Ursula Strandberg

Stratification, Composition, and Function of Marine Mammal Blubber: The Ecology of Fatty Acids in Marine Mammals

This study of vertical fatty acid profiles, based on analysis of 58 fatty acids sampled at 3‐mm intervals throughout the blubber column of a model marine mammal, the ringed seal (Pusa hispida), revealed three chemically distinct layers. The average depths of the outer and inner layers were quite consistent (∼1.5 and ∼1 cm, respectively). Consequently, the middle layer varied greatly in thickness, from being virtually absent in the thinnest animals to 2.5 cm thick in the fattest. The relative consistencies of the thickness and composition of the layers as well as the nature of the fatty acids making up each layer support the generally assumed function of the various layers: (1) the outer layer is primarily structural and thermoregulatory, (2) the inner layer is metabolically active with a fatty acid composition that is strongly affected by recent/ongoing lipid mobilization/deposition, and (3) the middle layer is a storage site that contracts and expands with food availability/consumption. The remarkable dynamics of the middle layer along with the discrete pattern of stratification found in the vertical fatty acid profiles have important implications for methodological sampling design for studies of foraging ecology and toxicology based on analyses of blubber of marine mammals.

Differing Daphnia magna assimilation efficiencies for terrestrial, bacterial, and algal carbon and fatty acids

There is considerable interest in the pathways by which carbon and growth-limiting elemental and biochemical nutrients are supplied to upper trophic levels. Fatty acids and sterols are among the most important molecules transferred across the plant-animal interface of food webs. In lake ecosystems, in addition to phytoplankton, bacteria and terrestrial organic matter are potential trophic resources for zooplankton, especially in those receiving high terrestrial organic matter inputs. We therefore tested carbon, nitrogen, and fatty acid assimilation by the crustacean Daphnia magna when consuming these resources. We fed Daphnia with monospecific diets of high-quality (Cryptomonas marssonii) and intermediate-quality (Chlamydomonas sp. and Scenedesmus gracilis) phytoplankton species, two heterotrophic bacterial strains, and particles from the globally dispersed riparian grass, Phragmites australis, representing terrestrial particulate organic carbon (t-POC). We also fed Daphnia with various mixed diets, and compared Daphnia fatty acid, carbon, and nitrogen assimilation across treatments. Our results suggest that bacteria were nutritionally inadequate diets because they lacked sterols and polyunsaturated omega-3 and omega-6 (omega-3 and omega-6) fatty acids (PUFAs). However, Daphnia were able to effectively use carbon and nitrogen from Actinobacteria, if their basal needs for essential fatty acids and sterols were met by phytoplankton. In contrast to bacteria, t-POC contained sterols and omega-6 and omega-3 fatty acids, but only at 22%, 1.4%, and 0.2% of phytoplankton levels, respectively, which indicated that t-POC food quality was especially restricted with regard to omega-3 PUFAs. Our results also showed higher assimilation of carbon than fatty acids from t-POC and bacteria into Daphnia, based on stable-isotope and fatty acids analysis, respectively. A relatively high (>20%) assimilation of carbon and fatty acids from t-POC was observed only when the proportion of t-POC was >60%, but due to low PUFA to carbon ratio, these conditions yielded poor Daphnia growth. Because of lower assimilation for carbon, nitrogen, and fatty acids from t-POC relative to diets of bacteria mixed with phytoplankton, we conclude that the microbial food web, supported by phytoplankton, and not direct t-POC consumption, may support zooplankton production. Our results suggest that terrestrial particulate organic carbon poorly supports upper trophic levels of the lakes.

Inferring phytoplankton community composition with a fatty acid mixing model

The taxon specificity of fatty acid composition in algal classes suggests that fatty acids could be used as chemotaxonomic markers for phytoplankton composition. The applicability of phospholipid-derived fatty acids as chemotaxonomic markers for phytoplankton composition was evaluated by using a Bayesian fatty acid-based mixing model. Fatty acid profiles from monocultures of chlorophytes, cyanobacteria, diatoms, euglenoids, dinoflagellates, raphidophyte, cryptophytes and chrysophytes were used as a reference library to infer phytoplankton community composition in five moderately humic, large boreal lakes in three different seasons (spring, summer and fall). The phytoplankton community composition was also estimated from microscopic counts. Both methods identified diatoms and cryptophytes as the major phytoplankton groups in the study lakes throughout the sampling period, together accounting for 54-63% of the phytoplankton. In addition, both methods revealed that the proportion of chlorophytes and cyanobacteria was lowest in the spring and increased towards the summer and fall, while dinoflagellates peaked in the spring. The proportion of euglenoids and raphidophytes was less than 8% of the phytoplankton biomass throughout the sampling period. The model estimated significantly lower proportions of chrysophytes in the seston than indicated by microscopic analyses. This is probably because the reference library for chrysophytes included too few taxa. Our results show that a fatty acid-based mixing model approach is a promising tool for estimating the phytoplankton community composition, while also providing information on the nutritional quality of the seston for consumers. Both the quantity and the quality of seston as a food source for zooplankton were high in the spring; total phytoplankton biomass was ;56 l gCL � 1 , and the physiologically important polyunsaturated fatty acids 20:5n-3 and 22:6n-3 comprised ;22% of fatty acids.

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

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 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.

Lake eutrophication and brownification downgrade availability and transfer of essential fatty acids for human consumption.

Fish are an important source of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) for birds, mammals and humans. In aquatic food webs, these highly unsaturated fatty acids (HUFA) are essential for many physiological processes and mainly synthetized by distinct phytoplankton taxa. Consumers at different trophic levels obtain essential fatty acids from their diet because they cannot produce these sufficiently de novo. Here, we evaluated how the increase in phosphorus concentration (eutrophication) or terrestrial organic matter inputs (brownification) change EPA and DHA content in the phytoplankton. Then, we evaluated whether these changes can be seen in the EPA and DHA content of piscivorous European perch (Perca fluviatilis), which is a widely distributed species and commonly consumed by humans. Data from 713 lakes showed statistically significant differences in the abundance of EPA- and DHA-synthesizing phytoplankton as well as in the concentrations and content of these essential fatty acids among oligo-mesotrophic, eutrophic and dystrophic lakes. The EPA and DHA content of phytoplankton biomass (mgHUFAg-1) was significantly lower in the eutrophic lakes than in the oligo-mesotrophic or dystrophic lakes. We found a strong significant correlation between the DHA content in the muscle of piscivorous perch and phytoplankton DHA content (r=0.85) as well with the contribution of DHA-synthesizing phytoplankton taxa (r=0.83). Among all DHA-synthesizing phytoplankton this correlation was the strongest with the dinoflagellates (r=0.74) and chrysophytes (r=0.70). Accordingly, the EPA+DHA content of perch muscle decreased with increasing total phosphorus (r2=0.80) and dissolved organic carbon concentration (r2=0.83) in the lakes. Our results suggest that although eutrophication generally increase biomass production across different trophic levels, the high proportion of low-quality primary producers reduce EPA and DHA content in the food web up to predatory fish. Ultimately, it seems that lake eutrophication and brownification decrease the nutritional quality of fish for human consumers.

A fatty acid based bayesian approach for inferring diet in aquatic consumers

We modified the stable isotope mixing model MixSIR to infer primary producer contributions to consumer diets based on their fatty acid composition. To parameterize the algorithm, we generated a ‘consumer-resource library’ of FA signatures of Daphnia fed different algal diets, using 34 feeding trials representing diverse phytoplankton lineages. This library corresponds to the resource or producer file in classic Bayesian mixing models such as MixSIR or SIAR. Because this library is based on the FA profiles of zooplankton consuming known diets, and not the FA profiles of algae directly, trophic modification of consumer lipids is directly accounted for. To test the model, we simulated hypothetical Daphnia comprised of 80% diatoms, 10% green algae, and 10% cryptophytes and compared the FA signatures of these known pseudo-mixtures to outputs generated by the mixing model. The algorithm inferred these simulated consumers were comprised of 82% (63-92%) [median (2.5th to 97.5th percentile credible interval)] diatoms, 11% (4-22%) green algae, and 6% (0-25%) cryptophytes. We used the same model with published phytoplankton stable isotope (SI) data for δ13C and δ15N to examine how a SI based approach resolved a similar scenario. With SI, the algorithm inferred that the simulated consumer assimilated 52% (4-91%) diatoms, 23% (1-78%) green algae, and 18% (1-73%) cyanobacteria. The accuracy and precision of SI based estimates was extremely sensitive to both resource and consumer uncertainty, as well as the trophic fractionation assumption. These results indicate that when using only two tracers with substantial uncertainty for the putative resources, as is often the case in this class of analyses, the underdetermined constraint in consumer-resource SI analyses may be intractable. The FA based approach alleviated the underdetermined constraint because many more FA biomarkers were utilized (n < 20), different primary producers (e.g., diatoms, green algae, and cryptophytes) have very characteristic FA compositions, and the FA profiles of many aquatic primary consumers are strongly influenced by their diets.

Terrestrial carbohydrates support freshwater zooplankton during phytoplankton deficiency

Freshwater food webs can be partly supported by terrestrial primary production, often deriving from plant litter of surrounding catchment vegetation. Although consisting mainly of poorly bioavailable lignin, with low protein and lipid content, the carbohydrates from fallen tree leaves and shoreline vegetation may be utilized by aquatic consumers. Here we show that during phytoplankton deficiency, zooplankton (Daphnia magna) can benefit from terrestrial particulate organic matter by using terrestrial-origin carbohydrates for energy and sparing essential fatty acids and amino acids for somatic growth and reproduction. Assimilated terrestrial-origin fatty acids from shoreline reed particles exceeded available diet, indicating that Daphnia may convert a part of their dietary carbohydrates to saturated fatty acids. This conversion was not observed with birch leaf diets, which had lower carbohydrate content. Subsequent analysis of 21 boreal and subarctic lakes showed that diet of herbivorous zooplankton is mainly based on high-quality phytoplankton rich in essential polyunsaturated fatty acids. The proportion of low-quality diets (bacteria and terrestrial particulate organic matter) was <28% of the assimilated carbon. Taken collectively, the incorporation of terrestrial carbon into zooplankton was not directly related to the concentration of terrestrial organic matter in experiments or lakes, but rather to the low availability of phytoplankton.

Spatial and length-dependent variation of the risks and benefits of consuming Walleye (Sander vitreus)

Restricted fish consumption due to elevated contaminant levels may limit the intake of essential omega-3 fatty acids, such as eicosapentaenoic (EPA; 20:5n-3) and docosahexaenoic (DHA; 22:6n-3) acids. We analyzed lake- and length-specific mercury and EPA+DHA contents in Walleye (Sander vitreus; Mitchell 1818) from 20 waterbodies in Ontario, Canada, and used this information to calculate the theoretical intake of EPA+DHA when the consumption advisories are followed. The stringent consumption advisory resulted in decreased EPA+DHA intake regardless of the EPA+DHA content in Walleye. Walleye length had a strong impact on the EPA+DHA intake mainly because it was positively correlated with the mercury content and thereby consumption advisories. The highest EPA+DHA intake was achieved when smaller Walleye (30-40cm) were consumed. The strong relationship between the consumption advisory and EPA+DHA intake enabled us to develop a more generic regression equation to estimate EPA+DHA intake from the consumption advisories, which we then applied to an additional 1322 waterbodies across Ontario, and 28 lakes from northern USA for which Walleye contaminant data are available but fatty acid data are missing. We estimate that adequate EPA+DHA intake (>250mgday-1) is achieved in 23% of the studied Ontario lakes, for the general population, when small (30-40cm) Walleye are eaten. Consumption of medium- (41-55cm), and large-sized (60-70cm) Walleye would provide adequate EPA+DHA intake from only 3% and 1% of the lakes, respectively. Our study highlights that mercury contamination, which triggers consumption advisories, strongly limits the suitability of Walleye as the sole dietary source of EPA+DHA to humans.

Decrease of Population Divergence in Eurasian Perch (Perca fluviatilis) in Browning Waters: Role of Fatty Acids and Foraging Efficiency

Due to altered biogeochemical processes related to climate change, highly colored dissolved organic carbon (DOC) from terrestrial sources will lead to a water “brownification” in many freshwater systems of the Northern Hemisphere. This will create deteriorated visual conditions that have been found to affect habitat-specific morphological variations in Eurasian perch (Perca fluviatilis) in a previous study. So far, potential drivers and ultimate causes of these findings have not been identified. We conducted a field study to investigate the connection between morphological divergence and polyunsaturated fatty acid (PUFA) composition of perch from six lakes across a gradient of DOC concentration. We expected a decrease in the prevalence of PUFAs, which are important for perch growth and divergence with increasing DOC concentrations, due to the restructuring effects of DOC on aquatic food webs. In general, rate of morphological divergence in perch decreased with increasing DOC concentrations. Proportions of specific PUFAs (22:6n-3, 18:3n-3, 20:5n-3, and 20:4n-6) identified to primarily contribute to overall differences between perch caught in clear and brown-water lakes tended to be connected to overall decline of morphological divergence. However, no overall significant relationship was found, indicating no severe limitation of essential fatty acids for perch inhabiting brown water lakes. We further broaden our approach by conducting a laboratory experiment on foraging efficiency of perch. Therefore, we induced pelagic and littoral phenotypes by differences in habitat-structure and feeding mode and recorded attack rate in a feeding experiment. Generally, fish were less efficient in foraging on littoral prey (Ephemeroptera) when visual conditions were degraded by brown water color. We concluded that browning water may have a strong effect on the forager’s ability to find particular food resources, resulting in the reduced development of evolutionary traits, such as habitat- specific morphological divergence.

Trophic upgrading via the microbial food web may link terrestrial dissolved organic matter to Daphnia

36 Direct consumption of allochthonous resources generally yields poor growth and reproduction in 37 zooplankton, but it is still unclear how trophic upgrading of terrestrial dissolved organic matter 38 (tDOM) via microbial food web may support zooplankton. We compared survival, somatic growth, 39 and reproduction of Daphnia magna fed with heterotrophic flagellate Paraphysomonas vestita and 40 three algal diets. Paraphysomonas was fed lake bacteria that used tDOM as a substrate to simulate 41 an allochthonous diet that zooplankton encounter in lakes. The highest survival, growth, and 42 reproduction was achieved with a diet of Cryptomonas, while Daphnia performance was the worst 43 when fed Microcystis. Paraphysomonas and Scenedesmus diets lead to intermediate growth and 44 reproduction. Cryptomonas contained high amounts of essential polyunsaturated fatty acids (PUFA) 45 and phytosterols that supported high somatic growth and reproduction, whereas poor performance 46 of Daphnia on cyanobacterial diet was most likely due to lack of sterols. Paraphysomonas 47 contained some phytosterols, but not in sufficient amounts, and also essential PUFA 48 (eicosapentaenoic and arachidonic acid) that enhance zooplankton growth and reproduction. Our 49 results indicate that tDOM-based microbial food web supports Daphnia performance even as a sole 50 food source, and may be important in providing zooplankton with essential biochemical 51 components when phytoplankton quantity or quality is low. 52 Introduction 53 The recognition that consumers in aquatic ecosystems may be fueled by terrestrial food 54 sources in addition to in-lake phytoplankton production has sparked vast amount of research during 55 the past three decades. The extent and possible pathways of consumer allochthony have been 56 studied both under laboratory conditions and in the field (e.g. Grey et al. 2001; Pace et al., 2004; 57 Berggren et al., 2014; Taipale et al., 2014). Field studies utilizing stable isotope ratios (C, N, H) 58 have found that large fraction of consumer biomass can be traced to allochthonous sources (Pace et 59 al., 2004; Berggren et al., 2014; Tanentzap et al. 2017, and references therein). Zooplankton 60 allochthony may also vary seasonally following the relative availability of phytoplankton and 61 allochthonous food sources (Grey et al. 2001). However, laboratory feeding experiments have 62 questioned the feasibility of high zooplankton allochthony. Although zooplankton (mainly 63 Daphnia) can survive on purely allochthonous diets, their growth efficiency, somatic growth rate 64 and reproductive output is very low on allochthonous compared to phytoplankton diets (Brett et al., 65 2009; Wenzel et al., 2012; Taipale et al., 2014). Consequently, high inputs of terrestrial carbon and 66 high consumer allochthony have been linked to low production of wild zooplankton (Kelly et al., 67 2014) and fish (Rask et al., 2014; Karlsson et al., 2015). 68 Most laboratory feeding trials testing consumer allochthony have been conducted using 69 terrestrial particulate organic matter (tPOM) as the food source. More than 90% of terrestrial 70 organic matter in lakes is in the dissolved form (DOM) (Kortelainen et al., 1993; Mattsson et al., 71 2005), and tPOM entering the lake in the shoreline or via river flow may rapidly sediment out of the 72 water column. Thus, pelagic consumers especially in large lakes may have limited access to tPOM. 73 Terrestrial DOM (tDOM) can be used as a substrate by bacteria, which can be grazed by 74 heterotrophic protists including flagellates and ciliates (the microbial loop) or directly by 75 zooplankton (Tranvik, 1992; Weisse 2004). Daphnia have been shown to benefit from tDOM 76 directly or via tDOM-supported bacteria when algae is limiting (McMeans et al., 2015). Previous 77 studies (Wenzel et al., 2012; Taipale et al., 2014) have found that Daphnia performance is better 78 when feeding on mixtures of phytoplankton and bacteria than on mixtures of phytoplankton and 79 tPOM, suggesting that DOM may be the more probable pathway for allochthonous organic matter 80 to enter the grazer food web. According to feeding experiments, bacteria alone cannot support 81 Daphnia growth and some taxa may even be toxic to Daphnia as a sole food source (Taipale et al., 82 2012; Freese and Martin-Creuzburg 2012). Few studies have been conducted on Daphnia 83 performance on diets of heterotrophic flagellates, but results have been variable (Sanders et al., 84 1996; Bec et al., 2003; 2006). 85 One of the reasons proposed why Daphnia has poor growth on allochthonous diets is their 86 lack of essential biomolecules, especially polyunsaturated fatty acids (PUFA) and sterols (Brett et 87 al., 2009; Taipale et al., 2014). Compared to many algae, tPOM contains very little PUFA, while 88 bacteria contain none (Lechevalier and Lechevalier 1988; Taipale et al., 2014). Some studies have 89 found that the fatty acid composition of heterotrophic flagellates depends on whether they feed on 90 algae or bacteria (Zhukova and Kharlamenko, 1999; Véra et al., 2001) while others conclude that 91 biosynthesis of lipids produces a consistent fatty acid (and sterol) composition in flagellates 92 irrespective of diet (Bec et al., 2010; Parrish et al., 2012). Bacteria, including cyanobacteria, also 93 lack sterols while phytoplankton contain various sterols in composition that is species-specific 94 (Taipale et al., 2016). The sterol composition of flagellates is poorly studied, but so far studies have 95 indicated that heterotrophic flagellates are capable of sterol synthesis (Klein Breteler et al., 1999; 96 Bec et al., 2006). In addition to concentrating PUFA and sterols present in their food e.g. by 97 selective retention, heterotrophic flagellates may enhance low quality bacterial or cyanobacterial 98 food for Daphnia by either biosynthesizing PUFA and sterols de novo or modifying dietary short99 chain PUFA to eicosapentaenoic acid (EPA, 20:5ω3) and docosahexaenoic acid (DHA, 22:6ω3). 100 This so called ‘trophic upgrading’ by heterotrophic flagellates has been observed in several studies 101 (Klein Breteler et al., 1999; Veloza et al., 2006; Bec et al., 2006; 2010). Also, indirect evidence of 102 trophic upgrading was obtained when increased abundance of Paraphysomonas vestita in a 103 decaying Microcystis culture was associated with rising EPA and DHA concentrations with a 104 concurrent decrease in short-chain PUFA prominent in Microcystis (Park et al., 2003). 105 Previous studies on Daphnia performance on allochthonous diets have used tPOM, single 106 strains of bacteria grown in artificial growth media, or heterotrophic flagellates growing on these 107 bacteria as a diet source (but see McMeans et al., 2015). We conducted a feeding experiment where 108 we constructed a simple microbial food web of tDOM (peat extract)-natural lake bacteria109 Paraphysomonas vestita to better simulate the pathway for allochthonous carbon to enter 110 zooplankton diets in lakes. We compared Daphnia somatic growth and reproduction on this 111 allochthonous diet to diets of three phytoplankton taxa (Cryptomonas, Scenedesmus, Microcystis) 112 known to vary in their quality as food for Daphnia. Our hypothesis was that 1) Daphnia survival 113 would be better on tDOM-based microbial diet than on pure bacterial diets (as seen in other studies) 114 and 2) Daphnia somatic growth and reproduction would be lower than on the algal diets. 115 116 Method 117 Experimental set up 118 We compared survival, growth and reproduction of Daphnia feeding on either diets of 119 algae or a diet of a heterotrophic flagellate that was grown on bacteria utilizing tDOM as a 120 substrate. For the experiment, we used Daphnia magna clone (DK-35-9), that originated from a 121 pond in North Germany and has been raised successfully in laboratory for several years. Prior to the 122 experiment Daphnia were reared several generations on Scenedesmus. Daphnia neonates (<24h 123 old) of multiple moms were pooled and randomly distributed among treatments (20 ind./treatment) 124 and some were used to determine Daphnia mean initial body weight. During the feeding 125 experiment, individual Daphnia were raised in 40mL vials in ADaM medium (Klüttgen et al., 126 1994). Daphnia were maintained on one of five different diets: no food, Cryptomonas marssonii, 127 Scenedesmus gracilis, Microcystis sp. (strain 130, unicellular, non-toxic) or the heterotrophic 128 flagellate Paraphysomonas vestita. The three algae were cultured in growth media optimal for each 129 of them (Table 1) in 14h:10h light:dark cycle at 20oC. The heterotrophic flagellate was grown in a 130 culture medium containing tDOM extracted from unfertilized garden peat (Kekkilä luonnonturve) 131 which was inoculated with lake bacteria (1 mL of 0.2 μm filtered lake water) a few days prior to 132 addition of the flagellate. Paraphysomonas was concentrated with gentle centrifugation, but the diet 133 given to Daphnia likely contained also bacteria. 134 The media was changed and the Daphnia fed every other day. We offered the food at non135 limiting concentration: 1.5 mg C L-1 on days 0-2, 2 mg C L-1 on day 4 and 5 mg C L-1 from day 6 136 onwards. Every day Daphnia were inspected and dead animals, and the number of offspring were 137 recorded. Sampling was conducted in the middle and in the end of the experiment (days 7 and 14), 138 and Daphnia (10 ind.) in each treatment were collected for measurements of length, weight and 139 subjected to fatty acid analysis. Due to difficulties in culturing the heterotrophic flagellate, we 140 ended the Paraphysomonas treatment already after 12 days. To facilitate the comparison with 141 Daphnia in algal diets that lasted 14 days, the eggs and embryos in Daphnia brood pouch in the 142 Paraphysomonas treatment on day 12 were included as “potential neonates” for day 14. 143 144 Fatty acid and sterol analysis 145 Prior to analy