Aquaculture 527 (2020) 735428

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Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture

Effects of probiotic supplementation on gut microbiota as well as metabolite T

profiles within Nile tilapia, Oreochromis niloticus
⁎ ⁎
Yun Xia, ErMeng Yu, Maixin Lu , Jun Xie
Key Laboratory of Tropical & Subtropical Fishery Resource Application & Cultivation, Ministry of Agriculture, Pearl River Fisheries Research Institute, Chinese Academy of
Fishery Sciences, ChinaCollege of Fisheries, Guangzhou 510380, China

A R T I C LE I N FO A B S T R A C T

Keywords: Lactococcus lactis JCM5805 plays an important role in tilapia farming to promote individual growth, immunity
JCM5805 and disease resistance. This study applied gas chromatography/mass spectrometry (GC/MS) and high-
GC/MS throughput sequencing in characterizing microbial composition as well as metabolite profiles within tilapia gut
Gut microbiota fed with control and JCM5805-suppmeneted diets. Our results showed that JCM5805 administration altered the
Metabolite profiles
composition of host gut microbiota. The significant metabolic pathways in terms of microbial function between
Tilapia
two groups included carbohydrate metabolism, nucleic acid metabolism, energy metabolism, and translation. In
addition, the functions of differential metabolites between two groups were mainly related to metabolism, ABC
transporter, metabolism of methionine and cysteine, amino acid biosynthesis, arachidonic acid metabolism,
protein digestion/absorption, and asthma. There was significant interdependence between metabolites and
microorganisms. Besides, JCM5805 distribution displayed significantly positive correlation with the up-reg-
ulation of metabolite L-cystine (CC > 0.90, CCP < 0.01), which was involved in body protein metabolism.
Findings in this study helped to understand the relationships of gut microbiota and metabolites in gut contents of
fish administered with probiotics, and highlighted the possible material basis and mechanism of JCM5805 in
affecting host growth and immunity.

1. Introduction
Vertebrates are associated with abundant and complicated micro-
bial communities colonized at the gastrointestinal (GI) tracts (Lin and
Zhang, 2017). Therefore, intestinal flora becomes the indivisible com-
ponent in digestive system. Intestinal flora has great influence on the
health of fish through irritating immune system and intestinal epithelial
development, and preventing the colonization of pathogenic micro-
organisms in the GI tract (Kokou et al., 2019; Li et al., 2016; Ringø
et al., 2016; Wang et al., 2018). Intestinal flora exerts a distinct part in
the regulation of host metabolism. For instance, those stodgy carbo-
hydrates are degradable through intestinal bacteria-mediated fermen-
tation, thus producing energy for microbial growth. In addition, this
process also produces the microbial end products that act as the sig-
naling molecules, inflammatory modulators and energy substrates
(Holmes et al., 2011). As a result, intestinal flora also affects the me-
tabolism in remote organs in the host, like brain, liver, muscle, and
adipose (Benakis et al., 2020; Ezra-Nevo et al., 2020). Increasing evi-
dence has revealed that, the associations of metabolic disturbances with
the alterations of intestinal flora composition represent the critical risk
factors in disease development (Nie et al., 2019). For instance, tri-
methylamine N-oxide produced by intestinal flora based on dietary
carnitine and choline is closely related to atherosclerosis in clinical
patients and animal models (Wang et al. 2011).
Probiotics refer to the living microorganisms with certain benefits
for the hosts if they are used in sufficient amounts (Doan et al., 2020).
To be specific, probiotics can enhance the immunity, accelerate diges-
tion, and protect against pathogens. As far as aquaculture is concerned,
probiotics decrease the incidences of bacterial pathogen-induced in-
fections (Wang et al., 2020). Besides, they are adopted to be the im-
munostimulants as well as growth promoters (Pérez-Sánchez, 2014;
LeBlanc et al., 2017), which effectively regulate gut microbiota. Con-
sequently, great interests are aroused to explore the relationship be-
tween the the intestinal flora-related metabolic alterations and the in-
testinal flora community under probiotics treatment. To this end,
metabolomics profiling based on mass spectrometry (MS) is an ap-
pealing approach, which is attributed to its great sensitivity, capability
of detecting molecules with different structures, quantitative ability,
wide dynamic range, and compatibility with other isolation methods
like liquid chromatography (Lu et al., 2012). Nonetheless, relative to

⁎
Corresponding authors.
E-mail addresses: mx-lu@163.com (M. Lu), xiejunhy01@126.com (J. Xie).

https://doi.org/10.1016/j.aquaculture.2020.735428
Received 19 February 2020; Received in revised form 29 April 2020
Available online 01 May 2020
0044-8486/ © 2020 Elsevier B.V. All rights reserved.
Y. Xia, et al. Aquaculture 527 (2020) 735428

studies on terrestrial vertebrates through LC/MS, little research is done
on the gut metabolome of fish.
Lactococcus lactis subsp. lactis JCM5805 was found in our previous
studies to positively affect the growth of nile tilapia, body immunity
together with the resistance to disease, and it also improves the gut
microbial composition in host (Xia et al., 2018; Xia et al., 2019). This
study adopted 16S rRNA gene sequencing in combination with liquid
chromatography-mass spectrometry (LC-MS) metabolomics for ana-
lyzing the effect of probiotics JCM5805 on gut microbiota and the re-
lated metabolite profiles. According to the results of metagenomic se-
quencing, JCM5805 administration dramatically changed the intestinal
microbial composition of Nile tilapia. Moreover, based on the non-
targeted metabolomic profiling, exposure to JCM5805 markedly af-
fected such fish, and the various changed metabolites were related to
alterations of intestinal microbiota. To sum up, findings in this study
reveal the possible mechanism of JCM5805 in affecting the intestinal
metabolism by changing the gut microbial structure, finally regulating
tilapia growth and immunity.
2. Materials and methods
2.1. Probiotics
JCM5805 was provided by China General Microbiological Culture
Collection Center (CGMCC). JCM5805 cultures were cultured within
the Brain Heart Infusion Broth (BHI) (OXOID) for 48 h under the
temperature of 30 °C in accordance with manufacturer protocols.
Afterwards, those cultures were subjected to 5 min of centrifugation at
5000g (Beckman Coulter, AK, USA). Then, the sterile distilled water was
used to rinse the pellets for twice, followed by freeze-drying and re-
suspension within PBS (supplemented with 2.7 mM KCl, 137 mM NaCl,
1.8 mM KH2PO4, 10.1 mM NaH2PO4, pH 7.4). Afterwards, the viable
cells were determined through the spread plate technology based on
cell contents determined with OD600, which showed linear proportion
to the viable cell number within the subsequent suspension. Eventually,
the OD600 values measured in cell suspension were corrected to the
sufficient value (CFU mL−1) prior to subsequent feed formulation ex-
periments.
Table 1 presents the formulation and major components in basal
Table 1
Formulation and calculated chemical compositions of the basal diet.
Ingredients Contents (g kg−1)
Fish meal 480
Soybean meal 220
Wheat flour 250
Adhesives 2 extract DNA from 250 mg contents in accordance with manufacturer
Soybean oil 20
Ca(H2PO4)2 20
VC phosphate ester 1
Choline chloride (50%) 3 each group for subsequent sequencing analysis.
Vitamin mixa 0.2
Mineral mixb 0.2
Crude protein 420
Crude lipid 73
Ash 95
Crude fiber 31
N free extract 279
a
Vitamin premix (g kg−1): thiamine, 0.438; riboflavin, 0.632;
pyridoxine·HCl, 0.908; D-pantothenic acid, 1.724; nicotinic acid,
4.583; biotin, 0.211; folic acid, 0.549; vitamin B-12, 0.001; inositol,
21.053; menadione sodium bisulfite, 0.889; retinyl acetate, 0.677;
cholecalciferol, 0.116; DL-α-tocopherol-acetate, 12.632.
b
Mineral premix (g kg−1): CoCl2·6H2O, 0.074; CuSO4·5H2O,
2.5; FeSO4·7H2O, 73.2; NaCl, 40.0; MgSO4·7H2O, 284.0; MnSO4·
H2O, 6.50; KI, 0.68; Na2SeO3, 0.10; ZnSO4·7H2O, 131.93; cellu-
lose, 501.09.
diet. These probiotics (1 × 106 CFU g−1) supplemented within basal
diet were applied to experimental tilapia, according to previous assay
on JCM5805 cultured within tilapia (Xia et al., 2019). In addition, the
pure basal diet was also given to control tilapia (C). Those experimental
diets were prepared in accordance with our prior work (Xia et al.,
2018). In brief, the dietary ingredient powders were sufficiently mixed
manually, then water and oil were added, and appropriate JCM5805
cells were then mixed for the formation of a soft dough. All diets were
prepared freshly everyday, encapsulated within the plastic bags, and
preserved under the temperature of 4 °C, so as to guarantee the pro-
biotic viability.
2.2. Fish husbandry and administration
The Nile tilapia for experiments were obtained from Gaoyao Fish
Farm of Pearl River Fisheries Research Institute (Guangzhou, China). If
necessary, ethyl 3-aminobenzoate methanesulfonate (MS-222) was
adopted for fish anesthesia. The normal young tilapia, which were fed
with basal diet, were screened and adapted for 2 weeks within the 750-
L tanks at a temperature of 28–29 °C under laboratory conditions. Later,
the smaller tilapia (4.68 ± 0.08 g) fed with normal diet, with no
disease or injury (a preliminary judgment was made based on the fish's
feeding situation, swimming status, and body surface status) were
randomly distributed into 6 50-L tanks, including 30 for every tank, and
3 replicates were set for every treatment. The feeding cycle adopted was
measured according to prior research (Xia et al., unpublished). During
the following 8 weeks, these fish were raised twice a day at 9:00 am and
4:00 pm, till apparent satiation. Those culture conditions were the same
as those adopted by Xia et al. (2018), and 50% water was replaced
every day.
2.3. Sample processing for microbiota and metabolomics.
Before dissection, each animal was given an excessive amount of MS
222 (Sigma, Germany) for euthanasia. The fish management and eu-
thanasia processes were carried out according to Wu et al. (2012).
Thereafter, the guts were collected aseptically from abdominal cavity,
the hindgut (distal 1/3 of the entire intestine) contents from 9 in-
dividuals (C1, C2 and C3 from C1 tank; C4, C5 and C6 from C2 tank; C7,
C8 and C from C3 tank; T1, T2 and T3 from T1 tank; T4, T5 and T6 from
T2 tank; T7, T8 and T from T3 tank) in every group were extracted and
collected into the sterile tube, and snap-frozen under liquid nitrogen,
followed by preservation under −80 °C prior to analysis.
2.4. Composition of gut microbial communities
The E.Z.N.A. Stool DNA kit (OMEGA, Bio-Tek, USA) was used to
instructions after light modifications (Giatsis et al., 2016). According to
the quality of DNA extraction, 5 samples were randomly selected from
GENEWIZ, Inc. (Suzhou, China) was responsible for preparing the
Illumina MiSeq libraries and implementing next-generation sequencing.
Each DNA sample was quantified using Qubit 2.0 Fluorometer
(Invitrogen, Carlsbad, CA, USA). Then, amplicons were generated using
MetaVx™ Library Preparation kit (GENEWIZ, Inc., South Plainfield, NJ,
USA) with 30–50 ng DNA. Later, the V3 and V4 hypervariable regions
of prokaryotic 16S rDNA were selected for amplicon generation and
subsequent taxonomic analysis (Lu et al., 2016). The subsequent sample
processing and analysis methods were the same as those in our pub-
lished studies (Xia et al., 2018).
2.5. Metabolite extraction and metabolite profiling analysis
A total of 5 fecal samples that were the same with sample for 16S
rDNA sequencing above in each group were subjected to thawing on ice

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Y. Xia, et al.
Fig. 1. Differential analysis on KEGG pathways in intestinal microbes. Note: C: control group; T: JCM5805 administration group.
under 4 °C. 100 μL sample was collected into the EP tube and extracted
by 300 μL methanol; afterwards, 20 μL internal reference was added
under 30 s of vortexing, followed by 10 min of ultrasonic treatment
within ice-cold water, 1 h incubation under −20 °Cfor protein pre-
cipitation, and 15 min centrifugation at 13000 rpm under 4 °C. Later,
the supernatants (200 μL) were transferred to the new 2 mL LC/MS
glass vial, 20 μL respective samples were collected and pooled to be the
QC samples. Finally, 200 μL supernatants were taken to carry out
UHPLC-QTOF-MS analyses.
The 1290 UHPLC system (Agilent Technologies) equipped with the
UPLC BEH Amide column (2.1*100 mm, 1.7 μm Waters) was ustilized
in combination with TripleTOF 5600 (Q-TOF, AB Sciex) for LC-MS/MS
analyses. Typically, the mobile phase was constituted by 25 mM
NH4OAc and 25 mM NH4OH contained within water (pH = 9.75) (A)
together with acetonitrile (B), which was adopted for the following
gradient elution: 95% B at 0 min; 65% B at 7 min; 40% B at 9 min; 95%
B at 9.1 min; and 95% B at 12 min, and it was delivered at the rate of
0.5 mL min-1, with the total volume of injection of 3 μL. The MS/MS
spectra were obtained using the Triple TOF mass spectrometer on the
information-dependent basis (IDA) in the process of LC/MS analysis.
Analyst TF 1.7 (AB Sciex), the software for acquisition, persistently
assesses the full-scan MS data in this mode, and it depends on the
preselected criteria to collect and trigger MS/MS spectral acquisition.
For every cycle, a total of 12 precursor ions with the intensity of > 100
were selected to fragment at 30 V collision energy (CE) (15 MS/MS
events, and the product ion accumulation time in every event was
50 ms). The following ESI source conditions were utilized: Curtain gas
as 35 Psi, ion source gases 1 and 2 as 60 and 60 Psi, source temperature
of 650 °C, Ion Spray Voltage Floating (ISVF) of −4000 V or 5000 V,
respectively, of negative or positive mode.
MS original data (.d) files were transformed into the format of
mzXML by ProteoWizard, and later XCMS 3.2 of R package was used for
data processing. A data matrix was generated based on those pre-
processed results, which was constituted by peak intensity, values of
massto-charge ratio (m/z), and retention time (RT). The CAMERA of R
package was utilized to annotate the peaks following the XCMS data
processing. Metabolites were identified using the in-house MS2 data-
base.
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Aquaculture 527 (2020) 735428
2.6. Statistical analysis
The metabolomic data intrinsic clusters were examined through
principal component analysis (PCA). The possible outliers were iden-
tified with the threshold of 95% confidence interval (CI) of each
sample. Additionally, the hierarchical clustering algorithm was em-
ployed to generate heat maps for visualizing the metabolite hetero-
geneity in dataset. Later, the intestinal microbiomic profiles between
treatment and control were compared using principal coordinate ana-
lysis (PCoA). The heterogeneous intestinal flora compositions were
evaluated through nonparametric test using Metastats (http://
metastats. cbcb.umd.edu/) according to previous description (White
et al., 2009). Eventually, the Pearson's correlation coefficient was
adopted to generate the correlation matrix between intestinal microbial
species and intestinal flora-associated metabolites.
3. Results
3.1. Response of the fish to the experimental diets
Our previous studies showed that the addition of JCM5805 was
beneficial to the growth of tilapia (Xia et al., 2018; Xia et al., 2019).
Throughout the breeding process, the fish did not show any adverse
reactions to the addition of JCM5805, and the immune data also con-
firmed this conclusion (Xia et al., 2018).
3.2. Differences in the bacterial community diversity
Fig. S1 (Supplemental Material) presents those main bacterial
components within fish guts fed with a probiotic or control diet
(GenBank accession number: SRX8020933-SRX8020942). Clearly, the
intestinal flora components mainly included the Proteobacteria,
Firmicutes, Fusobacteria, Actinobacteria, Cyanobacteria and
Bacteroidetes. Among them, the phylum Proteobacteria (75.30%)
showed the highest abundance within CK, whereas Firmicutes (76.73%)
was dominant in probiotic-fed fish.
Based on functional analysis, the gut bacterial community pathways
related to carbohydrate metabolism, Nucleotide metabolism, Energy
metabolism and Translation were altered in fish fed with different diets
(Fig. 1).
Y. Xia, et al. Aquaculture 527 (2020) 735428

Fig. 2. Summary of the variation in gut metabolite between each group. (A) Controls were separated from probiotic-treated tilapia in metabolite profiles by PCA. (B)
The OPLS-DA model for metabolic difference analysis was excellent, in which R2X = 0.575, R2Y = 0.999, and O2 = 0.907. (C) Probiotic exposure changed the
metabolic profile of fecal samples of fish, with 545 molecular features being significantly changed compared with controls (fold change > 1.5 and p < .05). (D)
Hierarchical clustering heat map constructed using molecular features with 1.5-fold changes (p < .05) shows a consistent clustering pattern within individual
groups. Note: C: control group; T: JCM5805 administration group.

3.3. Differences in metabolite profiles
Based on Fig. 2A, fish fed with probiotic and control were distin-
guishable according to the metabolite fingerprints; typically, the initial
2 PCA components (PC1 and PC2) were able to excellently separate fish
fed with a probiotic or control diet.
The results were further examined through orthogonal projections
to latent structures-discriminant analysis (OPLS-DA). In this metabo-
lites model, the orthogonal variables that were not related to catego-
rical variables were filtered out. Then, the non-orthogonal variables
and orthogonal variables were analyzed separately, so as to obtain the
differences and correlation information between different groups of
metabolites. The prediction parameters of the evaluation model were
R2X, R2Y and Q2, where R2X and R2Y represented the interpretation
rate of the built model to the X and Y matrix, Q2 stood for the pre-
diction ability of the model. The closer of these three indicators to 1
indicated the higher stability and reliability of the model. It was
considered as an effective model when Q2 > 0.5, and as an excellent
model when Q2 > 0.9. Subsequently, the metabolic difference analysis
model in this study was constructed, in which R2X was 0.575, R2Y was
0.999, and Q2 was 0.907. The model was excellent (Fig. 2B).
The combination of numerous intestinal microbes in feces sample,
together with the corresponding metabolites generates a favorable
biological sample for assessing the functional changes of intestinal
flora. The fold change (FC), VIP-value for OPLS-DA model, and P-value
upon Student's t-test were combined to screen the differential metabo-
lites. The screening criteria were as follows, FC > 1.50, VIP > 1 and
P-value < .05. Altogether 545 significantly different metabolites were
obtained, among which, 447 showed up-regulation, while 98 showed
down-regulation (Fig. 2C). Many metabolites that had > 1.5 FC be-
tween probiotic- and control diet-fed fish were found (Supplemental
Material, Table S1). The top 15 metabolites with the highest FC are
shown in Table 2. Among them, the up-regulated metabolites included
N-Acetyl-L-Cysteine, L-Cystine, Propinol adenylate, 5-Phosphoribosyl-

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Y. Xia, et al. Aquaculture 527 (2020) 735428

Table 2
Composition of the major top 15 known differential metabolites.
#ID Name Fold_change log2FC Pvalue VIP regulated

meta_13 Valeric acid 0.2140 −2.2241 0.0225 1.4991 down

meta_17 2-hydroxy-butanoic acid 2 0.2085 −2.2619 0.0089 1.4704 down
meta_23 Histamine 0.208497 −2.2619 0.0067 1.5243 down
meta_117 N-Acetyl-L-Cysteine 4.3439 2.1190 1.42E-05 1.8588 up
meta_686 L-Cystine 5.0098 2.3248 0.0465 1.2245 up
meta_48 ketoisocaproic acid 0.1939 −2.3665 0.0121 1.5944 down
meta_1513 Propinol adenylate 8.4409 3.0774 0.0011 1.7616 up
meta_2611 Soyasaponin 5.2317 2.3873 0.0047 1.6854 up
meta_336 5-Phosphoribosyl-1-amine 8.6182 3.1074 0.0084 1.6297 up
meta_232 O-Phospho-L-threonine 7.4197 2.8914 0.0080 1.6336 up
meta_394 2,4-Dinitroaniline 9.3993 3.2326 0.0207 1.5187 up
meta_642 Diphenhydramine hydrochloride 4.7057 2.2344 0.00062 1.8143 up
meta_528 Brassica oleracea Alkaloid 4.6047 2.2031 5.44E-05 1.8444 up
meta_709 Sinapinic acid-O-sulphate 4.8365 2.27340 0.0433 1.3491 up
meta_545 3-Hydroxytetradecanedioic acid 4.9576 2.3096 0.0011 1.7198 up

Fig. 3. Pathway classification map of differential metabolites.

1-amine, and O-Phospho-L-threonine; whereas metabolites Valeric acid,
2-hydroxy-butanoic acid 2, Histamine, and ketoisocaproic acid were
down-regulated.
Cluster analysis of the selected differential metabolites contributes
to further analyzing the trend of metabolite related to biological
processes in each group of samples. Fig. 2D exhibits the hierarchical
clustering heat map, as well as the akin clustering profiles for those
molecular features detected in every group. Clear separations of me-
tabolite profiles between control and probiotic-treated fish were ob-
served for the stool samples, with a large number of perturbed

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Y. Xia, et al.
Fig. 4. Inter-omic pearson correlation networks. Note: For every pair of each
operational taxonomic unit (OTU) and metabolite, the specific pair-wise
pearson correlation was determined. The circles represent OTU, and different
colors indicate different classifications (level on phylum).
molecular features.
The KEGG annotation results of differential metabolites were clas-
sified according to the type of pathways (Fig. 3), which mainly included
metabolism, organismal systems, environmental information proces-
sing, and human diseases. To be specific, they included metabolism,
ABC transporters, metabolism of methionine and cysteine, biosynthesis
of amino acid, protein digestion and absorption, arachidonic acid me-
tabolism, and asthma. (Supplemental Material, Table S1).
3.4. Associations of intestinal flora with metabolite profiles
For investigating those functional correlations of changes in in-
testinal flora with metabolite disturbances, the Pearson's correlation
coefficient was calculated to generate a correlation matrix (Fig. 4).
Clearly, the disturbed intestinal flora was related to the changed me-
tabolite profiles (CC > 0.9 or < −0.5, p < .01). Fig. 4 displays two
hundred typical intestinal flora-associated metabolites showing high
correlations with specific intestinal flora, thus revealing the functional
correlations of intestinal flora with metabolites. For instance, L-Cystine
(mate 1467), which increased by 5.0-fold within probiotic-fed fish,
showed positive correlation with OTU1 (JCM5805), but negative cor-
relation with f_Comamonadaceae. 1-Palmitoyl-2-hydroxy-sn-glycero-3-
phosphoethanolamine (mate 1467). In addition, it increased by 3.4-fold
in probiotic-treated fish, was positively correlated with g_Sphingo-
monas, but negatively correlated with g_Psychrobacter.
Coinertia analysis can identify successive axes of covariance be-
tween two datasets involving the same test subjects. In this study, co-
inertia analysis was performed using the coinertia function from the
ade4 R package, and was applied to eigenvalues of the metabolome and
microbiome. As shown in Fig. 5, the distributions of Firmicutes and
Proteobacteria were more extensive, which indicated that these types of
microorganisms were the main cause leading to the differential dis-
tribution of metabolites. According to the metabolic components and
the correlation analysis between differential metabolites and microbial
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Aquaculture 527 (2020) 735428
composition, some metabolites lacked the KEGG partition. Besides, the
analysis indicated that metabolites related to amino acid metabolism,
porphyrin and chlorophyll metabolism, metabolism of α-linolenic acid,
metabolism of nicotinamide and niacin, as well as sputum metabolism
were associated with the original differences between groups (Supple-
mental Material, Table S1). The above findings revealed significant
interdependence between metabolites and microorganisms.
4. Discussion
This study described for the first time the gut contents metabolic
profiling changes in tilapia fed probiotic diet using LC-MS. Metabolic
phenotypes revealed significant pattern differences between control
and probiotic-treated groups. In the OPLS-DA score plot, the control
group deviated from the probiotic-treated group, indicating that the
changes in gut content metabolite profiles might be attributed to the
probiotic exposure, and that the function of probiotic as im-
munostimulant and growth promoter might be reflected at the meta-
bolic level. LC-MS is currently the most important metabolomics ana-
lysis platform, and it is suitable for the detection of complex
metabolites in samples. Nonetheless, so far little research was done on
the gut contents metabolome of fish by LC-MS. For instance, the LC-MS
of gut contents was employed to identify the most influential metabo-
lites as the potential biomarkers of soybean meal-induced enteritis in
juvenile pearl gentian grouper (Zhang et al., 2019).
Previous research indicated that, the carps with identical feeding
habits had closer intestinal flora compositions and metabolite profiles
(Li et al., 2017). Besides, additional investigation indicated that, the
evolutionary distance was positively correlated with metabolite profiles
or intestinal flora (Li et al., 2017). Fish with genetic homogeneity had
different metabolites in the presence of structural difference in in-
testinal flora, which reflected the importance of intestinal microbiota
for host metabolism (Wu et al., 2015). The changed intestinal microbial
structure might lead to alterations of intestinal metabolite structures
(Ni et al., 2014). The application of JCM5805 changed the intestinal
microbial composition of tilapia, increased the amount of potential
intestinal probiotics, and reduced certain pathogenic bacterial dis-
tribution (Xia et al., 2018). KEGG annotations of differential metabo-
lites mainly included the metabolism, ABC transporters, metabolism of
methionine and cysteine, biosynthesis of amino acids, protein digestion
and absorption, arachidonic acid metabolism, and asthma. In addition,
KEGG functional analysis of microbial community showed that the
significantly differential metabolic pathways between the two groups
mainly included carbohydrate metabolism, Nucleotide metabolism,
Energy metabolism and Translation. Such results demonstrated the
presence of consistency between intestinal flora composition or its
function and intestinal metabolites. Accumulating evidence suggested
that, the application of JCM5805 altered the intestinal flora composi-
tion in tilapia, thereby changing the intestinal metabolism.
In mammals, including the teleosts order with the greatest amount
and highest evolutionary advance, as well as Perciformes that contain
seabream (Sparus aurata), sea bass (Dicentrarchus labrax), and Nile ti-
lapia (Oreochromis niloticus), both basophils and mast cells (MCs) have
represented the main histamine sources in response to environmental or
immunological stimuli through degranulation (Fang and Xiang, 2015;
Dezfuli et al., 2010). The intestinal epithelial layer is one of the main
distribution tissues of MCs. Our experiment detected that, histamine
was significantly down-regulated in intestinal metabolites of Nile ti-
lapia after administration of JCM5805. Histamine plays an extensive
role in numerous physiopathological processes, and it has the most
significant effects on gastric acid secretion, inflammation, and its role as
a neurotransmitter. It is suggested that, histamine exerts potent reg-
ulation on immune response through affecting a vast majority of im-
mune system cells, and by specifically enrolling them at the tissue sites.
Typically, the latter mechanism impacts the effector function, polar-
ization, activation, and maturation of these cells, thereby resulting in
Y. Xia, et al.
Fig. 5. Results graph of coinertia analysis. Note: The points in the picture represent different OTU, and the different colors stand for various classifications (at phylum
level).
the production of immediate APR and subsequently inducing chronic
inflammatory responses (Galindo-Villegas et al., 2016). In mammal GI
tract, the content of histamine may be affected by the inflammatory and
allergic responses, the changed degradative enzyme activities, micro-
bial processes, and dietary intake of host (Smolinska et al., 2014). In
immunology, histamine shows its modulating effect through binding
onto a specific histamine receptor subtype (Galindo-Villegas et al.,
2016). For example, in mouse macrophages, histamine exerts its effect
on H1R and H2R to limit Mycobacterium bovis bacillus growth through
producing IL-18 (Megyeri et al., 2006). On the contrary, histamine
exerts certain effect on the receptors of H1 and H2 in the model of
Escherichia coli-induced peritonitis, which damages the recruitment of
neutrophils, thus delaying bacterial elimination (Hori et al., 2002). In
existing vertebrate studies, histamine was distributed in the gut-brain
axis, and it played a role of neurotransmitter modulator through reg-
ulating the complicated feeding behavior (Provensi et al., 2016). Due to
the effects of histamine on food intake, it might be the novel approach
for nutritional and immunological management in the industry of
aquaculture (Galindo-Villegas et al., 2016). It was speculated that,
changes in intestinal histamine resulted from the diverse treatments in
this experiment were closely related to host immune and metabolic
regulation.
Administration of JCM5805 increased the distribution of metabolite
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Aquaculture 527 (2020) 735428
N-Acetyl-L-Cysteine in tilapia intestine. According to previous study,
oral administration of N-acetyl-L-cysteine (NAC) into hypothalamic
obese rats blocked dyslipidemia, pre-diabetes, as well as inflamed-
dysmetabolic liver development through effectively lowering the great
endogenous oxidative stress (OS) (Villagarcía et al., 2018). NAC
therapy effectively modulates adipogenesis and inhibits adipocyte lipid
accumulation (Boşgelmez and Güvendik, 2017; Pieralisi et al., 2016);
besides, NAC benefits liver lipogenesis. This was confirmed in the
morphological structure of tilapia liver after JCM5805 treatment
(Supplementary Fig. S2), which showed the significantly reduced adi-
pose tissue and amount of vacuoles.
Correlation analysis of intestinal microbes and metabolites showed
that, Proteobacteria had more p < .01 than other microbial species,
revealing a more important role of Proteobacteria in the production of
differential metabolites. In this study, JCM5805 was significantly po-
sitively correlated with the markedly up-regulated L-cystine in tilapia
intestine. L-cystine is the frequently used treatment for acute infectious
diseases, such as dysentery, typhoid and influenza, and other diseases
like asthma, which is consistent with the effects of JCM5805 on
mammals (Fujii et al., 2017; Shibata et al., 2016). When applied as a
feed additive, L-cystine promotes animal growth and development.
This, might be the direct and important evidence that JCM5805 im-
proved the host growth performance in this study. Additionally, this
Y. Xia, et al.
work also suggested that, microbiome was significantly correlated with
metabolome. The structure of such correlation may arise two ways: 1)
metabolite anabolism and catabolism through microbes; and 2) mi-
crobial growth inhibition and stimulation through metabolites. In fact,
diet variations have been extensively suggested to accompany with
changes in intestinal microbial composition, and the latter affects gut
metabolome (Claesson et al., 2012; Nicholson et al., 2012; Wu et al.,
2011). Nonetheless, it remains unclear about the associated metabolic
pathways and metabolites within the above processes. As a result, the
present work aimed to examine the feasibility to detect bioinformatics
signatures in any process, since the causal relationship of related data
was hardly be finalized.
The correlation network map indicated that, there were many
possibilities at different directions for the correlations between micro-
organisms and metabolites. For instance, the metabolic end product
might be positively correlated with OTU (subsequently the underlying
pathway) that was responsible for production and export, while nega-
tively correlated with OTU (subsequently the underlying pathway) for
import and processing within the downstream pathway. Such results
also suggested that, certain metabolites showed insignificant correla-
tion curves, because enzymes that produced these metabolites were
encoded in organisms. However, they were not related to metabolites
due to the lack of export, a process necessary for this correlation. In
addition, the network diagram demostrated that, some metabolites
were significantly positively correlated with the presence of certain
bacteria, while negatively correlated with other bacteria. This phe-
nomenon might suggest that, metabolites produced by the former had
an inhibitory effect on the latter, revealing that metabolites might drive
the flora structures, and regulate population competition in a direct or
indirect way.
5. Conclusions
In summary, the metabolomic approach is used in combination with
16S rRNA sequencing in this work for analyzing the JCM5805 effect on
intestinal flora as well as the metabolic profiles within tilapia. Based on
the sequencing results, JCM5805 administration remarkably altered
intestinal microbial composition. Meanwhile, according to metabo-
lomic analysis, some metabolites associated with different kinds of
metabolic pathways are substantially disturbed following probiotic
administration. Additionally, certain intestinal flora families show high
correlation with the changed gut microflora-related metabolites upon
correlation analysis. In conclusion, the above findings indicate that,
JCM5805 administration perturbs the abundance of intestinal bacteria,
and alters the intestinal microbial metabolic profiles, supporting the
hypothesis that JCM5805 affects gut flora community composition,
which changes intestinal metabolism, ultimately leading to the im-
provements of host growth and immunity.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
The current study was funded by the China Agriculture Research
System (CARS-46).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.aquaculture.2020.735428.
8
Aquaculture 527 (2020) 735428
References
Benakis, C., Martin-Gallausiaux, C., Trezzi, J.P., Melton, P., Liesz, A., Wilmes, P., 2020.
The microbiome-gut-brain axis in acute and chronic brain diseases. Curr. Opin.
Neurobiol. 61, 1–9. https://doi.org/10.1016/j.conb.2019.11.009.
Boşgelmez, I.İ., Güvendik, G., 2017. N-acetyl-L-cysteine protects liver and kidney against
chromium(VI)-induced oxidative stress in mice. Biol. Trace Elem. Res. 178 (1),
44–53. https://doi.org/10.1007/s12011-016-0901-2.
Claesson, M.J., Jeffery, I.B., Conde, S., Power, S.E., O’Connor, E.M., Cusack, S., Harris,
H.M.B., Coakley, M., Lakshminarayanan, B., O’Sullivan, O., Fitzgerald, G.F., Deane,
J., O’Connor, M., Harnedy, N., O’Connor, K., O’Mahony, D., van Sinderen, D.,
Wallace, M., Brennan, L., Stanton, C., 2012. Gut microbiota composition correlates
with diet and health in the elderly. Nat. 488, 178–184.
Dezfuli, B.S., Pironi, F., Giari, L., Noga, E., 2010. Immunocytochemical localization of
piscidin in mast cells of infected seabass gill. Fish Shellfish Immunol. 28, 476–482.
Doan, H.V., Hoseinifar, S.H., Ringø, E., Esteban, M.Á., Dadar, M., Dawood, M.A.O.,
Faggio, C., 2020. Host-associated. Probiotics: A Key Factor in Sustainable
Aquaculture. 28 (1), 16–42. https://doi.org/10.1080/23308249.2019.1643288.
Ezra-Nevo, G., Henriques, S.F., Ribeiro, C., 2020. The diet-microbiome tango: how nu-
trients lead the gut brain axis. Curr. Opin. Neurobiol. 62, 122–132. https://doi.org/
10.1016/j.conb.2020.02.005.
Fang, Y., Xiang, Z., 2015. Roles and relevance of mast cells in infection and vaccination. J.
Biomed. Res. 30. https://doi.org/10.7555/JBR.30.20150038.
Fujii, T., Jounai, K., Horie, A., Takahashi, H., Suzuki, H., Ohshio, K., Fujiwara, D.,
Yamamoto, N., 2017. Effects of heat-killed Lactococcus lactis subsp. lactis JCM 5805
on mucosal and systemic immune parameters, and antiviral reactions to influenza
virus in healthy adults; a randomized controlled double-blind study. J. Funct. Foods
35, 513–521.
Galindo-Villegas, J., Garcia-Garcia, E., Mulero, V., 2016. Role of histamine in the reg-
ulation of intestinal immunity in fish. Dev. Comp. Immunol. 64, 178–186.
Giatsis, C., Sipkema, D., Ramiro-Garcia, J., Bacanu, G.M., Abernathy, J., Verreth, J.,
Smidt, H., Verdegem, M., 2016. Probiotic legacy effects on gut microbial assembly in
tilapia larvae. Sci. Rep. 6, 33965. https://doi.org/10.1038/srep33965.
Holmes, E., Li, J.V., Athanasiou, T., Ashrafian, H., Nicholson, J.K., 2011. Understanding
the role of gut microbiome-host metabolic signal disruption in health and disease.
Trends Microbiol. 19, 349–359.
Hori, Y., Nihei, Y., Kurokawa, Y., Kuramasu, A., Makabe, K.Y., Terui, T., Doi, H., Satomi,
S., Sakurai, E., Nagy, A., Watanabe, T., Ohtsu, H., 2002. Accelerated clearance of
Escherichia coli in experimental peritonitis of histamine deficient mice. J. Immunol.
169, 1978–1983.
Kokou, F., Sasson, G., Friedman, J., Eyal, S., Ovadia, O., Harpaz, S., Cnaani, A., Mizrahi,
I., 2019. Core gut microbial communities are maintained by beneficial interactions
and strain variability in fish. Nat. Microbilo. 4, 2456–2465. https://doi.org/10.1038/
s41564-019-0560-0.
LeBlanc, J.G., Chain, F., Martin, R., Bermudez-Humaran, L.G., Courau, S., Langella, P.,
2017. Beneficial effects on host energy metabolism of short-chain fatty acids and
vitamins produced by commensal and probiotic bacteria. Microb. Cell Factories
16, 79.
Li, T.T., Long, M., Ji, C., Shen, Z.X., Gatesoupe, F.J., Zhang, X.J., Zhang, Q.Q., Zhang, L.L.,
Zhao, Y.L., Liu, X.H., Li, A.H., 2016. Alterations of the gut microbiome of largemouth
bronze gudgeon (Coreius guichenoti) suffering from furunculosis. Sci. Rep. 6, 30606.
https://doi.org/10.1038/srep30606.
Li, T.T., Long, M., Li, H., Gatesoupe, F.J., Zhang, X.J., Zhang, Q.Q., Feng, D.Y., Li, A.H.,
2017. Multi-Omics analysis reveals a correlation between the host phylogeny, gut
microbiota and metabolite profiles in cyprinid fishes. Front. Microbiol. 8, 454.
Lin, L., Zhang, J.Q., 2017. Role of intestinal microbiota and metabolites on gut home-
ostasis and human diseases. BMC Immunol. 18, 2.
Lu, Y., Chen, J., Zheng, J., Hu, G., Wang, J., Huang, C., Lou, L., Wang, X., Zeng, Y., 2016.
Mucosal adherent bacterial dysbiosis in patients with colorectal adenomas. Sci. Rep.
6, 26337. https://doi.org/10.1038/srep26337.
Lu, K., Knutson, C.G., Wishnok, J.S., Fox, J.G., Tannenbaum, S.R., 2012. Serum meta-
bolomics in a helicobacter hepaticus mouse model of inflammatory bowel disease
reveal important changes in the microbiome, serum peptides, and intermediary
metabolism. J. Proteome Res. 11, 4916–4926.
Megyeri, K., Buzás, K., Miczák, A., Buzás, E., Kovács, L., Seprényi, G., Falus, A., Mándi, Y.,
2006. The role of histamine in the intracellular survival of Mycobacterium bovis BCG.
Microbes Infect. 8, 1035–1044.
Ni, J., Yan, Q., Yu, Y., Zhang, T., 2014. Factors influencing the grass carp gut microbiome
and its effect on metabolism. FEMS Microbiol. Ecol. 87, 704–714. https://doi.org/10.
1111/1574-6941.12256.
Nicholson, J.K., Holmes, E., Kinross, J., Burcelin, R., Gibson, Glenn, Jia, W., Pettersson, S.,
2012. Host-gut microbiota metabolic interactions. Sci. 336, 1262–1267.
Nie, P., Li, Z., Wang, Y., Zhang, Y., Zhao, M., Luo, J., Du, S., Deng, Z., Chen, J., Wang, Y.,
Chen, S., Wang, L., 2019. Gut microbiome interventions in human health and diseases
(Review). Med. Res. Rev. 39 (6), 2286–2313. https://doi.org/10.1002/med.21584.
Pérez-Sánchez, T., Ruiz-Zarzuela, I., de Blas, I., Balcázar, J.L., 2014. Probiotics in aqua-
culture: a current assessment. Rev. Aquac. 6, 133–146. https://doi.org/10.1111/raq.
12033.
Pieralisi, A., Martini, C., Soto, D., Vila, M.C., Calvo, J.C., Guerra, L.N., 2016. N-acet-
ylcysteine inhibits lipid accumulation in mouse embryonic adipocytes. Redox Biol. 9,
39–44.
Provensi, G., Blandina, P., Passani, M.B., 2016. The histaminergic system as a target for
the prevention of obesity and metabolic syndrome. Neuropharmacology 106, 3–12.
Ringø, E., Zhou, Z., Gonzalez Vecino, J.L., Wadsworth, S., Romero, J., Krogdahl, Å.,
Olsen, R.E., Dimitroglou, A., Foey, A., Davies, S., Owen, M., Lauzon, H.L., Martinsen,
Y. Xia, et al.
L.L., De Schryver, P., Bossier, P., Sperstad, S., Merrifield, D., 2016. Effects of dietary
components on the gut microbiota of aquatic animals: a never-ending story? Aquac.
Nutr. 22, 219–282. https://doi.org/10.1111/anu.12346.
Shibata, T., Kanayama, M., Haida, M., Fujimoto, S., Oroguchi, T., Sata, K., Mita, N.,
Kutsuzawa, T., Ikeuchi, M., Kondo, M., Naito, K., Tsuda, M., Nishizaki, Y., Ishii, N.,
2016. Lactococcus lactis JCM5805 activates anti-viral immunity and reduces symp-
toms of common cold and influenza in healthy adults in a randomized controlled
trial. J. Funct. Foods 24, 492–500.
Smolinska, S., Jutel, M., Crameri, R., OMahony, L., 2014. Histamine and gut mucosal
immune regulation. Allergy 69, 273–281.
Villagarcía, H.G., Castro, M.C., Arbelaez, L.G., 2018. N-Acetyl-L-Cysteine treatment effi-
ciently prevented pre-diabetes and inflamed-dysmetabolic liver development in hy-
pothalamic obese rats. Life Sci. 199, 88–95.
Wang, A.R., Ran, C., Ringø, E., Zhou, Z.G., 2018. Progress in fish gastrointestinal mi-
crobiota research. Rev. Aquac. 10 (3), 626–640. https://doi.org/10.1111/raq.12191.
Wang, C., Chuprom, J., Wang, Y., Fu, L., et al., 2020. Beneficial bacteria for aquaculture:
nutrition, bacteriostasis and immunoregulation. J. Appl. Microbiol. 128 (1), 28–40.
https://doi.org/10.1111/jam.14383.
White, J.R., Nagarajan, N., Pop, M., 2009. Statistical methods for detecting differentially
abundant features in clinical metagenomic samples. PLoS Comput. Biol. 5, e1000352.
https://doi.org/10.1371/journal.pcbi.1000352.
Wu, G.D., Chen, J., Hoffmann, C., Bittinger, K., Chen, Y.Y., Keilbaugh, S.A., Bewtra, M.,
Knights, D., Walters, W.A., Knight, R., Sinha, R., Gilroy, E., Gupta, K., Baldassano, R.,
Aquaculture 527 (2020) 735428
Nessel, L., Li, H.Z., Bushman, F.D., Lewis, J.D., 2011. Linking long-term dietary
patterns with gut microbial enterotypes. Sci. 334, 105–108.
Wu, S., Wang, G., Angert, E.R., Wang, W., Li, W., Zou, H., 2012. Composition, diversity,
and origin of the bacterial community in grass carp intestine. PLoS One 7, e30440.
https://doi.org/10.1371/journal.pone.0030440.
Wu, S., Ren, Y., Peng, C., Hao, Y.T., Xiong, F., Wang, G.T., Li, W.X., Zou, H., Angert, E.R.,
2015. Metatranscriptomic discovery of plant biomass-degrading capacity from grass
carp intestinal microbiomes. FEMS Microbiol. Ecol. 91, fiv107. https://doi.org/10.
1093/femsec/fiv107.
Xia, Y., Lu, M.X., Chen, G., Cao, J.M., Gao, F.Y., Wang, M., Liu, Z.G., Zhang, D.F., Zhu,
H.P., Yi, M.M., 2018. Effects of dietary Lactobacillus rhamnosus JCM1136 and
Lactococcus lactis subsp. lactis JCM5805 on the growth, intestinal microbiota, mor-
phology, immune response and disease resistance of juvenile Nile tilapia, Oreochromis
niloticus. Fish Shellfish Immunol. 76, 368–379. https://doi.org/10.1016/j.fsi.2018.
03.020.
Xia, Y., Cao, J.M., Wang, M., Lu, M.X., Chen, G., Gao, F.Y., Liu, Z.G., Zhang, D.F., Ke, X.L.,
Yi, M.M., 2019. Effects of Lactococcus lactis subsp. lactis JCM5805 on colonization
dynamics of gut microbiota and regulation of immunity in early ontogenetic stages of
tilapia. Fish and Shellfish Immunol. 86, 53–63.
Zhang, W., Tan, B.P., Ye, G.L., Wang, J.X., Dong, X.H., Yang, Q.H., Chi, S.Y., Liu, H.Y.,
Zhang, S., Zhang, H.T., 2019. Identification of potential biomarkers for soybean meal-
induced enteritis in juvenile pearl gentian grouper, Epinephelus
lanceolatus♂ × Epinephelus fuscoguttatus♀. Aquaculture 512, 734337.