Fluorescence Lifetime Imaging Microscopy (FLIM) as a method to elucidate trends in marine planktonic symbioses
The Retaria are an abundant planktonic protist lineage that play a substantial role in biogeochemical cycling, primary production, and food web dynamics in open ocean ecosystems. Their impact is, in large part, due to their capacity to host photosynthetic endosymbionts. Despite the importance of these relationships in the global oceans, our knowledge about the diversity of photosymbionts associated with the planktonic Retaria is still limited due to the difficulty of collecting and analyzing these cells. Here we apply a new protocol to assess retarian photosymbiont diversity: Fluorescence Lifetime Imaging Microscopy (FLIM) is an easy-to-use method to distinguish between different sources of fluorescence during confocal microscopy. We applied FISH probes that hybridize with the most commonly reported retarian photosymbiont lineages to environmental plankton samples and used FLIM to confirm the presence or absence of our FISH probes in retarian cells. By analyzing the fluorescence lifetime of 92 photosymbionts, we found that the retarian photosymbiosis is far more complex than originally described, where Retaria commonly host multiple algal lineages per cell, and regularly host algal lineages outside of the two most common lineages. These efforts demonstrate that FLIM is an overlooked tool in the study of host-microbe associations, with significant potential to be used across diverse host lineages and environmental samples.