Bacterial Population in Intestines of the Black Tiger

Shrimp (Penaeus monodon) under Different Growth

Stages
Wanilada Rungrassamee1, Amornpan Klanchui1, Sage Chaiyapechara2, Sawarot Maibunkaew1,
Sithichoke Tangphatsornruang3, Pikul Jiravanichpaisal2, Nitsara Karoonuthaisiri1*
1 Microarray Laboratory, National Center for Genetic Engineering and Biotechnology, Khlong Luang, Pathum Thani, Thailand, 2 Aquatic Molecular Genetics and
Biotechnology Laboratory, National Center for Genetic Engineering and Biotechnology, Khlong Luang, Pathum Thani, Thailand, 3 Genomic Research Laboratory, National
Center for Genetic Engineering and Biotechnology, Khlong Luang, Pathum Thani, Thailand

Abstract
Intestinal bacterial communities in aquaculture have been drawn to attention due to potential benefit to their hosts. To
identify core intestinal bacteria in the black tiger shrimp (Penaeus monodon), bacterial populations of disease-free shrimp
were characterized from intestines of four developmental stages (15-day-old post larvae (PL15), 1- (J1), 2- (J2), and 3-month-
old (J3) juveniles) using pyrosequencing, real-time PCR and denaturing gradient gel electrophoresis (DGGE) approaches. A
total of 25,121 pyrosequencing reads (reading length = 442624 bases) were obtained, which were categorized by barcode
for PL15 (7,045 sequences), J1 (3,055 sequences), J2 (13,130 sequences) and J3 (1,890 sequences). Bacteria in the phyla
Bacteroides, Firmicutes and Proteobacteria were found in intestines at all four growth stages. There were 88, 14, 27, and 20
bacterial genera associated with the intestinal tract of PL15, J1, J2 and J3, respectively. Pyrosequencing analysis revealed
that Proteobacteria (class Gammaproteobacteria) was a dominant bacteria group with a relative abundance of 89% for PL15
and 99% for J1, J2 and J3. Real-time PCR assay also confirmed that Gammaproteobacteria had the highest relative
abundance in intestines from all growth stages. Intestinal bacterial communities from the three juvenile stages were more
similar to each other than that of the PL shrimp based on PCA analyses of pyrosequencing results and their DGGE profiles.
This study provides descriptive bacterial communities associated to the black tiger shrimp intestines during these growth
development stages in rearing facilities.

Citation: Rungrassamee W, Klanchui A, Chaiyapechara S, Maibunkaew S, Tangphatsornruang S, et al. (2013) Bacterial Population in Intestines of the Black Tiger
Shrimp (Penaeus monodon) under Different Growth Stages. PLoS ONE 8(4): e60802. doi:10.1371/journal.pone.0060802
Editor: Stefan Bereswill, Charité-University Medicine Berlin, Germany
Received October 29, 2012; Accepted March 3, 2013; Published April 5, 2013
Copyright: ß 2013 Rungrassamee et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was co-supported by the National Center for Genetic Engineering and Biotechnology (BIOTEC, grant numbers P-10-10722 and P-11-
00671) and the Thailand Research Fund (TRF, grant numbers DBG5380030 and TRG5480014). The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected]

Introduction Japan and Australia regions [9,10]. Commercial production of the

black tiger shrimp has been in a major decline due to higher
The intestinal tract is a complex ecosystem that harbors a incidents of viral and bacterial disease outbreaks [10,11]. Many
diverse bacterial community and this bacterial population has studies in crustaceans, including the black tiger shrimp, focused on
been shown to have profound influence on immunity, nutrient the development of probiotic applications to enhance disease
processing and protective processes in the host animal resistance and growth [12,13,14]. However, a probiotic approach
[1,2,3,4,5,6]. In the zebrafish model, the gut microbial community for the black tiger shrimp is challenging due to the lack of
plays a significant role in digestive tract development [7]. The understanding of natural microflora and factors contributing to the
colonization of microorganisms in germfree zebrafish modulates diversity of the bacterial population. The life cycle of penaeid
expression of host genes involved in nutrient absorption, mucosal shrimp includes egg, larval (nauplius, protozea and mysis), post-
modification and immunity [3]. Bacteria in intestines of other larval, juvenile and adult stages. Shrimp from the post-larval
aquatic animals have also been shown to contribute to host’s
through juvenile stages have been reported to have higher
immune system development, and the potential application of
bacterial disease susceptibility than those at the later stages
beneficial bacteria to aquaculture feeds, especially in fish and
[15,16,17,18]. Being aquatic species, shrimp are constantly
shellfish larviculture, has been increasingly investigated [8].
exposed to a variety of bacteria and viruses, which some can be
However, the knowledge about this bacterial community in these
pathogenic. Shrimp pathogens can orally enter, invade digestive
aquatic species is still limited. Thus, such an investigation will help
tract system and cause infection [19]. Although shrimp has innate
shedding light on beneficial bacteria that help maintaining the
immune system to battle against the pathogen’s invasion, it has
health of domestic animal stocks [5].
been speculated that bacterial community in shrimp intestines may
The black tiger shrimp (Penaeus monodon) is a prominent marine
play protective roles as natural barriers. Thus far, bacteria
shrimp with high economic value in Southeast Asia, the Sea of

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 Intestinal Bacteria in Shrimp Growth Stages

associated with the intestine of black tiger shrimp at the early Table 1. Sampling depth and biodiversity analysis of 16S
developmental stages have not yet been characterized. rRNA sequences from barcoded pyrosequencing of the black
Traditionally, bacterial populations in the intestines have been tiger shrimp intestines from different growth stages.
characterized by both culture-dependent and culture-independent
techniques based on 16S rRNA sequences [20,21,22]. The 16S
rRNA sequences from these approaches are then individually Growth stage1
sequenced using the Sanger sequencing method. Thus, the limited
PL15 J1 J2 J3
number of clones or DNA bands selected for sequencing might not
give a complete picture to represent actual bacterial ecology. To Sampling depth
provide comprehensive coverage of bacterial communities, a high- A total number of sequences 7,045 3,055 13,130 1,890
throughput pyrosequencing method has been employed to obtain (25,121)
a higher number of sequence-reads [23]. The method has recently OTUs (0.03 dissimilarity level) 514 97 298 167
gained popularity for an in-depth characterization of various Phylum 6 4 6 3
microbial communities such as those in the human [24,25,26],
Class 12 8 10 4
fermented food [27], soil [28,29,30,31], extreme environments
[32], the deep marine biosphere [33,34], wastewater treatment Family 52 15 24 14
bioreactors, [35] and plant biospheres [36,37]. Furthermore, to Genus 88 14 27 20
enable an analysis of multiple samples in parallel within a single Diversity indices
run, a barcode tag with a short unique nucleotide sequence was Good’s Coverage 0.96 0.98 0.98 0.94
added to distinguish each sample [31].
Shannon 3.46 0.58 0.67 2.86
This study provides the first report on intestinal bacterial
Chao1 1,016 277 896 482
populations in the black tiger shrimp at different growth stages
using pyrosequencing approach and the results were validated by 1
PL15 denotes 15-day-old post lava, whereas J1, J2 and J3 denote 1-, 2- and 3-
additional methods of real-time PCR and denaturing gradient gel month-old juvenile, respectively.
electrophoresis (DGGE). The rearing of black tiger shrimp from doi:10.1371/journal.pone.0060802.t001
post-larval to juvenile stages encounters high risk of bacterial
disease outbreak. In this work, we aim to identify bacterial Bayesian classifier with a confidence threshold of 80% [40],
populations that provide natural barriers to host shrimp in rearing phylum distribution of bacteria in intestines at four growth stages
facilities by characterization of bacterial communities in intestines varied from 3 to 6 phyla. The intestines of PL15 showed the
of 15-day-old post larval, 1-, 2- and 3-month-old juvenile stages. highest diversity of bacteria (88 genera), where J1, J2 and J3 had
The bacterial populations from different growth stages were also 14, 27 and 20 genera, respectively (Table 1).
compared to identify core bacteria-associated to the black tiger To estimate and compare bacterial diversity among growth
shrimp intestines throughout growth development in rearing stages, the OTUs from each library were used to calculate three
facilities. diversity indices: Good’s coverage, Shannon and Chao1 (Table 1).
Good’s coverage index estimates the percentage of total bacterial
Results and Discussion OTUs represented in a sample, in which the four growth stage
libraries had the values in a range of 0.94–0.98 (Table 1). When its
The microbiota and their influences on immune response, value approaches 1.0, it indicates that the obtained sequences from
disease resistance, and nutrition for the animal hosts have been each library represent the majority of bacterial sequences in the
intensively studied in many vertebrate model organisms [3,38,39]. sample [41]. Bacterial diversity was estimated by Shannon index,
However, less is known about the intestinal bacterial community in which a higher value means a greater bacterial diversity [42]. In
in other non-model organisms such as the black tiger shrimp. This this work, PL15 library had higher bacterial diversity (Shannon
study provides the first report on bacteria associated in intestines of index = 3.46) than that of J1, J2 and J3 libraries (0.58, 0.67, and
the black tiger shrimp by following four different growth stages of 2.86, respectively). Due to the lack of pyrosequencing data of
the same shrimp population. Three culture-independent ap- bacterial diversity in invertebrates, especially in aquatic organisms,
proaches, pyrosequencing, real-time PCR and denaturing gradient we compared our results to well-studied model animals, in which
gel electrophoresis (DGGE), were employed to characterize bacteria from juvenile intestines (Shannon index at 0.58–2.86)
bacterial diversity and community structure in shrimp intestines. show much lower diversity than those from humans (4.0) [43,44],
To determine bacterial populations, pyrosequencing of the 16S and mouse (5.5) [45]. Different host animal digestive systems and
rRNA gene amplicons were performed in intestine samples of 15- habitats, such as aquatic and terrestrial habitats, might contribute
day-old post larval (PL15), 1- (J1), 2- (J2) and 3-month-old (J3) to different levels of bacterial diversity. Lower bacterial diversity
juvenile shrimp. A total of 25,121 sequences of V3-6 regions of the might suggest that the habitat within the shrimp intestine was
16S rRNA gene were obtained from the barcoded pyrosequencing more dynamic with harsher conditions than mammalian hosts,
method and with an average read length of 442624 bases thus, fewer bacteria can adapt under such environments.
(Table 1). All sequences were sorted by a barcode for each library: To estimate true bacterial species richness, Chao1 index was
PL15 (7,045 sequences), J1 (3,055 sequences), J2 (13,130 calculated and its value reflects an estimated OTU for each sample
sequences) and J3 (1,890 sequences). Sequences were clustered [46]. Chao1 values were calculated in PL15 (1,016), J1 (277), J2
into operational taxonomic units (OTUs) at 0.03 dissimilarity (896) and J3 (482). All Chao1 values were higher than the observed
levels, where each OTU represents unique phylotype. The total OTUs in all growth stages, indicating that more bacterial species
number of OTUs was the highest in PL15 (514 OTUs), where J1, are still expected in shrimp intestines. In consistent to Chao1
J2 and J3 contained 97, 298, and 167 OTUs, respectively. analysis, rarefraction curves calculated at a distance of 0.03 did
Interestingly, number of OTUs decreased as shrimp were not reach saturation for the four growth stages even in the J2
developed to juvenile stage. When sequences were classified into library that contained .10,000 reads (Fig. S1). Both Chao1 and
taxonomic ranks using the Ribosomal Database Project (RDP) rarefraction curve analyses indicate that bacterial richness in the

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 Intestinal Bacteria in Shrimp Growth Stages

black tiger shrimp intestines are expected to be greater than from Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria have also
described in this study. The estimation of bacterial richness is a been found associated to Artemia nauplii [55]. Moreover, several
major challenge in various ecologies. For instance, rarefraction studies have shown that Artemia nauplii are one of major vectors to
curve analysis of bacterial communities in zebrafish intestines has introduce bacteria including pathogens to hatchery system
not been saturated, even when a range of 5,000 to 10,000 [56,57].
sequence reads were obtained [47]. To visualize bacterial composition in hierarchical orders, the
Bacterial pyrosequencing reads were classified to phyla classified pyrosequencing reads from all four growth stages were
Actinobacteria, Firmicutes, Bacteroidetes, Proteobacteria (class: Alpha-, Beta- used to construct pie charts to represent relative abundance for
and Gammaproteobacteria) (Fig. 1 and Table S1). Fusobacteria, each taxonomic node (Fig. 3) and the observed frequency of
Spirochaetes and unclassified bacteria were grouped as other pyrosequencing reads in each library was shown in Table S1. The
bacteria (Fig. 1A). Of these, Gammaproteobacteria (phylum Proteobac- pyrosequencing reads that could not be classified at genus level
teria) were the most abundant, accounting for more than 80% in were classified to the nearest match of the next taxonomic ranking
shrimp intestines from all growth stages (Fig. 1A). In addition to (Table S1). These unclassified sequences suggest that many
Proteobacteria, Bacteroidetes and Firmicutes were also found in all bacteria-associated with shrimp intestine were still uncultured or
growth stages (Fig. 1A), suggesting that these phyla constituted as might not be previously reported. The bacterial populations of
core intestinal bacteria in the black tiger shrimp. Bacteria in these PL15 were highly diverse and found in most of the taxonomic
phyla are also commonly found associated in intestines of many nodes, whereas those bacteria found in juvenile stages were more
other aquatic species [20,48,49]. In attempt to identify core closely related (Fig. 3). The top five most abundant bacteria genera
intrinsic bacterial groups in the black tiger shrimp’s intestine were compared among the four libraries (Fig. 4). In the PL15
regardless of rearing environment, bacterial community analysis in library, Photobacterium (34.4%) was the highest, followed by Vibrio
intestines of wild-caught black tiger shrimp is underway and results (6.1%), Acinetobacter (1.0%), Pseudomonas (0.9%) and Thalassobius
will be compared to this study to identify shared core bacterial (0.8%) (Fig. 4A). The J1 and J2 libraries contained similar
members. dominant genera: Vibrio (19.6% and 14.7%, respectively) and
To validate bacterial abundance from pyrosequencing results, Listonella (1.2% and 1.5%, respectively) (Fig. 4B and C). However,
bacterial groups in shrimp intestines were further analyzed by real- the J1 and J2 libraries differ in the minor bacteria. The J1 had
time PCR at the higher taxonomic levels (Fig. 1B). Bacteria were Escherichia/Shigella (0.2%), Bacillus (0.1%) and Aeromonas (0.1%),
detected in each sample using specific primers for Firmicutes, Alpha-, while the J2 contained 0.1% of Pseudomonas, Shewanella and
Beta- and Gammaproteobacteria, Actinobacteria and Bacteroidetes. From Photobacterium. In the J3 library, the bacterial population was
the real-time PCR result, the obtained relative abundance 32.9% Vibrio, 3.3% Labrenzia, 2.3% Silicibacter, 0.6% Listonella and
revealed that bacteria from class Gammaproteobacteria were domi- 0.5% Pseudoalteromonas (Fig. 4D). The bacterial genera shared
nant in the shrimp intestines from all growth stages, which was among the four growth stages were Vibrio, Photobacterium, Pseudo-
consistent with the pyrosequencing observation. The previous monas, and Listonella.
survey of bacterial population in the black tiger shrimp juveniles In addition to pyrosequencing and real-time PCR, bacterial
from the commercial farms also shows that Gammaproteobacteria was profiles in the four growth stages were also compared using DGGE
predominated in intestines [20]. analysis (Fig. 5). The DGGE profiles were clustered according to
Within class Gammaproteobacteria, the main bacterial genera were Pearson correlation, and it showed two distinct groups, PL15 and
from the family Vibrionaceae (Fig. 2 and Table S2). This family of the juvenile stages (Fig. 5A). DGGE profiles of J1, J2 and J3 were
bacteria are widely distributed in marine environment [50] and at 85% similarity level. Sequences from the dominant DGGE
has previously been reported to be found in high relative bands were similar to those of Vibrio species in all four stages, and
abundance in intestine of various aquatic marine organisms Photobacterium species was detected in PL15 (Fig. 5B). Principal
including the black tiger shrimp [21,22,48,49,51]. Photobacterium component analysis based on relative bacterial abundance from
species were the majority during the post-larval stage (80%) and pyrosequencing results showed the similar trend to DGGE profile
then the dominant bacterial group shifted to Vibrio species during analysis, in which the bacterial profile from PL15 intestines was in
the juvenile stages (Fig. 2). Other less abundant bacteria were a different cluster from those found in the juvenile stages (Fig. 6).
found sporadically. Fusobacteria were only found in the PL15 Although our DGGE analysis revealed only bacteria genera with
intestines, whereas Spirochaetes were only associated with J2 high abundance and may not provide an in-depth analysis of
intestines. Actinobacteria were found in both PL15 and J2 intestines. bacterial population, this method is proven to be useful for
Moreover, Listonella species were found only in the intestines of the comparison of bacteria community profiles in food and soil
juvenile stages, while the genera Labrenzia and Silicibacter were samples [27,58,59]. Our study also showed that bacterial patterns
found only in intestines of the J3 stage. from DGGE and pyrosequencing were congruence. The DGGE
The difference in bacterial composition in post-larva and analysis can therefore be used as a validation technique to confirm
juveniles might also be due to different feed conditions. In this the results from the pyrosequencing analysis, which can detect
study, bacterial populations were investigated in shrimp rearing bacterial genera with low abundance in a sample.
under commercial production, where post-larval shrimp were fed One other factor that influences on intestinal bacterial diversity
with live Artemia nauplii (brine shrimp) as nutritional supplement in is the selective pressure from the host such as resistance to host
addition to commercial feed pellets until they reach juvenile stage. immune system [60]. The shrimp digestive system is fully
Once shrimp reached juvenile stages, their diet is solely based on developed in the post-larval stage [61]. However, the immune
commercial pellets [52,53]. The gut layer in Penaeid shrimp is system of post-larval shrimp is not as developed as in juvenile
ectodermal origin, which regularly shreds off during the molting shrimp [19,62]. For example, penaedin, a shrimp antibacterial
period [54]. Therefore, bacteria from ingested food influence re- peptide, is expressed weakly in post-larval shrimp in comparison to
colonization of the shrimp gut’s microbial flora after shredding. juvenile shrimp [62]. This factor, in addition to the difference in
The shifting from the live feed in post-larval shrimp to commercial shrimp diets between different developmental stages might
pellets in juvenile shrimp might explain the difference in diversity contribute to a higher bacterial diversity post-larval shrimp
of bacterial composition in shrimp rearing ponds because bacteria [5,63]. The lower immune level in the post-larval stage resulted

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 Intestinal Bacteria in Shrimp Growth Stages

Figure 1. Frequency distribution of phylogenetic groups in intestines of different growth stages of the black tiger shrimp: 15-day-
old post-larva (PL15) and 1-, 2- and 3-month-old juveniles (J1, J2 and J3, respectively). (A) Percent distribution of bacterial phylum by
pyrosequencing analysis and (B) relative abundance of the six bacterial phylogenetic groups estimated by real-time PCR.
doi:10.1371/journal.pone.0060802.g001

in less harsh condition for bacteria to reside within [60,62], and this might allow establishment of resident bacterial popula-
resulting in different intestinal bacterial diversity from the harsher tion.
conditions in the juveniles. In contrast, the more developed In this study, Bacteroidetes, Firmicutes and Proteobacteria were core
immune system in juvenile shrimp imposes a higher selective phyla found in intestines of post-larvae and juvenile shrimp and
pressure for those bacteria that can resist the host immune system, selecting bacterial candidates from resident population might be
resulting in more closely related bacteria found in their intestines. another mean for successful probiotics development. Bacteria
The intrinsic developmental changes in transition to later growth associated with animal digestive tract might be beneficial to the
stage may exert selective pressure for those highly adapted bacteria host such as natural barriers against pathogen colonization
to host gut habitat [64,65]. Moreover, the digestive tract of a through competition for binding sites [66]. Our findings showed
juvenile shrimp has more surface area than that of the post-larvae, that Proteobacteria (class Gammaproteobacteria), particularly Vibrio

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 Intestinal Bacteria in Shrimp Growth Stages

Figure 2. Frequency distribution of selected genera with high abundance in Gammaproteobacteria from pyrosequencing analysis.
PL15 denotes 15-day-old post-larva (PL15) whereas J1, J2 and J3 denote 1-, 2- and 3-month-old juveniles, respectively.
doi:10.1371/journal.pone.0060802.g002

species dominated the intestines at all three juvenile stages, and Yellow Head Virus, Monodon Baculovirus, and Infectious
Photobacterium species was the highest in intestines at the post-larval Hypodermal and Hematopoietic Necrosis Virus [10]. Shrimp
stage. While some Vibrio species are reported as shrimp pathogens larvae were reared in cement tanks at 15 ppt salinity and fed 4
[17,67], there is some evidence suggesting beneficial roles of Vibrio times daily with Artemia nauplii and commercial feed with high
species to increase disease resistance in aquatic animal hosts [21]. protein formula (Starfeeds for PL containing 50% proteins, CPF,
Additionally, many studies have focused on the search for Thailand). Once the larvae were 15-days old, they were
beneficial bacteria for marine shrimp farming, and the indigenous transferred to a plastic-lined pond (20620 m) at 10–11 ppt salinity
bacteria found in shrimp intestines may be more effective for and fed 4 times daily with commercial feed (Starfeeds containing
probiotic candidates due to their abilities to survive and colonize 40% proteins) at the Marine Technology Research Center,
inside host shrimp. The main bacterial groups that have been Faculty of Marine Technology (Burapha University Chanthaburi
tested as probiotics in aqua-feeds are Vibrio, Pseudomonas, Bacillus Campus, Thailand). Shrimp at 15-day of post-larval, 1-, 2- and 3-
and Lactobacillus [13,68], and they were present in our survey of month-old juvenile stages (PL15, J1, J2 and J3, respectively) were
bacteria populations in intestines of post-larval and juvenile stages. collected 2 hours after the morning feeding. Intestines from post-
Although intestinal bacterial population of shrimp in their early larval stages were aseptically dissected using sterile needles under a
stage was highly diverse, we observed that they became more dissecting microscope, and intestines from juvenile stages were
progressively similar in juvenile stages. Understanding the dissected from the shrimp using sterile surgical blades and kept
relationship between intestinal bacterial communities and their frozen in liquid nitrogen. All intestine samples were stored at
influence on the health and survival of their hosts under bacterial 280uC until use.
pathogen invasion is essential. In the case of opportunistic
pathogens, if intestinal bacterial balance is maintained, the risk DNA Extraction and Purification
of disease outbreak in shrimp farms may be reduced. Bacterial The intestine samples were collected from the same shrimp
population dynamics in the presence of shrimp pathogens in the family, in which all of them were reared under the same
black tiger shrimp intestines will be further investigated. environment conditions. Our previous study shows that intestinal
Approaches to maintain intestinal bacterial balance especially in bacteria from individual shrimp rearing under the same environ-
early life stage will contribute to lower disease outbreak in shrimp mental conditions exhibit similar bacterial profile [20]. Therefore,
rearing facilities. Identification of the intestinal microflora in to obtain high efficiency of DNA extraction, 5 intestine samples
forming a protective barrier and out-competing pathogenic were pooled for juvenile shrimp. Due to relatively small mass of
bacteria in the black tiger shrimp will benefit the shrimp industry. PL15 intestine to those of juveniles, 50 intestines were pooled in
order to obtain a comparable total mass of intestines to juvenile
Materials and Methods intestines and to obtain adequate mass for subsequent experi-
ments. Genomic DNA was extracted using the QIAamp DNA
Animals and Intestine Sample Collection mini kit (Qiagen, Germany) according to the manufacturer’s
The black tiger shrimp (P. monodon) were hatched at the Shrimp instructions. The DNA purity and concentration was determined
Genetic Improvement Center (Surat Thani province, Thailand) by the NanoDrop (ND-8000) spectrophotometer and DNA
and routinely checked to ensure that they are free from specific- samples were kept at 220uC for subsequent analyses.
pathogens: Taura Syndrome Virus, White Spot Syndrome Virus,

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 Intestinal Bacteria in Shrimp Growth Stages

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 Intestinal Bacteria in Shrimp Growth Stages

Figure 3. Comparison of the bacterial compositions in shrimp intestines of four growth stages: 15-day-old post-larva (PL15) and 1-,
2- and 3-month-old juveniles (J1, J2 and J3, respectively). Pie charts of the hierarchical tree reflect relative abundance for each genus from
each library (red represents PL15, blue represents J1, green represents J2 and yellow represents J3).
doi:10.1371/journal.pone.0060802.g003

Figure 4. Top five most abundant bacterial genera in shrimp intestines of four growth stages: A) 15-day-old post-larva (PL15), B) 1-
month-old juveniles (J1), C) 2-month-old juveniles (J2) and D) 3-month-old juveniles (J3).
doi:10.1371/journal.pone.0060802.g004

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 Intestinal Bacteria in Shrimp Growth Stages

Figure 5. PCR-DGGE analysis of the predominant bacterial population in intestines of black tiger shrimp from different growth
stages: 15-day-old post-larva (PL15), 1-, 2- and 3-month-old juveniles (J1, J2 and J3, respectively) and S is in-house standard
marker. (A)Dendogram analysis of DGGE profile and (B) bacterial profiles and species similarity of selected DGGE bands.
doi:10.1371/journal.pone.0060802.g005

Pyrosequencing of Barcoded 16S rRNA Genes and Data 80% confidence threshold [40]. Operational taxonomy units
Analysis (OTUs) were clustered by Mothur using the average neighbor
To prepare DNA libraries for barcoded pyrosequencing, the algorithm at 0.03 dissimilarity level to classify to OTUs [71].
V3-6 region of 16S rRNA gene was amplified with the 338F and Shannon index and Chao1 biodiversity index values were
786R primer pair using high-fidelity DNA polymerase (Novagen, calculated for each shrimp library using Mothur. The coverage
Germany). The primers were modified with a barcode tag by index was calculated by 1– (n/N), where n is the number of
adding a sequence of 8 nucleotides to the 59 end of each primer phylotypes and N is the total number of sequences [41]. Only
pair (Table S2, underlined sequence). The PCR products were gel pyrosequencing reads with minimum frequency of five were used
purified with the Qiaquick Gel Extraction kit (Qiagen), ligated to to construct a hierarchical tree to visualize and compare relative
454-adapters (Table S2) and sequenced on the Genome Sequencer abundance of each genus from all growth stages. The taxonomic
Junior platform according to Roche protocol (454 GS Junior tree was constructed using MEGAN based on the results of
System, Roche, USA). taxonomical assignment by the RDP classifier [72].
The sequence data was processed using Mothur software to Principal component analysis was performed using R script to
remove low quality sequences [69]. In Mothur, poor quality compare bacterial community structures among the samples based
sequences were set as sequences with a length less than 200 bases, on relative abundance of various bacteria genera.
contained ambiguous bases and homopolymers greater than 6 All nucleotide sequence data in this study were deposited in
bases or did not have a barcode and a primer sequence. All GenBank database with accession numbers JX919344 to
sequences were checked for chimeras by UCHIME [70]. The JX926388 for the PL15 library, JX916289 to JX919343 for the
sequence data were analyzed by the RDP II Classifier with an

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 Intestinal Bacteria in Shrimp Growth Stages

PCR Amplification of 16S rRNA Genes and Denaturing

Gradient Gel Electrophoresis (DGGE)
PCR-DGGE profiles of intestine samples from four shrimp
stages were performed according as previously described with
some modifications [20,75,76]. Briefly, genomic DNA sample
(final concentration of 1 ng/ml) from each library was used as a
template for PCR reaction with primer pair 338F-GC clamps and
517R (Table S2). The PCR cycle parameters were 3 min initial
denaturation at 94uC, 30 cycles of 30 sec at 94uC, 1 min at 55uC
and 1.5 min at 72uC, and 10 min final extension at 72uC. The
presence of a 200-bp fragment was confirmed on a 1.5% agarose
gel electrophoresis. PCR-DGGE products were purified by illustra
GFX PCR DNA and gel purification kit according to supplier’s
manual (GE Healthcare, USA). Each PCR-DGGE product
(600 ng) was loaded directly onto an 8% polyacrylamide gel with
40% to 60% denaturant vertical gradient. The in-house standard
Figure 6. Principal component analysis of bacterial popula- ladders were loaded as a control of DGGE analysis. The
tions in intestines of different growth stages of the black tiger electrophoresis was performed at 80 V 60uC for 14 h using a
shrimp: 15-day-old post-larva (PL15) and 1-, 2- and 3-month- DCode DGGE Electrophoresis System (Bio-Rad Laboratories,
old juveniles (J1, J2 and J3, respectively). The principal Inc., USA). After electrophoresis, the gels were stained using
component analysis (PCA) was performed using R script to compare SYBR gold (Invitrogen, USA) and visualized under UV light gel
bacterial community structures among the samples based on relative
abundance of various bacteria genera.
doc system (Bio-Rad Laboratories, Inc.). All gel analyses were
doi:10.1371/journal.pone.0060802.g006 performed using InfoQuestTM Software (Bio-Rad Laboratories,
Inc.). DGGE bands were excised and cloned to pGEM-T
J1 library, JX926389 to JX939518 for the J2 library and according to supplier’s instruction (Promega). Plasmid was
JX939519 to JX941408 for the J3 library. extracted and submitted for sequencing. Sequence identity was
determined by BLASTN in the GenBank database.
Real-time PCR Analysis of Bacterial Phyla
To validate pyrosequencing results, specific bacterial groups Supporting Information
were determined by real-time PCR. Detection of bacterial taxa by Figure S1 Rarefaction analysis of 16S rRNA sequences
real-time PCR assay and primer specificity was previously from four black tiger shrimp growth stages: 15-day-old
developed [73,74]. Primer pairs for all bacteria, Firmicutes, post-larva (PL15) and 1-, 2- and 3-month-old juveniles
Betaproteobacteria, Gammaproteobacteria, Alphaproteobacteria, Actinobacteria (J1, J2 and J3, respectively). The operational taxonomic units
and Bacteroidetes were listed in Table S3. For each intestinal DNA (OTUs) were clustered at 0.03% dissimilarity level. Number of
sample, the real-time PCR were carried out in triplicates with IQ sequences refers to the number of pyrosequencing reads.
SYBR Green supermix (Bio-Rad Laboratories, Inc., USA) in (TIF)
CFX96 real-time detection system (Bio-Rad Laboratories, Inc.).
Each 20-ml reaction contained 100 ng of DNA template, 200 nM Table S1 Taxonomical assignments of 16S rRNA se-
of each primer, 10 ml of IQ SYBR green supermix and sterile quences from pyrosequencing using the RDP classifier
water to complete the final 20 ml volume. The real-time cycle with a confidence threshold of 80%.
parameters were initial denaturation at 95uC for 3 min, 40 cycles (DOC)
of denaturation at 95uC for 30 sec, annealing at 54uC for 20 sec Table S2 Oligonucleotide used in this study.
and extension at 72uC for 30 sec, fluorescent signal was detected (DOC)
at the end of each cycle. Melting curve analysis was performed
from 55uC to 95uC with continuous fluorescence reading every Table S3 Bacteria-specific primers used for real-time
0.5uC increment to validate specificity of PCR amplification. The PCR analysis.
relative folds of bacterial abundance were calculated as previously (DOC)
described [73]. Briefly, a plasmid DNA containing target sequence
was constructed from PCR amplification using DNA from Acknowledgments
Escherichia coli MG1655, Lactobacillus plantarum BCC9546, Afifella We thank Ms. Somjai Wongtripob and staff at the Shrimp Genetic
marina BCC40083, Kineococcus gynurae BCC26245, Burkholderia Improvement Center (SGIC, Thailand) and Dr. Vasin Yuvanatemiya and
tropica BCC26066 or Chryseobacterium indologenes BCC19188 as a Dr. Rachanimuk Preechaphol at Faculty of Marine Technology, Burapha
template with a taxon-specific primer pair (Table S3). A standard University at Chanthaburi for shrimp samples. We thank Dr. Verawat
curve was constructed using 10-fold serial dilution of a plasmid Champreda for kindly provide DGGE equipment, particularly Ms.
Thanaporn Laothanachareon for assisting with the initial DGGE setup
DNA (from 10 to 108 copy number) as a template in real-time
and Biotec Culture Collection for providing bacterial strains used for
PCR reaction. The real-time PCR was carried out in triplicates standard curve construction. We thank Dr. Samaporn Teeravechyan for
and a target copy number was calculated from the standard curve proofreading the manuscript. We are grateful to Prof. Morakot
equation. Relative abundance for each target bacteria in each Tanticharoen, Dr. Kanyawim Kirtikara and Dr. Sirawut Klinbunga for
sample was determined by normalizing with the abundance of their mentorships.
total bacteria in that sample, and percent relative abundance was
determined within six target taxa.

PLOS ONE | www.plosone.org 9 April 2013 | Volume 8 | Issue 4 | e60802

 Intestinal Bacteria in Shrimp Growth Stages

Author Contributions
Conceived and designed the experiments: WR SC PJ NK. Performed the
experiments: WR AK SM. Analyzed the data: WR AK NK. Contributed
reagents/materials/analysis tools: ST NK. Wrote the paper: WR SC NK.

References
1. Hooper LV, Midtvedt T, Gordon JI (2002) How host-microbial interactions 26. Ling Z, Kong J, Jia P, Wei C, Wang Y, et al. (2010) Analysis of oral microbiota
shape the nutrient environment of the mammalian intestine. Annu Rev Nutr 22: in children with dental caries by PCR-DGGE and barcoded pyrosequencing.
283–307. Microb Ecol 60: 677–690.
2. Li M, Wang B, Zhang M, Rantalainen M, Wang S, et al. (2008) Symbiotic gut 27. Roh SW, Kim KH, Nam YD, Chang HW, Park EJ, et al. (2010) Investigation of
microbes modulate human metabolic phenotypes. PNAS 105: 2117–2122. archaeal and bacterial diversity in fermented seafood using barcoded
3. Rawls JF, Samuel BS, Gordon JI (2004) Gnotobiotic zebrafish reveal pyrosequencing. ISME J 4: 1–16.
evolutionarily conserved responses to the gut microbiota. PNAS 101: 4596– 28. Lauber CL, Hamady M, Knight R, Fierer N (2009) Pyrosequencing-Based
4601. Assessment of Soil pH as a Predictor of Soil Bacterial Community Structure at
4. Brune A, Friedrich M (2000) Microecology of the termite gut: structure and the Continental Scale. Appl Environ Microbiol 75: 5111–5120.
function on a microscale. Curr Opin Microbiol 3: 263–269. 29. Roesch LF, Fulthorpe RR, Riva A, Casella G, Hadwin AK, et al. (2007)
5. Harris JM (1993) The presence, nature, and role of gut microflora in aquatic Pyrosequencing enumerates and contrasts soil microbial diversity. ISME J 1:
invertebrates: A synthesis. Microbial Ecol 25: 195–231. 283–290.
6. Xu J, Gordon JI (2003) Inaugural Article: Honor thy symbionts. PNAS 100: 30. Jones RT, Robeson MS, Lauber CL, Hamady M, Knight R, et al. (2009) A
10452–10459. comprehensive survey of soil acidobacterial diversity using pyrosequencing and
7. Bates JM, Mittge E, Kuhlman J, Baden KN, Cheesman SE, et al. (2006) Distinct clone library analyses. ISME J 3: 442–453.
signals from the microbiota promote different aspects of zebrafish gut 31. Parameswaran P, Jalili R, Tao L, Shokralla S, Gharizadeh B, et al. (2007) A
differentiation. Dev Biol 297: 374–386. pyrosequencing-tailored nucleotide barcode design unveils opportunities for
8. Gomez-Gil B, Roque A, Turnbull JF (2000) The use and selection of probiotic large-scale sample multiplexing. Nucleic Acids Res 35: e130.
bacteria for use in the culture of larval aquatic organisms. Aquaculture 191: 32. Hollister EB, Engledow AS, Hammett AJ, Provin TL, Wilkinson HH, et al.
259–270. (2010) Shifts in microbial community structure along an ecological gradient of
9. FAO (2003) Towards sustainable shrimp culture development: implementing the hypersaline soils and sediments. ISME J 4: 829–838.
FAO code of conduct for responsible risheries (CCRF). Fisheries Department, 33. Huber JA, Mark Welch DB, Morrison HG, Huse SM, Neal PR, et al. (2007)
Food and Agriculture Organization of the United Nations. Microbial population structures in the deep marine biosphere. Science 318: 97–
10. Flegel TW (2012) Historic emergence, impact and current status of shrimp 100.
pathogens in Asia. J Invertebr Pathol 110: 166–173. 34. Sogin ML, Morrison HG, Huber JA, Mark Welch D, Huse SM, et al. (2006)
Microbial diversity in the deep sea and the underexplored "rare biosphere".
11. Tanticharoen M, Flegel TW, Meerod W, Grudloyma U, Pisamai N (2009)
PNAS 103: 12115–12120.
Aquacultural biotechnology in Thailand: the case of the shrimp industry.
Int J Biotechnology 10: 588–603. 35. Zhang X, Yue S, Zhong H, Hua W, Chen R, et al. (2011) A diverse bacterial
community in an anoxic quinoline-degrading bioreactor determined by using
12. Rengpipat S, Phianphak W, Piyatiratitivorakul S, Menasveta P (1998) Effects of
pyrosequencing and clone library analysis. Appl Microbiol Biotechnol 91: 425–
a probiotic bacterium on black tiger shrimp Penaeus monodon survival and growth.
434.
Aquaculture 167: 301–313.
36. Kauserud H, Kumar S, Brysting AK, Norden J, Carlsen T (2011) High
13. Rengpipat S, Tunyanun A, Fast AW, Piyatiratitivorakul S, Menasveta P (2003)
consistency between replicate 454 pyrosequencing analyses of ectomycorrhizal
Enhanced growth and resistance to Vibrio challenge in pond-reared black tiger
plant root samples. Mycorrhiza 10.1007/s00572-011-0403-1 [doi].
shrimp Penaeus monodon fed a Bacillus probiotic. Dis Aquat Organ 55: 169–173.
37. Telias A, White J, Pahl D, Ottesen A, Walsh C (2011) Bacterial community
14. Browdy CL (1998) Recent developments in penaeid broodstock and seed diversity and variation in spray water sources and the tomato fruit surface. BMC
production technologies: improving the outlook for superior captive stocks. Microbiol 11: 81–93.
Aquaculture 164: 3–21.
38. Laycock G, Sait L, Inman C, Lewis M, Smidt H, et al. (2012) A defined
15. Lavilla-Pitogo CR, Leaño EM, Paner MG (1998) Mortalities of pond-cultured intestinal colonization microbiota for gnotobiotic pigs. Vet Immunol Immuno-
juvenile shrimp, Penaeus monodon, associated with dominance of luminescent pathol 149: 216–224.
vibrios in the rearing environment. Aquaculture 164: 337–349. 39. Bäckhed F, Ley RE, Sonnenburg JL, Peterson DA, Gordon JI (2005) Host-
16. Soto-Rodriguez SA, Gomez-Gil B, Lozano R, del Rio-Rodriguez R, Dieguez bacterial mutualism in the human intestine. Science 307: 1915–1920.
AL, et al. (2012) Virulence of Vibrio harveyi responsible for the ‘‘Bright-red’’ 40. Wang Q, Garrity GM, Tiedje JM, Cole JR (2007) Naive Bayesian classifier for
Syndrome in the Pacific white shrimp Litopenaeus vannamei. J Invertebr Pathol rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl
109: 307–317. Environ Microbiol 73: 5261–5267.
17. Jiravanichpaisal P, MIyazaki T, Limsuwan C (1994) Histopathology, Biochem- 41. Good IJ (1953) The population frequencies of species and the estimation of
istry, and Pathogenicity of Vibrio harveyi Infecting Black Tiger Prawn Penaeus population parameters. Biometrica 40: 237–264.
monodon. J Aquat Anim Health 6: 27–35. 42. Hill TC, Walsh KA, Harris JA, Moffett BF (2003) Using ecological diversity
18. Manefield M, Harris L, Rice SA, de Nys R, Kjelleberg S (2000) Inhibition of measures with bacterial communities. FEMS Microbiol Ecol 43: 1–11.
Luminescence and Virulence in the Black Tiger Prawn (Penaeus monodon) 43. Dethlefsen L, Huse S, Sogin ML, Relman DA (2008) The Pervasive Effects of an
Pathogen Vibrio harveyi by Intercellular Signal Antagonists. Appl Environ Antibiotic on the Human Gut Microbiota, as Revealed by Deep 16S rRNA
Microbiol 66: 2079–2084. Sequencing. PLoS Biol 6: e280.
19. Soonthornchai W, Rungrassamee W, Karoonuthaisiri N, Jarayabhand P, 44. Andersson AF, Lindberg M, Jakobsson H, B!1ckhed F, Nyr!ßn Pl, et al. (2008)
Klinbunga S, et al. (2010) Expression of immune-related genes in the digestive Comparative Analysis of Human Gut Microbiota by Barcoded Pyrosequencing.
organ of shrimp, Penaeus monodon, after an oral infection by Vibrio harveyi. Dev PLoS ONE 3: e2836.
Comp Immunol 34: 19–28. 45. Gillilland MG, 3rd, Erb-Downward JR, Bassis CM, Shen MC, Toews GB, et al.
20. Chaiyapechara S, Rungrassamee W, Suriyachay I, Kuncharin Y, Klanchui A, et (2012) Ecological succession of bacterial communities during conventionaliza-
al. (2011) Bacterial Community Associated with the Intestinal Tract of P. monodon tion of germ-free mice. Appl Environ Microbiol 78: 2359–2366.
in Commercial Farms. Microb Ecol DOI: 10.1007/s00248-011-9936-2. 46. Hughes JB, Hellmann JJ, Ricketts TH, Bohannan BJM (2002) Counting the
21. Oxley AP, Shipton W, Owens L, McKay D (2002) Bacterial flora from the gut of Uncountable: Statistical Approaches to Estimating Microbial Diversity. Appl
the wild and cultured banana prawn, Penaeus merguiensis. J Appl Microbiol 93: Environ Microbiol 68: 448.
214–223. 47. Roeselers G, Mittge EK, Stephens WZ, Parichy DM, Cavanaugh CM, et al.
22. Johnson CN, Barnes S, Ogle J, Grimes DJ, Chang Y-J, et al. (2008) Microbial (2011) Evidence for a core gut microbiota in the zebrafish. ISME J 5: 1595–
Community Analysis of Water, Foregut, and Hindgut during Growth of Pacific 1608.
White Shrimp, Litopenaeus vannamei, in Closed-System Aquaculture. J World 48. Kim DH, Brunt J, Austin B (2007) Microbial diversity of intestinal contents and
Aquacult Soc 39: 251–258. mucus in rainbow trout (Oncorhynchus mykiss). J Appl Microbiol 102: 1654–
23. Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, et al. (2005) Genome 1664.
sequencing in microfabricated high-density picolitre reactors. Nature 437: 376– 49. Navarrete P, Espejo RT, Romero J (2009) Molecular Analysis of Microbiota
380. Along the Digestive Tract of Juvenile Atlantic Salmon (Salmo salar L.). Microb
24. Costello EK, Lauber CL, Hamady M, Fierer N, Gordon JI, et al. (2009) Ecol 57: 550–561.
Bacterial community variation in human body habitats across space and time. 50. Thompson FL, Iida T, Swings J (2004) Biodiversity of Vibrios. Microbiol Mol
Science 326: 1694–1697. Biol R 68: 403–431.
25. Naqvi A, Rangwala H, Spear G, Gillevet P (2010) Analysis of multitag 51. Liu H, Wang L, Liu M, Wang B, Jiang K, et al. (2011) The intestinal microbial
pyrosequence data from human cervical lavage samples. Chem Biodivers 7: diversity in Chinese shrimp (Fenneropenaeus chinensis) as determined by PCR-
1076–1085. DGGE and clone library analyses. Aquaculture 317: 32–36.

PLOS ONE | www.plosone.org 10 April 2013 | Volume 8 | Issue 4 | e60802

 Intestinal Bacteria in Shrimp Growth Stages

52. Wilkenfeld JS, Lawrence AL, Kuban FD (1984) Survival, metamorphosis and 65. Palmer C, Bik EM, DiGiulio DB, Relman DA, Brown PO (2007) Development
growth of Penaeid shrimp larvae reared on a variety of algal and animal foods. of the Human Infant Intestinal Microbiota. PLoS Biol 5: e177.
J World Maricult Soc 15: 31–49. 66. Perez T, Balcazar JL, Ruiz-Zarzuela I, Halaihel N, Vendrell D, et al. (2010)
53. Chen H (1993) Recent Advances in Nutrition of Penaeus monodon. J World Host-microbiota interactions within the fish intestinal ecosystem. Mucosal
Aquacult Soc 24: 231–240. Immunol 3: 355–360.
54. Chang ES (1995) Physiological and biochemical changes during the molt cycle in 67. Austin B, Zhang XH (2006) Vibrio harveyi: a significant pathogen of marine
decapod crustaceans: an overview. J Exp Mar Biol Ecol 193: 1–14. vertebrates and invertebrates. Lett Appl Microbiol 43: 119–124.
55. Tkavc R, Ausec L, Oren A, Gunde-Cimerman N (2011) Bacteria associated with 68. Balcazar JL, de Blas I, Ruiz-Zarzuela I, Cunningham D, Vendrell D, et al.
Artemia spp. along the salinity gradient of the solar salterns at Eilat (Israel). FEMS (2006) The role of probiotics in aquaculture. Vet Microbiol 114: 173–186.
Microbiology Ecology 77: 310–321. 69. Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, et al. (2009)
56. Hoj L, Bourne DG, Hall MR (2009) Localization, abundance and community Introducing mothur: Open-Source, Platform-Independent, Community-Sup-
structure of bacteria associated with Artemia: Effects of nauplii enrichment and ported Software for Describing and Comparing Microbial Communities. Appl
antimicrobial treatment. Aquaculture 293: 278–285. Environ Microbiol 75: 7537–7541.
57. Lopez-Torres MA, Lizarraga-Partida ML (2001) Bacteria isolated on TCBS 70. Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R (2011) UCHIME
media associated with hatched Artemia cysts of commercial brands. Aquaculture
improves sensitivity and speed of chimera detection. Bioinformatics doi10.1093/
194: 11–20.
bioinformatics/btr381.
58. Leite AM, Mayo B, Rachid CT, Peixoto RS, Silva JT, et al. (2012) Assessment of
71. Kunin V, Engelbrektson A, Ochman H, Hugenholtz P (2010) Wrinkles in the
the microbial diversity of Brazilian kefir grains by PCR-DGGE and
rare biosphere: pyrosequencing errors can lead to artificial inflation of diversity
pyrosequencing analysis. Food Microbiol 31: 215–221.
59. Cleary DF, Smalla K, Mendonca-Hagler LC, Gomes NC (2012) Assessment of estimates. Environ Microbiol 12: 118–123.
variation in bacterial composition among microhabitats in a mangrove 72. Huson DH, Auch AF, Qi J, Schuster SC (2007) MEGAN analysis of
environment using DGGE fingerprints and barcoded pyrosequencing. PLoS metagenomic data. Genome Res 17: 377–386.
ONE 7: e29380. 73. Fierer N, Jackson JA, Vilgalys R, Jackson RB (2005) Assessment of soil microbial
60. Ley RE, Peterson DA, Gordon JI (2006) Ecological and evolutionary forces community structure by use of taxon-specific quantitative PCR assays. Appl
shaping microbial diversity in the human intestine. Cell 124: 837–848. Environ Microb 71: 4117–4120.
61. Ribeiro F (2010) Development morphology and oncogeny of penaeid prawns 74. Bacchetti De Gregoris T, Aldred N, Clare AS, Burgess JG (2011) Improvement
postlarva; Alday-Sanz V, editor. Nottingham, UK: Nottingham University Press. of phylum- and class-specific primers for real-time PCR quantification of
62. Jiravanichpaisal P, Puanglarp N, Petkon S, Donnuea S, Söderhäll I, et al. (2007) bacterial taxa. J Microbiol Methods 86: 351–356.
Expression of immune-related genes in larval stages of the giant tiger shrimp, 75. Muyzer G (1999) DGGE/TGGE a method for identifying genes from natural
Penaeus monodon. Fish & Shellfish Immunology 23: 815–824. ecosystems. Curr Opin Microbiol 2: 317–322.
63. Cahill MM (1990) Bacterial flora of fishes: A review. Microbiol Ecol 19: 21–41. 76. Muyzer G, de Waal EC, Uitterlinden AG (1993) Profiling of complex microbial
64. Lovett DL, Felder DL (1990) Ontogenetic Changes in Enzyme Distribution and populations by denaturing gradient gel electrophoresis analysis of polymerase
Midgut Function in Developmental Stages of Penaeus setiferus (Crustacea, chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol
Decapoda, Penaeidae). Biol Bull 178: 160–174. 59: 695–700.

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