Diuron

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et al. 2009 (link)), which in spawning experiments typically behaves as a male (S. F. Perez and J. R. Pringle, unpublished data).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.

Lugol’s iodine preserved cells were enumerated using a Beckman Coulter Multisizer™ 3 Coulter Counter® with a 50 µm aperture which allowed cell densities to be quantified with a relative standard deviation of 3%. Cell densities of selected samples were verified microscopically with a hemacytometer. Growth rates were calculated for each day of the experiment based upon changes in cell abundance according to the equation µ = Ln(N₂/N₁)/(t₂–t₁) where N₁ and N₂ equal the biomass at time 1 (t₁) and time 2 (t₂) respectively [54] . Nitrate was analyzed by reducing the nitrate to nitrite using spongy cadmium as per Jones [55] . Ammonium and phosphate were analyzed using techniques modified from Parsons et al[56] . Total dissolved N and P were analyzed using persulfate digestion techniques from Valderrama [57] . Urea was analyzed according to Price and Harrison [58] . These nutrient analyses provided 100±10% recovery of standard reference material (SPEX CertiPrep™) for nitrate, ammonium, phosphate, total dissolved N, and total dissolved P. Whole water samples were analyzed for the hepatoxin microcystin by first freezing samples at −80°C for 24 h and then lysing the cells using an Abraxis QuikLyse™ Cell Lysis kit for Microcystins/Nodularins ELISA Microtiter Plate according to the manufacturer’s instructions. Lysed samples were then analyzed with a colorimetric immunoassay using an Abraxis Microcystins/Nodularins (ADDA) ELISA Kit according to the manufacturer’s instructions [59] (link). This method provided an analytical precision of ±2% and a 96±2% recovery of spiked samples. Bulk alkaline phosphatase activity was measured for each replicate experimental sample on a Turner Designs TD-700 fluorometer (EM filter of 410–600 nm and EX filter of 300–400 nm) using 4-Methylumbelliferone phosphate (250-µM concentration) as the substrate [60] . Alkaline phosphatase activity measured by this assay has been shown to be significantly correlated with the expression of the gene encoding for alkaline phosphatase (phoX) in Microcystis aeruginosa LE-3 (p<0.005) [34] (link) and provided an analytical precision of ±4%. Maximum quantum efficiency of photosystem II (PSII) was estimated from in vivo (F_i) and DCMU (3,4-dichlorophenyl-1,1-dimethylurea)-enhanced in vivo fluorescence (F_m) of each replicate experimental sample on a Turner Designs TD-700 fluorometer (EM filter of >665 nm and EX filter of 340–500 nm). All readings were blank corrected using BG-11 media. DCMU blocks electron transfer between PSII and PSI and yields maximal fluorescence and previous studies have demonstrated that F_v/F_m can be a sensitive diagnostic of nutrient limitation, reaching a maximal value of ∼ 0.65 under nutrient replete conditions, and decreasing to less than half of that under nutrient limitation [61] (link).

For evaluation of CEF activity, P700⁺ re-reduction and the redox kinetics of P700 were determined according to previously described methods^{65 (link),66 (link)} with some modifications. Briefly, Synechococcus cells were cultured in BG11 medium at an initial OD₇₃₀ of 0.05 in column photobioreactors and cultivated in MC1000 under 30 °C and 50 μmol photons/m²/s bubbled with air. Cells in the mid-log phase were harvested and adjusted to approximately 6 × 10⁹ cells in 3 mL and were dark-acclimated for 20 min at room temperature (23 °C). P700 redox was monitored after the termination of AL illumination (2800 μE/m²/s for 35 s) under a background of FR light (Intensity level 20) using Dual-PAM 100 (Heinz Walz) (Dual PAM v1.19 software). The P700 levels were normalized by equating the absorbance minimum after the termination of AL illumination to 0 and equating the absorbance maximum of 3 s before the termination of AL illumination to one. The re-reduction of P700⁺ in darkness was measured by monitoring absorbance changes at 830 nm and using 875 nm as a reference. After dark-acclimated for 20 min, 20 μM DCMU was added to the cultures before the measurement. The P700 was oxidized by FR light (Intensity level 20) for 40 s, and the subsequent re-reduction of P700⁺ in the dark was monitored. The curves were normalized by equating the absorbance maximum of 1 s before termination of FR light to one. The initial rate of P700⁺ re-reduction was acquired from normalized curves by calculating the slope of the initial values after FR was turned off and the R² was more than 0.99.

Approximately 1.7 × 10⁹ cells in 3 mL were dark-acclimated for 20 min at room temperature (23 °C). And the fluorescence parameters F_v/F_m (maximum quantum yield) of PSII were measured using a Dual-PAM 100 (Heinz Walz) (Dual PAM v1.19 software). A measure light intensity of 19 μE/m²/s was used and the saturating light pulse was 6000 μE/m²/s (200 ms). The maximum chlorophyll fluorescence after adding 20 μM DCMU with AL (54 μE/m²/s) was recorded as the maximum F_m.