Aquaculture research, tập 41, số 11, 2010

Aquaculture Research, 2010, 41, 1553^1573

doi:10.1111/j.1365-2109.2010.02546.x

REVIEW ARTICLE
Role of gastrointestinal microbiota in fish
Sukanta K Nayak
Laboratory of Fish Pathology, Department of Veterinary Medicine, College of Bioresorece Sciences, Nihon University,
Kanagawa 252-8510, Japan
Correspondence: S K Nayak, Laboratory of Fish Pathology, Department of Veterinary Medicine, College of Bioresorece Sciences, Nihon
University, 1866 Kameino, Fujisawa, Kanagawa 252-8510, Japan. E. Mail: sukantanayak@redi¡mail.com

Abstract
The gastrointestinal (GI) tract of an animal consists
of a very complex and dynamic microbial ecosystem
that is very important from a nutritional, physiological
and pathological point of view. A wide range of
microbes derived from the surrounding aquatic environment, soil/sediment and feed are found to colonize
in the GI tract of ¢sh. Among the microbial groups,
bacteria (aerobic, facultative anaerobic and obligate
aneraobic forms) are the principal colonizers in the
GI tract of ¢sh, and in some ¢sh, yeasts are also
reported. The common bacterial colonizers in the GI
tract of freshwater and marine ¢sh include Vibrio,
Aeromonas, Flavobacterium, Plesiomonas, Pseudomonas, Enterobacteriaceae, Micrococcus, Acinetobacter,
Clostridium, Fusarium and Bacteroides, which may
vary from species to species as well as environmental
conditions. Besides, several unknown bacteria belonging to Mycoplasma, Arthrobacter, Brochothrix, Jeotgailbacillus, Ochrobactrum, Psychrobacter and Sejongia
species in the GI tract of di¡erent ¢sh species have
now been identi¢ed successfully using culture-independent techniques. Gnotobiotic and conventional studies indicate the involvement of GI microbiota in ¢sh

nutrition, epithelial development, immunity as well as
disease outbreak. This review also highlights the need
for manipulating the gut microbiota with useful bene¢cial microbes through probiotic, prebiotic and synbiotic concepts for better ¢sh health management.

Keywords: ¢sh, gastrointestinal tract, gnotobiotic, microbiota, probiotics, prebiotics
Introduction
The ¢rst observation on the occurrence of a gastrointestinal (GI) microorganism in any host was made

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Aquaculture Research r 2010 Blackwell Publishing Ltd

by Leewenhock in 1674 (DoBell 1932). However, the
comprehensive study of intestinal bacteria was
initiated only after the discovery of Escherichia in the
human GI tract, which laid the foundation of GI
microbiota in other species. With the advent of the
20th century, the distribution of GI microbiota has
been studied extensively in various animals for di¡erent purposes. The GI ‘microbiota’ usually refers to
a very complex and dynamic microbial ecosystem
that colonizes the GI tract of an animal. Furthermore,
with the developments in molecular and biotechnological tools, this complex ecosystem is beginning to
be unravelled in many species (Rastall 2004).
The mammalian GI tract contains an enormous
variety of aerobic and anaerobic microbes that interact in its complex ecosystem (Eckburg, Bik, Bernstein,
Purdom, Dethlefsen, Sargent, Gill, Nelson & Relman
2005; Nicholson, Holmes & Wilson 2005), but that of
¢sh is believed to be simpler and less in number than
that of endothermic animals (Trust & Sparrow 1974;
Horsley1977; Finegold, Suher & Mathisen1983; Sakata
1990). Until the 1970s, a concrete report on the existence of a stable indigenous microbiota in many aquatic animals was not available (Savage 1977;Yoshimizu,

Kimura & Sakai1980; RingÖ, Olsen, Mayhew & Myklebust 2003), but during the past few decades, substantial research has been carried out to characterize the
GI microbiota in a wide range of ¢sh species (RingÖ,
Strom & Tabachek 1995; RingÖ & Gatesoupe 1998;
RingÖ, Lodemel, Myklebust, Kaino, Mayhew & Olsen
2001; Ward, Steven, Penn, Methe & Detrich III 2009).
However, most of the earlier GI microbiota studies in
¢sh have emphasized the microbial spoilage, environmental relationship (Horsley 1973), their enzymatic
ability (Shcherbina & Kazlawlene 1971; Lindsay &
Harris 1980), studies on nutritional aspects (Moriarty
1990), monitoring of the changes in farms (Allen,

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Role of gastrointestinal microbiota in ¢sh S K Nayak

Austin & Colwell 1983) and antibiotic resistance
(Ogbondeminu & Olayemi 1993).
The GI microbiota serve a variety of functions in
the host and their importance in the nutrition and
health of the host by promoting nutrient supply, preventing the colonization of infectious agents, energy
homeostasis and maintenance of normal mucosal
immunity is well documented in mammals (Xu,
Bjursell, Himrod, Deng, Carmichael, Chiang, Hooper
& Gordon 2003; Nicholson et al. 2005; Delzenne &
Cani 2008). Although the presence of native GI
microbiota in ¢sh has been recognized, little is
known about the bacterial communities and their establishment, diversity and most importantly their
role in ¢sh nutrition and health. Therefore, an
attempt has been made to review the information

available for a better understanding of the GI
microbes and their possible functional roles in ¢sh.
Development and establishment of GI
microbiota of fish
The microbial colonization, establishment, composition and diversity in the GI tract of ¢sh is a complex
process and believed to be a re£ection of the microbial composition of the rearing water, diet and their
environment (Liston 1957; Geldreich & Clarke 1966;
Nieto,Toranzo & Barja1984; Buras, Duek, Niv, Hepher
& Sandbank 1987; Ogbondeminu 1993; Korsnes,
Nicolaisen, Skar, Nerland & Bergh 2006; RingÖ,
Sperstad, Myklebust, Refstie & Krogdahl 2006;
RingÖ, Sperstad, Myklebust, Mayhew & Olsen 2006;
Fjellheim, Playfoot, Skjermo & Vadstein 2007). Two
distinct groups, i.e. either allochthonous (transient)
and autochthonous (adherent), are usually found in
the GI tract of ¢sh. The latter group of bacteria, by virtue of their ability to tolerate the low pH in gastric
juices and resistance to the actions of bile acids, only
succeeded in colonizing in the epithelial surface of the
stomach, small and large intestine (Savage1989). These
bacteria can ¢rmly attach to the intestinal mucosa to
become the autochthonous microbiota of the host
(Yoshimizu, Kimura & Sakai 1976; Onarheim & Raa
1990; Sakata1990; Onarheim,Wiik, Burghardt & Stackebradt 1994). The other group of bacteria is present
transiently in the GI tract because they are not able to
colonize the mucus layer and/or the epithelial surface
(RingÖ & Birkbeck 1999). They either lack this ability
entirely or are so ine¡ective at it that they are outcompeted by other bacteria in the mucus/epithelium.
The initial colonization process is very complex
at larval and fry and mostly depend on the ¢sh type,

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Aquaculture Research, 2010, 41, 1553–1573

nutrients/food and surrounding conditions (Bignell
1984; Voveriene, Mickeniene & Syvokiene 2002). The
total bacterial load at the larval stage is low (approximately 102 CFU larva À 1) before active feeding (Munro, Barbour & Birkbeck 1994; Verner-Je¡reys, Shields,
Bricknell & Birkbeck 2003; Reid, Treasurer, Adam &
Birkbeck 2009), and this initial load is mostly derived
from the water by larvae to maintain osmotic balance
(Tytler & Blaxter 1988; Reitan, Natvik & Vadstein
1998). However, the number increases rapidly
(4105 CFU larva À 1) once the larvae start to feed
(Munro, Birkbeck & Barbour 1993; Munro et al.1994).
Furthermore, the microbial composition and density also vary in di¡erent regions of the GI tract of ¢sh
depending on the physico-chemical conditions of
gut. In ¢sh, a progressive increase in culturable bacterial levels from the stomach to the posterior intestine is often reported (Trust & Sparrow 1974;
MacDonald, Stark & Austin 1986; Molinari, Scoaris,
Pedroso, Bittencourt, Nakamura, Ueda-Nakamura,
Abreu Filho & Dias Filho 2003). Molinari et al. (2003)
recorded higher viable bacteria in both the anterior
and the posterior gut than in the stomach of semi-intensively cultured Oreochromis niloticus. However,
with the successful application of modern non-culturable techniques, the composition of bacteria in
the GI tract and their percentage may vary with respect to ¢sh. Recently, Navarrete, Espejo and Romero
(2009) recorded the average bacterial density to be
1 Â 107, 8 Â 106 and 5 Â 107 bacteria g À 1 in the
stomach, pyloric caeca and intestine, respectively, in
Salmo salar using epi£uorescence microscopy.
Factors a¡ecting the establishment of GI
microbiota

A series of exogenous and endogenous factors can
a¡ect the establishment and nature of the microbial
composition in the GI tract of ¢sh. The developmental
stage of ¢sh (Bell, Hoskins & Hodgkiss 1971; Sugita,
Enomoto & Deguchi 1982; Sugita,Tokuyama & Deguchi 1985), gut structure (Sera, Ishida & Kadota 1974;
Sugita et al. 1985), the surrounding environment like
ambient water temperature (Lesel & Peringer 1981;
Sugita, Oshima, Tamuar & Deguchi 1989), rearing
and farming conditions (Trust & Sparrow 1974; Trust
1975;Yoshimizu & Kimura 1976; Horsley1977; Sugita,
Isida, Deguchi & Kadota 1982; RingÖ & Strom 1994)
are very critical factors that a¡ect the initial colonization and the subsequent establishment process.
Besides, stress factors can signi¢cantly a¡ect the GI
microbiota (Lesel & Sechet 1982). When di¡erent

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Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 1553^1573


Aquaculture Research, 2010, 41, 1553^1573

types of chemicals, antibiotics, pollutants like pesticides, herbicides and insecticides enter into the digestive tract of an aquatic animal, they can drastically
a¡ect the composition of dominant GI microbiota and
may lead to the elimination of individual species from
the whole microbial community (Austin & Al-Zahrani
1988; Sugita, Fukumoto, Koyama & Deguchi 1988;
Sugita et al. 1989; Syvokiene & Mickeniene 2002;
Bakke-McKellep, Koppang, Gunnes, Sanden, Hemre,
Landsverk & Krogdahl 2007; Mickeniene & Syvokiene
2008; Navarrete, Mardones, Opazo, Espejo & Romero

2008).
Feed and feeding conditions considerably in£uence the composition of GI microbiota of ¢sh (Campbell & Buswell 1983; Sugita, Tsunohara, Fukumoto &
Deguchi1987; Syvokiene1989; Onarheim & Raa1990;
RingÖ 1993; RingÖ & Olsen 1999; Uchii, Matsui,
Yonekura, Tani, Kenzaka, Nasu & Kawabata 2006;
RingÖ, Sperstad, Myklebust, Refstie et al. 2006;
Martin-Antonio, Manchado, Infante, Zerolo, Labella,
Alonso & Borrego 2007), and during the larval stage,
the gut microbial £ora has been found to change
rapidly with respect to feed (Brunvold, Sandaa,
Mikkelsen, Welde, Bleie & Bergh 2007; Reid et al.
2009). A positive correlation of capelin roe diet with
Enterobacteriaceae in the GI microbiota of wild charr
irrespective of freshwater and seawater maintenance
was recorded by RingÖ and Strom (1994). However,
they have recorded the predominance of Aeromonas
in freshwater and theVibrio species under marine conditions from wild catch charr fed with a commercial
diet. Recently, RingÖ, Sperstad, Myklebust, Mayhew
et al. (2006) recorded the variation in GI microbiota
with respect to the diet. RingÖ and colleagues
observed the dominance of Gram-positive bacteria
belonging to Brochothrix and Carnobacterium species
in the GI tract of Gadus morhua fed with ¢sh meal,
while Psychrobacter species and Psychrobacter glacincola, Chryseobacterium and Carnobacterium species
dominated the GI tract when fed with bio-processed
soybean meal and standard soybean respectively.
Furthermore, the seasonal and day-to-day £uctuations in GI bacteria in di¡erent ¢sh species have also
been recorded (Sugita et al.1987; MacMillan & Santucci
1990; Spanggaard, Huber, Nielsen, Nielsen, Appel &
Gram 2000; Al-Harbi & Uddin 2004; Hagi, Tanaka,

Iwamura & Hoshino 2004). Variations in the total
viable GI bacterial counts from 1.6 Â 106 to
5.1 Â 107 CFU g À 1 intestine in summer, 3.1 Â 108 to
1.3 Â 109 CFU g À 1 intestine in autumn and 8.9 Â 105
to1.3 Â 107 CFU g À 1 intestine in winter were recorded
in hybrid tilapia (O. niloticus  Oreochromis aureus)

Role of gastrointestinal microbiota in ¢sh S K Nayak

(Al-Harbi & Uddin 2004). Similarly, MacMillan and
Santucci (1990) reported seasonal variations among
the bacterial species belonging to Escherichia coli, Klebsiella, Pseudomonas, Plesiomonas, Shigelloides, Streptococcus and Moraxella species in farm-raised Ictalurus
punctatus. In a yearlong study on the changes in lactic
acid bacteria (LAB) composition, Hagi et al. (2004)
found the predominance of Lactococcus lactis in summer (water temperature 420 1C) and Lactococcus ra⁄nolactis in winter (water temperature range 4^10 1C) in
the GI tract of Cyprinus carpio. Inter-individual variation and highest daily £uctuation of Bacteroides species
in the GI tract of ¢sh like C. carpio have also been
observed (As¢e,Yoshijima & Sugita 2003).

Microbial composition in the GI tract of ¢sh
The GI tract is a favourable ecologic niche for microorganisms, and like any other animals, a wide range
of microbes colonize in the GI tract of ¢sh (Skrodenyte-Arbaciauskiene 2007). However, information
on the type of bacterial composition in the GI tract
of ¢sh is often controversial (Izvekova & Lapteva
2004). Most of the earlier studies of ¢sh GI microbiota
have been derived from the homogenates of intestinal
content and/or faecal materials using culture-based
techniques using selective or non-selective isolation
media, followed by phenotypic characterization
using a series of conventional morphological and biochemical assays (Horsley 1977; Sakata, Sugita, Mitsuoka, Kakimoto & Kadota 1981; Sugita, Oshima,

Tamura & Deguchi 1983; Sakata 1989, 1990; Sugita
et al.1989; Cahill 1990; Onarheim & Raa1990; Zaman
& Leong 1994; RingÖ et al. 1995; Sivakami, Premkishore & Chandran 1996). However, conventional
methods are often time consuming and lack accuracy (As¢e et al. 2003) and sensitivity in characterizing certain fastidious and obligate anaerobes that
require special culture conditions. Therefore, culture-based study of GI microbiota of any animal often
leads to a very uncertain picture of the total microbial community residing inside the tract.
Nowadays, several novel molecular technologies
are being increasingly used for the analysis of
microbes present in the complex GI ecosystem of
animals. Molecular techniques based on genotypic
¢ngerprinting techniques such as colony hybridization with nucleic acid probes, pulsed ¢eld gel electrophoresis, ribotyping, polymerase chain reaction
(PCR), random ampli¢ed polymorphic DNA, multiplex-PCR, arbitrary primed-PCR and triplet arbitrary

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Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 1553^1573

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Role of gastrointestinal microbiota in ¢sh S K Nayak

primed-PCR, denaturing gradient gel electrophoresis
(DGGE), temporal temperature gradient gel electrophoresis, £uorescence in situ hybridization (FISH)
and also electron microscopy have been used to study
and characterize the microbes in the GI tracts of
many animals including ¢sh (Spanggaard et al.
2000;Walter, Hertel,Tannock, Lis, Munro & Hammes
2001; Holben,Williams, Saarinen, SÌrkilahti & Apajalahti 2002; Temmerman, Huys & Swings 2004; Kim,
Brunt & Austin 2007; Peter & Sommaruga 2008).
Most of the recent studies have successfully used

these techniques alone or in combination with conventional methods to characterize both culturable
and unculturable microbiota in the GI tract of ¢sh
(Huber, Spanggaard, Appel, Rossen, Nielsen & Gram
2004; Pond, Stone & Alderman 2006; Seppola, Olsen,
Sandaker, Kanapathippillai, Holzapfel & Ringo 2006;
Shiina, Itoi,Washio & Sugita 2006; Skrodenyte-Arbaciauskiene, Sruoga & Butkauskas 2006; Hovda, Lunestad, Fontanillas & Rosnes 2007; Namba, Mano &
Hirose 2007; Liu, Zhou,Yao, Shi, He, Holvold & Ringo
2008; Skrodenyte-Arbaciauskiene, Sruoga, Butkauskas & Skrupskelis 2008; Merri¢eld, Burnard, Bradley,
Davies & Baker 2009), and are presented in Table 1.
The culture-dependent and -independent studies
indicate that bacteria are the major microbial colonizer in the GI tract of ¢sh (MacDonald et al. 1986;
Spanggaard et al. 2000; Molinari et al. 2003; Pond
et al. 2006). Besides, yeast is also reported to colonize
in the GI tract of some ¢sh (Andlid,Vazquez-Juarez &
Gustafsson 1998; Gatesoupe 2007). Yeasts belonging
to Rhodotorula species are frequently found in the GI
tract of both marine and freshwater ¢sh while
Metschnikowia zobelii, Trichosporon cutaneum and
Candida tropicalisare are the dominant GI yeast species in marine ¢sh (Gatesoupe 2007). The GI microbiota of ¢sh mainly consists of aerobic or facultative
anaerobic microorganisms (Clements 1997; Bairagi,
Ghosh, Sen & Ray 2002; Saha, Roy, Sen & Ray 2006),
facultative as well as obligate anaerobes, especially
Cetobacterium somerae (previously classi¢ed as Bacteroides type A), Bacteroidaceae and Clostridium species (Cahill 1990; Sugita, Miyajima & Deguchi 1990;
Pond et al. 2006; Tsuchiya, Sakata & Sugita 2008).
The predominance of anaerobes in the GI tract of ¢sh
like gold ¢sh (Carrrasius auratus), Oncorhynchus
mykiss and O. niloticus has been recorded (Sakata,
Okabayashi & Kakimoto 1980; Sugita et al. 1989;
Spanggaard et al. 2000). Earlier, Trust, Bull, Currie
and Buckley (1979) reported equal numbers

(107 cells g À 1 of gut content) of anaerobic bacteria
(Bacteroides and Fusabacterium) and aerobic bacteria

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Aquaculture Research, 2010, 41, 1553–1573

(Aeromonas and Pseudomonas) in the GI tract of grass
carp (Ctenopharyngodon idella).
The total bacterial load in the GI tract of ¢sh is low in
comparison with warm-blooded animals and their
number often varies with age, nutrition and environment (RingÖ et al. 2003; Gomez & Balcazar 2008). In
¢sh, the approximate viable aerobic and anaerobic
bacteria usually vary from 104^109 to 6.6 Â 104^
1.6 Â 109 CFU g À 1 intestinal content respectively
(Skrodenyte-Arbaciauskiene 2007). Earlier culturedependent studies in faecal samples of ¢sh indicate
the aerobic and anaerobic bacterial load to be 108 and
105 bacteria g À 1 of faeces respectively (Trust 1975;
Trust et al. 1979; Trust & Sparrow 1974; Austin &
Al-Zahrani 1988). Recently, As¢e et al. (2003) reported
the variation in the total microbial cells in ¢ve
gold¢sh specimens to range from 9.6 Â 108 to
6.5 Â 1010 cells g À 1 of faeces by FISH. Considering the
fact that a large population of GI bacteria in ¢sh is unculturable (Romero & Navarrete 2006; Navarrete et al.
2009), the total bacterial load is higher as recorded
from the total culturable heterotrophic bacteria. For instance, Shiina et al. (2006), using direct microscopic
enumeration of bacteria with 4 0,6-diamidino-2-phenylindole, reported the total bacterial count to vary from
1.0 Â 104 to 1.4 Â 109 CFU g À 1 intestinal content in
contrast to 4.7 Â 1010^1.9 Â 1011 cells g À 1 intestinal
content in coastal ¢sh like Ditrema temmincki, Girella

punctata, Pseudolabrus japonicas, Sebastes pachycephalus,Takifugu niphobles and Thalassoma cupido. Similarly,
Sugita, Kurosaki, Okamura, Yamamoto and Tsuchiya
(2005) also reported that the total bacterial load
of each coastal ¢sh varied from 2.9 Â 109 to
3.0 Â 1010 cells g À 1 intestinal content through direct
counts regardless of the ¢sh species and their
feeding habitat, while the viable counts of intestinal
bacteria of these species ranged from 1.9 Â 103 to
4.2 Â 109 CFU g À 1 intestinal content. Recently, Navarrete et al. (2009) also recorded such type of di¡erences
in the total bacterial density in di¡erent regions of the
GI tract such as stomach, pyloric caeca and intestine of
S. salar using epi£uorescence microscopy as compared
with a culture-dependent technique.
The bacterial composition in the GI tract varies
from freshwater to marine water ¢sh, with the predominance of Gram-negative bacteria over Gram-positive bacteria in the intestine of several ¢sh species
(Sakata, Uno & Kakimoto 1984; RingÖ 1993; Hatha,
Kuruvilla & Cheriyan 2000). Aeromonads are mostly
associated with the GI tract of freshwater ¢sh (Sugita
et al. 1983; Sugita, Nakamura, Tanaka & Deguchi
1994; Wang, He, Live, Hu & Chen 1994; As¢e et al.

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Aquaculture Research, 2010, 41, 1553^1573

Role of gastrointestinal microbiota in ¢sh S K Nayak

Table 1 Di¡erent culture-dependent and -independent methods used to study the whole and/or the speci¢c gastrointestinal

microorganisms of ¢sh
Sl. No

Fish

Methods of study

References

1

Rainbow trout
(Oncorhynchus mykiss)

Merrifield et al. (2009)

2

Atlantic salmon (Salmo salar)

3

Atlantic Cod (Gadus morhua)

4

Grouper (Epinephelus coioides)

5

Antarctic notothenioid fish
(Notothenia coriiceps,
Chaenocephalus aceratus)
S. salar

Culture-based isolation, followed by partial 16S rRNA
gene sequencing
Culture-independent analysis of 16S rRNA gene
sequence by denaturing gradient gel electrophoresis
(DGGE)
Scanning electron microscopic study
Culture-based isolation and characterization by RFLP
analysis of 16S rRNA gene and intergenic spacer region
profiles
Culture-independent analysis by temporal temperature
gradient gel electrophoresis of the 16S rRNA gene and
intergenic spacer region profiles
Culture-based isolation and biochemical, physiological
characterization and partial sequence analysis of the rpoB
and 16S rRNA genes by DGGE
Culture-based isolation and biochemical and physiological
characterization, followed by 16S rRNA gene analysis
Culture-independent analysis of the 16S rRNA gene

6
7
8

9

Sea trout (Salmo trutta trutta),
S. salar
S. salar

10

Goldfish (Carassius auratus),
Common carp (Cyprinus carpio),
Mozambique tilapia (Oreochromis
mossambicus), Japanese catfish
(Silurus asotus), grass carp
(Ctenopharyngodon idella)
G. morhua

11

G. morhua

12

S. salar

13

O. mykiss

14

Senegalese sole
(Solea senegalensis)

15

C. carpio

16

O. mykiss

17

G. morhua

Navarrete et al. (2009)

Reid et al. (2009)

Sun et al. 2009
Ward et al. (2009)

Culture-independent analysis of the 16S rRNA gene by
DGGE
Culture-based isolation and characterization by 16S rRNA
gene analysis
Culture-based isolation and biochemical and physiological
characterization, followed by 16S rRNA gene analysis
Transmission electron microscopic study
Culture-based isolation and characterization by
biochemical reactions including API ZYM and API 20A
systems as well as by 16S rRNA gene analysis

Liu et al. (2008)

Culture-independent analysis of 16S rRNA gene analysis
by DGGE
Culture-based isolation and phenotypic characterization
along with 16S rRNA gene analysis
Culture-based isolation and characterization using API
20E and API 20NE systems along with 16S rRNA gene
analysis by DGGE
Culture-independent analysis of 16S rRNA gene by DGGE
Culture-based isolation and characterization by 16S rRNA
gene analysis
Culture-independent analysis of 16S rRNA gene by DGGE
and 16S rDNA clone library techniques
Culture-based isolation and biochemical characterization
using the API 20NE system and also by 16S rRNA gene
analysis
Culture-based isolation and phenotypical characterization
along with 16S rRNA gene analysis
Culture-based isolation and characterization using the
Biolog system, API strips and 16S rRNA gene analysis
Culture-independent analysis of 16S rRNA gene by
restriction fragment length polymorphism (RFLP)
Culture-based isolation and phenotypic characterization
and also by 16S rRNA gene analysis
Electron microscopic study

Brunvold et al. (2007)

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Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 1553^1573

Skrodenyte-Arbaciauskiene
et al. (2008)
Ringø, Sperstad, Kraugerud
and Krogdahl (2008)
Tsuchiya et al. (2008)

Fjellheim et al. (2007)
Hovda et al. (2007)

Kim et al. (2007)

Martin-Antonio et al. 2007

Namba et al. (2007)
Pond et al. 2006

Ringø et al. 2006a

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Role of gastrointestinal microbiota in ¢sh S K Nayak

Aquaculture Research, 2010, 41, 1553–1573

Table1 Continued
Sl. No

Fish

18

Coho Salmon
(Oncorhynchus kisutch)

19

20

21
22

23

24

25

26

27

28
29
30

Methods of study

Culture-based isolation and characterization by RFLP
analysis of the 16S rRNA gene
Culture-independent analysis of the 16S rRNA gene
by DGGE
G. morhua
Culture-based isolation and phenotypic characterization in
combination with random amplification of polymorphic –
DNA (RAPD) analysis
Takifugu niphobles
Culture-based quantitative study, Culture-independent
characterization of 16S rRNA gene analysis using the
clonal library method
River trout (Salmo trutta fario)
Culture-based isolation and characterization by partial 16S
rRNA gene sequence analysis
Japanese flounder
Culture-based isolation and characterization by
(Paralichthys olivaceus)
biochemical and physiological assays, followed by 16S
rRNA gene analysis
Bluegill (Lepomis macrochirus)
Culture-independent study by community-level
physiological profile analysis using Biolog microplates and
analysis of the 16S rRNA gene by TGGE
Culture-based isolation and characterization by RAPD
Deepbodied crucian carp
(Carassius uvieri), Channel catfish analysis of the 16S rRNA gene
(Ictalurus punctatus), Silver carp
(Hypophthalmichthys molitrix),

C. carpio
O. mykiss
Culture-based isolation and characterization by RAPD
analysis of the 16S rRNA gene
Culture-independent study by fluorescent in situ
hybridization (FISH) and DGGE
C. auratus, C. carpio
Culture-based isolation and characterization using the
O. mossambicus
whole-cell hybridization technique using rRNA-targeted
oligonucleotide probes
Culture-independent analysis of fecal samples by FISH
Atlantic halibut
Culture-based isolation and characterization using
(Hippoglossus hippoglossus)
biochemical and the Biolog GN bacterial identification
system as well as RFLP analysis of the 16S rRNA gene
and the partial 16S rDNA gene
S. salar
Culture-independent study by partial 16S rRNA gene
sequence analysis
Arctic charr (Salvelinus alpines)
Electron microscopic study
O. mykiss
Culture-based isolation and characterization using
biochemical nature as well as RAPD analysis of the 16S
rRNA gene

2003; Skrodenyte-Arbaciauskiene et al. 2008). In
freshwater ¢sh, Aeromonas, Pseudomonas and Bacteroides type A are major colonizers in the GI tract,

followed by Plesiomonas, Enterobacteriaceae, Micrococcus, Acinetobacter, Clostridium, Bacteroides type B
and Fusarium species (Trust et al. 1979; Lesel 1981;
Sugita, Sakata, Ishida, Deguihi & Kadota 1981;
Sugita et al. 1985). In contrast to freshwater ¢sh,
Vibrio, Pseudomonas, Achromobacter, Corynebacterium, Alteromonas, Flavobacterium and Micrococcus
species are predominant in the GI tract of most of
the marine ¢sh (Cahill 1990; Onarheim et al. 1994;

1558

References
Romero and Navarrete
(2006)

Seppola et al. (2006)

Shiina et al. (2006)

Skrodenyte-Arbaciauskiene
et al. (2006)
Sugita and Ito (2006)

Uchii et al. (2006)

Hagi et al. (2004)

Huber et al. (2004)

Asfie et al. (2003)

Verner-Jeffreys et al. (2003)

Holben et al. (2002)
Ringø et al. (2001)
Spanggaard et al. (2000)

Blanch, Alsina, Simon & Jofre 1997; Verner-Je¡reys
et al. 2003). Among other bacterial groups that
colonize in the GI tract of both freshwater and
marine ¢sh are LAB (Strom 1988; Strom & RingÖ
1993; Pilet, Dousset, Barre, Novel, Des mazeaud &
Piard 1995; Balcazar, de Blas, Ruiz-Zarzuela,Vendrell,
Girones & Muzquiz 2007). However, they are usually
not the dominant component of the GI microbiota
(RingÖ et al. 1995; Jankauskiene 2000a, b), but under
certain conditions like a pond culture system, they
can dominate, with an abundance as high as
1.1 Â 106 cells g À 1 ¢sh body weight, and can form a

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stable component of the GI tract of ¢sh (Syvokien
1989). A recent study on GI bacteria of ¢sh like
Notothenia coriiceps and Chaenocephalus aceratus,
which di¡er in their pelagic distribution and feeding
strategies, indicated the dominance of Vibrionanceae

(g-proteobacteria) like that in temperate teleost
species (Ward et al. 2009).
Application of a recent molecular technology has
provided a major breakthrough in the detection and
identi¢cation of the microbial composition in the GI
ecosystem of many animals including ¢sh. Among
the recent technologies, DGGE, a‘genetic ¢ngerprint’
method based on PCR ampli¢cation of 16S rDNA, has
been successfully used to study the dynamic behaviour of the dominant microbes in di¡erent environments (Gri⁄ths, Melville, Cook & Vincent 2001; Long
& Azam 2001; Sandaa, Magnesen,Torkildsen & Bergh
2003). PCR- and DGGE-based identi¢cation and
characterization provides an accurate picture of the
complexity of the GI microbiota of ¢sh (Simpson,
McCracken, White, Gaskins & Mackie 1999; Gri⁄ths
et al. 2001; Huber et al. 2004; Vanhoutte, Huys, De
Brandt, Fahey & Swings 2005; Brunvold et al. 2007;
Kim et al. 2007). Apart from culturableVibrio, Pseudomonas, Janthinobacterium and Acinetobacter species,
Hovda et al. (2007) successfully characterized predominant but slow-growing culturable bacteria such as
Lactobacillus fermentum, Photobacterium phosphoreum, Lactococcus and Bacillus species in the GI tract
of Atlantic salmon (S. salar) using PCR- and DGGEbased techniques. Similarly, Pond et al. (2006)
succeeded in characterizing the presence of a number of bacterial species like Stenotrophomonas maltophilia, Pseudomonas picketti, Ralstonia eutrophia and
b-Proteobacterium in the GI tract of O. mykiss using
16S rRNA technology.
Nowadays, many new uncultuturable bacteria are
being identi¢ed using molecular biological tools from
the GI tract of ¢sh from freshwater to marine type.
Several unknown bacteria like Gram-negative Acinetobacter (A. johnsoni), Chryseobacterium, Ochrobactrum, Psychrobacter (P. luti, P. fozii, P. glacincola, P.
psychrophilus and P. cibarius) and Sejongia species
(S. antarctica) and Gram-positive bacteria like Arthrobacter (A. agilis and A. psychrolactophilus), Brochothrix
(B. thermosphacta), Jeotgailbacillus (J. psychrophilus),

Microbacterium and Staphylococcus species (S. equorum
spp. linens) in S. salar (RingÖ, Sperstad, Myklebust,
Mayhew et al. 2006); Mycoplasma and Acinetobacter
species (A. junii) in S. salar (Holben et al. 2002); a and
b subclass of Proteobacteria in C. auratus (As¢e et al.
2003); Clostridium species (C. gasigenes) in O. mykiss

Role of gastrointestinal microbiota in ¢sh S K Nayak

(Pond et al. 2006);Tiedjeia arctica in wild river trout Salmo trutta fario (Skrodenyte-Arbaciauskiene et al. 2008),
Psychrobacter species; Delftia acidovorans, Burkholderia
cepacia and Erwinia carotovora in grouper (Epinephelus
coioides) (Sun, Yang, Ling, Chang & Ye 2009); and
A. aurescens and Janibacter species in O. mykiss (Merri¢eld et al. 2009) are now found to be part of the normal
microbiota in the GI tract of those ¢sh.
There still remain doubts about the complete
microbial composition and load in the GI of majority
¢sh species, and in the near future, culture-independent molecular tools may be able to provide a more
detailed picture of the true complexity in the GI tract
of di¡erent ¢sh species.

Role of GI microbiota in fish: gnotobiotic
approaches
Gnotobiotic models (animals cultured under axenic
conditions or with a known microbiota) are excellent
tools to study the role GI microbes in host (Marques,
Ollevier,Verstraete, Sorgeloos & Bossier 2006; Dierckens, Rekecki, Laureau, Sorgeloos, Boon, Van den
Broeck & Bossier 2009). Di¡erent gnotobiotic model
studies reveal the importance of GI microbiota in nutrient metabolism and absorption, xenobiotic metabolism, regulation of energy balance, epithelial
renewal, angiogenesis and development and maturation of the mucosal immune system (Falk, Hooper,

Midtvedt & Gordon 1998; Cebra 1999). Like other animals, gnotobiotic/germ-free technology is now developed in ¢sh (Dahm & Geisler 2006; Pham, Kanther,
Semova & Rawls 2008) and is also used to study evolutionarily conserved microbiota among vertebrates,
to monitor the microbial behaviour, interaction and
localization of microbes in the gut, as well as their
role in nutrition, epithelial development and immunity (Rawls, Mahowald, Ley & Gordon 2006; Rawls,
Mahowald, Goodman,Trent & Gordon 2007).
Gnotobiotic studies in ¢sh indicate the involvement of GI microbiota in epithelial di¡erentiation
and maturation. Bates, Mittg, Kuhlman, Baden,
Cheesman and Guillemin (2006) observed that the
di¡erentiation of gut epithelium is arrested by the
lack of brushborder intestinal alkaline phosphatase
activity and the maintenance of immature patterns
of glycan expression in the absence of the microbiota.
Alkaline phosphatase activity, which is a marker of
epithelial maturation, as well as mucous-secreting
goblet cells and hormone-secreting enteroendocrine
cells, the secretory cell lineages, are found to increase

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Role of gastrointestinal microbiota in ¢sh S K Nayak

signi¢cantly in the digestive tract of conventional Danio rerio larvae compared with their gnotobiotic
counterparts (Bates et al. 2006). Furthermore,
microbes are found to up-regulate the expression of
15 genes involved in DNA replication and cell division for epithelial proliferation (Rawls, Samuel & Gordon 2004). Similarly, a marked di¡erence in the

enterocytes has also been observed in germ-free and
conventional ¢sh. Rawls et al. (2004) observed consistent morphologic enterocytes in gnotobiotic D. rerio,
with the large supranuclear vacuoles ¢lled with clear
electron-lucent material and that of conventional ¢sh
¢lled with eosinophilic and electron-dense material.
In another study, Rekecki, Dierckens, Laureau, Boon,
Bossier and Van den Broeck (2009) observed variations with a slightly higher intestinal epithelium in
the midgut consisting of a cuboidal to columnar
epithelium in conventional larvae compared with
cuboidal to squamous epithelium in the midgut of
germ-free larvae of Dicentrarchus labrax after the
ninth day post hatching.
The GI microbiota plays a crucial role in the nutrition of the host and in ¢sh, they are involved in nutrient metabolism, especially in cholesterol metabolism
and tra⁄cking. It has been reported that gnotobiotic
D. rerio larvae failed in the uptake of protein macromolecules, with a signi¢cant di¡erence in the levels
of farnesyldiphosphate synthetase and apolipoprotein
B as compared with conventional larvae (Bates et al.
2006). Furthermore, the microbial upregulation of
apolipoprotein B, which plays a pivotal role in
intra- and extracellular cholesterol tra⁄cking, and
downregulation of the liver-speci¢c cholesterol
7a-hydrolase, which catalyses the ¢rst step in cholesterol catabolism and bile acid biosynthesis, indicate
the microbial modulation of cholesterol metabolism
and tra⁄cking (Rawls et al. 2004; Bates et al. 2006).
Besides these, gnotobiotic studies also indicate the
involvement of GI microbes in ¢sh immunity and also
in xenobitic metabolism. Microbial regulation of
glycoprotein production in the GI tract is reported in
D. labrax (Rekecki et al. 2009). Furthermore, microbiota are found to up-regulate the genes involved in
innate immunity parameters such as serum amyloid

A1, C-reactive protein, complement component 3,
angiogenin 4, glutathione peroxidase and myeloperoxidase (Rawls et al. 2004; Rawls et al. 2007). Although
gnotobiotic studies reveal some of the functional
roles of GI microbiota in ¢sh, the full extent to which
the microbiota in£uences gut development, local as
well as systemic immunity and homeostasis at the
cellular and molecular levels remains to be explored.

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Role of GI microbiota in immunity
The gut immune system, which is known as gut-associated lymphoid tissues (GALT), not only provides
defence against infectious agents but also regulates
immunity in the alimentary tract. The GI microbes
play a critical role in the development and maturation
of GALT, which in turn mediate a variety of host immune functions (Rhee, Sethupathi, Driks, Lanning &
Knight 2004). A complex and integrated interaction
between the epithelium, immune components in the
mucosa and microbes is responsible for the development and maturation of the gut-associated immune
system of the host. Gnotobiotic studies in di¡erent
animal models also support this notion (Umesaki &
Setoyama 2000; Peterson, McNulty, Guruge &
Gordon 2007). Several mechanisms are proposed for
the involvement of GI bacteria in the development of
GALT. Bacteria could stimulate B cell proliferation in
GALT through a classical antigen-speci¢c immune
response like protein A of Staphylococcus aureus and
protein L of Peptostreptococcus magnus (Nilson,

Solomon, Bjorck & Akerstrom 1992; Silverman &
Goodyear 2002) or by directly stimulating the innate
immune system (Medzhitov & Janeway 1997; Leadbetter, Rifkin, Hohlbaum, Beaudette, Shlomchik &
Marshak-Rothstein 2002). In ¢sh, GALT consists
principally of lymphocytes, eosinophil granular cells,
several types of granulocytes and plasma cells (Zapata & Amemiya 2000; Zapata, Diez, Cejalvo, Gutierrezde & Cortes 2006). The involvement of GI microbes in
the epithelial proliferation, maturation and immunity of ¢sh has already been discussed in the gnotobiotic studies (Rawls et al. 2004; Rekecki et al. 2009).
Similarly, the endocytosis of bacteria by epithelial
cells in the hindgut of immature larvae (Hansen,
Strom & Olafsen1992) as well as intact uptake of bacterial antigens in columnar epithelial cells in the
foregut, followed by their penetration into the gut
epithelium, have been recorded in ¢sh (Olafsen &
Hansen 1992). All these factors may directly or indirectly contribute to the development and stimulation
of the immune system.
The early exposure of the intestine to live bacteria
and subsequent colonization is very important for
the development of gut barrier. In ¢sh, dietary supplementation of useful microbes (probiotics) at early
developmental stages can be helpful in increasing the
subpopulations of speci¢c acidophilic granulocytes
(Picchietti, Mazzini, Taddei, Renna, Fausto, Mulero,
Carnevali, Cresci & Abelli 2007). Although probiotics
are often found in a transient state and persist for a

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certain period in the GI tract after the withdrawal of

feed in ¢sh, it is worth noting that dietary supplementation of probiotics can enhance both local
as well as systemic immunity in a wide range of ¢sh
species (Panigrahi, Kiron, Puangkaew, Kobayashi,
Satoh & Sugita 2005; Nayak, Swain & Mukherjee
2007; Panigrahi, Kiron, Satoh, Hirono, Kobayashi,
Sugita, Puangkaew & Aoki 2007; Picchietti et al.
2007; Aly, Ahmed, Ghareeb & Mohamed 2008;
Picchietti, Fausto, Randelli, Carnevali, Taddei, Buonocore, Scapigliati & Abelli 2009; Sharifuzzaman & Austin 2009; Son, Changa,Wu, Guu, Chiu & Cheng 2009).

Role of GI microbiota in nutrition
The importance of intestinal bacteria in the nutrition
and well-being of their hosts has been established in
several animals (Floch, Gorbach & Lucky 1970). The
ability to synthesize vitamins and essential growth
factors and digestive enzymes by GI microorganisms
has been demonstrated (Teply, Krehi & Elvehjem1947;
Uphill, Jalob & Lall 1977; Drasar & Barrow 1985;
Brock, Madigan, Martinko & Parker 1997).
The GI microbiota of hydrobionts has been reported to contribute to the nutrition and physiological processes of the host by producing vitamins,
amino acids, digestive enzymes and metabolites, similar to that of mammals (Syvokiene 1989; Cahill
1990; Sugita et al.1990; Sugita, Matsuo, Hirose, Iwato
& Deguchi 1997; Mickeniene 1999; SkrodenyteArbaeiauskiene 2000; Skrodenyte-Arbaeiauskiene
et al. 2006). Nevertheless, a wide range of enzymes
like carbohydrases, phosphatases, esterases, lipases
and peptidases, cellulase, lipase and proteases (Bairagi et al. 2002; Ramirez & Dixon 2003; Izvekova &
Lapteva 2004) produced by GI bacteria could be a
contributory source to digestive enzymes in ¢sh. The
presence of a high concentration of Aeromonas in the
GI tract can play an important role in digestion as
Aeromonas species secrete several proteases (Pemberton, Kidd & Schmidt 1997). Similarly, the p-nitrophenyl-b-n-acetylglucosaminide-, chitin-, cellulose- and

collagen-degrading ability of gut bacteria indicates
their possible involvement in the nutrition of ¢sh
(Shcherbina & Kazlawlene 1971; Lindsay & Harris
1980; Lesel, Fromageot & Lesel 1986; Macdonald
et al. 1986; Das & Tripathi 1991; Kar & Ghosh 2008).
Recent studies indicate that anaerobic bacteria
might play a role in the digestion and absorption of
nutrients (Ramirez & Dixon 2003). Anaerobic bacteria can contribute to ¢sh nutrition by supplying it with

Role of gastrointestinal microbiota in ¢sh S K Nayak

volatile fatty acids (Clements 1997). This is due to the
fact that volatile fatty acids, end products of anaerobic fermentation, are often reported in the intestines
of carp (C. carpio), shad (Dorosoma cepedianum) and
largemouth bass (Micropterus salmoides) (Smith,Wahl
& Mackie1996). Nevertheless, the ability of GI aerobic,
anaerobic and facultative aerobic bacteria to synthesize di¡erent vitamins and amino acids in ¢sh like
C. carpio, C. auratus, I. punctatus and O. nilotica is
noteworthy (Kashiwada & Teshima 1966; Teshima &
Kashiwada 1967; Limsuwan & Lovell 1981; Sugita
et al. 1989; Sugita, Miyajima & Deguchi 1991a; Sugita,
Takahashi, Miyajima & Deguchi 1991b). Among the
vitamins, the production of vitamin B12 by GI bacteria is well documented in ¢sh (Sugita et al. 1991a,b;
Sugita, Takahashi, Miyajima & Deguchi 1992). The
production of vitamin B12 di¡ers from species to
species and is correlated with the abundance of more
anaerobes as compared with aerobes in the GI tract.
Fish like O. nilotica produce more vitamin B12 as
compared with I. punctatus due to the presence of
more anaerobic bacteria in the gut of former ¢sh than

the latter (Sugita et al. 1990). Similarly, a signi¢cant
di¡erence in daily vitamin B12 synthesis in O. nilotica
(11.2 ng kg À 1 body weight) and I. punctatus
(1.4 ng kg À 1 body weight) has also been recorded by
Lovell and Limsuwan (1982).
In contrast to endothermic animals, the exact role
of gut microbiota in ¢sh nutrition is di⁄cult to conclude because of the complex and variable ecology of
the GI tract of ¢sh. Despite recent conventional and
gnotobiotic studies that indicate the possible involvement of GI bacteria in several physiological and
nutritional functions in ¢sh, more emphasis and/or
thorough research is required in order to establish
the nutritional importance of the gut microbiota.

Role of GI microbiota in disease
outbreak
The gut of an organism usually harbours a diverse
population of non-pathogenic, pathogenic and commensal bacteria, which can contribute signi¢cantly
to the overall health and disease outbreak in a host.
In a healthy animal, some microbiota are established
and others are transient in the intestine. There
occurs a proper balance between the endogenous
microbiota of the intestine and the host’s control
mechanism. However, if this balance is disturbed,
several pathogens present in the transient state can
establish lethal infections (Sekirov & Finlay 2009).

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The GI tract, as well as the skin and gills (Birkbeck &
RingÖ 2005), serves as a major route for entry of several pathogens to establish lethal infections (Sakai
1979; Chair, Dehasque, Vanpoucke, Nelis, Sorgeloos &
Be Leenheer 1994; Grisez, Chair, Sorgeloos & Ollevier
1996; Magarinos, Romalde, Noya, Barja & Toranzo
1996; Olsson, Joborn,Westerdahl, Blomberg, Kjelleberg
& Conway 1996; Romalde, Magarinos, Nunez, Barja &
Toranzo1996; RingÖ, Jutfelt, Kanapathippillai, Bakken,
Sundell, Glette, Mayhew, Myklebust & Olsen 2004).The
detailed bacterial translocation process and pathogenesis in the GI tract of ¢sh larvae and fry is a complicated process, which has been discussed in detail by
RingÖ, Myklebustd, Mayhew and Olsen (2007).
In aquaculture, the advent of intensive aquaculture practices led to the outbreak of diseases in all
forms of practices ranging from freshwater to marine
and warm water to cold water ¢sh (Nicolas, Robic &
Ansquer 1989; Youssef, E1-Timawy & Ahmed 1992;
Keskin, Keskin & Rosenrhal 1994; Press & Lillehaug
1995; Karunasagar & Karunasagar 1999). The GI
tract is believed to be the major route for the onset of
diseases like vibriosis, furunculosis, enteric septicaemia and aeromoniasis in ¢sh. A more recent in vitro
study based on the kinetics of the bacterial adhesion
to mucus also indicates that the GI tract is the one
portal of entry for pathogenicVibrio alginolyticus into
large yellow croaker Pseudosciaena crocea (Chen,Yan,
Wang, Zhuang & Wang 2008). Ransom, Lannan, Rohovec and Fryer (1984) reported Vibrio anguillarum
and Vibrio ordalii, pathogens in the pyloric caeca and
throughout the GI tract of naturally infected Paci¢c

salmon. Furthermore, pathogens after attachment
and/or colonization in the tract, can damage the intestinal lining by releasing extracellular enzymes
or toxins (RingÖ et al. 2004), and within a few hours,
establish a lethal infection in ¢sh. Pathogens like
Edwardsiella ictaluri and several other entero invasive
members of Enterobacteriaceae can infect within 25 h
by crossing the mucosal membrane in ¢sh (Baldwin
& Newton 1993).

Role of GI microbiota in fish health
management
For a very long time, it was believed that the activity of
intestinal microbiota in the host is correlated with
the longevity of host (Metchniko¡ 1901). In endothermic animals, the GI microbiota not only aids the digestive function but also acts as a protective barrier
against pathogens (Sissons 1989). These microbes in

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the gut can protect the host by depriving invading
pathogens of nutrients and secreting a range of antimicrobial substances. Fish often harbour a wide
range of bacteria in their intestine that have the ability to inhibit pathogens (Schroder, Clausen, Sandberg
& Raa 1980; Strom 1988; Onarheim & Raa 1990; Westerdahl, Olsson, Kjelleberg & Conway 1991; Olsson,
Westerdahl, Conway & Kjellberg 1992; Smith & Davey
1993; Westerdahl, Olsson, Conway & Kjellberg 1994;
Austin, Struckey, Robertson, E¡endi & Gri⁄th 1995;
Bergh 1995; Sugita, Shibuyu, Shimooka & Deguchi
1996; Joborn, Oisson, Westerdahl, Conway & Kjelleberg 1997; Sugita, Matsuo, Hirose, Iwato & Deguchi
1997; Olsson, Joborn, Westerdahl, Blomberg, Kjelleberg & Conway 1998; Sugita, Ishigaki, Iwai, Suzuki,

Okano, Matsuura, As¢e, Aono & Deguchi 1998;
Robertson, Dowd, Burrels, Williams & Austin 2000;
Sugita, Okano, Suzuki, Iwai, Mizukami, Akiyama &
Matsuura 2002).
Sugita, Hirose, Matsuo and Deguchi (1998) found
that 2.7% of GI bacteria of freshwater ¢sh inhibited different pathogens, while earlier, Sugita et al. (1996)
found 3.2% of GI bacterial isolates in freshwater ¢sh
to be e¡ective against Aeromonas. Similarly,Westerdahl
et al. (1991) recorded a signi¢cantly higher proportion
of GI bacteria (28%) of turbot (Scophthalmus maximus)
that could inhibit pathogens like Listonella anguillarum. A study in Senegalese sole (Solea senegalensis) indicates an increase in the percentage of antagonistic
bacteria in the gut once the larvae start to feed, and
after 6 weeks, almost 40% of the GI microbiota was
antagonistic against pathogens like L. anguillarum
and Photobacterium damselae (Makridis, Martins, Vercauteren, Van Driessche, Decamp & Dinis 2005). Besides, bacteria with a broad-spectrum inhibitory
activity, i.e. e¡ective against a wide range of pathogens,
are also found in the GI tract of ¢sh. Carnobacterium
species, a bacterium often isolated from the GI tract of
salmonids, is found to inhibit several pathogens like
Aeromonas hydrophila, Aeromonas salmonicida, Flavobacterium branchiophilum, P. damselae, V. anguillarum
and Streptococcus milleri (Robertson et al. 2000). Similar types of broad-spectrum antagonistic activity are
also exhibited by Weissella hellenica, a Gram-positive
GI LAB bacterium isolated from Japanese £ounder
(Paralichthys olivaceus) (Cai, Benno, Nakase & Oh1998).

Manipulation of GI microbiota in fish
Nowadays, considerable attention is being focused on
the manipulation of intestinal microbial composition

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and their activities through dietary supplementation
to improve the overall health status of the host organism. Health-promoting bene¢cial microorganisms
(probiotics), non-digestible substances that selectively stimulate the growth of one or limited healthpromoting bacteria in the intestine of the host
(prebiotics) and/or a combination of both (synbiotics)
are routinely supplemented in feed for better health
management in many animals including ¢sh/shell¢sh. Their roles in nutrition and growth, immunity,
intestinal balance and disease resistance in aquatic
animals have been reviewed extensively recently
(Kesarcodi-Watson, Kaspar, Lategan & Gibson 2008;
Merri¢eld, Dimitroglou, Foey, Davies, Baker, BÖgwald, Castex & RingÖ 2010; RingÖ, Olsen, Gifstad,
Dalmo, Amlund, Hemre & Bakke 2010).

Probiotics concept
Probiotics are bene¢cial microbes that help to improve
the overall health status of the host organism. The
term, probiotic, simply means ‘for life’, originating from
the Greek words ‘pro’ and ‘bios’ (Gismondo, Drago &
Lombardi 1999). The application of probiotics in aquaculture practices has already gained momentum, and
nowadays, numerous microorganisms, both from indigenous and exogenous sources, are used as probiotics (Gatesoupe 1999; Gomez-Gil, Rogue & Turnbull
2000; Ahilan, Shine & Santhanam 2004; Salinas,
Cuesta, Esteban & Meseguer 2005; Ghosh, Sinha &
Sahu 2007; Buntin, Chanthachum & Hongpattarakere
2008; Kesarcodi-Watson et al. 2008). The commonly
used probiotics in ¢sh culture practices belong to
Saccharomyces, Clostridium, Bacillus, Enteroccus,

Lactobacillus, Shewanella, Leuconostoc, Lactococcus,
Carnobacterium, Aeromonas and several other species.
In aquaculture, probiotics are administered by feed
and/or as a water additive. However, supplementation
of probiotics through feed is a better method for the
establishment and successful colonization of the
probiont in the GI tract of ¢sh (Gildberg, Mikkelsen,
Sandaker & RingÖ 1997; Joborn et al. 1997; Robertson
et al. 2000). On the other hand, the suspension or the
bioencapsulation method is best suitable for ¢sh
larvae (Gatesoupe 1991, 1993). Likewise, the co-supplementation of probiotics even at a low concentration with live carriers like rotifers is also found to
be e¡ective (Gatesoupe 1993). Gatesoupe (1993)
recorded better survival of the larvae of S. maximus
by feeding them with Bacillus-enriched rotifers.
However, the quantitative and qualitative properties

Role of gastrointestinal microbiota in ¢sh S K Nayak

of the bacterial biota in live food have to be adjusted
to avoid negative e¡ects in order to accomplish successful colonization in the intestinal tract of ¢sh larvae (Keskin et al.1994; Munro, Henderson, Barbour &
Birkbeck 1999). Nevertheless, certain probiotics,
when used as a water additive, can exert several bene¢cial e¡ects in the host. Increased survival and production of channel cat¢sh (I. punctatus) by Bacillus
species (Queiroz & Boyd 1998), improved growth and
immunity of O. niloticus by Bacillus subtilis and
Rhodopseudomonas palustris (Zhou, Tian, Wang & Li
2009), protection of O. mykiss against V. anguillarum
by Pseudomonas £uorescencs (Gram, Melchiorsen,
Sanggard, Hubber & Nielsen 1999) and other Pseudomonas species (Spanggaard, Huber, Nielsen, Sick, Pipper, Martinussen, Slierendrecht & Gram 2001) are
recorded by direct addition of these bacteria to water.
Probiotics can help increase growth by enhancing

the feed conversion e⁄ciency, and confer protection
against harmful bacteria by competitive exclusion,
production of organic acids (formic acid, acetic acid,
lactic acid), hydrogen peroxide and several other compounds such as antibiotics, bacteriocins, siderophores
and lysozymes (Austin et al. 1995; Sugita et al. 1996;
Gildberg et al. 1997; Gibson 1999; Gram et al. 1999).
Besides these, probiotics can also e¡ectively trigger
the piscine immune system as already described
elsewhere in the text.

Prebiotics concept
Prebiotics are usually non-digestible oligosaccharides used as food ingredients to enhance the composition of certain endogenous health-promoting
bacteria in the GI tract of the host (Gibson & Roberfroid 1995; Mussatto & Mancilha 2007). Prebiotics
help in generating speci¢c microbiota like Bi¢dobacter and Lactobacillus species in the host (Houdijk,
Bosch,Verstegen & Berenpas 1998; Torrecillas, Makol,
Caballero, Montero, Robaina, Real, Sweetman,Tort &
Izquierdo 2007; Costalos, Kapiki, Apostolou &
Papathoma 2008). Fructo-oligosaccharide, galactooligosaccharides, mannan-oligosaccharides (MOS),
xylo-oligosaccharides (XOS), inulin, lactulose and
lactosucrose are the common prebiotics that are
being used in di¡erent animals and humans (Teitelbaum & Walker 2002; White, Newman, Cromwell
& Lindemann 2002; Tuohy, Rouzaud, Bruck &
Gibson 2005).
Prebiotics are found to stimulate the growth of speci¢c intestinal bacteria in ¢sh (Mahious, Gatesoupe,

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Role of gastrointestinal microbiota in ¢sh S K Nayak

Hervi, Metailler & Ollevier 2006; Dimitroglou,
Merri¢eld, Moate, Davies, Spring, Sweetman & Bradley
2009). Furthermore, dietary supplementation of prebiotics like MOS leads to improved growth and immunity in C. carpio (Staykov, Denev & Spring 2005), O.
mykiss (Staykov, Spring, Denev & Sweetman 2007)
and D. labrax (Torrecillas et al. 2007), and immunity
and disease resistance in hybrid striped bass (Morone
chrysops  Morone saxatilis) (Li & Gatlin III 2004,
2005). Nevertheless, enhancement of digestive enzymes like protease and amylase in allogynogenetic
crucian carp (C. auratus gibelio) by supplementation
of XOS (Xu,Wang, Li & Lin 2009) and increase in the
thickness of intestinal tunica muscularis by lactosucrose in red sea bream (Pagrus major) (Kihara, Ohba &
Sakata1995) have also been recorded. However, there
are some concerns associated with the use of prebiotics in aquaculture practices. Several pathogens as
well as opportunistic bacteria can utilize a wide
range of carbohydrates and can eventually pose
health hazards by proliferating inside the gut by metabolizing the prebiotics (Gatesoupe 2005). Similarly,
another major concern for prebiotics is that high concentrations of prebiotics can be harmful as evidenced
from the damaging e¡ect of inulin at a high concentration on the enterocytes of Salvelinus alpinus (Olsen, Myklebust, Kryvi, Mayhew & RingÖ 2001).
The success of probiotics and prebiotics has led to
the concept of ‘synbiotics’, which refer to nutritional
supplements combining probiotics and prebiotics to
form a symbiotic relationship. The synbiotic combination of Enterococcus faecalis and MOS was found to
exhibit several bene¢ts such as better immune responses and survival against V. anguillarum in ¢sh
like O. mykiss (Rodriguez-Estrada, Satoh, Haga, Fushimi & Sweetman 2009). Similarly, positive synergistic
e¡ects with higher immune responses and disease
resistance by feeding a probiotic Bacillus strain
with isomaltooligosaccharides to shrimp were also

reported (Li, Tan & Mai 2009). However, the growth
enhancement and health improvement of ¢sh/shell¢sh by promoting the growth of certain microbes in
the GI tract through prebiotics and/or synbiotics is a
bene¢cial and rational strategy beyond any doubt but
their use in aquaculture is still in its infancy (ELDakar, Shalaby & Saoud 2007; Ho¡mann 2009).
Conclusion
Over the years, substantial literature has been accumulated, albeit sometimes con£icting, on the nature
of the GI bacterial communities in di¡erent ¢sh spe-

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Aquaculture Research, 2010, 41, 1553–1573

cies. Although successful application of molecular
techniques has shown considerable complexity of
the microbial ecosystem with several new bacteria
in the GI tract of ¢sh, unculturable bacteria are still
poorly characterized in a wide range of ¢sh species.
The conventional and gnotobiotic studies indicate
the involvement of GI microbiota of ¢sh in several important biological functions such as physiological,
nutritional and immunological processes. However,
it is necessary to establish the detailed mechanisms
that govern the dynamic microbial community of
gut and their e¡ects on each other as well as on the
host at the molecular level. Furthermore, molecularand genomic-based knowledge of the composition
and functions of the GI microbiota of ¢sh will
certainly help to develop strategies for better health
management through the manipulation of microbial
ecosystem of gut with suitable probiotics/prebiotics/
synbiotics.

Acknowledgment
The author is grateful to Professor T. Nakanishi,
Laboratory of Fish Pathology, Department of Veterinary Medicine, Nihon University, Japan, for his novel
motivation to write this article.

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doi:10.1111/j.1365-2109.2009.02453.x

New production strategy for silver perch (Bidyanus

bidyanus); over-wintering fingerlings in a tank-based
recirculating aquaculture system
David A Foley1,2, Stuart J Rowland1, Geo¡rey GlennWilson2, Paul Winters1, Mark Nixon1 & Charlie Mifsud1
1

NSW Department of Industry and Investment, Grafton Aquaculture Centre, Grafton, NSW, Australia

2

Cotton Catchment Communities Cooperative Research Centre, School of Environmental and Rural Science, University of

New England, Armidale, NSW, Australia
Correspondence: D A Foley, NSW Department of Industry and Investment, Grafton Aquaculture Centre, PMB 2, Grafton, NSW 2460,
Australia. E-mail:

Abstract

Introduction

Slow growth and losses to bird predation and infectious diseases in winter can compromise the pro¢tability of silver perch farming. To evaluate over-wintering
silver perch (Bidyanus bidyanus) in a recirculating
aquaculture system (RAS), ¢ngerlings (38 g) were
stocked in either cages in a pond at ambient temperatures (10^21 1C) or tanks in the RAS at elevated temperatures (19^25 1C) and cultured for 125 days. Mean
survival (96%), ¢nal weight (146 g), speci¢c growth
rate (1.07% day À 1) and production rate (28.1kg m À 3)
of ¢sh in the RAS were signi¢cantly higher than for
¢sh over-wintered in cages (77%, 73 g, 0.53% day À 1,
11.1kg m À 3). Fish from both treatments were then
reared in cages for a further 129 days. Final mean
weight of ¢sh originally over-wintered in the RAS was
426 g, while ¢sh over-wintered in cages were only
273 g. To determine optimal stocking densities, ¢ngerlings (11.8 g) were stocked at 500, 1000 or 1500 ¢sh
m À 3 in tanks in the RAS and cultured for 124 days.
Survival was not a¡ected, but growth was signi¢cantly slower and feed conversion ratio higher at
1500 ¢sh m À 3 compared with 500 or1000 ¢sh m À 3.
Results demonstrate that over-wintering silver perch
in an RAS can produce large ¢ngerlings for grow-out
in early spring. This strategy could eliminate bird
predation, reduce losses to diseases and shorten the
overall culture period.

The Australian native, warm water silver perch
(Bidyanus bidyanus Mitchell) is an excellent species
for intensive culture in earthen ponds, and an industry producing around 500 tonnes per annum has
developed in Australia (Rowland, Allan, Clark, Hollis
& Pontifex 1994; Rowland 1995a, 1998, 2009;

Rowland, Allan, Hollis & Pontifex 1995). Fish reach
market size in 16^24 months using a three-phase
production system involving hatchery, ¢ngerling
and grow-out phases (Rowland 1995b). Fingerlings
are routinely over-wintered in ponds at ambient
temperatures, but at water temperatures below 15 1C,
feeding activity and growth are reduced, immune
responses are suppressed and temperature ranges are
ideal for some virulent parasitic organisms (Rowland
1995b, 2009; Rowland, Landos, Callinan, Allan, Read,
Mifsud, Nixon, Boyd & Tully 2007). The pro¢tability of
some pond-based silver perch farms has been compromised by slow growth, and low survival due to bird
predation and outbreaks of the infectious diseases
ichthyophthiriosis and saprolegniosis during winter
(Rowland 2009).
Recent research has shown that cage culture has
potential for the production of ¢ngerlings and
market-sized silver perch (Rowland, Allan, Hollis &
Pontifex 2004; Rowland, Mifsud, Nixon & Boyd
2006). Cage culture has many advantages over pond
culture including reduced bird predation and more
e⁄cient feeding, grading, health management and
harvesting (Beveridge 1996). Cages could provide a
relatively low-cost and £exible form of ¢sh culture
that can be readily adapted to existing water bodies

Keywords: silver perch, cage culture, production
strategy, recirculating aquaculture system, integrated aquaculture, over-winter ¢ngerlings, stocking density

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Aquaculture Research, 2010, 41, 1574^1581

such as irrigation storages and channels (Beveridge
1996; Chua & Tech 2002). Opportunities exist for the
integration of silver perch culture with established
agricultural industries (Gooley, De Silva, Hone,
McKinnon & Ingram 2000; Rowland & Allan 2006;
Rowland et al. 2006). As a part of a project to evaluate
the potential for integration of aquaculture with
irrigation industries in Australia, research is being
carried out to determine optimal growth and
welfare conditions for the cage culture of silver perch
(Rowland & Allan 2006). However, there is a challenge to develop an e¡ective production strategy to
overcome the poor performance and survival at low
water temperatures, as well as possible low water
volumes in many irrigation storages during winter.
The use of tank-based, recirculating aquaculture systems (RAS) could improve survival and growth in
winter, and shorten the grow-out period by providing
advanced ¢ngerlings at the beginning of the growing
season in early spring (Rowland 1995b; Rowland,
O’Connor, Ogburn & Lyall 1998). Recirculating aquaculture systems could also provide an alternative culture technique to ponds and cages on irrigation
farms during the winter period.
There is much interest in tank culture in Australia,
particularly in RAS (Larkin 2000), and a grow-out
industry is developing based on the freshwater

species Murray cod (Maccullochella peelii peelii), barramundi (Lates calcarifer) and eels (Anguilla spp.)
(Ingram, De Silva & Gooley 2005; O’Sullivan &
Savage 2009). Recirculating aquaculture systems
require much less land and water than pond culture, water temperatures can be controlled and
facilities can be located close to markets (Masser,
Rakocy & Losordo 1999). However, RAS require high
capital investment, the technology is not well known
and many RAS around the world have not realized
their planned production or have failed due to
poor management and/or design (Masser et al. 1999;
Halachmi, Simon & Hallerman 2004; Timmons &
Ebeling 2007).
Species-speci¢c research is needed to determine if
silver perch survive and grow in RAS, and to identify
optimal growing conditions. Stocking density is of
fundamental importance in intensive aquaculture,
and has a signi¢cant e¡ect on the performance
of silver perch in ponds and cages (Rowland et al.
1994, 1995, 2004; Rowland et al. 2006; Rowland
1995a, b). In order to investigate the use of RAS as
an alternative over-wintering strategy for silver
perch, the e¡ects of stocking density in the RAS need
to be evaluated.

Over-wintering silver perch ¢ngerlings D A Foley et al.

The aims of this study were to (i) compare the
performance of silver perch ¢ngerlings cultured at
elevated water temperatures in RAS tanks to ¢ngerlings cultured in cages at ambient temperatures;
(ii) determine if any growth advantage in overwintered ¢ngerlings is maintained during the growout phase; and (iii) determine the e¡ects of stocking

density on the performance of ¢ngerlings in the RAS.

Materials and methods
General
Experiments were run at the NSW Department of
Industry and Investment’s Grafton Aquaculture
Centre (GAC).Water from the Clarence River (salinity
0^0.1g L À 1) was stored in a large earthen reservoir,
and ¢ltered to 80 mm using sand and cartridge ¢lters
before use in the RAS.Water quality monitoring, ¢sh
husbandry, ¢sh health and management of ponds
followed Rowland (1995b) and Rowland et al. (2007).
A Horiba U-10 meter (Horiba, Kyoto, Japan) was used
to monitor water temperature, dissolved oxygen
(DO) pH, conductivity and salinity. Total ammonia^
nitrogen was determined using Nessler’s reagent.
After harvest and before each stocking, ¢sh were
quarantined for 7 days in a 9000 L tank and treated
continuously with NaCl at 5 g L À 1 to ensure that they
were free of ectoparasites, to prevent fungal infection
and to reduce stress (Selosse & Rowland 1990;
Mifsud & Rowland 2008). Fish were then anaesthetized
using 20 mg L À 1 benzocaine (ethyl-r-aminobenzoate;
Sigma-Aldrich, Shanghai, China), randomly selected,
counted and stocked at assigned densities into 1m3
cages or tanks. Cages were made from 12 to 44 mm
stretched mesh-sized knotless mesh and were placed
in £oating pontoons attached to a ¢xed walkway in a
0.32 ha earthen pond that was 2 m deep. The pond
was aerated using two 1hp paddlewheel aerators for

a least 11h day À 1 between 21:00 and 08:00 hours
and the aerators were positioned to ensure that an
even current £owed through each cage.

RAS
The RAS had a total water volume of 16 000 L. There
were 12 black, circular, ¢breglass tanks, each of 1m3
capacity, with central drainage. Central 50 mm
standpipes allowed the removal of water and wastes,
and prevented ¢sh from escaping. Water entered
the tanks through submerged manifolds at £ow rates
of 10^15 L min À 1. The tanks were subjected to a

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Over-wintering silver perch ¢ngerlings D A Foley et al.

natural photoperiod and were aerated continuously
with di¡used air, via air-stones centrally located in
each tank. E¥uent water from each rearing tank
£owed into two, 500 L swirl separators where larger
solids were removed. A 50 L drum ¢lter then removed
suspended solids 480 mm before the water entered a
1000 L sump. A 60 L foam fractionator between the
sump and the main water line removed ¢ne and
dissolved solids. Water was pumped to a mezzanine

level via a UV sterilization unit. An 11kW heater^
chiller unit (Accent, Chipping Norton, NSW, Australia) was used to elevate water temperature during
winter. The bio-¢lter consisted of a 1000-L tank
with a rotating arm that sprayed water over 1m3 of
‘ovi-£ow’ bio-balls, which had a speci¢c surface area
of 402 m2 m À 3. Water then £owed into a 2000-L
header tank before returning to the rearing tanks by
gravity. Salinity was maintained at 2^3 g L À 1 NaCl to
prevent nitrite poisoning (Losordo, Masser & Rakocy
1998). Total alkalinity and pH were maintained by the
daily addition of 0.8^1.0 kg of sodium bi-carbonate.
A 110 kVA generator provided back-up power to
the RAS.

Fingerlings for experiments
To provide ¢ngerlings with a common culture history
before over-wintering, weaned silver perch fry (mean
weight 2.3 g) were stocked at a density of 500 ¢sh m À 3 into 12 cages of 12 mm stretched mesh and cultured for 99 days. When pond water temperatures
dropped below 20 1C in autumn, ¢ngerlings were harvested, weighed, counted and quarantined for 7 days.

Experiment 1: over-wintering in cages and
RAS
Fingerlings (mean weight 38 g) were stocked at a density of 200 ¢sh m À 3 into either 1m3 cages (19 mm
mesh) in a pond or1m3 tanks in the RAS and cultured
for125 days. There were eight replicate cages and eight
tanks. Fifty ¢sh from each cage or tank were sampled
each month, weighed and the mean weights and
biomasses were estimated. Daily feed rations, based
on mean body weight, were adjusted accordingly.
Fingerlings (o100 g) in cages were fed a commercial

¢ngerling diet (2^3 mm pellet size) containing 52%
protein, 17 MJ kg À 1 energy and 12% lipid (Ridley
Aqua-Feed, Narangba, Qld, Australia). Fingerlings in
the RAS were fed the same ¢ngerling diet, but when
their mean weight exceeded 100 g, they were then fed

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Aquaculture Research, 2010, 41, 1574^1581

a larger pellet (4 mm) commercial grow-out diet with
45% protein, 17 MJ kg À 1 energy and 10% lipid (Ridley
Aqua-Feed). Fish were fed a restricted ration according
to recommendations by Rowland, Allan, Mifsud,
Nixon, Boyd and Glendenning (2005). Water quality
variables including temperature were recorded twice
daily,5 days week À 1. After125 days, when pond water
temperatures rose above 20 1C, ¢sh were harvested
from cages and the RAS and the survival rate, mean
weight, speci¢c growth rate [SGR 5 ln ¢nal weight
(g) À ln initial weight (g)/days  100; % day À 1],
absolute growth rate (AGR 5 g ¢sh À 1 day À 1), production rate (kg m À 3) and feed conversion ratio
(FCR 5weight of feed/biomass gain) were determined
for each replicate and treatment.

Experiment 2: on-growing in cages after
winter
Fingerlings (mean weight 85 g) that had been overwintered in cages were randomly selected and stocked
into 1m3 cages (19 mm mesh) at a density of 100 ¢sh m À 3, and ¢sh over-wintered in the RAS (mean weight
165 g) were randomly selected and stocked into 1m3

cages (44 mm mesh) at a density of 100 ¢sh m À 3.
There were six replicate cages for each treatment. Fingerlings that had been over-wintered in cages were fed
the ¢ngerling diet as in Experiment 1, until mean
weights exceeded 100 g, after which the commercial
grow-out diet described in Experiment1was used. Fingerlings over-wintered in the RAS remained on the
commercial grow-out diet. Fish were fed a restricted
ration, as recommended by Rowland et al. (2005),
5 days week À 1. Fish were sampled monthly, weighed,
and the mean weights and biomasses were estimated
and the daily feed rations were adjusted accordingly.
Fish were harvested after 129 days and performance
parameters were determined as for Experiment 1.

Experiment 3: stocking density in RAS
Silver perch ¢ngerlings (11.8 g) were stocked into
RAS tanks at densities of 500,1000 or1500 ¢sh m À 3.
There were three replicate tanks for each density.
Fish were fed the ¢ngerling diet (Experiment 1) to
satiation three times each day (08:30, 12:30 and 16:30
hours), 5 days week À 1. Management of RAS water
quality including pH and ammonia was as for Experiment 1. Water quality variables, including temperature and DO, were recorded at 09:00 and 14:30 daily,
5 days week À 1. A random sample, equal to 10% of

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