METHOD DEVELOPMENT OF WHOLE ORGANISM DNA EXTRACTION TECHNIQUES FOR DIET CHARACTERIZATION OF DAPHNIA MAGNA USING 16S AND 18S RDNA AND NEXT-GENERATION SEQUENCING.

The role of zooplankton in linking producers to higher trophic levels is essential to understanding carbon and nutrient cycling. New approaches are needed for diet characterization of freshwater zooplankton because fatty acid profiles, stable isotopes, pigments, and molecular work on dissected guts are not practical for smaller, freshwater, organisms. Recently, whole organism extraction of prey DNA has been demonstrated on estuarine copepods. Here, we develop a whole organism DNA extraction technique for the freshwater cladoceran, Daphnia magna, to assess diet through 16S rDNA sequences in controlled laboratory experiments. Lab raised D. magna were placed in feeding microcosms containing three monoculture strains of Synechococcus for 24 h in varying ratios, monocultures of each taxon, and a eukaryote, Chlorella, control. We amplified the 16S rDNA V3-V4 region after DNA extraction, then purified our target fragment by excising the band from an agarose gel. We have found this step important for successful sequencing due to the large amount of non-target Daphnia mitochondrial DNA that was being amplified. We then sequenced the V3-V4 amplicons using Illumina MiSeq and compared them to the sequences of the three feeding microcosms. The D. magna showed feeding preference towards Synechocystis sp. over Synechococcus sp. We successfully demonstrated the effectiveness of this new technique for assessing diet through whole organism extraction. This method will be a tool in assessing trophic cycling of carbon and nutrients in freshwater ecosystems.

Primary Presenter: Ciaran Edwards, Niagara University (ciaranowls@gmail.com)

Authors:

Samantha Wrobel, Niagara University  (swrobel@mail.niagara.edu)

Cassandra Marnocha, Niagara University  (cmarnocha@niagara.edu)

William Edwards, Niagara University  (wje@niagara.edu)

Coleen Edwards, Niagara University  (cte@niagara.edu)