A novel menthol-DCMU bleaching method for foraminifera: Generating aposymbiotic hosts for symbiosis research

Predicting the response and resilience of coral reefs to climate change can be achieved through better understanding the cellular symbiosis between coral reef holobionts and their associated endosymbiotic algae. Larger benthic foraminifera (LBF) are key calcium carbonate producers, of which two species were investigated for their suitability for menthol bleaching. The LBF Amphistegina lobifera, hosting diatoms, and Sorites orbiculus, hosting dinoflagellates of the family Symbiodiniaceae. This study aimed to rapidly generate symbiont-free (aposymbiotic) hosts via treatment with menthol and DCMU. The first experiment, Menthol Concentration Comparison (MCC), aimed to find a non-lethal and effective dose for both species. The second experiment, Menthol-bleaching Ecophysiology Assessment (MEA), used a larger sample size of both species to test the response to one concentration 0.19 mmol L-1 and measured growth, motility (an indicator for overall fitness) and mortality over a 4-week time frame. Menthol led to an aposymbiotic state in 100% of A. lobifera and only minimally impacted its motility and mortality. The method was effective for S. orbiculus, where an aposymbiotic state, defined as no visible remains of symbiont cells inside the host at the end of the experimental period, occurred in 66% of specimens of the MCC experiment. Growth was strongly impacted by the bleaching protocol in both species, allowing no new calcite to be formed during the acute exposure. This method can be applied for testing aspects of symbiosis establishment in LBF as well as their potential to take up different symbionts in a short-to medium time frame.