# Small Things that Make a Big Difference: Single-Cell Transcriptomic of Nanociliates Reveals Genes Potentially Involved in Mixotrophy

## TL;DR

This study explores the genes of marine nanociliates to understand their mixotrophic abilities, combining photosynthesis and digestion.

## Contribution

The study identifies genes related to both photosynthesis and digestion in nanociliates, suggesting a broader basis for mixotrophy than previously thought.

## Key findings

- Two nanociliates clustered with Tintinnida, while three clustered with Oligotrichida based on phylogenetic analysis.
- Photosynthesis-related genes were found in all transcriptomes, along with genes for phagosome, lysosome, and metabolic pathways.
- The findings suggest that mixotrophy in nanociliates involves more than just photosynthesis-related genes.

## Abstract

Nanociliates play an important role in the microbial food web of oligotrophic marine systems as grazers of picoplankton on one side, and as prey for microplankton, on the other. However, knowledge on their taxonomy, phylogeny, and trophic strategies is very limited, as well as their potential role as mixotrophs. In the present study, we investigated the transcriptomes of five marine planktonic nanociliates isolated from the Eastern Mediterranean Sea. Our aim was the following: (i) to characterize the phylogenetic placement of these cells using concatenated phylotranscriptomic and (ii) to identify genes potentially involved in mixotrophy by focusing on both photosynthesis and digestion-related genes (phagosome, lysosome). Phylogenetic reconstruction revealed that two cells clustered with Tintinnida, while the other three clustered with Oligotrichida. Reciprocal best hits (RHBs) BlastP analysis indicated the presence of genes related to photosynthesis across all the transcriptomes, while the detection of genes associated with phagosome, lysosome, and generic metabolic pathways provided a more informative insight into the mechanism of mixotrophy. These findings suggest that photosynthesis-related genes alone may not be sufficient indicators of mixotrophic potential in nanociliates and highlight the importance of considering additional cellular pathways involved in phagotrophy. Moreover, these transcriptomes will help to establish a basis for the evaluation of differential gene expression in Oligotrichida, Choreotrichida, and Tintinnida, and a step stone for mixotrophic investigation.

The online version contains supplementary material available at 10.1007/s00248-025-02575-4.