Morphological and molecular analyses of season-specific responses of freshwater ciliate communities to top-down and bottom-up experimental manipulations

In aquatic microbial food webs, ciliates represent an important trophic link in the energy transfer from prokaryotes, algae, and heterotrophic nanoflagellates (HNF) to higher trophic levels. However, the trophic role of abundant small ciliates (< 20 µm) is not clearly understood. To unveil their trophic linkages, we conducted two experiments manipulating both top-down and bottom-up controlling factors, thus modulating the trophic cascading and bacterial prey availability for protists during contrasting spring and summer seasons with samples collected from a freshwater meso-eutrophic reservoir. Water samples were size fractionated, to modify food web complexity, i.e. 10-µm, 20-µm and unfiltered control and amended with bacterial prey additions. The samples were analyzed by morphological and sequencing techniques. The bacterial amendments triggered strong ciliate growth following the peaks of HNF in the 10-µm and 20-µm treatments, reflecting a trophic cascading from HNF to raptorial prostome ciliates (Balanion planctonicum and Urotricha spp.) in spring. In summer, HNF and ciliates peaked simultaneously, suggesting the important trophic cascade also from bacteria to bacterivorous scuticociliates (Cyclidium glaucoma and Cinetochilum margaritaceum) and HNF. In spring, unfiltered treatments showed stronger ciliate top-down control by zooplankton than in summer. The sequence analysis revealed season-specific manipulation-induced shifts in ciliate communities and their large cryptic diversity. However, morphological and molecular analyses also revealed considerable discrepancies in the abundance of major ciliate taxa. The ciliate communities responded to our experimental manipulations in season-specific fashions, thereby highlighting the different roles of ciliates as an intermediate trophic link between prokaryotes and higher trophic levels.