Generating Symbiotic and Aposymbiotic Sea Anemones

All animals were from clonal population CC7 (Sunagawa et al. 2009 (link)), which in spawning experiments typically behaves as a male (S. F. Perez and J. R. Pringle, unpublished data). For experiments performed at Stanford, the stock cultures were grown in a circulating artificial sea water (ASW) system at ∼25° with 20 to 40 µmol photons m⁻² s⁻¹ of photosynthetically active radiation on an ∼12-hr light/12-hr dark (12L:12D) cycle and fed freshly hatched brine shrimp nauplii approximately twice per week. To generate aposymbiotic anemones, animals were placed in a separate polycarbonate tub and subjected to several repetitions of the following process: cold-shocking by addition of 4° ASW and incubation at 4° for 4 hr, followed by 1–2 days of treatment at ∼25° in ASW containing the photosynthesis inhibitor Diuron (Sigma-Aldrich D2425) at 50 µM (lighting approximately as noted above). After recovery for several weeks in ASW at ∼25° in the light (as noted above) with feeding (as noted above, with water changes on the days following feeding), putatively aposymbiotic anemones were inspected by fluorescence microscopy to confirm the complete absence of dinoflagellates (whose bright chlorophyll autofluorescence is conspicuous when they are present).
For experiments performed at Cornell, anemones were grown in incubators at 25° in ASW in 1-liter glass bowls and fed (as noted above) approximately three times per week. Symbiotic anemones were kept on a 12L:12D cycle at 18 to 22 µmol photons m⁻² s⁻¹ of photosynthetically active radiation. Aposymbiotic animals were generated by exposing anemones under the same lighting and feeding regimen to 50 µM Diuron in ASW, with daily water changes, for ∼30 d or until the anemones were devoid of algae, as confirmed by fluorescence microscopy. After bleaching, aposymbiotic anemones were maintained in the dark for ∼2 yr (with feeding as noted above) before experimentation.