Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Related Publications
• Related Datasets
• Parameters
• Instruments
• Project Information
• Funding

Transects ranged from 24 to 86 km in length were sampled along the Newport Hydrographic (NH) line as well as the Trinidad Head (TR) line during the winters (February-March) and summers (July-August) of 2018 and 2019 (winter 2018 transect sample size was n=2 per location due to weather days while summer 2018 – summer 2019, n=4-5). Located off Newport, Oregon, the NH Line has been sampled since 1961 (Peterson and Miller, 1975), while the TR line off northern California has been sampled since 2007.

DNA sampling for microbes was carried out from surface (5-10 meters depth) seawater samples (500-1000 mL, n=2 per station, 6 stations per transect), collected by CTD, that were size fractionated on 47 mm 1.6 µm GF/A filters (Whatman) followed by 47 mm 0.2 µm Supor polyethersulfone filters (Pall Corporation)  using a peristaltic pump. Filter membranes were moved to bead-beater tubes and frozen immediately at -20 ˚C and stored at -80 ˚C. DNA extraction was done using the DNeasy Plant Tissue Mini Kit (Qiagen) with the following modifications. Samples were lysed by bead beating with 0.55 mm and 0.25 mm sterile glass beads at 30 Hz for 2 minutes after addition of lysis buffer, freeze-fractured 3 times, incubated with Proteinase K (VWR Chemicals, Solon, OH, USA) at 20 mg/mL for 1 hour at 55 ˚C, and incubated with RNase A at 100 mg/mL for 10 minutes at 65 ˚C. PCR was performed in triplicate on 1 ng of DNA with the primer pair 515F‐Y/806R amplified the 16S rRNA V4 hypervariable region with conditions as published (Parada et al. 2016) using golay barcodes on the forward primers as in the EMP protocols. Reactions were performed with the QuantaBio 5Prime HotMasterMix (Qiagen Beverly, MA, USA). The Agilent High Sensitivity Kit in the Bioanalyzer (Agilent Technologies, Waldbronn, Germany) confirmed amplicon size. Triplicate PCR reactions from each sample were pooled then purified by magnetic beads. Each final pooled sample was paired-end sequenced with Illumina MiSeq v.3 (Illumina, San Diego, USA).    

Amplicon sequence variants (ASVs) of seawater samples were identified based on 16S rRNA gene sequence reads that were processed using dada2 and phyloseq. Sequences were quality controlled using filterAndTrim() with truncLen set to 190 (forward reads) and 160 (reverse reads), maxEE was set to 3, and maxN set to 0 to eliminate low quality base calls. Forward and reverse primers were trimmed from all reads. Error learning, sample inference, and merging of paired-end reads were done with dada2 default settings to yield unique amplicon sequence variants (ASVs). Chimeric ASVs were removed with the "consensus" method. The reference database "RefSeq-RDP16S_v2_May2018" was used to assign taxonomy to the ASVs. phyloseq was used to connect ASV sequence counts per sample to taxonomic data and metadata. Sequence abundances were standardized to the median sequencing depth of all samples (“standardized relative abundance”) without rarefying. Raw sequence data can be accessed from the NCBI Sequence Read Archive at https://www.ncbi.nlm.nih.gov/bioproject/PRJNA999694/.

NSF Award Abstract:
The oceans are dominated by microscopic plants and animals (microorganisms) that are at the base of the food web and drive energy and carbon cycles on global scales. Soft jellylike animals called gelatinous grazers specialize in feeding on microorganisms using nets made out of mucus. Gelatinous grazers are abundant in the ocean and have high feeding rates on microorganisms so could have a very strong influence on the abundance and diversity of microorganisms and could change how oceanic food webs are currently understood. However, gelatinous grazers are very fragile and patchy in their distributions so it has been difficult to determine the magnitude and dynamics of these important predator-prey relationships on a meaningful scale using traditional approaches, thus they have typically been disregarded in food web studies. Learning more about the predator-prey relationship between gelatinous grazers and microorganisms will improve understanding of the structure, mechanics, and dynamics of the ocean's food web, which is a critical economic and ecosystem resource on Earth. This project is determining grazing rates by gelatinous animals on microbes to inform food web models. The project also trains students to communicate, disseminate, and interpret scientific findings. These broader impacts goals will be attained through partnerships at the University of Oregon (Applied Scientific Communication) and Portland State University (Advanced Technical Writing), training of 1 PhD student, 2 undergraduates, and 4 science communication interns, and development of a week-long workshop and establish student mentorship relationships towards production of communication products.

The project integrates laboratory and oceanographic approaches to address several specific aspects of the predator-prey relationship between gelatinous grazers and ocean microorganisms. Five distinct types of gelatinous grazers, each with different feeding morphologies and life history, will be studied in an oceanographic setting with an abundant and diverse natural microbial population. These target organisms include pelagic tunicates (salps, appendicularians, doliolods and pyrosomes) and thecosome pteropods. The approach quantifies: 1) grazing rates in the natural ocean environment, 2) particle selectivity with a focus on size and shape and, 3) the morphological and hydrodynamic properties of feeding that underlie the measured grazing rates and particle selection. The project uses a variety of techniques including sampling via SCUBA diving, laboratory experiments, high speed/high resolution videography, flow cytometry, and DNA sequencing techniques.