Microb Ecol

DOI 10.1007/s00248-017-0967-1

MICROBIOLOGY OF AQUATIC SYSTEMS

Bacterial Signatures of BRed-Operculum^ Disease in the Gut

of Crucian Carp (Carassius auratus)
Tongtong Li 1,2 & Huan Li 1,2 & Franois-Jol Gatesoupe 3 & Rong She 4 & Qiang Lin 1,2 &
Xuefeng Yan 1,2 & Jiabao Li 1,2 & Xiangzhen Li 1,2

Received: 8 November 2016 / Accepted: 16 March 2017

# Springer Science+Business Media New York 2017

Abstract Fish gut microbiota play important roles in fish
immunity, nutrition, and the adaptation to environmental
changes. To date, few studies have focused on the interactions
among environmental factors, fish diseases, and gut microbi-
ota compositions. We compared the gut bacterial communities
of healthy crucian carps (Carassius auratus) with those of
individuals affected by Bred-operculum^ disease and corre-
sponding water and sediment microbiota in four fish farm
ponds. Distinct gut bacterial communities were observed in
healthy and diseased fish. The bacterial communities of dis-
eased fish were less diverse and stable than those of healthy
individuals. The differences in bacterial community composi-
tions between diseased and healthy fish were explained by the
changes in the relative abundances of some specific bacterial
OTUs, which belonged to the genera such as Vibrio,
Aeromonas, and Shewanella, and they were prevalent in dis-
eased fish, but rare or even absent in environmental samples.
Tongtong Li and Huan Li contributed equally to this work.
Electronic supplementary material The online version of this article
(doi:10.1007/s00248-017-0967-1) contains supplementary material,
which is available to authorized users.
* Xiangzhen Li
lixz@cib.ac.cn
1
Key Laboratory of Environmental and Applied Microbiology, CAS,
Chengdu Institute of Biology, Chinese Academy of Sciences,
Chengdu 610041, China
2
Environmental Microbiology Key Laboratory of Sichuan Province,
Chengdu Institute of Biology, Chinese Academy of Sciences,
Chengdu 610041, China
3
NUMEA, INRA, University of Pau and Pays Adour, 64310 Saint Pe
sur Nivelle, France
4
Inspection Center, Tongwei Co., Ltd, Chengdu 610041, China
Water temperature and ammonia concentration were the two
most important environmental factors that impacted gut mi-
crobiota in diseased fish. These results highlighted the surge
of some potential pathogens as bacterial signatures that were
associated with Bred-operculum^ disease in crucian carps.
Keywords Gut microbiota . Crucian carp . BRed-operculum^
disease . Water temperature . Ammonia concentration
Introduction
Gut microbiota are integral components of the hosts, which
play important roles in host nutrition and health [13]. The
loss of microbial diversity may cause gut ecosystem less sta-
ble and healthy [4]. However, the gut microbiota also harbor
opportunistic pathogens. The overgrowth of these potential
pathogens may cause intestinal dysbiosis, possibly due to
the deficiency in the host immune defense system or the dam-
age to intestinal mucosal barrier [57]. The pathogens and
their harmful products (such as endotoxin) can then migrate
from the intestinal lumen through the epithelial mucosa to
infect internal tissues [8]. Previous investigations have dem-
onstrated the translocation of pathogenic Vibrio salmonicida,
Vibrio fischeri, Vibrio anguillarum, and Aeromonas
salmonicida in the gut of fish, with intestinal cell damages
caused by pathogenic A. salmonicida and V. anguillarum
[912]. Therefore, the gastrointestinal tract has been proposed
as a principal pathway for pathogen invasion in fish [13, 14].
Many gastrointestinal diseases are associated with the shifts of
gut microbiota in mammals and lower vertebrates, such as fish
[1519], and this stimulates the interests in understanding how
gut microbial communities are assembled and how they im-
pact host fitness. Such studies have been increasingly carried
out in fish gut microbiota using next generation sequencing

technique. However, most of the previous studies focused on
healthy fish to investigate the factors that may contribute to
shape gut microbiota, such as diet, behavior, and genotype of
the host [7, 2022]. Few of them focused on the interactive
relationships among gut microbiota, fish diseases, and envi-
ronmental drivers [18, 23].
Teleosts are in direct contacts with the aquatic environ-
ment, and the exposure to complex and dynamic environmen-
tal microbiota may influence their health [19]. Aquatic micro-
biota are strongly shaped by water quality. Further, the gut
bacterial communities of fish also change considerably in re-
sponse to the dynamics of environmental factors, e.g., water
temperature and salinity [2426]. Temperature shock or ex-
cess in ammonia and nitrite in the pond environment leads to
the deterioration of water quality, and finally impair the host
immune function seriously and further increases the suscepti-
bility of fish to disease [2730]. Since host immunity may
alter gut microbial compositions [31], water quality can mod-
ulate the assembly of gut microbiota through its influence on
fish immunity [29, 30, 3234]. Understanding the relation-
ships among the occurrences of some specific microbes in fish
gut and the concomitant changes in water quality and disease
occurrences may help us to develop efficient measures to pre-
vent and treat fish diseases.
Crucian carp (Carassius auratus) is one of the oldest cul-
tured fish in the world. The production of this fish species in
China reached 2.6 million tons in 2014, accounting for about
10.4% of the total annual yield of freshwater cultured fish
[35]. However, various disease outbreaks are frequent in
high-density culture conditions. In recent years, a novel bac-
terial disease occurs among crucian carp farms in China. The
disease caused huge economic loss in freshwater fish farming
in Jiangsu province. Because of the typical symptoms of
bright red gills, the disease was called Bred-operculum^ by
local fishermen. The cumulative mortality could reach 60%
during the epidemic in summer, and the conventional bacteri-
ological methods failed to identify the causative agent of the
disease. Some symptoms are similar to those of the hemor-
rhagic septicemia that affected crucian carp in the early
nineties in the same region, which was attributed to
Aeromonas punctata and non-cholera Vibrio with
cultivation-dependent methods [36]. There were other reports
about bacterial hemorrhagic septicemia affecting Carassius
auratus [3740], but virus also caused similar diseases
[4143]. In order to improve health and productivity in
crucian carp farming, it is essential to characterize the gut
microbiota in correlation with such a disease and the environ-
mental factors that may influence its composition and stability.
The present study focused on the relationships between the
emergence of the Bred-operculum^ disease and the gut micro-
biota in crucian carp cultured in farm ponds. Using 16S rRNA
gene Miseq sequencing, we compared the gut microbiota in
diseased and healthy fish to identify the key bacterial
Li T. et al.
phylotypes that linked with the disease and to determine the
environmental factors that may influence the composition and
diversity of fish gut microbiota.
Materials and Methods
Sample Collection and DNA Extraction
Severe Bred-operculum^ disease outbreaks in cultured crucian
carp ponds occurred from the May to August 2015, in
Yancheng city, Jiangsu province, China. Diseased crucian
carp showed clinical signs of a bright red and swollen gill
and an increased production of mucus in gill. Clear yellow
to blood-tinged fluid was presented in the body cavities of
abdominal distention individuals. The liver and kidney
showed that tumefaction, hemorrhage, lack of elasticity, and
gall bladder were dark and enlarged. The digestive tract did
not contain food, and hemorrhages were observed on the in-
testinal wall. The healthy fish were active and feeding normal-
ly, and bacteria were not detected by bacteriological culture
from the liver. The fish were reared in the pond, in which the
water depth and surface were approximately 1.7 m and 1 ha,
respectively. The fish were fed twice a day with commercial
feed (moisture 12.5%; crude protein 33.0%; crude fiber
7.5%; crude fat 3.0%; crude ash 15.0%; calcium 0.4
1.6%; phosphate 1.0%; salt 0.31.2%; lysine 1.5%;
Tongwei Group Co., Ltd., China). A total of 36 diseased fish
(D) showing typical clinical symptoms and 47 healthy fish (H)
were sampled in four ponds (CB, CN, S, and W) from May to
August 2015. Three ponds were sampled twice from May to
June (CB) and from June to August (S and W), respectively.
The sampled fish averaged 132.7 36.2 g and 18.99 1.7 cm
in body weight and length, respectively.
Prior to dissection, all fish were euthanized with an over-
dose of MS 222 (Sigma, Germany). All procedures for han-
dling and euthanasia of fish were done according to the pro-
cedures described by Li et al. [18]. All the samples were
placed in an ice box and sent to the laboratory within 24 h.
DNA extraction from gut samples was according to Li et al.
[18] with some modification. Excised gut of 200 mg was
placed into a sterile 2-mL tube with 200 mg of 0.1-mm glass
beads, 600 L of lysis solution, and 10 L Proteinase-K so-
lution (Qiagen, Germany), homogenized for 40 s in a bead
beater (Biospec Products), and centrifuged at 4000g for
5 min to eliminate the pellet of debris. The supernatant was
collected for isolating DNA with the QIAamp Fast DNA Stool
Mini Kit (Qiagen, Germany) according to the manufacturers
instructions.
In addition, the pond water and surface sediment samples
from different sites in the same pond were taken at the same
time of a day (ca. 13:30 h) with fish samples. Pond water was
sampled at a depth of approximately 50 cm to the top water
BRed-Operculum^ Disease and Gut Microbiota
layer, and then filtered through a 0.2 m hydrophilic polye-
thersulfone membrane filter (47 mm diameter, Pall, Lane
Cove, Australia). Sediment samples were collected using a
Petersen grab, and only the unconsolidated surface sediments
were collected. The ponds with disease outbreaks at sampling
were termed as diseased ponds in this study (Table S1).
DNA extraction from the filter membrane and sediment
was also performed using the QIAamp Fast DNA Stool
Mini Kit (Qiagen, Germany). NanoDrop 2000
Spectrophotometer (Thermo Scientific, USA) was used to
check the concentration and quality of extracted DNA.
Extracted DNA was diluted to 10 ng/L and stored at
40 C for downstream use. We also measured physicochem-
ical parameters of water from each pond, including tempera-
ture, pH, transparency, dissolved oxygen (DO), total
ammonia-nitrogen (NH 3-N), nitrite-nitrogen (NO 2 -N),
nitrate-nitrogen (NO3-N), and soluble reactive phosphate
(PO43) concentrations according to Boyd, Tucker [44].
PCR Amplification and MiSeq Sequencing
Universal primer 515F (5-GTGYCAGCMGCCGCGGTA-
3) and 909R (5-CCCCGYCAATTCMTTTRAGT-3) with
12 nt unique barcode at 5-end of 515F were used to amplify
the V4-V5 hypervariable region of bacterial 16S rRNA gene
[45]. Each sample was amplified in duplicate and with a 25 L
reaction system containing 1 Ex Taq PCR buffer, 10 pM of
each primer, 0.25 U Takara Ex Taq (all TaKaRa
Biotechnology Co., Ltd., Dalian, China), and 10 ng genomic
DNA. The following program included 3 min at 94 C,
followed by 30 cycles of 94 C for 30 s, 56 C for 30 s, and
72 C for 30 s, and finally, 10 min at 72 C. All the samples
were sequenced using Illumina Miseq platform (MiSeq
Reagent Kit V.2, 500 cycles) at the Environmental Genome
Platform of Chengdu Institute of Biology, Chinese Academy
of Sciences. The raw sequences were deposited at NCBI/EBI/
DDBJ Sequence Read Archive (Accession No. DRA004779).
Statistical and Bioinformatics Analysis
The raw sequence data were processed using QIIME Pipeline-
Version 1.7.0 (http://qiime.org/) [46]. Chimeras were removed
using the Uchime algorithm [47, 48]. The overlapping paired-
end reads were merged using the FLASH-1.2.8 software [49].
T h e m e rg ed se q ue n c es w i t h hi g h- q ua l i t y ( r ea d s
length > 300 bp, without ambiguous base BN,^ and average
base quality score > 30) were used for further analysis. All
sequences were trimmed and assigned to each sample based
on their barcodes (barcode mismatches = 0). Taxonomy was
assigned using the Ribosomal Database Project classifier, with
a confidence threshold of 80% [50]. The sequences classified
as Bunassigned^ and Barchaea^ were removed. The remaining
sequences were clustered into operational taxonomic units
(OTUs) at 97% identity threshold using CD-HIT [51].
Singleton sequences were also filtered out. The number of
reads was normalized to 2786 reads per sample, and alpha-
diversity (number of observed OTUs) and beta diversity
(BrayCurtis distance) of bacterial communities were calcu-
lated. The rarefaction curves were generated from the remain-
ing numbers of OTUs.
Nonmetric multidimensional scaling (NMDS) based on
BrayCurtis distance was used to visualize the variation of
bacterial structure across different groups using the R vegan
package [52]. The heatmap was constructed using the
heatmap 2 function of the R gplots package [53]. Statistical
testing among variations in bacterial community compositions
(BrayCurtis distance metric) was carried out using analysis
of similarity (ANOSIM). Linear discriminant analysis coupled
with effect size (LEfSe) was utilized to identify the bacterial
taxa differentially represented between groups with the signif-
icance threshold of 0.05 for the alpha parameter for the
KruskalWallis test among classes and the cut-off logarithmic
LDA score of 2.0 [54]. In order to test the relationships be-
tween bacterial communities, the discriminating abundant
taxa, and various water physicochemical factors, multiple re-
gressions were performed using the envfit function in the R
package (NMDS ordination of the microbial community on a
matrix of BrayCurtis) and spearmans correlation analysis
[52]. Significances of differences between groups (alpha di-
versity and alpha variability of bacterial community) were
evaluated by one-way analysis of variance (one-way
ANOVA) followed by Tukeys post hoc test (SPSS, version
19.0).
To predict the functional profiles of microbial communi-
ties, OTUs were picked using a closed reference (Greengenes
ver. 13.5) at 97% sequence similarity, with normalization to
the control for differences in 16S rDNA copy numbers among
OTUs, and their metagenomic contributions were predicted
using PICRUSt based on the Kyoto Encyclopedia of Genes
and Genomes (KEGG) pathways [5557]. The pertinence of
the metagenome predictions was assessed by computing NSTI
(Nearest Sequenced Taxon Index).
Determination of Ecosystem Stability
Ecosystem stability is measured specifically by comparing
squared coefficients of variation of alpha diversity [58]: lower
variability signifies higher ecosystem stability. The alpha var-
iability ( = CV2species) of bacterial community in each gut
samples was calculated based on the method of Wang, Loreau
[58]. In this framework, the alpha variability represents the
local shifts of species diversity within a meta-community.
The observed species (Vspecies) in the bacterial community of
each gut sample was used to calculate alpha variability. In
order to assess the relationship between biodiversity and the
 Li T. et al.

stability of fish gut microbiota, linear regression analysis was diversity indices in the healthy fish were significantly higher
carried out between observed species and alpha variability. than those in the diseased fish (Fig. 1a, ANOVA, p < 0.05).
Alpha variability is used to reflect ecosystem stability at
local scale in meta-community. Our data showed that the al-
pha variability of gut bacterial communities increased signif-
Results
icantly in the diseased fish (diseased, 0.20 0.034; healthy,
0.068 0.046; ANOVA, F = 17.25, p = 0.009). In addition,
Overall Microbial Community Composition
the decrease in bacterial biodiversity correlated with the in-
crease in alpha variability, indicating a positive correlation
A total of 131 samples from 4 ponds (CB, CN, S and W) were
between alpha bacterial diversity and the stability of fish gut
collected from May to August, 2015, including 83 gut sam-
microbiota (Fig. 1b).
ples (36 diseased fish and 47 healthy fish), 24 water samples
NMDS plot was used to compare the similarity in the bac-
(10 from diseased pond and 14 from normal pond), and 24
terial community compositions of gut (diseased and healthy
sediment samples (10 from diseased pond and 14 from normal
fish) and environmental samples (water and sediment). Gut
pond) (Table S1). After initial quality filtering, chimera
bacterial communities were remarkably distinct from their
checking, and OTU picking, 34,285 non-singleton OTUs
surrounding environmental communities (ANOSIM,
were identified at the 97% similarity level. For the down-
r = 0.60, p = 0.001), whereas there was also a clear grouping
stream alpha and beta diversity analyses, sequences were nor-
of the samples from sediment and water (Fig. 1c, d, ANOSIM,
malized to the depth of 2786 sequences. Although the rarefac-
r = 0.68, p = 0.001). Samples from diseased and healthy fish
tion curves of observed OTUs did not reach asymptote
formed two distinct clusters (ANOSIM, r = 0.60, p = 0.001).
(Fig. S1A), the Shannon diversity index, reflecting both rich-
The sampling location had effects on the clustering patterns of
ness and evenness, plateaued in all samples. This indicated
gut microbial communities from both healthy and diseased
that the bacterial diversities presenting in these samples were
fish (Fig. 1d, ANOSIM, r = 0.36, p = 0.001, and r = 0.15,
enough to yield stable and unbiased estimates of phylotype
p = 0.004, respectively), but sampling time only had signifi-
richness (Fig. S1B).
cant impacts on the clustering pattern of healthy fish (Fig. 1c,
Roughly, 70% of total reads were annotated at genus level.
ANOSIM, r = 0.64, p = 0.001, and r = 0.06, p = 0.21,
Cetobacterium, Vibrio, unclassified_Cyanobacteria, GpXIII,
respectively).
Aeromonas, GpIIa, Paludibacter, Shewanella, and
Bacteroides represented approximately 43% of the sequences,
Differences in Bacterial OTUs and Functions
constituting the dominant genera in intestinal and environ-
mental microbiome. Linear discriminant analysis effect size
We also performed LEfSe to identify specific OTUs (relative
(LEfSe) was performed to identify the abundant bacterial gen-
abundance >0.30%) that were differentially distributed be-
era that were significantly differentiated between diseased and
tween diseased and healthy fish. The diseased fish showed
healthy/normal samples, including gut, sediment, and water
overgrowth of Vibrio OTU6580, Vibrio OTU50048,
samples. Nine abundant genera (relative abundance >0.30%;
Paludibacter OTU424, Aeromonas OTU5314, and
e.g., Vibrio, Aeromonas, Bacteroides, and Shewanella) were
Shewanella OTU82306, together with the depression of
overrepresented in diseased fish, while depressing the relative
Cetobacterium OTU43215, Clostridium XI OTU12412,
abundances of Cetobacterium, Clostridium sensu stricto,
GpXIII OTU190, and Cyanobacteria OTU78 (Fig. 2a, b).
GpXIII, and unclassified_Cyanobacteria (Table S2). Possible
The presumptive functions of the gut microbial community
disease indicators might correspond to GpIIa, Ilumatobacter,
of fish were also examined by predicting the metagenome
Methylotenera, and unclassified_Cyanobacteria, which were
using PICRUSt based on 16S rRNA sequences. The nearest
among the detected bacteria enriched in the water of diseased
sequenced taxon index (NSTI) mean value was 0.12 0.04 in
ponds (relative abundance >0.10%; Table S3). For the sedi-
the fish gut samples. For comparison, Langille et al. [55]
ment samples, no genera were observed to be significantly
found that Human Microbiome Project samples had NSTI
more abundant in the diseased ponds than those in the healthy
mean value of 0.03 0.2. These values were higher in other
ponds (data not shown).
mammalian guts (0.14 0.06) and much higher in soil com-
munities (0.17 0.02). Thus, the fish gut samples provide
Differences in Bacterial Community Diversity reasonable datasets to examine predictions from PICRUSt.
and Structure Similarly, we used LEfSe to explore the differences in predict-
ed prevalence of bacterial functions between gut microbiota in
Overall, higher bacterial community diversity indices were diseased and healthy fish. Two functional trends were inferred
observed in the environmental samples, compared to those from the comparison by identifying two sets of 17 gene fam-
from fish guts (Fig. 1a, ANOVA, p < 0.05). The alpha ilies, the presumptive prevalence of which appeared different
BRed-Operculum^ Disease and Gut Microbiota
Fig. 1 Alpha diversity, alpha variability, and community structures of
fish gut and environmental samples. Alpha diversity is measured by the
number of observed OTUs (a). The boxes represent the interquartile range
(IQR), from the first and third quartiles, and the inside bold line represents
the median. The dotted lines extending vertically from the boxes
(whiskers) denote the lowest and highest values within 1.5 IQR from
the first and third quartiles. Groups indicated with the same letter are
in the two groups of bacterial communities. The most discrim-
inable genes corresponded to bacterial motility proteins and
chemotaxis, and they were more abundant in diseased fish
samples (Fig. 2c, d).
In addition, 28 abundant OTUs were overrepresented in the
water samples of diseased ponds identified by LEfSe (e.g.,
GpIIa OTU9226, Cyanobacteria OTU78, and Methylotenera
OTU7952; relative abundance >0.10%; Fig. S2). The most
abundant OTUs in intestinal samples were not or feebly de-
tected in the environmental samples (Fig. S3A). The percent-
age of the abundant OTUs (relative abundance >0.30%) in
diseased and healthy fish were 76.70 and 55.87%, respective-
ly. However, the relative abundances of these OTUs were
13.77 and 8.51% in water samples of the diseased and normal
ponds, respectively, and only 4.59 and 2.74% in correspond-
ing sediment samples. Among the most abundant OTUs in
not significantly different at = 0.05 using Tukeys post hoc test. b
Linear regression relationship between microbial biodiversity (number
of observed OTUs) and alpha variability. NMDS plot shows the
microbial community structures of samples collected at different
sampling time (c) and location (d). Pairwise community distances were
determined based on BrayCurtis distance. H represents healthy and D
represents diseased fish
diseased fish, some specific OTUs belonged to the genus
Vibrio, Aeromonas, and Shewanella, which were likely oppor-
tunistic bacteria that are pathogenic to fish [5961]. These
OTUs were also present in some healthy fish with low abun-
dances, but most of them were rare or even absent in the
environmental samples (Fig. S3b).
Relationships Between Bacterial Community
Compositions and Environmental Parameters
Various water physicochemical factors, including temperature
(Tep), pH, transparency (Trans), dissolved oxygen (DO), total
ammonia-nitrogen (NH 3-N), nitrite-nitrogen (NO 2 -N),
nitrate-nitrogen (NO3-N), and soluble reactive phosphate
(PO43), were measured in each pond (Fig. S4). The impacts
of these factors on the bacterial communities of fish and water
 Li T. et al.

Fig. 2 The most differentially

abundant OTUs (relative
abundance >0.30%) and
predicted function between the
diseased and healthy fishes
identified by LEfSe. The left
histogram shows the linear
discriminant analysis scores
computed for the most
discriminating OTUs (a) and gene
functions (c), based on their
relative abundances in the gut
microbiota of crucian carp. The
right heatmap shows the
distributions of the most
differentially distributed OTUs
(b) and gene functions (d) based
on the relative abundances of
OTUs (rows) and gene functions
(rows) after z score transformation

were tested using multiple regression models based on NMDS
ordination scores (Fig. 3). We found that all water physico-
chemical factors were significantly correlated to the gut bac-
terial composition of fish. Tep and inorganic nitrogen (e.g.,
NH3-N, NO2-N, and NO3-N) were positively correlated
with the microbial communities of diseased fish, while DO,
pH, Trans, and PO43 were positively associated with the gut
microbiota of healthy fish (Fig. 3a). Similarly for water sam-
ples, positive associations were observed between Tep, NH3-
N, and the microbial communities from diseased ponds, while
BRed-Operculum^ Disease and Gut Microbiota
Fig. 3 The relationships between water physicochemical factors and the
bacterial compositions in fish gut (a) and water (b) identified using
NMDS. Arrows indicate multiple regressions between NMDS
ordination scores and environmental variables, with orientation and
length indicating the changing direction and correlation strength,
respectively. The arrows with red and blue indicate significant and non-
significant correlations with the ordination, respectively
pH and DO were positively correlated to the microbial com-
munities of normal ponds (Fig. 3b). Tep and NH3-N strongly
correlated the microbial community of fish, while two most
important factors shaping the community composition of wa-
ter were Tep and DO.
In addition, the correlations between those abundant genera
in fish gut (mentioned in Table S2) and water physicochemical
factors were estimated in order to understand the influence of
environmental factors on the gut microbiota. The most
abundant genera in diseased fish (e.g., Vibrio, Aeromonas,
Bacteroides, and Shewanella) were positively correlated with
Tep and inorganic nitrogen and negatively correlated with pH
and DO (Fig. S5A). Most of the genera abundant in water
samples from diseased ponds (unclassified_Cyanobacteria,
GpIIa, Methylotenera, Conexibacter, and Methylocystis) were
positively correlated to Tep and NH3-N or NO2-N and neg-
atively correlated with DO and pH (Fig. S5B).
Discussion
Hemorrhagic septicemia, including the Bred-operculum^ dis-
ease, frequently occurs under intensive aquaculture in China.
Due to the lack of comparative research between microbial
communities of healthy and diseased fish, little is known
about the potential bacterial pathogens associated with Bred-
operculum^ disease. For the first time, this study explored the
relationship between Bred-operculum^ disease and fish gut
bacterial communities. Bacterial signatures of red-operculum
disease were observed in the gut of crucian carp (Carassius
auratus). These results may provide a theoretical reference to
bacterial markers in the intestine for preventing and treating
fish diseases in the future.
Red-Operculum Disease Significantly Changed Gut
Microbiota
Many investigations demonstrate the important roles of gut
microbiota in fish health [1, 2, 18, 19, 62, 63]. In addition,
the intestine may host opportunistic pathogens, and it is evi-
denced as a port of entry for several experimental infections
[9, 11, 13]. Diversity is important in all ecosystems for pro-
moting stability and performance. Microbial diversity, in par-
ticular, gut microbial diversity, may be considered as a bio-
marker of host health and metabolic capacity [4]. This study
provided further evidence that the alpha diversity of gut mi-
crobiota was significantly reduced in the diseased fish
(Fig. 2a). A significantly higher alpha variability of bacterial
community in the diseased fish indicated less ecosystem sta-
bility, compared to healthy fish. Such loss of microbial diver-
sity has been observed under environmental stress [4, 58].
Thus, less diversity and stability of gut microbiota in fish
may be associated with diseases.
In addition, the structure of the fish gut bacterial commu-
nity was primarily influenced by the disease, even though the
sampling location and time had significant impacts on the
clustering patterns (Fig. 1c, d). Both bacteria and virus may
cause hemorrhagic septicemia in crucian carp [36, 37], and
pathogenic bacteria seem involved in the etiology of spring
viremia in common carp [64]. The oral route is evidenced for
both types of microbes [41, 6567], and the intestinal defenses
are crucial against such infections. Intestinal cellular damages

are among the most common symptoms in fishes affected by
the Bred-operculum^ disease. Similar to the intestinal cellular
damages, it is unclear whether microbial community change is
a cause or a consequence of the disease. However, the shift in
gut bacterial community may provide some signature charac-
teristics of the Bred-operculum^ disease. The overgrowth of
intestinal opportunistic bacteria, e.g., Vibrio, Aeromonas, and
Shewanella, and the depression of beneficial bacteria, e.g.,
Cetobacterium, in diseased fish are likely linked to the occur-
rence of the disease.
Water Temperature and Inorganic N Are Important
in Shaping Fish Gut Microbiota
Fish commensal microbiota can be affected by environmental
factors [22, 2426, 33, 68]. Previous studies revealed that
salinity and temperature influenced the composition of fish
gut bacteria [22, 25, 26, 68]. In addition, the homeostasis of
fish bacterial community was extensively disturbed by envi-
ronmental stress (high density and hypoxia), and the abun-
dance of beneficial bacteria decreased after stress exposure,
while the proportion of potentially pathogenic bacteria in-
creased [33]. Our data pointed out that water temperature
and ammonia strongly influenced the gut community compo-
sition of fish. As ectotherms, many fish have relatively narrow
ranges of temperature in which they are able to thrive and
grow. This is why temperature has long been termed as the
Bmaster factor^ in the biology of fishes for its governance over
physiological processes, such as oxygen consumption, as well
as ammonia and carbon dioxide excretion, which increase
with temperature in many fish species [6971]. Carbon diox-
ide is the most common cause of acidity in water [72]. DO and
pH in pond water are lower, while ammonia concentration is
higher in summer, compared with other seasons, in correlation
with changes in fish metabolism.
Ammonia was another key factor that influences gut mi-
crobiota. High ammonia concentration may result in poor
growth and feed conversion and increase stress and suscepti-
bility to bacterial infections and diseases [73]. Ammonia can
be oxidized by bacteria into nitrite (NO2), which remains
toxic to fish by decreasing oxygen transport in blood [74],
unless it is further oxidized into nitrate. High concentration
of dissolved ammonia and nitrite or sudden temperature
change can harm the immune function of fish, and thus in-
crease the susceptibility to diseases [68, 73, 7577].
Water temperature was also the most significant factor that
influenced the microbial community of pond water (Fig. 3b).
In summer, the concentration of dissolved inorganic nitrogen
increased (NH3-N, NO2-N, and NO3-N), most likely due to
fast mineralization of protein-rich feedstuff and suspended
organic matter (e.g., feces) in water system under high tem-
perature. The consequences of fast mineralization of organic
matter result in inorganic N release, oxygen consumption, and
Li T. et al.
pH reduction in water system. The hot season corresponded to
the main Bred-operculum^ disease outbreaks, with distinct gut
bacterial compositions between summer and spring (Fig. 1c).
Cyanobacteria OTU78, 764 and GpIIa OTU275, 9226
(Cyanobacteria) were dominant in water samples from dis-
eased ponds (Fig. S2). Associated with the water temperature
increase, the presence of high concentrations of inorganic ni-
trogen generated favorable conditions for the proliferation of
Cyanobacteria (blue-green algae), which were ubiquitous in
both pond water and fish gut (Fig. S3) [78, 79]. Moreover, the
dominance of aquatic Cyanobacteria results in the increase of
cyanobacteria toxins release and the levels of carbon dioxide,
but the reduction of DO, PO43 concentrations, and pH.
Finally, these environmental factors lead to the deterioration
of water quality and may further influence the gut microbial
community by affecting the immune system of fish [8082].
This study suggests the importance of manipulating inorganic
N, DO, pH, and PO43 et al., in rearing water to prevent and
treat the occurrence of Bred-operculum^ disease in aquacul-
ture practice.
We found that the genus Methylotenera was more abundant
in water samples of diseased ponds than normal ponds
(Table S3 and Fig. S2). Some members of this genus are
known as methylotrophic bacteria, which have been frequent-
ly found to be associated with harmful blooms of freshwater
cyanobacteria [83]. The strong correlations between the levels
of inorganic nitrogen (positive), PO 4 3 , DO, and pH
(negative) and the predominance of OTUs in GpIIa,
unclassified_Cyanobacteria and Methylotenera might be di-
rect consequences of the metabolism of these microorganisms
(Fig. S5B). This may help to explain why the Bred-
operculum^ disease was common and seasonal.
Bacterial Signatures of Red-Operculum Disease in the Gut
of Crucian Carp
Cetobacterium (Fusobacteria) was the most abundant genus in
fish gut, and the relative abundance of this genus was much
higher in healthy fish than those in diseased fish (Table S2).
The highly abundant OTUs of Cetobacterium (OTU735,
4731, and 43215) were closely related to Cetobacterium
somerae with similarities of 9599% using BLAST search.
Their total relative abundance was higher than 33% in healthy
fish in contrast to 18.2% in the diseased fish. Other studies
also observed high proportion of Cetobacterium in fish guts.
Some of them are beneficial to the host by producing vitamin
B12 and butyrate [84, 85], and their antibacterial properties
[86, 87].
The OTUs belonging to genus Vibrio, Aeromonas, and
Shewanella (Proteobacteria) were overrepresented in the gut
microbiota of diseased fish. The abundances of these bacterial
taxa correlated positively with temperature and inorganic ni-
trogen (e.g., NH3-N, NO2-N, and NO3-N) and negatively
BRed-Operculum^ Disease and Gut Microbiota
with pH and DO (Fig. 3b and Fig. S3A). These genera are
known to include main opportunistic bacteria pathogenic to
fish [5961, 88]. Moreover, these bacteria have high-adhesive
capability that can help to colonize the surface of the intestinal
mucosa, and the gut might be a primary location for stress-
induced infections by pathogenic strains [89]. The preserva-
tion of Bmicrobial balance^ in the gut is critical for health. In
diseased fish, the likely overgrowth of opportunistic patho-
genic bacteria diminished the relative importances of other
taxa and affected the host immune system and health by caus-
ing the imbalance of gut microbial communities. Our results
suggested that the dominance of these microbes belonging to
Vibrio, Aeromonas, and Shewanella in fish gut microbiota
might be associated with Bred-operculum^ disease, though
more evidences are needed.
Interestingly, the members of Vibrio, Aeromonas, and
Shewanella were more frequently present in some of the
healthy fishes, but not in environmental samples (Fig. S3).
This suggested that these pathogenic strains are usually pres-
ent in the healthy gut, which is consistent with a previous
study about Aeromonas hydrophila and Aeromonas
salmonicida [13, 18]. The presence of these microorganisms
in the healthy fish indicates that they may act not only as
opportunistic pathogens, but also as important players of other
functions, e.g., cellulose digestion or polyunsaturated fatty
acid production [7, 9092]. The Brare biosphere^ might sim-
ply be the products of historically ecological change with the
potential for rare taxa to become dominant in responses to the
shifts in environmental conditions, when local or global
changes favor their growth [93]. Environmental stressors,
such as hypoxia, inorganic nitrogen pollutants, or changes in
temperature and pH, may weaken hosts immune system and
allow opportunistic pathogens to cause diseases by colonizing
or invading the intestinal mucosa. Then, the disruption of
ecological equilibrium in the gut leads to the overgrowth of
pathogenic intestinal bacteria [73, 75, 76].
Stress can alter the intestinal function in fish. Some patho-
genic strains in fish gut can attack membrane surfaces, trans-
locate across the intestinal wall, and propagate the disease
[77]. The PICRUSt functional prediction further supported
that the genes involved in cell motility (bacterial chemotaxis
and motility proteins), membrane transport (bacterial secretion
system), and signaling molecules and interaction (bacterial
toxins) were significantly more abundant in diseased fish
(Fig. 2c, d), implicating that these genes are required for many
pathogenic species to colonize and invade a host [94].
Therefore, dysbiosis and the transformation of micro-
ecological niches in fish gut might be associated with the
occurrence of the Bred-operculum^ disease in response to en-
vironmental stresses. However, the functional categories of
genes were only predicted in this study, and their presence
and activity must be tested further using multiple approaches,
e.g., metatranscriptome.
In summary, this study showed the interactive relationships
among fish gut microbiota, Bred-operculum^ disease, and en-
vironmental factors, e.g., water temperature and ammonia
concentration. The likely overgrowth of the opportunistic
pathogens might play an important role in the occurrence of
Bred-operculum^ disease. These signature bacteria are mem-
bers of the genera Vibrio, Aeromonas, and Shewanella, and
many of them have been documented as pathogenic strains to
fish. Water temperature and ammonia concentration (quickly
mineralized from protein-rich foodstuff at warm water tem-
perature in summer) were recognized as two most important
factors that impacted gut microbiota in diseased fish. These
phenomena highlight the need for manipulating the water
quality in rearing ponds according to seasonal changes, for
example, manipulating water inorganic N, DO, pH, and
PO43 et al., in the framework of suitable strategies for better
health management in aquaculture. The present study pro-
vides significant insights into the etiology of Bred-operculum^
disease and help us to design effective prevention and control
strategies in intensive aquaculture practice.
Acknowledgments This work was supported by Sichuan Province
Science and Technology Project (2017SZ0004, 2017JY0231), Tongwei
Co., Ltd., China, and China Biodiversity Observation Networks (Sino
BON).
Authors Contributions Xiangzhen Li, Tongtong Li, and Huan Li
conceived the research. Tongtong Li, Huan Li, and Xuefeng Yan per-
formed the experiments. Tongtong Li wrote the manuscript. Tongtong
Li, Xiangzhen Li, Huan Li, and Franois-Jol Gatesoupe edited the man-
uscript. She Rong, Qiang Lin, Xuefeng Yan, and Jiabao Li contributed
sampling, reagents, or data analysis pipeline. All authors reviewed and
accepted the manuscript.
Compliance with Ethical Standards
Ethical Approval Compliance with the ethics committee of the
Chengdu Institute of Biology, Chinese Academy of Sciences, and the
methods used in this study were carried out in accordance with the
approved guidelines.
Conflict of Interest The authors declare that they have no conflicts of
interest.
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