Biodiversity of yeasts in the gut of insects

The main aim of this project is the investigation of biodiversity of yeasts and yeast-like fungi isolated from gut of insects. These habitats are widely unexplored and therefore it is supposed that new yeast species and genera can be detected. Especially pests living on Fusarium-contaminated grain are interested in the course of this project since these insects could have had contact with mould-contaminated plants for a long time, thus symbioses with mycotoxin degrading microorganisms in the intestine could have been evolved. At least 25% of the world`s food crops are contaminated with mycotoxins (FAO, Food Outlook 1996, 5/6) and therefore it is necessary to find ways for decontamination to protect humans and animals from carcinogenic, immunosuppressive and genotoxic effects of these mycotoxins. In further projects the isolated, mycotoxin-transforming strains can be investigated regarding their applicability as mycotoxin detoxifying feed additive or they can serve as donor organism for the gene sequence of the respective detoxifying enzymes, which can be cloned into the plant genome with the aim to prevent mycotoxin accumulation. During this project yeast diversity will be investigated in two ways: First - without enrichment - by using denaturing gradient gel electrophoresis (DGGE) to get an overview of the entire yeast population (culturable and non culturable) and second - after strain isolation - by applying genotypical (rDNA sequencing, RAPD-PCR) methods. DGGE analysis will be performed using the ITS 1-2 region amplified with fungal specific primer. RAPD-PCR (random amplified polymorphic DNA analysis) will provide information to distinguish different species immediately after isolation and to confirm identification results obtained after partial sequencing of the D1/D2 region of the 26S rDNA. Traditional morphological and physiological tests will be used only for new species delimitations. Additionally a partial sequencing of the 18S rDNA will be used for euascomycetous yeasts. In some cases the whole sequences of the 18S rDNA are needed to perform phylogenetic analyses. Further aims are gathering information about yeast phylogeny and development of yeast endocytobiosis, discovering new symbiontic relationships and comparing yeast flora during different developing phases of the insects. Finally the isolates will be tested for their potential to transform or degrade mycotoxins.

A yeast strain isolated from the hindgut of the lower termite Mastotermes darwiniensis (Mastotermitidae) was found to represent a new member of the genus Trichosporon. Trichosporon mycotoxinivorans is closely related to T. loubieri on the basis of the phylogenetic trees based on the D1/D2 region of 26S rDNA, an approx. 600 bp fragment of the 18S rDNA and both ITS regions. However, the two species differ at nine positions in the D1/D2 region of 26S rDNA. The IGS1 region of T. mycotoxinivorans is 401 bp long (T. loubieri 430 bp). T. mycotoxinivorans is distinguished from T. loubieri by its ability to assimilate inulin and galactitol, and its inability to grow at 40C. The name of this newly isolated strain refers to an important characteristics of T. mycotoxinivorans to detoxify mycotoxins such as ochratoxin A and zearalenone. Therefore this strain can be used for the deactivation of the respective mycotoxins in animal feeds. Thirtytwo yeast isolates were cultured from guts or excrements of 3 different pests of corn or from the stem of healthy corn. The strains were analyzed using MSP-PCR (micro/minisatellite-primed polymerase chain reaction), sequences of the D1/D2 region of the large subunit rDNA and a 979 bp long part of the actin gene (act-1). They seem to belong to three groups that are all sister groups of Metschnikowia pulcherrima, M. fructicola and M. chrysoperlae. A new species, Metschnikowia andauensis (HA 1657T) is described. In contrast to M. pulcherrima and M. fructicola, M. andauensis is well separated in the act-1 phylogenetic tree too. A new yeast, Cryptococcus zeae (type strain HB 1207T) is described. Six strains were isolated from corn and pests of corn in Austria. MSP-PCR fingerprints showed that the strains are members of the same species. Phylogenetical analyses of domains D1/D2 26S rDNA and ITS 1 - 5,8S - ITS 2 sequences showed Cryptococcus zeae to have the closest relationship to Cryptococcus luteolus. The new species is separable from the closest relative C. luteolus using only two physiological tests. It shows positive results in the L-sorbose assimilation test, whereas C. luteolus assimilates this carbon source delayed or not at all; similarly, it assimilates D-tryptophan as sole nitrogene source, whereas C. luteolus does not. Denaturing gradient gel electrophoresis (DGGE) and cloning were used to detect unculturable yeasts. Using this methods we could only find yeast species which were already found by the classical isolation method.