DOI: 10.25338/b8qw4g
title: Phytoplankton prey of an abundant estuarine copepod identified in situ using DNA metabarcoding

description: Plankton trophic interactions play a crucial role in ecosystem processes.
 Diet analysis using high-throughput sequencing methods such as
 metabarcoding can provide new insight where traditional methods have been
 limited. We used 16S ribosomal RNA genemetabarcoding to identify
 phytoplankton fromthe guts of the copepod Pseudodiaptomus forbesi and in
 seston from the Cache Slough Complex, a tidal freshwater reach of the San
 Francisco Estuary. Cyanobacteria, assumed to have low nutritional value
 for copepods, were detected in all copepod samples and comprised the
 highest relative read abundance in metabarcoding results. Di erential
 abundance analysis, used to compare representation of operational
 taxonomic units between copepod and seston samples, showed that two
 filamentous taxa (a streptophyte and a cyanobacterium) were most
 overrepresented in copepod samples, whereas cryptophytes and most
 ochropytes (diatoms and related taxa) were underrepresented in copepod
 samples. These findings could reflect unexpected feeding patterns or
 trophic upgrading. Understanding the capabilities and limitations of DNA
 metabarcoding is key to its use in diet analysis and integration with
 traditional approaches.
description: Copepods and seston (available prey) were collected by boat
 during eight sampling events at five locations (Fig. 1) in June and
 October 2015, a drought year, as part of a broader study (Kimmerer et
 al., 2018). Of 23 sampling events throughout the CSC (not
 shown), eight sampling events had sufficient P. forbesi adults for diet
 analysis. Groups of 5 copepods per sampling event were processed whole
 because their small size precluded dissecting guts intact. Seston was
 collected by Van Dorn sampler from ~1 m depth and prefiltered through a
 sterile 150 µm nylon mesh sieve to remove larger pieces of detritus and
 minimize filter clogging. Samples were filtered using syringe or vacuum
 filtration (&lt;100 mm Hg) onto a 1 µm pore, 47mm diameter
 polycarbonate filter (Whatman) which was transferred to a sterile tube
 using sterile forceps (n =2 to 3 filters per sampling event). Copepods
 maintained without food were used to as negative controls to determine if
 non-ingested organisms were detected, and laboratory-fed copepods were
 used to confirm successful detection of known prey. 
 DNA was extracted from copepods and whole filters using E.Z.N.A.
 Tissue DNA Kit (Omega Biotek) and amplified using16S rRNA gene V3-V4
 region primers (5'-CCTACGGGNGGCWGCAG-3' and
 5'-GACTACHVGGGTATCTAATCC-3'). Library preparation followed
 Illumina guidelines. Libraries were sequenced on two Illumina MiSeq runs
 at loading concentrations from 6 to 12 pM using 2 x 250 V2 or 2 x 300 V3
 chemistry with PhiX genomic library loaded at 20%. Samples were
 demultiplexed using Illumina MiSeq Control Software v2.2 MiSeq Reporter
 v2.6 and analyzed in MacQIIME v1.9.1 (Caporaso et al.,
 2010). Quality-filtered sequences (Phred score at least 20) were
 aligned in MacQIIME using PyNAST (Caporaso et al.,
 2010) and clustered into Operational Taxonomic Units (OTUs) at
 97% genetic similarity using UCLUST (Edgar, 2010). OTUs with fewer than 10
 total reads were removed from the data. Taxonomy was assigned using
 Greengenes v13_8 (DeSantis et al.,
 2006) and PhytoREF (Decelle et
 al., 2015). 
description: Pilot data from 18S rRNA gene sequencing from a subset of samples
 is included in this dataset. Sequencing and analysis followed 16S rDNA
 methods except library preparation followed Stoeck et al. 2010 and OTUs were identified using the SILVA reference database v. 11https://doi.org/10.1093/bioinformatics/btq4611 (Pruesse et al., 2007; Quast et al., 2013).