aqrep

Safety evaluation for the use of Bacillus amyloliquefaciens in freshwater

fish cultures
Yibin Yang a, b, 1, Jingjin Xia a, 1, Yongtao Liu a, b, Jing Dong a, b, Ning Xu a, b, Qiuhong Yang a, b,
Shun Zhou a, b, Xiaohui Ai a, b, *
a
Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, PR China
b
Key Laboratory of Control of Quality and Safety for Aquatic Products, Ministry of Agriculture, Beijing 100141, PR China

A R T I C L E I N F O A B S T R A C T

Keywords: The primary objective of this work was to assess the effect of Bacillus amyloliquefaciens on the microbiota in
Ammonia polyculture tanks stocked with grass carp, gibel carp and sliver carp. The B. amyloliquefaciens showed high
Illumina-MiSeq biosafety toward freshwater fish. The addition of B. amyloliquefaciens to the tanks significantly reduced ammonia,
Microbial diversity
total nitrogen, total phosphorus and chemical oxygen demand (p ＜ 0.05) although nitrites increased (p ＜ 0.05).
Bio-safety
Bacterial diversity in the tanks was greatest in the un-inoculated tanks where Proteobacteria, Actinobacteria,
Cyanobacteria, Verrucomicrobia and Bacteroidetes dominated. In the presence of added B. amyloliquefaciens,
Proteobacteria and Bacteroidetes levels increased while Actinobacteria decreased. The classes and genera be­
tween controls and treatment groups differed although Bacillus levels did not increase in the inoculated tanks. We
conclude that supplementation with B. amyloliquefaciens in aquaculture water had many advantages but also
potential safety risks.

1. Introduction microbial ecosystems, damage to the natural environment as well as the

presence of residual antibiotics in fish products. This also presents risks
Aquaculture has undergone rapid growth during the last two decades to human health and reduced fish immunity due to their adverse effects
and now accounts for 43 % of the world’s fish production (Moffitt and on fish gastrointestinal microflora (Liu et al., 2017, 2012).
Cajas-Cano, 2014). The continuous demand for aquaculture products One solution to these problems is biological control using inoculation
and associated economic benefits has led to a rapid expansion of of probiotic bacteria. This has been successful in improving water
intensive aquaculture systems in developing countries (Dawood and quality, inhibiting potential pathogens and reducing the frequency of
Koshio, 2016). Production has risen from 45.5 million to 110.2 million necessary water exchange in tanks (Crab et al., 2012). Biological control
tons over the last 12 years with a first-sale production value of $234.5 also represents a low cost and convenient alternative to more traditional
billion (Pauly and Zeller, 2017). However, environmental pollution and methods of aquaculture management (Hoseinifar et al., 2018). Pro­
disease outbreaks have been increasing leading to massive fish mortality biotics have been successfully used in aquatic system including the
and economic losses (Wang et al., 2019). For example, aquaculture addition of photosynthetic bacteria to stimulate shrimp and fish growth,
wastewater contains high levels of nitrogen and phosphorus compounds enhance fish larvae survival and to improve scallop production (Wang,
that can cause a serious oxygen deficit, eutrophication or algal blooms, 2007; Shiung et al., 2018). Some Bacillus strains such as Bacillus subtilis
water deterioration and disease outbreaks (Ali et al., 2005; Cao et al., are also being used in aquaculture. These bacteria isolated from fish or
2007). Infection with Aeromonas hydrophila causes red fin disease and shrimp are antagonistic to the pathogen A. hydrophila and can decrease
hemorrhagic septicemia in freshwater fish and antibiotics and disin­ ammonia levels in water (Zhang et al., 2019). Ammonia and nitrites can
fectants are commonly used to prevent these types of disease outbreaks be removed from water through nitrification by the sequential actions of
(Bebak et al., 2015). However, the overuse of antibiotics in aquaculture ammonia oxidizing and nitrite oxidizing bacteria (Velusamy and
has resulted in the rapid spread of antibiotic-resistant pathogens in Krishnani, 2013). The combined use of Bacillus and molasses increased

* Corresponding author at: Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, PR China.
E-mail address: [email protected] (X. Ai).
1
Co-first authors.

https://doi.org/10.1016/j.aqrep.2021.100822
Received 22 March 2021; Received in revised form 5 August 2021; Accepted 5 August 2021
Available online 9 August 2021
2352-5134/© 2021 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/).
Y. Yang et al. Aquaculture Reports 21 (2021) 100822

the diversity and enhanced the development of beneficial microbial 08:00pm to 09:00am and 18:00 to 19:00.
communities while inhibiting pathogens in culture of Pacific white During the 15-day experimental period, water quality parameters
shrimp (Hu et al., 2017). including dissolved oxygen (DO), pH and temperature were measured
Probiotics are either derived from the intestines of host animals or daily using an HQ40D Portable Multi Meter (Hach, Loveland, CO, USA).
isolated from their natural growth environments in Chinese aquaculture Ammonia nitrogen, nitrite and nitrate nitrogen, total nitrogen, total
systems (Chi et al., 2014; Wu et al., 2014; Hao et al., 2017; Wang et al., phosphorus and chemical oxygen demand (COD) were measured using
2017). Moreover, aquatic animals are different from terrestrial animals kits purchased from Hubei Tusuo Technologies. according to standard
and humans. The strains that are safe to terrestrial animals and humans methods (Chinese NEPA, 2012). Water samples were collected using a
are not necessarily safe in aquaculture. Because aquatic animals have horizontal water sampler in center of each tank 15 cm below the surface
complex relationships with the surrounding environment (Verschuere and samples were stored at 4 ◦ C and analyzed within 24 h. At the end of
et al., 2000), the safety of probiotics used by aquatic animals is not the experiments, tank water was filtered through a 0.22 μm membrane
completely different from that used by terrestrial animals and human and stored at − 80 ℃ until DNA extraction.
probiotics. Therefore, the safety of potential aquaculture probiotics Microbial DNA was extracted from water samples using the E.Z.N.A.
should be tested in model systems before use. soil DNA Kit (Omega Bio-Tek, Norcross, GA, USA) according to the
Strains of Bacillus amyloliquefaciens have been used in aquaculture as manufacturer’s protocol. DNA concentrations were measured using UV
a phytopathogen biocontrol agent and are effective through production spectroscopy with Nanodrop 2000 instrument (Thermo Scientific, Wil­
of the bioactive lipopeptides fengycin, surfactin and iturin (Li et al., mington, DE, USA) and DNA quality was checked by electrophoresis
2016; Kalai-Grami et al., 2014; Alvarez et al., 2012). However, eff ;ects through 1% agarose gels. The V3-V4 hypervariable regions of bacterial
of B. amyloliquefaciens on the microbiota present in aquaculture practice 16S rRNA genes were amplified with primers 338 F (5′ -ACTCCTACGG­
have not been determined. To provide a reference for the safe applica­ GAGGCAGCAG-3′ ) and 806R (5′ -GGACTACHVGGGTWTCTAAT-3′ ) as
tion of B. amyloliquefaciens we used high-throughput sequencing to previously described (Xu J 2018). PCR reactions were carried out in a
examine microbial community diversity and structure in a freshwater GeneAmp 9700 thermocycler (Applied Biosystems, Waltham, MA, USA)
fish integrated system and examined alterations in the chemical using the following protocol: 95 ◦ C 3 min, 27 cycles of 30 s at 95 ◦ C, 30 s
composition of the culture water. at 55 ◦ C, and 45 s at 72 ◦ C and a final step of 72 ◦ C for 10 min. PCR
reactions were performed in triplicate in 20 μL mixtures using a FastPFU
2. Materials and methods polymerase kit (Transgen Biotech, Beijing, China) that included 4 μL of 5
× Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of
2.1. Bacteria preparation FastPfu Polymerase, 0.2 μL BSA and 10 ng of template DNA. The
resulting amplicons were extracted from a 2％ agarose gels after elec­
B. amyloliquefaciens was isolated from pond sludge and preserved at trophoresis using an AxyPrep DNA Gel Extraction Kit (Axygen Bio­
the Yangtze River Fisheries Research Institute (Wuhan, China). The sciences, Union City, CA, USA) and quantified using QuantiFluor-ST
selected strain was cultured for 24 h at 37℃ and the optical density of (Promega, Madison, WI, USA).
the cells was adjusted to OD600nm = 1.0 (approximately 5 × 108 cfu/
mL). Then 2 L of bacterial fluid was centrifuged at 4000 r/min for 2.4. Illumina MiSeq sequencing
10 min at 4℃, suspended in 20 mL of sterilized 0.85％ NaCl and washed
twice with the same volume of the solution. The experimental group Purified amplicons were pooled in equimolar and paired-end
received a corresponding volume of suspended bacterial cells that was sequenced (2 × 300) on an Illumina MiSeq platform (Illumina, San
added to tanks and controls received the suspending vehicle alone. Diego, CA, USA) according to the standard protocols at Majorbio Bio-
Pharm Technology (Shanghai, China). The raw reads were deposited
2.2. Bio-safety assessment into the NCBI Sequence Read Archive (SRA) database accession number
SRP8518516.
The tanks (400 L) were filled with 300 L of fresh water collected
from rearing ponds located in Jingzhou, China (30◦ 16′ 0.95′′ N- 2.5. Processing of sequencing data and analysis
112◦ 18′ 27.65′′ E). Prior to experiments, the 3 fish species grass carp,
gibel carp and silver carp were acclimatized together in 3 tanks for 14 Raw fastq files were demultiplexed, quality-filtered and merged
days. Fish showing overt signs of disease or malnutrition were removed. using Trimmomatic (http://www.usadellab.org/cms/?page=trimmoma
Fish used for experimental procedures were weighed and the masses of tic) and FLASH (https://ccb.jhu.edu/software/FLASH/) using the
grass, gibel and silver carp used were 11.0 ± 1.2 g, 2.6 ± 0.4 g and following criteria: (i) the reads were truncated at any site receiving an
7.6 ± 0.3 g, respectively. Both the experimental and the control group average quality score <20 over a 50 bp sliding window. (ii) Primers
had three replicates each and every group contained 10 of each species. were exactly matched allowing 2 nucleotide mismatches and reads
Suspended bacterial cells (3 mL) were added to begin experiments and containing ambiguous bases were removed (iii) sequences with >10 bp
controls received the same volume of 0.85 % NaCl. The fish were fed overlaps were merged according to their overlap sequence. Operational
normally during the duration of the experiments and survival was taxonomic units (OTUs) were clustered with 97 % similarity cutoff using
recorded. The basal diet consisted of 30 % crude protein, 5.5 % crude UPARSE (Version 7.1 (http://drive5.com/uparse/) and chimeric se­
lipid, and 12 % ash, 10 % moisture, 6% crude fiber and 36.5 % nitrogen quences were identified and removed using UCHIME (http://www.dri
free extract (Provided by the Yangtze River Fisheries Research Institute). ve5.com/usearch/manual/uchime_algo.html). The taxonomy of each
16S rRNA gene sequence was analyzed with the RDP Classifier algorithm
2.3. Water quality and microbial community analyses (http://rdp.Cme.msu.edu/) against the Silva SSU 16S rRNA database
(https://www.arb-silva.de/) using a confidence threshold of 70 %.
The experimental period was 15 days and 8 grass carp, 4 gibel carp Before the genetic analyses, the assumptions of a normal distribution
and 2 silver carp were stocked into each of six experimental tanks. The and homogeneity of variance were checked. One-way ANOVA was used
culture water in the tanks was neither aerated nor exchanged during the to compare the water quality parameters at each sampling time using the
experiment. The control group was fed with commercial feed and the SPSS 20.0 program. The microbial community diversity and construc­
treated group was fed with commercial feed in addition to tion were conducted using the i-Sanger platform (http://www.i-sanger.
B. amyloliquefaciens at 1 × 1011 cfu/m3 per for 7 days. All the experi­ com/). The Predict functional genes were compared by using Phyloge­
mental fish were fed 2％ of their body weight twice a day between netic Investigation of Communities by Reconstruction of Unobserved

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Y. Yang et al. Aquaculture Reports 21 (2021) 100822

Fig. 1. Effects of B. amyloliquefaciens on water quality in a freshwater fish polyculture system.

a. Ammonia nitrogen, b. Nitrate nitrogen, c. Nitrite nitrogen, d. Total phosphorus, e. Total nitrogen, f. Chemical oxygen demand.*, p < 0.05 * *, p < 0.01 compared
with control. C, control, no bacteria added; T, treatment group, B. amyloliquefaciens added.

States and P＜0.05 was considered to indicate a statistically significantly 3.2. Water properties
difference.
B. amyloliquefaciens had an effect on the water quality in freshwater
3. Results fish polyculture system. Compared with the controls, ammonia, total
nitrogen and phosphorus and COD significantly decreased in the treat­
3.1. Assessment of biological safety for B. amyloliquefaciens ment groups. Specifically, ammonia decreased by 41.6–50.7% (p＜0.05)
during days 9–15 (Fig. 1a). Total phosphorus decreased by 21.8–30.9%
The addition of B. amyloliquefaciens was harmless to the freshwater (p＜0.05) on days 6–12 (Fig. 1d). Total nitrogen decreased by 49 % (p＜
fish and we observed neither mortality nor morbidity during the 14 day 0.05) on day 15 and COD decreased by 20 and 15 % (p＜0.05) on days
post-challenge observation. The high dosage of B. amyloliquefaciens 12 and 15, respectively (Fig. 1e and f). However, nitrite significantly
added to the tanks indicated that this bacterium has a high biosafety increased (p < 0.05) from days 6–12 (Fig. 1c) while nitrate did not
level for freshwater fish. change significantly (Fig. 1b).

Table 1
α-diversity indices of bacterial communities identified in this study.
Sample ID OTUs Shannon Simpson Chao Ace coverage
a b b a a
T 511 ± 11.66 3.04 ± 0.61 0.15 ± 0.04 422.45 ± 26.87 417.72 ± 24.91 99.8％
C 549 ± 8.87a 3.88 ± 0.19a 0.05 ± 0.01a 445.29 ± 10.36a 448.59 ± 12.45a 99.8％

Values are expressed as mean ± SD during the experimental period of 15 days. C, no B. amyloliquefaciens addition; T, B. amyloliquefaciens added. In each column, diff ;
erent superscript letters indicate significant diff ;erences at the P < 0.05 level (one− way ANOVA).

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3.3. Effects of B. amyloliquefaciens on microbial diversity

Illumina analysis of six culture water samples yielded a total of

512,979 high-quality 16S rDNA sequences reads. To estimate the di­
versity, we identified OTUs corresponding to 3％ sequence divergence.
The α-diversity evaluated community richness (Ace, Chao) and com­
munity diversity (Shannon, Simpson) at the same sequencing depth.
Each sample was more than 99％of the coverage indicating that the
depth of the sequences was sufficient. The Ace and Chao values and the
OTU indices indicated that bacterial richness was not significantly
different between groups. However, the Simpson and Shannon indices
revealed a lower microbial diversity for the treatment groups (Table 1).
The rarefaction curves of these samples approached plateaus indicating
that highly diverse microbial communities were present in each sample.
The rarefaction curves also illustrated a lower microbial diversity in the
treatment group (Fig. 2).

Fig. 2. Rarefaction results for the control (C) and treatment (T) groups. 3.4. Effects of B. amyloliquefaciens on microbial community structure
C, no bacteria added; T, B. amyloliquefaciens added.
In total, we found 25 bacterial phyla that dominated and Proteo­
bacteria accounted for 45.45％ of total sequence reads followed by
Actinobacteria (17.12％), Bacteroidetes (12.07％), Cyanobacteria

Fig. 3. Relative abundance of 16 s rDNA bacterial OTUs in control and treatment groups.
A. Phylum level; B. Class level; C. genus level; D. Difference of community distribution at the genus level between treatment and control groups. (<1％ abundance of
the phyla or genera were merged).

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Fig. 4. Similarity analysis between control and treatment groups.

A. Venn diagrams of shared OTUs between treatment and control groups. B. Principal coordinates analyses (PCoA) of microbial communities. C, no bacteria added; T,
B. amyloliquefaciens added.

(9.84％), Verrucomicrobia (5.62％), Acidobacteria (4.45％), Chloroflexi between the groups (75.5％). In addition, we found 55 (9.1 %) and 93
(1.95％), Firmicutes (1.07％) and Planctomycetes (1.24％). Both (15.4 %) unique OTUs from the treatment and controls, respectively
experimental and control groups possessed similar bacterial commu­ (Fig. 4A). These results indicated that the both groups have the same
nities. Specifically, the treatment group consisted of Proteobacteria water source.
(55.03％), Bacteroidetes (16.01％), Actinobacteria (8.03％), Cyano­ We used principal coordinate analysis (PCoA) to identify the primary
bacteria (11.23％), Verrucomicrobia (4.69％) and Acidobacteria factors driving community composition differences. We identified two
(3.84％). These dominant bacterial phyla of both groups contributed different clusters for controls and treatment groups at the phylum and
approximately 98％ of all observed sequence readings. The only obvious OTU levels. These formed two different clusters and were separated
difference was a decrease in the number of Actinobacteria, Chloroflexi, along principal coordinate axis 1 (PC1) at the phylum level and along
Firmicutes, Planctomycetes and an increase in Bacteroidetes and Pro­ PC2 for the OTUs (Fig. 4B). These results indicated that application of
teobacteria in the treatment group (Fig. 3A). B. amyloliquefaciens had strong effects on the bacterial community
At the class level, the most abundant bacterial taxa were α- Proteo­ structures during the 15-day experimental period.
bacteria and β-Proteobacteria, Actinobacteria, Cyanobacteria, Sphin­
gobacteria, Acidobacteria and Verrucomicrobia. In the treatment group, 3.6. Taxa differences and predicted functions
the relative of abundance of the class Betaproteobacteria (42.38 vs 29.8
%) was increased compared with controls. In the Bacteroidetes phylum, To further investigate the taxonomic distribution and differentially
the Sphingobacteria (13.97 vs 3.11 %) were elevated over controls while dominant bacterial clades we used a linear discriminant analysis (LDA,
the Flavobacteria (0.65 vs 3.94 %) were lower. Moreover, the control LEfSe) to compare the abundance of bacterial compositions from
group was enriched for the class Actinobacteria (Fig. 3B). domain to the species level. We identified 44 differentially abundant
In all the samples we screened, 250 genera were shared between in taxa. The most significantly different bacterial groups were in the Fla­
all samples. The first 35 dominant genera in all samples were chosen and vobacteria and SBR2076 classes, the orders Micrococales, Flavobacter­
the comparison of their abundance was analyzed using the hierar­ iales and Acidimicrobiales, the families Chthoniobacterales,
chically clustered heatmap (Fig. 3C). The treatment group was domi­ Chitinophagaceae, Acidimicrobiaceae and env-ops-17, the genera Ter­
nated by Hydrogenophaga, Haliscomenobacter, Terrimicrobium, rimicrobium, Methylophilus, the CL50029 marine-group, Sed­
Haloferula, Alsobacter and Novosphingobium. The control group was iminibacterium, Paludibacter and RRDOLa011B as well as
dominated by Aeromonas, Flavobacterium, Acinetobacter, Pseudomonas, Polynucleobacter cosmopolitanus (Fig. 5). These results indicated that
hgcl_clade and Rivicola (Fig. 3C and D). The abundance of Bacillus was supplementation with B. amyloliquefaciens caused alterations in the
similar between the treatment and control groups (Fig. 3D). Although microbial composition of this freshwater fish polyculture system.
some taxa were unclassified at the genus level, the abundance of the Functional inferences from 16S rDNA indicated that the relative
dominant genera of the groups differed (Fig. 3C). abundance of bacteria with varied metabolic modules had no significant
differences between the treatment and control groups (P＞0.05) (Fig. 6).
This suggested that the culture water environment possessed similar
3.5. Similarity between communities possibilities in the ability of group self-purification despite a significant
decrease in biodiversity (P＜0.05).
We analyzed bacterial community similarities and identified 549 and
511 OTUs that clustered at 97％ similarity in the treatment and controls,
respectively. There were 604 different OTUs and 456 were shared

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Fig. 5. LEfSe analysis of bacterial communities with LDA scores > 3.0.
Cladograms represent the phylogenetic distribution of microbial lineages associated with the C and T groups. Circles represent phylogenetic levels from kingdom to
species. Labels in the dotted box indicate kingdom and species levels of communities. The diff ;erentially abundant clades of each group are represented by colors in
cladograms and the LDA scores of these clades indicate the degree of statistical and biological diff ;erences. C, no bacteria added; T, B. amyloliquefaciens added.

4. Discussion nitrifying bacteria or anabolized by heterotrophic bacteria (Avnimelech,

1999). B. subtilis SC02 and Bacillus licheniformis BSK-4 could also
The use of probiotics, especially of Bacillus strains, in aquaculture is decrease the nitrogen level in grass carp culture water (Zhang et al.,
increasingly being considered as an ecofriendly approach to alleviate 2013; Liang et al., 2015). However, in that study, the nitrite concen­
health related problems in aquatic animals. We confirmed the non- tration in the treatment group was significantly higher than controls
pathogenicity of our test strain before application (Sahu et al., 2008). from days 6–12. This was most likely associated with the oxidative
We found no fish mortality or morbidity during the 14-day experimental metabolism of ammonia due to imbalances in nitrifying bacterial ac­
test and this finding agreed with a previous study (Austin et al., 1995). tivity (Wang et al., 2004). In addition, chemical water quality in a
The addition of B. amyloliquefaciens to our culture system signifi­ freshwater fish polyculture system was not improved using three com­
cantly reduced the levels of ammonia, total nitrogen and phosphorus mercial microbial products (Tang et al., 2016). These authors indicated
and COD compared with controls. This result was not surprising since that the function of the microbial products might be dependent on the
B. amyloliquefaciens group strains have the ability to metabolize diverse environmental background in which they are used (Tang et al., 2016).
carbon sources both aerobically and anaerobically (Chun et al., 2019). The present study demonstrates that B. amyloliquefaciens signi­
Bacillus cereus S1 could also degrade organic matter and significantly ficantly aff ;ected bacterial community diversity and composition in an
reduce COD in shrimp aquaculture systems (Lalloo et al., 2008). integrated aquaculture system. The α-diversity and the rarefaction
Ammonia and nitrite can be converted into nitrate by autotrophic curves illustrated a lower level of microbial diversity in the treatment

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biosphere, especially polysaccharides and hydrocarbons (Tsagaraki

et al., 2018). Their presence is therefore tied to water quality
improvement.
Overall, Bacillus abundance did not increase in our treatment groups
suggesting that B. amyloliquefaciens did not multiply in large numbers.
This result is consistent with a previous report that Bacillus could not
colonize a native bacterial community in a freshwater fish polyculture
system (Zhou et al., 2017). The addition of both Bacillus and molasses
altered the bacterial community but did not increase the Bacillus
numbers in a shrimp culture system (Hu et al., 2017). Our results also
provide a reference for the question of whether the application of
microecological agents in aquaculture will result in species invasion.

Declaration of Competing Interest

Fig. 6. Predicted functions of bacterial OTUs based on KEGG modules.
C, no bacteria added; T, B. amyloliquefaciens added. The authors report no declarations of interest.

group. As we all know, the higher the biodiversity, the higher the sta­ Acknowledgements
bility of the ecosystem and the resistance to water (Briones and Raskin,
2003). Our results are inconsistent with studies using B. subtilis SC02 This work was supported by Major innovation projects in Hubei
that increased microbial diversity in similar aquaculture systems (Zhang Province [No. 2019ABA077] and the Central Public-interest Scientific
et al., 2013). On the other hand, the addition of the Gram negative Institution Basal Research Fund, CAFS [No. 2017HY-ZD0505].
bacteria parasite Bdellovibrio significantly reduced the diversity of water
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