Agriculture Reference
In-Depth Information
at the mucosal brush border. In a limited number of recent studies, next-generation sequencing
(NGS) approaches have been used to generate extensive libraries to extend our knowledge
of the 'rare biosphere'. This review provides an overview of the techniques which have
been used in evaluations of fish gut microbiota, the techniques with most relevance to future
studies, and discusses the benefits and drawbacks of each approach with reference to the
microbial populations present in the GI tract of fish.
5.1 CULTURE-DEPENDENT APPROACHES
Historically the general approach to study the microbiota of the gastrointestinal (GI) tract of
fish has been the use of conventional culture based methods (Cahill 1990; Ringø and Gate-
soupe 1998). This consists of sampling gut material and spreading gut homogenates on either
selective or general purpose agar. The incubation temperature used should (although this has
not always been the case) reflect the host's environmental conditions and thus the incuba-
tion duration should be longer when assessing the gut microbiota of cold water fish species
where the growth rates of the indigenous gut microbes are likely to be slower. For example,
some studies have utilized incubation periods of up to 4 weeks at 12
∘
C when assessing the
gut microbiota of Atlantic cod (
Gadus morhua
L.) (Ringø
et al
. 2006a) and Atlantic salmon
(
Salmo salar
L.) (Ringø
et al
. 2006b; 2008). Common incubation conditions for rainbow trout
(
Oncorhynchus mykiss
) intestinal communities on general purpose media are 15-20
∘
Cfor
7-14 days (Spanggaard
et al
. 2000; Huber
et al
. 2004; Pond
et al
. 2006; Merrifield
et al
.
2009a; 2009b; Navarrete
et al
. 2010). In contrast, higher temperatures and shorter incuba-
tion periods are appropriate for warm water fish species; for example, 25
∘
C for 1-7 days in
cyprinids (Al-Harbi and Uddin 2008; Sugita
et al
. 2009; Kühlwein
et al
. 2013) and 28-30
∘
C
for 1-7 days in tilapia (
Oreochromis
spp.) (Al-Harbi and Uddin 2003; 2004; 2005; Ferguson
et al
. 2010; Standen
et al
. 2013). It should be noted of course that incubation periods, but not
temperatures, should be adjusted when using selective media, as appropriate for the bacterial
group(s) being investigated. This is commonly the case for the isolation of lactic acid bacteria
(LAB) from cold water fish where it is often necessary to incubate for longer periods than
required for general purpose media (e.g. Pond
et al
. 2006).
After an appropriate incubation period colonies are counted and a representative num-
ber of colonies are re-streaked for isolation. Traditionally, in older studies, isolates would be
identified by biochemical assays and phenotypic observations, but identification using these
techniques lacks sufficient discrimination power and in the last decade isolate identification has
been undertaken using molecular methods such as 16S rRNA sequencing (Hagi
et al
. 2004;
Ringø
et al
. 2006a; 2006b; 2008; Merrifield
et al
. 2009a). In addition, randomly amplified
polymorphic DNA (RAPD) analysis has been used to identify selected groups such as probi-
otics or LAB (Balcázar
et al
. 2007a; 2007b; 2009; Vendrell
et al
. 2008; Avella
et al
. 2010).
Although there are numerous studies using a cultivation based approach, most of them
are limited to using one or two types of media, typically general purpose; few studies have
used several media within one investigation. Rare examples include Mudarris and Austin
(1988) who used 14 media, Schulze
et al
. (2006) who used seven general and two selective
media, and Yazawa
et al
. (1988) who used 11 different types of media. A number of early
reviews have presented information based on the bacterial populations in the fish GI tract from
culture-dependent observations (Cahill 1990; Ringø and Birckbeck 1999;
Chapter 4
).
However, as is often the case with microbial communities from environmental samples,
the gut microbiota of fish has been reported to be of low cultivability (Table 5.1). Although
Search WWH ::
Custom Search