Quantification of Coral Chlorophyll UsingShimadzu UV-VIS 2450 Spectrophotometer v1
Authors: Mclachlan Rowan , G Grottoli Andrea , Vega Thurber Rebecca 
Published: October 2024

Most corals living in shallow, tropical waters have a symbiotic relationship with unicellular dinoflagellate algae belonging to the family Symbiodiniaceae. These algal cells are located within membrane-bound vacuoles in gastrodermal cells in the oral layer of the host coral and thus, are referred to as endosymbiotic. These endosymbiotic algae can photosynthetically fix carbon due to the presence of chlorophyll a and c2 photopigments within their chloroplast organelles. The overall photosynthetic capacity of corals is affected by the concentration of total chlorophyll pigments per endosymbiotic algal cell and the density of endosymbiotic algal cells in the coral host. When corals are exposed to stressful environmental conditions, including prolonged periods of elevated seawater temperature or high ultraviolet radiation, the symbiotic relationship between corals and algae is disrupted. During this process, known as coral bleaching, the concentration of total chlorophyll pigments per endosymbiotic algal cell can decrease (i.e., photopigment degradation) and/or the density of endosymbiotic algal cells in the coral host can decrease (i.e., algal loss) (e.g. Porter et al. 1989; Lesser 1997; Grottoli et al. 2006). Both processes result in the coral colony appearing pale or white (i.e., bleached), and photosynthesis is reduced.Chlorophyll concentration is the second most commonly used metric for assessing coral bleaching status after Symbiodiniaceae density (McLachlan et al 2020). This protocol outlines a method of quantifying the concentration of total chlorophyll pigments (i.e., chlorophyll a and c2) per unit of biomass (i.e., micrograms of chlorophyll per gram dry weight) of Scleractinian coral samples that have been ground into a homogenous paste consisting of aragonite skeleton, coral host tissue, and endosymbiotic Symbiodiniaceae cells. This procedure employs a double solvent extraction (using 100% acetone) and uses a Shimadzu UV-VIS 2450 spectrophotometer to measure the photopigment absorbance at three wavelengths of light (630 nm, 663 nm, and 750 nm). If you are using a different make and model of spectrophotometer, please fork this protocol and replace the "Measuring Chlorophyll absorbance" steps with instructions appropriate to the spectrophotometer you are using. The concentration of chlorophyll a and c2 pigments are calculated using the equations from Jeffrey & Humphrey (1975) for dinoflagellates using 100% acetone solvent. There are nine steps to this protocol that are completed over five days: 1) Preparation of ground coral samples 2) Day 1: First acetone extraction 3) Day 2: Measuring Chlorophyll absorbance from first extraction 4) Day 2: Second acetone extraction 5) Day 3: Measuring chlorophyll absorbance from second extraction 6) Day 3: Filter and dry the chlorophyll samples 7) Day 4: Weigh dried pans then combust the dried samples 8) Day 5: Weigh burnt pans 9) Calculations This protocol was written by Dr. Rowan McLachlan and was reviewed by co-authors (Dr. Andréa Grottoli and Dr. Rebecca Vega Thurber). Acknowledgments I would like to thank Dr. Kimberly H. Halsey (Oregon State University) for kindly allowing me access to her UV-Vis Spectrophotometer and Vaishnavi Padaki (Oregon State University) for training me to use this instrument and facilitating access to the laboratory.