Supplementary information for Altermatt et al. Methods in Ecology and Evolution. DOI: 10.1111/2041-210X.12312
“Big answers from small worlds: a user's guide for protist microcosms as a model system in ecology and evolution”
Altermatt F, Fronhofer EA, Garnier A, Giometto A, Hammes F, Klecka J, Legrand D, Mächler E, Massie TM, Pennekamp F, Plebani M, Pontarp M, Schtickzelle N, Thuillier V & Petchey OL
3.2 Density manipulation
Density manipulations are an important technique used in microcosm studies using protists to answer questions related to population dynamics (Gause 1934b; Gause 1934a) and density regulation (Luckinbill & Fenton 1978), but also dispersal (Hauzy et al. 2007; Fellous et al. 2012; Fronhofer & Altermatt 2014; Fronhofer, Kropf & Altermatt 2014; Pennekamp et al. 2014), life history evolution (Luckinbill 1979) and cooperative behaviours and sociality in microbes (Chaine et al. 2010).
As long as densities are manipulated within the range zero to carrying capacity (K), it is sufficient to grow cultures to K and subsequently dilute them. In case of density manipulations beyond K, or if reaching K takes a long time for slowly growing species, there are two methods to concentrate cells, namely centrifugation and reverse filtration. Centrifugation of cultures is the standard procedure to concentrate cells, if necessary to levels far beyond carrying capacity (orders of magnitude). Luckinbill & Fenton (1978) used hand centrifugation for their tests of population regulation, whereas Warren & Spencer (1996) concentrated cultures of various bacterivorous protists using centrifugation at 1000 rpm for 5 min. Fjerdingstad et al. (2007) used centrifugation to concentrate cultures and remove nutrients from the culture for a starvation experiment. They centrifuged cultures of T. thermophila at 2000 rpm for three minutes and repeated this procedure four times. Unfortunately, most studies so far state rotations per minute, which translate however into different g-forces according to the diameter of the rotating axis and the different types of centrifuges (swing-head versus fixed). Reporting g forces is therefore recommended to guarantee comparisons among studies. Centrifugation exposes cells to considerable physical stress. Thus, care has to be taken that the manipulation does not introduce artefacts into the experimental design or has other unwanted side effects that may be confounded with the effect of the density manipulation.
An alternative for concentration is reverse filtration, whereby the medium is filtered out and where the supernatant containing the cells is retained. This method has the advantage that it is less stressful to the cells, but only about 2- to 4-fold concentrations are possible.
Equipment for centrifugation
Appropriate tubes for centrifugation (resisting the physical forces acting on the tubes during the procedure)
Equipment for reverse filtration
Vacuum aspirator or disposable hand held syringes
Filters with pore sizes smaller than the protists of interest (e.g., ≤1 µm) that can be attached to a vacuum aspirator or to disposable hand held syringes
- Medium/water to re-suspend cell pellet
Place medium with the protists into the appropriate centrifugation tube.
Centrifuge the tubes for 2 minutes at appropriate rpm / g.
Quickly remove the supernatant.
Re-suspend protist cells in the remainder of medium or some replacement liquid depending on the goal.
Quickly proceed with the processing of the cultures, given that a small medium volume with high individual numbers will quickly deplete the remaining oxygen.
Place medium with the protists into an appropriate tube, e.g., 50 mL of protist culture.
Start removing medium by putting the tip of the filter into the medium and creating a vacuum pressure (either with vacuum pump or with the disposable syringe), such that medium is sucked through the filter out of the protist culture.
Importantly, the process of filtration needs to be done carefully and slowly (generally >30 s for removing 50% of the medium in a 50 mL culture), such that protists do not get stuck on the filter but remain in the supernatant.
Dispose the filtrate, and keep the supernatant with the protists at a concentrated density.
The total volume of medium (of initial culture) divided by volume of the supernatant gives the level of concentration (e.g., 50 mL of initial culture, 12.5 mL of supernatant and 37.5 mL of discarded filtrate give a 4-fold concentration of the culture).
Chaine, A.S., Schtickzelle, N., Polard, T., Huet, M. & Clobert, J. (2010) Kin-based recognition and social aggregation in a ciliate. Evolution, 64, 1290-1300.
Fellous, S., Duncan, A., Coulon, A.l. & Kaltz, O. (2012) Quorum Sensing and Density-Dependent Dispersal in an Aquatic Model System. PLoS ONE, 7, e48436.
Fjerdingstad, E., Schtickzelle, N., Manhes, P., Gutierrez, A. & Clobert, J. (2007) Evolution of dispersal and life history strategies - Tetrahymena ciliates. BMC Evolutionary Biology, 7, 133.
Fronhofer, E.A. & Altermatt, F. (2014) Eco-evolutionary dynamics during experimental range expansions. Nature Communications, in review.
Fronhofer, E.A., Kropf, T. & Altermatt, F. (2014) Density-dependent movement and the consequences of the Allee effect in the model organism Tetrahymena. Journal of Animal Ecology, in press.
Gause, G.F. (1934a) Experimental analysis of Vito Volterra’s mathematical theory of the struggle for existence. Science, 79, 16-17.
Gause, G.F. (1934b) The Struggle for Existence. Dover Publications, Mineaola, N.Y.
Hauzy, C., Hulot, F.D., Gins, A. & Loreau, M. (2007) Intra- and interspecific density-dependent dispersal in an aquatic prey-predator system. Journal of Animal Ecology, 76, 552-558.
Luckinbill, L.S. (1979) Selection and the r/K Continuum in Experimental Populations of Protozoa. The American Naturalist, 113, 427-437.
Luckinbill, L.S. & Fenton, M.M. (1978) Regulation and environmental variability in experimental populations of protozoa. Ecology, 59, 1271-1276.
Pennekamp, F., Mitchell, K.A., Chaine, A. & Schtickzelle, N. (2014) Dispersal propensity in Tetrahymena thermophila ciliates—a reaction norm perspective. Evolution, 68, 2319-2330.
Warren, P.H. & Spencer, M. (1996) Community and food-web responses to the manipulation of energy input and disturbance in small ponds. Oikos, 75, 407-418.