Isolation and Genetic Identification of Metazoan Cells From Seawater

Abstract

Environmental DNA (eDNA) metabarcoding typically relies on collecting and characterising a pool of mixed, fragmented DNA from environmental samples for species identification. Here, we introduce environmental metazoan cells (emCells), representing whole individual cells shed by macro-organisms into aquatic ecosystems, and report on a method to successfully isolate and amplifying short amplicons to determine species identity. Using a custom fish probe and a novel multi-factor fluorescence-activated cell sorting (FACS) protocol on mesocosm water samples, we successfully enriched for target emCells, as confirmed by shifts in population density using FACS and imaging flow cytometry. Imaging flow cytometry demonstrated dual nuclear and mitochondrial staining of whole single cells, while multiplexed PCR assays (targeting both mitochondrial and nuclear DNA) confirmed the effective enrichment of fish emCells, with one-quarter of sorted cells identified as fish. Sequences obtained from isolated emCells matched known species in the mesocosm, validating our approach. Despite efforts to exclude non-target cells, diverse single-cell eukaryotes were also recovered, highlighting the need for additional strategies to enrich for target emCells given the abundance and diversity of off-target particles present in aquatic environments, which will be especially important for real-world environments. Isolation and analysis of emCells could provide a versatile complementary approach to current eDNA methodologies by providing genomic information that normally requires direct sampling from live organisms.