Dataset: Compound-specific isotope analysis of amino acids (CSIA-AA) from a subset of siphophore samples collected during four research cruises on the R/V Wester Flyer in the California Current Ecosystem between 2019 and 2021

Samples were collected in the central and southern California Current. Most samples were collected in the Monterey Bay region, but a subset of samples were collected in southern California in 2020 and 2021. Samples were collected from between 0 to 3,000 meters depth. Samples were collected on four cruises across three years. All cruises were on the R/V Western Flyer. Dr. Steven Haddock (haddock@mbari.org) was the Chief Scientist on all cruises. Cruises occurred in March 2019 (Cruise ID: WF0319), January 2020 (Cruise ID: WF0120), July 2020 (Cruise ID: WF0720), and July 2021 (Cruise ID: WF0721). Sample locations and dates are provided as columns in the data file.

Siphonophores were collected using three methods: (1) a remotely operated vehicle, (2) blue water diving, and (3) a midwater trawl.

(1) We used the Remotely Operated Vehicle (ROV) Doc Ricketts (https://www.mbari.org/technology/rov-doc-ricketts/) to collect siphonophores, which is an electro-hydraulic vehicle that operates between 200 and 4000 meters. The vehicle was fitted with high-definition video cameras, environmental data instrumentation (e.g. depth, temperature, salinity, and oxygen sensors), and suction and detritus samples to collect in-tact siphonophore specimens. ROV collections occurred during daylight hours.

(2) Siphonophores were collected by blue water diving between 0 and 20 meters during daylight hours. Blue water diving techniques followed the guidelines in the following publication: Haddock, Steven HD, and John N. Heine. "Scientific blue-water diving." (2005). From Haddock and Heine (2005): "In a typical blue-water dive, working divers are connected to a surface platform (and indirectly to each other) by tethers attached to a central hub, which is tended by a safety diver. This hub is connected to a down-line, providing a vertical point of reference. A surface float allows the divers to drift freely through the upper water-column, focusing on their work while the safety diver acts as a buddy for everyone."

(3) A Tucker Trawl with a frame area: 2 square meters (m²), mesh size: 500 micrometers (μm) was towed obliquely for ~2 hours between 900 meters and the surface at night.

Upon collection, siphonophores were identified to the finest taxonomic level, which was either genus or species. For some genera, there are likely undescribed and/or cryptic species (e.g., Apolemia) and for these taxa, genera-level identifications were used. All siphonophores were rinsed with DI water and frozen at -80°C until further processing. Siphonophore tissues were weighed, lyophilized, packaged into tin capsules for bulk isotope analysis, and analyzed at the University of Hawaii's Isotope Geochemistry Facility.

For bulk stable isotope analysis, siphonophore samples were analyzed using a Costech (Valencia, CA, USA) elemental combustion system coupled to a Thermo-Finnigan Delta XP isotope ratio mass spectrometer with N2 standard for nitrogen and Vienna Pee Dee Belemnite for carbon. The bulk stable isotope data are available in a separate BCO-DMO dataset (see 'Related Datasets').

A subset of samples was selected for compound-specific isotope analysis of amino acids (CSIA-AA). CSIA-AA was also conducted at the University of Hawaii's Isotope Geochemistry Facility using acid hydrolysis followed by derivatization (see Popp et al. (2007) and Hannides et al. (2013) for details). Derivatives were analyzed using a Thermo-Finnigan Delta V Plus isotope ratio mass spectrometer (IRMS), interfaced with a Thermo Trace GC gas chromatograph via GC-C III combustion furnace (980°C), reduction furnace (680°C), and a liquid nitrogen cold trap. Samples were injected (split/splitless injector, splitless mode) with a 180°C injector temperature and a constant helium flow rate of 1.4 milliliters per minute (mL min-1). For quality control, we analyzed an amino acid suite, with known δ15N values of 14 amino acids, every 3-4 sample injections. Internal reference compounds, L-2-Aminoadipic acid and L-(+)-Norleucine of known nitrogen isotopic composition, were co-injected with samples and suites and used as a measure of accuracy and instrument precision. Samples for CSIA-AA are typically analyzed in triplicate runs. Our samples, however, required six runs to obtain peaks for all amino acids (AAs) due to the inordinate relative abundance of glycine compared to all other amino acids. It is unclear why glycine peaks were large, although we note that the relative abundances of the different AAs can vary depending on the taxa and tissue type observed. It is unknown whether this is common for siphonophores since there are no other published siphonophore CSIA studies. Glycine peaks were so large that the chromatography surrounding glycine was deleteriously affected when injecting volumes large enough to detect all AAs of interest. To overcome this, we analyzed samples in triplicate at injection volumes that allowed for good chromatography around glycine, and then again in triplicate at a larger injection volume to allow smaller AAs to be detected while back flushing the large glycine peak out of the chromatogram. We obtained well-defined peaks for 14 amino acids, which were grouped into standard 'trophic' and 'source' categories based on previous studies.

Related Resources:
​Some of the siphonophores collected in this dataset were also used for metabarcoding. Those data are published Damian-Serrano, et al. (2022) (doi: 10.1371/journal.pone.0267761)

Illumina sequencing data files can be found in the NCBI BioProject PRJNA733192 (https://www.ncbi.nlm.nih.gov/bioproject/733192)

Prey 18S reference database enhancement sequences are available in NCBI (accession numbers between MZ333540 - MZ333629)

Other data, intermediary files, and all code can be found in the GitHub repository: https://github.com/dunnlab/siphweb_metabarcoding (see DOI: doi.org/10.1371/journal.pone.0267761)