PAM fluorometry of chlorophyll fluorescence

Chlorophyll fluorescence can be used to calculate a number of different photosynthetic metrics in a non-invasive way and consequently can be used as a proxy for the health of endosymbiont populations. Over the duration of both experiments maximum dark-adapted quantum yield (Fv/Fm) of chlorophyll a fluorescence was measured at least 30 min after sunset for dark adaption to assess the efficiency of photochemistry. All photochemical measurements were made using an imaging pulse-amplitude-modulated (PAM) fluorometer with MAXI head (Waltz, Effeltrich, Germany). The minimum fluorescence (Fo) was measured with a weak pulse of light, followed by saturating pulses of 2700 μmol m⁻² s⁻¹ of PAR for 0.8 s to establish the maximal fluorescence (Fm). Variable fluorescence yield (Fm—Fo) is then used to calculate the dark-adapted maximum quantum yield (Fv/Fm). Fragments were immediately returned to mesocosms following measurement. In Experiment 1, measurements of quantum yield were taken on Days 3–24. In Experiment 2, measurements of quantum yield were taken on Days 1, 3 and 5–20.

In addition to quantum yield, induction and recovery curves were determined on coral fragments in Experiment 2 on Days 6, 12 and 15–20. As a more sensitive physiological measure than dark-adapted yield, this measurement examines the ability of the endosymbiont to acclimate to and recover from short-term light stress. This measurement was taken to record any physiological differences that may not be shown through typical quantum yield measurements. Specifically, the induction and recovery kinetic recording type on the PAM fluorometer (Waltz, Effeltrich, Germany) was used to examine the ability of endosymbiont PSII to dissipate excess light energy and recover from light stress. Following dark adaption for at least 30 min, minimum chlorophyll fluorescence (Fo) was determined using blue measuring light (Intensity 2) and maximum chlorophyll fluorescence (Fm) was determined by applying a pulse (0.72 s) of saturating light (Intensity 5, ~2800 μmol quanta m⁻² s⁻¹) allowing calculation of the dark-adapted maximal quantum yield of PSII (Fv/Fm). For the induction curve, actinic illumination (254 μmol quanta m⁻² s⁻¹, intensity 6) was switched on and 15 saturating pulses of PAR (~2800 μmol quanta m⁻² s⁻¹, Intensity 5, 0.72 s) were applied at 20 s intervals for 5 min. During the recovery phase, a further 16 saturation pulses were applied within a 7-min period without actinic illumination, where time between each pulse exponentially increased. Imaging-PAM fluorometry was then used to determine photo-kinetic parameters, such as the maximal quantum yield of PS II (Fv/Fm), and the effective quantum yield at the end of the induction. Light levels were measured using a LI-190SA Quantum Sensor with a LI-250A Light Meter (LI-COR^® Inc., NE, USA). Each coral fragment was immediately returned to its respective treatment following PAM measurements. At the end of the experiment, these fragments were returned to the reef flat.