Journal of Fish Diseases 2017 doi:10.1111/jfd.

12624

Changes in the gut microbiome of the Chinese mitten crab

(Eriocheir sinensis) in response to White spot syndrome
virus (WSSV) infection

Z F Ding , M J Cao, X S Zhu, G H Xu and R L Wang

Jiangsu Key Laboratory for Biofunctional Molecules, College of Life Sciences and Chemistry, Jiangsu Second Normal
University, Nanjing, China

microbiome were closely associated with the sever-

Abstract
ity of WSSV infection and that indicator taxa
Intestinal microorganisms play important roles in could be used to evaluate the crab health status.
maintaining host health, but their functions in
Keywords: 16S rRNA, Eriocheir sinensis.
aquatic animal hosts have yet to be fully eluci-
gut microbiome, white spot syndrome virus.
dated. The Chinese mitten crab, Eriocheir sinensis,
is one such example. We attempted to identify
the shift of gut microbiota that occurred in
response to infection of white spot syndrome virus Introduction
(WSSV), an emerging viral pathogen in the crab
The Chinese mitten crab, Eriocheir sinensis, is one
aquaculture industry. The microbiota may exert
of the most economically important crustaceans in
some control over aspects of the viral pathogene-
freshwater aquaculture production in China. The
sis. We investigated the changes in composition
aquaculture of E. sinensis is a rapidly developing
and structure of the crab gut microbiome during
industry, with an annual output worth ~US $4 bil-
various WSSV infection stages of 6 h post-infec-
lion in Jiangsu Province, China (Meng et al. 2014).
tion (hpi) and 48 hpi, using a 16S rRNA
Recently, serious outbreaks of white spot syndrome
approach on the MiSeq Illumina sequencing plat-
virus (WSSV) have caused catastrophic losses in the
form. Four phyla (Firmicutes, Proteobacteria,
crab harvest (Ding et al. 2015). WSSV is an extre-
Tenericutes and Bacteroidetes) were most dominant
mely lethal and contagious pathogen responsible
in the gut of E. sinensis regardless of the WSSV
for massive mortalities reported in the cultivation
infection stages. However, further analysis revealed
of crustaceans worldwide and has been associated
that over 12 bacterial phyla, 44 orders and 68
with the severe economical impact registered in dif-
families were significantly different in abundance
ferent countries where its occurrence has been
at various states of WSSV infection. Several
reported (Oidtmann & Stentiford 2011). However,
intriguing aspects of E. sinensis gut bacteria that
the correlation between the viral pathogen and host
had not been previously reported were also uncov-
intestinal microbiome as beneficial agents against
ered, such as class Mollicutes was dominant here,
the virus remains to be revealed. It is important for
but absent in crabs from Yangtze River estuary
aquaculture that the issues regarding the analysis of
and Chongming Islands. Overall, this study pro-
correlations and outcome be explored.
vided the first evidence that changes in gut
Increasing evidence has revealed a close associa-
tion between intestinal bacterial communities and
Correspondence Z F Ding, Jiangsu Key Laboratory for Biofunc- animal health. Characterization of the variations
tional Molecules, College of Life Sciences and Chemistry, Jiangsu in intestinal microflora between healthy and dis-
Second Normal University, 77 West Beijing Road, Nanjing 210013,
eased hosts can provide an initial step towards
China
(e-mails: ding@jssnu.edu.cn; dingzf@yeah.net) better disease prediction and therapy (Boutin et al.

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2013). Intestinal microflora may assist hosts by
promoting nutrient absorption, stimulating the
immune response and improving disease resis-
tance, thereby crucially affecting the health status
of the individual (Clemente et al. 2012). How-
ever, most studies of intestinal microbial commu-
nities have focused on mammals, marine
invertebrates and fishes (Meziti et al. 2010; Berry
et al. 2012; Chaiyapechara et al. 2012; Clemente
et al. 2012; Li et al. 2012), whereas far fewer
studies have focused on crustaceans, including
crabs, despite the fact that crabs are major agricul-
tural products with high economic value
worldwide.
Interactions between bacteria and their host rep-
resent a full continuum from pathogenicity to
mutualism and are no longer considered a two-
component system but rather a complex network
of associations (Boutin et al. 2013). Therefore, to
explore the complexity of host–bacteria interac-
tions, experimental methods with high resolution
are needed. Despite their usefulness, denaturing
gradient gel electrophoresis (DGGE), cloning or
culture-based methods have shown limitations in
accurately characterizing complex bacterial com-
munities. The emergence of high-throughput
sequencing technologies now provides high-defini-
tion tools to deeply investigate complex taxonomic
assemblages in bacterial communities (Caporaso
et al. 2012; Boutin et al. 2013). This allows char-
acterizing both the composition (i.e. diversity) and
the structure (i.e. abundance of species) of highly
diverse bacterial communities.
In this study, we used a 16S rRNA approach
on the MiSeq Illumina sequencing platform to
investigate the structure dynamics of gut bacterial
communities in the freshwater crab E. sinensis
subject to challenge with WSSV. This study pre-
sented for the first time that major changes in the
crab gut bacterial community coincided with virus
infection.
Materials and methods
Experimental animals
Ninety Chinese mitten crabs (E. sinensis) with a
mean body weight of 91.42  8.76 g (48 males
and 42 females) were used for the experiment. After
being removed from traps that were placed in Lake
Gucheng, in Nanjing city of China, the crabs were
brought into the laboratory where they were
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acclimated in a recirculating holding system at
about 27 29 °C, pH 7.1–7.3, for 2 weeks. This
holding system had its own filtration unit to main-
tain adequate water quality. Hemolymph of the
crabs was sampled aseptically and examined initially
by real-time PCR assays for the detection of WSSV
(Ding et al. 2016a), microsporidium (Ding et al.
2016b) and spiroplasma (another common patho-
gen in E. sinensis; Ding et al. 2014). Crabs negative
for these pathogens were chosen for the
experiments.
Challenge experiment
WSSV was propagated in vivo by inoculation of
clarified gill homogenates from naturally infected
E. sinensis. The WSSV solution, filtered through a
0.22-lm filter and serially diluted to
100 copies lL 1 with PBS (pH 7.2), was used as
inocula (Ding et al. 2016a). The 90 crabs were
randomly divided into two groups: 20 crabs in a
control group and 70 crabs in the infection group.
The two groups were housed separately. After
1 week of starvation, crabs in the infection group
were intramuscularly injected with WSSV at a
dose of 103 copies (10 lL) per crab, while 20
crabs in the control group were intramuscularly
injected with 10 lL PBS.
Sampling
Ten crabs injected with PBS were sampled as con-
trols, and ten crabs from the infection group were
sampled at 6 h post-infection (hpi) and 48 hpi,
respectively. All of the crabs were subjected to
cold-temperature anaesthesia by being placed in a
freezer ( 20 °C) for 5–10 min. Their body sur-
faces were then washed thoroughly using sterile
water and disinfected with 75% ethanol, and dis-
sected using sterile lancets and forceps. All dissect-
ing tools were dropped in alcohol and then flame-
sterilized between each individual sample. The
extracted gut were immediately placed in 1.5-mL
sterilized Eppendorf tubes on dry ice and then
stored in a 80 °C freezer until further analysis.
Histological analysis and electron microscopy
observations
For histology, gut of the crabs were also fixed in
4% paraformaldehyde solution for 24 h and then
transferred to 70% ethanol. After dehydration in a
 Journal of Fish Diseases 2017
graded ethanol series to absolute ethanol, samples
were embedded in paraffin. Five 10-lm-thick sec-
tions were obtained from each piece in different
orientations and planes using a manual rotary
microtome (Leica RM2235). Sections were then
stained with haematoxylin and eosin (H&E) and
examined using a light microscope (Nikon,
Eclipse 80i).
For transmission electron microscopy (TEM),
10 lL of the hemolymph from the crab E. sinensis
was fixed with 2.5% glutaraldehyde and then col-
lected on formvar-coated copper grids for 1 min.
The wet residues were immediately stained with
sodium phosphotungstate (2%) for 30 s. The grid
was air-dried before examination using a transmis-
sion electron microscope (Hitachi H-7650).
PCR amplification and 16S rDNA sequencing
Microbial DNA was extracted from the 30 gut
samples using the MicroElute Genomic DNA Kit
(Omega) following the manufacturer’s instruc-
tions. Once the sample DNA was prepared, PCR
was used with unique bar-coded primers to
amplify the hypervariable region 3 (V3) and V4
of the 16S rRNA gene, creating an amplicon
library from the metacommunity DNA samples
(Kozich et al. 2013). The oligonucleotide primers
used for the amplification were as follows: the for-
ward primer 319F (ACTCCTACGGGAGGCAG
CAG) and the reverse primer 806R (GGACTAC
HVGGGTWTCTAAT; Kozich et al. 2013). Each
DNA template was amplified separately in a final
volume of 30 lL containing 5 lL template DNA,
3 lL 10 9 STR buffer (including dNTP, Mg2+,
Promega), 0.2 lL Taq DNA polymerase, 2 lL
primers mix (2 lM) and 19.8 lL sterile water.
Amplifications were performed starting with a 30-
s template denaturation step at 98 °C, followed
by 35 cycles of denaturation at 98 °C for 10 s,
annealing at 55 °C for 30 s, extension at 72 °C
for 45 s and final extension at 72 °C for 10 min.
The PCR products (approximately 469-bp pre-
dicted product size) were visualized by UV illumi-
nation and normalized by AxyPrepTM Mag PCR
Normalizer (Axygen Biosciences), which allowed
for the skipping of the quantifications step regard-
less of the PCR volume submitted for sequencing.
The amplicons were prepared for sequencing with
AMPure XT beads (Beckman Coulter Genomics),
and the size and quantity of the amplicon library
were assessed on the LabChip GX (Perkin Elmer)
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Z Ding et al. Gut microbiota after WSSV infection
and with the Library Quantification Kit for Illu-
mina (Kapa Biosciences), respectively. The PhiX
Control library (v3; Illumina) was combined with
the amplicon library (expected at 30%; Hu et al.
2016). The libraries were clustered to a density of
approximately 570 K mm 2 and sequenced on
300PE MiSeq runs.
Data analysis
The sequence reads were filtered by QIIME
(Quantitative Insights Into Microbial Ecology,
http://qiime.org/tutorials/processing_illumina_da
ta.html) quality filters. The CD-HIT pipeline was
used for picking operational taxonomic units
(OTUs) through making OTU tables. Sequences
were assigned to OTUs at 97% similarity. Repre-
sentative sequences were chosen for each OTU,
and taxonomic data were then assigned to each
representative sequence using the RDP (Riboso-
mal Database Project) classifier (Caporaso et al.
2011). To estimate alpha diversity (diversity
within the samples), three metrics were calculated:
Shannon index, Simpson index and the observed
OTUs metric as the count of unique OTUs found
in the sample (Vishnivetskaya et al. 2011). To
estimate the beta diversity (differences between the
samples), principal coordinates analysis (PCoA)
was conducted with the software package of
Fathom Toolbox for Matlab (http://www.marine.
usf.edu/user/djones/). A UPGMA (unweighted
pair group method with arithmetic mean) tree
was also constructed from the pairwise distance
matrix, using the QIIME (Quantitative Insights
Into Microbial Ecology) program. To reveal the
shared OTUs among samples, a Venn diagram
was drawn with VennDiagram package in R envi-
ronment (http://www.r-project.org). Nonparamet-
ric t-test and Fisher’s exact test were applied to
determine significant difference between two
groups. All statistical analyses and plotting were
carried out with SigmaPlot 11.0 (Systat Software
Inc.). Values of P < 0.05 were considered signifi-
cant (95% confidence interval).
Results
Histopathology and electron microscopy
observations
Infections by WSSV in the crabs were confirmed
by histology and negative staining analysis. No
 Journal of Fish Diseases 2017
pathological changes could be observed in the
control crabs (data not shown), whereas the exper-
imentally infected crabs showed typical
histopathological changes associated with WSSV
infection in the gut (Fig. 1a). Infected nuclei were
hypertrophied with marginalized chromatin and
contained inclusion bodies that stained intensely
eosinophilic. Hemolymph from experimentally
infected crabs examined by TEM also showed the
presence of WSSV virions (Fig. 1b).
Total Illumina sequence reads, quality
trimming and OTU designation
A total of 907 110 raw sequence reads from 30
samples were generated on the Illumina Miseq
sequencing platform, including 213 711 raw
reads, 101.3 M base from control group; 392 891
raw reads, 186.23 M base from 6 hpi group; and
300 508 raw reads, 142.44 M base from 48 hpi
(Fig. 2a). After trimming, 878 168 quality
sequence reads were used for further bioinformat-
ics analyses. Within these reads, 206 789
sequences clustered into 176 980 OTUs from the
control group; 380 615 sequences clustered into
301 678 OTUs from 6 hpi group; 290 764
sequences clustered into 227 871 OTUs from
48 hpi group. All OTUs were clustered at a 97%
sequence similarity from the trimmed sequences
of the respective samples using UCLUST (Edgar
2010; Koo et al. 2015). There were statistically
(a)
Figure 1 (a) Histopathological findings of the hindgut from WSSV-infected Eriocheir sinensis. Hindgut showed evident signs of
infection with hypertrophied nuclei displaying marginalized chromatin (thick arrow) and eosinophilic inclusion bodies (thin arrow);
the upper left corner showed magnification of the frame area. Scale bar = 50 lm. (b) Electron micrograph of negatively stained
WSSV virions in its hemolymph, Scale bar = 10 lm.
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Z Ding et al. Gut microbiota after WSSV infection
significant differences (P < 0.001, t = 4.752
with 18 degrees of freedom, 95% confidence
interval for difference of means) among the three
groups, confirming they hosted different OTUs
abundances. A significant increase in the number
of OTUs was observed at 6 hpi (P < 0.001), fol-
lowed by a distinct decline at 48 hpi (P < 0.05),
but still significantly higher than the controls
(P < 0.01), suggesting a change in the gut envi-
ronment (Fig. 2b).
A total of 1142 OTUs were shared by all sam-
ples, whereas the numbers of unique OTUs were
11 704 in the control group, 21 059 at 6 hpi and
16 153 at 48 hpi, respectively (Fig. 3).
Diversity indices (Simpson and Shannon; Key-
lock 2005) and observed species indicated a rela-
tively low diversity within the control samples,
with moderate diversity in the 48 hpi; and high
diversity in the 6 hpi samples (Fig. 4a–c). The
unweighted UniFrac analysis of PCoA (Fig. 4d)
and UPGMA clustering (Fig. 4e) implied that the
WSSV infection may have a significant influence
on the gut microbiome, due to the distinct clus-
ters depended mostly on the infection process.
Microbial composition and structure
The abundances of taxa were identified to the
most resolvable taxa (phylum, class, order, family
and genus). Across all 30 samples from three
groups, 97.69% of the phylotypes belonged to just
(b)
 Journal of Fish Diseases 2017 Z Ding et al. Gut microbiota after WSSV infection

Raw sequence reads

(a) Base (M) (b)
5e+5 200 45 000
**
** *
180 40 000
4e+5
Raw sequence reads

35 000
160

No. of OTUs
3e+5

Base (M)
30 000
140
25 000
2e+5
120
20 000
1e+5
100 15 000

0 80 10 000
Control 6 hpi 48 hpi Control 6 hpi 48 hpi

Figure 2 (a) Raw sequence reads and base generated from the gut of Eriocheir sinensis at different stages of WSSV infection using a
16S rRNA approach on the MiSeq Illumina sequencing platform; (b) comparisons of the total operational taxonomic units (OTUs)
of the gut samples at different WSSV infection states. All OTUs clustered at a 97% sequence similarity from the trimmed sequences
of the respective samples. *P < 0.05; **P < 0.01.

Figure 3 The Venn diagram showing the

number of unique and shared operational
taxonomic units (OTUs) of gut bacteria at
different stages of WSSV infection in the
Chinese mitten crab (Eriocheir sinensis).

four core phyla: Firmicutes (36.98%), Proteobacte-
ria (30.9%), Tenericutes (19.2%) and Bacteroidetes
(10.6%; Fig. 5a,b). The four dominant phyla
were relatively similar distributed in each sample
at different states of WSSV infection, but with
different abundance and variation trend. The
other divisions that have been consistently found
were Actinobacteria, Spirochaetes, Verrucomicrobia
and TM6 (a candidate phylum without cultured
representatives; Fig. 5b).

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 Journal of Fish Diseases 2017 Z Ding et al. Gut microbiota after WSSV infection

Figure 4 Microbial diversity comparisons of the crab Eriocheir sinensis at different WSSV infection states. (a) Shannon index, (b)
observed species, (c) Simpson index and (d) unweighted UniFrac-based principal coordinates analysis (PCoA) plot (the control,
6 hpi and 48 hpi were indicated in red, orange and blue, respectively). (e) UPGMA (unweighted pair group method with arithmetic
mean) clustering based on all OTUs from the gut samples.

As the ‘core’ microbiome, Proteobacteria and
Bacteroidetes in the crab gut upregulated signifi-
cantly at 6 hpi and reached the highest abundance
at 48 hpi. However, Firmicutes and Tenericutes
showed significant decrease in the abundance fol-
lowing the WSSV infection and displayed the
lowest composition at 48 hpi (P < 0.001, q = 0;
Fig. 5b,c; Table S1). As the ‘secondary’ or ‘rare’
OTUs, Actinobacteria was identified from seven
crabs in the control group (7/10, 0.1–5.7%), six
crabs in the 6 hpi group (6/10, 0.1%), while only
two crabs at 48 hpi (2/10, 0.1%; Fig. 5c); Spiro-
chaetes was detected from nine crabs in the control
group (9/10, 0.1–3.7%), while only identified
from five crabs in the 48 hpi group (5/10, 0.1–
2.7%; Fig. 5c).
At the class level, the abundance of Gammapro-
teobacteria, Epsilonproteobacteria and Bacteroidia
increased significantly following WSSV infection,
achieving the highest numbers at 48 hpi
(P < 0.001, q < 0.0001). In contrast, Mollicutes,
Alphaproteobacteria and Bacilli displayed an obvi-
ous decrease in abundance and reached the lowest
number at 48 hpi (Fig. 6a–c; Table S2).
Additionally, 55 bacterial orders changed signif-
icantly in abundance at 6 hpi, 45 orders at 48 hpi
and 44 orders differed significantly between 6 and
48 hpi (Fig. 7a; Table S3). Among them,
Aeromonadales, Campylobacterales and Bacteroidales
showed significant increase in the abundance fol-
lowing the WSSV infection; however, Bacillales
displayed gradual decrease (Fig. 7b).
At the family level, 96 bacterial families
were significantly different in abundance between
the control and 6 hpi groups; 87 families
between the control and 48 hpi; and 68 families
changed pronouncedly between 6 and 48 hpi
(Table S4).

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Figure 5 Structure and composition of the gut bacterial communities of Eriocheir sinensis at different stages of WSSV infection on
phylum level of taxonomy. (a) appearing in each sample (N = 10), (b) means representing as three groups and (c) the changes in
abundance of dominant bacterial phyla at different WSSV infection states. Abundances were based on the proportional frequencies
of those DNA sequences that could be classified at the phylum level.

the gut bacteria of aquatic invertebrates (Goffredi

Discussion
Intestinal microorganisms influence various host
functions including immunity, disease resistance,
development and nutrition (Kwong & Moran
2016). The effect of the intestinal microorganisms
on the host–pathogen interaction is clearly of great
interest in disease control in aquaculture. Thus far,
the shift of the intestinal bacterial profile during
the microbial infection process is not well under-
stood. A limited number of studies in crustaceans
include shrimps (Chaiyapechara et al. 2012),
prawns (Tzeng et al. 2015; Mente et al. 2016),
crabs (Givens et al. 2013; Chen et al. 2015) and
lobsters (Meziti et al. 2010; O’Rorke et al. 2012)
and in most cases are focused on development,
habitats, digestion and nutrition. In this study, we
investigated for the first time the changes in gut
bacterial populations at two different stages of
WSSV infection in E. sinensis using an Illumina-
based high-throughput sequencing method.
Several types of association between the gut bac-
teria and aquatic host animals such as ingested,
transient and resident populations are possible,
and many factors such as the internal structure,
diet, host conditions, habitat and season can affect
et al. 2014; Zhang et al. 2016). This study
revealed similar dominant bacteria population to
the previous studies done in the Atlantic blue
crab, Callinectes sapidus (Givens et al. 2013).
Crabs used in this research were farmed in Lake
Gucheng, in Nanjing city of China. In a study of
E. sinensis from another Lake Tai (286 km from
Lake Gucheng), the microbiome contained five
phyla: Proteobacteria (38.3%), Bacteroidetes
(33.6%), Tenericutes (20.3%), Firmicutes (7.0%)
and TM7 (0.8%; Chen et al. 2015). These were
the dominant bacterial taxa in both studies, but
they differed in abundance between locations.
Moreover, the TM7 was absent in our study, and
only 0.3% of TM6 were identified. The microbial
composition in E. sinensis gut was different from
that found in black tiger shrimp (Penaeus mon-
odon), which were more heavily dominated by
Proteobacteria (more than 80%), followed by Fir-
micutes and Bacteroidetes (Rungrassamee et al.
2014). These dominant bacteria may pose central
roles in gut functions or were well adapted to the
digestive tract.
Some intriguing aspects of crab gut bacteria
were also uncovered. In this study, class Mollicutes

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Figure 6 Structure and composition of the gut bacterial communities of Eriocheir sinensis at different stages of WSSV infection on
class level of taxonomy. (a) appearing in each sample (N = 10), (b) means representing as three groups and (c) the changes in abun-
dance of dominant bacterial classes at different WSSV infection states. Abundances were based on the proportional frequencies of
those DNA sequences that could be classified at the class level.

Figure 7 Structure and composition of the gut bacterial communities of Eriocheir sinensis at different stages of WSSV infection
on order level of taxonomy. (a) Abundances of the dominant orders in the gut of Eriocheir sinensis at different WSSV infection
states (N = 10 in each group). (b) The changes in abundance of dominant bacterial orders at different WSSV infection states.
Orders with abundances <1% were not considered. Unclassified meant that the taxa were not assigned to any known order in
the current database.

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(phylum Tenericutes) was dominant in the gut
microbiota of E. sinensis; however, it was absent
in crabs from other habitats, such as Yangtze
River estuary and the Chongming Islands (Li
et al. 2007; Chen et al. 2015). Several less abun-
dant Bacteroidetes phylotypes, like Mangrovibac-
terium (2.9%), Dysgonomonas (1.4%), were also
only present in our study, but absent in E. sinensis
both from Lake Tai and Chongming Islands
(Chen et al. 2015). These discrepancies may result
from diet or habitat differences. Representatives
from the class Epsilonproteobacteria (Proteobacteria)
inhabit many ecological niches, both terrestrial
and marine, and may perform a diversity of meta-
bolic functions (Gupta 2006). Members of
Epsilonproteobacteria have been associated as epi-
bionts of crustaceans such as Kiwa puravida (Gof-
fredi et al. 2014), and as gut microbial inhabitants
in reared Norway lobster Nephrops norvegicus
(Meziti, Mente & Kormas 2012) as well as
hydrothermal vent-dwelling shrimp, Rimicaris
exoculata (Durand et al. 2010). The commonality
of the occurrence of Epsilonproteobacteria in inver-
tebrates may indicate a mutual benefit between
the bacterial taxa and the host, perhaps at the
physiological and nutritional levels (Hakim et al.
2015). However, none of these previous studies
have investigated the role of Epsilonproteobacteria
in host–virus interactions. We monitored the
dynamics of Epsilonproteobacteria during WSSV
infection process and our findings implied that
members of Epsilonproteobacteria may play an
important role in antiviral protection.
Regarding the dramatic changes in the intestinal
bacterial community from healthy to diseased
crabs, one of the most important questions is
whether sensitive assemblages could be served as
indicators of host health. In this study, we found
that the more than 87 bacterial families were sig-
nificantly different in abundance between healthy
and diseased crabs. Importantly, we can make
educated guesses regarding functionality based on
our knowledge of the biology and ecology of these
families. For example, the abundances of Lacto-
bacillaceae and Bacillaceae significantly decreased
in WSSV-infected crabs. Species affiliated with
these families are known to inhibit the adherence
of enteropathogens (Clemente et al. 2012) and
stimulate the immune response (Xiong et al.
2015). A substantial reduction in these species
could lead to pathogen-induced infection. Indeed,
members of these families have been widely
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Z Ding et al. Gut microbiota after WSSV infection
applied in controlling crustacean disease. Members
of Bacillus, Bifidobacterium, Enterococcus, Lacto-
bacillus, Lactococcus, Nitrosomonas, Paracoccus and
Streptococcus can be important probiotics in crus-
tacean aquaculture (Bentzon-Tilia, Sonnenschein
& Gram 2016).
Crustaceans suffer from many bacterial diseases
(Wang 2011). In the current study, several asymp-
tomatic pathogens such as Acinetobacter, Aeromonas,
Arcobacter, Cyanobacteria, Flavobacterium, Pseu-
domonas and Vibrio were identified in E. sinensis
from the control group, but the abundances were
low. Members of Arcobacter (phylum Proteobacte-
ria) are common enteropathogens and have been
detected in the intestinal and faecal samples of
different farmed animals (Collado & Figueras
2011). We found that the order Arcobacter
accounted for only 0.3% of the gut bacteria in
the control group. However, a sharp increase in
its abundance was observed after WSSV infection.
Similarly, Aeromonas enteropelogenes, causing diar-
rhoea in humans and presenting the same
pathogenicity factors featured by A. hydrophila,
was also common but in low abundance; it signifi-
cantly increased with WSSV infection. The same
dynamics was also observed in the toxic Cyanobac-
teria. Taken together, it was possible that these
opportunistic pathogens were commonly present
in the gut of healthy crabs, but may become infec-
tious when the hosts became stressed via an indi-
rect suppression of the host’s immune system.
Many factors could contribute to the individual
bacterial population variations within groups as
observed by some unique OTUs in the present
experiment (Fig. 4). The use of mixed family
post-larvae, feeding status and the health status of
an individual could potentially affect the gut bac-
teria of crabs (Chaiyapechara et al. 2012). Further
studies should focus more on the individual varia-
tions in the same treatments and associate the
importance of these bacterial groups with specific
aspects of the host–virus interactions.
Acknowledgements
We thank Prof. Jeffrey D. Shields (Virginia Insti-
tute of Marine Science, The College of William
& Mary) for valuable comments and suggestions.
We thank the anonymous reviewers and the edi-
tors for suggestions that have helped improve the
quality and focus on this MS. This work was sup-
ported by grants from the Natural Sciences
 Journal of Fish Diseases 2017
Foundation of China (NSFC No. 31402332),
Natural Science Fund of Colleges and Universities
in Jiangsu Province (16KJA180005,
16KJB180006) and Project for Aquaculture of
Jiangsu Province (Y2016-29).
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Supporting Information
Additional Supporting Information may be found
in the online version of this article:
Table S1. Statistical comparisons of the abun-
dances of gut bacteria at different stages of WSSV
infection on phylum level of taxonomy.
Table S2. Statistical comparisons of the abun-
dances of gut bacteria at different states of WSSV
infection on class level.
Table S3. Comparisons of the abundances of
gut bacteria at different WSSV infection stages on
order level.
Table S4. Comparisons of the abundances of
gut bacteria at different WSSV infection states on
family level.
Received: 27 November 2016
Revision received: 30 January 2017
Accepted: 31 January 2017