Selective transfer of polyunsaturated fatty acids from phytoplankton to planktivorous fish in large boreal lakes

Science of the Total Environment 536 (2015) 858–865

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Selective transfer of polyunsaturated fatty acids from phytoplankton to planktivorous fish in large boreal lakes Ursula Strandberg a,⁎, Minna Hiltunen a, Elli Jelkänen a, Sami J. Taipale a, Martin J. Kainz b, Michael T. Brett c, Paula Kankaala a a b c

Department of Biology, University of Eastern Finland, Box 111, FI-80101 Joensuu, Finland WasserCluster, Biological Station Lunz, Dr. Carl Kupelwieser Prom. 5, A-3293 Lunz am See, Austria Department of Civil and Environmental Engineering, University of Washington, Box 352700, Seattle, WA 98195, USA

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• We analyzed the transfer of polyunsaturated fatty acid (PUFA) in pelagic food web • The trophic transfer of PUFA along the food chain was selective • Docosahexaenoic acid (DHA) was strongly enriched in the food chain • DHA accounted for about 30% of total fatty acids in zooplanktivorous fish • The proportion of C18 PUFA decreased with increasing trophic level

a r t i c l e

i n f o

Article history: Received 20 May 2015 Received in revised form 2 July 2015 Accepted 3 July 2015 Available online xxxx Editor: D. Barcelo Keywords: Boreal lake Phytoplankton Zooplankton Planktivorous fish Copepods Cladocerans Coregonus albula Osmerus eperlanus

a b s t r a c t Lake size influences various hydrological parameters, such as water retention time, circulation patterns and thermal stratification that can consequently affect the plankton community composition, benthic–pelagic coupling and the function of aquatic food webs. Although the socio-economical (particularly commercial fisheries) and ecological importance of large lakes has been widely acknowledged, little is known about the availability and trophic transfer of polyunsaturated fatty (PUFA) in large lakes. The objective of this study was to investigate trophic trajectories of PUFA in the pelagic food web (seston, zooplankton, and planktivorous fish) of six large boreal lakes in the Finnish Lake District. Docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and α-linolenic acid (ALA) were the most abundant PUFA in pelagic organisms, particularly in the zooplanktivorous fish. Our results show that PUFA from the n-3 family (PUFAn-3), often associated with marine food webs, are also abundant in large lakes. The proportion of DHA increased from ~4 ± 3% in seston to ~32 ± 6% in vendace (Coregonus albula) and smelt (Osmerus eperlanus), whereas ALA showed the opposite trophic transfer pattern with the highest values observed in seston (~11 ± 2%) and the lowest in the opossum shrimp (Mysis relicta) and fish (~2 ± 1%). The dominance of diatoms and cryptophytes at the base of the food web in the study lakes accounted for the high amount of PUFAn-3 in the planktonic consumers. Furthermore, the abundance of copepods in the large lakes explains the effective transfer

⁎ Corresponding author at: Chemistry and Biology, Ryerson University, 350 Victoria St., Toronto M5B 2K3, Canada.

http://dx.doi.org/10.1016/j.scitotenv.2015.07.010 0048-9697/© 2015 Elsevier B.V. All rights reserved.

U. Strandberg et al. / Science of the Total Environment 536 (2015) 858–865

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of DHA to planktivorous fish. The plankton community composition in these lakes supports a fishery resource (vendace) that is very high nutritional quality (in terms of EPA and DHA contents) to humans. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Large lakes are important ecosystems for maintaining biodiversity as well as providing a wide variety of ecosystem services for humans (Holmlund and Hammer, 1999; Krantzberg and deBoer, 2006; Vadeboncoeur et al., 2011). Commercial freshwater fisheries are usually focused on large lakes, which are also important for recreation and commercial navigation (Holmlund and Hammer, 1999; Krantzberg and deBoer, 2006). Lake size and morphometry have direct effects on several physicochemical parameters, such as vertical temperature gradients, water retention time and water circulation patterns, which together with catchment characteristics influence e.g. nutrient cycling, the trophic state, and food web structure. For example, plankton community composition (Lepistö and Rosenström, 1998), food chain length (Post et al., 2000) and carbon fluxes scale with lake size (Kortelainen et al., 2004; Brett et al., 2012). Although the ecological and hydrological processes of large lakes have been extensively studied (Nõges, 2008), research on the trophic transfer of polyunsaturated fatty acids (PUFA) through food webs of large lakes (surface area N 100 km2) is very limited (Smyntek et al., 2008). Current knowledge on the composition and trophic dynamics of PUFA in freshwater lakes is almost entirely based on small or moderately sized lakes (Ahlgren et al., 1996; Kainz et al., 2004; Müller-Navarra et al., 2004; Ravet et al., 2010; Lau et al., 2012). In aquatic ecology, PUFA are attracting increasing attention because studies indicate that PUFA from the n-3 family (PUFAn-3) may limit consumer production (Müller-Navarra et al., 2004). Animals in general cannot synthesize PUFA de novo (Cook and McMaster, 2004), and must obtain these molecules from their diets to support somatic growth and reproduction (Tocher, 2003; Brett et al., 2009; Martin-Creuzburg et al., 2009). Production of PUFA by algae is taxon-specific and the composition and availability of PUFA in food webs is determined by the community structure of the primary producers (Sushchik et al., 2004; Galloway and Winder, 2015; Strandberg et al., 2015). Although pelagic bacteria may be an important link in transferring carbon to zooplankton, bacteria contain only trace amounts of PUFA and thus the importance of the microbial loop in conveying PUFA to upper trophic level consumers is negligible (Taipale et al., 2014). The availability of PUFA to secondary consumers in the pelagic food web is not only determined by the taxonomic composition of producers but also by the community composition of the zooplankton (Persson and Vrede, 2006; Burns et al., 2011). Taxon-specific differences in fatty acid (FA) composition are much larger than intraspecific variation due to differences in the diet composition and/or environmental conditions (Hiltunen et al., 2015). Variation in phytoplankton and zooplankton community composition according to physical characteristics of lakes, allochthonous organic matter content and trophic state (Kortelainen, 1993; Lepistö and Rosenström, 1998) suggest that the FA in the pelagic food web of large lakes may differ from the more intensively studied smaller and shallower lakes (Müller-Navarra et al., 2004; Lau et al., 2012). Most studies on trophic transfer of PUFA have been carried out at the phytoplankton–herbivore interface (e.g. Ravet et al., 2010; Burns et al., 2011; Gladyshev et al., 2011). Studies focusing on fish are much less common (Kainz et al., 2004). Compared to marine fish, freshwater fish typically contain less PUFAn-3 and are more enriched in PUFA from the n-6 family (PUFAn-6), leading to a lower n-3/n-6 ratio (Ahlgren et al., 1994; Ahlgren et al., 1996). The difference between the abundance of PUFAn-3 and PUFAn-6 between marine and freshwater fish presumably originates from concurrent differences in their diets. Even in large

lakes, the majority of freshwater fish (up to 93% of species) utilize benthic resources in the littoral zone that are considered to be abundant in PUFAn-6 and have a low n-3/n-6 ratio (Vadeboncoeur et al., 2011; Lau et al., 2012). Extending the knowledge of the trophic transfer of PUFA to large lakes is also societally relevant due to the importance of commercial fisheries in these ecosystems (Holmlund and Hammer, 1999). The aim of this study was to evaluate the significance of the commercially important vendace (Coregonus albula) as a source of eicosapentaenoic acid (EPA or 20:5n-3) and docosahexaenoic acid (DHA or 22:6n-3) for humans in this region, and to track the trophic transfer of PUFA in the pelagic food web of large lakes. Detailed understanding of the transfer patterns of these molecules from phytoplankton to fish is essential to identify and preserve the availability of EPA and DHA to higher trophic level consumers, including humans. Vendace is an obligate zooplankton specialist, and is therefore an excellent model to evaluate the composition and trophic transfer of PUFA in the pelagic ecosystem of large lakes (Viljanen, 1983; Northcote and Hammar, 2006). 2. Material and methods The pelagic food web of large oligo-mesotrophic lakes (surface area N 100 km2) were sampled for FA and stable isotope (SI) analyses in three seasons: spring, summer and autumn. The following members of the food web were sampled: seston (primary producers, bacteria and small heterotrophic flagellates), zooplankton (copepods, cladocerans, the opossum shrimp Mysis relicta and dipteran larvae Chaoborus spp.), and two highly zooplanktivorous fish species, vendace and smelt (Osmerus eperlanus). Vendace is a small-sized (total length 10–25 cm) and short-lived coregonid inhabiting large to medium-sized boreal lakes, mainly in northern Europe (Viljanen, 1983; Northcote and Hammar, 2006). Vendace is the most important species for freshwater fisheries in Finland accounting for over half of all inland fisheries; the value of the catch was estimated to be 6.5 million euros in 2012 (Anonymous, 2013). The species is abundant in oligo-mesotrophic lakes with low to moderate humic content and prefers well-oxygenated cooler water layers (Rask et al., 1999). Smelt were sampled opportunistically in spring and summer as a reference to check the similarity of the FA profile in another zooplanktivorous species. Although the data is presented the results are not discussed in detail due to the low sample number. Total phosphorus concentrations of these lakes ranged between ~5 and 12 μg L−1, and the chlorophyll a concentration between ~ 2 and 6 μg L−1 (Hiltunen et al., 2015; Strandberg et al., 2015). Dissolved organic carbon (DOC) concentration ranged from ~ 5 to 10 mg C L−1, and correlated positively with water color (~ 15–57 mg Pt L− 1) and total nitrogen concentration (~230–640 μg L−1). Seston (n = 21), zooplankton (n = 214) and planktivorous fish (vendace n = 71, and smelt n = 9) were collected for FA and stable isotope (SI) analyses from six lakes (eight sampling points) in the Vuoksi Lake District in Eastern Finland (Fig. 1). In five lakes (Lake Kallavesi, Lake Suvasvesi, Lake Paasivesi, Lake Orivesi and Lake Pyhäselkä) samples were collected during three seasons: spring (late May–early June), summer (late July– early August) and autumn (late September) in 2011. Lake Karjalan Pyhäjärvi was sampled only once in summer 2012. Details on the lakes can be found elsewhere (Hiltunen et al., 2015; Strandberg et al., 2015). For seston FA analyses, lake water was pumped from the euphotic zone (0–4 m depth), sieved through a net (50 μm mesh size) to exclude zooplankton, and filtered through glass fiber filters (~0.7 μm; Whatman

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U. Strandberg et al. / Science of the Total Environment 536 (2015) 858–865

Fig. 1. Map of the Finnish Lake district with the sampling points (one sampling point per lake, with the exception of Orivesi and Suvasvesi, where we sampled at two locations). Major towns in the region are marked with black squares.

GF/F filters) for phospholipid-derived FA analyses (PLFA; Strandberg et al., 2015). For seston SI analyses, filtered lake water (~40 L) was concentrated with tangential filtration (Millipore, Pellicon P2GV PPC05 cassette, pore size 0.2 μm) and sub-samples (100 mL) were freeze-dried and about 1 mg was placed into small aluminum cups. Zooplankton were collected using horizontal (Hydro-Bios Multi Plankton Sampler, mesh size 100 um) or vertical (200 μm) net hauls and stored in − 20 °C until analysis. Zooplankton samples were briefly thawed and organisms were sorted into herbivorous cladocerans, Chaoborus spp., Mysis relicta, cyclopoids, Eudiaptomus spp., Heterocope sp., and Limnocalanus macrurus, and freeze-dried for subsequent FA and SI analyses. Vendace and smelt were caught by trawling nets (from local fishermen). Fish were held on ice during transportation to the laboratory and then immediately frozen at − 20 °C. Dorsal samples were taken from thawed fish and freeze-dried prior to FA and SI analyses (samples were taken from the same fish for both analyses). 2.1. Fatty acid analyses The FA analyses for seston and zooplankton are described elsewhere (Hiltunen et al., 2015; Strandberg et al., 2015). Briefly, the FA composition in the seston was analyzed as PLFA (Strandberg et al., 2015), which excludes detrital FA and better reflects the potential food particles, particularly for selective feeders such as copepods (DeMott, 1986; Knisely and Geller, 1986). For zooplankton and the dorsal muscle tissue of fish, total lipids were extracted with a chloroform:methanol (2:1 v/v) solution, after which the extracted lipids were transmethylated under acidic conditions (1% H2SO4 in methanol) and heated for 16 h at 50 °C. FA methyl esters were analyzed gas chromatographically using mass selective detection (details in Hiltunen et al., 2015; Strandberg et al., 2015). 2.2. Stable isotope analyses Stable nitrogen isotopes in freeze-dried seston (b50 μm), zooplankton, and homogenized fish muscle tissues (~0.7–1.0 mg) were used to evaluate the approximate trophic relationship between the different

food web components, as consumers are typically enriched in 15N in comparison to their diet (Peterson and Fry, 1987). The SI were analyzed with a Carlo-Erba Flash 1112 series Element Analyzer connected to a Thermo Finnigan Delta Plus Advantage IRMS at the University of Jyväskylä, Finland. These samples were compared to the NBS-22 standard using fish muscle (for zooplankton and fish), and potato leaves (for seston) as laboratory-working standards. The precision of δ15N analyses was 0.3‰ for all samples.

2.3. Data analysis The FA composition of seston and zooplankton showed substantial temporal variation, which was larger than spatial variation (Hiltunen et al., 2015; Strandberg et al., 2015). To represent the general FA composition of the pelagic food web during the entire sampling period, the data from all the sampled seasons and all lake basins was combined. The lipid content of vendace muscle tissue was calculated assuming that FA comprise 70% of all lipids in fish muscle (Ahlgren et al., 1996). Multidimensional scaling (MDS) was used to visualize the FA profiles among different taxonomic groups and permutational multivariate analysis of variance (PERMANOVA; Anderson et al., 2008) was used to test for taxonomic differences in FA composition. The taxonomic identity was used as a random factor with ten levels, i.e. taxonomic groups: vendace, smelt, Mysis, Limnocalanus, Heterocope, Eudiaptomus, cyclopods, Chaoborus, cladocerans and seston. Ordination was based on Euclidean distances, and the stress value represents the goodness of fit for the ordination. The lower the stress the better the data fits in the plot. Stress value b 0.2 is considered to be acceptable, while plots with stress values N 0.2 are close to random (Clarke, 1993). The significance of PERMANOVA analyses was determined using unrestricted permutation of the raw data (9999 permutations) with type III sums of squares, (Anderson et al., 2008). Correlation coefficients (Pearson's r) of the FA and the MDS 1 and MDS2 were also calculated. Multivariate tests were conducted with PRIMER 6.0 with the PERMANOVA + add on. Other statistical analyses were performed using the IBM SPSS Statistics 21 program package.

U. Strandberg et al. / Science of the Total Environment 536 (2015) 858–865

3. Results PUFA were abundant in the pelagic food webs of the studied lakes; the group means (seston, zooplankton, fish) of PUFA varied from 37 to 63% of total FA and the PUFAn-3 were more abundant than PUFAn-6 (Table 1). The PUFAn-3 accounted for ~ 26–54% of total FA and ~ 70– 88% of total PUFA. The lowest proportion of PUFAn-3 was found in cladocerans, and the highest in the calanoid copepod Limnocalanus. The proportion of C18, C20 and C22 PUFAn-3 varied between different taxonomic groups. In seston, cladocerans and Chaoborus C18 and C20 PUFAn-3, specifically 18:3n-3, 18:4n-3 and 20:5n-3, were more abundant than C22 PUFAn-3 (Table 1). In copepods and fish C22 PUFA were the most abundant PUFAn-3. The proportion of C18 PUFAn-3 was ≤ 4% of total FA in fish and Mysis, and ≤8% in Limnocalanus and Heterocope. The group means of saturated FA (SFA; excluding iso- and anteiso branched and odd chained SFA) accounted for ~ 17–34% of total FA and short length monounsaturated FA (LC-MUFA, with 14–18 carbons in the acyl chain) accounted for ~ 9–26% of total FA. The proportion of longer chained MUFA (sum of C20 and C24 MUFA) was ≤1% of total FA in all the taxonomic groups. The sum of odd chained and iso- and anteiso-branched FA, biomarkers for heterotrophic bacteria (BFA), varied from ~ 1% in vendace to ~ 4% in cyclopoids. Isoprenoid FA (4,8,12TMTD and phytanate) accounted for ~0–1% of total FA in the food web.

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The MDS ordination indicated that the compositional differences between the food web components were primarily related to the trophic position and taxa (Fig. 2). Differences in the FA composition of seston (primary producers, heterotrophic bacteria and small heterotrophic flagellates), different zooplankton groups and fish were significant, and the taxonomic grouping explained ~ 72% of the observed variation in the FA composition of the pelagic food web (PERMANOVA PseudoF9,305 = 80.588, P = 0.001). The remaining variation is likely due to seasonal and spatial differences in the FA composition (see Hiltunen et al., 2015; Strandberg et al., 2015 for details). The PUFA with the highest effect on the sample ordination were DHA and ALA. The Pearson correlation between DHA and the MDS 1 was 0.99 (Table 2), which results from the low values in the seston and high values in fish (see below for more details). Other major FA that also contributed to the distribution of the samples in the two-dimensional space were stearidonic acid (SDA or 18:4n-3), 16:1n-7, 16:0, 18:1n-9 and linoleic acid (LIN or 18:2n-6; Fig. 2, Table 2). 18:1n-9 and 16:0 were mainly responsible for the distribution of the samples along MDS 2. The mean δ15N value in the seston was ~ 3.2‰ (± 2.3‰) and in cladocerans ~ 5.1‰ (± 2.3‰). In Mysis, Chaoborus, cyclopoids, Eudiaptomus, and Heterocope the mean δ15N value varied from 8.7 to 10.0‰. In Limnocalanus the mean δ15N was 11.1‰ (±2.9‰; Table 1). The mean δ15N value in vendace was 10.0‰ (± 1.3‰) and in smelt

Table 1 FA and δ15N of seston, herbivorous cladocerans, dipteran larvae Chaoborus, cyclopoids, calanoid copepods: Eudiaptomus, Heterocope and Limnocalanus, macrocrustacean Mysis relicta and two planktivorous fish: vendace and smelt. Only those FA are presented that accounted for ≥0.5% of all FA at least in one group. FA %

14:0 4,8,12-TMTD i15:0 15:0 16:0 16:1n-9 16:1n-7 16:1n-5 ai17:0 16:2n-4 17:0 Phytanate 16:4n-3 18:0 18:1n-9 18:1n-7 18:2n-6 18:3n-3 18:4n-3 18:5n-3 20:1n-9 20:2n-6 20:4n-6 20:3n-3 20:4n-3 20:5n-3 22:5n-6 22:4n-3 22:5n-3 22:6n-3 24:1n-9 24:4n-3 24:5n-3 SFA⁎ MUFA PUFA PUFAn-3 PUFAn-6 n-3/n-6 Stable isotope ‰ δ15N

Seston N = 21

Cladocerans N = 35

Chaoborus N = 19

Cyclopoids N = 40

Eudiaptomus N = 42

Heterocope N = 22

Limnocalanus N = 37

Mysis N = 19

Vendace N = 71

Smelt N=9

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

4.0 0.0 0.7 0.3 20.3 0.3 11.0 1.6 0.1 0.7 0.2 0.0 0.5 6.9 2.7 3.1 2.1 11.4 15.0 2.0 0.0 0.4 1.5 0.0 0.0 8.9 0.8 0.0 0.0 3.9 0.0 0.0 0.0 31.2 18.8 47.2 41.7 8.6 10.8 N = 21 3.2

2.6 0.0 0.4 0.4 4.8 0.2 2.9 1.0 0.1 0.4 0.2 0.0 0.4 4.2 0.9 2.0 0.9 2.4 3.8 2.3 0.0 0.4 1.3 0.0 0.1 4.0 0.9 0.0 0.0 3.4 0.0 0.0 0.0 7.8 4.4 9.2 4.9 2.7 5.1

6.2 0.1 1.0 0.8 21.7 0.9 8.3 0.9 0.4 0.4 1.1 0.2 0.1 5.0 8.6 6.0 5.2 7.7 5.4 0.1 0.0 0.0 4.9 0.0 0.1 12.2 0.1 0.0 0.0 0.5 0.0 0.0 0.0 32.9 24.7 36.7 26.1 10.2 2.8 N = 14 5.1

2.0 0.2 0.4 0.2 3.3 0.3 2.6 0.4 0.3 0.3 0.4 0.2 0.1 1.5 2.4 1.9 1.3 1.7 2.2 0.2 0.0 0.1 2.4 0.1 0.2 3.9 0.1 0.0 0.0 0.5 0.0 0.0 0.0 4.8 5.0 7.6 6.3 3.2 1.2

2.7 0.0 0.4 0.5 21.0 0.1 3.0 0.2 0.4 0.1 1.2 0.0 0.0 6.1 14.3 4.0 5.2 8.5 2.1 0.0 0.1 0.6 3.3 0.1 0.2 18.0 0.4 0.0 0.0 5.5 0.0 0.0 0.0 29.8 21.6 44.0 34.5 9.4 3.8 N=5 10.0

1.4 0.1 0.1 0.1 3.3 0.1 1.0 0.1 0.2 0.1 0.2 0.1 0.0 3.0 2.3 0.6 0.9 1.5 0.7 0.0 0.1 1.5 0.9 0.2 0.2 1.4 0.2 0.0 0.1 1.7 0.0 0.0 0.0 5.0 2.5 4.1 2.9 2.4 0.8

5.8 0.0 0.9 0.9 21.2 0.2 5.7 0.3 0.5 0.3 1.7 0.8 0.0 6.5 3.5 2.4 4.5 8.1 5.8 0.0 0.2 0.1 1.6 0.4 0.9 9.1 1.1 0.0 0.8 13.9 0.2 0.0 0.0 33.5 12.5 46.6 39.0 7.3 5.7 N = 16 9.9

2.3 0.1 0.6 0.4 6.1 0.1 4.8 0.1 0.4 0.3 1.3 0.5 0.1 3.2 0.8 0.8 1.2 3.0 4.0 0.0 0.2 0.1 1.0 0.3 0.6 2.5 0.6 0.0 0.6 3.1 0.2 0.0 0.0 8.8 4.9 9.4 8.3 2.4 1.8

5.8 1.4 0.8 0.8 20.8 0.1 3.3 0.3 0.5 0.3 1.1 0.8 0.1 4.8 3.9 1.8 3.8 8.5 7.6 0.1 0.1 0.3 1.8 0.0 0.1 11.0 2.3 0.0 0.1 15.3 0.5 0.0 0.0 31.3 9.9 51.2 42.7 8.2 5.5 N = 13 9.1

1.4 0.8 0.4 0.9 4.9 0.1 1.4 0.1 0.4 0.2 0.4 0.4 0.1 2.2 1.3 0.6 0.9 2.6 4.1 0.1 0.1 0.2 0.9 0.0 0.1 1.8 0.7 0.0 0.1 4.2 0.3 0.0 0.0 6.9 3.1 5.0 4.6 1.8 1.3

5.0 0.1 0.5 0.7 19.1 0.1 2.5 0.2 0.3 0.0 1.2 0.0 0.0 5.7 3.7 3.8 2.4 4.9 1.7 0.0 0.2 0.2 3.4 0.0 0.1 18.0 1.5 0.0 0.2 23.0 0.4 0.0 0.0 29.8 10.9 55.5 48.0 7.5 6.9 N=8 9.0

1.1 0.1 0.2 0.4 3.2 0.1 1.1 0.1 0.1 0.1 0.2 0.1 0.0 1.2 0.8 1.0 0.5 1.7 0.9 0.0 0.1 0.1 0.8 0.0 0.1 3.2 1.3 0.0 0.2 4.8 0.2 0.0 0.0 4.9 2.6 4.3 4.7 2.0 2.2

3.2 1.3 0.3 0.4 12.1 0.3 3.5 0.2 0.1 0.0 0.6 0.1 0.0 2.0 5.4 3.8 4.1 6.0 2.9 0.0 0.5 1.4 1.9 2.7 2.0 14.9 1.6 0.9 0.9 20.6 0.6 1.2 1.5 17.2 14.3 62.7 53.7 9.0 6.8 N = 16 11.1

1.8 0.8 0.1 0.6 5.1 0.3 1.6 0.1 0.2 0.1 0.8 0.2 0.0 1.2 1.7 0.9 1.3 2.4 1.2 0.0 0.4 0.9 0.7 1.8 1.1 3.7 0.6 1.1 0.7 5.7 0.2 1.5 1.6 7.8 3.1 7.2 6.1 3.0 2.8

3.4 0.1 0.2 0.5 22.9 0.1 7.8 0.2 0.2 0.2 0.7 0.1 0.0 2.0 14.1 3.3 1.7 2.2 0.9 0.0 0.6 0.5 3.7 0.1 0.1 17.6 0.5 0.0 0.0 15.1 0.0 0.0 0.0 28.2 26.1 42.7 36.1 6.4 6.4 N=8 8.7

1.3 0.1 0.2 0.1 4.5 0.1 5.3 0.2 0.1 0.3 0.3 0.2 0.0 1.4 1.8 0.6 0.7 1.8 1.1 0.0 0.2 0.2 1.6 0.1 0.2 2.7 0.2 0.0 0.1 4.7 0.0 0.0 0.0 4.1 5.5 5.4 5.0 2.3 2.6

1.8 0.1 0.1 0.3 24.3 0.1 1.7 0.2 0.1 0.0 0.4 0.0 0.0 6.2 5.9 2.0 1.8 2.3 1.6 0.0 0.0 0.0 4.7 0.0 0.6 9.2 1.8 0.0 1.0 32.8 0.1 0.0 0.1 23.1 12.8 62.2 50.5 11.7 5.9 N = 71 10.0

1.1 0.1 0.2 0.2 3.0 0.1 1.2 0.1 0.1 0.1 0.1 0.0 0.0 1.1 2.0 0.6 0.9 1.1 1.5 0.0 0.1 0.1 0.9 0.1 0.4 1.3 0.5 0.0 0.4 7.2 0.1 0.0 0.1 2.7 3.6 4.3 3.7 2.6 1.2

1.2 0.0 0.1 0.7 18.0 0.0 1.5 0.3 0.1 0.0 0.4 0.0 0.0 4.0 8.4 2.7 2.0 2.4 0.7 0.0 0.0 0.2 6.2 0.1 0.3 14.6 3.4 0.0 0.7 31.7 0.3 0.0 0.0 32.3 10.0 56.0 47.6 8.3 4.6 N=9 12.3

0.5 0.1 0.1 0.9 2.0 0.0 0.6 0.2 0.1 0.0 0.1 0.0 0.0 1.0 2.6 0.5 0.5 0.6 0.4 0.0 0.0 0.1 1.8 0.2 0.2 2.2 1.0 0.0 0.4 5.3 0.3 0.0 0.0 3.0 3.6 4.0 4.2 1.4 1.2

2.3

2.3

⁎ Excluding branched and odd chained saturated FA.

1.6

2.4

4.2

2.3

2.9

2.2

1.3

1.6

862

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Fig. 2. Multidimensional scaling ordination of different taxonomic groups, based on 33 FA. Taxonomic groups are separated with different colors and seasons with different symbols. FA that accounts at least 1% of all FA in the food web and have a strong correlation (r ≥ 0.6) between either of the dimension are presented as vectors. Direction of the vector indicates increasing proportions of the FA and the length of the vector the strength of the correlation (see Table 2 for the correlation coefficients).

12.3‰ (±1.6‰; Table 1). The trophic transfer patterns of PUFAn-3 were related to the molecular structure of the FA; in general the proportion of EPA and DHA increased with increasing δ15N, while C18PUFA (ALA and SDA) decreased with increasing δ15N (Fig. 3). The proportion of DHA increased in the planktivorous food chain, with the exception of filterfeeding cladocerans and Chaoborus (Fig. 3). The proportion of DHA in cladocerans was lower than in seston, and the variation due to sampling location and season was very low. In Chaoborus DHA did not significantly differ from that of seston. Other zooplankton groups and the two planktivorous fish were strongly enriched in 22:6n-3 compared to seston (Fig. 3). The proportion of DHA increased 8-fold from the seston to fish. The mean total lipid content of vendace muscle tissue varied between 2.5% and 4.8%. The trophic enrichment of EPA was not as strong as that of DHA (Fig. 3). The mean percentage of EPA in seston was 8.9%, and highest proportions were found in Chaoborus, Mysis and Heterocope (~ 18% of all FA). Interestingly, the proportion of EPA in the two fish species was different; ~ 15% of total FA in smelt, and ~ 9% in vendace. The proportions of DHA, SDA and ALA were similar between smelt and vendace (Table 1). The PUFAn-6 were generally more abundant in the consumers than in the seston (Table 1). The most abundant PUFAn-6 in the pelagic food web were arachidonic acid (ARA or 20:4n-6) and LIN (Table 1). Taxonomic differences in the proportion of PUFAn-6 were less pronounced than for PUFAn-3. However, LIN and ARA showed some taxon-specific transfer patterns. In seston, ARA accounted for ~ 1.5% of all FA and in consumers varied between ~ 1.6% and 6.2%. In consumers the lowest Table 2 Correlation coefficients (Pearson's r) of the FA and the dimensions (MDS 1 and 2). Only those FA that account at least 1% of all FA and correlate strongly (r ≥ 0.6) with MDS 1 or MDS 2 are presented. FA

MDS 1

MDS 2

16:0 16:1n-7 18:1n-9 18:2n-6 18:3n-3 18:4n-3 22:6n-3

0.21 −0.66 −0.17 −0.60 −0.77 −0.64 0.99

0.77 −0.02 0.58 −0.08 −0.29 −0.43 −0.10

mean value of ARA (b2%) was detected in cyclopoids, Eudiaptomus and Limnocalanus, while the highest values were detected in fish and cladocerans. On average 18:2n-6 accounted for b 6% of all FA, and no clear trophic enrichment was detected. C16 PUFA were minor components in organisms of the studied pelagic food webs and were predominantly detected in seston, and occasionally in trace amounts in zooplankton. The most abundant C16 and C18 MUFA in the food web were 16:1n-7 and 18:1n-9. The highest proportion of 16:1n-7 was found in the seston and the lowest proportions in vendace and smelt (Table 1). Proportion of 18:1n-9 varied between taxa; the lowest proportion of 18:1n-9 was found in seston (~3%), and highest values in Chaoborus and Mysis relicta, ~ 14% (Table 1). The most abundant SFA in all the samples was 16:0.

4. Discussion This study showed that similar to marine food webs, the pelagic food webs of large lakes can be highly enriched in PUFAn-3; ~37 - 63% of total FA in the pelagic food web were PUFA. The highest proportions of PUFAn-3 were observed in the planktivorous fish, vendace and smelt. In vendace, DHA accounted for ~ 33% of all FA, indicating that this zooplanktivorous salmonid, with muscle lipid content of 2.5–4.8%, can constitute an important source of DHA for subsequent consumers, including humans. High proportions of DHA in vendace (~ 29%) have been previously reported from Lake Kallavesi and Lake Suvasvesi (Ågren et al., 1987; Muje et al., 1989). Such high DHA proportions are comparable to marine fish (even exceeding some species; Budge et al., 2002). In marine fish DHA ranged from ~ 10 to 30% depending on the species and lipid content (Budge et al., 2002). The species that had high lipid content (N5%) typically contained proportionally less DHA than species with lower lipid content (Budge et al., 2002). The proportion of DHA in freshwater fish is highly variable and likely linked with the resource utilization and total lipid content of the muscle tissue as is in marine fish (Ahlgren et al., 1994). Contrary to most salmonids, vendace remain zooplanktivorous throughout their life (Viljanen, 1983; Northcote and Hammar, 2006), and our results indicate that their dependence on pelagic resources leads to high levels of DHA. The availability of PUFA in the pelagic food web is largely determined by the phytoplankton community composition (Strandberg

U. Strandberg et al. / Science of the Total Environment 536 (2015) 858–865

16

7

20

14

6

12 10 8 6 4

18:2n-6 (% of total FA)

15 18:4n-3 (% of total FA)

18:3n-3 (% of total FA)

863

10

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5 4 3 2 1

2 0

0

0 0

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Seston Cladocerans Chaoborus Cyclopoids Eudiaptomus Heterocope Limnocalanus Mysis Vendace Smelt

20 35

6

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22:6n-3 (% of total FA)

20:5n-3 (% of total FA)

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8 15

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Fig. 3. Contrasting trophic transfer of C18 (ALA, SDA and LIN), C20 (EPA and ARA) and C22 PUFA (DHA) in the pelagic food web of large lakes.

et al., 2015). Diatoms and cryptophytes, both being rich in PUFAn-3, dominated the phytoplankton community in sampled lakes (Strandberg et al., 2015). Diatoms are typically enriched in EPA, while cryptophytes contain a wide array of n-3 PUFA: ALA, SDA, EPA as well as DHA (Taipale et al., 2013). PUFAn-6 are considerably less abundant in phytoplankton than PUFAn-3, although in chrysophytes PUFAn-6 may account for ~ 20% of total FA (Taipale et al., 2013). Diatoms typically predominate in oligo- and mesotrophic large lakes with extensive turbulence (Lepistö and Rosenström, 1998). Furthermore, the flagellate taxa, such as cryptophytes and chrysophytes are favored in moderately humic lakes with reduced light penetration (Lepistö and Rosenström, 1998). Spring diatom blooms are common in boreal lakes (Lepistö and Rosenström, 1998), however in our study lakes diatoms remained abundant throughout the sampling period, with only a modest increase in cyanobacteria and green algae in the warmer months (Strandberg et al., 2015). Even if phytoplankton community composition ultimately determines the availability of PUFA for consumers, not all PUFA are transferred equally effectively through the food chain. The trophic transfer of PUFA in the pelagic food web of the large lakes was highly selective, and the trophic transfer pattern of PUFAn-3 depended on the molecular structure of the PUFA. In the seston, the most abundant PUFA were the C18 PUFA: ALA and SDA that accounted for ~ 26% of total FA. The proportion of C 18 PUFA decreased with increasing trophic level, while the C20 and C22 PUFAn-3 (predominantly EPA and DHA) were enriched up the food chain. Despite the temporal variation in the composition of PUFA in seston, zooplankton and fish, the enrichment of DHA throughout the pelagic food web was strong. C18 PUFA are abundant in many freshwater algae, while in consumers EPA and DHA are considered to be the physiologically active forms of

PUFAn-3 (Tocher, 2003; Martin-Creuzburg et al., 2009), thus accounting for their trophic enrichment. The contrasting trophic transfer of C18, C20 and C22 PUFAn-3 in the pelagic food web indicates taxon-specific feeding strategies (filter vs. selective feeding) and/or selective FA metabolism (i.e. selectivity in retention, biosynthesis, catabolism and modification of FA) in consumers. The accumulation of C20 PUFA in cladocerans is due to preferential retention of dietary EPA and ARA (Taipale et al., 2011), as well as active retroconversion of C22 PUFA to C20 PUFA (Strandberg et al., 2014). Similarly, the retroconversion of C22 PUFA to shorter chained analogs may partly explain the scarcity of DHA and docosapentaenoic acid n-6 (DPAn-6) in filter feeding cladocerans. Filter feeding cladocerans are not very selective in food ingestion but will discriminate food particles according to size (DeMott, 1986). Filter feeding zooplankton will ingest detritus, however in the large lakes phytoplankton accounted for on average N 90% of the zooplankton diet in the summer and autumn (Galloway et al., 2014). Copepods feed selectively and avoid ingestion of detrital particles (DeMott, 1986; Knisely and Geller, 1986). Particle size is an important discriminating factor, but also palatability, the food quality in terms of “food” vs. “non-food” or “toxic food” and motility of the prey affects feeding patterns (DeMott, 1986; Knisely and Geller, 1986). Compared to cladocerans copepods are at a higher trophic position (based on their δ 15N signatures), possibly because of consumption of microzooplankton such as rotifers and copepod nauplii. The trophic enrichment of C20 and C22 PUFA and the concurrent decline of C18 PUFA in the food web also may indicate conversion of C 18 PUFA to longer chained analogs. Both cladocerans and copepods seem to be able to elongate and desaturate C18 PUFA, but this metabolic pathway is not considered to be effective enough to support optimal growth and reproduction (Bell et al., 2007).

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Vendace are known to feed extensively on both cladocerans and copepods (Viljanen, 1983; Northcote and Hammar, 2006), presumably also in our study area since the δ15N of vendace should be higher if they solely consumed copepods. However, it is well established that cladocerans contain very little DHA, while freshwater copepods in general are rich in DHA (Persson and Vrede, 2006; Smyntek et al., 2008; Ravet et al., 2010; Burns et al., 2011; Hiltunen et al., 2015). Elongation and desaturation of shorter chained analogs (C18 PUFA or EPA) could account for the high proportion of DHA in vendace. It is uncertain if this metabolic pathway is efficient enough to explain the high values observed in the vendace, although another coregonid (Coregonus lavaretus maraena) has been shown to biosynthesize DHA from shorter chained analogs (Watanabe et al., 1989). High DHA synthesis rate in vendace would suggest small intraspecific variation due to differences in the food web structure and diet composition. However, the FA composition of vendace muscle tissue varies among lakes (Ågren et al., 1987; Muje et al., 1989; Linko et al., 1992). In Lake Säkylän Pyhäjärvi (Southwestern Finland), DHA accounted for only ~13% of total FA and the mean n-3/n-6 ratio was 3.6 (Linko et al., 1992) compared to the 5.9 found in vendace from lakes in the Eastern Finland. The DHA content in vendace was linked to the abundance of DHA in zooplankton, and the spatial variation in the vendace FA composition reflects concurrent differences in the diet. The lower DHA content in the zooplankton in Lake Säkylän Pyhäjärvi was related to the abundance of cladocerans in the zooplankton community (Linko et al., 1992). It is most likely that direct dietary intake of DHA is the most important source of DHA in vendace, and DHA is effectively retained in the body. The trophic transfer of PUFA throughout the food web does not necessarily correlate with the retention of PUFA at the taxon level. For instance, in zooplankton the proportion of ARA increased during the summer feeding period (Hiltunen et al., 2015), indicating that ARA is highly retained in the zooplankton, which is in accordance with previous studies (Kainz et al., 2004; Taipale et al., 2011). However, no clear trophic enrichment of ARA was observed across the trophic levels. Instead, the proportion of ARA was highly taxa-specific. ARA is a PUFAn6 that has specific physiological functions in consumers, e.g. it is a precursor of local hormones, and may be particularly important for the early development of fish (Tocher, 2003). Compared to PUFAn-3, the proportion of PUFAn-6 was much lower (5–12% of total FA); the highest proportions of PUFAn-6 were found in cladocerans and smelt. In cladocerans, the proportions of PUFA were not significantly different from those previously reported in smaller boreal lakes (Lau et al., 2012), suggesting that lake size may not strongly affect PUFA retention of cladocerans. The calanoid copepods, particularly the Limnocalanus macrurus and Heterocope spp. were enriched in PUFAn-3 and their n-3/n-6 ratio was higher, than previously reported for copepods in small lakes (Lau et al., 2012). Similarly, Mysis relicta as well as the planktivorous fish had a high n-3/n-6 ratio. The large body size and high lipid content together with the abundance of PUFAn-3 suggest that Limnocalanus, Heterocope and Mysis may be key taxa in conveying the physiologically important PUFA to planktivorous fish in these large lakes. Post glacial rebound separated large areas from the ancient Baltic proper, isolating marine species in inland lakes. Due to their dependence on the cold, well-oxygenated hypolimnia, these cold-adapted glacial relicts are only present in large and relatively deep lakes (Väinolä and Rockas, 1990). Changes in the planktonic community composition as a result of eutrophication or the introduction of invasive species may affect the composition and transfer of PUFA in the food web. Large-scale changes in the food web in Lake Ontario have been attributed to the invasion of dreissenid mussels (Paterson et al., 2014). Rainbow smelt (Osmerus mordax), alewife (Alosa pseudoharengus) and slimy sculpin (Cottus cognatus) increased their reliance for littoral resources as a response to the invasion, and the dietary shift was reflected in the FA composition of the fishes (Paterson et al., 2014). The lakes in the current study have been relatively protected from

invasive species due to low water temperature, an isolated northern location and low concentrations of nutrients and major ions (Pienimäki and Leppäkoski, 2004). In addition to increasing the risk of species invasions, increased water temperature and prolonged growing season as a consequence of climate change are anticipated to benefit cyanobacteria in boreal lakes, particularly if accompanied with increased nutrient loading (Anneville et al., 2015). Cyanobacteria do not synthesize C 20 and C22 PUFA and many taxa also have low n-3/n-6 ratios, potentially decreasing the availability of PUFAn-3 to consumers. Increased water temperatures would also threaten the survival of cold-adapted zooplankton, e.g. Limnocalanus. The ongoing brownification of boreal lakes due to increased loading of terrestrial organic matter from the catchment area influences the physical and chemical environment (e.g. the light regime, nitrogen concentration). Brownification and high content of dissolved organic matter in lakes has also been suggested to decrease primary production (Thrane et al., 2014), but the PUFA dynamics in seston may not be that straightforward (Gutseit et al., 2007). Compositional changes in the phytoplankton community, specifically increased proportions of flagellates, may increase the amount of PUFA in the food web (Gutseit et al., 2007). However, low digestibility and defenses against grazing (e.g. toxins, large size or tendency to form large colonies) of algae may limit the trophic transfer of PUFA. For instance, the raphidophyte Gonyostomum semen, an abundant source of EPA, is common in many humic lakes but due to its large size G. semen are not effectively grazed by smaller zooplankton (Gutseit et al., 2007; Johansson et al., 2013). Brownification of lakes can also decrease water visibility, which may shift fish that have plastic feeding habits to a more benthic diet (Estlander et al., 2010). Vendace is an obligate planktivore that cannot easily shift its feeding habits. However, ecosystem-scale changes in the food web structure and function could alter the PUFA composition of vendace from large lakes, which are currently a valuable source of DHA for secondary consumers.

5. Conclusion Selective trophic transfer of PUFA in large lakes results in high proportions of PUFAn-3, particularly DHA, in vendace. In general the proportion of C18 PUFA declined in the food chain while C20 and C22 PUFA increased. Presumably, the reasons for the trophic enrichment of C20 and C22PUFA are the selective incorporation and mobilization of PUFA, and possibly also bioconversion of C18 PUFA to C20 and C22 PUFA (Watanabe et al., 1989; Bell et al., 2007). Calanoid copepods as well as mysids, which are abundant in these oligo-mesotrophic large lakes (Rahkola-Sorsa, 2008), likely account for the effective trophic transfer of DHA to vendace. In an effort to keep these aquatic ecosystems productive and efficient in transferring PUFA through the pelagic food chain all the way to humans, caution is required to prevent lakes from being degraded by eutrophication, brownification and/or spread of invasive species.

Acknowledgments We would like to acknowledge the staff of R/V Muikku: Matti Jalkanen and Jukka Kettunen, as well as Kari Ratilainen and Tuomo Nielsen (University of Eastern Finland), Jukka Kerman (Lake Suvasvesi), Esa Hirvonen and Markku Gavrilov (Finnish Game and Research Institute) for their fishing efforts and Jarmo Mononen (Pohjois-Savon Kalatalouskeskus; Lake Kallavesi) for delivering the samples. We would like to thank Sean Yeung for the help with field sampling and fish fatty acids analyses, and Stefanie Hixson for providing helpful comments on the manuscript. We also thank the anonymous reviewers for their constructive comments. The study was supported by the Academy of Finland grant 139786 to P. Kankaala.

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