Ecological Archives E095-223-A1

Sanni L. Aalto, Tarmo Ketola, Katja Pulkkinen. 2014. No uniform associations between parasite prevalence and environmental nutrients. Ecology 95:2558–2568. http://dx.doi.org/10.1890/13-2007.1

Appendix A. Description of the study site, sampling procedure, and chemical analysis.

Study site

Lake Mekkojärvi (61º13´ N, 25º8´ E) is small (0.35 ha) humic lake in Southern Finland in the Evo forest area. The lake has a maximum depth of 4.3 m and mean depth of 3 m and high concentration of dissolved organic carbon (DOC; 20 to 45 mg C/L; Taipale et al. 2008). In winter the lake is covered with ice, and mostly anoxic due to the shallowness and high organic content of water, preventing establishment of fish populations. Open water season starts usually in May and ends in October -November. After the spring turnover, surface water warms up rapidly leading to temperature, oxygen and nutrient gradients. Lake has a narrow oxic and euphotic epilimnion (0.5–1 m) and main nutrients (free nitrogen and phosphorus) are rapidly consumed in the epilimnion during the spring phytoplankton bloom (Salonen and Lehtovaara 1992). The annual primary production of the phytoplankton is below 10 mg C/m2 and decreases from spring to autumn (Salonen et al. 2005). In epilimnion, cryptophytes (Cryptomonas spp.) and chrysophytes (Mallomonas spp.) are the most abundant phytoplankton taxa. In meta- and upper hypolimnion, prasinophyte Scourfieldia cordiformis and flagellate chlorophytes (Chlamydomonas spp.) are rather abundant throughout the open water season (Arvola et al.1992). The bacterial biomass increases from spring to autumn being highest during autumnal mixing (Taipale et al. 2009). Most abundant groups (in terms of abundance and biomass) are anaerobic photoautotrophic green sulphur bacteria (e.g., Chlorobium sp.) and metanotrophic bacteria (Type I MOB) in the oxic-anoxic boundary layer and in hypolimnion (Taipale et al. 2009, Karhunen et al. 2013). Heterotrophic bacteria affiliated to Actinobacteria and Polynucleobacter spp. are less abundant but met throughout the open water season (Taipale et al. 2011).

Predation pressure towards D. longispina is low, because the lake lacks zooplanktivorous fish and the main predators are Chaoborus larvae at densities ca. 300 ind/m2 and Notonecta sp.(Salonen and Lehtovaara 1992).

Sampling procedure and chemical analyses

From the epilimnion and metalimnion, we pooled two replicate lake water samples within each layer and from hypolimnion we derived a pooled sample collected from three discrete depths (1.2–1.8, 1.8–2.4 and 2.4–3.0 m).

Water samples for inorganic nitrogen (NH4, NO2+NO3) and phosphorus (PO4), stored frozen prior analyses, were filtered through glass fiber filters (Whatman GF/F) and measured with QuickChem 8000 analyzer (LaChat instruments, Loveland, CO, USA). Total nitrogen (TN) and total phosphorus (TP) of the seston were measured from the unfiltered water samples respectively after digestion by alkaline persulphate. Alkalinity, pH, conductivity and water color were analyzed using the validated routine methods of the Finnish Standard Association (www.sfs.fi). For chlorophyll a determinations, a 0.5 L water sample from each layer was filtered through a glass fiber filter (Whatman GF/F). Pigments were extracted in ethanol and their absorptions measured with a spectrophotometer (UV-240, Shimadzu, Kyoto, Japan) at 665 nm and 750 nm and the concentration of chlorophyll a was calculated according to Lorenzen (1967). Seston N and P concentrations can be used as proxies of the stoichiometric quality of the food sources in Lake Mekkojärvi (Järvinen and Salonen 1998).

For determining the stoichiometry (%C, %N) and stable isotope values of carbon and nitrogen (δ13C and δ15N) of Daphnia, animals were picked into tubes, freeze-dried and weighed into tin cups. Generally, two to three replicates were prepared from one sample tube, each with dry mass approximately 0.5 mg. Samples were analyzed with a Carlo-Erba Flash 1112 series Elemental Analyzer connected to a DELTAplus Advantage IRMS (Thermo Finnigan, Waltham, MA, USA) and run against IAEA standard NBS-22 using dried and homogenized fish muscle as internal laboratory working standards. δ13C values were corrected for lipids according to Syväranta and Rautio (2010). For determining %P, freeze-dried Daphnia were combusted (450ºC, 4 h), diluted in 0.2N H2SO4 and measured with a QuickChem 8000 analyzer.

Literature cited

Arvola L., K. Salonen, P. Kankaala, and A. Lehtovaara. 1992. Vertical distributions of bacteria and algae in a steeply stratified humic lake under high grazing pressure from Daphnia longispina. Hydrobiologia 229:253–269.

Järvinen M., and K. Salonen. 1998. Influence of changing food web structure on nutrient limitation of phytoplankton in a highly humic lake. Canadian Journal of Fisheries and Aquatic Sciences 55:2562–2571.

Karhunen J., L. Arvola, S. Peura, and M. Tiirola. 2013. Green sulphur bacteria as a component of the photosynthetic plankton community in small dimictic humic lakes with an anoxic hypolimnion. Aquatic microbial ecology 68:267–272.

Lorenzen C. J. 1967. Determination of chlorophyll and pheo-pigments: spectrophotometric equations. Limnology and Oceanography 12:343–346.

Salonen K., T. Hammar, P. Kuuppoleinikki, U. Smolander, and A. Ojala. 2005. Robust parameters confirm predominance of heterotrophic processes in the plankton of a highly humic pond. Hydrobiologia 543:181–189.

Salonen K., and A. Lehtovaara. 1992. Migrations of haemoglobin-rich Daphnia longispina in a small, steeply stratified, humic lake with an anoxic hypolimnion. Hydrobiologia 229:271–288.

Syväranta, J., and M. Rautio 2010. Zooplankton, lipids and stable isotopes: importance of seasonal, latitudinal, and taxonomic differences. Canadian Journal of Fisheries and Aquatic Sciences 67:1721–1729.

Taipale S. J., P. Kankaala, M. W. Hahn, R. I. Jones, and M. Tiirola. 2011. Methane-oxidizing and photoautotrophic bacteria are major producers in a humic lake with a large anoxic hypolimnion. Aquatic Microbial Ecology 64:81–95.

Taipale S., P. Kankaala, M. Tiirola, and R. I. Jones. 2008. Whole-lake DI13C additions reveal seasonal shifts between multiple food source contributions to zooplankton diet. Ecology 89:463–474.

Taipale S., R. I. Jones, and M. Tiirola. 2009. Vertical diversity of bacteria in an oxygen-stratified humic lake, evaluated using DNA and phospholipid analyses. Aquatic Microbial Ecology 55:1–16.


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