Incubation and grazing effects on spirotrich ciliate diversity inferred from molecular analyses of microcosm experiments

Abstract

We used an experimental approach of analyzing marine microcosms to evaluate the impact of both predation (top-down) and food resources (bottom-up) on spirotrich ciliate communities. To assess the diversity, we used two molecular methods–denaturing gradient gel electrophoresis (DGGE) and high-throughput sequencing (HTS). We carried out two types of experiments to measure top-down (adult copepods as predators) and bottom-up effects (phytoplankton as food resources) on the spirotrich ciliates. We observed both strong incubation effects (untreated controls departed from initial assessment of diversity) and high variability across replicates within treatments, particularly for the bottom-up experiments. This suggests a rapid community turn-over during incubation and differential susceptibility to the effects of experimental manipulation. Despite the variability, our analyses reveal some broad patterns such as (1) increasing adult copepod predator abundance had a greater impact on spirotrich ciliates than on other microbial eukaryotes; (2) there was no evidence for strong food selection by the dominant spirotrich ciliates.

Top-Down and Bottom-Up Controls on Microeukaryotic Diversity (i.e., Amplicon Analyses of SAR Lineages) and Function (i.e., Metatranscriptome Analyses) Assessed in Microcosm Experiments

“…To perform our microcosm experiment, we used four cultures: three phytoplankton (prey) cultures Tetraselmis chui, Isochrysis galbana, and Phaeodactylum tricornutum; and a culture of Acartia tonsa (copepod, predators). The species of phytoplankton used are common in aquaculture application, represented various size, shape, pigment composition, and lineages, and have been used successfully in the past (McManus et al, 2004(McManus et al, , 2012Grattepanche et al, 2019). All phytoplankton were from the culture collection of the National Marine Fisheries Service Laboratory in Milford, CT, United States, and were grown in F/2 medium prior to experiments.…”

“…We used dialysis tubing to perform our microcosm experiment and incubated samples in a sea table with circulating water from collections sites to minimize impact of isolation, following the approach of Grattepanche et al (2019). The dialysis tubing is composed of a cellulose membrane that is pervious to molecules <12,000 molecular weight (product D9402, Sigma), allowing for exchange of nutrients during incubation in sea tables.…”

“…For each of the phytoplankton abundance conditions, including controls, we added zero (z0), 5 (z1), or 10 copepods (z2) to triplicate microcosm for each treatment (three sets of dialysis tubing per phytoplankton/zooplankton combination). Based on insights from a preliminary study (Grattepanche et al, 2019), the microcosms were incubated for 3 days and then the total contents of each bag were collected for nucleic acids extractions. We used a 3-day incubation to minimize cascade effects and maximize nano-and microsized plankton impact based on insights from Grattepanche et al (2019).…”

“…Based on insights from a preliminary study (Grattepanche et al, 2019), the microcosms were incubated for 3 days and then the total contents of each bag were collected for nucleic acids extractions. We used a 3-day incubation to minimize cascade effects and maximize nano-and microsized plankton impact based on insights from Grattepanche et al (2019). We also collected duplicates of the initial in situ water sample and the in situ sample plus high phytoplankton abundance added (t0p3) at the beginning of the experiment.…”

“…For the RNA prep, residual DNA was removed using the Turbo DNA-free kit (Invitrogen, CA, United States). This approach (both filtration and extraction) was tested on previous samples and resulted in good DNA/RNA quality (Tucker et al, 2017;Sisson et al, 2018;Grattepanche et al, 2019).…”