Evaluating acidotropic dyes for detecting mixotrophy in protists: Insights from cultures and field communities

Mixotrophic protists combine photoautotrophic primary production with heterotrophic phagotrophy, and distinctly impact nutrient cycling and microbial food web dynamics in aquatic environments. Despite their biogeochemical importance, detecting and quantifying mixotrophic presence and grazing in situ remains challenging, preventing a comprehensive understanding of their ecology and biogeography. Fluorescently labeled particle (FLP) incubations are commonly used to quantify mixotroph abundance and ingestion but may underestimate activity due to prey and size preferences of grazers. Acidotropic dyes that stain acidic vacuoles associated with phagotrophy have emerged as an alternative to FLP incubations for estimating mixotroph abundance, yet have not been thoroughly tested among a diverse suite of marine eukaryotes. Here, we evaluate the effectiveness and specificity of two dyes, LysoTracker Green and LysoSensor Blue, in laboratory cultures and natural marine communities. In laboratory cultures, both dyes correctly did not stain one photoautotrophic species. However, LysoSensor failed to stain several known mixotrophs, indicating false negatives, while both dyes stained photoautotrophic diatoms, indicating false positives. In the field, LysoTracker staining broadly tracked with FLP-derived results in the North East Shelf (NES) and the diatom-rich California Current System (CCS). Both methods indicated lower mixotroph abundance and proportion in the CCS, suggesting acidotropic dyes may more reliably reflect mixotrophy in the field than in monoculture. This study highlights the utility and limitations of acidotropic dyes for detecting mixotrophy and underscores the importance of incorporating community composition and complementary grazing estimates for reliable interpretation.