Methodological precision of in situ and in vitro algal density measurements in the model cnidarian, Exaiptasia diaphana

Abstract

In cnidarian symbiosis research, studying algal uptake, maintenance, and expulsion typically requires quantification of algal density in host tissue. Multiple methods are used to measure algal density including in vitro cell counts of holobiont homogenate and in situ cell counts of tentacle clippings. The relative precision of both types of measurement has not previously been reported for the model cnidarian Exaiptasia diaphana in the fully symbiotic state. The objective of this study was to evaluate the precision of in vitro and in situ algal density measurement protocols using light, fluorescent, and confocal microscopy and an automated cell counter. In situ algal density was quantified as algal area fraction (%) using confocal images of tentacle clippings mounted on two types of slides. In vitro algal density of holobiont homogenate was quantified as algal cells/µl of holobiont homogenate using an automated cell counter and a hemocytometer viewed using light and fluorescent microscopy. Triplicate measurements of each method for ten anemones were collected and the coefficient of variation was calculated and compared across the ten anemones within each method. The algal density measurements were equally precise when they were obtained by quantifying in vitro cell counts using a hemocytometer and when they were obtained by quantifying in situ cell counts. While both light and fluorescent microscopy yielded similar measurement precision of in vitro cell counts, use of a fluorescent microscope was more efficient and convenient than use of a light microscope, and both methods required terminal sampling. Conversely, in situ methods required more sophisticated equipment (namely a confocal microscope) but involved non-terminal sampling. An automated cell counter was ineffective for in vitro quantification of algal density, although the potential utility of this technology warrants future attempts using a more robust algal cell purification process that could include filtering homogenate prior to analysis. This study demonstrated that in vitro and in situ methods yield estimates of algal density with comparable precision, which is information that researchers can use for future studies when making decisions about methodology.