1. Introduction

In honey bees, the gut is the primary location for digestion and food processing, as well as the site of infection for a variety of pathogens, including Paenibacillus larvae (de Graaf et al., 2013), Ascosphaera apis  (Jensen et al., 2013) Nosema ceranae (Fries et al., 2013), and probably many of the honey bee viruses (de Miranda et al., 2013). Biologists have increasingly recognized that gut microorganisms play a beneficial role in many aspects of the health of animals, animals as diverse as mammals and insects. The role of symbiotic gut microbes in digestion, resistance to infectious disease, and the general health of honey bees, both individual bees and the colony at large, is an intriguing area of research where much is still to be learned.  In this chapter we present a number of basic protocols for investigating the microbial communities found in honey bee guts.

Traditional microbiological studies, from the time of Pasteur, have relied on the axenic (pure) isolation of individual microbes, which were then characterized based on their metabolic, biochemical, and morphological characteristics. These culture-based studies remain the foundation of microbiology. However, for most environmental samples, estimates of microbial density based on microscopic counts of cell numbers tend to be far higher than estimates based on colony-forming units on culture plates (Staley and Konopka, 1985). Furthermore, it has long been known that many microbes cannot be cultured in the laboratory, or require specialized conditions yet to be discovered, but no real solution to this problem existed for environmental microbiology until molecular sequencing technology became available. Using sequencing techniques, Rappé and Giovannoni (2003) show that the microorganisms readily cultured from a given environment are only a tiny subset of the species actually living there. Typically, only about 1% of bacteria from a given habitat will grow in culture (Staley and Konopka, 1985). With attention to specific aspects of the culture media and atmospheric conditions, more organisms might grow, but consistently, many organisms sampled from most environments do not appear in lab cultures (Stevenson et al., 2004). Therefore, good estimates of "what is really there" in microbiology depend on non-culture-based studies.  Non-culture-based studies may be followed by culturing efforts so that the microbes of interest can be isolated, and thus better described chemically and morphologically, and to conduct experiments with them. In addition, molecular methods such as fluorescent in situ hybridization (FISH) can be used to locate specific microorganisms in their precise locations within a sample; for example, FISH can be used to identify where certain microbes occur in the honey bee gut, or where infection takes place for a pathogen. 

Numerous researchers have performed studies on organisms cultured from bee guts and the hive, documenting a variety of metabolic and functional activities (Gilliam and Prest, 1972, 1987; Gilliam and Valentine, 1974; Gilliam et al., 1974; Gilliam, 1978; Evans and Armstrong, 2006). However, non-culture-based surveys have presented a contrasting view of the dominant members of the honey bee gut microbiota, revealing that readily cultured, fully aerobic organisms comprise only a small portion of the diversity of microbes present. A set of eight major taxa dominate the honey bee gut environment, and these fall within the Gammaproteobacteria, Betaproteobacteria, Alphaproteobacteria, Lactobacillales, and Actinomycetes.  These eight bacterial taxa, which correspond to the typical definition of bacterial species, have been found in A. mellifera worldwide (Jeyaprakash et al., 2003; Mohr and Tebbe, 2006; Babendreier et al., 2007; Cox-Foster et al., 2007; Martinson et al., 2011; Cornman et al., 2012; Engel et al., 2012; Li et al., 2012; Martinson et al., 2012; Moran et al., 2012; Sabree et al., 2012; Tian et al., 2012). Close relatives of some of these taxa have been found in other Apis species in Asia (Ahn et al., 2012; Li et al., 2012) and in many species of bumble bees (Bombus) (Koch and Schmid-Hempel, 2011, 2012; Koch et al., 2012, 2013). These surveys used a variety of sequencing methodologies, yet consistently retrieved a similar set of organisms.

The primary molecule currently used for surveying microbial diversity and verifying taxonomic identities is the small subunit ribosomal RNA, which is referred to as the 16S rRNA in Bacteria and Archaea, and the 18S rRNA in Eukaryota. This molecule is present in all cells and provides a molecular label for a particular species or taxon, and can be compared against public databases to determine whether a sampled sequence corresponds to previously studied organisms (McDonald et al., 2012). The microbial community in a bee gut can be determined by extracting the DNA, using targeted PCR to amplify the bacterial rRNA genes present, followed by high throughput sequencing technologies such as Illumina, 454 or others (Sogin et al., 2006; Tringe and Hugenholtz, 2008), often called next-generation sequencing (NGS) technologies. Bioinformatic searches are then used to compare the resulting sequences to those previously identified and stored in publically available databases.  We present protocols for these methods.

Sequencing technologies are becoming increasingly cost-effective while also yielding improved data quality, primarily through an increase in the length of the sequencing read. Longer and more accurate sequences give higher quality information for identifying microbial taxa. Because these technologies are evolving rapidly, we give a generalized overview for the methods, recognizing that details of techniques will change as these technologies, and the methods for analysing the results, evolve. Our recommendations are for specific techniques that are likely to remain static for some time, such as DNA extraction, PCR, and a general approach for analysing microbial communities.

The 16S rRNA can also be used to design in situ hybridization probes.  Many ribosomes are present in the cytoplasm of each cell, and probes corresponding to diagnostic regions of the rRNA gene can be used to selectively label and visualize specific cells containing the corresponding RNA sequences. In addition, the 16S rRNA can be used to identify bacteria isolated using culture-based methods. DNA can be extracted from isolated colonies of the bacteria, amplified using selective PCR, followed by sequencing. Again, a bioinformatic search can then be used to compare the unknown sequence to those previously identified and stored in publically available databases.

Metagenomics includes several approaches useful for studying functions of gut biota, but is not included among our listed protocols. The original use of the word "metagenomics" referred to cloning relatively large fragments of DNA from community DNA samples, and then attempting to screen these cloned fragments for functional activities (Handelsman, 2004). A primary limitation of this approach is that genes underlying many functional activities maybe present but not expressed, although metagenomics can be an effective method for detecting certain functions, such as antibiotic resistance (Tian et al., 2012). Since major members of the bee microbiota can be cultured, the study of cultured isolates may offer a more robust approach for finding functional capabilities. Currently, metagenomics generally refers to the use of deep sequencing of total genomic contents of a microbial community to identify and compare the prevalence of functional genes, such as those for enzymes associated with cellulose or pectin degradation (e.g. Warnecke et al., 2007; Brulc et al., 2009; Engel et al., 2012).  Typically, metagenomic sequencing is combined with a 16S rRNA gene sequencing approach, with the former being used for inferring functional capabilities, and the latter used to infer community membership and diversity. The study by Engel et al. (2012) is the only example to date of using deep sequencing metagenomics to understand functions of the bee gut microbiota. Metagenomic methods encompass rapidly evolving approaches to environmental microbiology. Since standard methodologies are still under development at this time, these were not included here.

We provide here standard protocols for studying bee gut bacteria, including NGS methods for surveying bacterial diversity, FISH microscopy to precisely locate where these microorganisms occur in the insect gut, and culturing methods for known gut symbionts. Although we focus on bacteria, our methods can be extended to other microorganisms and viruses, with appropriate changes in the oligonucleotide primers used for diversity surveys and FISH.