| Abstract [eng] |
Unicellular protozoa are an integral part of aquatic ecosystems. However, the seasonal prevalence and viability of parasitic species, particularly members of the genus Sarcocystis, in environmental samples remain poorly understood. The aim of this master’s thesis was to evaluate the viability and species diversity of unicellular parasites in water samples and to investigate the dynamics of DNA copy numbers across different seasons using real-time PCR. The study was conducted at Bukiškis pond, located in the Vilnius district. Over a two- year period (2024–2026), water samples were collected monthly from three distinct locations within the pond. To determine the species diversity of unicellular protozoa, eDNA cloning and sequencing methods were applied using the universal 18S rRNA genetic marker. The analysis revealed that various eukaryotic microorganisms were prevalent in the studied water samples, the majority of which belonged to the SAR supergroup. A combination of propidium monoazide (PMA) treatment and standard PCR was used to assess the viability of the unicellular protozoa. It was determined that temperature has a statistically significant effect on the viability of these microorganisms. As the temperature decreased from 22◦ C to 4◦ C and -20◦ C, viability decreased by approximately 15% and 22%, respectively. The prevalence of S. cruzi and S. bovifelis in the water body during different months of the year was evaluated using nested PCR with species-specific cox1 gene primers. S. cruzi DNA was detected in samples from 10 out of 12 months (83.3%), while S. bovifelis was identified in 8 out of 12 months (66.7%). August was the only month in which DNA from neither species was detected. Quantitative analysis was performed using real-time PCR (qPCR) with SYBR Green dye. The highest DNA copy numbers for both species were recorded in autumn; however, one- way ANOVA results revealed that these seasonal differences were statistically non-significant (p > 0.05). It was concluded that the prevalence of Sarcocystis sporocysts in the water body is a random process independent of seasonality, driven by local factors such as the diet and activity near the shoreline of definitive hosts, as well as meteorological conditions. |
