Detailed Stratification Patterns and Vertical Layering of Lipids in Seal Blubber

dissertations

Ursula Strandberg Detailed stratification

patterns and vertical blubber seal in of lipids layering and vertical patterns stratification | Detailed Strandberg | No 65 | Ursula layering of lipids in seal blubber – source of ecophysiological information Fatty acids are among the most analyzed components in marine mammal blubber, and increasingly employed Ursula Strandberg in ecological and physiological studies. However, the interpretation of Detailed stratification the results from these analyses is not straightforward, as the fatty acids patterns and vertical and other lipids are stratified in the blubber. This thesis documents the layering of lipids detailed stratification patterns of lipids in blubber, discusses the physiological and ecological implications in seal blubber of the biochemical stratification of – source of ecophysiological information blubber and provides a suggestion for standardized sampling procedure.

Publications of the University of Eastern Finland Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences Dissertations in Forestry and Natural Sciences No 65

isbn 978-952-61-0764-6 issn 1798-5668 issnl 1798-5668 isbn 978-952-61-0765-3 (pdf) issn 1798-5676 (pdf) URSULA STRANDBERG

Detailed stratification patterns and vertical layering of lipids in seal blubber

– source of ecophysiological information

Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences No 65

Academic Dissertation To be presented by permission of the Faculty of Science and Forestry for public examination in the Auditorium N100 in Natura Building at the University of Eastern Finland, Joensuu, on May 11, 2012, at 12 o’clock noon.

Department of Biology Kopijyvä Joensuu, 2012 Editors: Prof. Pertti Pasanen Prof. Matti Vornanen, Prof. Kai Peiponen Distribution: Eastern Finland University Library / Sales of publications [email protected]; http://www.uef.fi/kirjasto tel. +358-50-3058396

ISBN: 978-952-61-0764-6 ISSN: 1798-5668 ISSNL: 1798-5668 ISBN: 978-952-61-0765-3 (PDF) ISSN: 1798-5676 (PDF) Author’s address: University of Eastern Finland Department of Biology P.O. Box 111 80101 JOENSUU FINLAND email: [email protected]

Supervisors: Docent Reijo Käkelä, Ph.D. University of Helsinki Department of Biosciences Physiology and Neuroscience P.O. Box 65 00014 University of Helsinki FINLAND email: [email protected]

Professor Matti Vornanen, Ph.D. University of Eastern Finland Department of Biology P.O. Box 111 80101 JOENSUU FINLAND email: [email protected]

Reviewers: Associate professor Heather Koopman, Ph.D. University of North Carolina Wilmington Biology & Marine Biology NC 28403 Wilmington USA email: [email protected]

Professor Seppo Saarela, Ph.D. University of Oulu Department of Biology P.O. Box 3000 FIN-90014 Oulu FINLAND email: [email protected]

Opponent: Professor Esa Hohtola, Ph.D. University of Oulu Department of Biology P.O. Box 3000 FIN-90014 Oulu email: [email protected]

ABSTRACT

Fatty acids are among the most analyzed components in marine mammal blubber, and increasingly employed in a variety of ecological and physiological studies, particularly in the evaluation of trophic interactions. It has become evident that the interpretation of the results from fatty acid analyses is not straightforward, as the fatty acid composition is stratified throughout the blubber layer. The general distribution of fatty acids in blubber is known, but knowledge on the detailed stratification patterns of fatty acids in seal blubber is scarce. However, for the correct interpretation of results from fatty acid analyses, information on the detailed stratification patterns and metabolism of fatty acids in the blubber is essential. The main objectives of this thesis were to i) document the detailed fatty acid stratification pattern in the blubber of closely related phocid seals, ii) to investigate how depth-specific lipid and fatty acid compositions may be associated with different blubber functions (mainly energy storage and thermoregulation) and iii) to deduce how the results could be applied in the development of standardized sampling procedures of seal blubber. In order to investigate the importance of endogenous and exogenous factors for the biochemical composition and stratification of blubber, blubber samples were collected from closely related seal populations and species from different habitats; two ringed seal subspecies, a marine (the Arctic ringed seal, Pusa hispida hispida, Phh) and a freshwater subspecies (the Saimaa ringed seal, Pusa hispida saimensis, Phs), and also from the Baikal seal (Pusa sibirica, Ps), a freshwater seal that is a close relative of the ringed seal. The entire blubber layer was dissected vertically into 3 mm thick sequential subsamples, and each of them was separately analyzed for fatty acids (Phh, Phs, Ps), triacylglycerols (Phs, Ps) and main phospholipids (Phs; phosphatidylcholine; PC, sphingomyelin; SM, and phosphatidyl-ethanolamine). The analyzed lipid classes are structurally and functionally different. Phospholipid species composition provides information on the biochemical composition of the membranes, whereas the analyses of triacylglycerol and total fatty acids represent mainly the composition of storage lipids. The blubber was found to be vertically stratified, and on the basis of biochemical composition, could be divided into separate layers: superficial, middle and deep blubber. The superficial blubber had a high degree of fatty acid ̇9-desaturation, suggesting high Ǌ9-desaturase activity, but may also result from selective incorporation of Ǌ9-desaturated fatty acids in the superficial blubber. The Ǌ9-desaturase has been linked to regulation of lipid metabolism and adaptation to low tissue temperatures. The superficial blubber also demonstrated a high SM/PC ratio, which has been associated with insulin insensitivity and impaired lipid metabolism. These findings suggest that the superficial blubber might primarily serve as thermoregulatory tissue and have low lipolytic activity, which could enable the maintenance of a sufficiently thick blubber layer for insulation, even during negative energy balance, e.g. during lactation. The thickness of the superficial blubber was surprisingly stable in all the studied individuals, despite the differences in e.g. diet, age and reproductive status. The middle blubber seems to expand and shrink with food availability; thus the middle blubber can be defined as an energy deposit. The high variability in lipid composition in the deep blubber suggests that this layer is metabolically very active and is probably strongly affected by recent lipid mobilization/deposition. The observed biochemical layering is most likely associated with functional specialization of the blubber layers. Furthermore the biochemical and functional layering of the seal blubber is expected to affect deposition, distribution and mobilization of many lipophilic substances in the tissue, e.g. lipid soluble vitamins and organic pollutants. Preface

There are many people who helped me throughout this thesis. First I wish to thank my supervisors. I would like to thank Reijo Käkelä for providing the opportunity to do this thesis and also for his encouragement and guidance during the study. Matti Vornanen and Markku Keinänen are also warmly acknowledged for their willingness to help in administrative and methodological issues. I also wish to express my sincere gratitude to the entire staff and faculty of the Department of Biology at the University of Eastern Finland, for providing excellent conditions for my research.

I also wish to thank my colleagues: Heikki Hyvärinen for our inspiring and encouraging conversations, and Kit M. Kovacs, Christian Lydersen, Mervi Kunnasranta, and Anne Käkelä for their comments and guidance. The co-authors Otto Grahl- Nielsen, Elena Averina, Tero Sipilä, Jouni Koskela, Larisa Radnaeva and Evgeniya Pintaeva are also gratefully acknowledged. I would also like to acknowledge Christina Lockyer, who kindly offered me a place to stay during my research visit. I remember fondly my short time in Tromsø.

The Maj and Tor Nessling Foundation and the Finnish Doctoral Programme in Environmental Science and Technology (EnSTe) are gratefully acknowledged for their financial support.

I also heartily thank all my friends who provided the frequently needed and always appreciated distraction from the thesis; I will cherish our moments together. I also wish to thank my family, particularly my parents, Sonja and Sture who have always supported me in everything I do. Finally I wish to thank Jasmin, Jemina, Aada and Luca, whose joy in life is contagious. LIST OF ABBREVIATIONS

AVA Arterio-venous anastomose Cn FA Fatty acid with n carbons ̇9-DI ̇9-desaturation index FA Fatty acid FAME Fatty acid methyl ester MUFA Monounsaturated fatty acid NG Numerical gradient PC Phosphatidylcholine PE Phosphatidylethanolamine PUFA Polyunsaturated fatty acid SFA Saturated fatty acid SI Relative stratification index SM Sphingomyelin TAG Triacylglycerol LIST OF ORIGINAL PUBLICATIONS

This thesis is based on data presented in the following articles, referred to by the Roman numerals I-IV

I Strandberg U, Käkelä A, Lydersen C, Kovacs KM, Grahl- Nielsen O, Hyvärinen H, Käkelä R. Stratification, composition, and function of marine mammal blubber: the ecology of fatty acids in marine mammals. Physiological and Biochemical Zoology 81: 473-485, 2008.

II Strandberg U, Sipilä T, Koskela J, Kunnasranta M, Käkelä R. Vertical fatty acid profiles in blubber of a freshwater ringed seal – Comparison to a marine relative. Journal of Experimental Marine Biology and Ecology. 407: 256-265, 2011.

III Strandberg U, Kunnasranta M, Sipilä T, Käkelä R. Identification and function of neutral and polar lipids in the blubber of Saimaa ringed seal (Pusa hispida saimensis). Manuscript.

IV Averina E, Strandberg U, Radnaeva L, Pintaeva E, Grahl- Nielsen O, Käkelä R. Vertical stratification of fatty acids and triacylglycerols in the blubber of the Baikal seal: Adults, pups and foetuses. Submitted manuscript.

Publications are reprinted with the kind permission of Elsevier and University of Chicago Press. AUTHOR’S CONTRIBUTION

Strandberg U. did most of the biochemical analyses for papers I- III, and was responsible for the data analysis and writing of the original manuscripts I-III. She participated in the biochemical and data analyses for paper IV and also contributed to the preparation of paper IV. Contents

1 Introduction...... 13 1.1 Blubber morphology ...... 14 1.2 Blubber lipids ...... 16 1.2.1 Fatty acids ...... 17 1.2.2 Fatty acid metabolism ...... 20 1.2.3 Storage lipids ...... 23 1.2.4 Membrane lipids ...... 25 1.3 Lipids in marine mammal research...... 27 1.4 Background information on the studied species ...... 29 1.5 Objectives of the study ...... 31

2 Material and methods ...... 33 2.1 Blubber samples ...... 33 2.2 Fatty acid analysis ...... 34 2.3 Lipid species analysis ...... 34 2.4 Data analysis ...... 35

3 Results and discussion ...... 39 3.1 Interspecific variation of blubber lipids...... 39 3.1.1 Fatty acids ...... 39 3.1.2 Triacylglycerols ...... 40 3.2 Biochemical stratification of blubber...... 44 3.2.1 General stratification of different fatty acids...... 44 3.2.2 Depth-specific changes in the fatty acid composition ...... 46 3.2.3 Indication of variable Ǌ9-desaturase activity...... 50 3.2.4 Stratification of membrane lipids ...... 52 3.2.5 Comparing the fatty acid composition of blubber and potential prey...... 53 3.3 Biochemical and functional layering of blubber ...... 54 3.4 Suggestions for blubber sampling procedure ...... 56

4 Conclusion ...... 57

5 References...... 61

1 Introduction

Marine mammals are a diverse assemblage of species from three orders: Carnivora, Cetacea and Sirenia (Rice, 1998; Berta et al., 2006). Pinnipeds (seals, sea lions, walruses) belong to the order Carnivora, and comprise of three families: Phocidae (true or earless seals), Otaridae (eared seals or fur seals and sea lions) and Odobenidae (walruses). Whales (order Cetacea) are divided into two suborders comprising of several families: the Odontoceti (toothed whales, 10 families) and Mysticeti (baleen whales, 4 families). Manatees (Trichechidae) and the dugong (Dugongidae) are within the order Sirenia. Polar bears (Ursus maritimus) and sea otters (Enhydra lutris), both from the order Carnivora, are also categorized as marine mammals. Despite the variable ancestry, pinnipeds, cetaceans and sirenians possess similar aquatic adaptations, and one of the major adaptations is the evolvement of a blubber layer. The blubber is a specialized subcutaneous adipose tissue that is morphologically and functionally different from the adipose tissue of terrestrial mammals (Pond, 1999). Both types of adipose tissue are important for energy storage, but blubber also serves as the main thermoregulatory tissue. Terrestrial mammals, as well as polar bears and sea otters, rely on a dense fur coat for insulation and their subcutaneous adipose tissue does not contribute significantly to thermoregulation (Pond et al., 1992; Pond, 1999). Consequently, the subcutaneous adipose tissue of polar bears and sea otters is similar to that of terrestrial mammals and is not considered to be blubber. In addition to storing energy and maintaining thermal balance, the blubber affects the formation of body shape, thus influencing the streamlining, locomotion and buoyancy of marine mammals. Blubber forms a continuous layer across the body trunk, but the thickness of the blubber layer is species-specific and highly variable depending on age, reproductive and nutritional status and location on the body (Ryg et al., 1988, 1990a; Rosen &

13 Renouf, 1997; Koopman et al., 2002; Mellish et al., 2007). Variation in nutritional status is reflected in blubber mass, e.g. during fasting the mean rate of mass loss in male southern elephant seals (Mirounga leonina) is about 10 kg/day, with over 60% of the loss due to decreases in blubber (Slip et al., 1992). In phocid seals, seasonal breeding is associated with reductions in energy intake and blubber content, and the changes in the blubber thickness are more pronounced in sexually mature adult seals than in immature juveniles (Ryg et al., 1990b; Mellish et al., 2007). On average the blubber content of mature ringed seals (Pusa hispida) is 40%–50% of body mass at the beginning of breeding season and about 30% of body mass at the end of molting (Ryg et al., 1990b). The decline of the blubber content is most pronounced in sexually mature females, mainly deriving from the high energetic cost of lactation (Ryg et al., 1990b). As blubber provides thermal insulation, a sufficiently thick layer of blubber has to be maintained, even during negative energy balance (Ryg et al., 1988). An excessive decrease in blubber thickness compromises its thermoregulatory function, with consequent loss of energy as heat. This could introduce a continuous cycle of increased energy demand and further loss of blubber due to impaired thermal insulation. Additionally, the decrease of blubber may also impair buoyancy and streamlining. Although the different functions of blubber and the variability in the thickness of the blubber layer are well recognized, there is little information as to how the potentially conflicting functions of blubber (particularly thermoregulation and energy expenditure) are manifested in its biochemical composition (Koopman et al., 2002).

1.1 BLUBBER MORPHOLOGY The blubber of marine mammals is a specialized type of white adipose tissue with a high density of connective tissue fibers (collagen and elastin); in small cetaceans the collagenous fibers account for 20–80% of the blubber area (Parry et al., 1949; Hamilton et al., 2004; Struntz et al., 2004; Montie et al., 2008). The high fiber density gives the blubber a firm and fibrous structure which also differentiates it from other types of white

14 adipose tissue. The content of connective fibers in the subcutaneous adipose tissue of terrestrial mammals is markedly lower than in the blubber of marine mammals; < 1% of wet weight in Arctic foxes (Vulpes lagopus) and polar bears (Pond et al., 1992; Ramsay et al., 1992; Pond et al., 1995). Blubber is also morphologically diverse; the size, number and shape of adipocytes (fat cells that contain a large lipid droplet) and the density of fibers depend on the blubber depth and location on the body (Koopman et al., 2002; Hamilton et al., 2004; Struntz et al., 2004; Montie et al., 2008). It is suggested that the morphological diversity is connected with differences in the functions of blubber, such that the blubber tissue is functionally compartmentalized into metabolically active and structural components (Koopman et al., 2002; Montie et al., 2008). Structural blubber is characterized by lower lipid content and a higher proportion of collagenous fibers, and is less affected by the nutritional status than metabolically active adipose tissue (Koopman et al., 2002; Montie et al., 2008). In the harbor porpoise (Phocoena phocoena) the size and number of adipocytes in the tailstock blubber and the superficial blubber (immediately below the skin) were stable under various nutritional conditions, suggesting low metabolic activity (Koopman et al., 2002). Also, in the bottlenose dolphin (Tursiops truncatus), the superficial blubber had a high density of fibers and smaller sized adipocytes, suggesting that the superficial blubber serves mainly as structural tissue (Struntz et al., 2004; Montie et al., 2008). The number and size of adipocytes in the deep blubber were affected by the nutritional status; the adipocytes in the deep blubber shrank and even disappeared in emaciated harbor porpoises, while the number and size of adipocytes in the superficial blubber remained unaffected (Koopman et al., 2002). Data on the distribution of connective tissue in seal blubber is limited, but in the leopard seal (Hydrurga leptonyx) the connective fibers were distributed relatively uniformly throughout the blubber (Gray et al., 2006). Marine mammal blubber contains arterio-venous anastomoses (AVAs), specialized shunts that interconnect

15 arterioles and venules (Parry, 1949; Bryden, 1978). The AVAs are important in thermoregulation, as heat loss via the peripheral circulation can be adjusted by regulating the diameter of AVAs. The redistribution of blood flow to peripheral tissues generates a temperature gradient in the blubber; skin temperature is 15–35 °C lower than the core temperature, and it fluctuates according to the ambient temperature (Irving & Hart, 1957; Hart & Irving, 1959; Meagher et al., 2008; Noren et al., 2008). In ice-cold water (0 °C) the temperature of the superficial blubber of the harp seal (Pagophilus groenlandicus) and harbor seal (Phoca vitulina) was less than 5 °C (Irving & Hart, 1957; Hart & Irving, 1959).

1.2 BLUBBER LIPIDS In the mammalian body, lipids have three main functions: they serve as energy storage (storage lipids), constituents of cell membranes (membrane lipids) and messengers of cellular signal transduction (Vanhaesebroeck et al., 2001; van Meer et al., 2008). Additionally, lipids in cellular membranes affect the function of membrane proteins via specific protein-lipid interactions (Dowhan & Boganov, 2002). Lipids consist of a broad group of naturally occurring molecules that have various structures and functions; lipids include, for example, fatty acids, waxes, eicosanoids, triacylglycerols, glycerophospholipids, sphingolipids and sterols. Traditionally lipids are considered to be compounds that are readily dissolved in organic solvents, but this is not a very specific or precise definition. Christie & Han (2010) defined lipids as “fatty acids and their derivatives, and substances related biosynthetically or functionally to these compounds”. This definition includes gangliosides (highly complex glycosphingolipids), although the gangliosides are more water-soluble than lipids in general (Christie & Han, 2010). An even more specific classification system for lipids was suggested by the LIPID MAPS consortium: “For the purpose of classification, we define lipids as hydrophobic or amphipathic small molecules that may originate entirely or in part by carbanion-based condensations of thioesters (fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, saccharo- lipids, and polyketides) and/or by carbocation-based 16 condensations of isoprene units (prenol lipids and sterol lipids)” (Fahy et al., 2009).

1.2.1 Fatty acids Most commonly determined lipids in blubber analyses are total fatty acids, the majority of which represent cleavage products from storage lipids (in phocid seals from triacylglycerols). Fatty acids are carboxylic acids with a hydrocarbon chain that varies in length (number of carbon atoms) and degree of unsaturation (Fig. 1). The degree of fatty acid unsaturation is defined by the number of double bonds in the carbon chain; saturated fatty acids (SFA) do not contain any double bonds, while unsaturated fatty acids contain one (i.e. monounsaturated fatty acids, MUFA) or several double bonds (i.e. polyunsaturated fatty acids, PUFA; Table 1). Fatty acids in seal blubber are typically even-numbered, unbranched and consist of 14-22 carbons, although small proportions of shorter, longer, odd-chained and branched fatty acids are frequently detected (Table 1; Käkelä et al., 1995; Andersen et al., 2004; Thiemann et al., 2007; Wheatley et al., 2007; Cooper et al., 2009). Most of the branched fatty acids are considered to be of bacterial origin (e.g. iso17:0), or derived from the algal chlorophyll (isoprenoid fatty acids such as phytanate). In certain small odontocetes, e.g. the harbor porpoise, Hector’s dolphin (Cephalorhynchus hectori) and the northern right-whale dolphin (Lissodelphis borealis), the blubber contains significant proportions (2–40%) of endogenously synthesized short chained branched fatty acids, such as isovaleric acid (iso5:0), but this fatty acid has not been detected in seal blubber (Litchfield et al., 1975; Koopman et al., 1996; Koopman et al., 2003; Thiemann et al, 2008). In general, the blubber of ringed seals contains 28–47% of PUFA, 7–15% of SFA, 39–53% of ǂ C18MUFA and up to 18% of > C18 MUFA (data from seals of various ages and from both sexes, and different subspecies or populations; Käkelä et al., 1993; Käkelä & Hyvärinen, 1996; Grahl-Nielsen et al., 2003; Thiemann et al., 2007; Cooper et al., 2009). There is considerable variation in fatty acid composition (termed the fatty acid “profile”) among

17 different populations and age classes, and a significant part of the variation is thought to result from differences in the foraging patterns, diet, and also from the nutritional and reproductive status (Beck et al., 2007; Thiemann et al., 2007; Cooper et al., 2009; Newland et al., 2009; Tucker et al., 2009). The largest compositional difference in the blubber of ringed seals is found between freshwater and marine populations (Käkelä et al., 1993; Käkelä & Hyvärinen, 1996; Grahl-Nielsen et al., 2003, Thiemann et al., 2007; Cooper et al., 2009). The >C18 MUFA (mainly 20:1n-9 and 22:1n-11), are abundant in the marine environment but rare in freshwater food webs, and thus the proportion of 20:1n-9 and 22:1n-11 is very low in the blubber of freshwater ringed seals but may comprise up to 18% of total fatty acids in marine ringed seals (Käkelä et al., 1993; Käkelä & Hyvärinen, 1996; Grahl- Nielsen et al., 2003; Thiemann et al., 2007; Cooper et al., 2009). The abundance of 20:1 and 22:1 fatty acids in marine food webs originates from certain calanoid copepods that store lipids as wax esters, whereas freshwater zooplankton typically do not contain these fatty acids (Morris, 1984; Muje et al., 1989).

n-7 ȴ9 carboxyl group terminal methyl end

9-cis-Hexadecenoic acid 16:1n-7 Palmitoleic acid

Figure 1. Structure and nomenclature of palmitoleic acid (16:1n-7). The systematic name 9-cis-hexadecenoic acid states that the fatty acid contains 16 carbons in a straight chain and 1 double bond (in cis-conformation) at the 9th carbon from the carboxyl end (i.e. at ̇9 carbon). Palmitoleic is the common name and the shorthand abbreviation (16:1n-7) indicates the number of acyl carbons and double bonds; the position of the double bond is calculated from the methyl end (the 7th carbon or n-7). Either the common name or the shorthand abbreviation is used throughout the thesis.

In addition to the spatial and temporal variability among different individuals, the fatty acid composition of blubber is vertically stratified. The superficial blubber, immediately below

18 Table 1. Fatty acid shorthand abbreviation, common name and systematic name derived from the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature (1967). Approximate melting points for the fatty acids are also presented, when available (Anonymous 2002, 2012).

Melting Fatty acid Common name Systematic name point °C

14:0 Myristic acid tetradecanoic acid 54

16:0 Palmitic acid hexadecanoic acid 63

18:0 Stearic acid octadecanoic acid 69

14:1n-5 Myristoleic acid cis-9-tetradecenoic acid -4

16:1n-7 Palmitoleic acid cis-9-hexadecenoic acid 0

16:1n-9 cis-7-hexadecenoic acid

18:1n-9 Oleic acid cis-9-octadecenoic acid 14

18:1n-7 cis-Vaccenic acid cis-11-octadecenoic acid 15 all-cis-9,12-octadecadienoic 18:2n-6 Linoleic acid -7 acid all-cis-9,12,15- 18:3n-3 Į-Linolenic acid -10 octadecatrienoic acid all-cis-6,9,12,15- 18:4n-3 Stearidonic acid -57 octadecatetraenoic acid

20:1n-9 Gondoic acid cis-11-eicosenoic acid 24

20:1n-11 Gadoleic acid cis-9-eicosenoic acid 24

22:1n-11 Cetoleic acid cis-11-docosenoic acid 33

Eicosatetraenoic acid all-cis-8,11,14,17- 20:4n-3 (ETA) eicosatetraenoic acid Arachidonic acid (ARA all-cis-5,8,11,14- 20:4n-6 -49 or AA) eicosatetraenoic acid Eicosapentaenoic acid all-cis-5,8,11,14,17- 20:5n-3 -54 (EPA) eicosapentaenoic acid Docosapentaenoic acid all-cis-7,10,13,16,19- 22:5n-3 (DPA) docosapentaenoic acid n-6Docosapentaenoic all-cis-4,7,10,13,16- 22:5n-6 acid (n6DPA) docosapentaenoic acid Docosahexaenoic acid all-cis-4,7,10,13,16,19- 22:6n-3 -44 (DHA) or cervonic acid docosahexaenoic acid

the epidermis, contains proportionally more ǂC18MUFA, while the deep blubber next to the muscle is more enriched with SFA and >C18MUFA (Käkelä & Hyvärinen, 1996; Wheatley et al., 2007; Grahl-Nielsen et al, 2011a). Lipids need to be maintained

19 in a semi-fluid state and the low tissue temperature in the superficial blubber (discussed in the blubber morphology section) prevents the deposition of large proportions of fatty acids with high melting points (i.e. SFA or long-chain MUFA). The compositional differences between the superficial and deep blubber have been documented for several seal species, for example the ringed seal, the southern elephant seal and the Weddell seal (Leptonychotes weddellii), but information on the depth-specific changes in the fatty acid composition has been scarce (Käkelä & Hyvärinen, 1996; Best et al., 2003; Wheatley et al., 2007). The compositional changes in the blubber may occur linearly throughout the entire blubber layer or in phases at specific blubber depths. Knowledge on the depth-specific changes in the fatty acid composition and the detailed stratification pattern throughout the blubber, and how this varies across individuals and over time within individuals is important for the assessment of the connection between the biochemical composition and functions of blubber.

1.2.2 Fatty acid metabolism Endogenous synthesis of fatty acids from non-fatty acid precursors yields primarily the saturated fatty acid 16:0, which may be further modified. The main modification products are 16:1n-7 (Ǌ9-desaturation in the endoplasmic reticulum), or chain elongation to 18:0, which may be further Ǌ9-desaturated to 18:1n-9 (Fig. 2). In humans the de novo lipogenesis (endogenous synthesis of lipids) is generally low and down-regulated by high dietary intake of fats, particularly polyunsaturated fatty acids (Hellerstein et al., 1991). De novo lipogenesis is mainly observed when the diet is rich in carbohydrates and low in fats (Hellerstein et al., 1991). Seals generally feed on fish and large crustaceans which are deficient in carbohydrate but rich in protein and fat (Henderson & Tocher, 1987; Ventura, 2006). Although amino acids may serve as substrates for lipogenesis, it is unlikely that de novo lipogenesis is an important source of fatty acids for seals; it is more likely that most of the fatty acids are obtained from the diet.

20 ¨9 desaturase 14:0 14:1n-5 14:1n-9 14:1n-7

peroxisomal chain shortening

16:0 16:1n-7 16:1n-5 16:1n-9

elongase

18:0 18:1n-9 18:1n-7 18:1n-5

20:1n-9 20:1n-7 20:1n-5 20:0 20:1n-11

22:1n-9 22:1n-7 22:1n-11 22:0

Figure 2. Modification pathways of saturated and monounsaturated fatty acids, modified after Cook & McMaster (2002). The predominant substrates for the Ǌ9- desaturase are 16:0 and 18:0 (pathways marked with red rectangular).

The fatty acid composition of storage lipids in blubber is strongly influenced by the dietary fatty acids, but does not exactly match that of the diet (Grahl-Nielsen & Mjaavatten, 1991; Budge et al., 2004; Iverson et al., 2004; Grahl-Nielsen et al., 2011a). The fatty acid composition of blubber is affected by variable digestion and absorption efficiency of dietary fatty acids, modifications of fatty acids (mainly in the liver but also in adipose tissue) and selective fatty acid metabolism in adipose

21 tissue (Raclot, 2003; Budge et al., 2004; Mu & Høy, 2004). Fatty acid modification pathways include desaturation (introduction of a double bond at a fixed position of the acyl chain), and elongation or shortening (partial Ά-oxidation) of the acyl chain. Data on fatty acid modifications in marine mammals are limited, but Budge et al. (2004) showed that in the grey seal (Halichoerus grypus), dietary 16:0 is partly desaturated to 16:1n-7 by the ̇9- desaturase. The ̇9-desaturase introduces a double bond into the ̇9-carbon of the acyl chain (i.e. between the 9th and 10th carbon from the carboxylic end, Fig. 1). Similar to 16:0, stearic acid (18:0) may serve as a substrate for ̇9-desaturase, yielding 18:1n-9 (Ntambi & Miyazaki, 2004; Fig. 2). Highly unsaturated PUFA from the n-3 and n-6 series (mainly 20:4n-6, 20:5n-3 and 22:6n-3) are considered to be essential fatty acids, since mammals are unable to endogenously synthesize these fatty acids, although they are invaluable in various physiological processes, e.g. cell membrane fluidity and function, development of the central nervous system and visual acuity, insulin and the immune function (Jump, 2001; Nakamura & Nara, 2004). Mammals lack the necessary enzymes (Ǌ12- and Ǌ15-desaturase) needed for endogenous synthesis of n-6 and n-3 PUFA, and thus must obtain these fatty acids or their precursors from the diet (Jump, 2001; Nakamura & Nara, 2004). Modification pathways of PUFA in mammals include (Fig. 3): conversion of 18:3n-3 (΅-linolenic acid) to 20:5n-3 (EPA) and 18:2n-6 (linoleic acid) to 20:4n-6 (ARA) via alternating desaturations and chain elongations, and further to C22 PUFA via the Sprecher pathway (Sprecher et al., 1995). The Ǌ5- and Ǌ6- desaturases catalyze synthesis of PUFA by introducing an additional double bond between the pre-existing double bond and the carboxylic end of the acyl chain. Long chain fatty acids are shortened in peroxisomes via partial Ά-oxidation, and the elongation of the fatty acid chain by 2 carbon units is performed predominantly in endoplasmic reticulum by elongases, located in complexes with the desaturases (Reddy & Hashimoto, 2001; Jakobsson et al., 2006).

22 18:3n-3 18:2n-6 18:1n-9

¨6 desaturase

18:4n-3 18:3n-6 18:2n-9

elongase

20:4n-3 20:3n-6 20:2n-9

¨5 desaturase

20:5n-3 20:4n-6 20:3n-9

elongase

22:5n-3 22:4n-6

elongase

(24:5n-3) (24:4n-6) The Sprecher pathway ¨6 desaturase

(24:6n-3) (24:5n-6)

peroxisomal chain shortening

22:6n-3 22:5n-6

Figure 3. Modification processes of PUFA in mammals. The arrow on the left indicates the direction of the modifications. The C24 PUFA in parenthesis are intermediate products which are poorly esterified into lipids, but are preferably converted to C22 PUFA. Figure modified after Sprecher et al. (1995), and Cook & McMaster (2002).

1.2.3 Storage lipids In phocid seals, excess energy is stored in blubber as triacylglycerol (TAG; Henderson et al., 1994). A TAG molecule comprises three fatty acids incorporated into a glycerol backbone. The physical properties of TAG are largely determined by the composition of the fatty acid triplet. TAGs

23 with saturated fatty acids have higher melting points and are structurally more rigid than TAGs with unsaturated acyl chains. Previous studies have concluded that in seal blubber, PUFA are generally esterified at the sn-1 or sn-3 position of TAG, and C16 and C18 MUFA and SFA are the predominant fatty acids at the sn-2 position (Brockerhoff, 1966; Brockerhoff et al., 1966). This positional distribution of fatty acids in TAG differs from that found in fish; in fish the polyunsaturated fatty acids are preferentially esterified at the sn-2 position (Brockerhoff, 1966; Brockerhoff et al., 1966). TAG is the most widely spread storage lipid in marine mammals, but in certain odontocetes, namely sperm whales (Kogia sp. and Physeter macrocephalus) and beaked whales (Ziphiidae), wax esters account for 60–100% of total lipids in blubber (Koopman, 2007; Walton et al., 2008). In addition, the blubber of harbor porpoises and a few species of dolphins, e.g. hector’s dolphin and the long-finned pilot whale (Globicephala melas), contains small amounts of wax esters, predominantly in the superficial blubber, although TAG is the main storage lipid (Koopman, 2007). The reason for the deposition of wax esters in the blubber is unclear, but may relate to the different physiological properties of wax esters in connection with ecological and physiological adaptations to deep-diving and energy metabolism (Koopman, 2007). As well as in deep-diving odontoces, wax esters are typically found in deep-diving polar zooplankton and fish species (Lee et al., 2006). However, deep- diving pinnipeds, such as the northern elephant seal (Mirounga angustirostris), store lipids as TAG. The northern elephant seals experience large annual changes in energy balance (connected with reproduction and molting; Costa & Ortiz, 1982), and it may be that hydrolysis of wax esters is not able to respond to the rapid changes in energy demand (Koopman, 2007). Wax esters are long chain fatty alcohols esterified to fatty acids, and compared to TAG, wax esters are generally more stable and seem to have a slower turnover rate (Sargent et al., 1981; Lee et al., 2006).

24 1.2.4 Membrane lipids The proportion of polar lipids in adipose tissues is exceedingly small, typically < 1% of total lipids (Body, 1988; Kotronen et al., 2010). Nevertheless, the polar lipids are critical constituents of all cellular membranes and also function as cellular messengers, enzyme activators, and precursors for local tissue hormones such as eicosanoids (Dowhan & Bogdanov, 2002; van Meer et al., 2008). The lipid composition of cellular membranes is highly tissue- and species-specific (Body et al., 1988; Henderson et al., 1994), but the most abundant and widely distributed classes of mammalian membrane lipids are glycerol-based phospholipids (glycerophospholipids), sphingolipids and sterols (mainly cholesterol; Body, 1988; Parrish et al., 1997; Zeghari et al., 2000a). Glycerophospholipids are comprised of a glycerol backbone with two fatty acids esterified at the sn-1 and sn-2 positions, and a phosphate at the sn-3 position. A polar molecule (e.g. choline, ethanolamine or serine) is attached to the phosphate moiety, producing a polar head group. The most common glycerophospholipids in mammalian cell membranes are phosphatidylcholine (PC) with a choline headgroup, and phosphatidylethanolamine (PE) with an ethanolamine attached to the phosphate. Sphingolipids are sphingosine (an amino alcohol) derivates with a functional head group attached to the sphingosine, such as choline in sphingomyelin (SM). SM is the most important sphingolipid in mammalian cell membranes, and its distribution is confined to the outer leaflet of the plasma membrane (Gulati et al., 2010). Unlike the glycerophospholipids, the fatty acid moiety in SM is saturated or monounsaturated, which gives the molecule a more rigid structure. Data on the membrane lipid composition of marine mammal blubber adipocytes are limited, but in adipose tissues of humans (subcutaneous adipose tissue) and rats (epididymal and perirenal adipose tissue), the most common membrane lipids were PC (30–45% of total phospholipids) and PE (20–30% of total phospholipids; Body, 1988; Parrish et al., 1997; Zeghari et al., 2000a). The proportion of SM varied from 8 to 25% of total phospholipids. In membrane lipids in Mediterranean monk seal

25 (Monachus monachus) liver, heart and muscle, the most common polar lipid classes were PC (30–40% of phospholipids) and PE (20–30% of phospholipids; Henderson et al., 1994). PC and PE were also the most abundant glycerophospholipids in erythrocytes and platelets of northern and southern elephant seals, the Antarctic fur seal (Arctocephalus gazella) and the harp seal (Nelson, 1970; Fayolle et al., 2000). Thus, at the level of lipid classes, the main building blocks of the membranes are similar across mammals. The glycerophospholipids and sphingolipids are amphipathic; the polar head group of the phospholipids is more hydrophilic than the non-polar hydrocarbon tails. This amphipathicity is the physical basis for the formation of biomembranes (Dowhan & Bogdanov, 2002). In aqueous environments the amphipathic lipid molecules self-associate into a two layered sheet; the polar (hydrophilic) head groups face the water phases on either side of the sheet and the nonpolar (hydrophobic) fatty acid tails point at the center of the bilayer. Biological membranes are not merely static barriers in cells but their lipid composition directly affects the function of membrane proteins (receptors, transporters, channels), and thus also a myriad of cellular processes (van Meer et al., 2008). The lipid bilayer may exist in various relatively fluid and solid phases, depending on the molecular structure of the membrane lipids and the environmental conditions (van Meer et al., 2008). The membrane phases determine the orientation and mobility of membrane lipids and thus affect the functions of the membrane proteins therein. Double bonds in cis-configuration bend the fatty acid chain, preventing the dense packing of molecules and resulting in a liquid disordered phase. Lipid molecules comprised of long saturated hydrocarbon chains (as in sphingomyelin) are densely packed in membranes, producing solid-like gel phases in the membrane that affect the mobility and functions of lipids and proteins (Slotte, 1999). The amount of cholesterol embedded between the fatty acid chains modulates the physical properties and phase behavior of cellular membranes. The addition of cholesterol to a highly

26 ordered membrane introduces a liquid ordered phase, where the acyl chains are highly ordered, but some lateral movement is allowed (distinguishing it from the gel phase). Cholesterol is a neutral lipid, composed of four hydrocarbon rings with a hydrocarbon and a hydroxyl group attached to it. The distribution of cholesterol in the plasma membrane is closely associated with that of SM and other sphingolipids, and the current view is that biological membranes are laterally segregated into sphingolipid/ cholesterol rich microdomains called rafts (Simons & Ikonen, 2000; Gulati et al., 2010). It has been suggested that the large head groups of sphingolipids shield the highly hydrophobic cholesterol from the aqueous media (Gulati et al., 2010). Furthermore, cholesterol is also preferentially distributed between long chain saturated acyl chains, which are predominant in sphingolipids, but rare in glycerophospholipids (Brown & London, 2000). These sphingolipid/ cholesterol rich domains may be involved in the regulation of several cellular functions, such as lipid metabolism, by modulating the activity of membrane receptors, pumps and channels (Zeghari et al., 2000a, 2000b; Al-Makdissy et al., 2003; Ikonen & Vainio, 2005; Vainio et al., 2005; Bastiani & Parton, 2010).

1.3 LIPIDS IN MARINE MAMMAL RESEARCH The blubber of marine mammals has been employed in a wide range of physiological, biochemical and ecological studies, e.g. in studies on adaptations to the aquatic environment (particularly energy metabolism, thermoregulation and locomotion), determination of trophic interactions, and biomonitoring of lipophilic pollutants (e.g. Irving & Hart, 1957; Molyneux & Bryden, 1975; Helle et al., 1976; Ryg et al., 1988; Käkelä et al., 1997, Aquilar et al., 2002; Koopman et al., 2002; Noren et al., 2003; Iverson et al., 2004). Blubber lipids have also provided information on population/stock identification and age estimation (Smith et al., 1996; Thiemann et al., 2007; Herman et al., 2008, 2009). Analyses of the fatty acid composition of blubber are commonly applied to studies on the foraging strategies and diet of marine mammals (Iverson et al., 1997; 27 Bradshaw et al., 2003; Cooper et al., 2009). Determination of the foraging strategies and diet is inherently difficult in these animals as the catch and consumption of prey takes place under the surface. Nonetheless, knowledge of the foraging patterns and diet preferences of seals is important, not only for understanding of the aquatic food web dynamics but also for management and conservation policies (e.g. in conflicts with fisheries). The feeding behavior of seals has been assessed by analyzing prey remains in the digestive tract or in fecal samples, but these techniques overestimate the importance of prey with large and robust hard parts which resist digestion better than small soft bodied species (reviewed by Pierce & Boyle, 1991). Also, such results provide information only on the most recent meal, which may not represent long-term diet. It has been suggested that application of molecular biomarkers, mainly fatty acids and stable isotopes, and also analyses of prey DNA in the scat, overcomes some of the problems encountered in the traditional hard part analyses (Hobson et al., 1996; Budge et al., 2006; Casper et al., 2007). The basic idea of the fatty acid biomarker approach is that the dietary fatty acids are predictably incorporated into blubber, and the diet composition of the predator can be resolved by comparing the fatty acid composition of blubber to that of the potential prey items (Iverson et al., 1997; Bradshaw et al., 2003; Iverson et al., 2004; Cooper et al., 2009). The analysis of blubber fatty acid composition has been widely employed in qualitative and quantitative estimates of diet (Bradshaw et al., 2003; Walton & Pomeroy, 2003; Iverson et al., 2004; Cooper et al., 2009; Newland et al., 2009; Tucker et al., 2009). However, the clarity of results from studies on different species at different times or locations has been variable, and it has been suggested that the results may be confounded by the stratification of fatty acids and fluctuating fatty acid metabolism (Best et al., 2003; Grahl-Nielsen et al., 2011a).

28 1.4 BACKGROUND INFORMATION ON THE STUDIED SPECIES The study animals were phocid seals from marine and freshwater environments (Fig. 4): the Arctic ringed seal Pusa hispida hispida (Svalbard, Norway), the Saimaa ringed seal Pusa hispida saimensis (Lake Saimaa, Finland) and the Baikal seal Pusa sibirica (Lake Baikal, Russia). The distribution of the Arctic ringed seal is circumpolar, while the distributions of the Saimaa ringed seal and the Baikal seal are restricted to Lake Saimaa and Lake Baikal, respectively.

Figure 4. Map of the sampling locations of the studied seals: Svalbard, Lake Saimaa and Lake Baikal.

The Arctic and Saimaa ringed seals are subspecies, and the Baikal seal is a close relative of the ringed seal (Palo & Väinölä, 2006). These seals are small sized; adults weigh on average

29 about 50–70 kg and have a standard length of about 130–150 cm (Lydersen & Gjertz, 1987; Sipilä & Hyvärinen, 1998). The ringed seals and the Baikal seal are considered to be opportunistic feeders with preferences for few particular prey types. Small fish (length 5–20 cm) are the predominant prey for all three species (Thomas et al., 1982; Weslawski et al., 1994; Kunnasranta et al., 1999). Additionally, the Arctic ringed seal and the Baikal seal feed on macro-crustaceans; e.g. Parathemisto libellula, Thysanoessa inermis and Pandalus borealis are important prey items for the Arctic ringed seal, and stable isotopes analysis indicated that the Baikal seal feeds on the pelagic amphipod Macrohectopus branickii (Weslawski et al., 1994; Yoshii et al., 1999). In Lake Saimaa the only abundant macro-crustacean Mysis relicta does not seem to be an important prey item for the Saimaa ringed seal (Kunnasranta et al., 1999). The Arctic ringed seal, the Saimaa ringed seal and the Baikal seal live in completely different habitats, with different food web structures. The Arctic ringed seal is a marine subspecies, while the Saimaa ringed seal and the Baikal seal live in freshwater environments. Furthermore, the freshwater bodies of Lake Saimaa and Lake Baikal are very different. Lake Saimaa is a shallow lake (mean depth 12 m, max depth 85 m) with a heterogeneous shoreline and numerous islands, while Lake Baikal is the deepest lake in the world (mean depth 744 m, max depth 1642 m) with large areas of open water. In Lake Baikal 64% of the flora and fauna are endemic (e.g. abundant pelagic fish genus Comephorus), and the fatty acid composition in the Lake Saimaa and Lake Baikal food webs demonstrate significant differences, which are also reflected in the fatty acid composition of the seal blubber (Muje et al., 1989; Käkelä et al., 1993; Ju et al., 1997; Grahl-Nielsen et al., 2005; Grahl-Nielsen et al., 2011b). Analyses of the detailed stratification pattern of blubber lipids in closely related seals inhabiting completely different environments provide an opportunity to evaluate the impacts of endogenous and environmental factors on the depth- specific fatty acid composition and metabolism of the seal blubber.

30 1.5 OBJECTIVES OF THE STUDY The objectives of this thesis were to determine the biochemical stratification of blubber in seals that are closely related but inhabit different environments and to evaluate the impact of exogenous (dietary fatty acids) and endogenous (e.g. energy balance, age) factors on the stratification patterns of lipids. Specific indices were employed to investigate the lipid stratification patterns among seals with different fatty acid, TAG and phospholipid compositions. The original papers contain species-specific data on these stratification patterns, including discussions on factors that influence the stratification pattern, particularly blubber thickness and age. In this summary my goal was to summarize the papers and to compare directly the stratification patterns among the different seal species. The interspecific comparison of the lipid and fatty acid stratification patterns makes it possible to address the factors (endogenous vs. environmental) that contribute to the formation of the depth- specific differences in lipid and fatty acid composition in the blubber. Additionally, the possible metabolic variations throughout the blubber were evaluated by analyzing the main lipid composition of the membranes. Significant depth-specific differences in the lipid composition of membranes may indicate concurrent differences in lipid metabolism, since the lipid composition of plasma membrane has been documented as a factor that influences the function of membrane proteins (Zeghari et al., 2000a, 2000b; Al-Makdissy et al., 2003; Ikonen & Vainio, 2005; Vainio et al., 2005).

2 Material and methods

2.1 BLUBBER SAMPLES Blubber samples were collected from Arctic ringed seal (N=25), Saimaa ringed seal (N=37) and Baikal seal (N=22) of varying age and from both sexes. The Saimaa ringed seal is critically endangered, and the blubber samples were collected during the years 1996–2008 from fresh carcasses (mostly by-caught), and subsequently the age distribution was biased towards seals younger than 1 year old (II, III). The Arctic ringed seals were shot in Svalbard in April 2000 (I) and the Baikal seal pups were caught on ice in April 2005, and older Baikal seals (aged 3–18 yrs) were netted in October 2005 (IV). The sampled individuals were highly variable in terms of age, as well as reproductive and nutritional status. The samples size (particularly for lipid species in the Saimaa ringed seal, III) did not allow the direct comparison of different categories; instead the approach of this thesis is to evaluate larger scale differences and similarities in the stratification patterns of fatty acids in the blubber among species and subspecies. The blubber samples from the Arctic and Saimaa ringed seal were obtained ventrally (over the sternum), and from the Baikal seal the blubber samples were cut mid-dorsally (at about 2/5 of the distance from snout to tail). Although the blubber samples from the Baikal seals were cut dorsally, we assumed that the fatty acid profile would be similar in both locations, as no differences were found between the vertical fatty acid profiles of ventral and dorsal blubber in the Arctic ringed seal (details in I). The blubber of the Arctic and Saimaa ringed seals was dissected into large blocks (about 20 cm×20 cm), with skin and some muscle still attached to the blubber, and stored at -20 °C for analysis. The Baikal seals were skinned prior to the cutting of the blubber block. The edges of the frozen blubber block were removed (from the lateral sides only) and 3 mm thick sequential subsamples were dissected

33 vertically from the centre of the blubber block, after which each subsample was analyzed separately (Fig. 5).

Figure 5. Schematic drawing of the sampling procedure of blubber subsamples. The entire blubber layer was dissected vertically into 3 mm thick sequential sections. The blubber was kept frozen with liquid nitrogen to prevent redistribution and mixing of semi-fluid lipids during sampling. The number of subsamples for each individual was dependent on the total blubber thickness.

2.2 FATTY ACID ANALYSIS Fatty acid methyl esters were prepared for the gas chromatographic analysis via direct transmethylation of fatty acids. The blubber subsamples were heated (90 °C) in acidic methanol to produce fatty acid methyl esters (FAME). The FAMEs were analyzed by gas chromatography with FID and MS detection (I, II, III, IV). In most cases the method for the analysis with FID detection was as follows: split injection (split ratio 1:20), inlet temperature 250 °C, the initial oven temperature (180 °C) was held for 8 min and then ramped at 3 °C /min to a final temperature of 210 °C, which was held for 25 min (I, II, III). Detailed information on the preparation of FAMEs and the used GC method can be found in the original papers. The peaks were identified by authentic standards and mass spectrometry.

2.3 LIPID SPECIES ANALYSIS For the analysis of TAG and phospholipid species and cholesterol, the total lipids were extracted from each subsample according to Folch et al. (1975; III, IV). Aliquots of extracts were evaporated under N2 stream, and redissolved into peroxide-

34 tested chloroform/methanol (1:2, v/v). Total phospholipid (i.e. total PO4 in the lipid extract) and cholesterol were determined by standard spectrophotometric methods (Bartlett & Lewis, 1970; Gamble et al., 1978). The TAG and phospholipid species were analyzed by electrospray ionization mass spectrometry (ESI-MS). Prior to the analysis NH4OH was added to give a 1% solution in order to support ionization and to prevent formation of sodium adducts. The TAG species were detected in positive ionization mode as (M+NH4)+ ions (Duffin & Henion, 1991). The fatty acid composition of isobaric TAG species was determined by tandem mass spectrometry (MS/MS) and a fingerprinting method that detected neutral losses of all possible natural fatty acids (Han & Gross, 2001, 2005). The major TAG molecular species for each isobaric TAG peak were calculated on the basis of the ion abundances for released fatty acid fragments. The species composition of the main phospholipid classes, i.e. PC, SM and PE, were selectively detected with class-specific MS/MS scanning modes (Brügger et al., 1997; Hsu et al., 1998). In the positive ion mode, the PC and SM species are precursors of m/z 184 (which corresponds to the choline head group), for the PE species the fragment indicating the ethanolamine head group is neutral, and thus the lipids were detected as a neutral loss of 141 amu. The mass spectra were corrected for overlap of isotopic patterns, and the lipids species were quantified on the basis of the responses of the standards by LIMSA (LIpid Mass Spectrum Analysis) software (Haimi et al., 2006).

2.4 DATA ANALYSIS For the evaluation of lipid stratification patterns in seal blubber with varying fatty acid and TAG species composition, a set of indices was employed: relative stratification index (SI), ̇9- desaturation index (̇9-DI), numerical gradient (NG), and Euclidean distance. The relative stratification index was calculated according to Olsen & Grahl-Nielsen (2003) from fatty acid molar percentage in the outermost subsample (0-3 mm from the skin, Fo) and the deepest blubber subsample (innermost 3 mm, Fi) as follows: SI= (Fo – Fi)/[(Fo + Fi)/2] (I, II, IV). The SI enables the comparison of the stratification of fatty 35 acids with different abundances. The selected 20 fatty acids represent the abundant (>1.5 mol% in at least one of the samples) saturated, monounsaturated, and polyunsaturated fatty acids of varying chain length. Negative SI-values indicate that the fatty acid is relatively more abundant in the innermost subsample while positive SI-values indicate enrichment in the outermost blubber sample. Similar relative stratification index was also calculated for TAG species (III). ̇9-Desaturation index (DI) was calculated for each subsample and ̇9-DI stratification pattern was used for the definition of superficial blubber (I, II, III, IV). The ̇9-DI is the ratio of C14, C16 and C18 MUFA to the corresponding SFA (Käkelä & Hyvärinen, 1996). ̇9-desaturation of SFA is one of the main modification processes for increasing the fluidity of lipids as a response to low tissue temperatures (Tiku et al., 1996; Trueman et al., 2000), and it has been demonstrated that the ̇9-DI is high in the superficial blubber immediately below the skin and low in the deep blubber next to the skin (Käkelä & Hyvärinen, 1996). It is likely that both Ǌ9-desaturation of SFA and selective deposition of Ǌ9- MUFA (mainly 16:1n-7 and 18:1n-9) contribute to the calculated Ǌ9-DI value in blubber. To evaluate the depth-specific changes in the fatty acid composition, numerical gradients of fatty acid mol% were calculated for blubber subsamples (Schey, 2005). Fatty acids that exceeded 0.1mol% were included in the analysis of numerical gradient (I, II, IV). The computations were done with MATLAB 2010a using the function “gradient.m” (MathWorks, Natick, MA). The NG value corresponds to compositional changes between adjacent blubber subsamples; a high value indicates greater differences. Euclidean distances were calculated between the fatty acid molar percentages of blubber subsamples and potential prey items (Eq.1); large values indicate greater differences in the fatty acid composition (II). The Euclidean distances were also calculated between the blubber samples of the two ringed seal subspecies to evaluate whether compositional differences between the subspecies were similar throughout the entire

36 blubber column. The purpose of these calculations was to estimate the absolute compositional difference between the tissue samples, and all the identified fatty acids were included in the analysis. Euclidean distance is a simple measure of the distance between two objects that does not correct for different scaling of variables (Quinn & Keough, 2002). The impact of different variables (fatty acid mol%) on the Euclidean distance value is directly dependent on the variance of the variable; variables with large variance weigh more, while less abundant fatty acids with low variance have a smaller impact on the Euclidean distance.

n 2 (1) d x, y = xi-yi i=1 ൫ ൯ ඩ෍൫ ൯

Additionally, SM/PC ratio was calculated to illustrate vertical changes in the membrane lipid composition, which may be related to metabolic differences in the blubber (III). For statistical analyses the following software were used: SPSS (17.0.1) and R (2.12.1). Differences between means were tested by parametric (paired t-test) and non-parametric methods (Mann-Whitney U-test, Wilcoxon signed rank test), depending on the data. Details can be found in the original papers.

3 Results and discussion

3.1 INTERSPECIFIC VARIATION OF BLUBBER LIPIDS

3.1.1 Fatty acids The blubber fatty acid compositions of the studied seal species were in accordance with those reported previously (Käkelä & Hyvärinen, 1996; Grahl-Nielsen et al., 2005). The predominant fatty acids in the two ringed seal subspecies and in the Baikal seal blubber were 14:0, 16:0, 16:1n-7, 18:1n-9, 18:1n-7, 20:5n-3, 22:5n-3 and 22:6n3, accounting for 50–85% of total fatty acids (I, II, III, IV). The most abundant fatty acids were generally similar in all the analyzed seals, but the relative proportions of fatty acids varied significantly, i.e. the blubber fatty acid composition of the closely related species from marine and freshwater environments were different (Fig. 6). The greatest differences were observed in the abundance of C20 and C22 MUFA and scarcity of n-6 PUFA in the Arctic ringed seal blubber (Fig. 6). The high proportion of C20 and C22 MUFA in marine seals originates from calanoid copepods that store lipids as wax esters, common in the marine environment but rare in freshwater environments (Morris, 1984, Muje et al., 1989). The higher proportion of C18 n-6 PUFA in the blubber of the lacustrine seals corresponds with the higher proportions of n-6 PUFA in freshwater food webs (Muje et al., 1989; Grahl-Nielsen et al., 2011b). The scarcity of n-6 PUFA in marine food webs probably results from the dominance of diatoms (Bacillariophyceae) in the phytoplankton community; diatoms are typically more enriched in n-3 PUFA than n-6 PUFA (Dunstan et al., 1993). Green algae (Chlorophyceae) are an abundant source of n-6 PUFA, and consequently freshwater food webs that are dominated by green algae are generally rich in n-6 PUFA (Thompson, 1996). In addition, the contribution of terrestrial n-6 PUFA (e.g. via terrestrial invertebrates) is

39 typically higher in freshwater lakes than in marine environments (Napolitano, 1999). Between the two species of freshwater seals, the most prominent difference in the fatty acid composition was the abundance of C18 MUFA in the Baikal seal (particularly 18:1n-9), whereas in the Saimaa ringed seal blubber the shorter chained, C16 MUFA were the predominant MUFA (Fig. 6). The differences in the proportion of C16 and C18 MUFA between the freshwater seals most likely arise from the differences in the fatty acid composition of the potential prey fish (Muje et al., 1989, Grahl-Nielsen et al., 2011b). 18:1n-9 comprises 25-50% of the fatty acid composition in golomyanka (Comephorus baicalensis), a common prey fish for the Baikal seal (Ju et al., 1997; Kozlova & Khotimchenko, 2000, Grahl-Nielsen et al., 2011b). For comparison, the proportions of 18:1n-9 (containing small amounts of 18:1n-11) in the potential prey fish of the Saimaa ringed seal, i.e. vendace (Coregonus albula), ruffe (Gymnocephalus cernuus), perch (Perca fluviatilis), roach (Rutilus rutilus), and smelt (Osmerus eperlanus), are only 7–15% (II). Additionally, the lipid content of adult golomyanka is very high, 30–40% of wet weight (Ju et al., 1997; Kozlova & Khotimchenko, 2000). The abundance of 18:1n-9 in the diet of the Baikal seal is reflected in the fatty acid composition of the blubber, and the high variation in the proportion of 18:1n-9 probably reflects significant differences in foraging strategies among different Baikal seal individuals (IV).

3.1.2 Triacylglycerols The species composition of TAG molecules was analyzed in Saimaa ringed seal and Baikal seal blubber (Fig. 7). As expected, the TAG composition reflected the fatty acid composition in the blubber. High proportions of oleic acid (18:1n-9) in the Baikal seal blubber resulted in high proportions of monounsaturated TAG (the FA triplet comprising of monounsaturated fatty acids), particularly 52:3 (mainly 16:1/18:1/18:1) and 54:3 (18:1/18:1/18:1). In the Baikal seal blubber the proportion of both 18:1n-9 and monounsaturated TAG species showed large standard deviations, demonstrating great individual variation.

40 The Saimaa ringed seal blubber contained proportionally more unsaturated TAGs, which typically contained one polyunsaturated fatty acid, e.g. 56:7 (18:1/18:1/20:5 or 16:1/18:1/22:5), 54:7 (16:1/18:1/20:5 or 16:0/16:1/22:6) and 56:8 (16:1/18:1/22:6 or 18:1/18:2/20:5). The general TAG species composition in the Saimaa ringed seal blubber was more diverse (i.e. the number of detected TAG species was higher). In particular, the TAG species composition of the deep blubber was more diverse in the Saimaa ringed seal than in the Baikal seal. The diversity of TAG species may be associated with the abundance of PUFA. In terrestrial mammals, which had low dietary intake of PUFA, the TAG diversity in adipose tissue was lower than in the blubber of the seal species in this study (Perona et al., 2000; Suzuki et al., 2008). Similarly, a high TAG diversity has been documented in sardines (Sardine pilchardus), which are also rich in PUFA (Perona & Ruiz-Gutiérrez, 1999). The fatty acid composition in the TAG showed higher plasticity than previously thought (Brockerhoff, 1966; Brockerhoff et al., 1966). Conventional analysis of the positional distribution of fatty acids in the TAG of blubber suggested that PUFA are mainly esterified at the sn-3 and sn-1 position, and the short-chain saturated and monounsaturated fatty acids favor the sn-2 position (Brockerhoff, 1966; Brockerhoff et al. 1966). The distribution of fatty acids in the TAG is mainly determined by the available fatty acids and the activity of acyltransferases, enzymes that catalyze the stepwise addition of fatty acids to the glycerol (Coleman & Lee, 2004; Kazuharu & Reue, 2009). The substrate preferences vary among different acyltransferases, and it has also been suggested that the enzyme activities may be species/tissue specific and also modulated by the available fatty acid pool (Coleman & Lee, 2004; Kazuharu & Reue, 2009). The differences in the substrate preferences and activities of acyltransferases may explain the observed plasticity in the TAG fatty acid distribution of the seal blubber.

41 35 A) Superficial blubber a Arctic ringed seal N=25 Saimaa ringed seal N=37 Baikal seal N=19 30 a a 25 a b 20 b Molar% 15

a 10 b bb b a a a bb c ab 5 bb b b a b c a a c b a a b c b c a a a b b a a b ab bc b ab b a a 0 *** *** *** *** *** * * * * * *** * * *** * *** 14:0 16:0 18:0 14:1n-5 16:1n-7 16:1n-9 18:1n-7 18:2n-6 18:3n-3 18:4n-3 20:1n-9 20:4n-3 20:4n-6 20:5n-3 22:5n-3 22:5n-6 22:6n-3 20:1n-11 22:1n-11 18:1n-9(11)

35 B) Deep blubber Arctic ringed seal N=25 Saimaa ringed seal N=37 Baikal seal N=19 30

ab 25

b 20 a b a a Molar% c 15 b a b 10 ab ab a a b b a a 5 a ab b b b b c b a b b a b b b b c b a c bc a a ab a a a b c bb b b a a 0 b 14:0 16:0 18:0 14:1n-5 16:1n-7 16:1n-9 18:1n-7 18:2n-6 18:3n-3 18:4n-3 20:1n-9 20:4n-3 20:4n-6 20:5n-3 22:5n-3 22:5n-6 22:6n-3 20:1n-11 22:1n-11

18:1n-9(11) Fatty acid

Figure 6. Mol% of 20 abundant saturated, monounsaturated and polyunsaturated fatty acids in the A) superficial and B) deep blubber of Arctic and Saimaa ringed seals, and the Baikal seal. Error bars represent ±SD. All individuals examined (except the Baikal seal fetuses) are included in the figure. Mean fatty acids values among species with a different letter differ significantly (P < 0.05, U-test). Superficial (panel A) and deep (panel B) blubber were tested separately. Asterix (*) next to the X-axis in panel A denotes those fatty acids for which mol% differs significantly between superficial and deep blubber within species (P < 0.05, Wilcoxon signed rank test).

42 10 A) Superficial blubber Baikal seal (N=11) Saimaa ringed seal (N=6)

8 a

6 a * * * * a

Molar% a 4 a a a a a * * * a a 2 *

0 TAG 48:3 TAG 48:2 TAG 50:3 TAG 50:2 TAG 52:6 TAG 52:5 TAG 52:4 TAG 52:3 TAG 52:2 TAG 54:8 TAG 54:7 TAG 54:6 TAG 54:5 TAG 54:4 TAG 54:3 TAG 56:8 TAG 56:7 TAG 56:6 TAG 58:8 TAG 10 B) Deep blubber

6 Molar% 4 a a a a a a 2 a

0 TAG 48:3 TAG 48:2 TAG 50:3 TAG 50:2 TAG 52:6 TAG 52:5 TAG 52:4 TAG 52:3 TAG 52:2 TAG 54:8 TAG 54:7 TAG 54:6 TAG 54:5 TAG 54:4 TAG 54:3 TAG 56:8 TAG 56:7 TAG 56:6 TAG 58:8 TAG

TAG

Figure 7. Mol% of the most abundant TAG species in the blubber of the Baikal seal and the Saimaa ringed seal (III, IV). Error bars represent ±SD. A letter (a) indicates significant difference between species (P < 0.05, U-test), and the asterix (*) indicates differences between the superficial and deep blubber for both species separately (P < 0.05, Wilcoxon signed rank test).

43 3.2 BIOCHEMICAL STRATIFICATION OF BLUBBER

3.2.1 General stratification of different fatty acids The stratification of fatty acids in the blubber was most pronounced in the Arctic ringed seal. The strong stratification of the Arctic ringed seal blubber was mostly influenced by the abundance of C20 and C22 MUFA in the deep blubber (Fig. 6), which is in accordance with previous studies on the ringed seal and other marine mammal species (Fredheim et al., 1995, Koopman et al., 1996; Käkelä & Hyvärinen, 1996; Best et al., 2003; Olsen & Grahl-Nielsen, 2003; Grahl-Nielsen et al., 2005). It has been suggested that the temperature gradient throughout the blubber is a major factor contributing to fatty acid stratification (Käkelä & Hyvärinen, 1996). The stratification of SFA and MUFA in Arctic ringed seal blubber was linearly correlated with the melting point of these fatty acids (I). The AVAs and counter-current heat exchange system preserves heat in the deep blubber, and the tissue temperature in superficial blubber and skin are only marginally higher than the ambient temperature (Irving & Hart, 1957; Hart & Irving, 1959; Meagher et al., 2008; Noren et al., 2008). The melting points of C20 and C22 MUFA are high (see table 1), and thus these fatty acids cannot be deposited in large quantities in the superficial blubber, in which tissue temperature is low and fluctuates according to the ambient temperature (Irving & Hart, 1957; Hart & Irving, 1959). Although the proportion of C20 and C22 MUFA is very low in the blubber of the Baikal seal and the Saimaa ringed seal, the stratification of fatty acids was evident in these species, suggesting that C20 and C22 MUFA are not the only driving force for fatty acid stratification in the blubber of phocid seals. The general stratification of the most abundant MUFA and SFA were similar in the studied seals regardless of habitat; i.e. monounsaturated C14 and C16 accumulate in the superficial blubber and the SFA in the deep blubber (Fig. 8). Although in the Baikal seal the relative stratification index of 20:1n-11 was positive (Fig. 8), the molar percentage is very low in the superficial blubber (Fig. 6), and thus this fatty acid does not

44 contribute significantly to the overall fatty acid stratification in the blubber. The stratification of PUFA was variable among the studied seals and the stratification was not connected with the fatty acid melting points, which is not surprising since the melting points of PUFA are well below zero Celsius (see table 1). More likely the stratification of PUFA is mainly affected by variation in dietary fatty acids and lipid metabolism. Ontogenetic changes in the vertical stratification of fatty acids were detected in the seals and the degree of fatty acid stratification in the blubber was low in pups (ǂ1 year old individuals). Only the Saimaa ringed seal and Baikal seal samples included pups. It is unclear why the pups showed a lower degree of stratification, but it is possible that i) the blubber layer in pups is built up quickly with little depth-specific selection, ii) the modification of fatty acid is slow, and/or iii) the annual fluctuations in blubber thickness (associated with the reproductive cycle) reinforce the stratification of fatty acids. In addition, the ontogenetic differences in fatty acid stratification may be connected with growth-related changes in blubber morphology and thermal properties (Dunkin et al., 2005). The ontogenetic differences in the fatty acid stratification patterns were most pronounced in the Baikal seal blubber (IV). The proportions and stratification patterns of 18:1n-9 differed significantly between >10-year-old and 2-10-year-old Baikal seals (IV). The proportion of 18:1n-9 was similar in the superficial blubber of both age categories, but in the >10-year- old seals the proportion of 18:1n-9 remained high also in the deep blubber. Thus in this respect the blubber was less stratified in the >10-year-old Baikal seals. These ontogenetic differences in the degree of stratification due to the differences in the stratification of 18:1n-9 may emerge from differences in foraging patterns. The high proportions of 18:1n-9 in the deep blubber of the older and larger Baikal seals may indicate recent feeding on mature golomyanka, as other potential prey fish (such as Comephorus dybowskii and the omul Coregonus autumnalis migratorius), and the macro-crustacean Macrohectopus have a lower lipid content and proportion of 18:1n-9 than the

45 golomyanka (Thomas et al., 1982; Morris, 1984; Ju et al., 1997; Kozlova & Khotimchenko, 2000).

2.0 Saturated Monounsaturated Polyunsaturated fatty acids fatty acids fatty acids 1.5

1.0

0.5

0.0

-0.5

-1.0 Relative stratification index

-1.5 Arctic ringed seal (N=20) Saimaa ringed seal (N=7) Baikal seal (N=5) -2.0 14:0 16:0 18:0 14:1n-5 16:1n-9 16:1n-7 18:1n-7 20:1n-9 18:2n-6 18:3n-3 18:4n-3 20:4n-6 20:4n-3 20:5n-3 22:5n-6 22:5n-3 22:6n-3 20:1n-11 22:1n-11 18:1n-9+11 Fatty acid

Figure 8. Relative stratification index of 20 abundant saturated, monounsaturated and polyunsaturated fatty acids in the blubber of Arctic and Saimaa ringed seals and Baikal seal (ǂ1 year old seals, and Baikal seals older than 10 year are excluded from the figure, see text and IV for explanation). The relative stratification index enables the comparison of fatty acids of varying proportions; negative values indicate relatively larger proportions in the deep blubber and positive values indicate relatively larger proportions in the superficial blubber. Error bars represent ±SD.

3.2.2 Depth-specific changes in the fatty acid composition The numerical gradients of fatty acids were calculated throughout the blubber column for all seal species; a high NG value indicates great compositional differences between adjacent subsamples. The vertical profiles of the NG values illustrate the combined stratification pattern of fatty acids

46 throughout the blubber, and specify whether the stratification of fatty acids in blubber is linear or whether the changes are more pronounced at specific blubber depths. In addition, the NG values permit comparison of the fatty acid stratification patterns among seal species with completely different fatty acid compositions. The high NG values in the deep blubber of the Arctic ringed seal demonstrate large vertical changes in the fatty acid composition, which are partly due to the substantial change in the molar% of long-chain MUFA, particularly C20 and C22 MUFA (Fig. 9A). Levels of C20 and C22 MUFA in Saimaa ringed seal and Baikal seal blubber were negligible, which partly explains the lower compositional changes in the deep blubber (Fig. 9A and B). Nevertheless, the NG values also increased slightly in the deep blubber of the Saimaa ringed seal and the Baikal seal. For all the studied seals, the compositional changes in the deep blubber were high in individuals with thin blubber (< 30 mm; I, II, IV). It is likely that the large compositional changes in the deep blubber adjacent to the muscle are due to restricted deposition of fatty acid with high melting points (SFA and long- chain MUFA) in the superficial blubber, as well as active and selective fatty acid metabolism (incorporation/mobilization of fatty acids) in the deep blubber, which remolds the fatty acid composition (Raclot & Groscolas, 1995; Käkelä & Hyvärinen, 1996; Raclot, 2003; Wheatley et al., 2008). Selective metabolism of fatty acids may be associated with their different physiological roles and functions. For instance, physiologically important PUFAs are precursors for local tissue hormones and important modulators of membrane function. These findings suggest that fluctuations in metabolism, energy balance (e.g. due to reproductive state) and/or diet composition may first be detected in the deepest 1 cm of the blubber in the studied species/subspecies. In the Arctic ringed seal a second peak in the NG values was detected at the depth of about 1.5 cm from the skin, indicating large compositional changes (Fig. 9A). Approximately at the same depth, a peak in NG values was also detectable in the >1-

47 18 18 Arctic ringed seal (N=20) A Baikal seal >10yrs (N=6) B Saimaa ringed seal (N=7) 16 Baikal seal (N=5) 16

14 14

12 12

10 10 a-b

8 8

6 6 a-b; b-b 4 4 Sum of fatty acid numerical gradients acid numerical of fatty Sum a-b; s-b; b-b a-b a-b a-b; s-b; b-b a-b; b-b a-b;b-b a-b a-b 2 2 a-b a-s 0 a-b a-s a-s a-s;s-b a-s; a-b 0 0-3 3-6 6-9 0-3 3-6 6-9 9-12 9-12 12-15 15-18 18-21 21-24 24-27 27-30 30-33 33-36 36-39 39-42 42-45 45-48 48-51 51-54 54-57 12-15 15-18 18-21 21-24 24-27 27-30 30-33 33-36 36-39 39-42 42-45 45-48

Distance from the skin (mm)

Figure 9. Panel A shows vertical profiles of fatty acid numerical gradients (NG) in the blubber of two ringed seals (Arctic and Saimaa) and Baikal seal (ǂ 10 yrs old). For clarity, the vertical profile of NG in the blubber of Baikal seals older than 10 years is presented in a separate panel (B). High NG values indicate large differences in the fatty acid composition of adjacent subsamples. Error bars represent ±SE. Letters indicate the depths at which the means differ significantly between Arctic and Saimaa ringed seals (a-s), between Arctic ringed seal and Baikal seal (a-b), and between Saimaa ringed seals and Baikal seals (s-b; P < 0.05, U-test). In panel B the letters indicate the depths at which the means differ between Baikal seals older than 10 years and Arctic ringed seals (a-b), Saimaa ringed seal (s-b) and Baikal seals younger than 10 years (b-b; P < 0.05, U-test). year-old Saimaa ringed seals, and in the blubber of 2-10-year-old Baikal seals (Fig. 9A). The high compositional changes (NG value) form a border separating the superficial and deeper blubber layers, and on the basis of biochemical composition, the thickness of the superficial blubber was estimated to be about 1.5 cm. The fatty acid stratification pattern was significantly different in the Baikal seals older than 10 years; the NG values were the highest in the superficial blubber and declined to a depth of about 1.5 cm and remained constantly low throughout

48 18 18 Baikal seal (N=3) A Baikal seal (N=5) B 16 Saimaa ringed seal (N=8) 16 Saimaa ringed seal (N=20)

14 14

12 * 12 * 10 10 8 8 6 6 * 4 * Sum of fatty acid numerical gradients * 4 2

0 2

0 0-3 3-6 6-9 0-3 3-6 6-9 9-12 9-12 12-15 15-18 18-21 21-24 24-27 27-30 30-33 33-36 36-39 39-42 42-45 45-48 49-51 12-15 15-18 18-21 21-24 24-27 27-30 30-33 33-36 36-39 39-42 42-45 45-48 49-51

Distance from the skin (mm)

Figure 10. Vertical profiles of fatty acid numerical gradients (NG) in the blubber of Saimaa ringed seal pups and Baikal seal pups (ǂ 1 year old) with total blubber thickness ǂ 30mm (Panel A), and > 30 mm (Panel B). High NG values indicate large differences in the fatty acid compositions of adjacent subsamples. Error bars represent ±SD. Asterix (*) indicates the depths at which the means differ significantly between species (P < 0.05 U-test).

the rest of the blubber column (Fig. 9B). Thus, in the old, large- sized Baikal seals, the compositional differences between adjacent subsamples were most pronounced in the superficial blubber, but modest in deeper blubber. The superficial blubber was still compositionally distinguishable from the deep blubber sections, but no border was recognizable between middle and deeper blubber, which could indicate stable energy balance and diet composition. The assumption of a stable energy balance is supported by the timing of the sample collection. The blubber samples of Baikal seal were collected at the end of October, when the phocid seals are accumulating energy in preparation for the energetic challenges of reproduction (especially lactation

49 in females) and molting (Ryg et al., 1990a; Lydersen & Kovacs, 1999; Mellish et al., 2007). In pups (< 1-year-old seals) the depth-specific compositional changes throughout the blubber were not as pronounced as in the older seals (Fig. 10A, 10B; II, IV). Similarly, it has been documented that in harbor porpoises the degree of fatty acid stratification of the entire blubber is related to the age of the individual (Koopman et al., 1996). Although the overall fatty acid stratification was less pronounced in pups, large compositional changes were still detected in the deep blubber of pups with thin blubber (total blubber thickness < 30 mm; Fig. 10A).

3.2.3 Indication of variable Ǌ9-desaturase activity The ̇9-desaturation index (ratio of C16 and C18 MUFA to the corresponding SFA) was high in the superficial blubber of all seal species/subspecies (Fig. 11A). Relatively higher proportions of C16 and C18 MUFA and concomitant lower proportions of corresponding SFA in the superficial blubber have also been reported in previous studies on fatty acid stratification in the blubber of marine mammals (Fredheim et al., 1995; Koopman et al., 1996; Käkelä & Hyvärinen, 1996; Best et al., 2003; Olsen & Grahl-Nielsen, 2003; Grahl-Nielsen et al., 2005). The high ̇9-desaturation index in superficial blubber is probably connected with the low tissue temperatures in the superficial blubber; induced ̇9-desaturation of SFA in cold environment can sustain lipids in their proper semi-fluid state (Tiku et al., 1996; Hsieh & Kuo, 2005). A double bond in cis-configuration weakens short-distance attractions between adjacent acyl chains and lowers the fatty acid melting point (Cook & McMaster, 2002). Thus, ̇9-desaturation has been considered to be a biochemical mechanism for cold acclimatization (Tiku et al., 1996; Trueman et al., 2000; Hsieh & Kuo, 2005). The high ̇9-DI values in the superficial blubber may indicate elevated ̇9- desaturase activity (Sjögren et al., 2008), but selective incorporation of ̇9-desaturated fatty acids may also influence the values. In grey seals active ̇9-desaturation has been

50 confirmed in feeding trials employing 3H labeled fatty acids (Budge et al. 2004). In ringed seals, the ̇9-DI in the superficial blubber correlated with age (Fig. 12), and the ̇9-desaturation index declined with increasing blubber depth in all seals older than 1 year. The decline in ̇9-DI was most pronounced down to a depth of about 1.5 cm from the skin (Fig. 11A).

12 12 '9-Desaturation index A Saimaa ringed seal B

Arctic ringed seal (N=20) '9-DI (N=9) 1.4 10 Baikal seal (N=11) 10 SM/PC (N=5) Saimaa ringed seal (N=9)

1.2 8 8

1.0 6 6 SM/PC

0.8 4 9-desaturation index 9-desaturation 4 '

0.6 2 2

0.4 0 0 0-3 3-6 6-9 0-3 3-6 6-9 9-12 9-12 12-15 15-18 18-21 21-24 24-27 27-30 30-33 33-36 36-39 39-42 42-45 45-48 48-51 51-54 54-57 12-15 15-18 18-21 21-24 24-27 27-30 30-33 33-36 36-39 39-42 42-45 45-48 48-51 51-54 54-57

Distance from the skin (mm)

Figure 11. Vertical profile of ̇9-DI in the blubber of the two ringed seal subspecies and the Baikal seal (Panel A), and SM/PC ratio and ̇9-DI in the blubber of the Saimaa ringed seal (Panel B). Error bars represent ±SD. Pups (ǂ 1year old) excluded from the figures, except for SM/PC in which all analyzed samples are included.

The stratification pattern of ̇9-DI suggests that the low tissue temperature of superficial blubber affects the biochemical composition down to a depth of about 1.5 cm from the skin, corresponding with the vertical profiles of the NG values. The observed vertical profile of ̇9-DI throughout the blubber may also reflect changes in lipid metabolism. The ̇9-desaturase seems to participate in the regulation of lipid metabolism; in mice, inhibition of Ǌ9-desaturase activity was manifested in

51 increased metabolic rate and upregulation of the genes involved in lipid oxidation (for review see Miyazaki & Ntambi, 2003; Flowers & Ntambi, 2008). It has been suggested that elevated levels of ̇9-desaturase in muscle tissue partition fatty acids towards storage rather than oxidation (Hulver et al., 2005). Additionally, elevated levels of ̇9-desaturase activity in human adipose tissue have been linked with insulin resistance (Sjögren et al., 2008). Although, the function and activity of Ǌ9-desaturase in marine mammals has not been studied in detail, the importance of Ǌ9-desaturase in lipid metabolism in terrestrial and aquatic vertebrates (e.g. humans, mice, domestic pigs and fish) encourages investigating the role of Ǌ9-desaturase in marine mammals (Tiku et al., 1996; Trueman et al., 2000; Miyazaki & Ntambi 2003; Hsieh & Kuo, 2005; Hulver et al., 2005; Flowers & Ntambi, 2008; Sjögren et al., 2008).

16 r 2 = 0.77 14 y = 5.2519+0.2892x p < 0.001 12

-DI 8 ' 6 Lake Saimaa, Male n=17 Svalbard, Male n=30 4 Baltic Sea, Male n=5 Lake Saimaa, Female n=16 2 Svalbard, Female n=10 Baltic Sea, Female n=4 0 0 5 10 15 20 25 30 35 40 Age (years)

Figure 12.ȱ ̇9-Desaturation index in the superficial blubber of three ringed seal subspecies: the Arctic ringed seal, the Saimaa ringed seal and the Baltic ringed seal as a function of age. Data for the Baltic ringed seal are from a previous study (Käkelä & Hyvärinen, 1996).

52 3.2.4 Stratification of membrane lipids The membrane lipids were stratified in the blubber of the Saimaa ringed seal; the superficial blubber contained relatively more SM and cholesterol than the deep blubber (III). In the superficial blubber the mean value of SM/PC ratio was 1.2 (SD 0.3), and the ratio declined with increasing blubber depth (III). Interestingly, the vertical profile of the SM/PC ratio resembled that of the ̇9-DI (Fig. 11B). A high level of SM and other sphingolipids in the membrane is typically accompanied by high proportions of cholesterol, which was also reported in the blubber of the Saimaa ringed seal (III). The current view is that the preference for sphingolipids and cholesterol to co-exist produces lateral phase separation in the plasma membrane and high sphingolipid-cholesterol clusters are segregated into specific microdomains, called rafts (Simons & Ikonen, 2000; Örtegren et al., 2004). The rafts in plasma membranes seem to regulate (among other cellular functions) the transport and accumulation of fatty acids in adipocytes (Brown & London, 2000; Al-Makdissy et al., 2003; Pilch et al., 2007; Worgall, 2007; Bastiani & Parton, 2010). The proportion of SM in adipocyte membranes has also been connected with impaired function of the insulin receptors (Zeghari et al., 2000a, 2000b; Li et al., 2011). Vertical changes in the membrane SM/PC ratio and the concurrent changes in the amount of cholesterol may indicate metabolic differences throughout blubber, which may aid in sustaining a layer of superficial blubber that is thick enough for insulation under variable nutritional conditions.

3.2.5 Comparing the fatty acid composition of blubber and potential prey Euclidean distances were calculated between Saimaa ringed seal blubber subsamples and the potential prey fish (II). The similarity between the composition of the blubber and that of the prey fish was not uniform throughout the blubber. The fatty acid composition of the superficial blubber resembled the fish species the least. Furthermore, the superficial blubber layers of the Saimaa and Arctic ringed seal were surprisingly similar in

53 composition, considering the significant differences in the fatty acids composition of the marine and freshwater food web. This suggests that the fatty acid composition of the superficial blubber may be endogenously controlled, and the influence of dietary fatty acids in this layer is limited.

3.3 BIOCHEMICAL AND FUNCTIONAL LAYERING OF BLUBBER Based on the analysis of the different indices, the blubbers of Arctic and Saimaa ringed seal, and the Baikal seal are biochemically stratified into different layers (I, II, III, IV). The stratification is less pronounced in pups than in seals older than 1 year. Blubber is a multifunctional tissue, and the different functions (thermoregulation, energy storage, etc.) are very likely associated with the depth-specific composition of lipids. Previous biochemical and morphological studies have suggested functional differences between the blubber immediately below the skin and the deep blubber next to the muscle (Koopman et al., 2002; Montie et al., 2008). The vertical profiles of polar membrane lipids, specifically the SM/PC ratio, combined with the similarity of the stratification patterns of the selected fatty acid indices among the seals, support the hypothesis that the superficial blubber may be metabolically less active and function mainly as thermoregulatory tissue. The thickness of the superficial blubber (about 1.5 in the studied seals) was not affected by the total blubber thickness and may represent the minimum blubber thickness needed for sufficient insulation in these species. Measurements of the insulative value and conductivity of the superficial blubber are needed in order to evaluate further the importance of superficial blubber in insulation. The lipid composition in the deep blubber showed large variation across individual seals, suggesting variable and active fatty acid metabolism. Previous morphological and biochemical studies have similarly suggested that the deep blubber is metabolically the most active layer (Koopman et al., 2002; Montie et al., 2008). Based on the indices reported here, the

54 deepest 1 cm next to the muscle seems to be biochemically separated from the rest of the blubber. Biochemically distinct deep blubber was detected in all the species, but not in all individuals. The deep blubber was biochemically most recognizable in the pups with thin blubber (ǂ 3cm) and in the Arctic ringed seal, while in Baikal seals older than 10 years, the fatty acid composition in middle and deep blubber was more homogeneous. The differences may be associated with seasonal changes in energy balance connected with reproductive cycle and molting (Field et al., 2005). Arctic ringed seal samples were collected at the end of April and at the onset of increased fatty acids mobilization from the blubber to support the increased energy demand of reproduction and molting (Ryg et al., 1990a; Beck & Smith, 1995; Thordarson et al., 2007). The Baikal seal blubber samples were collected outside of the breeding season (in October), when phocid seals are not under strain due to the increased energy demand, and typically have a thick blubber layer (Ryg et al., 1990a; Beck & Smith, 1995, Thordarson et al., 2007). It is expected that active fatty acid metabolism will be found in pups and thin individuals, and the fact that the compositional differences were greatest in the deepest blubber of thin individuals suggests depth-specific changes in the metabolic activity of the blubber. In addition to changes in energy balance, the compositional changes in the deep blubber may be associated with changes in foraging patterns, as seasonal changes in diet composition have been documented in ringed seals (Lowry et al., 1980; Ryg et al., 1990b). The deep blubber may not be biochemically recognizable in seals with a stable nutritional status and small variations in diet composition. The pelagic food web of Lake Baikal is relatively simple with few prey species (Yoshii et al., 1999), and the >10-year-old Baikal seals contained large proportions of 18:1n-9, suggesting that the diet or the nutritional status has not changed recently and that the predominant prey is most likely golomyanka.

55 3.4 SUGGESTIONS FOR BLUBBER SAMPLING PROCEDURE The results of this study can be applied directly to the development of standardized blubber sampling procedures for studies on foraging strategies and diet in phocid seals. The results indicate that superficial blubber reflects the dietary fatty acid composition poorly. The fatty acid composition of superficial blubber in the Saimaa ringed seal resembled that of the Arctic ringed seal more than that of any of the potential prey fish (II). The superficial blubber seems to function mainly as a thermoregulatory tissue; thus collecting subsamples only from the superficial blubber should be avoided in studies on trophic interactions. Furthermore, blubber from very thin individuals (with little if any thermoneutral middle and deep blubber) may not provide a representative sample for diet analysis. The thickness of superficial blubber was about 1.5 cm, even in seals with thin blubber (in these animals total blubber thickness < 3 cm), and thus a significant proportion (50% or more) of the blubber was comprised of tissue with restricted dietary information. In addition to the relatively large proportion of thermoregulatory blubber in thin individuals, the evaluation of long term diet from the fatty acid analyses of the entire blubber layer may be biased by fluctuations in nutritional status and lipid metabolism. The intraspecific variation in the storage lipid composition (both TAG species and fatty acids) was highest in the deep blubber (I, II, III). Individual variation in nutritional status and specific fatty acid intake/mobilization most likely produce the observed high compositional variation in the deepest centimeter of the blubber. This could open up the possibility of evaluating recent changes in the diet and/or energy balance. The middle blubber, between the superficial and deep blubber, could provide the most representative sample of long term diet, since the fatty acid composition does not seem to be influenced by the low tissue temperature or variable fatty acid metabolism.

56 4 Conclusion

The lipid composition of marine mammal blubber has proven to be an abundant source of ecological information, but endogenous factors, mainly variable lipid metabolism and thermoregulatory adaptations, can affect the composition and distribution of lipids in the blubber. These factors must be considered when evaluating the data from studies employing marine mammal blubber, e.g. in analysis of foraging patterns and diet composition. It should be emphasized that the depth- specific differences in fatty acid composition of the blubber also provide the opportunity to investigate in more detail the endogenous factors mentioned above (i.e. variable lipid metabolism and thermoregulatory adaptations). For instance, the insulation capacity of superficial blubber may be related to the fatty acid and lipid composition, or the nutritional status of individuals may be evaluated on the basis of biochemical variations in the deep blubber. The biochemical and functional layering of the blubber also probably affects the distribution and metabolism of lipophilic compounds, such as lipid soluble vitamins and persistent organic pollutants (POP) in the blubber (Debier et al., 2002, 2003). Vitamin A has been employed in health assessments of seals, and POP concentrations of blubber are frequently used in environmental biomonitoring (Käkelä et al., 1997; Aguilar et al., 2002; Mos et al., 2007). Previous studies have documented that lipophilic compounds are not homogenously distributed throughout the blubber layer and that there is a need for standardization of sampling procedures (Lydersen et al., 2002; Debier et al., 2003; Kleivane et al., 2004). The detailed stratification patterns of the lipophilic compounds in the blubber would significantly add to our understanding of the metabolic fate and deposition patterns of these compounds in marine mammals and help in reducing methodological data variation due to different blubber sampling procedures.

57 This study demonstrated that the blubber of small-sized phocid seals is biochemically layered, and that the pattern of layering is similar among the studied seals irrespective of the differences in the blubber fatty acid composition. All the studied species were closely related, and the fatty acids were generally strongly stratified throughout the blubber. One factor that probably has a strong impact on the stratification pattern of fatty acids in blubber is thermoregulatory needs. The studied species are among the smallest pinnipeds and also inhabit a cold environment. Small sized pinnipeds are susceptible to greater heat loss because of their larger surface-to-volume ratio, and this may be one reason for the strong stratification of fatty acids in the blubber. The blubber of walruses (Odobenus rosmarus rosmarus) was found to be less stratified, most likely because of their larger body size and several centimeters thick epidermis, which provides extra insulation (Skoglund et al., 2010). However, the stratification pattern seems also to be affected by depth-specific and selective metabolism, and if the deep blubber is metabolically the most active layer, the seasonal changes in energy balance (associated with reproduction and molting) probably induce stratification and layering of fatty acids in blubber. Diet influences the blubber fatty acid composition, and thus the interspecific differences in the fatty acid composition of seal blubber were pronounced. Marine food webs are rich in long chain monounsaturated fatty acids (>C18 MUFA), and the abundance of these fatty acids strengthened the fatty acid stratification in the Arctic ringed seal blubber. Although the degree of fatty acid stratification was less pronounced in the freshwater seals, the detailed stratification patterns among the studied seal species were similar. In particular, the fatty acid composition of the superficial blubber seemed to be relatively stable, while the fatty acid composition in the deeper blubber layers is likely to be influenced more by the diet. The thickness of the superficial blubber was estimated to be about 1.5 cm in all the studied species, regardless of total blubber thickness.

58 The composition of the membrane lipids (specifically the SM/PC ratio) suggests that metabolic activity may be lowest in the superficial blubber. If the superficial blubber expresses lower lipolytic activity, it may help to sustain blubber for insulation during negative energy balance. Biochemically the layer of postulated high metabolic activity next to the muscle was most recognizable in seals with thin blubber, particularly in pups (possibly due to the switch from nursing to independent feeding, and/or the post-weaning fast). This suggests that recent changes in the nutritional status and diet composition could be evaluated on the basis of significant compositional changes in the deep blubber (deepest centimeter next to the muscle). The middle blubber could be categorized as a storage layer that expands and contracts according to the nutritional status. Similar functional layering has been documented for bottlenose dolphins; the blubber was morphologically stratified into three layers, which may have variable lipid dynamics (Montie et al., 2008). The exact depths and degree of layering probably vary depending on the species and environmental conditions, which warrants detailed biochemical studies of adipose tissue in a variety of species from different environments. The novel findings on the variability in membrane lipid composition throughout the blubber and the links with depth- specific differences in lipid metabolism should be studied further, as this would provide valuable information on lipid metabolism in marine mammal blubber. Comprehensive knowledge on the function and dynamics of the blubber layer is essential not only for physiological and ecophysiological studies on marine mammals, but also for ecological studies utilizing the blubber tissue.

5 References

Aguilar A, Borrell A & Reijnders P J H (2002) Geographical and temporal variation in levels of organochlorine contaminants in marine mammals. Marine Environmental Research 53: 425- 452. Al-Makdissy N, Younsi M, Pierre S, Ziegler O & Donner M (2003) Sphingomyelin/cholesterol ratio: an important determinant of glucose transport mediated by GLUT-1 in 3T3-L1 preadipocytes. Cellulat Signalling 15: 1019-1030. Andersen S M, Lydersen C, Grahl-Nielsen O & Kovacs K M (2004) Autumn diet of harbour seals (Phoca vitulina) at Prins Karls Forland, Svalbard, assessed via scat and fatty acid analyses. Canadian Journal of Zoology 82: 1230-1245. Anonymous (2002) Physical constants of organic compounds. In Lide D R (Ed) Handbook of Chemistry and Physics (83rd Edn). CRC, Boca Raton, pp. 3-1 – 3-764. Anonymous (2012) Lipid bank for web database. The Japanese Conference on the Biochemistry of Lipids. http://lipidbank.jp/. Bartlett E M & Lewis D H (1970) Spectrophotometric determination of phosphate esters in the presence and absence of orthophosphate. Analytical Biochemistry 36: 159– 167. Bastiani M & Parton R G (2010) Caveolae at a glance. Journal of Cell Science 123: 3831-3836. Beck G G & Smith T G (1995) Distribution of blubber in northwest Atlantic harp seal, Phoca groenlandica. Canadian Journal of Zoology 73:1991-1998. Beck C A, Iverson S J, Bowen W D & Blanchard W (2007) Sex differences in grey seal diet reflect seasonal variation in foraging behavior and reproductive expenditure: evidence from quantitative fatty acid signature analysis. Journal of Animal Ecology 76: 490-502.

61 Berta A, Sumich J L & Kovacs K M (2006) Marine mammals – evolutionary biology (2nd Edn). Elsevier. USA. Best N J, Bradshaw C J A, Hindell M A & Nichols P D (2003) Vertical stratification of fatty acids in the blubber of southern elephant seals (Mirounga leonina): implications for diet analysis. Comparative Biochemistry and Physiology B 134: 253- 263. Body D R (1988) The lipid composition of adipose tissue. Progress in Lipid Research 27:39-40. Bradshaw C J A, Hindell M A, Best N J, Phillips K L, Wilson G & Nichols P D (2003) You are what you eat: describing the foraging ecology of southern elephant seals (Mirounga leonina) using blubber fatty acids. Proceedings of the Royal Society of London B 270: 1283-1292. Brockerhoff H (1966) Fatty acid distribution patterns of animal depot fats. Comparative Biochemistry and Physiology 19: 1-12. Brockerhoff H, Hoyle R J & Huang P C (1966) Positional distribution of fatty acids in the fats of a polar bear and a seal. Canadian Journal of Biochemistry 44: 1519-1525. Brown D A & London E (2000) Structure and function of sphingolipid- and cholesterol-rich membrane rafts. The Journal of Biological Chemistry 275: 17221-17224. Bryden M M (1978) Arteriovenous anastomoses in the skin of seals. III The harp seal Pagophilus groenlandicus and the hooded seal Cystophora cristata (Pinnipedia: Phocidae). Aquatic Mammals 6: 67-75. Brügger B, Erben G, Sandhoff R, Wieland F T & Lehmann W D (1997) Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionisation tandem mass spectrometry. Proceedings of the National Academy of Sciences of the United States of America 94: 2339– 2344. Budge S M, Cooper M H & Iverson S J (2004) Demonstration of the deposition and modification of dietary fatty acids in pinniped blubber using radiolabelled precursors. Physiological and Biochemical Zoology 77: 682-687.

62 Budge S M, Iverson S J & Koopman H N (2006) Studying trophic ecology in marine ecosystems using fatty acids: a primer on analysis and interpretation. Marine Mammal Science 22: 759- 801. Casper R M, Jarman S N, Deagle B E, Gales N J & Hindell M A (2007) Detecting prey from DNA in predator scats: A comparison with morphological analysis, using Arctocephalus seals fed a known diet. Journal of Experimental Marine Biology and Ecology 347: 144-154. Christie W W & Han X (2010) Lipid analysis – Isolation, separation, identification and lipodomic analysis (4th Edn). The Oily Press, Great Britain. Coleman R A & Lee D P (2004) Enzymes of triacylglycerol synthesis and their regulation. Progress in Lipid Research 43:134-176. Cook H W & McMaster C R (2002) Fatty acid desaturation and chain elongation in eukaryotes. In Vance D E & Vance J E (Eds) Biochemistry of lipids, lipoproteins and membranes (4th Edn). Elsevier, The Netherlands, pp. 181-204. Cooper M H, Budge S M, Springer A M & Sheffield G (2009) Resource portioning by sympatric pagophilic seals in Alaska: monitoring effects of climate variation with fatty acids. Polar Biology 32: 1137-1145. Costa D P & Ortiz C L (1982) Blood chemistry homeostasis during prolonged fasting in the northern elephant seal. American Journal of Physiology 242: R591-R595. Debier C, Pomeroy P P, Van Wouwe N, Mignolet E, Baret P V & Larondelle Y (2002) Dynamics of vitamin A in grey seal (Halichoerus grypus) mothers and pups throughout lactation. Canadian Journal of Zoology 80: 1262-1273. Debier C, Pomeroy P P, Dupont C, Joiris C, Comblin V, Le Boulengé E, Larondelle Y & Thomé J-P (2003) Dynamics of PCB transfer from mother to pup during lactation in UK grey seals Halichoerus grypus: differences in PCB profile between compartments of transfer and changes during lactation period. Marine Ecology Progress Series 247: 249-256.

63 Dowhan W & Bogdanov M (2002) Functional roles of lipids in membranes. In Vance D E & Vance J E (Eds) Biochemistry of lipids, lipoproteins and membranes (4th Edn). Elsevier, The Netherlands, pp. 1-35. Duffin K L & Henion J D (1991) Electrospray and tandem mass spectrometric characterizartion of acylglycerol mixtures that are dissolved in nonpolar solvents. Analytical Chemistry 63: 1781-1788. Dunkin R C, McLellan W A, Blum J E & Pabst D A (2005) The ontogenetic changes in the thermal properties of blubber from Atlantic bottlenose dolphin Tursiops truncatus. Journal of Experimental Biology 208: 1469-1480. Dunstan G A, Volkman J K, Barrett S M, Leroi J-M & Jeffrey S W (1993) Essential polyunsaturated fatty acids from 14 species of diatom (Bacillariophyceae). Phytochemistry 35: 155-161. Fahy E, Subramaniam S, Murphy R C, Nishijima M, Raetz C R H, Shimizu T, Spener F, van Meer G, Wakelam M J O & Dennis E A (2009) Update of the LIPID MAPS comprehensive classification system for lipids. Journal of Lipid Research 50: S9– S14. Fayolle C, Leray C, Ohlmann P, Gutbier G, Cazenave J-P, Gachet C & Groscolas R (2000) Lipid composition of blood platelets and erythrocytes of southern elephant seal (Mirounga leonina) and antarctic fur seal (Arctocephalus gazelle). Comparative Biochemistry and Physiology B 126: 39-47. Field I C, Bradshaw C J A, Burton H R & Hindell M A (2005) Juvenile southern elephant seal exhibit seasonal differences in energetic requirements and use of lipids and protein stores. Physiological and Biochemical Zoology 78: 491-504. Flowers M T & Ntambi J M (2008) Role of stearoyl-coenzyme A desaturase in regulating lipid metabolism. Current Opinion in Lipidology 19: 248-256. Folch J M, Lees M & Sloane-Stanley G H (1957) A simple method for the isolation and purification of total lipides from animal tissue. The Journal of Biological Chemistry 226: 497–509. Fredheim B S, Holen S, Ugland K I & Grahl-Nielsen O (1995) Fatty acid composition in blubber, heart and brain from

64 phocid seals. In Blix A S, Walløe L, Ulltang Ø (Eds) Whales, seals, fish and man. Elsevier, The Netherlands, pp. 153-168. Gamble W, Vaughan M, Kruth H S & Avigan J (1978) Procedure for the determination of free and total cholesterol in micro- and nanogram amounts for studies with cultured cells. Journal of Lipid Research 19: 1068–1070. Grahl-Nielsen O & Mjaavatten O (1991) Dietary influence on fatty acid composition of blubber fat of seals as determined by biopsy: a multivariate approach. Marine Biology 110: 59-64. Grahl-Nielsen O, Andersen M, Derocher A E, Lydersen C, Wiig O & Kovacs K M (2003) Fatty acid composition of the adipose tissue of polar bears and of their prey: ringed seals, bearded seals and harp seals. Marine Ecology Progress Series 265: 275- 282. Grahl-Nielsen O, Halvorsen A-K, Bodoev N, Averina L, Radnaeva L, Pronin N, Käkelä R & Petrov E (2005) Fatty acid composition of the blubber of the Baikal seal, Phoca sibirica, and its marine relative, the ringed seal, Phoca hispida. Marine Ecology Progress Series 305: 261-274. Grahl-Nielsen O, Haug T, Lindstrøm U & Nilssen K T (2011a) Fatty acids in harp seal blubber do not necessarily reflect their diet. Marine Ecology Progress Series 426: 263-276. Grahl-Nielsen O, Averina E, Pronin N, Radnaeva L & Käkelä R (2011b) Fatty acid profiles in different fish species in Lake Baikal. Aquatic Biology 13: 1-10. Gray R, Canfield P & Rogers T (2006) Histology of selected tissues of leopard seal and implications for functional adaptations to an aquatic lifestyle. Journal of Anatomy 209: 179-199. Gulati S, Liu Y, Munkacsi A B, Wilcox L & Sturley S L (2010) Sterols and sphingolipids: Dynamic duo or partners in crime? Progress in Lipid Research 49: 353-365. Haimi P, Uphoff A, Hermansson M & Somerharju P (2006) Software tools for analysis of mass spectrometric lipidome data. Analytical Chemistry 78: 8324-8331. Hamilton J L, Dillaman R M, McLellan W A & Pabst A D (2004) Structural fiber reinforcement of keel blubber in harbor

65 porpoise (Phocoena phocoena). Journal of Morphology 261: 105- 117. Han X & Gross R W (2001) Quantitative analysis and molecular species fingerprinting of triacylglyceride molecular species directly from lipid extracts of biological samples by electrospray ionization tandem mass spectrometry. Analytical Biochemistry 295: 88-100. Han X & Gross R W (2005) Shotgun lipidomics: electrospray ionization mass spectrometric analysis and quantitation of cellular lipidomes directly from crude extracts of biological samples. Mass Spectrometry Reviews 24: 367-412. Hart J S & Irving L (1959) The energetics of harbour seals in air and in water with special considerations of seasonal changes. Canadian Journal of Zoology 37: 447-457. Helle E, Olsson M & Jensen S (1976) DDT and PCB Levels and Reproduction in Ringed Seal from the Bothnian Bay. Ambio 5: 188-189. Hellerstein M K, Christiansen M, Kaempfer S, Kletke C, Wu J, Reid J S, Mulligan K, Hellerstein N S & Shackleton C H L (1991) Measurement of de novo hepatic lipogenesis in humans using stable isotopes. The Journal of Clinical Investigation 87: 1841-1852. Henderson J R, Kalogeropoulos N & Alexis M N (1994) The lipid composition of selected tissues from a Mediterranean monk seal, Monachus monachus. Lipids 29: 577-582. Henderson J R & Tocher D R (1987) The lipid composition and biochemistry of freshwater fish. Progress in Lipid Research 26:281-347. Herman D P, Matkin C O, Ylitalo G M, Durban J W, Hanson M B, Dahlheim M E, Straley J M, Wade P R, Tilbury K L, Boyer R H, Pearce R W & Krahn M M (2008) Assessing age distributions of killer whale Orcinus orca populations from the composition of endogenous fatty acids in their outer blubber layers. Marine Ecology Progress Series 372: 289-302. Herman D P, Ylitalo G M, Robbins J, Straley J M, Gabriele C M, Clapham P J, Boyer R H, Tilbury K L, Pearce R W & Krahn M M (2009) Age determination of humpback whales Megaptera

66 novaeangliae through blubber fatty acid compositions of biopsy samples. Marine Ecology Progress Series 392. 277-293. Hobson K A, Schell D M, Renouf D & Noseworthy E (1996) Stable carbon and nitrogen fractionation between diet and tissues of captive seals: Implications for dietary reconstruction involving marine mammals. Canadian Journal of Fisheries and Aquatic Sciences 53: 528-533. Hsieh S L & Kuo C-M (2005) Stearoyl-CoA desaturase expression and fatty acid composition in milkfish (Chanos chanos) and grass carp (Ctenopharyngodon idella) during cold acclimation. Comparative Biochemistry and Physiology B 141: 95- 101. Hsu F-F, Bohrer A & Turk J (1998) Electrospray ionization tandem mass spectrometric analysis of sulfatide. Determination of fragmentation patterns and characterization of molecular species expressed in brain and in pancreatic islets. Biochimica et Biophysica Acta 1392: 202–216. Ikonen E & Vainio S (2005) Lipid microdomains and insulin resistance: Is there a connection? Science STKE 268:pe3. Irving L & Hart J S (1957) The metabolism and insulation of seals as bare-skinned mammals in cold water. Canadian Journal of Zoology 35: 293-495. IUPAC-IUB Commission on Biochemical Nomenclature (1967) The nomenclature of lipids. Journal of Lipid Research 8:523-528. Iverson S J, Frost K J & Lowry L F (1997) Fatty acid signatures reveal fine scale structure of foraging distribution of harbor seals and their prey in Prince William Sound, Alaska. Marine Ecology Progress Series 151: 255-271. Iverson S J, Field C, Bowen W D & Blanchard W (2004) Quantitative fatty acid signature analysis: a new method of estimating predator diets. Ecological Monographs 74: 211-235. Jakobsson A, Westerberg R & Jacobsson A (2006) Fatty acid elongases in mammals: their regulation and roles in metabolism. Progress in Lipid Research 45: 237-249. Jump D B (2001) The biochemistry of n-3 polyunsaturated fatty acids. The Journal of Biological Chemistry 277: 8755-8758.

67 Ju S-J, Kucklick J R, Kozlova T & Harvey H R (1997) Lipid accumulation and fatty acid composition during maturation of three pelagic fish species in Lake Baikal. Journal of Great Lakes Research 23: 241-253. Kazuharu T & Reue K (2009) Biochemistry, physiology, and genetics of GPAT, AGPAT, and lipin enzymes in triglyceride synthesis. American Journal of Physiology - Endocrinology and Metabolism 296: E1195-1209. Kleivane L, Severinsen T, Lydersen C, Berg V & Skaare J U (2004) Total blubber burden of organochlorine pollutants in phocid seals: methods and suggested standardization. Science of the Total Environment 320: 109-119. Koopman H N, Iverson S J & Gaskin D E (1996) Stratification and age-related differences in blubber fatty acids of the male harbour porpoise. Journal of Comparative Physiology B 165: 628- 639. Koopman H N, Pabst D A, McLellan W A, Dillaman R M & Read A J (2002) Changes in blubber distribution and morphology associated with starvation in the harbor porpoise (Phocoena phocoena): evidence for regional differences in blubber structure and function. Physiological and Biochemical Zoology 75: 498-512. Koopman H N, Iverson S J & Read A J (2003) High concentration of isovaleric acid in the fats of odontocetes: variation and patterns of accumulation in blubber vs. stability in the melon. Journal of Comparative Physiology B 173: 247-261. Koopman H N (2007) Phylogenetic, ecological, and ontogenetic factors influencing the biochemical structure of the blubber of odontocetes. Marine Biology 151: 277-291. Kotronen A, Seppänen-Laakso T, Westerbacka J, Kiviluoto T, Arola J, Ruskeepää A-L, Yki-Järvinen H & Oreši² M (2010) Comparison of lipid and fatty acid composition of the liver, subcutaneous and intra-abdominal adipose tissue, and serum. Obesity 18: 937-944. Kozlova T A & Khotimchenko S V (2000) Lipids and fatty acids of two pelagic cottoid fishes (Comephorus spp.) endemic to

68 Lake Baikal. Comparative Biochemistry and Physiology B 126: 477-485. Kunnasranta M, Hyvärinen H, Sipilä T & Koskela, J T (1999) The diet of the Saimaa ringed seal Phoca hispida saimensis. Acta Theriologica 44: 443–450. Käkelä R, Hyvärinen H & Vainiotalo P (1993) Fatty acid composition in liver and blubber of the Saimaa ringed seal (Phoca hispida saimensis) compared with that of the ringed seal (Phoca hispida botnica) and grey seal (Halichoerus grypus) from the Baltic. Comparative Biochemistry and Physiology B 105: 553- 565. Käkelä R, Ackman R G & Hyvärinen H (1995) Very long-chain polyunsaturated fatty acids in the blubber of ringed seals (Phoca hispida sp) from Lake Saimaa, Lake Ladoga, the Baltic sea and Spitsbergen. Lipids 30: 725-731. Käkelä R & Hyvärinen H (1996) Site-specific fatty acid composition in adipose tissue of several northern aquatic and terrestrial mammals. Comparative Biochemistry and Physiology B 115: 501-514. Käkelä R, Hyvärinen H & Käkelä A (1997) Vitamins A1 (retinol), A2 (3,4-didehydroretinol) and E (΅-tocopherol) in the liver and blubber of lacustrine and marine ringed seals (Phoca hispida sp). Comparative Biochemistry and Physiology B 116: 27- 33. Lee R F, Hagen W & Kattner G (2006) Lipid storage in marine zooplankton. Marine Ecology Progress Series 307: 273-306. Li Z, Zhang H, Liu J, Liang C-P, Li Y, Li Y, Teitelman G, Beyer T, Bui H H, Peake D A, Zhang Y, Sanders P E, Kuo M-S, Park T- S, Cao G & Jiang X-C (2011) Reducing plasma membrane sphingomyelin increases insulin sensitivity. Molecular and Cellular Biology 31: 4205-4218. Litchfield C, Greenberg A J, Caldwell D K, Caldwell M C, Sipos J C & Ackman R G (1975) Comparative lipid patterns in acoustical and non-acoustical fatty tissues of dolphins, porpoises and toothed whales. Comparative Biochemistry and Physiology B 50: 591-597.

69 Lowry L L, Frost K J & Burns J J (1980) Variability in the diet of ringed seals, Phoca hispida, in Alaska. Canadian Journal of Fisheries and Aquatic Sciences 37: 2254-2261. Lydersen C & Gjertz I (1987) Population parameters of ringed seals (Phoca hispida Schreber, 1775) in the Svalbard area. Canadian Journal of Zoology 65: 1021-1027. Lydersen C & Kovacs K M (1999) Behaviour and energetics of ice-breeding, North Atlantic phocid seals during the lactation period. Marine Ecology Progress Series 187: 265-281. Lydersen C, Wolkers H, Severinsen T, Kleivane L, Nordøy E S & Skaare J U (2002) Blood is a poor substrate for monitoring pollution burdens in phocid seals. Science of the Total Environment 292: 193-203. Meagher E M, McLellan W A, Westgate A J, Wells R S, Blum J E & Pabst D A (2008) Seasonal patterns of heat loss in wild bottlenose dolphins (Tursiops truncatus). Journal of Comparative Physiology B 178: 529-543. Mellish J-M, Horning M & York A E (2007) Seasonal and spatial blubber depth changes in captive harbor seals (Phoca vitulina) and Steller’s sea lion (Eumetopias jubatus). Journal of Mammalogy 88: 408-414. Miyazaki M & Ntambi J M (2003) Role of stearoyl-coenzyme A desaturase in lipid metabolism. Prostaglandins, Leukotrienes and Essential Fatty Acids 68: 113-121. Molyneux G S & Bryden M M (1975) Arteriovenous anastomoses in the skin of the Weddell seal, Leptonychotes weddelli. Science 189: 1100-1102. Montie E W, Garvin S R, Fair P A, Bossart G D, Mitchum G B, McFee W E, Speakman T, Starczak V R & Hahn M E (2008) Blubber morphology in wild bottlenose dolphins (Tursiops truncatus) from the Southeastern United States: influence of geographic location, age class, and reproductive state. Journal of Morphology 269: 496-511. Morris R J (1984) The endemic faunae of Lake Baikal: Their general biochemistry and detailed lipid composition. Proceedings of the Royal Society of London B 222: 51-78.

70 Mos L, Tabuchi M, Dangerfield N, Jeffries S J, Koop B F & Ross P S (2007) Contaminant-associated disruption of vitamin A and its receptor (retinoic acid receptor ΅) in free-ranging harbour seals (Phoca vitulina). Aquatic Toxicology 81: 319-328. Mu H & Høy C-E (2004) The digestion of dietary triacylglycerols. Progress in Lipid Research 43: 105-133. Muje P, Ågren J J, Lindqvist O V & Hänninen O (1989) Fatty acid composition of vendace (Coregonus albula L.) muscle and its plankton feed. Comparative Biochemistry and Physiology B 92: 75-79. Nakamura M T & Nara T Y (2004) Structure, function, and dietary regulation of delta 6, delta 5, and delta 9 desaturases. Annual Review of Nutrition 24: 345-376. Napolitano G E (1999) Fatty acids as trophic and chemical markers in freshwater ecosystems. In Arts M T & Wainman B C (Eds) Lipids in freshwater ecosystems. Springer, New York, pp. 21- 44. Nelson G J (1970) The lipid composition of the blood of marine mammals – I. Young elephant seals, Mirounga angustirostis and harp seals, Pagophilus groenlandicus. Comparative Biochemistry and Physiology 34: 109-116. Newland C, Field I C, Nichols P D, Bradshaw C J A & Hindell M (2009) Blubber fatty acid profiles indicate dietary resource partitioning between adult and juvenile southern elephant seals. Marine Ecology Progress Series 384: 303-312. Noren D P, Crocker D E, Williams T M & Costa D P (2003) Energy utilization in northern elephant seal (Mirounga angustirostris) pups during the postweaning fast: size does matter. Journal of Comparative Physiology B 173: 443-454. Noren S R, Pearson L E, Davis J, Trumble S J & Kanatous S B (2008) Different thermoregulatory strategies in nearly weaned pup, yearling, and adult Weddell seals (Leptonychotes weddelli). Physiological and Biochemical Zoology 81: 868-879. Ntambi J M & Miyazaki M (2004) Regulation of stearoyl-CoA desaturases and role in metabolism. Progress in Lipid Research 43: 91-104.

71 Olsen E & Grahl-Nielsen O (2003) Blubber fatty acids of minke whale: stratification, population identification and relation to diet. Marine Biology 142: 13-24. Palo J U & Väinölä R (2006) The enigma of the landlocked Baikal and Caspian seals addressed through phylogeny of phocine mitochondrial sequences. Biological Journal of the Linnean Society 88: 61-72. Parrish C C, Myher J J, Kuksis A & Angel A (1997) Lipid structure of rat adipocyte plasma membranes following dietary lard and fish oil. Biochimica et Biophysica Acta 1323: 253-262. Parry D A (1949) The structure of whale blubber, and discussion of its thermal properties. Quarterly Journal of Microscopical Science 90: 13-25. Perona J S & Ruiz-Gutiérrez V (1999) Characterization of the triacylglycerol molecular species of fish oil by reversed-phase high performance liquid chromatography. Journal of Liquid Chromatography and Related Technologies 22: 1699-1714. Perona J S, Portillo M P, Macarulla M T, Tueros A I & Ruiz- Gutiérrez V (2000) Influence of different dietary fats in triacylglycerol deposition in rat adipose tissue. British Journal of Nutrition 84: 765-774. Pierce G J & Boyle P R (1991) A review of methods for diet analysis in piscivorous marine mammals. Oceanography and Marine Biology: An Annual Review 29: 409-486. Pilch P F, Souto R P, Liu L, Jedrychowski M P, Berg E A, Costello C E & Gygi S P (2007) Cellular spelunking: exploring adipocyte caveolae. Journal of Lipid Research 48: 2103-2111. Pond C M (1999) Physiological specialisation of adipose tissue. Progress in Lipid Research 38: 225-248. Pond C M, Mattacks C A & Prestrud P (1995) Variability in the distribution and composition of adipose tissue in wild arctic foxes (Alopes lagopus) on Svalbard. Journal of Zoology 236: 593- 610. Pond C M, Mattacks C A, Colby R H & Ramsay M A (1992) The anatomy, chemical composition, and metabolism of adipose

72 tissue in wild polar bears (Ursus maritimus). Canadian Journal of Zoology 70: 326-341. Quinn G P & Keough M J (2002) Experimental Design and Data Analysis. Cambridge University Press, United Kingdom. Raclot T & Groscolas R (1995) Selective mobilization of adipose tissue fatty acids during energy depletion in the rat. Journal of Lipid Research 36:2164-2173 Raclot T (2003) Selective mobilization of fatty acids from adipose tissue triacylglycerols. Progress in Lipid Research 42: 257-288. Ramsay M A, Mattacks C A & Pond C M (1992) Seasonal and sex differences in the structure and chemical composition of adipose tissue in wild polar bears (Ursus maritimus). Journal of Zoology 228: 533-544. Reddy J N & Hashimoto T (2001) Peroxisomal Ά-oxidation and peroxisome proliferator-activated receptor ΅: an adaptive metabolic system. Annual Review of Nutrition 21:193-230. Rice D W (1998) Marine Mammals of the World. Systematics and Distribution. Society for Marine Mammalogy Special Publication 4: 1-231. Rosen D A S & Renouf D (1997) Seasonal changes in blubber distribution in Atlantic harbour seals: indications of thermodynamic considerations. Marine Mammal Science 13: 229-240. Ryg M, Smith T G & Øritsland N A (1988) Thermal significance of the topographical distribution of blubber in ringed seals (Phoca hispida). Canadian Journal of Fisheries and Aquatic Sciences 45: 985-992. Ryg M, Smith T G & Øritsland N A (1990a) Estimating the blubber content of phocid seals. Canadian Journal of Fisheries and Aquatic Sciences 47: 1223-1227. Ryg M, Smith T G & Øritsland N A (1990b). Seasonal changes in body mass and body composition of ringed seals (Phoca hispida) on Svalbard. Canadian Journal of Zoology 68: 470-475. Sargent J R, Gatten R R & Henderson R J (1981) Marine wax esters. Pure and Applied Chemistry 53: 867-871. Schey H M (2005) Div, grad, curl and all that: an informal text on vector calculus (4th edn). W W Norton, New York.

73 Simons K & Ikonen E (2000) How cells handle cholesterol. Science 290: 1721-1726. Sipilä T & Hyvärinen H (1998) Status and biology of Saimaa (Phoca hispida saimensis) and Ladoga (Phoca hispida ladogensis) ringed seal. In: Heide- Jorgensen M P & Lydersen C (Eds) Ringed seals in the north Atlantic. The North Atlantic Marine Mammal Commission (NAMMCO) Scientific Publications, No 1. pp. 83-99. Sjögren P, Sierra-Johnson J, Gertow K, Rosell M, Vessby B, de Faire U, Hamsten A, Hellenius M-L & Fisher R M (2008) Fatty acid desaturases in human adipose tissue: relationship between gene expression, desaturation indexes and insulin resistance. Diabetologia 51: 328-335. Skoglund E G, Lydersen C, Grahl-Nielsen O, Haug T & Kovacs K M (2010) Fatty acid composition of the blubber and dermis of adult male Atlantic walruses (Odobenus rosmarus rosmarus) in Svalbard, and their potential prey. Marine Biology Research 6: 239-250. Slip D J, Gales N J & Burton H R (1992) Body mass loss, utilisation of blubber and fat, and energetic requirements of male southern elephant seals, Mirounga leonina, during moulting fast. Australian Journal of Zoology 40: 235-243. Slotte J P (1999) Sphingomyelin–cholesterol interactions in biological and model membranes. Chemistry and Physics of Lipids 102: 13-27. Smith R J, Hobson K A, Koopman H N & Lavigne D M (1996) Distinguishing between populations of fresh- and salt-water harbour seals (Phoca vitulina) using stable-isotope ratios and fatty acid profiles. Canadian Journal of Fisheries and Aquatic Sciences 53: 272-279. Sprecher H, Luthria D L, Mohammed B S & Baykousheva S P (1995) Reevaluation of the pathways for the biosynthesis of polyunsaturated fatty acids. Journal of Lipid Research 36: 2471- 2477. Struntz D J, McLellan W A, Dillaman R M, Blum J E, Kucklick J R & Pabst D A (2004) Blubber development in bottlenose dolphins (Tursiops truncatus). Journal of Morphology 259: 7-20.

74 Suzuki S, Ishikawa S, Arihara K & Itoh M (2008) Molecular species-specific differences in the composition of triacylglycerols of mouse adipose tissue and diet. Nutrition Research 28: 258-262. Thiemann G W, Iverson S J & Stirling I (2007) Variability in the blubber fatty acid composition of ringed seals (Phoca hispida) across the Canadian Arctic. Marine Mammal Science 23: 241- 261. Thiemann G W, Iverson S J & Stirling I (2008) Variation in blubber fatty acid composition among marine mammals in the Canadian Arctic. Marine Mammal Science 24: 91-111. Thomas J, Pastukhov V, Elsner R & Petrov E (1982) Phoca sibirica. Mammalian Species 188:1-6. Thompson Jr G A (1996) Lipids and membrane function in green algae. Biochimica et Biophysica Acta 1302:17-45. Thordarson G, Vikingsson G A & Hersteinsson P (2007) Seasonal variation in body condition of adult male hooded seals (Cystophora cristata) in Skjalfandi-Bay, northeast Iceland. Polar Biology 30: 379-386. Tiku P E, Gracey A Y, Macartney A I, Beynon R J & Cossins A R (1996) Cold-induced expression of delta 9-desaturase in carp by transcriptional and posttranslational mechanisms. Science 271: 815-818. Trueman R J, Tiku P E, Caddick M X & Cossins A R. (2000) Thermal thresholds of lipid restructuring and Ǌ9 –desaturase expression in the liver of carp (Cyprinus carpio L.). Journal of Experimental Biology 203: 641-650. Tucker S, Bowen W D, Iverson S J & Stenson G B (2009) Intrinsic and extrinsic sources of variation in the diets of harp and hooded seals revealed by fatty acid profiles. Canadian Journal of Zoology 87: 139-151. Vainio S, Bykov I, Hermansson M, Jokitalo E, Somerharju P & Ikonen E (2005) Defective insulin receptor activation and altered lipid rafts in Niemann-Pick type C disease hepatocytes. Biochemical Journal 391: 465-472. Vanhaesebroeck B, Leevers S J, Ahmadi K, Timms J, Katso R, Driscoll P C, Woscholski R, Parker P J & Waterfield M D

75 (2001) Synthesis and function of 3-phosphorylated inositol lipids. Annual Review of Biochemistry 70: 535-602. van Meer G, Voelker D R & Feigenson G W (2008) Membrane lipids: where they are and how they behave. Nature Reviews Molecular Cell Biology 9: 112–124. Ventura M (2006) Linking biochemical and elemental composition in freshwater and marine crustacean zooplankton. Marine Ecology Progress Series 327:233-246. Walton M & Pomeroy P (2003) Use of blubber fatty acid profiles to detect inter-annual variation in the diet of grey seals Halichoerus grypus. Marine Ecology Progress Series 248: 257-266. Walton M J, Silva M A, Magalhães S M, Prieto R & Santos R S (2008) Fatty acid characterization of lipid fractions from blubber biopsies of sperm whales Physeter macrocephalus located around the Azores. Journal of the Marine Biological Association of the United Kingdom 88: 1109-1115. Wheatley K E, Nichols P D, Hindell M A, Harcourt R G & Bradshaw C J A (2007) Temporal variation in the vertical stratification of blubber fatty acids alters diet predictions for lactating Weddell seals. Journal of Experimental Marine Biology and Ecology 352: 103-113. Wheatley K E, Nichols P D, Hindell M A, Harcourt R G & Bradshaw C J T (2008) Differential mobilization of blubber fatty acids in lactating Weddell seals: Evidence for selective use. Physiological and Biochemical Zoology 81: 651-662. Weslawski J M, Ryg M, Smith T G & Øritsland N A (1994) Diet of ringed seals (Phoca hispida) in a fjord of west Svalbard. Arctic 47: 109-114. Worgall T S (2007) Sphingolipids: major regulators of lipid metabolism. Current Opinion in Clinical Nutrition and Metabolic Care10: 149-155. Yoshii K, Melnik N G, Timoshkin O A, Bondarenko N A, Anoshko P N, Yoshioka T & Wada E (1999) Stable isotope analyses of the pelagic food web in Lake Baikal. Limnology and Oceanography 44: 502-511. Zeghari N, Vidal H, Younsi M, Ziegler O, Drouin P & Donner M (2000a) Adipocyte membrane phospholipid and PPAR-J

76 expression in obese women: relationship to hyperinsulinemia. American Journal of Physiology - Endocrinology and Metabolism 279: E736-E743. Zeghari N, Younsi M, Meyer L, Donner M, Drouin P & Ziegler O (2000b) Adipocyte and erythrocyte plasma membrane phospholipid composition and hyperinsulinemia: a study in nondiabetic and diabetic obese women. International Journal of Obesity 24: 1600-1607. Örtegren U, Karlsson M, Blazic N, Blomqvist M, Nystrom F H, Gustavsson J, Fredman P & Strålfors P (2004) Lipids and glycosphingolipids in caveolae and surrounding plasma membrane of primary rat adipocytes. European Journal of Biochemistry 271: 2028-2036.

77 dissertations

Ursula Strandberg Detailed stratification

Publications of the University of Eastern Finland Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences Dissertations in Forestry and Natural Sciences No 65

isbn 978-952-61-0764-6 issn 1798-5668 issnl 1798-5668 isbn 978-952-61-0765-3 (pdf) issn 1798-5676 (pdf)