Fluorescence Lifetime Imaging Microscopy (FLIM) reveals the diversity and complexities of an open ocean photosymbiosis

Abstract

The Retaria are an abundant marine 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 planktic Retaria is still limited due to the difficulty of collecting and analyzing these cells. Here we used Fluorescence in situ Hybridization (FISH) in combination with Fluorescence Lifetime Imaging Microscopy (FLIM) to assess the frequency and abundance of common algal photosymbiont lineages. We applied FISH probes that hybridize with the most commonly reported retarian photosymbiont lineages to environmental plankton samples and used FLIM to distinguish chlorophyll autofluorescence of retarian symbionts from FISH probe signal. By analyzing the fluorescence lifetime of 92 photosymbionts across 71 hosts, 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. Similarly, the most prevalent photosymbiont species to open ocean Retaria (the dinoflagellate Brandtodinium nutricula) was only present in 34% of hosts according to our analyses. These efforts demonstrate that FLIM is an overlooked tool in the study of host-microbe associations, with significant potential to be used to distinguish chlorophyll autofluorescence from FISH probe signal in environmental samples.