As a contribution to the Norwegian Nansen Legacy project (arvenetternansen.com), samples were collected during the summer expedition Q3 (5 to 27 August 2019) and the winter expedition Q4 (28 November to 17 December 2019) onboard RV Kronprins Haakon in the northern Barents Sea (Table 1 and Figure 1). During Q3, sampling was carried out from south to north, while on Q4 the transect was sampled from north to south. Detailed information about sampling during Q3 can be found in Kohlbach et al. (2021).

During Q3, Calanus copepods (C. glacialis Jaschnov, 1955; C. hyperboreus Krøyer, 1838; C. finmarchicus Gunnerus, 1770) and Themisto amphipods (Themisto libellula Liechtenstein, 1822; T. abyssorum Boeck, 1870) were collected at six stations (Kohlbach et al., 2021) and during Q4 at eight stations (Table 1 and Figure 1) with MIK nets (1200 μm with 500 μm cod end), WP3 nets (1000 μm), Macroplankton trawls (multiple mesh sizes, 8 mm bottom of the net), Bongonets (180 μm), Multinets (180 μm) and WP2 nets (90 μm). Samples were sorted into species (Supplementary Figures 1,2) and stage/size groups (Table 2) onboard the ship and immediately frozen at −80°C until further processing. The different Calanus species were differentiated based on morphology and prosome lengths according to Kwasniewski et al. (2003). Small species/individuals were pooled by stage/size group in order to obtain sufficient sample material for analyses (Table 2).

During Q3, pelagic particulate organic matter (PPOM) was collected at six stations (P1, P2, P4, P5, P6, and P7) with Niskin bottles attached to a CTD rosette at the depth of the chlorophyll (Chl) a maximum (between 14 and 73 m). During Q4, water samples were collected at five stations (P1, P4, P5, P6, and P7) at 20 m depth. Volumes of 1.2 to 3 L of seawater were filtered via a vacuum pump through pre-combusted Whatman GF/F filters (3 h, 550°C) and PPOM filters were stored at −80°C until further processing. Chlorophyll a concentrations reached up to 2.6 μg L^–1 during summer and were generally below 0.05 μg L^–1 during winter (Vader et al., 2021).

During both sampling campaigns, ice-associated POM (IPOM) was collected at two stations, respectively, (Q3: P6_ice, P7_ice; Q4: P5_ice, P7_ice). The bottom 10 cm of the ice cores were melted in the dark without the addition of filtered seawater. For each sample, between 570 and 640 mL of melted ice sample was filtered by a vacuum pump through pre-combusted 47 mm GF/F filters and filters were stored at −80°C until further processing. Mean Chl a concentrations (n = 2 each) in the bottom 3 cm of the sea ice were higher during the summer sampling (P6_ice: 0.4 μg L^–1, P7_ice: 2.3 μg L^–1) compared to the winter ice cores (P5_ice: 0.04 μg L^–1, P7_ice: 0.3 μg L^–1; Vader et al., 2021).

Lipid classes and FAs were analyzed at the Alfred Wegener Institute, Bremerhaven, Germany. Details on sample preparation, analysis and lab equipment can be found in Kohlbach et al. (2021). Briefly, total lipids were extracted from the freeze-dried samples using a modified procedure from Folch et al. (1957) with dichloromethane/methanol (2:1, v/v). Lipid class analysis of the zooplankton was performed directly on the extracted lipids (Graeve and Janssen, 2009) via high performance liquid chromatography. Lipids were separated into neutral (= storage lipids) and polar lipid classes (= membrane lipids; Supplementary Table 1).

Total lipids were converted into fatty acid methyl esters (FAMEs) by transesterification in methanol, containing 3% concentrated sulfuric acid, and separated via gas chromatography (Kattner and Fricke, 1986). Total lipids were estimated using an internal standard (C23:0). FAs are expressed by the nomenclature A:Bn-X, where A represents the number of carbon atoms, B the amount of double bonds, and X gives the position of the first double bond starting from the methyl end of the carbon chain. The proportions of individual FAs are expressed as mass percentage of the total FA content.

Dry weights and percentage total lipids per dry weight are summarized in Table 2.

To study changes in carbon-source composition of the zooplankton species from summer to winter, we investigated relative proportions of marker FAs that can inform about carbon-source preferences of the consumers. The FAs 16:1(n-7), 16:4(n-1), and 20:5(n-3) are mainly produced by diatoms (= diatom-associated FAs), and the FAs 18:4(n-3) and 22:6(n-3) (= dinoflagellate-associated FAs) are predominantly produced by dinoflagellates and the prymnesiophyte Phaeocystis (Falk-Petersen et al., 1998; Reuss and Poulsen, 2002; Dalsgaard et al., 2003). FA ratios of 16:1(n-7)/16:0, ΣC16/ΣC18 and 20:5(n-3)/22:6(n-3) > 1 can indicate a dominance of diatom-produced versus dinoflagellate-produced carbon in an algal community or carbon pool of a consumer. Calanus spp. biosynthesize the monounsaturated long-chained FAs 20:1 and 22:1 (all isomers), which then can indicate the importance of Calanus spp. as a food source for a consumer (Sargent and Falk-Petersen, 1988; Falk-Petersen et al., 1990). The FA 18:1(n-7) derives from the elongation of 16:1n-7 in algae (Falk-Petersen et al., 1990) whereas 18:1(n-9) can be produced by the consumers (Graeve et al., 1994; Nyssen et al., 2005), and ratios of 18:1(n-9)/18:1(n-7) (carnivory index) can further inform about the degree of carnivory in a consumer (Graeve et al., 1997; Falk-Petersen et al., 2000a; Auel et al., 2002).

PPOM was analyzed on triplicates for each station (Q3: total n = 18, Q4: total n = 15). Q3 IPOM was analyzed on duplicates (total n = 4) and Q4 IPOM on individual samples (total n = 2).

Highly branched isoprenoids and sterols were analyzed at the University of Plymouth, United Kingdom. Details on sample preparation, analysis and lab equipment can be found in Kohlbach et al. (2021). Briefly, total lipids were extracted with chloroform/methanol (2:1, v/v) and saponified with 20% potassium hydroxide in water/methanol (1:9, v/v). Non-saponifiable lipids were purified by open column chromatography (SiO₂). The sea ice algae-associated HBIs IP₂₅ (m/z 350.3) and IPSO₂₅ (m/z 348.3), and the pelagic HBIs III and IV (m/z 346.3), were analyzed by gas chromatography mass spectrometry (GC-MS). HBIs were quantified by integrating individual ion responses in single-ion monitoring mode, and normalizing these to the corresponding peak area of the internal standard and an instrumental response factor obtained from purified standards (Belt et al., 2012).

Sterols were eluted from the same silica column using hexane:methylacetate (4:1,v/v). Sterol fractions were derivatized using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and analyzed by GC-MS. Individual sterols were identified by comparison of the mass spectra of their trimethylsilyl-ethers with published data (Belt et al., 2018). The main identifiable sterols were those commonly synthesized by plants (hereafter: phytosterols): brassicasterol (24-methylcholesta-5,22E-dien-3β–ol; m/z 470), sitosterol (24-ethylcholest-5-en-3β–ol; m/z 396), chalinasterol (24-methylcholesta-5,24(28)-dien-3β–ol; m/z 470) and campesterol (24-methylcholest-5-en-3β–ol; m/z 382) and those that are synthesized by both plants and animals (hereafter: zoosterols): cholesterol (Cholest-5-en-3β–ol; m/z 458), desmosterol (Cholesta-5,24-dien-3β–ol; m/z 343) and dehydrocholesterol (Cholesta-5,22E-dien-3β–ol; m/z 327).

Statistical significance of differences in trophic markers between summer and winter in POM and the zooplankton were assessed using unpaired Student’s t-tests. A statistical threshold of α = 0.05 was chosen, and results with p ≤ 0.05 were considered significant. All measures of statistical variation are reported as means ±1 SD. Prior to statistical analysis, the data were verified for normality of distribution. For testing, lipid class and FA data were transformed by applying an arcsine square root function to meet normality requirements for parametric statistics (Legendre and Legendre, 2012). All statistical analyses and data visualization were run in R v.3.4.3 (R Core Team, 2017), using the R package ggplot2 (Wickham, 2016).

In all species, the relative proportions of the neutral lipids, mainly wax esters (WEs) and triacylglycerols (TAGs), during summer (mean 66% in T. libellula to 97% in C. hyperboreus) and winter (mean 79% in T. libellula to 98% in C. hyperboreus) exceeded the proportions of the polar lipids, mainly phosphatidylethanolamines (PEs) and phosphatidylcholines (PCs) during summer (mean 3% in C. hyperboreus and C. finmarchicus to 34% in T. libellula) and winter (mean 2% in C. hyperboreus to 21% in T. libellula). The lipid content of the Calanus copepods consisted almost entirely of WEs during both seasons (mean ≥79%; Figure 2). In T. libellula, WEs and TAGs contributed equally to the neutral lipid fraction in winter, while in T. abyssorum, TAGs had higher relative contributions than WEs during both summer and winter (Figure 2A). In all species, the proportions of WEs were higher during winter compared to summer (significant difference in C. hyperboreus). In contrast, proportions of TAGs were higher during summer compared to winter in all species except for T. libellula (significant difference in T. abyssorum). The relative contributions of the polar lipids PC and PE showed no significant seasonal differences within each species, but were somewhat higher during winter compared to summer in T. abyssorum (Figure 2B). Relative proportions of individual lipid classes can be found in Supplementary Table 1.

In both seasons, PPOM and all three Calanus species did not contain detectable quantities of the sea ice algae-associated HBIs IP₂₅ and IPSO₂₅ (Figure 5A). In both amphipod species, mean sea ice algae-associated HBI concentrations were slightly higher in winter compared to summer. Mean concentrations of the pelagic/MIZ HBIs III and IV were slightly lower in winter than in summer in C. glacialis, C. hyperboreus, and T. libellula, but lower in summer than in winter in C. finmarchicus and T. abyssorum (Figure 5B).

The mean ratio of zoosterols/phytosterols increased in all zooplankton species from summer to winter (Table 4), but there was large variability in individual sterols among the zooplankton between the seasons. In C. glacialis, all phyto- and zoosterols, except for cholesterol, had higher averaged values in summer compared to winter (Figure 6A). In C. hyperboreus, chalinasterol content in winter was about 30% of that in summer, while averaged levels of the other phytosterols were more similar between the seasons, and all zoosterols were higher during winter (Figure 6B). In C. finmarchicus, all phytosterols and zoosterols had higher averaged levels in winter than in summer (Figure 6C). All phytosterols, except for brassicasterol, were similar in summer and winter in T. libellula (Figure 6D), while in T. abyssorum, all phytosterols were less abundant in winter than in summer (Figure 6E). In both amphipods, the zoosterol content was relatively stable between the seasons.

Congruent with other studies (Kattner et al., 1989; Scott et al., 2000; Falk-Petersen et al., 2009), C. hyperboreus had the highest WE levels (mean 97% in summer and 90% in winter related to total lipids) among the copepods during both seasons, which can be attributed to their superior ability to synthesize and store high-energy long-chained FAs and alcohols (20:1 and 22:1 isomers) (Ackman et al., 1974; Sargent and Falk-Petersen, 1988). In comparison with the other two Calanus species, C. hyperboreus showed similar FA composition and FA marker ratios during the polar day and night (Table 3 and Figure 4). Moreover, the total lipid content per dry weight was similar between summer and winter in C. hyperboreus (Table 2), indicating that this species was in similar body condition between the seasons and well adapted to the food-poor winter conditions.

In C. glacialis and C. hyperboreus, WE levels were higher in winter than in summer (Figure 2A), reflecting the significance of long-term storage lipids for successful overwintering in species that are directly dependent on the highly seasonal algal production (Albers et al., 1996; Lee et al., 2006; Falk-Petersen et al., 2009). Concomitantly, relative proportions of the faster metabolized TAGs decreased in these species in December, likely already utilized in the days and weeks prior to the winter sampling. In C. finmarchicus by contrast, the relative proportions of individual neutral and polar lipids varied little from summer to winter, indicating species-specific differences in seasonal lipid dynamics among the copepods (Figures 2A,B and Supplementary Table 1).

As found before (Søreide et al., 2008; Cleary et al., 2017), the marker FA ratios suggested the importance of diatom- over dinoflagellate-associated carbon in the diets of all three Calanus species during both seasons (Table 3). In C. glacialis and C. hyperboreus, the overall phytosterol content declined from late summer to early winter (Figures 6A,B), most noticeably in chalinasterol, however, variability across the sampling area was large. The decrease in phytosterols in C. glacialis and C. hyperboreus was not evident in C. finmarchicus, further pointing to differences in overwintering activity and strategies between the Calanus species. However, phyto- and zoosterol contents varied largely between the sampling stations within one species so that differences between the species might not only be species-specific but also driven by spatial variability. Pelagic HBIs were also slightly lower in C. glacialis and C. hyperboreus during winter (Figure 5B). Given that diatoms are the major producers of marine sterols (Stonik and Stonik, 2015; Jaramillo-Madrid et al., 2019), these changes likely reflected a shift away from intake of fresh phytoplankton during summer, as the primary production declined. The lower proportions of the diatom-associated FAs 16:4(n-1) and 20:5(n-3) in winter compared to summer in all three copepods (Figure 4A) further reflected the seasonal decrease in diatom contribution to their diet (lower Chl a concentrations in water column and sea ice, lower proportions of diatom-associated FAs in PPOM and IPOM; Figure 3).

In C. finmarchicus, the carnivory index 18:1(n-9)/18:1(n-7) and the averaged ratio of zoosterols to phytosterols were higher in winter than in summer (Tables 3, 4). The other two Calanus species also showed higher ratios of zoosterols/phytosterols during winter, but ratios of 18:1(n-9)/18:1(n-7) varied little between the seasons in C. hyperboreus and were even lower in winter compared to summer in C. glacialis (Tables 3, 4). While proportions of 18:1(n-9) can potentially increase during starvation (Graeve et al., 1997), these species-specific differences could also indicate that the C. finmarchicus investigated in our study were actually active during the winter sampling, which was also reported for Calanus spp. in Svalbard fjords (Daase et al., 2018), and relied more on degraded heterotrophic material or heterotrophic prey due to the low quantity and quality of POM during winter (Søreide et al., 2008; Hobbs et al., 2020). For example, winter-active C. glacialis may be able to prey on small copepods, and their eggs and nauplii that are available during winter (Hobbs et al., 2020), as well as early life stages of chaetognaths or ctenophores (Cleary et al., 2017). Calanus individuals that were not able to accumulate enough lipids during the productive season might be required to stay active during winter (Hobbs et al., 2020), and search for food near the surface or ice-water interface (Pedersen et al., 1995), while individuals with large lipid stores can descend into deeper water layers where food but also predators are scarce (Wold et al., 2011; Freese et al., 2017; Schmid et al., 2018; Kvile et al., 2019). Seasonal changes in the trophic markers can, to some degree also reflect ontogenetic differences in the copepods, and consequently feeding behavior and food composition driven by size or possibly sex. The samples collected during summer contained individuals of the copepodid stages CIV and CV as well as female adults, and the samples collected during winter contained stages CIV, CV, female, and male individuals (Table 2), however, due to the low sample size of each life stage it is not possible to reveal the influence of ontogenetic variability in this study.

Failure to detect the sea ice algae-associated HBIs in all three Calanus species during both seasons (Figure 5A) suggests that their carbon sources were likely predominantly of pelagic origin. Ice algae can be essential for reproduction and development of early Calanus life stages in spring (Niehoff et al., 2002; Søreide et al., 2010; Daase et al., 2013), but abundant pelagic food during spring and summer, large lipid reservoirs and dormancy during winter possibly render alternative or additional ice-associated carbon sources unnecessary. However, the known HBI-producing ice algal taxa Haslea and Pleurosigma (Brown et al., 2014a,b; Limoges et al., 2018) are not usually amongst the major ice algal species, and indeed were not detected in the sea-ice communities during both seasons (or their abundances were below the detection limit of the Utermöhl settling method). This is in agreement with previous taxonomic studies showing that the known IP₂₅-producing species (Haslea crucigeroides, H. spicula, H. kjellmanii, and Pleurosigma stuxbergii var rhomboides) typically represent <1% of the sea ice diatom assemblages in Svalbard waters (von Quillfeldt, 2000). Therefore, we cannot rule out the possibility that the copepods were grazing on sea-ice algae containing HBI producers, but which were below the detection limit or were grazing on non-HBI-producing ice algal taxa. We should emphasize, however, that both amphipods and other pelagic zooplankton such as the filter-feeding pteropods Clione limacina and Limacina helicina contained sympagic HBIs during summer (Kohlbach et al., 2021), pointing to differences in feeding depth layers and/or feeding mechanisms between the taxa, and showing that HBI-producing diatoms were present at some point in the sampling area.

The two pelagic amphipods varied in their lipid class (Figures 2A,B) and FA compositions (Figures 4A,B) as well as seasonal variability of these parameters, which supports the notion that the two species differ in their lipid storage modes, carbon source composition and consequently their trophic interactions within the Arctic food web (Auel et al., 2002).

During both seasons, concentrations of pelagic HBIs were higher(Figure 5B), whereas 18:1(n-9)/18:1(n-7) carnivory ratios were lower in T. libellula compared toT. abyssorum (Table 3), suggestingthat T. abyssorum occupied a higher trophic level than T. libellula, in agreement with the literature (Auel et al., 2002). In both amphipod species, heterotrophic prey had a greater contribution to their food composition in winter versus summer based on the higher carnivory ratios of 18:1(n-9)/18:1(n-7) and zoosterols to phytosterol ratios (Tables 3, 4). To some extent, the observed seasonal differences likely reflect ontogenetic changes in carbon and prey composition (Noyon et al., 2012), e.g., in T. libellula from early life stages during summer (average dry weight 3.5 mg ind.^–1) to larger predatory T. libellula individuals during winter (average dry weight 43.4 mg/ind^–1).

The significantly higher relative proportions of the marker FA 18:4(n-3) in T. abyssorum during summer (Figure 4B) reflected the higher concentrations and availability of dinoflagellates and/or Phaeocystis, in agreement with higher relative proportions of 18:4(n-3) in summer PPOM compared to winter PPOM (Figure 3), and further suggests the greater importance of phytoplankton and flagellate-based prey in their diet during the polar day. Our results correspond to previous studies (Mayzaud and Boutoute, 2015), suggesting continuous, more carnivorous, feeding during winter for both species (Dale et al., 2006; Kraft et al., 2013).

In both amphipods, WE levels were higher during winter (Figure 2A), pointing to an increased predation on wax ester-rich species during the polar night. In the case of T. libellula, these seasonal differences in WEs as well as calanoid copepod-associated FAs reflected the greater importance of Calanus copepods in their winter diet (Supplementary Table 2). Significantly higher levels of the diatom-associated FA 16:1(n-7), an important contributor to the FA pool of Calanus spp., in T. libellula during winter further highlights the significance of these copepods for the successful overwintering of T. libellula. The significantly higher relative proportions of the polyunsaturated FAs (PUFAs) 20:5(n-3) and 22:6(n-3) in T. libellula in summer compared to winter (Figures 4A,B) could again point to ontogenetic differences in feeding habits, as the rather omnivorous younger stages (length group 5–20 mm) collected during summer probably fed more intensely on PUFA-rich phytoplankton/ice algae than the mostly carnivorous adult stages of T. libellula (length group 10–40 mm) (Noyon et al., 2009, 2012). However, these PUFAs are mainly incorporated into polar lipids (Stübing et al., 2003), and lower PUFA concentrations in T. libellula in winter might be explained by the larger proportion of storage lipids during the polar night (Supplementary Table 1).

In T. abyssorum, zooplankton other than Calanus spp. might have been equally important prey items due to similar relative proportions of calanoid copepod-associated FAs in both seasons. Calanus copepods are generally an important food item for both amphipods (Auel et al., 2002; Dalsgaard et al., 2003; Marion et al., 2008; Noyon et al., 2009), but T. abyssorum has been reported to also prey on other copepods such as Metridia and appendicularians (Dalpadado et al., 2008). Information on feeding modes and diet composition during the polar night is generally scarce, but during summer and autumn, T. abyssorum has been found to have a greater diversity of food items in comparison to T. libellula which fed mostly on copepods (Auel et al., 2002; Dalpadado et al., 2008). For carnivorous species, such as the two hyperiid amphipods, seasonality in primary production has a less severe impact on their feeding behavior in comparison to herbivorous copepods since their preferred prey is basically available throughout the year (Hagen and Auel, 2001; Hirche and Kosobokova, 2011).

Themisto libellula is associated with Arctic waters, while T. abyssorum is more abundant in subarctic (Atlantic) waters. Moreover, T. libellula has been observed to be distributed closer to the surface (<50 m) which could give them easier access to sea ice-associated prey than T. abyssorum that has a more mesopelagic lifestyle (>200 m) (Dalpadado et al., 2001; Auel et al., 2002; Dalpadado, 2002). However, concentrations of sea ice algae-associated HBIs were similar between the two species (Figure 5A), only slightly higher in T. abyssorum, indicating an at least comparable trophic relationship to the ice-associated ecosystem for both species. The lack of sea ice algae-associated HBIs in the copepods further reveals that both amphipod species obtained sympagic metabolites by feeding on other sympagic prey. The presence versus absence of sea ice-derived HBIs in amphipods and copepods, respectively, likely reflects differences in their feeding location (under ice versus water column) and/or feeding behavior. Furthermore, it is possible that the juvenile amphipods during summer were feeding on sinking ice algal aggregates in the mid-water column (marine snow), as suggested for pteropods and appendicularians (Kohlbach et al., 2021), as young Arctic and Antarctic Themisto have been observed to feed partly on phytoplankton (Noyon et al., 2012; Watts and Tarling, 2012). Sea ice algae-associated HBIs were somewhat more concentrated in the winter compared to the summer Themisto spp., but concentrations varied strongly among the individuals (Figure 5A). On one hand, this could again reflect ontogenetic differences in food composition, but could on the other hand, be the result of the very opportunistic feeding style of these amphipods (Havermans et al., 2019).

Highly branched isoprenoids concentrations in Barents Sea fauna during winter, spanning pelagic and benthic invertebrates and fish, were in a similar order of magnitude as in our study (Figures 5A,B; Brown et al., 2015). Compared to HBI concentrations in Antarctic krill Euphausia superba, however, the levels of both sea ice algae-associated and pelagic HBIs in the amphipods were distinctly lower in our study. This could reflect hemispheric differences in the abundance of HBI-producing algae as for example Arctic diatoms producing IP₂₅ have, despite their ubiquitous distribution throughout the Arctic, a relatively low contribution (< 5%) to the overall sympagic diatom community (Brown et al., 2014b). Given the low level of sea ice-associated primary production and that the available ice algae are not accessible for grazers during winter, HBIs detected in the winter samples could also, to some degree, reflect the importance of benthic food sources for the amphipods as HBI-producing diatoms have been found to be present in benthic sediments and bottom waters rather than sea ice and upper water layers during winter (Brown et al., 2015). Both amphipod species have been observed close to the seafloor (Vinogradov, 1999), however, the extent that they rely on benthic food sources remains unclear.