ReviewA new view of the fish gut microbiome: Advances from next-generation sequencing
Introduction
In-depth knowledge of community membership as well as the structure and relationships between resident microbes (microbiota) and their hosts can provide insight into both the function and dysfunction of the host organism. Comprising a large diversity of approximately 28,000 species, fish make up nearly half of all vertebrate species and exhibit a wide variety of physiologies, ecologies and natural histories (Wong and Rawls, 2012). Thus, fish represent an important group for understanding the variety and nature of symbioses in vertebrate gut microbial communities (Nayak, 2010). The digestive tract of fish receives water and food that are populated with microorganisms from the surrounding environment; these microbes undoubtedly affect the resident microbiota. Correlations between changes in the composition and activity of the fish gut microbiota with fish physiology and disease have been proposed, increasing the scientific community's interest in this field of research. From this perspective, a comprehensive and detailed view of the fish gut microbiota, including phylogenetic composition as well as the genetic and metabolic potential, is essential to understand the dynamics and possible mechanisms of the cause/effect relationships between gut microbiota and physiology (Austin, 2006, Austin, 2011). As numerous studies have indicated that culture-dependent techniques possess dubious sensitivity and often detect only a limited fraction of microbial communities (Austin, 2006, Kim et al., 2007, Namba et al., 2007, Wu et al., 2010, Wu et al., 2012a, Lan and Love, 2012, Larsen et al., 2013), several methods for culture-independent microbial analysis have been developed within the past few decades (Head et al., 1998, Muyzer and Smalla, 1998) and applied in studies of the fish gut. Molecular methods provide faster results and novel, high-resolution insights into the structure and diversity of microbial communities within the digestive tracts of freshwater and marine fish (Austin, 2006, Kim et al., 2007, Namba et al., 2007, Wu et al., 2010, Wu et al., 2012a, Lan and Love, 2012, Larsen et al., 2013).
Despite these recent findings, the cost, technical difficulties and low coverage associated with Sanger sequencing have limited the ability to analyse a large number of samples. Recently, however, rapid and low-cost approaches for next-generation sequencing (NGS) technologies have been introduced to study the composition and genetic potential of densely populated microbial communities such as gut microbiota (Foster et al., 2012). Within the past few years, these techniques have been applied to analyse the composition and functional properties of fish microbial communities. Fish microbiota studies have most frequently utilised the 454/Roche pyrosequencing (e.g., the Roche 454 FLX Titanium and FLX +) and Illumina technologies (e.g., the Illumina MiSeq and HiSeq 2000). Unlike the Sanger sequencing approach, these NGS platforms provide a larger number of reads in a single run, enabling the rapid and cost-effective acquisition of in-depth and accurate sequence data and allowing for the detection of both dominant and low-abundance (rare) microbial community members (Roeselers et al., 2011, Wu et al., 2012b, Star et al., 2013, Wong et al., 2013). With the emergence and availability of NGS techniques for studying complex microbial ecosystems and the growing appreciation of the importance of the indigenous microbiota of fish, this review highlights the potential of NGS platforms for the analysis of fish gut microbial ecology.
Section snippets
NGS platforms and technologies
Although capillary sequencers such as the ABI 3730 (Applied Biosystems, USA) continue to be used for small-scale sequencing (Metzker, 2010, Zhou et al., 2010) in fish microbiota studies, there is an increasing trend towards the use of NGS platforms. While there are several different NGS technologies, one benefit common to all of them is the ability to rapidly generate large volumes of sequencing data in parallel. Table 1 compares the main characteristics of NGS sequencers to emphasise the
Caveats and limitations of NGS approaches
As with any other method, NGS techniques also have their limitations. Most of these drawbacks, such as the effect of DNA extraction and primer selection on the interpretation of bacterial diversity data, are common to other culture-independent molecular methods and have been reviewed elsewhere (Kuczynski et al., 2012). Some limitations inherent to the NGS approaches include the short reads, which pose difficulties when assembling and mapping to reference sequences, particularly at repetitive
Application of NGS technologies in recent studies of fish gut-associated microbiota
One approach to studying animal behaviour involves the examination of gut microbial communities. Many studies on humans, mice, chickens and termites have successfully correlated gut microbial communities with host physiology, nutrition and growth (Ye et al., 2014). It has been well documented that in aquatic animals, the gut microbiota is responsible for the digestion of algal cells, the production of amino acids and the secretion of inhibitory compounds that protect against colonisation of the
Future lines of research: Moving from phylogenetic characterisation to functional metagenomics
Although studies applying NGS techniques to provide greater in-depth knowledge of the fish gut microbiota are continually increasing in number, there are still some issues that warrant further examination. For example, one overriding problem is whether it is possible to differentiate members of the indigenous (fish) microbiota from transients that could be present in the water film around the fish or in the water/food within the digestive tract. How can NGS methods be used to distinguish these
Conclusions
Characterisation of the diverse microbial communities that are ubiquitous in any environment represents a significant approach towards a better understanding of the factors that determine the composition of the microbiota and their respective roles in the ecosystem. The role of microorganisms in the ecosystem is also of great relevance for developing effective strategies to manage and manipulate microbial communities. This review provides information regarding how new nucleic acid-based
Conflict of interest statement
The authors have no conflict of interest to declare.
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