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Genetics of Aquatic Organisms 5(1), 19-28
http://doi.org/10.4194/2459-1831-v5_1_03
Published by Central Fisheries Research Institute (SUMAE) Trabzon, Turkey.
P R O O F
R E S E A R C H P A P E R
Comparison of Symbiotic Bacterial Community of Soft Corals
Sarcophyton and Sinularia of the Hainan Province, (South China
Sea, China)
Hao Lu1,2,#, Alireza Asem1, #, Lu Wang3, Weidong Li4,*, PeiZheng Wang5
1Hainan Tropical Ocean University, College of Fisheries and Life Science, 572000 Sanya, China.
2Hainan University, College of Ocean, 570228 Haikou, China.
3Jiangsu Provincial Freshwater Fisheries Research Institute, 210000 Nanjing, China.
4Hainan University, College of Ecology and Environment, Haikou, China.
5Hainan Tropical Ocean University, College of Ecology and Environment, Sanya, China.
#These authors contributed equally to this work.
Article History
Received 27 October 2020
Accepted 29 November 2020
First Online 30 November 2020
Corresponding Author
Tel.: +86089866213606
E-mail: 542148880@qq.com
Keywords
Sarcophyton
Sinularia
16S rRNA
Microbiome
Soft corals
Abstract
Changes in the microbial community associated with environmental impacts can lead
to opportunistic infections, coral disease and death. Diversity analysis and community
comparison were performed on 23 collected soft Coral specimens from South China
See surrounding Hainan Province (China) based on Illumina MiSeq sequencing. The
results showed that Proteobacteria was the main symbiotic bacteria in soft corals. In
the same geographical location, the diversity and abundance of symbiotic bacteria in
the genus Sinularia are higher than genus Sarcophyton. Unlike Sinularia, the genus
Sarcophyton is more inclined to Tenericutes. Furthermore, the same coral species has
different bacterial community structure in different environments. The temperature
difference between sampling points at 2 ℃ is the main factor affecting the results. A
large number of Endozoicomonas found in stone corals have not become the dominant
bacteria associated with soft corals. Coral-related pathogenic bacteria were not found
in this investigation. This study provided a baseline for future studies of soft coral
microbiomes, and assessment of functions of host metabolites and soft coral
holobionts. Our result documented that same coral species in each locality represent
identical pattern of bacterial diversity and community
Introduction
Alcyonacea is an order of Coelenterata, Anthozoa,
and Octocorallia. The sea area near Hainan Island is rich
in soft coral resources, and their symbiotic microbes
have become an important target for the study of
marine natural products (Hassan et al., 2019). Symbiotic
microorganisms play an important role in driving the
nutritional transformation and community succession of
coral reef ecosystems and provide new ideas for
scientists to develop new marine drugs (Sang et al.,
2019).
As an important part of the symbiotic microbial
system, symbiotic bacteria play a key role in the material
cycle (Lesser et al., 2004), energy flow (Mao-Jones et al.,
2010), and healthy growth of coral ecosystems
(Mahmoud & Kalendar, 2016). During coral bleaching,
some nitrogen-fixing bacteria in mucus can replace the
algae to feed coral organisms. For example, Oculinary
patagonica in the Mediterranean Sea can synthesize
organic matter and supply coral tissue to help corals
survive during the crisis of lost zooxanthella (Teplitski &
Ritchie, 2009). Moreover, probiotics can also promote
the ecological balance of flora in or around the host
(Merrifield et al., 2010). The coral symbiotic bacteria
community is a complex dynamic combination, and this
complex community structure is susceptible to many
factors. Regional differences, eutrophication, diseases
(Rosenberg et al., 2009) and other driving forces can
lead to changes in coral symbiotic microbial community
20
GenAqua 5: 19-28 (2021)
structure. For example, McKew et al. (2012) found that
the same species of corals in the Caribbean and
Indonesian seas have different commensal bacterial
community, and the diversity and abundance of
symbiotic bacteria are significantly different. Thurber et
al. (2009) pointed out that temperature, dissolved
organic carbon content, and acidity can affect the
symbiotic bacterial community structure of corals.
Furthermore, the composition of the coral symbiotic
bacteria is affected by environmental factors. Thus, the
structure of the coral symbiotic bacteria community can
better elucidate the growth condition of the corals. In a
study of coral symbiotic bacteria under healthy and
bleached conditions, Yu et al. (2019) found that some
pathogens (Vibrio, Pseudospirilum, Alteromonas, and
Coxiella) are present in albino individuals at high rates.
Guest et al. (2016) pointed out that the breaking of the
balance of microbial structure would increase the
susceptibility of corals to disease, as well as the stability
of the community or the biomarker that indicates the
risk of bleaching.
Environmental factors can alter relationships
among coral hosts and its bacterial community.
Neulinger et al. (2008) suggested there was no indicative
dissimilarly between the bacterial communities of deep-
sea stony coral Lophelia from different localities. In
contrast, bacterial diversity of Porites and Acropora
from two geographical localities in Caribbean Sea
(Mexico) and Indo-Pacific (Indonesia) were significantly
different (McKew et al. 2012). Additionally, samples of
soft coral Scleronephthya gracillimum from different
geographically sites represented distinguished
differentiation in bacterial diversity (Seonock et al.
2017). It is evident that environmental factors can alter
the diversity structure of bacteria at different
geographical areas.
Based on high throughput sequencing of extender,
which has been widely used in the study of microbial
diversity of coral symbiosis, more microbial groups have
been found. Liang et al. (2017) obtained a high-
throughput sequencing library of 16S rRNA gene to
analyze the bacterial community structure of various
massive and dendritic corals collected from the Xinyi
Reef in the Nansha Islands of the South China Sea. They
obtained the symbiotic bacteria database of the Nansha
reef-building corals in China. The commensal bacterial
species have their own preferences in terms of their
choice of corals to inhabit (Liang et al., 2017). In this
study, the bacterial community structures of 23 soft
coral samples collected from Ganzhe island (18 °45 °N,
110 °30 °E), Dazhou Island (18 °40 °N, 110 °28 °E), and
Ximao Island (18 °15 °N, 109 °22'E) were analyzed by 16s
rRNA gene high throughput sequencing. Although
bacterial community of stony coral have been studied
well, there is a lack of information about soft coral. This
study performed to explore the diversity of bacterial
community with different localities from South China
Sea (Hainan Province coral reefs, China) in two common
host soft corals genera (Sarcophyton and Sinularia). The
main aim is to investigate diversity of symbiotic bacterial
associated with localities and coral species.
Materials and Methods
Coral Collection
23 soft coral specimens of Sinularia and
Sacoprhyton were collected from the coral reef areas in
Ganzhe Island (18°45'31.21"N; 110°29'56.98"E), Dazhou
Island (18°40'43.87"N; 110°28'45.68"E), and Ximao
Island (18°14'9.03"N; 109°22'27.06"E) (Figure 1). The
sampling distance was more than 10 meters, and three
samples were collected per individual. The collected
specimens were washed with aseptic seawater and
placed in aseptic plastic bags. All specimens were stored
briefly at low temperature (0 °C–4 ℃) until laboratory
DNA extraction. The coral species were identified in
laboratory and compared with bone needles and COI +
igr1+ msh1 barcode (Zhou et al., 2019). Species
information is shown in Table 1.
DNA Extraction
The crown tissue of Sarcophyton (2.5×2.5 cm) and
finger tissue of Sinularia (2-3 cm) were cut with a pair of
scissors. The total DNA (about 50 mg, including tissue
and mucus) was extracted from marine animal genomic
DNA extraction kit (Tsingke, Guangzhou, China). The
Table 1. Coral species list in this study
Location
Total No
Genus
Species
No
Abb.
Ximao Island (XI)
6
Sarcophyton
S. trocheliophorum
3
XI_Satr01; XI_Satr02; XI_Satr03
Sinularia
S. grandilobata
2
XI_Sigr01; XI_Sigr02
S. querciformis
1
XI_Siqu03
Ganzhe Island (GI)
8
Sarcophyton
S. crassum
1
GI_Sacr04
S. glaucum
2
GI_Sagl05; GI_Sagl09
S. cherbonnieri
2
GI_Sach06; GI_Sach10
Sinularia
S. wanannensis
3
GI_Siwa10; GI_Siwa11; GI_Siwa12
Dazhou Island (DI)
9
Sarcophyton
S. ehrenbergi
2
DI_Saeh07; DI_Saeh11
S. crassum
1
DI_Sacr08
Sinularia
S. maxima
2
DI_Sima04; DI_Sima06
S. humilis
1
DI_Sihu05
S. querciformis
3
DI_Siqu07; DI_Siqu08; DI_Siqu09
21
GenAqua 5: 19-28 (2021)
instructions on the kit were strictly followed. The
prepared DNA is stored at -80°C after quality detection.
PCR Amplification and Illumina MiSeq Sequencing
Using DNA as template, the V3-V4 variable region
of bacterial 16SrRNA gene was amplified by PCR with
forward primer 338F(5'-ACTCCTACGGGAGGCAGCAG-3')
and reverse primer 806R(5'-
GGACTACHVGGGTWTCTAAT-3') (Mori et al., 2014; Xu et
al., 2016). PCR was carried out in a total volume of 50 μl
containing 48 μl Taq polymerase (2× Easy Taq® PCR
SuperMix, Code#AS111 +dye, TransGen Biotech CO.,
Ltd. CHN), 1 μl solution of DNA and 0.5 μl of each primer.
PCR amplification was carried out following Liang et al
(2017). The quality of PCR products were considered
using 2% agarose gel and it was purified by Gel
extraction kit (Axygen Scientific, Inc; CA, USA) following
its instruction manual before sequencing. Finally, the
duplicate samples were combined and the PE2×300
library was constructed according to the standard
operating procedures of the Illumina MiSeq platform.
Sequencing was performed on the Illumina Miseq PE300
platform (Majorbio, Shanghai, China).
Data Analysis
According to the method of Liang et al. (2017), raw
dataset were standardized using the software
Trimmomatic v.0.39 (Bolger et al., 2014) to eliminate the
reads with low quality and homopolymer inserts (<20).
The remaining high-quality sequences were subjected to
OTU cluster analysis and taxonomic analysis utilizing the
RDP (Ribosomal Database Project) by USEARCH
(UPARSE) (Woo et al., 2017). Based on the results of OTU
cluster analysis, the diversity of single specimen (Alpha
diversity) was obtained by Alpha diversity index analysis
of sample clustering results by software Mothur version
v.1.30.1with 1000 iterations (Schloss et al., 2011). The
coverage, abundance, and diversity of microbial
Figure 1. Sample collection location. Ganzhe Island (GI), Dazhou Island (DI), and Ximao Island (XI). Map data ©2020 Google.
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GenAqua 5: 19-28 (2021)
community were reflected by Good's species coverage
(Coverage), community richness (Ace), and community
diversity (Shannon) index. Taxonomy was identified and
compared via SILVA database (Quast et al., 2013)
following Qiime platform. Additionally, the Principal
Coordinate Analysis (PCoA) was performed using the
beta diversity index the OTUs level (Liang et al., 2017).
Results
Twenty-three specimen raw reads were obtained
by high throughput sequencing, with a total of 1 175 650
valid sequences. The effective sequences of each
specimen were more than 27 000, and the length of the
sequences was 421–460 bp. The coverage index for all
specimen databases was greater than 99%, and the
sequencing results accurately reflected the true flora of
the specimens. The specimen alpha-diversity index was
listed in Table 2.
The average OTU number of genus Sinularia in
both Ximao Island and Ganzhe Island was significantly
higher than that of genus Sarcophyton, and the average
OTU number of genus Sinularia in Dazhou Island was
slightly lower than that of genus Sarcophyton. Nine
specimens had OTU numbers greater than 500, of which
six specimens were from genus Sinularia and three from
genus Sarcophyton. On the contrary, less than 300 OTU
samples were from the genus genus Sarcophyton. The
values of Shannon index of all specimens were between
1.24 and 5.10 which the GI_Sagl09 specimen and the
XI_Sigr02 specimen were respectively the minimum and
maximum values. The difference of the large numerical
value indicated a great difference in the diversity of the
symbiotic bacteria in different specimens. The
difference of community richness ACE index between
specimens was about the same as that of the Shannon
index, except for a few cases such as the XI_Satr02 and
XI_Satr03 specimens.
A total of 46 bacterial phyla were detected in 23
examined coral specimens which 24 bacterial phyla
were shared among all coral hosts (Figure 2). The
dominant phyla of all specimens were Proteobacteria
(39.99%), Tendericutes (22.35%), Firmicutes (14.13%),
Bacteroidetes (9.58%) and Acinetobacteria (8.29%)
(Figure 3). After cluster analysis of each specimen, the
abundance of symbiotic Proteobacteria was found to be
the highest in the genus Sinularia from Dazhou Island.
Its abundance was more than 60% (Figure 4). The
specimen with the highest abundance was Si06 (92.8%).
Proteobacteria was detected in other specimens, and
the minimum abundance was still more than 10%.
Unlike the genus Sinularia, the symbiotic bacteria of the
Sarcophyton genus were more abundant. In addition to
the large number of Proteobacteria, Bacteroidetes were
maintained at a higher abundance (more than 30%) in
the three specimens of the Sarcophyton genus from
Ximao Island. The remaining nine specimens of genus
Sarcophyton were rich in Tenericutes. In addition,
Cyanobacteria were also found in all specimens, and
their abundance was the highest in the DI_Saeh07
specimens (13.5%).
At the class level (Figure 5), Mollicutes,
Gammaproteobacteria, and Alphaproteobacteria were
the most abundant in all specimens. Among them,
Mollicutes was more abundant in the genus
Sarcophyton than genus Sinularia, whereas
Table 2. Numbers of sequences, operational taxonomic units (OTUs) (97%) and diversity estimates of bacteria associated with
different corals
Location
Abb.
No. of. Seq
OTUs
ACE
Chao
Coverage
Shannon
Ximao Island
XI_Satr01
36530
478
591.26
591.06
0.996304
3.55
XI_Satr02
33589
344
435.80
459.12
0.997023
3.49
XI_Satr03
47633
356
471.77
469.29
0.997733
3.08
XI_Sigr01
43586
866
892.66
913.63
0.998555
4.94
XI_Sigr02
35024
942
972.04
987.02
0.997887
5.10
XI_Siqu03
83226
659
681.71
696.14
0.999519
5.03
Ganzhe Island
GI_Sacr04
72077
279
290.18
289.91
0.999778
3.00
GI_Sagl05
77683
324
333.60
347.75
0.999743
2.70
GI_Sach06
88922
324
332.75
347.75
0.999775
2.02
GI_Sagl09
41178
220
230.54
239.00
0.999514
1.24
GI_Sach10
74655
352
356.14
358.00
0.999826
2.29
GI_Siwa10
77457
518
528.61
528.00
0.999793
5.01
GI_Siwa11
58289
392
397.96
401.75
0.999777
4.35
GI_Siwa12
31863
309
324.56
337.88
0.999310
4.73
Dazhou Island
DI_Saeh07
58289
520
553.43
567.53
0.999262
3.61
DI_Sacr08
54505
648
658.70
660.83
0.999596
4.92
DI_Saeh11
27271
495
504.33
512.65
0.999083
4.06
DI_Sima04
33513
548
674.00
641.44
0.995554
2.93
DI_Sihu05
36534
424
583.70
593.03
0.995867
2.74
DI_Sima06
37338
398
499.86
501.71
0.996759
2.50
DI_Siqu07
32416
587
639.41
646.07
0.997100
3.06
DI_Siqu08
43343
450
482.38
498.89
0.998639
2.85
DI_Siqu09
50729
606
629.26
651.23
0.999034
3.89
23
GenAqua 5: 19-28 (2021)
Figure 2. Venn diagram analysis. Different colors represent different groups (differnet islands), overlapping parts represent unique
phyla in multiple groups (differnet islands), no overlapping parts represent phyla specific to the grouping (differnet islands), and
numbers represent the corresponding number of phyla.
Figure 3. Microbial commiunity pieplot on phylum level: All samples. “others” represent the bacterial phyla with abundances of
less than 0.01%.
Figure 4. Bacterial composition profiles. Taxonomic classification of bacterial reads retrieved from different coral species on phylum
level. “others” represent the bacterial phyla with abundances of less than 0.01%.
24
GenAqua 5: 19-28 (2021)
Gammaproteobacteria were more abundant in genus
Sinularia than genus Sarcophyton. Mollicutes comprised
81.4% of the total bacterial population in the GI_Sagl09
specimen, but was only 9.6% in the most highly
abundant specimen DI_Siqu09 in the genus Sinularia.
Gammaproteobacteria has a maximum abundance
values of 72.6% and 17.1% in genus Sinularia and genus
Sarcophyton respectively.
Totally identified bacterial were divided in 408
families (Figure 6) whereas 336 and 345 belonged to
Sarcophyton and Sinularia, respectively.
Spiroplasmataceae, Rhodobacteraceae, and
Halomonadaceae are dominant flora with abundance
percentages of 20.8%, 9.5%, and 4.0%, respectively.
Rhodobacteracea is a potential pathogen of coral. In
addition, the abundance of symbiotic bacteria showed
high similarity in the same coral genus at the same
sampling point. For example, Rhodobacteracea was
most abundant in the genus Sarcophyton collected from
Ximao Island, whereas Spiroplasmataceae were most
abundant in genus Sarcophyton collected from Ganzhe
Island. Unlike genus Sarcophyton, symbiotic bacteria in
all specimens that were collected from Ximao Island and
Ganzhe Island showed high diversity and do not contain
major bacterial families.
PCoA analysis obviously showed seven separated
groups (Figure 7). Although result cannot display a
relationship between bacterial diversity and localities, it
can be generally documented that in each locality
bacterial characteristics of same coral species were
clustered in separated group.
Discussion
At present, stony coral has been investigated by
many experts for its unique reef-building capacity (Lee
et al., 2012; Li et al., 2013; Li et al., 2014). Although soft
corals have high potential medicinal value (Li et al.,
2019), there is no equivalent research report. This study
collected two genera from three coral reefs of Ganzhe
Island, Dazhou Island, and Ximao Island of Wanning City,
Hainan Province, and a total of 23 soft coral specimens
from nine species. A database of 23 commensal bacteria
was obtained.
Significant differences in symbiotic bacteria in
different species of stony corals have been confirmed
(Hong et al., 2009). This study also found that this
variability also exists in soft corals, and the three sea
areas in this survey all showed this. There were
significant differences in the abundance and diversity of
symbiotic bacteria between different genera, but the
differences in abundance and diversity of symbiotic
bacteria in different species of a genus were not obvious
(Table 2). The reason was unclear. Using the same
method, Liang et al. (2017) found 55 bacteria phyla in 25
stone coral specimens, whereas in 23 soft coral
specimens in this study, we detected 46 phyla. More
bacterial symbiosis leads to greater uncertainty.
Gardner et al. (2013) pointed out that for each
additional species, the probability of instability increases
by 2n-1. Pollock et al. (2019) found that the reduction of
coral bacterial community is more beneficial to coral
immune function. Although this finding does not prove
that the gradual replacement of the coverage of stony
corals by soft corals in the waters around Hainan Island
was due to the existence of fewer symbiotic bacteria,
the symbiotic relationship between bacteria and genus
Sarcophyton or genus Sinularia was more stable than
that of bacteria and stony corals.
Consistent with previous studies, the dominant
symbiotic bacteria of soft corals in the South China Sea
is Proteobacteria (39.99%), but its abundance in soft
Figure 5. Bacterial composition profiles. Taxonomic classification of bacterial reads retrieved from different coral species on Class
level. “others” represent the bacterial phyla with abundances of less than 0.01%.
25
GenAqua 5: 19-28 (2021)
Figure 6. Bacterial composition profiles. Taxonomic classification of bacterial reads retrieved from different coral species on Family
level. “others” represent the bacterial phyla with abundances of less than 0.01%.
Figure 7. Principal co-ordinates analysis (PCoA) plot based on the OTU level from 25 coral species. The scatter plot is of principal
coordinate 1 (PC1) vs. principal coordinate 2 (PC2). PC1 and PC2 represent the principal factors affecting bacterial composition
associated with corals.
26
GenAqua 5: 19-28 (2021)
corals is slightly lower than stony corals (52.56%).
Compared with previous studies on symbiotic bacteria
of stony corals, the presence of a large number of
Tenericutes in the soft corals of the genus Sarcophyton
genus has not been reported in studies on coral
symbiotic bacteria near Hainan Island (Figure 4). Weiler
et al. (2018) argue that Teneriquets has a relationship
with cold-water corals that play an important role in the
nitrogen cycle. However, in the genus Sinularia of the
same waters, the abundance of such bacteria was lower,
and even some species did not contain Teneriquets. At
the same time, we found a large number of Vibrio fortis
in the Sinularia genus in Dazhou Island (Figure S1). Many
Vibrio species are considered to be potential coral
pathogens, such as Vibrio shiloi (Kushmaro, Fine, &
Rosenberg, 1966), Vibrio coralliilyticus (Ben-Haim,
Zicherman-Keren, & Rosenberg, 2003), Vibrio carchariae
(Ritchie & Smith, 1995), and Vibrio alginolyticus (Cervino
et al., 2004). However, no report has indicated that V.
fortis is also a coral pathogen. García‐Amado et al.
(2011) also confirmed that V. fortis is a common bacteria
in seawater living at specific depths and a natural
component of microbial community in marine redox
environments. Sarcophyton and Sinularia have their
own symbiotic bacteria, which are different from those
of stony coral, thereby providing a theoretical basis for
the further study of the genetic evolution of soft corals.
Chan et al. (2019) found that different coral species
have different bacterial community, and environmental
conditions are the main driving forces for early coral
microbial community. This study confirmed that the
same genus or the same species of soft corals that had
different geographical locations, contained various
bacterial community. Unlike the previous results on the
genus Sarcophyton in the Red Sea (Lee et al., 2012),
Endozoicomonas is not a dominant bacterial species and
has low abundance among the Sarcophyton corals in the
waters around Hainan. Symbiotic zooxanthellae can
affect the abundance of Endozoicomonas (Pantos et al.,
2015), and the composition of different zooxanthellae
may be one of the causes of this variation. The impact of
different geographical environments on coral symbiotic
bacteria is multifaceted. First, Woo et al. (2017) believed
that differences in latitude are potential causes of coral
symbiotic bacteria diversity. When latitude changes
exceed 10°, it should be considered as an important
factor affecting the results. In this study, the three
sampling points are located near the 18° north latitude,
which we believe is no longer a factor affecting the
bacteria. Secondly, the composition of coral-associated
bacteria is influenced by temperature and season (Hong
et al., 2009). Based on the sampling time concentrated
in September, a temperature difference of 2 °C exists
between the temperatures at the Sanya sampling point
and that at Wanning sampling point, which may lead to
the richness in symbiotic bacteria in cold water corals,
such as Tenericums, as seen in the specimens from
Dazhou Island and Ganzhe Island.
In a specific area, the unique breeding of corals
often affects their symbiotic bacterial community. For
example, Stylophora pistillata allows microorganisms to
be transferred vertically from the parent to the offspring
(Sharp, Distel, & Paul, 2012). Human activities, such as
diving and fisheries, are also factors that influence the
microbial composition of coral reef ecosystems (Kelly et
al., 2014). However, we are not sure whether these
incentives are direct factors influencing the
geographical differences in soft coral symbiotic bacteria.
Thus, direct experimental evidence is needed. Future
studies on soft coral-associated microbes should
consider host reproductive strategies, metabolites, and
environmental factors.
In conclusion, although each locality represents
special bacterial diversity, same coral species in each
locality represent similar pattern of bacterial
community.
Figure S1. Vibrio fortis composition profiles.
27
GenAqua 5: 19-28 (2021)
Acknowledgments
This work was supported by Hainan Province
Science and Technology Department Key Research and
Development Program (ZDYF2019154).
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