Experimental set-up
The experiment was performed in the MOBICOS mesocosm facility, a container-based laboratory platform34 located by the river Holtemme in Wernigerode, central Germany (51° 49′ 00.7″ N, 10° 43′ 29.26″ E). See Weitere et al.35 for detailed water quality data at this station. Each experimental unit consisted of a rectangular flume (62 cm long, 14 cm high and 8 cm wide) constantly supplied with water from the river Holtemme, with a flow rate of 1000 L h−1 per flume. The water was filtered by a self-cleaning filter with a mesh size of 50 µm in order to remove larger particles without removing most unicellular organisms. The water level in each flume was 7.5 cm. At the bottom of each flume was a tray containing 30 white ceramic tiles (2.3 × 2.3 cm), disposed in three rows of ten tiles each, and a smaller tray containing nine additional tiles, disposed in three rows of three tiles each. The tiles served as substrates for periphyton growth. Vertical nets were placed at both ends of each flume to prevent grazers from leaving the experimental facility.
The study consisted of a fully factorial experiment, in which two levels of phosphorus supply (high, P+, versus low, P−) were crossed with two levels of light intensity above the flumes (high, L+, versus low, L−) and with grazer presence (G+) and absence (G−), for a total of eight treatments: P+L+G+, P+L+G−, P+L−G+, P+L−G−, P−L+G+, P−L+G−, P−L−G+, and P−L−G−. In the P− treatments, the water flowing in the flumes was kept at ambient P concentration, which was below detection limit (< 3 µg L−1 soluble reactive phosphorus, SRP). In the P+ treatments, a concentration of 100 µg P L−1, typically observed in eutrophic streams36, was achieved in each flume by pumping a constant supply of dissolved KH2PO4 with a peristaltic pump. The light (PAR) intensity above the flumes, produced by LED lamps, was 111.8 µmol m−2 s−1 in the L+ treatments and 46.6 µmol m−2 s−1 in the L− treatments, in a 14:10 h light:dark cycle. In the G+ treatments, eight individuals of the pulmonate snail A. fluviatilis, collected in the Holtemme river, were added to each flume. Each of the eight treatments was replicated three times, for a total of 24 flumes.
Experimental procedure
Natural periphyton was pre-cultivated for two weeks in the experimental flumes, in the respective phosphorus and light treatments (see previous section), from 27 May to 10 June 2019 before adding grazers. On 10 June, three periphyton-covered tiles were selected from each flume for initial sampling. Periphyton was scraped off each tile, homogenised in tap water and filtered onto pre-combusted GF/F glass fibre filters for dry mass, elemental and pigment analyses (see following sections).
On 11 June, eight juvenile A. fluviatilis individuals with shell lengths of 2.5–5 mm were added to each G+ flume. The average snail shell length for each flume was between 3.5 and 4.1 mm. Subsequently, the snails were allowed to graze on periphyton for 8 weeks. During this grazing phase, dead or lost snails were replaced with new snails, which were marked with nail polish. Snails that had been added to the experiment after the first 10 days of the grazing phase were not included in the final snail size measurements (see next paragraph). Nevertheless, loss of snails during the experiment was minor, with a maximum of three snails per flume lost after the first 10 days.
On 25 June (day 14 of the grazing phase) and on 17 July (day 36 of the grazing phase), three tiles were selected from each flume for intermediate sampling. Periphyton was scraped off each tile, homogenised in tap water and filtered onto pre-combusted GF/F glass fibre filters for dry mass determination. However, the data collection on 25 June was compromised by a sediment accident that previously occurred in the morning of the same day. A high sediment load was discharged into the Holtemme River due to the cleaning of an upstream basin. Although the water pumped into the MOBICOS facility was cleaned of particles larger than 50 µm with a self-cleaning filter, a large amount of fine sediments entered the experimental facility and shut down the filter for approximately four hours. Due to this immediate impact, data collected on 25 June were excluded from the final analyses. Some sediments remained in the flumes for the rest of the experiment, but periphyton was re-established before the next sampling date. Each experimental flume was affected by the sediments and no systematic effect on specific treatments occurred. Therefore, the event was considered as environmental variability, which is part of the concept of mesocosm experiments, and not as bias to the manipulations on top of the enviromental variability.
On 7 August (day 57 of the grazing phase), the experiment was terminated by removing all grazers from the flumes. Snail shell length was measured with a dial caliper. In addition, snails were freeze-dried and their soft bodies were weighed with a microbalance. The remaining periphyton was scraped off each tile, homogenised in tap water and filtered onto pre-combusted glass fiber filters for dry mass, elemental and pigment analyses.
Periphyton dry mass and C:P analysis
For dry mass analysis, periphyton samples were filtered onto pre-weighed GF/F glass fibre filters, which were dried at 60 °C for 24 h and weighed with a microbalance to the nearest µg. Periphyton C content was measured with a Vario EL Cube elemental analyser (Elementar Analysensysteme GmbH, Hanau, Germany). For the analysis of periphyton particulate P, filters were transferred to a solution of 9% potassium peroxodisulfate and 0.9% sulphuric acid and heated at 100 °C for one hour in a DigiPREP Block Digestion System (SCP Science, Quebec, Canada). P analysis was subsequently performed with the molybdate-ascorbic acid method37 with a DR5000 UV–Vis spectrophotometer (Hach, Düsseldorf, Germany).
Pigment extraction and analysis
For pigment extraction, filters were transferred into 96% ethanol, left at room temperature for 2 h and stored overnight at − 20 °C. The freezing–thawing cycle was performed twice, and the sample tubes were subsequently placed in an ultrasound bath for 1 min and centrifuged to remove filter residues. The samples were transferred into vials and analyzed via high performance liquid chromatography with a Thermo Scientific UltiMate 3000 HPLC System (Dionex, Thermo Fisher Scientific Corporation, Waltham, MA, USA). Pigments were separated with a reverse phase YMC C30 column. The two solvents used were composed of (A) 45:20:30:5 methanol: acetonitrile:water:ion pair reagent (ammonium acetate + tetrabutylammonium acetate) and (B) 30:50:20 methanol:acetonitrile:ethyl acetate. The flow gradient was the following: 0–4 min: 80% solvent A, 20% solvent B; 35–75 min: 100% solvent B; 77–80 min: 80% solvent A, 20% solvent B. The flow rate was 0.2 ml min-1 and the column oven was set at 35 °C.
Pigment measurements were used to estimate the community composition of periphyton with CHEMTAX (version 1.95, Wright and Mackey, Hobart, Australia)38,39. The pigment:chlorophyll a ratio matrix for oligotrophic environments from Schlüter et al.21 was used as input ratio matrix for the P− treatments, whereas the matrix for meso-eutrophic environments21 was used for the P+ treatments. From the input matrix, CHEMTAX generated 60 ratio matrices for each treatment. The six matrices (10%) with the lowest residual root mean square were averaged to create a final ratio matrix for each treatment, which was run repeatedly until the ratios and root mean square were stable.
Statistical analyses
Statistical analyses were performed in R (R Core Team, version 3.6.1, 2019). All data were checked and approved for normal distribution with a Shapiro–Wilk’s test and for homogeneity of variance with a Levene’s test.
Two-way ANOVAs were used to determine the interactive effects of phosphorus and light on periphyton dry mass, C:P ratio and taxonomic composition (measured as diatom abundance) at the beginning of the grazing phase. The interactive effects of phosphorus addition, light and grazing over time on periphyton dry mass during the grazing phase were determined with a linear mixed effects model using phosphorus, light, grazing and time as fixed effects and flume identity as random effect. To analyse the effects of top-down (i.e. grazing) and bottom-up factors (i.e. light and phosphorus) both separately and together over time, the interactions tested in the model were time × grazing, time × phosphorus, time × light, time × phosphorus × light, and time × phosphorus × light × grazing. The individual effects of phosphorus, light and grazing on periphyton dry mass were additionally included in the model.
The interactive effects of phosphorus, light and grazing on periphyton C:P ratio and taxonomic composition at the end of the experiment were determined with three-way ANOVAs. Finally, the interactive effects of phosphorus and light on snail shell length and soft body mass were determined with two-way ANOVAs, where the average shell length and soft body mass for each flume were counted as a replicate. All two-way and three-way ANOVAs were followed by Tukey’s HSD post-hoc tests.
Source: Ecology - nature.com
