DOI: 10.5285/e8792e69-c9ae-4d54-a0a0-622005f325ad
title: Trophic marker composition of Calanus hyperboreus (Copepoda) sampled under sea ice and throughout the water column of the Central Arctic Ocean during the MOSAiC expedition (2019/2020)

description: Calanus hyperboreus dominates the copepod biomass in the high Arctic. It forms an important intermediate trophic level in the Central Arctic food web, grazing on algae and protists and serving as prey for a large range of other zooplankton, fish and seabirds. Their unique lipids (20:1, 22:1 fatty acids and fatty alcohols) can be traced within the Arctic megafauna from seals to whales and polar bears, as these energy-rich lipids are crucial body reserves for the dark season.

During the MOSAiC expedition in the Central Arctic Ocean (CAO, 2019-2020), C. hyperboreus adult females (AF) and subadult copepodites stages (CV) were sampled weekly to fortnightly. A range of nets were used to sample either horizontally underneath the sea ice or vertically from maximum 2000 m through the water column. Onboard, ~10 AF and ~20 CV of C. hyperboreus were sorted from each catch, photographed, rinsed with freshwater to remove salt and frozen at -80C for subsequent analysis of their total dry mass (DM), lipid content and a suite of trophic markers, including bulk stable isotopes (BSI), phytosterols (PS), total fatty acids (TFA), total fatty alcohols (TFAlc), and highly-branched isoprenoids (HBI). During the time of their seasonal descent at the end of summer, vertical sampling of C. hyperboreus was intensified and additional parameters were analysed, e.g. the FA and FAlc composition of their storage lipids (neutral lipids, NLFA, NLFAlc) and membrane lipids (polar lipids, PLFA, PLFAlc), the carbon isotopic composition of key FA and FAlc (CSIA-FA; CSIA-FAlc), and the tissue density. By combining this array of trophic markers, valuable information about the body conditions and feeding history of these copepods can be linked to their life cycle and vertical distribution. 

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 compares the trophic marker composition of C. hyperboreus from the surface vs. deep ocean to understand drivers, benefits, and risks of their seasonal migration in the CAO. The manuscript focusses mainly on the copepod descent in late summer and the changes in body conditions and trophic marker composition over the winter months. 

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 C. hyperboreus was carried out in August-September 2020 (Leg 5) 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 (2C) 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 C. hyperboreus AF and CV were gently lifted from the water by placing forceps under their antennae. Once ~10 AF or ~20 CV were collected, the copepods 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 -80C. In rare cases, either AF or CV were missing from a certain depth stratum and no sample was prepared. 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 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) fore 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 '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 copepods, the dry mass was estimated and each sample was divided into three parts: one subsample for BSI analysis, one subsample for lipid analysis and one subsample for density measurements. 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 TFA, TFAlc, PS and HBI con...(54)
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 um mesh size), Nansen Net (mouth area: 0.28 m2, 150 um mesh size), Ring Net (mouth area: 1 m2, 150 μm mesh size), ROV Net ('Beast', mouth area: 0.24 m2, 150 um 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 um film thickness, Agilent Technologies, USA) supplied with a splitless injector and a flame ionization detector using temperature programming.

NLFA, NLFAlc, PLFA, PLFAlc: FAME and FAlc were quantified using a gas chromatograph (7890A, Agilent Technologies, USA) equipped with a DB-FFAP capillary column (30 m, 0.25 mm I.D., 0.25 um film thickness) running a temperature programme (oven temperature 80-240C) with helium as carrier gas. Samples were injected in solvent vent mode by a programmable temperature vaporiser injector and detected by flame ionization.

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 um film thickness).

PS: Sterols 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.

Pycnometer: The pycnometer (BELSORP-max, Microtrac MRB) uses helium gas to fill the voids around solid structures for high precision volume measurements. In the first step, the empty sample cell (Model 010-20002-0-0; 0.5 cm3) was immersed in a thermostatic bath and the space volume of the cell was measured across six temperatures between -2C and 3 C to supply a temperature-specific 'blank' volume. The temperature of the thermostatic bath was maintained via an open circulating bath (Julabo, UK) filled with a water-glycol based fluid (Thermal G, Julabo, UK). 
In the second step, dried Calanus tissue was added to the sample cell and the space volume of the cell was measured for the same temperatures as the blank. At the end, the sample cell was weighed with and without animal tissue using a high-precision digital balance (Mettler Toledo, XP 504, d = 0.1 mg). The density of the tissue was obtained from the quantity of tissue divided by the difference in volumetric capacity between the blank sample cell and the cell with tissue.
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 four 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? for both d13C and d15N values. The δ13C values of the individual FAMEs were calibrated by analysing the certified standard FAMEs 14:0 (certified d13C value: -29.98 ppt, measured d13C value: -29.54 ppt) and 18:0 (certified d13C value: -23.24 ppt, measured d13C value: -23.29 ppt) at regular intervals (~ every five samples). The analytical error was +/- 0.3 ppt 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 ppt (representing the standard deviation of 10 analyses).

The pycnometers' measuring accuracy can be as high as +/- 0.02% (BELSORP, Instruction Manual Ver.1.3.4), but generally drops with lower sample mass. To accumulate sufficient mass (usually 15-38 mg dry mass), samples had to be compiled based on species, developmental stage, and sampling depth. Between 5 and 18 subsamples from the trophic marker samples contributed to one sample for density measurements, representing 50 to several hundred specimens. For each temperature between -2 and 3C, four measurements were taken, each based on 12 individual runs. Then the temperature was increased by 1C, with a 70-minute pause to allow for stabilisation of the new temperature within the sample cell. These 'blank' measurements were carried out three times, at the beginning, middle and end of the lab working period. The standard deviation for the three blank measurements varied between 0.003 and 0.008% for the six temperatures.