Abstract

For historical reasons, much of the information available on the intestinal microbiota of fish is based on the use of conventional culture-dependent methods. This has consisted of sampling gut material and spreading gut homogenates on selective or general purpose agar, followed by incubation, colony counting and subsequent identification, typically by phenotypic/biochemical tests or during the last decade 16S rRNA sequencing. As is often the case with microbial communities from environmental samples, the gut microbiota of fish has been reported to be of low cultivability: cultivability using general purpose culture media has been reported to represent <0.1% of the total microbial community in the gastrointestinal (GI) tract of some fish species. A wide range of molecular ecology techniques are available based on the sequence variability of the 16S and 23S rRNA genes, and over the last 10-12 years such approaches have become more commonly used to investigate the gut microbiomes of fish species. The molecular based approaches used have depended on the aim of the studies: (1) clone libraries have been used to identify the microbiota composition; (2) fingerprinting methods such as denaturing gradient gel electrophoresis (DGGE) and temporal temperature gradient electrophoresis (TTGE) have been used to analyse microbial community structure and diversity; (3) quantitative real-time PCR (qPCR) or fluorescent in situ hybridization (FISH) have been used to determine the abundance of particular taxa or total microbial levels; and (4) FISH and immunohistochemistry have been used to assess bacterial-host interactions 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.