Abstract
The uses of fluorescent microscopy and fluorescent probes, such as the metabolically activated probe CellTracker™ Green CMFDA (CTG), have become common in studies of living Foraminifera. This metabolic requirement, as well as the relatively quick production of the fluorescent reaction products, makes CTG a prime candidate for determining mortality in bioassay and other laboratory experiments. Previous work with the foraminifer Amphistegina gibbosa, which hosts diatom endosymbionts, has shown that the species is capable of surviving both acute chemical exposure and extended periods of total darkness by entering a low-activity dormant state. This paper explores the use of CTG and fluorescent microscopy to determine mortality in such experiments, as well as to explore the physiology of dormant foraminifers. The application of CTG was found to be complicated by the autofluorescence of the diatom symbionts, which masks the signal of the CTG, as well as by interactions between CTG and propylene glycol, a chemical of interest known to cause dormancy. These complications necessitated adapting methods from earlier studies using CTG. Here we present observations on CTG fluorescence and autofluorescence in A. gibbosa following both chemical exposure and periods of total darkness. While CTG can indicate vital activity in dormant foraminifers, complications include underestimates of total survival and recovery, and falsely indicating dead individuals as live due to rapid microbial colonization. Nonetheless, the brightness of the CTG signal in dormant individuals exposed to propylene glycol supports previously published results of survival patterns in A. gibbosa. Observations of CTG fluorescence in individuals kept for extended periods in aphotic conditions indicate uptake of CTG may begin within 30 min of exposure to light, suggesting darkness-induced dormancy and subsequent recovery can occur on short time scales. These results suggest that CTG accurately reflects changes associated with dormancy, and can be useful in laboratory experiments utilizing symbiont-bearing foraminifers.
Highlights
Fossil foraminiferal shells have been key tools in paleontological applications for more than a century
The specific goals of this paper were the following: (a) to adapt methods utilizing CTG to observations of A. gibbosa in laboratory toxicity experiments; (b) to use these methods to determine if CTG fluorescence is a valid tool for distinguishing mortality versus survival in A. gibbosa that may be dormant, including both toxicity- and darkness-induced dormancy; and (c) to determine what fluorescence microscopy can reveal about the activity of dormant individuals
Based on non-parametric ANOVA, significant differences in coverage were only observed in Experiment 1 (72-hr recovery after propylene glycol (PG) exposure, df = 92, p = 0.03) between the 1.5% PG and control treatments (p = 0.02), 3% PG treatment (0.01) and 8% PG treatment (0.01)
Summary
Fossil foraminiferal shells have been key tools in paleontological applications for more than a century. In the past half century, shell assemblages have become widely used tools in environmental monitoring and assessment, and live foraminifers are increasingly being. How to cite this article Ross and Hallock (2018), Challenges in using CellTracker Green on foraminifers that host algal endosymbionts. Over the past 20+ years, foraminiferal assemblages and selected populations have become increasingly used to assess and monitor environmental conditions associated with coral reefs, which are in decline worldwide (e.g., De’ath et al, 2012; Perry et al, 2013; others). Hallock (2000) summarized the potential and benefits of using reef-dwelling larger benthic foraminifers (LBF) as indicators of water quality conducive to coral-reef accretion. Experimental approaches have included studies of growth rates (Hallock, Forward & Hansen, 1986), photosynthetic activity (e.g., Talge & Hallock, 2003; Méndez-Ferrer, Hallock & Jones, 2018), prevalence of morphological anomalies (e.g., Prazeres, Uthicke & Pandolfi, 2016), symbiont loss (e.g., Hallock et al, 1995), and, most recently, proteomics (e.g., Prazeres et al, 2011; Stuhr et al, 2018) and antioxidant capacity (i.e., Prazeres, Uthicke & Pandolfi, 2016; Stuhr et al, 2017)
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