Evaluation of laser scanning confocal microscopy as a method for characterizing reef-building coral tissue thickness and Symbiodiniaceae fluorescence

Abstract

Predicting the sensitivity of reef-building corals to disturbance, including bleaching, requires an understanding of physiological responses to stressors, which may be limited by destructive sampling and the capacity of common methodologies to characterize early life history stages. We developed a new methodology using laser scanning confocal microscopy (LSCM) to measure and track the physiological condition of corals. In a thermal stress experiment, we used LSCM to track coral condition during bleaching in adults and juveniles of two species, Montipora capitata and Pocillopora acuta. Depth of fluorescence in coral tissues provides a proxy measure of tissue thickness, whereas Symbiodiniaceae population fluorescence relates to both population density and chlorophyll a content. In response to thermal stress, there were significant shifts in tissue thickness and Symbiodiniaceae fluorescence with differences between life stages. This method is particularly well suited for detecting shifts in physiological condition of living corals in laboratory studies, especially in small juvenile colonies.

Preconditioning improves bleaching tolerance in the reef‐building coral Pocillopora acuta through modulations in the programmed cell death pathways

“…The bleaching rate was expressed as the gradual loss of symbiont-derived signal to host-derived signal over time (Figure 1c). The use of live laser-scanning confocal microscopy has been previously established to evaluate coral bleaching and it showed that the host-derived signal relates to the tissue thickness, and algal-derived signal mirrors both Symbiodinium cell density and chlorophyll α content (Huffmyer et al, 2020).…”

Feeding and thermal conditioning enhance coral temperature tolerance in juvenilePocillopora acuta

“…Feeding increases calcification and growth, tissue biomass and symbiont carbon fixation in adult colonies [4,12,20] and here we show that juvenile colonies also respond to feeding by increasing tissue thickness and symbiont fluorescence. Enhanced symbiont fluorescence (related to increased cell density and/or photosynthetic pigments [61]) could potentially result in elevated levels of symbiont photosynthesis and translocation of photosynthates [62] in fed juveniles that would increase coral energy storage and serve as a key factor in promoting tissue and skeletal growth [32,58,71]. In addition to the positive effects of feeding on growth and biomass, fed juvenile P. acuta demonstrated higher thermal tolerance.…”

“…Briefly, autofluorescence of both the coral host and Symbiodiniaceae was used to characterize tissue thickness and Symbiodiniaceae fluorescence, respectively. First, the average depth of tissue fluorescence (μm) was measured on each colony as a proxy of tissue thickness [61] on three cross sections (n = 3 measurements per cross section) of three-dimensional rendered models. Second, the average intensity of fluorescence emitted by Symbiodiniaceae was measured on each colony (n = 3 measurements per colony), and these values were calibrated to relative intensity (RI; Red InSpeck Microscope Image Intensity Calibration Kit, Molecular Probes, Sigma-Aldrich, St. Louis, MO, USA).…”

“…Second, the average intensity of fluorescence emitted by Symbiodiniaceae was measured on each colony (n = 3 measurements per colony), and these values were calibrated to relative intensity (RI; Red InSpeck Microscope Image Intensity Calibration Kit, Molecular Probes, Sigma-Aldrich, St. Louis, MO, USA). Previous work has shown that Symbiodiniaceae fluorescence measured by LSCM is positively related to both Symbiodiniaceae chlorophyll a content and population cell densities [61], and therefore, in this study, we use fluorescence as a proxy measure for these characteristics, which are related to the potential for energy acquisition and translocation by the symbiont population [62].…”

“…Open Sci. 8: 210644 following skeletal decalcification) and Symbiodiniaceae cell density using a hemocytometer, respectively (electronic supplementary material, figure S3), using methods modified from [61]. We chose to validate this methodology using measurements of fed and unfed colonies as previous research has demonstrated the strong effect of feeding on coral physiology [4] that allowed us to ground-truth LSCM measurements.…”