DOI: 10.5285/b5c898b1-98e8-483a-838e-456741891d62
title: Trophic marker composition of Arctic zooplankton and Polar cod sampled under sea ice and throughout the water column of the Central Arctic Ocean during the MOSAiC expedition (2019/2020)

description: At the basis of the marine Arctic food web, there are several carbon sources including ice-associated (sympagic) algae that live primarily in sea ice, melt ponds or underneath sea ice; pelagic algae that live primarily in open waters; terrestrial detritus that got incorporated into sea ice forming on the Siberian Shelf and being transported with the transpolar drift across the Central Arctic Ocean (CAO); and detritus that derives with currents from the Atlantic ocean. Copepods and amphipods are diverse and often biomass-dominant zooplankton groups in the CAO that include taxa specialised in feeding on algae and small heterotrophs, on sinking detritus or other zooplankton. Gelatinous (cnidaria, appendicularia) and semi-gelatinous (chaetognaths) taxa, ostracods, pteropods, euphausiids and pelagic decapods are other important zooplankton groups in the CAO that likewise feed on a range of food sources. With the loss of Arctic sea ice, the relative importance of ice-associated carbon in the Arctic food web became a central research topic, and multiple trophic marker approaches have been developed to distinguish between pelagic and sympagic carbon transfer to higher trophic levels.
During the MOSAiC expedition in the CAO (2019-2020), zooplankton was sampled weekly to fortnightly. A range of nets were used to sample either horizontally underneath the sea ice or vertically from a maximum depth of 2000 m to the surface. Onboard, abundant zooplankton taxa were sorted from each catch, photographed, rinsed with freshwater to remove salt and frozen at -80 degrees Celsius for subsequent analysis of their total dry mass (DM), lipid content, lipid classes and a suite of trophic markers, including bulk stable isotopes (BSI), phytosterols (PS), total fatty acids (TFA), total fatty alcohols (TFAlc), highly-branched isoprenoids (HBI) and the carbon isotopic composition of key FA and FAlc (CSIA-FA; CSIA-FAlc). We had ~10 target species that were sampled in all seasons (the copepods C. hyperboreus, C. glacialis, Metridia longa, the ice amphipods Apherusa glacialis and Eusirus spp., the pelagic amphipods Themisto abyssorum and T. libellula, the euphausiid Thysanoessa spp., chaetognaths and the shrimp H. glacialis). Further zooplankton taxa were collected when available in the net catches and time permitted. Additionally, Polar cod was collected in early and late summer.
The initial separation of the various trophic markers was carried out at the University of Plymouth. After estimating the total DM, subsamples for BSI were sent to the Littoral, Environment and Societies Joint Research Unit stable isotope facility (CNRS - University of La Rochelle, France) for analysis. Three internal standards were added to the samples used for lipid analysis to quantify the TFA, TFAlc, PS and HBI content. As a first step, the total lipid content of the animals was extracted in dichloromethane : methanol. The lipid samples were split into two equal subsamples, one was sent to the Alfred-Wegener-Institute (AWI) in Bremerhaven/Germany for FA and FAlc analyses and the second was used for PS and HBI analyses in Plymouth. 
This dataset is linked to a manuscript that assesses trophic relationships in the CAO to understand the carbon fluxes in the current Arctic food web and to predict potential changes in a future ice-free Arctic. 

Contributions by KS were funded by the UK's Natural Environment Research Council MOSAiC Thematic project SYM-PEL: "Quantifying the contribution of sympagic versus pelagic diatoms to Arctic food webs and biogeochemical fluxes: application of source-specific highly branched isoprenoid biomarkers"/ (NE/S002502/1). CJA, RGC, CEG, KMS and RJ were funded by the US National Science Foundation Office of Polar Programs (OPP-1824447 and OPP-1824414).
description: The MOSAiC cruise: The MOSAiC (Multidisciplinary drifting Observatory for the Study of Arctic Climate; tag MOSAiC20192020 and the Project ID: AWI_PS122_00) expedition represents the first year-round interdisciplinary study of the atmosphere, the sea ice, the ocean, the ecosystem, and biogeochemical processes during the transpolar drift across the CAO, and was a unique opportunity for intensive field sampling (Shupe et al. 2022; Rabe et al. 2022; Nicolaus et al. 2022, Fong et al. 2024). The observational year was divided into 5 legs: Leg 1 started on October 4, 2019 with the setup of the first Central Observatory (CO1) and installations on the research icebreaker RV Polarstern north of the Laptev Sea (Krumpen et al. 2020). The winter Leg 2 and spring Leg 3 continued the work on CO1, before RV Polarstern had to leave the floe, for logistical reasons, on May 16, 2020. The vessel returned to the original ice floe on June 19, 2020, but at a different location some hundred meters away. Leg 4 continued the drift with the new CO2 over the summer until the disintegration of the floe in the Fram Strait on July 31, 2020. During Leg 5, RV Polarstern travelled back into the ice and started the setup of CO3 on August 21, 2020, near the North Pole. The third drift ended on September 20, 2020, when the vessel started the return voyage.

Water column sampling (vertically): High resolution vertical sampling of zooplankton was carried out using a Multi Net. The following five depth intervals were sampled: 2000-1000, 1000-500, 500-200, 200-50 and 50-0 m. The net was equipped with a calibrated electronic flow meter measuring the volume of filtered seawater (m3) for each sample. On deck, each cod end was emptied into a 5 L jar and stored in the cold-room (2 °C) until the catch was sorted. For the sorting, the catch was spread into a shallow plastic tray that was kept in an ice bath to prevent warming. Healthy zooplankton were gently lifted from the water by using forceps. The number of individuals pooled for one trophic marker sample varied between ~40 small copepods (e.g. Metridia longa) and one large decapod (Hymenodora glacialis). For the Polar cod, liver and muscle tissue were analysed separately. Once sufficient specimens were collected, they were placed on a slide, photographed (Leica M125 or Wild M5 microscopes), dipped in freshwater to remove the salt, placed in a 7 ml glass vial and frozen at -80 °C. Additional samples were taken with the Ring Net, either from the upper ocean (100-0 m or 200-0 m) or deeper water (2000-200 m) using a closing device. At times when the ice hole next to the ship was not available, a Nansen Net or the Ring Net were deployed from the sea ice, either at Ocean City (Leg 3) or from various locations using a tripod and hauling manually from 100 m (Leg 5).

Under sea ice sampling (horizontally): During all seasons, a net was attached to the under-ice remotely operated vehicle (ROV) 'Beast' (ROV Net, Katlein et al. 2017) for sampling 3 depth strata: the ice-ocean interface, 10 m and 50 m under the ice. The ROV was deployed from the ROV hut, several hundred meters away from RV Polarstern. Any net catches taken on the ice were transferred into a 5 L jar, stored in a cool box and brought to the ship using a pulka. 

Laboratory analyses: The initial separation of the various trophic markers was carried out at the University of Plymouth. After freeze-drying and homogenising the zooplankton and Polar cod, the dry mass was estimated and each sample was divided into two parts: one subsample for bulk stable isotopes (BSI) analysis and one subsample for lipid analysis. The samples for BSI were placed in tin capsules, compacted, and sent to the Littoral, Environment and Societies Joint Research Unit stable isotope facility (CNRS - University of La Rochelle, France) for analysis. Three internal standards were added to the samples used for lipid analysis to quantify the total fatty acids (TFA), total fatty alcohols (TFAlc), phytosterols (PS) and highly-branched isoprenoids (HBI) content. As a first step, the total lipid content of the animals was extracted in dichloromethane : methanol 2:1 (v/v). The cell-free lipid extracts were transferred to pre-weighed vials, evaporated to dryness under N2-atmosphere and weighed. Thereafter, the lipid samples were split into two halves, one was sent to the Alfred-Wegener-Institute (AWI) in Bremerhaven for FA and FAlc analyses and the second was used for PS and HBI analyses in Plymouth. At the AWI, the lipid extracts were divided for two sets of compounds: FA and FAlc in total lipid, and compound-specific stable isotope analysis (CSIA) on key FA and FAlc in total lipids. For the first, the samples were converted into fatty acid methyl esters (FAME) and analysed using an Agilent 6890N gas chromatograph. The Clarity chromatography software system (DataApex, Czech Republic) was used for chromatogram data evaluation. FAME were quantified via the internal standard, Tricosanoic acid methyl ester (23:0) to estimate the total amount of FA and FAlc per mg extracted lipid. Additionally, we provide the mass percentage composition of the TFA and TFAlc, considering 39 individual FA and 11 individual FAlc. The carbon isotopic compositions of abundant FA and FAlc from the total lipid samples was analysed using a GC-c-IRMS system, equipped with a Trace GC Ultra gas chromatograph, a GC Isolink and Delta V Plus isotope ratio mass spectrometer, connected via a Conflo IV interface (Thermo Scientific Corporation, Germany). The delta13C values of the individual FAMEs were calibrated by analysing the certified standard FAMEs 14:0 and 18:0 at regular intervals.
In addition to the FA and FAlc, five phytoplankton-produced sterols were analysed: epi-brassicasterol (Brass), chalinasterol (Chal), beta-sitosterol (Sito), campesterol (Camp) and cholesterol (Chole). After saponification with 20% potassium hydroxide in water:methanol, sterols were extracted with hexane and purified by open-column chromatography (SiO2) using hexane : methylacetate (4:1,v:v) as solvent. Sterol fractions were derivatised with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA, 70°C, 1 h) and analysed using a gas chromatograph (Agilent 7890A GC), coupled to a mass selective detector (Agilent 5975 mass spectrometry), fitted with an Agilent HP-5ms column with auto-splitless injection. Individual sterols were identified by comparison of the mass spectra of their trimethylsilylethers with published data (Rontani et al. 2018). Quantification of individual sterols was carried out in SIM mode utilising 5alpha-Androstan-3beta-ol (m/z 333) as an internal standard.
description: In-situ sample collection: A range of nets were used for zooplankton sampling: Multi Net (HYDROBIOS MultiNet 'Midi'; mouth area: 0.25 m2, 150 micrometre mesh size), Nansen Net (mouth area: 0.28 m2, 150 micrometre mesh size), Ring Net (mouth area: 1 m2, 150 micrometre mesh size), ROV Net ('Beast', mouth area: 0.24 m2, 150 micrometre mesh size). During transit to and from the CAO, animals were occasionally sampled from the ship's seawater intake at 11 m depth. The Multi Net was deployed from RV 'Polarstern' to successively sample five depth strata from 2000 m to the surface. The Nansen Net was deployed from the sea ice (Ocean City) to sample one depth stratum between 800 m and the surface, using an electric winch and a time-depth recorder. The Ring Nets were either deployed from 'Polarstern' to sample one depth stratum between 2000 m and the surface, or from the sea ice for shallow, manual casts. The ROV Net was attached to the remotely operated vehicle (ROV) for horizontally tows directly under the ice, at 10 m or 50 m.

BSI: The samples were analysed with a continuous flow isotope ratio mass spectrometer (Delta V Plus with a Conflo IV interface, Thermo Scientific, Bremen, Germany) interfaced with an elemental analyser (EA Isolink, Thermo Scientific, Milan, Italy).
TFA and TFAlc: FAME were quantified using an Agilent 6890N gas chromatograph (Agilent Technologies, USA) with a DB-FFAP capillary column (60 m, 0.25 mm I.D., 0.25 micrometre film thickness, Agilent Technologies, USA) supplied with a splitless injector and a flame ionization detector using temperature programming.
CSIA: Carbon isotopic compositions were determined for key FA and FALc using a GC-c-IRMS system, equipped with a Trace GC Ultra gas chromatograph, a GC Isolink and Delta V Plus isotope ratio mass spectrometer, connected via a Conflo IV interface (Thermo Scientific Corporation, Germany). The FAMEs were injected in splitless mode and separated on a DB-FFAP column (60 m, 0.25 mm I.D., 0.25 micrometre film thickness). 
HBI and PS: The HBIs IP25 and IPSO25 and the sterols epi-brassicasterol, beta-sitosterol, chalinasterol, campesterol and cholesterol were quantified using a gas chromatograph (7890A, Agilent Technologies, USA), coupled to a mass selective detector (Agilent 5975 mass spectrometry), fitted with an Agilent HP-5ms column with auto-splitless injection.
Chromatogram data evaluation: The Clarity chromatography software system (version 8.8.0, Data Apex, Czech Republic) was used to quantify FA and FAlc; and the GC/MSD Productivity ChemStation software (version 7.01.01.2317, Agilent Technologies, USA) was used for sterols and HBIs.
description: Due to restrictions on ship-time, replicate net tows were not feasible. The same is true for the sorting of animals onboard: to cover all the selected target species and parameters in a timely manner, only one sample per species and developmental stage was prepared for trophic marker analysis. From the dried material only one sample was prepared for each BSI and lipid analysis, but a small back-up of dried material was kept in case one of the analyses failed. A very few BSI samples had to be repeated due to problems with the autosampler, while all lipid analyses were carried out successfully at first attempt. Therefore, the back-up material was subsequently used for tissue density analysis. The FA and FAlc profiles were compared to both commercial- (Supelco 37 Component FAME mix, Supelco, Germany) and self-produced standards (e.g. Arctic algae standard, bacteria standard, Calanus spp. standard), and were identified accordingly. In a few cases, samples were also analysed with the mass spectrometer and peaks were identified via (1) the mass of the compound, (2) the retention time of the compound and (3) the equivalent chain length method. For sterols, cultured algae of known sterol composition were used to clarify the identity of the five phytosterol peaks. Therefore, the mass spectra of their trimethylsilylethers were compared with published data. For several samples, parallel analysis of the extracts was carried out at the AWI, benefitting from the long-term experience in sterol identification by our colleague Dr. Kirsten Fahl. The uncertainty of the reported isotope-delta values (BSI) was evaluated as the standard deviation of repeated measurements (n = 5) for the reference material, USGS61 and USGS63 (Geological Survey, Reston, VA, USA), based on their assigned carbon and nitrogen isotope-delta values and standard uncertainties. For our measurements the uncertainty did not exceed 0.10 per mille for both delta13C and delta15N values. The delta13C values of the individual FAMEs were calibrated by analysing the certified standard FAMEs 14:0 (certified delta13C value: -29.98 per mille, measured delta13C value: -29.54 per mille) and 18:0 (certified delta13C value: -23.24 per mille, measured delta13C value: -23.29 per mille) at regular intervals (~ every five samples). The analytical error was ± 0.3 per mille for both 14:0 and 18:0 (representing 1 standard deviation of 10 analyses each). Furthermore, for quality assurance and analytical precision of the determined carbon stable isotope composition, the laboratory standard 23:0 was measured intermittently during the sample runs with an analytical error of ± 0.4 per mille (representing the standard deviation of 10 analyses).