Flow cytometry with cell sorting and sequencing as a tool for the study of the Humboldt Current krill stomach microbiota

Abstract

Euphausiids (or krill) are important contributors to marine biomass and key players in marine pelagic trophic webs. Euphausiids stomachs represent a specific niche for microbes that participate in the digestion of the host dietary components. To date, methods for the study of the diversity and function of these microorganisms remain complex. Often, bacterial ribosomal sequences obtained from lysates of stomachs are overrepresented by organisms from the surrounding environment. Flow cytometry with cell sorting (FC-CS) have become a powerful technique to study microbial community structure but also for the study of population genomics of gut-associated bacteria, even at a single-cell level. In this study, we used FC-CS and sequencing of the bacterial 16S rRNA gene to study the microorganisms inhabiting the stomach of the Humboldt Current krill, Euphausia mucronata. This approach was complemented with DNA extraction and sequencing from whole lysate stomachs as described for other crustacean species. Non-specific amplification was not retrieved in the polymerase chain reaction (PCR) from cells sorted, opposite to the observed using the DNA from the whole lysate. Sequences obtained from the whole stomach DNA were enriched in picocyanobacteria, meanwhile, sequences retrieved from cells sorted belonged almost exclusively to Balneola sp. of the new phylum, Balneolaeota. This study represents, to our knowledge, the first report of Balneola sp. in the stomach for any organism inhabiting the Humboldt Current System (HCS). Our results suggest that the stomach-associated microbiota can be characterized by FC-CS and sequencing by manual scraping of the stomach coupled with the DNA extraction and sequencing. This work represents a baseline for similar studies of other mesozooplankton groups. The implementation of this technique might complement future studies on host-microbes interaction and their implications on the marine pelagic food web.