**3. Methods for investigating biodiversity of the yeasts from GIT**

From the cited references it is obvious, the biodiversity studies depend very much on the applied method. However, this is beyond the scope of this chapter to provide very detailed description of all possible methods that could be used for studies on yeasts' diversity. Nor calculation of the different biodiversity indices is in the focus of the paragraph. This paragraph is meant to provide short discussion on the existing possibilities, their limitations and advantages, and provide the reader with some input for consideration which methods he or she would choose for his/her studies.

Any application of either method mentioned below requires correct sampling of the material. Studying the biodiversity of the yeasts harbouring the GIT the dominating yeasts are in focus of most studies, as well as their abundance and changes of the abundance in time and in relation to the diet. For these purposes faecal or digesta samples have been collected from large animals (Urubschurov et al., 2008; 2011) or whole intestines from e.g. insects have been dissected (Suh et al., 2004b; 2005a; Nguyen et al., 2007). Whereas rain worms, termites or other small animals can provide the whole GIT for the studies, only part of contents of wall of the GIT can be studied in large animals. Therefore the choice of sampling is the first bottle neck in the studies on yeasts biodiversity in the GIT. Following proceedings such as homogenization, concentration or dilution of the samples must be hereby additionally considered.

Biodiversity of Yeasts in the Gastrointestinal

Ecosystem with Emphasis on Its Importance for the Host 293

for studying the yeast biodiversity in the GIT of animals. Pyrosequencing is a rapid method providing up to several thousands of sequences per sample in just few days. Followed by bioinformatics processing, alignment to known species is performed resulting not only in a phylogenetic tree but also in description of the species diversity. Unknown species can be also detected in this way. The high cost provides the limitation for the wide application of this method; however it is to expect that in the near future the high-throughput sequencing

A microarray has been recently developed allowing characterization of pig GIT bacterial community, targeting over 800 phylotypes (Pérez Gutiérrez, 2010). Microarrays for yeasts would need to be developed to provide further molecular tool for studying the biodiversity

Studying diversity of yeasts harbouring the GIT of animals would be incomplete without consideration of the role that these microorganisms play for the host. For years the yeasts harbouring the GIT of animals and humans have been considered rather as harmful to the host's health. Indeed, there are some species belonging to *Candida*, *Cryptococcus*, *Malassezia*, *Trichosporon* and *Geotrichum* that could be pathogenic to members of the Animal kingdom (Fidel et al., 1999; Girmenia et al., 2005; Cabañes, 2010). Furthermore, many researchers have evaluated yeasts in association with various diseases and if they found representatives of this group they acted against them applying medical treatment (Schulze & Sonnenborn, 2009). However, there is just as little known about yeasts harbouring the GIT of healthy animals to understand their importance there, and growing evidences appear for their role in the proper function and survival of the host. In fact, the current knowledge about yeasts in the digestive tract of vertebrates is still based on the findings from 50's and 70's of the XXth Century; therefore there is a great demand for the scientific evaluation in this field. As enough reports exist concerning pathogenic yeasts, in this paragraph a possible positive

There are nice reviews (e.g. Phaff & Starmer, 1987; Ganter, 2006) pronouncing a yeast-insect relationship. Gatesoupe (2007) gave an insight into the ecology of yeasts naturally occurring in the intestinal tract of fish, and thereby emphasized a possible importance of yeasts to the

Similarily to the probiotic strains of *Saccharomyces cerevisiae* (Buts, 2009), the cells of some intestinal yeasts could have a trophic effects since they provide a source of B vitamins, proteins, trace minerals and essential amino acids. Besides, the major portion (> 90%) of the yeast cell walls comprise of polysaccharides such as β-glucans, mannans and chitin, which composition and structure are specific for individual yeast (Latgé, 2007). In many human and animals studies, β-glucans and mannans have been comprehensively investigated; they may play important diverse roles for the host immune system and exhibit antimicrobial activity against bacteria thereby influencing the establishment of the intestinal microbiota and promising to promote host's health. Therefore, several studies concentrated on use of the live or dead yeast cells in human and animal nutrition as supplements or as a remedy for acute diarrhoea in humans (Bekatorou et al., 2006; Buts & De Keyser, 2006; Fleet, 2007; Buts, 2009). Furthermore, due to production of several enzymes, some yeast species, e.g. found in the gut of termites (Schäfer et al., 1996; Molnar et al., 2004) and beetles (Suh et al., 2003), are able to degrade hemicelluloses that are being the main carbohydrates of

will be as expensive as the other commonly used molecular tools.

impact of yeasts on the gut ecology and host health will be discussed.

and its changes caused by different extrinsic factors.

**4. Role of yeasts in GIT** 

host.

Among the methods applied for investigating the biodiversity of yeasts harbouring the GIT of animals, cultivation and morphological and/or biochemical identification have been the most often used for more than 150 years. However, these methods bear limitations such as the choice of the right cultivation medium, pH, temperature and moisture. Furthermore the yeast species that need more time for growth and are at lower abundance in the community cannot be identified in this way. It has been accepted that every ecosystem consists, next to cultivable organisms, also of viable but non-cultivable (VBNC) microorganisms, that contemporarily cannot be cultivated in laboratory because of nutrient limitation or lack of optimal living conditions (Edwards, 2000). This is why only approximately 1% of yeast species could be described so far (Kurtzman & Fell, 2006). Sabouraud agar is the medium most commonly used for cultivation of yeasts from clinical or ecological samples (Odds, 1991), however many others have been used for industrial purposes, providing alternatives for cultivation of more demanding species (King et al., 1986; Jarvis & Williams, 1987; Fleet, 1990; Deak, 1991). It is to remember, that various species can give similar colonies, and the same species can grow in a different way under different conditions. Cultivated species can be however observed under microscope, what helps for identification of the isolates. Spectrophotometric methods such as MALDI-TOF could also provide fast and good tool for identification of the isolates. Molecular methods can be also applied for identification of isolates, e.g. pyrosequencing of target genes (Borman et al., 2009; 2010). Further development of modified media and combinations of temperature, pH, aerobic/anaerobic conditions and moisture would probably increase the number of isolated yeasts, it is however laborious and very time consuming.

Cultivation-independent methods which have been used for the last two decades provided the researches with fast and specific tools for the biodiversity studies. Polymerase chain reaction (PCR), DNA-DNA hybridization or fluorescence in situ hybridization (FISH) applying probes targeting the RNA allow in theory detection of 1 single colony present in a sample population. Further separation of the specific DNA fragments performing denaturing or temperature gradient gel electrophoresis (DGGE / TGGE) allows studying the diversity of the complex community (Cocolin et al., 2002; Prakitchaiwattana et al., 2004; Molnar et al., 2008). Other molecular methods could be also applied for identification of members of a community, e.g. terminal restriction fragment length polymorphism (T-RFLP), amplified fragment length polymorphism (AFLP), multiple-locus variable number tandem repeat analysis (MLVA) (e.g. Tiedje et al., 1999; Gemmer et al., 2002). These methods are very specific, allowing targeting of specified species and thus quantification of the yeasts and calculating the biodiversity. The largest limitation for methods based on PCR is the low sensitivity, as the practice shows only 1-2% of the community can be detected in this way (Macnaughton et al., 1999). Furthermore, fingerprint methods have the bias combined to the fact that amplicons form different species with sequences of similar energetic profile may migrate to the same positions; multiple gene copies with slight sequence differences may give multiple bands for one strain or species; finally some species are phylogeneticaly very similar (Lachance et al., 2003; Janczyk et al., 2006; Borman et al., 2010). The design of probes for direct targeting needs knowledge on the sequence of the target gene and differences between species.

Pyrosequencing and other high-throughput methods provide a fast and very efficient tool for identification of the members of the complex populations. Metagenome analyses targeting the D1/D2 domain of the 26S rRNA gene or the internal transcribed regions (ITSs) allow distinction of the yeasts (Kurtzman & Fell, 2006) and seem to be very suitable methods for studying the yeast biodiversity in the GIT of animals. Pyrosequencing is a rapid method providing up to several thousands of sequences per sample in just few days. Followed by bioinformatics processing, alignment to known species is performed resulting not only in a phylogenetic tree but also in description of the species diversity. Unknown species can be also detected in this way. The high cost provides the limitation for the wide application of this method; however it is to expect that in the near future the high-throughput sequencing will be as expensive as the other commonly used molecular tools.

A microarray has been recently developed allowing characterization of pig GIT bacterial community, targeting over 800 phylotypes (Pérez Gutiérrez, 2010). Microarrays for yeasts would need to be developed to provide further molecular tool for studying the biodiversity and its changes caused by different extrinsic factors.
