**4. Role of yeasts in GIT**

292 The Dynamical Processes of Biodiversity – Case Studies of Evolution and Spatial Distribution

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

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

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

however laborious and very time consuming.

between species.

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

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 host.

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

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herbivorous diet, and also detoxify toxins that can appear in the feed. The possibility cannot be excluded that some yeasts harbouring GIT of herbivorous animals may produce extracellular enzymes (e.g. exohemicellulases, exocellulases) or show endocellulolytic activity, and thereby contribute to their digestion by braking down complex, indigestible fibre into simple carbohydrates.

It is still a prevalent opinion, that yeasts harbouring the digestive tract of animals have only minor importance for the host. The main scientific argument up to date is the negligible quantity of yeasts. Nevertheless, yeasts may be of physiological relevance, even though they are present to a much lesser extent than bacteria. In fact, yeasts could provide a relevant biomass, as their have a cell volume 30- to 100-fold higher than bacteria (Gatesoupe, 2007). Commensal yeasts may interact with intestinal bacteria and due to this interplay affect microbial diversity and host organism. An example of such yeasts-bacteria interrelationship provides the study of Urubschurov et al. (2011) who examined changes of yeasts and major bacterial groups (lactobacilli, enterobacteria and enterococci) in the faeces of piglets after weaning. They observed that the increase of yeasts number, where the dominating species was *Kazachstania slooffiae*, significantly correlated with the increase of lactobacilli and decrease of enterobacteria numbers. Other studies hypothesized that specific yeasts frequently occurred in high quantity at the digestive tract of lacewings (Woolfolk & Inglis, 2004; Woolfolk et al., 2004) and mosquitoes (Ricci et al., 2011a; 2011b) and were symbiotically related to the host.

These first indications need further confirmation but they already show that the yeasts cannot be considered negligible any more.
