the gut microbiota of fish

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The Gut Microbiota of Fish
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DOI: 10.1002/9781118897263.ch4
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4 The Gut Microbiota of Fish
Jaime Romero1, Einar Ringø2and Daniel L. Merrifield3
1Instituto de Nutrición y Tecnología de los Alimentos (INTA), Universidad de Chile,
Santiago, Chile
2Norwegian College of Fishery Science, UiT The Arctic University of Norway,
Tromsø, Norway
3School of Biological Sciences, Plymouth University, UK
ABSTRACT
Animals harbour a complex microbial community, consisting of bacteria, yeast, viruses,
archaeans and protozoans, in their gastrointestinal (GI) tract. These microbes inuence
various host functions including development, digestion, nutrition, disease resistance and
immunity. One important aim of GI microbiota studies therefore is to give a scientic basis
for developing effective strategies for manipulating GI microbial communities to promote
the host health and improve productivity. This chapter reviews the current knowledge on
the microbiota composition in several sh species, emphasizing the compilation of results
reported regarding the most frequently observed bacterial genera and phyla in marine and
freshwater species. This also includes descriptions of the microbiota in early stages of
development, the inuence of environmental and host factors on the establishment of the
bacterial populations that become part of the gut microbiota, and the importance of these
microbial communities on host health, development and nutrition.
4.1 INTRODUCTION
In the classic description, the complex community of microorganisms inhabiting body sites in
which surfaces and cavities are open to the environment is termed the microbiota; previously
this was called the microora or microbial biota. Moreover, the epithelial surfaces of sh and
all other vertebrates are colonized at birth by large numbers of microorganisms (microbiota)
that form commensal or mutual relationships with their hosts (Spor et al. 2011). The majority
of these microbes reside in the digestive tract, where they inuence a broad range of host
biological processes. The vertebrate gut harbours a coevolved consortium of microbes that play
critical roles in the development and health of this organ. This microbial community can be
subcategorized into two major groups. One group simply passes through the lumen with food
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Aquaculture Nutrition: Gut Health, Probiotics and Prebiotics, First Edition. Edited by Daniel Merrield and Einar Ringø.
© 2014 John Wiley & Sons, Ltd. Published 2014 by John Wiley & Sons, Ltd.

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76 Aquaculture Nutrition: Gut Health, Probiotics and Prebiotics
Fig. 4.1 Scanning electron micrograph of the anterior intestinal mucosa of rainbow trout; a pair of
autochthonous bacterial rods is present in close association with the mucosal brush border. Scale bar =
2μm. (Source: Merrifield et al. 2009. Reproduced with permission of John Wiley & Sons.)
or digesta (the allochthonous microbiota), whereas the other group is potentially resident and
intimately associated with host tissues (the autochthonous microbiota; Figure 4.1) (Ringø and
Birkbeck 1999). The normal microbiota has also been dened as the community of microbes
present in most individuals of a population or a species that, despite continual contact with
different tissues, cause no harm to the host (Berg 1996).
In previous investigations to study the microbiota of the GI tract of shes, the general
approach has been the use of conventional culture based methods (Cahill 1990; Ringø and
Birkbeck 1999). However, it has been reported that these methods present several disadvan-
tages since the number and species of bacteria detected are affected mainly by the culture
conditions and the culture media used, particularly certain fastidious and obligate anaerobes
(Spanggaard et al. 2000). These conventional methods are time consuming and lack accuracy
in isolate identication. Early in the 1990s, Cahill (1990) reviewed the current knowledge
concerning the bacterial communities in shes, at that time mostly based on culture-dependent
observations. The description provided in that review was mainly based on biochemical identi-
cation of the microorganisms, which has restricted discrimination power and may lack proper
denitions of relationships between aquatic-environmental microorganisms and sh micro-
biota. The lack of cultivability of the majority of the indigenous bacteria in many aquatic
environments, including the GI tract of aquatic animals, is becoming increasingly apparent
(Amann et al. 1995; as discussed in Chapter 5). For example, in Atlantic salmon (Salmo
salar L.), coho salmon (Oncorhynchus kisutch) or yellowtail (Seriola lalandi), cultivable bac-
teria (using tryptic soy agar (TSA) incubated at 10 days at 17 ∘C) represent ≤1% of the
total bacteria (Romero and Navarrete 2006; Navarrete et al. 2009; Aguilera et al. 2013). To

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The Gut Microbiota of Fish 77
study such environmental samples, several culture-independent molecular techniques have
been developed. These methods have allowed the identication of microorganisms without iso-
lation and the determination of the phylogenetic afliation of community members, revealing
the enormous extent of microbial diversity. The analysis of DNA extracted directly from a com-
plex environmental sample provides a powerful and relatively bias-free alternative approach
towards characterizing a microbial community (Nayak 2010). Typically, fragments of 16S or
18S ribosomal genes are selectively amplied by PCR to provide information on prokaryotic
and eukaryotic communities, respectively (Navarrete et al. 2010a). Patterns of diversity and
relative abundance of amplied DNA fragments can then be assayed using several strategies
(see Chapter 5). Massive sequencing technologies have revolutionized this eld by allowing
direct sequencing of millions of DNA molecules from a single sample (Qin et al. 2010). It is
thus now possible to obtain unbiased qualitative and quantitative reconstructions of complete
microbial communities – including both cultivable and uncultivable representatives – within
reasonable time frames and at affordable cost. Molecular techniques have been successfully
applied in dozens of studies proling the GI microbial communities of sh. Several attempts
have been made to describe the microbiota in a number of important aquacultured sh species.
Molecular methods based on PCR amplication of DNA extracted from frozen samples have
typically been the favoured approach and have proven to be efcient in studying the GI bac-
terial community of shes (Grifths et al. 2001; Jensen et al. 2004; Romero and Navarrete
2006; Hovda et al. 2007; Kim et al. 2007). Recently, studies have begun to analyse the sh
gut microbiota using massive sequencing strategies (e.g. van Kessel et al. 2011; Roeselers
et al. 2011; Desai et al. 2012; Wu et al. 2012). It is anticipated that as this technology becomes
more accessible it will signicantly improve our knowledge of the sh gut microbiota, enabling
identication of the rare biosphere and community metabolic pathways.
The ultimate goal of these studies is to provide a scientic basis for developing effective
strategies for manipulating gut microbial communities to promote animal health and improve
productivity. To achieve this goal, the principles governing microbiota composition (assembly)
and maintenance within the intestine must be understood. The vast majority of these studies
have focused on the bacterial communities and to a lesser extent yeast; very little information
is available for the viral, archaean and protozoan populations in the GI tract of sh.
4.1.1 Current knowledge of the gut microbiota in fish
Our current knowledge of the microbiota composition is derived from a compilation of
information in numerous reports; most of them focused on farmed sh, and among these the
salmonids have received much attention. Figure 4.2 summarizes the most commonly reported
bacterial phyla in salmonids, based on the review of Nayak (2010). Proteobacteria and
Firmicutes are the most frequently reported phyla in the salmonid gut microbiota, suggesting
that members of these bacterial classes are especially well adapted to conditions in the sh
intestine or their surrounding aquatic environment. Interestingly, studies in salmonids show
that some particular bacterial genera can be predominant in the microbiota composition; for
example, Pseudomonas can represent more than 60% of the community when ribosomal
amplicons are cloned and sequenced (Navarrete et al. 2009). The dominance of a particular
bacterial group has been observed in salmonid guts using similar culture-independent meth-
ods. Holben et al. (2002) reported that some genera were highly abundant in reared Atlantic
salmon from two different locations: in a Scottish hatchery, Mycoplasma corresponded to
81% of clones retrieved, whereas in a Norwegian hatchery, Acinetobacter accounted for 55%.

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78 Aquaculture Nutrition: Gut Health, Probiotics and Prebiotics
0246
Number of reports
Phylum reported in salmonids
Proteobacteria
Firmicutes
Actinobacteria
Bacteroidetes
Fusobacteria
Tenericutes
Deinococcus-Thermus
810 12
Fig. 4.2 Bacterial phyla observed in the gut microbiota of salmonids. (Source: Nayak 2010.
Reproduced with permission of John Wiley & Sons.) For colour detail see Plate 9.
Although other genera were also present, their abundance was closer to 2%. Interestingly, in
wild salmon (entirely carnivorous), the abundance of Mycoplasma was 96% of the clones
analysed. Similarly, Pond et al. (2006) described the intestinal microbiota of rainbow trout
(Oncorhynchus mykiss Walbaum) by using a cloning approach. They reported only two major
groups among 200 clones analysed, which corresponded to Clostridium and Aeromonas.
Furthermore, Kim et al. (2007) reported that Clostridium dominated the gut microbiota in
rainbow trout analysed by denaturing gradient gel electrophoresis (DGGE). The carnivorous
diet of salmon may explain in part the low number of taxa observed, since a recent study
indicated that diet inuences the bacterial diversity of the digestive tract. In this report,
a more comprehensive analysis of vertebrate gut microbiota (albeit mostly mammalian)
indicates that bacterial diversity increases from carnivore to omnivore to herbivore (Ley
et al. 2008). This has recently been observed in Antarctic sh, among which the omnivorous
Notothenia coriiceps (yellowbelly rockcod or bullhead notothen) exhibits greater diversity
than the exclusively carnivorous Chaenocephalus aceratus (blackn or Scotia Arc icesh)
(Ward et al. 2009). This may indicate that increasing herbivory in sh leads to gut microbiota
diversication, as observed in mammals.
Descriptions of microbiota from wild sh have also been reported, especially in herbivorous
sh and also in some habitats of ecological interest. Recently, the microbiota of three sh from
a coral reef was reported (Smriga et al. 2010). The studied sh corresponded to different diets
representing two trophic levels: Chlorurus sordidus (parrotsh) is a herbivore that consumes
primarily endolithic and epilithic algae; Lutjanus bohar (two-spot red snapper) is a top preda-
tor that consumes shes and crustaceans; and Acanthurus nigricans (whitecheek surgeonsh)