ИСТИНА

The present study examines ultrastructural changes observed in filamentous fungus Podospora anserina in the course of long-term laboratory evolution. Our scientific group succeeded in finding experimental conditions that triggers irreversible changes in P. anserina phenotype. Acceptable conditions are continuous mycelia cultivation in liquid nutrient medium with the use of orbital shaker [1]. We furthermore demonstrated positive selection of de novo mutations in our model system [2]. Despite the fact that most of acquired mutations belong to different genomic sites, phenotypic changes cover the same morphological aspects in independent P. anserina lines. Eight lines derived from two initial P. anserina strains of wild type were removed from agar containing to liquid medium in the year of 2012. Since then continuous growth of all lines is maintained by simultaneous serial passages. The evolutionary experiment is still in progress. Ultrastructural study let us conclude that during the short period right after the experiment initiation P. anserina subjected to the stress, but after that adapted to new conditions of growth. Fungal cells in the course of short adaptation phase had thick double layered or multilayered cell walls and additionally were protected with fibrillous covers. Cytoplasm demonstrated high electron density making it quite difficult to trace intracellular components. Mitochondria showed rounded profiles, they were small and numerous probably because of fragmentation. Nuclei were comparatively small. Mycelia adaptation was characterized by dramatic increase of biomass yield. At the ultrastructural level adapted lines had some distinctive features, the most important of which are: thin cell walls (mainly single-layered) having no additional protection, big nuclei which diameter frequently was almost equal to hyphal width, elongated mitochondrial profiles with parallel cristae. Electron density of cytoplasm decreased making perfectly visible all cell structures. Two and a half years after experiment start the new distinctiveness of P. anserina intracellular life was noticed. Cellular vacuoles became to capture, absorb and degrade huge parts of cytoplasm that is not usual for P. anserina wild type strains. It is possible that this is the way for the fungus to realize recycling of intracellular materials faster. The most surprising characteristic of P. anserina under conditions of growth in liquid medium was formation of special double-membrane vesicles. Their length ranges between 130 and 760 nm, width varies between 100 and 500 nm (the average values are 336±20 nm and 222±12 nm respectively). The experiment continuation led to enrichment of P. anserina cells by the vesicles of such kind. After five years of growth in liquid large double-membrane vesicles became common for experimental lines as a result of which we could follow their way of formation. Plasma membrane invaginates very deeply into cytoplasm, curves and forms hemisphere with electron-translucent content (Fig. 1). After closure the vesicles migrate into different parts of cell cytoplasm. In fungi double-membrane vesicles originated from the cell membrane were occasionally noticed by classical [3] and modern [4] authors, but their functional purpose is debatable and essentially unknown. It may be more delicate and sophisticated mechanism of recycling. Mitochondria often could be found near forming double-membrane vesicle indicating that this process may need some energy. P. anserina mycelial isolates being transferred from adapted experimental cultures to solid medium (standard conditions) demonstrates all modifications acquired by the appropriate submerged culture. On the level of ultrastructure isolates are indistinguishable from submerged mycelia. The reported study was funded by RFBR, research project № 18-04-01349 a.