**2.1.5 Yeasts in the digestive tract of beetles**

At the present time, the most yeasts were isolated from the digestive tract of beetles (*Coleoptera*). Shifrine & Phaff (1956) collected bark beetles (*Dendroctonus* and *Ips*) and their larvae from the various coniferus trees in Northern California. After sterilization of the outer surface, the beetles were dissected and yeasts were isolated from internal parts of the insects. Total of 169 yeast strains could be assigned to 13 species. *Candida silvicola* (41.4%; teleomorph *Hansenula holstii*), *Hansenula capsulata* (21.3%), *Pichia pinus* (18.9%) and *Candida curvata* (8.9%) were frequently found; other species (e.g. *C. parapsilosis, C. mycoderma, C. rugosa*, *Cryptococcus diffiuens*) were rarely (from 0.6 to 1.8%) isolated. Some yeasts as *Candida* (*Torulopsis*) *nitratophila*, *C.* (*T.*) *melibiosum*, *Rhodotorula crocea* and *C. silvicola* were described by these authors as new species. Recently, Rivera et al. (2009) provided an account of yeasts associated with alimentary tract of *Dendroctonus* beetles. Yeasts (403 strains) were isolated from different parts of intestine, the midgut (anterior & posterior) and the hindgut, as well as from the ovaries, eggs and frass of the beetles collected from pine trees at 34 locations in Mexico, Cuatemala and the USA. Based on the sequence analysis of several DNA regions (18S, 26S rRNA genes and ITS1) and phenotypical characteristics, the yeasts were reletated to three genera: *Candida* spp. (*C. ernobii*, *C. piceae, C. membranifaciens*, *C. lessepsii*, *C. arabinofermentans* and *C. oregonensis*), *Pichia* spp. (*P. americana, P. guilliermondii, P. scolyti, P. mexicana, P. glucozyma* and *P. canadensis*) and *Kurashia* spp. (*K. capsulata* and *K.* cf. *molischiana*). The exact numbers of the yeast strains isolated from the different gut sections have not been provided by the autors, however, they indicated that yeasts were present in eggs, ovaries and frass to much lesser extent than in the guts. For instance, *P. americana, C.* 

Lacewings (*Neuroptera*: *Chrysopidae*) are one of the predators admitted as biological control agents of pests. During the scanning and transmission electron microscopical studies, a large numbers of yeast cells were observed within lacewings' alimentary tract (Woolfolk et al., 2004; Woolfolk & Inglis, 2004; Chen et al., 2006). Woolfolk & Inglis (2004) investigated yeasts in the different parts: diverticulum, foregut, midgut, and hindgut of digestive tract of 24 lacewing adults (*Chrysoperla rufilabris*) collected at two field locations in Mississippi. With the exception of 7 insects that were yeasts-free, lacewings harboured a high concentration (≈ 103 colony forming units; CFU) of yeasts distributed in the all analysed gut sections; however the highest (5.4x105 CFU/g) density was in diverticulum. In total 752 yeasts were isolated in the study and arranged in five groups based on their phenotypic properties; some specimens were randomly chosen from each group for further genotyping analysis. Interestingly, 89% of the isolates were identified as *Metschnikowia pulcherrima* and the remaining 11% involved either *Cryptococcus victoriae* or *Cryptococcus luteolus* or strains that could not be assigned by the authors to any known species. Sometimes, closely related yeast species could be separated only according to the genotypic characterization, while they were showing similar physiological properties (Kurtzman & Fell, 2006) as it was the case in the study of Suh et al. (2004a). These authors isolated 14 yeasts from digestive tract of *Chrysoperla* spp. which were closely related to *M. pulcherrima*, however sufficiently variable in the D1&D2 domains of the 26S rRNA gene of the large subunit (LSU) to represent three new species: *Metschnikowia chrysoperlae, Candida picachoensis* and *Candida pimensis*. Recently, several new yeasts of *Metschnikowia* and *Candida* (see table 1) were discovered in the gut of

At the present time, the most yeasts were isolated from the digestive tract of beetles (*Coleoptera*). Shifrine & Phaff (1956) collected bark beetles (*Dendroctonus* and *Ips*) and their larvae from the various coniferus trees in Northern California. After sterilization of the outer surface, the beetles were dissected and yeasts were isolated from internal parts of the insects. Total of 169 yeast strains could be assigned to 13 species. *Candida silvicola* (41.4%; teleomorph *Hansenula holstii*), *Hansenula capsulata* (21.3%), *Pichia pinus* (18.9%) and *Candida curvata* (8.9%) were frequently found; other species (e.g. *C. parapsilosis, C. mycoderma, C. rugosa*, *Cryptococcus diffiuens*) were rarely (from 0.6 to 1.8%) isolated. Some yeasts as *Candida* (*Torulopsis*) *nitratophila*, *C.* (*T.*) *melibiosum*, *Rhodotorula crocea* and *C. silvicola* were described by these authors as new species. Recently, Rivera et al. (2009) provided an account of yeasts associated with alimentary tract of *Dendroctonus* beetles. Yeasts (403 strains) were isolated from different parts of intestine, the midgut (anterior & posterior) and the hindgut, as well as from the ovaries, eggs and frass of the beetles collected from pine trees at 34 locations in Mexico, Cuatemala and the USA. Based on the sequence analysis of several DNA regions (18S, 26S rRNA genes and ITS1) and phenotypical characteristics, the yeasts were reletated to three genera: *Candida* spp. (*C. ernobii*, *C. piceae, C. membranifaciens*, *C. lessepsii*, *C. arabinofermentans* and *C. oregonensis*), *Pichia* spp. (*P. americana, P. guilliermondii, P. scolyti, P. mexicana, P. glucozyma* and *P. canadensis*) and *Kurashia* spp. (*K. capsulata* and *K.* cf. *molischiana*). The exact numbers of the yeast strains isolated from the different gut sections have not been provided by the autors, however, they indicated that yeasts were present in eggs, ovaries and frass to much lesser extent than in the guts. For instance, *P. americana, C.* 

**2.1.4 Yeasts in the digestive tract of lacewings** 

other members of the *Neuroptera*, too (Nguyen et al., 2006; 2007).

**2.1.5 Yeasts in the digestive tract of beetles** 

*ernobii* and the strains related to the one *Candida* sp. were prevalent in all parts of gut and frass and *P. guilliermondii* and *C. ernobii* were cultured most frequently from the posterior midgut.

In relation to high number of the yeast isolates (richness) described above, comparatively low yeast diversity was found in the assemblage of *Dendroctonus* beetles. It thereby underlined the impact of the host and/or environmental factors on the yeasts diversity. Nevertheless, examination of yeasts harbouring the GIT of beetles from 27 families reviled a huge variety of yeasts (Suh et al., 2005a).

Fig. 1. Schematic representation of the different yeast taxa belonging to the two phyla *Ascomycota* and *Basidiomycota* of the Dikarya subkingdom isolated from the intestinal tract of insects during the study of Suh et al. (2005a).

During three-years-period, Suh et al. (2005a) isolated about 650 yeasts from the gut of diverse beetles collected from the south-eastern USA and Panama. Sequence analyses of the D1&D2 domains of LSU rRNA gene revealed 290 single species belonging to at least 27 taxa (Fig. 1.); the great majority of which were ascomycetous and some basidiomycetous yeasts. It is noteworthy that nearly 200 yeasts determined throughout the study were considered by the authors to represent new, not yet described species. In the meantime, some of them (table 1) were characterized by Suh, Nguyen, Blackwell and their co-workers. Based on their observation Suh et al. (2005a) suggested that almost each beetle species may be a host for at least one unknown yeast species. In the last decades, describing of many novel species of yeasts isolated from the gut of insects corroborates this supposition.

Biodiversity of Yeasts in the Gastrointestinal

al., 2005).

*Kazachstania slooffiae*

*Issatchenkia orientalis*

*Pichia fermentans*

> *Candida tropicalis*

Fig. 2. Dominated yeasts isolated from the gut of 39 d old piglets, kept at experimental (EF)

*Trichosporon asahii*

and commercial farm (CF), in the study of Urubschurov at al. (2008).

*Candida glabrata*

Ecosystem with Emphasis on Its Importance for the Host 289

digesta samples collected from stomach and rectum from 98 (of 100 examined) pigs. Apart from the *C. slooffiae* which was detectable in 75% of pigs, yeast species such as *C. krusei*, *S. telluris*, *C. albicans*, *C. glabrata, C. tropicalis, C. parapsilosis* and *C. pintolopesii* (60%, 26%, 9%, 4%, 3%, 3% and 2% respectively) were isolated, too. Also in this study the appearance of yeasts was variable within a part of the GIT and among examined animals. Thus, stomach was generally colonized by yeasts at lesser intense than rectum. In most animals, *C. slooffiae*  and *C. krusei* were detected both in stomach and rectum, while just in a few cases the yeasts could be found only in a single part of the GIT. *C. slooffiae* and its closely related species: *S. telluris* and *C. pintolopesii* have been newly molecularly investigated and based on multigene sequence analyses they were assigned to the teleomorphic genus *Kazachstania* (Kurtzman et

Recently, Urubschurov et al. (2008) described yeasts' diversity in the gut of piglets around weaning which were reared at two facilities: at experimental farm (EF) with improved husbandry conditions than at commercial farm (CF). Most piglets, 33 at CF and 35 at EF, were weaned at 28 days (d) of age and fed with the same diet until 39 d in both farms. A number of piglets, namely 18 at CF and 9 at EF, were left by the sows without additional feeding. All piglets were sacrificed at 39 d of age and digesta samples from GIT were collected. D1&D2 domains of 26S rRNA gene from 173 yeast isolates obtained from 95 piglets were sequenced. The alignment to known sequences revealed close relationship to 17 species, of which the most dominated are presented in figure 2. Urubschurov et al. (2008) observed distinction of yeasts variety between both facilities that were proven by calculation of different similarity and diversity indices. In piglets from CF *Galactomyces geotrichum*, *Kazachstania slooffiae* and *Candida catenulata* were the most abundant ones and the other were present only at low abundances. Unlike at CF, at EF two species, namely *K. slooffiae* and *C. glabrata* were found to be the most dominating ones and the others were rarely isolated. Some of the other species could be found in piglets either only at the EF (*P. fermentans*, *C. tropicalis, C. oleophila*, *C. parapsilosis*, *P. guilliermondii*, *Rh. mucillaginosa*, *T. montevideense*) or at the CF (*C. silvae* and *P. farinose*). This study provided evidence for association of *K. slooffiae* with the porcine GIT. *K* (*C.*) *slooffiae* was found for the first time in 6 of 252 examined horses (Van Uden & Carmo-Sousa, 1957a), however, due to frequent occurrence and high concentration in the porcine digestive tract it can be considered to be specific for pigs.

> *Kazachstania slooffiae*

EF CF

*Galactomyces geotrichum*

> *Candida catenulata*

> > *Trichosporon coremiiforme*

*Trichosporon asahii*

Nowadays, there are over one million of accepted insect species; however, their number has yearly increased and is still largely undiscovered (Chapman, 2009). Thus, it can be supposed that the number of yeasts would tremendously rise, even if only the intestinal tracts of the currently known insects were explored.
