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 (HNFs) 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 HNFs in the 10 and 20 µm treatments, reflecting a trophic cascading from HNFs to raptorial prostome ciliates (Balanion planctonicum and Urotricha spp.) in spring. In summer, HNFs and ciliates peaked simultaneously, suggesting the important trophic cascade also from bacteria to bacterivorous scuticociliates (Cyclidium glaucoma and Cinetochilum margaritaceum) and HNFs. 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. Ciliates represent an important trophic link in aquatic microbial food webs. In this study, we used the food web manipulation techniques to reveal their complex trophic interactions during seasonally different plankton scenarios occurring in spring and summer. Manipulating top-down controlling factors (grazing pressure of micro- and metazooplankton grazers) and bottom-up factors (an availability of bacterial prey) shaped distinctly the complexity and dynamics of natural plankton communities and thus yielded significant changes in ciliate community dynamics. The experimentally simplified plankton and ciliate communities responded to our manipulations in season-specific fashions, reflected in different roles of ciliates as an intermediate trophic link between prokaryotes and higher trophic levels. This study also demonstrates that the combination of molecular and morphological analyses is essential to gain more realistic insights into the structure of ciliate community and for providing robust, ecologically meaningful results.