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

Publications of the University of Eastern Finland

Dissertations in Forestry and Natural Sciences

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 stratificationpatterns and verticallayering of lipidsin seal blubber– source of ecophysiological information

Fatty acids are among the most ana-

lyzed components in marine mammal

blubber, and increasingly employed

in ecological and physiological stud-

ies. However, the interpretation of

the results from these analyses is not

straightforward, as the fatty acids

and other lipids are stratified in the

blubber. This thesis documents the

detailed stratification patterns of

lipids in blubber, discusses the physi-

ological and ecological implications

of the biochemical stratification of

blubber and provides a suggestion for

standardized sampling procedure.

disser

tation

s | No

65 | Ur

sula S

tra

nd

berg

| Deta

iled stra

tifica

tion

pa

tterns a

nd

vertical la

yering of lip

ids in

seal b

lub

ber

Ursula StrandbergDetailed stratificationpatterns and vertical

layering of lipids in seal blubber

– source of ecophysiological

information

URSULA STRANDBERG

Detailed stratificationpatterns and vertical

layering of lipidsin seal blubber

– source of ecophysiological information

Publications of the University of Eastern FinlandDissertations in Forestry and Natural Sciences

No 65

Academic DissertationTo be presented by permission of the Faculty of Science and Forestry for public

examination in the Auditorium N100 in Natura Building at the University ofEastern Finland, Joensuu, on May 11, 2012, at 12 o’clock noon.

Department of Biology

KopijyväJoensuu, 2012

Editors: Prof. Pertti PasanenProf. Matti Vornanen, Prof. Kai Peiponen

Distribution:Eastern Finland University Library / Sales of publications

julkaisumyynti@uef.fi; http://www.uef.fi/kirjastotel. +358-50-3058396

ISBN: 978-952-61-0764-6ISSN: 1798-5668

ISSNL: 1798-5668ISBN: 978-952-61-0765-3 (PDF)

ISSN: 1798-5676 (PDF)

Author’s address: University of Eastern FinlandDepartment of BiologyP.O. Box 11180101 JOENSUUFINLANDemail: ursula.strandberg@uef.fi

Supervisors: Docent Reijo Käkelä, Ph.D.University of HelsinkiDepartment of BiosciencesPhysiology and NeuroscienceP.O. Box 6500014 University of HelsinkiFINLANDemail: reijo.kakela@helsinki.fi

Professor Matti Vornanen, Ph.D.University of Eastern FinlandDepartment of BiologyP.O. Box 11180101 JOENSUUFINLANDemail: matti.vornanen@uef.fi

Reviewers: Associate professor Heather Koopman, Ph.D.University of North Carolina WilmingtonBiology & Marine BiologyNC 28403 WilmingtonUSAemail: koopmanh@uncw.edu

Professor Seppo Saarela, Ph.D.University of OuluDepartment of BiologyP.O. Box 3000FIN-90014 OuluFINLANDemail: seppo.saarela@oulu.fi

Opponent: Professor Esa Hohtola, Ph.D.University of OuluDepartment of BiologyP.O. Box 3000FIN-90014 Ouluemail: esa.hohtola@oulu.fi

ABSTRACT

Fatty acids are among the most analyzed components in marinemammal blubber, and increasingly employed in a variety ofecological and physiological studies, particularly in theevaluation of trophic interactions. It has become evident that theinterpretation of the results from fatty acid analyses is notstraightforward, as the fatty acid composition is stratifiedthroughout the blubber layer. The general distribution of fattyacids in blubber is known, but knowledge on the detailedstratification patterns of fatty acids in seal blubber is scarce.However, for the correct interpretation of results from fatty acidanalyses, information on the detailed stratification patterns andmetabolism of fatty acids in the blubber is essential. The mainobjectives of this thesis were to i) document the detailed fattyacid stratification pattern in the blubber of closely relatedphocid seals, ii) to investigate how depth-specific lipid and fattyacid compositions may be associated with different blubberfunctions (mainly energy storage and thermoregulation) and iii)to deduce how the results could be applied in the developmentof standardized sampling procedures of seal blubber.

In order to investigate the importance of endogenous andexogenous factors for the biochemical composition andstratification of blubber, blubber samples were collected fromclosely related seal populations and species from differenthabitats; two ringed seal subspecies, a marine (the Arctic ringedseal, Pusa hispida hispida, Phh) and a freshwater subspecies (theSaimaa ringed seal, Pusa hispida saimensis, Phs), and also fromthe Baikal seal (Pusa sibirica, Ps), a freshwater seal that is a closerelative of the ringed seal. The entire blubber layer wasdissected vertically into 3 mm thick sequential subsamples, andeach of them was separately analyzed for fatty acids (Phh, Phs,Ps), triacylglycerols (Phs, Ps) and main phospholipids (Phs;phosphatidylcholine; PC, sphingomyelin; SM, andphosphatidyl-ethanolamine). The analyzed lipid classes arestructurally and functionally different. Phospholipid speciescomposition provides information on the biochemicalcomposition of the membranes, whereas the analyses of

triacylglycerol and total fatty acids represent mainly thecomposition of storage lipids.

The blubber was found to be vertically stratified, and on thebasis of biochemical composition, could be divided into separatelayers: superficial, middle and deep blubber. The superficialblubber had a high degree of fatty acid 9-desaturation,suggesting high 9-desaturase activity, but may also result fromselective incorporation of 9-desaturated fatty acids in thesuperficial blubber. The 9-desaturase has been linked toregulation of lipid metabolism and adaptation to low tissuetemperatures. The superficial blubber also demonstrated a highSM/PC ratio, which has been associated with insulininsensitivity and impaired lipid metabolism. These findingssuggest that the superficial blubber might primarily serve asthermoregulatory tissue and have low lipolytic activity, whichcould enable the maintenance of a sufficiently thick blubberlayer for insulation, even during negative energy balance, e.g.during lactation. The thickness of the superficial blubber wassurprisingly stable in all the studied individuals, despite thedifferences in e.g. diet, age and reproductive status. Themiddle blubber seems to expand and shrink with foodavailability; thus the middle blubber can be defined as anenergy deposit. The high variability in lipid composition in thedeep blubber suggests that this layer is metabolically very activeand is probably strongly affected by recent lipidmobilization/deposition. The observed biochemical layering ismost likely associated with functional specialization of theblubber layers. Furthermore the biochemical and functionallayering of the seal blubber is expected to affect deposition,distribution and mobilization of many lipophilic substances inthe 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 ReijoKäkelä for providing the opportunity to do this thesis and alsofor his encouragement and guidance during the study. MattiVornanen and Markku Keinänen are also warmlyacknowledged for their willingness to help in administrativeand methodological issues. I also wish to express my sinceregratitude to the entire staff and faculty of the Department ofBiology at the University of Eastern Finland, for providingexcellent conditions for my research.

I also wish to thank my colleagues: Heikki Hyvärinen for ourinspiring and encouraging conversations, and Kit M. Kovacs,Christian Lydersen, Mervi Kunnasranta, and Anne Käkelä fortheir comments and guidance. The co-authors Otto Grahl-Nielsen, Elena Averina, Tero Sipilä, Jouni Koskela, LarisaRadnaeva and Evgeniya Pintaeva are also gratefullyacknowledged. I would also like to acknowledge ChristinaLockyer, who kindly offered me a place to stay during myresearch visit. I remember fondly my short time in Tromsø.

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

I also heartily thank all my friends who provided the frequentlyneeded and always appreciated distraction from the thesis; Iwill cherish our moments together. I also wish to thank myfamily, particularly my parents, Sonja and Sture who havealways supported me in everything I do. Finally I wish to thankJasmin, Jemina, Aada and Luca, whose joy in life is contagious.

LIST OF ABBREVIATIONS

AVA Arterio-venous anastomoseCn FA Fatty acid with n carbons

9-DI 9-desaturation indexFA Fatty acidFAME Fatty acid methyl esterMUFA Monounsaturated fatty acidNG Numerical gradientPC PhosphatidylcholinePE PhosphatidylethanolaminePUFA Polyunsaturated fatty acidSFA Saturated fatty acidSI Relative stratification indexSM SphingomyelinTAG 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: theecology of fatty acids in marine mammals. Physiological andBiochemical 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 ringedseal – Comparison to a marine relative. Journal ofExperimental 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 theblubber 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 andtriacylglycerols in the blubber of the Baikal seal: Adults,pups and foetuses. Submitted manuscript.

Publications are reprinted with the kind permission of Elsevierand 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 theoriginal manuscripts I-III. She participated in the biochemicaland data analyses for paper IV and also contributed to thepreparation of paper IV.

Contents1 Introduction ............................................................................................ 131.1 Blubber morphology ......................................................................... 141.2 Blubber lipids .................................................................................. 161.2.1 Fatty acids ....................................................................................... 171.2.2 Fatty acid metabolism ...................................................................... 201.2.3 Storage lipids ................................................................................... 231.2.4 Membrane lipids .............................................................................. 251.3 Lipids in marine mammal research................................................... 271.4 Background information on the studied species ................................ 291.5 Objectives of the study ..................................................................... 31

2 Material and methods .......................................................................... 332.1 Blubber samples ............................................................................... 332.2 Fatty acid analysis ........................................................................... 342.3 Lipid species analysis ....................................................................... 342.4 Data analysis ................................................................................... 35

3 Results and discussion ......................................................................... 393.1 Interspecific variation of blubber lipids............................................. 393.1.1 Fatty acids ....................................................................................... 393.1.2 Triacylglycerols ............................................................................... 403.2 Biochemical stratification of blubber ................................................. 443.2.1 General stratification of different fatty acids ..................................... 443.2.2 Depth-specific changes in the fatty acid composition ........................ 463.2.3 Indication of variable 9-desaturase activity .................................... 503.2.4 Stratification of membrane lipids ..................................................... 523.2.5 Comparing the fatty acid composition of blubber and potential

prey.................................................................................................. 533.3 Biochemical and functional layering of blubber ................................ 543.4 Suggestions for blubber sampling procedure .................................... 56

4 Conclusion .............................................................................................. 57

5 References .............................................................................................. 61

13

1 Introduction

Marine mammals are a diverse assemblage of species from threeorders: Carnivora, Cetacea and Sirenia (Rice, 1998; Berta et al.,2006). Pinnipeds (seals, sea lions, walruses) belong to the orderCarnivora, and comprise of three families: Phocidae (true orearless seals), Otaridae (eared seals or fur seals and sea lions)and Odobenidae (walruses). Whales (order Cetacea) are dividedinto two suborders comprising of several families: theOdontoceti (toothed whales, 10 families) and Mysticeti (baleenwhales, 4 families). Manatees (Trichechidae) and the dugong(Dugongidae) are within the order Sirenia. Polar bears (Ursusmaritimus) and sea otters (Enhydra lutris), both from the orderCarnivora, are also categorized as marine mammals. Despite thevariable ancestry, pinnipeds, cetaceans and sirenians possesssimilar aquatic adaptations, and one of the major adaptations isthe evolvement of a blubber layer. The blubber is a specializedsubcutaneous adipose tissue that is morphologically andfunctionally different from the adipose tissue of terrestrialmammals (Pond, 1999). Both types of adipose tissue areimportant for energy storage, but blubber also serves as themain thermoregulatory tissue. Terrestrial mammals, as well aspolar bears and sea otters, rely on a dense fur coat for insulationand their subcutaneous adipose tissue does not contributesignificantly to thermoregulation (Pond et al., 1992; Pond, 1999).Consequently, the subcutaneous adipose tissue of polar bearsand sea otters is similar to that of terrestrial mammals and is notconsidered to be blubber. In addition to storing energy andmaintaining thermal balance, the blubber affects the formationof body shape, thus influencing the streamlining, locomotionand buoyancy of marine mammals.

Blubber forms a continuous layer across the body trunk, butthe thickness of the blubber layer is species-specific and highlyvariable depending on age, reproductive and nutritional statusand location on the body (Ryg et al., 1988, 1990a; Rosen &

14

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 southernelephant seals (Mirounga leonina) is about 10 kg/day, with over60% of the loss due to decreases in blubber (Slip et al., 1992). Inphocid seals, seasonal breeding is associated with reductions inenergy intake and blubber content, and the changes in theblubber thickness are more pronounced in sexually matureadult seals than in immature juveniles (Ryg et al., 1990b; Mellishet al., 2007). On average the blubber content of mature ringedseals (Pusa hispida) is 40%–50% of body mass at the beginning ofbreeding season and about 30% of body mass at the end ofmolting (Ryg et al., 1990b). The decline of the blubber content ismost pronounced in sexually mature females, mainly derivingfrom the high energetic cost of lactation (Ryg et al., 1990b).

As blubber provides thermal insulation, a sufficiently thicklayer of blubber has to be maintained, even during negativeenergy balance (Ryg et al., 1988). An excessive decrease inblubber thickness compromises its thermoregulatory function,with consequent loss of energy as heat. This could introduce acontinuous cycle of increased energy demand and further loss ofblubber due to impaired thermal insulation. Additionally, thedecrease of blubber may also impair buoyancy andstreamlining. Although the different functions of blubber andthe variability in the thickness of the blubber layer are wellrecognized, there is little information as to how the potentiallyconflicting functions of blubber (particularly thermoregulationand energy expenditure) are manifested in its biochemicalcomposition (Koopman et al., 2002).

1.1 BLUBBER MORPHOLOGYThe blubber of marine mammals is a specialized type of whiteadipose tissue with a high density of connective tissue fibers(collagen and elastin); in small cetaceans the collagenous fibersaccount 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 fibrousstructure which also differentiates it from other types of white

15

adipose tissue. The content of connective fibers in thesubcutaneous adipose tissue of terrestrial mammals is markedlylower than in the blubber of marine mammals; < 1% of wetweight in Arctic foxes (Vulpes lagopus) and polar bears (Pond etal., 1992; Ramsay et al., 1992; Pond et al., 1995). Blubber is alsomorphologically diverse; the size, number and shape ofadipocytes (fat cells that contain a large lipid droplet) and thedensity of fibers depend on the blubber depth and location onthe body (Koopman et al., 2002; Hamilton et al., 2004; Struntz etal., 2004; Montie et al., 2008). It is suggested that themorphological diversity is connected with differences in thefunctions of blubber, such that the blubber tissue is functionallycompartmentalized into metabolically active and structuralcomponents (Koopman et al., 2002; Montie et al., 2008).Structural blubber is characterized by lower lipid content and ahigher proportion of collagenous fibers, and is less affected bythe nutritional status than metabolically active adipose tissue(Koopman et al., 2002; Montie et al., 2008). In the harborporpoise (Phocoena phocoena) the size and number of adipocytesin the tailstock blubber and the superficial blubber (immediatelybelow the skin) were stable under various nutritionalconditions, suggesting low metabolic activity (Koopman et al.,2002). Also, in the bottlenose dolphin (Tursiops truncatus), thesuperficial blubber had a high density of fibers and smallersized adipocytes, suggesting that the superficial blubber servesmainly as structural tissue (Struntz et al., 2004; Montie et al.,2008). The number and size of adipocytes in the deep blubberwere affected by the nutritional status; the adipocytes in thedeep blubber shrank and even disappeared in emaciated harborporpoises, while the number and size of adipocytes in thesuperficial blubber remained unaffected (Koopman et al., 2002).Data on the distribution of connective tissue in seal blubber islimited, but in the leopard seal (Hydrurga leptonyx) theconnective fibers were distributed relatively uniformlythroughout the blubber (Gray et al., 2006).

Marine mammal blubber contains arterio-venousanastomoses (AVAs), specialized shunts that interconnect

16

arterioles and venules (Parry, 1949; Bryden, 1978). The AVAs areimportant in thermoregulation, as heat loss via the peripheralcirculation can be adjusted by regulating the diameter of AVAs.The redistribution of blood flow to peripheral tissues generatesa temperature gradient in the blubber; skin temperature is 15–35°C lower than the core temperature, and it fluctuates accordingto 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) wasless than 5 °C (Irving & Hart, 1957; Hart & Irving, 1959).

1.2 BLUBBER LIPIDSIn the mammalian body, lipids have three main functions: theyserve as energy storage (storage lipids), constituents of cellmembranes (membrane lipids) and messengers of cellular signaltransduction (Vanhaesebroeck et al., 2001; van Meer et al., 2008).Additionally, lipids in cellular membranes affect the function ofmembrane proteins via specific protein-lipid interactions(Dowhan & Boganov, 2002). Lipids consist of a broad group ofnaturally occurring molecules that have various structures andfunctions; lipids include, for example, fatty acids, waxes,eicosanoids, triacylglycerols, glycerophospholipids, sphingo-lipids and sterols. Traditionally lipids are considered to becompounds that are readily dissolved in organic solvents, butthis is not a very specific or precise definition. Christie & Han(2010) defined lipids as “fatty acids and their derivatives, andsubstances related biosynthetically or functionally to thesecompounds”. This definition includes gangliosides (highlycomplex glycosphingolipids), although the gangliosides aremore water-soluble than lipids in general (Christie & Han,2010). An even more specific classification system for lipids wassuggested by the LIPID MAPS consortium: “For the purpose ofclassification, we define lipids as hydrophobic or amphipathicsmall molecules that may originate entirely or in part bycarbanion-based condensations of thioesters (fatty acyls,glycerolipids, glycerophospholipids, sphingolipids, saccharo-lipids, and polyketides) and/or by carbocation-based

17

condensations of isoprene units (prenol lipids and sterol lipids)”(Fahy et al., 2009).

1.2.1 Fatty acidsMost commonly determined lipids in blubber analyses are totalfatty acids, the majority of which represent cleavage productsfrom storage lipids (in phocid seals from triacylglycerols). Fattyacids are carboxylic acids with a hydrocarbon chain that variesin length (number of carbon atoms) and degree of unsaturation(Fig. 1). The degree of fatty acid unsaturation is defined by thenumber of double bonds in the carbon chain; saturated fattyacids (SFA) do not contain any double bonds, while unsaturatedfatty acids contain one (i.e. monounsaturated fatty acids,MUFA) or several double bonds (i.e. polyunsaturated fattyacids, PUFA; Table 1). Fatty acids in seal blubber are typicallyeven-numbered, unbranched and consist of 14-22 carbons,although small proportions of shorter, longer, odd-chained andbranched fatty acids are frequently detected (Table 1; Käkelä etal., 1995; Andersen et al., 2004; Thiemann et al., 2007; Wheatleyet al., 2007; Cooper et al., 2009). Most of the branched fatty acidsare considered to be of bacterial origin (e.g. iso17:0), or derivedfrom the algal chlorophyll (isoprenoid fatty acids such asphytanate). In certain small odontocetes, e.g. the harborporpoise, Hector’s dolphin (Cephalorhynchus hectori) and thenorthern right-whale dolphin (Lissodelphis borealis), the blubbercontains significant proportions (2–40%) of endogenouslysynthesized short chained branched fatty acids, such asisovaleric acid (iso5:0), but this fatty acid has not been detectedin seal blubber (Litchfield et al., 1975; Koopman et al., 1996;Koopman et al., 2003; Thiemann et al, 2008). In general, theblubber of ringed seals contains 28–47% of PUFA, 7–15% of SFA,39–53% of C18MUFA and up to 18% of > C18 MUFA (data fromseals of various ages and from both sexes, and differentsubspecies 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 fattyacid composition (termed the fatty acid “profile”) among

18

different populations and age classes, and a significant part ofthe variation is thought to result from differences in the foragingpatterns, diet, and also from the nutritional and reproductivestatus (Beck et al., 2007; Thiemann et al., 2007; Cooper et al.,2009; Newland et al., 2009; Tucker et al., 2009). The largestcompositional difference in the blubber of ringed seals is foundbetween freshwater and marine populations (Käkelä et al., 1993;Käkelä & Hyvärinen, 1996; Grahl-Nielsen et al., 2003, Thiemannet al., 2007; Cooper et al., 2009). The >C18 MUFA (mainly 20:1n-9and 22:1n-11), are abundant in the marine environment but rarein freshwater food webs, and thus the proportion of 20:1n-9 and22:1n-11 is very low in the blubber of freshwater ringed sealsbut may comprise up to 18% of total fatty acids in marine ringedseals (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 websoriginates from certain calanoid copepods that store lipids aswax esters, whereas freshwater zooplankton typically do notcontain these fatty acids (Morris, 1984; Muje et al., 1989).

Figure 1. Structure and nomenclature of palmitoleic acid (16:1n-7). The systematicname 9-cis-hexadecenoic acid states that the fatty acid contains 16 carbons in astraight chain and 1 double bond (in cis-conformation) at the 9th carbon from thecarboxyl end (i.e. at 9 carbon). Palmitoleic is the common name and the shorthandabbreviation (16:1n-7) indicates the number of acyl carbons and double bonds; theposition 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 amongdifferent individuals, the fatty acid composition of blubber isvertically stratified. The superficial blubber, immediately below

terminalmethyl end

9 carboxyl groupn-7

9-cis-Hexadecenoic acid16:1n-7Palmitoleic acid

19

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

Fatty acid Common name Systematic nameMeltingpoint

°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

18:2n-6 Linoleic acid all-cis-9,12-octadecadienoicacid -7

18:3n-3 -Linolenic acid all-cis-9,12,15-octadecatrienoic acid -10

18:4n-3 Stearidonic acid all-cis-6,9,12,15-octadecatetraenoic acid -57

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

20:4n-3 Eicosatetraenoic acid(ETA)

all-cis-8,11,14,17-eicosatetraenoic acid

20:4n-6 Arachidonic acid (ARAor AA)

all-cis-5,8,11,14-eicosatetraenoic acid -49

20:5n-3 Eicosapentaenoic acid(EPA)

all-cis-5,8,11,14,17-eicosapentaenoic acid -54

22:5n-3 Docosapentaenoic acid(DPA)

all-cis-7,10,13,16,19-docosapentaenoic acid

22:5n-6 n-6Docosapentaenoicacid (n6DPA)

all-cis-4,7,10,13,16-docosapentaenoic acid

22:6n-3 Docosahexaenoic acid(DHA) or cervonic acid

all-cis-4,7,10,13,16,19-docosahexaenoic acid -44

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

20

in a semi-fluid state and the low tissue temperature in thesuperficial blubber (discussed in the blubber morphologysection) prevents the deposition of large proportions of fattyacids with high melting points (i.e. SFA or long-chain MUFA).The compositional differences between the superficial and deepblubber have been documented for several seal species, forexample the ringed seal, the southern elephant seal and theWeddell seal (Leptonychotes weddellii), but information on thedepth-specific changes in the fatty acid composition has beenscarce (Käkelä & Hyvärinen, 1996; Best et al., 2003; Wheatley etal., 2007). The compositional changes in the blubber may occurlinearly throughout the entire blubber layer or in phases atspecific blubber depths. Knowledge on the depth-specificchanges in the fatty acid composition and the detailedstratification pattern throughout the blubber, and how thisvaries across individuals and over time within individuals isimportant for the assessment of the connection between thebiochemical composition and functions of blubber.

1.2.2 Fatty acid metabolismEndogenous synthesis of fatty acids from non-fatty acidprecursors yields primarily the saturated fatty acid 16:0, whichmay be further modified. The main modification products are16:1n-7 ( 9-desaturation in the endoplasmic reticulum), or chainelongation to 18:0, which may be further 9-desaturated to18:1n-9 (Fig. 2). In humans the de novo lipogenesis (endogenoussynthesis of lipids) is generally low and down-regulated by highdietary intake of fats, particularly polyunsaturated fatty acids(Hellerstein et al., 1991). De novo lipogenesis is mainly observedwhen the diet is rich in carbohydrates and low in fats(Hellerstein et al., 1991). Seals generally feed on fish and largecrustaceans which are deficient in carbohydrate but rich inprotein and fat (Henderson & Tocher, 1987; Ventura, 2006).Although amino acids may serve as substrates for lipogenesis, itis unlikely that de novo lipogenesis is an important source offatty acids for seals; it is more likely that most of the fatty acidsare obtained from the diet.

21

16:0

18:0

20:0

14:0

18:1n-9

20:1n-9

16:1n-7

18:1n-7

14:1n-514:1n-7

16:1n-9

20:1n-7

16:1n-5

18:1n-5

9 desaturase

14:1n-9

20:1n-5

peroxisomal chain shortening

elongase

22:022:1n-11

22:1n-722:1n-9

20:1n-11

Figure 2. Modification pathways of saturated and monounsaturated fatty acids,modified after Cook & McMaster (2002). The predominant substrates for the 9-desaturase are 16:0 and 18:0 (pathways marked with red rectangular).

The fatty acid composition of storage lipids in blubber isstrongly influenced by the dietary fatty acids, but does notexactly 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 byvariable digestion and absorption efficiency of dietary fattyacids, modifications of fatty acids (mainly in the liver but also inadipose tissue) and selective fatty acid metabolism in adipose

22

tissue (Raclot, 2003; Budge et al., 2004; Mu & Høy, 2004). Fattyacid modification pathways include desaturation (introductionof a double bond at a fixed position of the acyl chain), andelongation or shortening (partial -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 (Halichoerusgrypus), dietary 16:0 is partly desaturated to 16:1n-7 by the 9-desaturase. The 9-desaturase introduces a double bond intothe 9-carbon of the acyl chain (i.e. between the 9th and 10th

carbon from the carboxylic end, Fig. 1). Similar to 16:0, stearicacid (18:0) may serve as a substrate for 9-desaturase, yielding18:1n-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 beessential fatty acids, since mammals are unable to endogenouslysynthesize these fatty acids, although they are invaluable invarious physiological processes, e.g. cell membrane fluidity andfunction, development of the central nervous system and visualacuity, insulin and the immune function (Jump, 2001; Nakamura& Nara, 2004). Mammals lack the necessary enzymes ( 12- and

15-desaturase) needed for endogenous synthesis of n-6 and n-3PUFA, and thus must obtain these fatty acids or their precursorsfrom 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) and18:2n-6 (linoleic acid) to 20:4n-6 (ARA) via alternatingdesaturations and chain elongations, and further to C22 PUFAvia the Sprecher pathway (Sprecher et al., 1995). The 5- and 6-desaturases catalyze synthesis of PUFA by introducing anadditional double bond between the pre-existing double bondand the carboxylic end of the acyl chain. Long chain fatty acidsare shortened in peroxisomes via partial -oxidation, and theelongation of the fatty acid chain by 2 carbon units is performedpredominantly in endoplasmic reticulum by elongases, locatedin complexes with the desaturases (Reddy & Hashimoto, 2001;Jakobsson et al., 2006).

23

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

1.2.3 Storage lipidsIn phocid seals, excess energy is stored in blubber astriacylglycerol (TAG; Henderson et al., 1994). A TAG moleculecomprises three fatty acids incorporated into a glycerolbackbone. The physical properties of TAG are largelydetermined by the composition of the fatty acid triplet. TAGs

6 desaturase

5 desaturase

elongase

peroxisomal chain shortening

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

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

elongase

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

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

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

6 desaturase

elongase

(24:5n-3) (24:4n-6)

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

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

The Sprecherpathway

24

with saturated fatty acids have higher melting points and arestructurally more rigid than TAGs with unsaturated acyl chains.Previous studies have concluded that in seal blubber, PUFA aregenerally esterified at the sn-1 or sn-3 position of TAG, and C16

and C18 MUFA and SFA are the predominant fatty acids at thesn-2 position (Brockerhoff, 1966; Brockerhoff et al., 1966). Thispositional distribution of fatty acids in TAG differs from thatfound in fish; in fish the polyunsaturated fatty acids arepreferentially esterified at the sn-2 position (Brockerhoff, 1966;Brockerhoff et al., 1966).

TAG is the most widely spread storage lipid in marinemammals, 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 inblubber (Koopman, 2007; Walton et al., 2008). In addition, theblubber of harbor porpoises and a few species of dolphins, e.g.hector’s dolphin and the long-finned pilot whale (Globicephalamelas), contains small amounts of wax esters, predominantly inthe superficial blubber, although TAG is the main storage lipid(Koopman, 2007). The reason for the deposition of wax esters inthe blubber is unclear, but may relate to the differentphysiological properties of wax esters in connection withecological and physiological adaptations to deep-diving andenergy metabolism (Koopman, 2007). As well as in deep-divingodontoces, wax esters are typically found in deep-diving polarzooplankton and fish species (Lee et al., 2006). However, deep-diving pinnipeds, such as the northern elephant seal (Miroungaangustirostris), store lipids as TAG. The northern elephant sealsexperience large annual changes in energy balance (connectedwith reproduction and molting; Costa & Ortiz, 1982), and it maybe that hydrolysis of wax esters is not able to respond to therapid changes in energy demand (Koopman, 2007). Wax estersare long chain fatty alcohols esterified to fatty acids, andcompared to TAG, wax esters are generally more stable andseem to have a slower turnover rate (Sargent et al., 1981; Lee etal., 2006).

25

1.2.4 Membrane lipidsThe proportion of polar lipids in adipose tissues is exceedinglysmall, typically < 1% of total lipids (Body, 1988; Kotronen et al.,2010). Nevertheless, the polar lipids are critical constituents ofall cellular membranes and also function as cellular messengers,enzyme activators, and precursors for local tissue hormonessuch as eicosanoids (Dowhan & Bogdanov, 2002; van Meer et al.,2008). The lipid composition of cellular membranes is highlytissue- and species-specific (Body et al., 1988; Henderson et al.,1994), but the most abundant and widely distributed classes ofmammalian membrane lipids are glycerol-based phospholipids(glycerophospholipids), sphingolipids and sterols (mainlycholesterol; Body, 1988; Parrish et al., 1997; Zeghari et al., 2000a).Glycerophospholipids are comprised of a glycerol backbonewith two fatty acids esterified at the sn-1 and sn-2 positions, anda 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 glycero-phospholipids in mammalian cell membranes arephosphatidylcholine (PC) with a choline headgroup, andphosphatidylethanolamine (PE) with an ethanolamine attachedto the phosphate. Sphingolipids are sphingosine (an aminoalcohol) derivates with a functional head group attached to thesphingosine, such as choline in sphingomyelin (SM). SM is themost important sphingolipid in mammalian cell membranes,and its distribution is confined to the outer leaflet of the plasmamembrane (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 mammalblubber adipocytes are limited, but in adipose tissues of humans(subcutaneous adipose tissue) and rats (epididymal andperirenal adipose tissue), the most common membrane lipidswere PC (30–45% of total phospholipids) and PE (20–30% oftotal phospholipids; Body, 1988; Parrish et al., 1997; Zeghari etal., 2000a). The proportion of SM varied from 8 to 25% of totalphospholipids. In membrane lipids in Mediterranean monk seal

26

(Monachus monachus) liver, heart and muscle, the most commonpolar lipid classes were PC (30–40% of phospholipids) and PE(20–30% of phospholipids; Henderson et al., 1994). PC and PEwere also the most abundant glycerophospholipids inerythrocytes and platelets of northern and southern elephantseals, the Antarctic fur seal (Arctocephalus gazella) and the harpseal (Nelson, 1970; Fayolle et al., 2000). Thus, at the level of lipidclasses, the main building blocks of the membranes are similaracross mammals.

The glycerophospholipids and sphingolipids areamphipathic; the polar head group of the phospholipids is morehydrophilic than the non-polar hydrocarbon tails. Thisamphipathicity is the physical basis for the formation ofbiomembranes (Dowhan & Bogdanov, 2002). In aqueousenvironments the amphipathic lipid molecules self-associateinto a two layered sheet; the polar (hydrophilic) head groupsface the water phases on either side of the sheet and the non-polar (hydrophobic) fatty acid tails point at the center of thebilayer. Biological membranes are not merely static barriers incells but their lipid composition directly affects the function ofmembrane proteins (receptors, transporters, channels), and thusalso a myriad of cellular processes (van Meer et al., 2008). Thelipid bilayer may exist in various relatively fluid and solidphases, depending on the molecular structure of the membranelipids and the environmental conditions (van Meer et al., 2008).The membrane phases determine the orientation and mobility ofmembrane lipids and thus affect the functions of the membraneproteins therein. Double bonds in cis-configuration bend thefatty acid chain, preventing the dense packing of molecules andresulting in a liquid disordered phase. Lipid moleculescomprised of long saturated hydrocarbon chains (as insphingomyelin) are densely packed in membranes, producingsolid-like gel phases in the membrane that affect the mobilityand functions of lipids and proteins (Slotte, 1999). The amountof cholesterol embedded between the fatty acid chainsmodulates the physical properties and phase behavior ofcellular membranes. The addition of cholesterol to a highly

27

ordered membrane introduces a liquid ordered phase, wherethe acyl chains are highly ordered, but some lateral movement isallowed (distinguishing it from the gel phase).

Cholesterol is a neutral lipid, composed of four hydrocarbonrings with a hydrocarbon and a hydroxyl group attached to it.The distribution of cholesterol in the plasma membrane isclosely associated with that of SM and other sphingolipids, andthe current view is that biological membranes are laterallysegregated into sphingolipid/ cholesterol rich microdomainscalled rafts (Simons & Ikonen, 2000; Gulati et al., 2010). It hasbeen suggested that the large head groups of sphingolipidsshield the highly hydrophobic cholesterol from the aqueousmedia (Gulati et al., 2010). Furthermore, cholesterol is alsopreferentially distributed between long chain saturated acylchains, which are predominant in sphingolipids, but rare inglycerophospholipids (Brown & London, 2000). Thesesphingolipid/ cholesterol rich domains may be involved in theregulation of several cellular functions, such as lipidmetabolism, by modulating the activity of membrane receptors,pumps and channels (Zeghari et al., 2000a, 2000b; Al-Makdissyet al., 2003; Ikonen & Vainio, 2005; Vainio et al., 2005; Bastiani &Parton, 2010).

1.3 LIPIDS IN MARINE MAMMAL RESEARCHThe blubber of marine mammals has been employed in a widerange of physiological, biochemical and ecological studies, e.g.in studies on adaptations to the aquatic environment(particularly energy metabolism, thermoregulation andlocomotion), determination of trophic interactions, andbiomonitoring 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 alsoprovided information on population/stock identification andage estimation (Smith et al., 1996; Thiemann et al., 2007; Hermanet al., 2008, 2009). Analyses of the fatty acid composition ofblubber are commonly applied to studies on the foragingstrategies and diet of marine mammals (Iverson et al., 1997;

28

Bradshaw et al., 2003; Cooper et al., 2009). Determination of theforaging strategies and diet is inherently difficult in theseanimals as the catch and consumption of prey takes place underthe surface. Nonetheless, knowledge of the foraging patternsand diet preferences of seals is important, not only forunderstanding of the aquatic food web dynamics but also formanagement and conservation policies (e.g. in conflicts withfisheries). The feeding behavior of seals has been assessed byanalyzing prey remains in the digestive tract or in fecal samples,but these techniques overestimate the importance of prey withlarge and robust hard parts which resist digestion better thansmall soft bodied species (reviewed by Pierce & Boyle, 1991).Also, such results provide information only on the most recentmeal, which may not represent long-term diet. It has beensuggested that application of molecular biomarkers, mainlyfatty acids and stable isotopes, and also analyses of prey DNA inthe scat, overcomes some of the problems encountered in thetraditional hard part analyses (Hobson et al., 1996; Budge et al.,2006; Casper et al., 2007). The basic idea of the fatty acidbiomarker approach is that the dietary fatty acids arepredictably incorporated into blubber, and the diet compositionof the predator can be resolved by comparing the fatty acidcomposition 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 acidcomposition has been widely employed in qualitative andquantitative estimates of diet (Bradshaw et al., 2003; Walton &Pomeroy, 2003; Iverson et al., 2004; Cooper et al., 2009; Newlandet al., 2009; Tucker et al., 2009). However, the clarity of resultsfrom studies on different species at different times or locationshas been variable, and it has been suggested that the results maybe confounded by the stratification of fatty acids and fluctuatingfatty acid metabolism (Best et al., 2003; Grahl-Nielsen et al.,2011a).

29

1.4 BACKGROUND INFORMATION ON THE STUDIEDSPECIESThe study animals were phocid seals from marine andfreshwater environments (Fig. 4): the Arctic ringed seal Pusahispida hispida (Svalbard, Norway), the Saimaa ringed seal Pusahispida saimensis (Lake Saimaa, Finland) and the Baikal seal Pusasibirica (Lake Baikal, Russia). The distribution of the Arcticringed seal is circumpolar, while the distributions of the Saimaaringed seal and the Baikal seal are restricted to Lake Saimaa andLake Baikal, respectively.

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

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

30

about 50–70 kg and have a standard length of about 130–150 cm(Lydersen & Gjertz, 1987; Sipilä & Hyvärinen, 1998). The ringedseals and the Baikal seal are considered to be opportunisticfeeders with preferences for few particular prey types. Smallfish (length 5–20 cm) are the predominant prey for all threespecies (Thomas et al., 1982; Weslawski et al., 1994; Kunnasrantaet al., 1999). Additionally, the Arctic ringed seal and the Baikalseal feed on macro-crustaceans; e.g. Parathemisto libellula,Thysanoessa inermis and Pandalus borealis are important preyitems for the Arctic ringed seal, and stable isotopes analysisindicated that the Baikal seal feeds on the pelagic amphipodMacrohectopus branickii (Weslawski et al., 1994; Yoshii et al.,1999). In Lake Saimaa the only abundant macro-crustaceanMysis relicta does not seem to be an important prey item for theSaimaa ringed seal (Kunnasranta et al., 1999).

The Arctic ringed seal, the Saimaa ringed seal and the Baikalseal live in completely different habitats, with different foodweb structures. The Arctic ringed seal is a marine subspecies,while the Saimaa ringed seal and the Baikal seal live infreshwater environments. Furthermore, the freshwater bodies ofLake Saimaa and Lake Baikal are very different. Lake Saimaa isa shallow lake (mean depth 12 m, max depth 85 m) with aheterogeneous shoreline and numerous islands, while LakeBaikal is the deepest lake in the world (mean depth 744 m, maxdepth 1642 m) with large areas of open water. In Lake Baikal64% of the flora and fauna are endemic (e.g. abundant pelagicfish genus Comephorus), and the fatty acid composition in theLake Saimaa and Lake Baikal food webs demonstrate significantdifferences, which are also reflected in the fatty acidcomposition of the seal blubber (Muje et al., 1989; Käkelä et al.,1993; Ju et al., 1997; Grahl-Nielsen et al., 2005; Grahl-Nielsen etal., 2011b). Analyses of the detailed stratification pattern ofblubber lipids in closely related seals inhabiting completelydifferent environments provide an opportunity to evaluate theimpacts of endogenous and environmental factors on the depth-specific fatty acid composition and metabolism of the sealblubber.

31

1.5 OBJECTIVES OF THE STUDYThe objectives of this thesis were to determine the biochemicalstratification of blubber in seals that are closely related butinhabit different environments and to evaluate the impact ofexogenous (dietary fatty acids) and endogenous (e.g. energybalance, age) factors on the stratification patterns of lipids.Specific indices were employed to investigate the lipidstratification patterns among seals with different fatty acid, TAGand phospholipid compositions. The original papers containspecies-specific data on these stratification patterns, includingdiscussions on factors that influence the stratification pattern,particularly blubber thickness and age. In this summary my goalwas to summarize the papers and to compare directly thestratification patterns among the different seal species. Theinterspecific comparison of the lipid and fatty acid stratificationpatterns 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 theblubber. Additionally, the possible metabolic variationsthroughout the blubber were evaluated by analyzing the mainlipid composition of the membranes. Significant depth-specificdifferences in the lipid composition of membranes may indicateconcurrent differences in lipid metabolism, since the lipidcomposition of plasma membrane has been documented as afactor that influences the function of membrane proteins(Zeghari et al., 2000a, 2000b; Al-Makdissy et al., 2003; Ikonen &Vainio, 2005; Vainio et al., 2005).

33

2 Material and methods

2.1 BLUBBER SAMPLESBlubber samples were collected from Arctic ringed seal (N=25),Saimaa ringed seal (N=37) and Baikal seal (N=22) of varying ageand from both sexes. The Saimaa ringed seal is criticallyendangered, and the blubber samples were collected during theyears 1996–2008 from fresh carcasses (mostly by-caught), andsubsequently the age distribution was biased towards sealsyounger than 1 year old (II, III). The Arctic ringed seals wereshot in Svalbard in April 2000 (I) and the Baikal seal pups werecaught on ice in April 2005, and older Baikal seals (aged 3–18yrs) were netted in October 2005 (IV). The sampled individualswere highly variable in terms of age, as well as reproductive andnutritional status. The samples size (particularly for lipid speciesin the Saimaa ringed seal, III) did not allow the directcomparison of different categories; instead the approach of thisthesis is to evaluate larger scale differences and similarities inthe stratification patterns of fatty acids in the blubber amongspecies and subspecies. The blubber samples from the Arcticand Saimaa ringed seal were obtained ventrally (over thesternum), and from the Baikal seal the blubber samples were cutmid-dorsally (at about 2/5 of the distance from snout to tail).Although the blubber samples from the Baikal seals were cutdorsally, we assumed that the fatty acid profile would be similarin both locations, as no differences were found between thevertical fatty acid profiles of ventral and dorsal blubber in theArctic ringed seal (details in I). The blubber of the Arctic andSaimaa ringed seals was dissected into large blocks (about 20cm×20 cm), with skin and some muscle still attached to theblubber, and stored at -20 °C for analysis. The Baikal seals wereskinned prior to the cutting of the blubber block. The edges ofthe frozen blubber block were removed (from the lateral sidesonly) and 3 mm thick sequential subsamples were dissected

34

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

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

2.2 FATTY ACID ANALYSISFatty acid methyl esters were prepared for the gaschromatographic analysis via direct transmethylation of fattyacids. The blubber subsamples were heated (90 °C) in acidicmethanol to produce fatty acid methyl esters (FAME). TheFAMEs were analyzed by gas chromatography with FID andMS detection (I, II, III, IV). In most cases the method for theanalysis with FID detection was as follows: split injection (splitratio 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 afinal temperature of 210 °C, which was held for 25 min (I, II, III).Detailed information on the preparation of FAMEs and the usedGC method can be found in the original papers. The peaks wereidentified by authentic standards and mass spectrometry.

2.3 LIPID SPECIES ANALYSISFor the analysis of TAG and phospholipid species andcholesterol, the total lipids were extracted from each subsampleaccording to Folch et al. (1975; III, IV). Aliquots of extracts wereevaporated under N2 stream, and redissolved into peroxide-

35

tested chloroform/methanol (1:2, v/v). Total phospholipid (i.e.total PO4 in the lipid extract) and cholesterol were determinedby standard spectrophotometric methods (Bartlett & Lewis,1970; Gamble et al., 1978). The TAG and phospholipid specieswere 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 formationof sodium adducts. The TAG species were detected in positiveionization mode as (M+NH4)+ ions (Duffin & Henion, 1991). Thefatty acid composition of isobaric TAG species was determinedby tandem mass spectrometry (MS/MS) and a fingerprintingmethod that detected neutral losses of all possible natural fattyacids (Han & Gross, 2001, 2005). The major TAG molecularspecies for each isobaric TAG peak were calculated on the basisof the ion abundances for released fatty acid fragments. Thespecies composition of the main phospholipid classes, i.e. PC,SM and PE, were selectively detected with class-specific MS/MSscanning modes (Brügger et al., 1997; Hsu et al., 1998). In thepositive ion mode, the PC and SM species are precursors of m/z184 (which corresponds to the choline head group), for the PEspecies the fragment indicating the ethanolamine head group isneutral, and thus the lipids were detected as a neutral loss of 141amu. The mass spectra were corrected for overlap of isotopicpatterns, and the lipids species were quantified on the basis ofthe responses of the standards by LIMSA (LIpid Mass SpectrumAnalysis) software (Haimi et al., 2006).

2.4 DATA ANALYSISFor the evaluation of lipid stratification patterns in seal blubberwith varying fatty acid and TAG species composition, a set ofindices was employed: relative stratification index (SI), 9-desaturation index ( 9-DI), numerical gradient (NG), andEuclidean distance. The relative stratification index wascalculated according to Olsen & Grahl-Nielsen (2003) from fattyacid molar percentage in the outermost subsample (0-3 mmfrom 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

36

acids with different abundances. The selected 20 fatty acidsrepresent the abundant (>1.5 mol% in at least one of thesamples) saturated, monounsaturated, and polyunsaturatedfatty acids of varying chain length. Negative SI-values indicatethat the fatty acid is relatively more abundant in the innermostsubsample while positive SI-values indicate enrichment in theoutermost blubber sample. Similar relative stratification indexwas also calculated for TAG species (III).

9-Desaturation index (DI) was calculated for eachsubsample and 9-DI stratification pattern was used for thedefinition of superficial blubber (I, II, III, IV). The 9-DI is theratio of C14, C16 and C18 MUFA to the corresponding SFA (Käkelä& Hyvärinen, 1996). 9-desaturation of SFA is one of the mainmodification processes for increasing the fluidity of lipids as aresponse to low tissue temperatures (Tiku et al., 1996; Truemanet al., 2000), and it has been demonstrated that the 9-DI is highin the superficial blubber immediately below the skin and lowin the deep blubber next to the skin (Käkelä & Hyvärinen, 1996).It is likely that both 9-desaturation of SFA and selectivedeposition of 9- MUFA (mainly 16:1n-7 and 18:1n-9) contributeto the calculated 9-DI value in blubber.

To evaluate the depth-specific changes in the fatty acidcomposition, numerical gradients of fatty acid mol% werecalculated for blubber subsamples (Schey, 2005). Fatty acids thatexceeded 0.1mol% were included in the analysis of numericalgradient (I, II, IV). The computations were done with MATLAB2010a using the function “gradient.m” (MathWorks, Natick,MA). The NG value corresponds to compositional changesbetween adjacent blubber subsamples; a high value indicatesgreater differences.

Euclidean distances were calculated between the fatty acidmolar percentages of blubber subsamples and potential preyitems (Eq.1); large values indicate greater differences in the fattyacid composition (II). The Euclidean distances were alsocalculated between the blubber samples of the two ringed sealsubspecies to evaluate whether compositional differencesbetween the subspecies were similar throughout the entire

37

blubber column. The purpose of these calculations was toestimate the absolute compositional difference between thetissue samples, and all the identified fatty acids were includedin the analysis. Euclidean distance is a simple measure of thedistance between two objects that does not correct for differentscaling of variables (Quinn & Keough, 2002). The impact ofdifferent variables (fatty acid mol%) on the Euclidean distancevalue is directly dependent on the variance of the variable;variables with large variance weigh more, while less abundantfatty acids with low variance have a smaller impact on theEuclidean distance.

d x, y = xi-yi2

n

i=1

(1)

Additionally, SM/PC ratio was calculated to illustrate verticalchanges in the membrane lipid composition, which may berelated 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 testedby parametric (paired t-test) and non-parametric methods(Mann-Whitney U-test, Wilcoxon signed rank test), dependingon the data. Details can be found in the original papers.

39

3 Results and discussion

3.1 INTERSPECIFIC VARIATION OF BLUBBER LIPIDS

3.1.1 Fatty acidsThe blubber fatty acid compositions of the studied seal specieswere in accordance with those reported previously (Käkelä &Hyvärinen, 1996; Grahl-Nielsen et al., 2005). The predominantfatty acids in the two ringed seal subspecies and in the Baikalseal 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 similarin all the analyzed seals, but the relative proportions of fattyacids varied significantly, i.e. the blubber fatty acid compositionof the closely related species from marine and freshwaterenvironments were different (Fig. 6). The greatest differenceswere observed in the abundance of C20 and C22 MUFA andscarcity of n-6 PUFA in the Arctic ringed seal blubber (Fig. 6).The high proportion of C20 and C22 MUFA in marine sealsoriginates from calanoid copepods that store lipids as waxesters, common in the marine environment but rare infreshwater environments (Morris, 1984, Muje et al., 1989). The higher proportion of C18 n-6 PUFA in the blubber of thelacustrine seals corresponds with the higher proportions of n-6PUFA in freshwater food webs (Muje et al., 1989; Grahl-Nielsenet al., 2011b). The scarcity of n-6 PUFA in marine food websprobably results from the dominance of diatoms(Bacillariophyceae) in the phytoplankton community; diatomsare typically more enriched in n-3 PUFA than n-6 PUFA(Dunstan et al., 1993). Green algae (Chlorophyceae) are anabundant source of n-6 PUFA, and consequently freshwaterfood webs that are dominated by green algae are generally richin n-6 PUFA (Thompson, 1996). In addition, the contribution ofterrestrial n-6 PUFA (e.g. via terrestrial invertebrates) is

40

typically higher in freshwater lakes than in marineenvironments (Napolitano, 1999). Between the two species offreshwater seals, the most prominent difference in the fatty acidcomposition was the abundance of C18 MUFA in the Baikal seal(particularly 18:1n-9), whereas in the Saimaa ringed seal blubberthe shorter chained, C16 MUFA were the predominant MUFA(Fig. 6). The differences in the proportion of C16 and C18 MUFAbetween the freshwater seals most likely arise from thedifferences in the fatty acid composition of the potential preyfish (Muje et al., 1989, Grahl-Nielsen et al., 2011b). 18:1n-9comprises 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 etal., 2011b). For comparison, the proportions of 18:1n-9(containing small amounts of 18:1n-11) in the potential prey fishof the Saimaa ringed seal, i.e. vendace (Coregonus albula), ruffe(Gymnocephalus cernuus), perch (Perca fluviatilis), roach (Rutilusrutilus), and smelt (Osmerus eperlanus), are only 7–15% (II).Additionally, the lipid content of adult golomyanka is veryhigh, 30–40% of wet weight (Ju et al., 1997; Kozlova &Khotimchenko, 2000). The abundance of 18:1n-9 in the diet ofthe Baikal seal is reflected in the fatty acid composition of theblubber, and the high variation in the proportion of 18:1n-9probably reflects significant differences in foraging strategiesamong different Baikal seal individuals (IV).

3.1.2 TriacylglycerolsThe species composition of TAG molecules was analyzed inSaimaa ringed seal and Baikal seal blubber (Fig. 7). As expected,the TAG composition reflected the fatty acid composition in theblubber. High proportions of oleic acid (18:1n-9) in the Baikalseal blubber resulted in high proportions of monounsaturatedTAG (the FA triplet comprising of monounsaturated fattyacids), 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 both18:1n-9 and monounsaturated TAG species showed largestandard deviations, demonstrating great individual variation.

41

The Saimaa ringed seal blubber contained proportionally moreunsaturated TAGs, which typically contained onepolyunsaturated fatty acid, e.g. 56:7 (18:1/18:1/20:5 or16:1/18:1/22:5), 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 speciescomposition in the Saimaa ringed seal blubber was more diverse(i.e. the number of detected TAG species was higher). Inparticular, the TAG species composition of the deep blubberwas more diverse in the Saimaa ringed seal than in the Baikalseal. The diversity of TAG species may be associated with theabundance of PUFA.

In terrestrial mammals, which had low dietary intake ofPUFA, the TAG diversity in adipose tissue was lower than inthe blubber of the seal species in this study (Perona et al., 2000;Suzuki et al., 2008). Similarly, a high TAG diversity has beendocumented in sardines (Sardine pilchardus), which are also richin PUFA (Perona & Ruiz-Gutiérrez, 1999).

The fatty acid composition in the TAG showed higherplasticity than previously thought (Brockerhoff, 1966;Brockerhoff et al., 1966). Conventional analysis of the positionaldistribution of fatty acids in the TAG of blubber suggested thatPUFA are mainly esterified at the sn-3 and sn-1 position, and theshort-chain saturated and monounsaturated fatty acids favor thesn-2 position (Brockerhoff, 1966; Brockerhoff et al. 1966). Thedistribution of fatty acids in the TAG is mainly determined bythe available fatty acids and the activity of acyltransferases,enzymes that catalyze the stepwise addition of fatty acids to theglycerol (Coleman & Lee, 2004; Kazuharu & Reue, 2009). Thesubstrate preferences vary among different acyltransferases, andit has also been suggested that the enzyme activities may bespecies/tissue specific and also modulated by the available fattyacid pool (Coleman & Lee, 2004; Kazuharu & Reue, 2009). Thedifferences in the substrate preferences and activities ofacyltransferases may explain the observed plasticity in the TAGfatty acid distribution of the seal blubber.

42

Figure 6. Mol% of 20 abundant saturated, monounsaturated and polyunsaturatedfatty 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 theBaikal seal fetuses) are included in the figure. Mean fatty acids values among specieswith a different letter differ significantly (P < 0.05, U-test). Superficial (panel A) anddeep (panel B) blubber were tested separately. Asterix (*) next to the X-axis in panel Adenotes those fatty acids for which mol% differs significantly between superficial anddeep blubber within species (P < 0.05, Wilcoxon signed rank test).

14:0

16:0

18:0

14:1

n-5

16:1

n-7

16:1

n-9

18:1

n-9(

11)

18:1

n-7

18:2

n-6

18:3

n-3

18:4

n-3

20:1

n-9

20:1

n-11

22:1

n-11

20:4

n-3

20:4

n-6

20:5

n-3

22:5

n-3

22:5

n-6

22:6

n-3

Mol

ar%

0

5

10

15

20

25

30

35 Arctic ringed seal N=25Saimaa ringed seal N=37Baikal seal N=19

Fatty acid

14:0

16:0

18:0

14:1

n-5

16:1

n-7

16:1

n-9

18:1

n-9(

11)

18:1

n-7

18:2

n-6

18:3

n-3

18:4

n-3

20:1

n-9

20:1

n-11

22:1

n-11

20:4

n-3

20:4

n-6

20:5

n-3

22:5

n-3

22:5

n-6

22:6

n-3

Mol

ar%

0

5

10

15

20

25

30

35

a bab

abba

bc

a

b

c

abc

a

b

ab

ab

ab

a

b b

a

bc

abb

a

b ca a

bb bb b ba a

bc

a ab

a ab

b ba

bb

a

B) Deep blubber

A) Superficial blubber

Arctic ringed seal N=25Saimaa ringed seal N=37Baikal seal N=19

a bb abb

abcab

a

aa

b

a

a

b

aab

bb

a

bc

abba

a

bc a ab b b b ab a a

bc

a

bc ab c

ab b

* ** * ** *** * ** * ** * * * * * * * * * * *** * * * *

43

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

TAG

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

Mol

ar%

0

2

4

6

8

10

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

Mol

ar%

0

2

4

6

8

10Baikal seal (N=11)Saimaa ringed seal (N=6)

B) Deep blubber

A) Superficial blubber

a

a

a

a aa

a

a

a

a

a

a

a

aa

a

a

a a

*

* *

* *

*

*

*

44

3.2 BIOCHEMICAL STRATIFICATION OF BLUBBER

3.2.1 General stratification of different fatty acidsThe stratification of fatty acids in the blubber was mostpronounced in the Arctic ringed seal. The strong stratification ofthe Arctic ringed seal blubber was mostly influenced by theabundance of C20 and C22 MUFA in the deep blubber (Fig. 6),which is in accordance with previous studies on the ringed sealand 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). Ithas been suggested that the temperature gradient throughoutthe blubber is a major factor contributing to fatty acidstratification (Käkelä & Hyvärinen, 1996). The stratification ofSFA and MUFA in Arctic ringed seal blubber was linearlycorrelated with the melting point of these fatty acids (I). TheAVAs and counter-current heat exchange system preserves heatin the deep blubber, and the tissue temperature in superficialblubber and skin are only marginally higher than the ambienttemperature (Irving & Hart, 1957; Hart & Irving, 1959; Meagheret al., 2008; Noren et al., 2008). The melting points of C20 and C22

MUFA are high (see table 1), and thus these fatty acids cannotbe deposited in large quantities in the superficial blubber, inwhich tissue temperature is low and fluctuates according to theambient temperature (Irving & Hart, 1957; Hart & Irving, 1959).Although the proportion of C20 and C22 MUFA is very low in theblubber of the Baikal seal and the Saimaa ringed seal, thestratification of fatty acids was evident in these species,suggesting that C20 and C22 MUFA are not the only driving forcefor fatty acid stratification in the blubber of phocid seals. Thegeneral stratification of the most abundant MUFA and SFAwere similar in the studied seals regardless of habitat; i.e.monounsaturated C14 and C16 accumulate in the superficialblubber and the SFA in the deep blubber (Fig. 8). Although inthe Baikal seal the relative stratification index of 20:1n-11 waspositive (Fig. 8), the molar percentage is very low in thesuperficial blubber (Fig. 6), and thus this fatty acid does not

45

contribute significantly to the overall fatty acid stratification inthe blubber. The stratification of PUFA was variable among thestudied seals and the stratification was not connected with thefatty acid melting points, which is not surprising since themelting points of PUFA are well below zero Celsius (see table 1).More likely the stratification of PUFA is mainly affected byvariation in dietary fatty acids and lipid metabolism.Ontogenetic changes in the vertical stratification of fatty acidswere detected in the seals and the degree of fatty acidstratification in the blubber was low in pups ( 1 year oldindividuals). Only the Saimaa ringed seal and Baikal sealsamples included pups. It is unclear why the pups showed alower degree of stratification, but it is possible that i) the blubberlayer in pups is built up quickly with little depth-specificselection, ii) the modification of fatty acid is slow, and/or iii) theannual fluctuations in blubber thickness (associated with thereproductive cycle) reinforce the stratification of fatty acids. Inaddition, the ontogenetic differences in fatty acid stratificationmay be connected with growth-related changes in blubbermorphology and thermal properties (Dunkin et al., 2005). Theontogenetic differences in the fatty acid stratification patternswere most pronounced in the Baikal seal blubber (IV). Theproportions and stratification patterns of 18:1n-9 differedsignificantly between >10-year-old and 2-10-year-old Baikalseals (IV). The proportion of 18:1n-9 was similar in thesuperficial blubber of both age categories, but in the >10-year-old seals the proportion of 18:1n-9 remained high also in thedeep blubber. Thus in this respect the blubber was less stratifiedin the >10-year-old Baikal seals. These ontogenetic differences inthe degree of stratification due to the differences in thestratification of 18:1n-9 may emerge from differences in foragingpatterns. The high proportions of 18:1n-9 in the deep blubber ofthe older and larger Baikal seals may indicate recent feeding onmature golomyanka, as other potential prey fish (such asComephorus dybowskii and the omul Coregonus autumnalismigratorius), and the macro-crustacean Macrohectopus have alower lipid content and proportion of 18:1n-9 than the

46

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

Figure 8. Relative stratification index of 20 abundant saturated, monounsaturated andpolyunsaturated fatty acids in the blubber of Arctic and Saimaa ringed seals andBaikal seal ( 1 year old seals, and Baikal seals older than 10 year are excluded from thefigure, see text and IV for explanation). The relative stratification index enables thecomparison of fatty acids of varying proportions; negative values indicate relativelylarger proportions in the deep blubber and positive values indicate relatively largerproportions in the superficial blubber. Error bars represent ±SD.

3.2.2 Depth-specific changes in the fatty acid compositionThe numerical gradients of fatty acids were calculatedthroughout the blubber column for all seal species; a high NGvalue indicates great compositional differences betweenadjacent subsamples. The vertical profiles of the NG valuesillustrate the combined stratification pattern of fatty acids

Fatty acid

14:0

16:0

18:0

14:1

n-5

16:1

n-9

16:1

n-7

18:1

n-9+

11

18:1

n-7

20:1

n-11

20:1

n-9

22:1

n-11

18:2

n-6

18:3

n-3

18:4

n-3

20:4

n-6

20:4

n-3

20:5

n-3

22:5

n-6

22:5

n-3

22:6

n-3

Rel

ativ

e str

atifi

catio

n in

dex

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

Arctic ringed seal (N=20)Saimaa ringed seal (N=7)Baikal seal (N=5)

Saturatedfatty acids

Monounsaturatedfatty acids

Polyunsaturated fatty acids

47

throughout the blubber, and specify whether the stratification offatty acids in blubber is linear or whether the changes are morepronounced at specific blubber depths. In addition, the NGvalues permit comparison of the fatty acid stratification patternsamong seal species with completely different fatty acidcompositions. The high NG values in the deep blubber of theArctic ringed seal demonstrate large vertical changes in the fattyacid composition, which are partly due to the substantial changein the molar% of long-chain MUFA, particularly C20 and C22

MUFA (Fig. 9A). Levels of C20 and C22 MUFA in Saimaa ringedseal and Baikal seal blubber were negligible, which partlyexplains the lower compositional changes in the deep blubber(Fig. 9A and B). Nevertheless, the NG values also increasedslightly in the deep blubber of the Saimaa ringed seal and theBaikal seal.

For all the studied seals, the compositional changes in thedeep blubber were high in individuals with thin blubber (< 30mm; I, II, IV). It is likely that the large compositional changes inthe deep blubber adjacent to the muscle are due to restricteddeposition of fatty acid with high melting points (SFA and long-chain MUFA) in the superficial blubber, as well as active andselective fatty acid metabolism (incorporation/mobilization offatty acids) in the deep blubber, which remolds the fatty acidcomposition (Raclot & Groscolas, 1995; Käkelä & Hyvärinen,1996; Raclot, 2003; Wheatley et al., 2008). Selective metabolism offatty acids may be associated with their different physiologicalroles and functions. For instance, physiologically importantPUFAs are precursors for local tissue hormones and importantmodulators of membrane function. These findings suggest thatfluctuations in metabolism, energy balance (e.g. due toreproductive state) and/or diet composition may first bedetected in the deepest 1 cm of the blubber in the studiedspecies/subspecies.In the Arctic ringed seal a second peak in the NG values wasdetected at the depth of about 1.5 cm from the skin, indicatinglarge compositional changes (Fig. 9A). Approximately at thesame depth, a peak in NG values was also detectable in the >1-

48

Figure 9. Panel A shows vertical profiles of fatty acid numerical gradients (NG) in theblubber of two ringed seals (Arctic and Saimaa) and Baikal seal ( 10 yrs old). Forclarity, the vertical profile of NG in the blubber of Baikal seals older than 10 years ispresented in a separate panel (B). High NG values indicate large differences in thefatty acid composition of adjacent subsamples. Error bars represent ±SE. Lettersindicate the depths at which the means differ significantly between Arctic and Saimaaringed seals (a-s), between Arctic ringed seal and Baikal seal (a-b), and betweenSaimaa ringed seals and Baikal seals (s-b; P < 0.05, U-test). In panel B the lettersindicate the depths at which the means differ between Baikal seals older than 10 yearsand Arctic ringed seals (a-b), Saimaa ringed seal (s-b) and Baikal seals younger than10 years (b-b; P < 0.05, U-test).

year-old Saimaa ringed seals, and in the blubber of 2-10-year-oldBaikal seals (Fig. 9A). The high compositional changes (NGvalue) form a border separating the superficial and deeperblubber layers, and on the basis of biochemical composition, thethickness of the superficial blubber was estimated to be about1.5 cm. The fatty acid stratification pattern was significantlydifferent in the Baikal seals older than 10 years; the NG valueswere the highest in the superficial blubber and declined to adepth of about 1.5 cm and remained constantly low throughout

Distance from the skin (mm)

0-3

3-6

6-9

9-12

12-1

515

-18

18-2

121

-24

24-2

727

-30

30-3

333

-36

36-3

939

-42

42-4

545

-48

Sum

of f

atty

aci

d nu

mer

ical

gra

dien

ts

0

2

4

6

8

10

12

14

16

18Arctic ringed seal (N=20)Saimaa ringed seal (N=7)Baikal seal (N=5)

0-3

3-6

6-9

9-12

12-1

515

-18

18-2

121

-24

24-2

727

-30

30-3

333

-36

36-3

939

-42

42-4

545

-48

48-5

151

-54

54-5

7

0

2

4

6

8

10

12

14

16

18Baikal seal >10yrs (N=6)A B

a-s

a-s

a-s;

s-b

a-s

a-s;

a-b

a-b

a-b

a-b;

b-b

a-b;

s-b;

b-b

a-b;

s-b;

b-b

a-b;

b-b

a-b;

b-b

a-b

a-b

a-b a-b

a-b

49

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

the rest of the blubber column (Fig. 9B). Thus, in the old, large-sized Baikal seals, the compositional differences betweenadjacent subsamples were most pronounced in the superficialblubber, but modest in deeper blubber. The superficial blubberwas still compositionally distinguishable from the deep blubbersections, but no border was recognizable between middle anddeeper blubber, which could indicate stable energy balance anddiet composition. The assumption of a stable energy balance issupported by the timing of the sample collection. The blubbersamples of Baikal seal were collected at the end of October,when the phocid seals are accumulating energy in preparationfor the energetic challenges of reproduction (especially lactation

0-3

3-6

6-9

9-12

12-1

515

-18

18-2

121

-24

24-2

727

-30

30-3

333

-36

36-3

939

-42

42-4

545

-48

49-5

1

Sum

of f

atty

aci

d nu

mer

ical

gra

dien

ts

0

2

4

6

8

10

12

14

16

18Baikal seal (N=5)Saimaa ringed seal (N=20)

Distance from the skin (mm)

0-3

3-6

6-9

9-12

12-1

515

-18

18-2

121

-24

24-2

727

-30

30-3

333

-36

36-3

939

-42

42-4

545

-48

49-5

1

0

2

4

6

8

10

12

14

16

18Baikal seal (N=3)Saimaa ringed seal (N=8)

A B

*

* **

*

50

in females) and molting (Ryg et al., 1990a; Lydersen & Kovacs,1999; Mellish et al., 2007).

In pups (< 1-year-old seals) the depth-specific compositionalchanges throughout the blubber were not as pronounced as inthe older seals (Fig. 10A, 10B; II, IV). Similarly, it has beendocumented that in harbor porpoises the degree of fatty acidstratification of the entire blubber is related to the age of theindividual (Koopman et al., 1996). Although the overall fattyacid stratification was less pronounced in pups, largecompositional changes were still detected in the deep blubber ofpups with thin blubber (total blubber thickness < 30 mm; Fig.10A).

3.2.3 Indication of variable 9-desaturase activityThe 9-desaturation index (ratio of C16 and C18 MUFA to thecorresponding SFA) was high in the superficial blubber of allseal species/subspecies (Fig. 11A). Relatively higher proportionsof C16 and C18 MUFA and concomitant lower proportions ofcorresponding SFA in the superficial blubber have also beenreported in previous studies on fatty acid stratification in theblubber of marine mammals (Fredheim et al., 1995; Koopmanet 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 probablyconnected with the low tissue temperatures in the superficialblubber; induced 9-desaturation of SFA in cold environmentcan sustain lipids in their proper semi-fluid state (Tiku et al.,1996; Hsieh & Kuo, 2005). A double bond in cis-configurationweakens short-distance attractions between adjacent acyl chainsand lowers the fatty acid melting point (Cook & McMaster,2002). Thus, 9-desaturation has been considered to be abiochemical mechanism for cold acclimatization (Tiku et al.,1996; Trueman et al., 2000; Hsieh & Kuo, 2005). The high 9-DIvalues in the superficial blubber may indicate elevated 9-desaturase activity (Sjögren et al., 2008), but selectiveincorporation of 9-desaturated fatty acids may also influencethe values. In grey seals active 9-desaturation has been

51

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

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

The stratification pattern of 9-DI suggests that the low tissuetemperature of superficial blubber affects the biochemicalcomposition down to a depth of about 1.5 cm from the skin,corresponding with the vertical profiles of the NG values. Theobserved vertical profile of 9-DI throughout the blubber mayalso reflect changes in lipid metabolism. The 9-desaturaseseems to participate in the regulation of lipid metabolism; inmice, inhibition of 9-desaturase activity was manifested in

Distance from the skin (mm)

0-3

3-6

6-9

9-12

12-1

515

-18

18-2

121

-24

24-2

727

-30

30-3

333

-36

36-3

939

-42

42-4

545

-48

48-5

151

-54

54-5

7

9-de

satu

ratio

n in

dex

0

2

4

6

8

10

12

Arctic ringed seal (N=20)Baikal seal (N=11)Saimaa ringed seal (N=9)

0-3

3-6

6-9

9-12

12-1

515

-18

18-2

121

-24

24-2

727

-30

30-3

333

-36

36-3

939

-42

42-4

545

-48

48-5

151

-54

54-5

7

0

2

4

6

8

10

12

SM/P

C

0.4

0.6

0.8

1.0

1.2

1.49-DI (N=9)SM/PC (N=5)

A B9-Desaturation index Saimaa ringed seal

52

increased metabolic rate and upregulation of the genes involvedin lipid oxidation (for review see Miyazaki & Ntambi, 2003;Flowers & Ntambi, 2008).

It has been suggested that elevated levels of 9-desaturase inmuscle tissue partition fatty acids towards storage rather thanoxidation (Hulver et al., 2005). Additionally, elevated levels of

9-desaturase activity in human adipose tissue have beenlinked with insulin resistance (Sjögren et al., 2008). Although,the function and activity of 9-desaturase in marine mammalshas not been studied in detail, the importance of 9-desaturasein lipid metabolism in terrestrial and aquatic vertebrates (e.g.humans, mice, domestic pigs and fish) encourages investigatingthe role of 9-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).

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

Age (years)

0 5 10 15 20 25 30 35 40

-DI

0

2

4

6

8

10

12

14

16

Lake Saimaa, Male n=17Svalbard, Male n=30Baltic Sea, Male n=5Lake Saimaa, Female n=16Svalbard, Female n=10Baltic Sea, Female n=4

r 2 = 0.77y = 5.2519+0.2892xp < 0.001

53

3.2.4 Stratification of membrane lipidsThe membrane lipids were stratified in the blubber of theSaimaa ringed seal; the superficial blubber contained relativelymore SM and cholesterol than the deep blubber (III). In thesuperficial blubber the mean value of SM/PC ratio was 1.2 (SD0.3), and the ratio declined with increasing blubber depth (III).Interestingly, the vertical profile of the SM/PC ratio resembledthat of the 9-DI (Fig. 11B). A high level of SM and othersphingolipids in the membrane is typically accompanied byhigh proportions of cholesterol, which was also reported in theblubber of the Saimaa ringed seal (III). The current view is thatthe preference for sphingolipids and cholesterol to co-existproduces lateral phase separation in the plasma membrane andhigh sphingolipid-cholesterol clusters are segregated intospecific microdomains, called rafts (Simons & Ikonen, 2000;Örtegren et al., 2004). The rafts in plasma membranes seem toregulate (among other cellular functions) the transport andaccumulation 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 adipocytemembranes has also been connected with impaired function ofthe insulin receptors (Zeghari et al., 2000a, 2000b; Li et al., 2011).Vertical changes in the membrane SM/PC ratio and theconcurrent changes in the amount of cholesterol may indicatemetabolic differences throughout blubber, which may aid insustaining a layer of superficial blubber that is thick enough forinsulation under variable nutritional conditions.

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

54

composition, considering the significant differences in the fattyacids composition of the marine and freshwater food web. Thissuggests that the fatty acid composition of the superficialblubber may be endogenously controlled, and the influence ofdietary fatty acids in this layer is limited.

3.3 BIOCHEMICAL AND FUNCTIONAL LAYERING OFBLUBBERBased on the analysis of the different indices, the blubbers ofArctic and Saimaa ringed seal, and the Baikal seal arebiochemically stratified into different layers (I, II, III, IV). Thestratification is less pronounced in pups than in seals older than1 year. Blubber is a multifunctional tissue, and the differentfunctions (thermoregulation, energy storage, etc.) are very likelyassociated with the depth-specific composition of lipids.Previous biochemical and morphological studies havesuggested functional differences between the blubberimmediately below the skin and the deep blubber next to themuscle (Koopman et al., 2002; Montie et al., 2008). The verticalprofiles of polar membrane lipids, specifically the SM/PC ratio,combined with the similarity of the stratification patterns of theselected fatty acid indices among the seals, support thehypothesis that the superficial blubber may be metabolically lessactive and function mainly as thermoregulatory tissue. Thethickness of the superficial blubber (about 1.5 in the studiedseals) was not affected by the total blubber thickness and mayrepresent the minimum blubber thickness needed for sufficientinsulation in these species. Measurements of the insulative valueand conductivity of the superficial blubber are needed in orderto evaluate further the importance of superficial blubber ininsulation.

The lipid composition in the deep blubber showed largevariation across individual seals, suggesting variable and activefatty acid metabolism. Previous morphological and biochemicalstudies have similarly suggested that the deep blubber ismetabolically the most active layer (Koopman et al., 2002;Montie et al., 2008). Based on the indices reported here, the

55

deepest 1 cm next to the muscle seems to be biochemicallyseparated from the rest of the blubber. Biochemically distinctdeep blubber was detected in all the species, but not in allindividuals. The deep blubber was biochemically mostrecognizable in the pups with thin blubber ( 3cm) and in theArctic ringed seal, while in Baikal seals older than 10 years, thefatty acid composition in middle and deep blubber was morehomogeneous. The differences may be associated with seasonalchanges in energy balance connected with reproductive cycleand molting (Field et al., 2005). Arctic ringed seal samples werecollected at the end of April and at the onset of increased fattyacids mobilization from the blubber to support the increasedenergy demand of reproduction and molting (Ryg et al., 1990a;Beck & Smith, 1995; Thordarson et al., 2007). The Baikal sealblubber samples were collected outside of the breeding season(in October), when phocid seals are not under strain due to theincreased energy demand, and typically have a thick blubberlayer (Ryg et al., 1990a; Beck & Smith, 1995, Thordarson et al.,2007). It is expected that active fatty acid metabolism will befound in pups and thin individuals, and the fact that thecompositional differences were greatest in the deepest blubberof thin individuals suggests depth-specific changes in themetabolic activity of the blubber. In addition to changes inenergy balance, the compositional changes in the deep blubbermay be associated with changes in foraging patterns, as seasonalchanges in diet composition have been documented in ringedseals (Lowry et al., 1980; Ryg et al., 1990b). The deep blubbermay not be biochemically recognizable in seals with a stablenutritional status and small variations in diet composition. Thepelagic food web of Lake Baikal is relatively simple with fewprey species (Yoshii et al., 1999), and the >10-year-old Baikalseals contained large proportions of 18:1n-9, suggesting that thediet or the nutritional status has not changed recently and thatthe predominant prey is most likely golomyanka.

56

3.4 SUGGESTIONS FOR BLUBBER SAMPLING PROCEDUREThe results of this study can be applied directly to thedevelopment of standardized blubber sampling procedures forstudies on foraging strategies and diet in phocid seals. Theresults indicate that superficial blubber reflects the dietary fattyacid composition poorly. The fatty acid composition ofsuperficial blubber in the Saimaa ringed seal resembled that ofthe Arctic ringed seal more than that of any of the potential preyfish (II). The superficial blubber seems to function mainly as athermoregulatory tissue; thus collecting subsamples only fromthe superficial blubber should be avoided in studies on trophicinteractions. Furthermore, blubber from very thin individuals(with little if any thermoneutral middle and deep blubber) maynot provide a representative sample for diet analysis. Thethickness of superficial blubber was about 1.5 cm, even in sealswith thin blubber (in these animals total blubber thickness < 3cm), and thus a significant proportion (50% or more) of theblubber was comprised of tissue with restricted dietaryinformation. In addition to the relatively large proportion ofthermoregulatory blubber in thin individuals, the evaluation oflong term diet from the fatty acid analyses of the entire blubberlayer may be biased by fluctuations in nutritional status andlipid metabolism. The intraspecific variation in the storage lipidcomposition (both TAG species and fatty acids) was highest inthe deep blubber (I, II, III). Individual variation in nutritionalstatus and specific fatty acid intake/mobilization most likelyproduce the observed high compositional variation in thedeepest centimeter of the blubber. This could open up thepossibility of evaluating recent changes in the diet and/orenergy balance. The middle blubber, between the superficialand deep blubber, could provide the most representative sampleof long term diet, since the fatty acid composition does not seemto be influenced by the low tissue temperature or variable fattyacid metabolism.

57

4 Conclusion

The lipid composition of marine mammal blubber has proven tobe an abundant source of ecological information, butendogenous factors, mainly variable lipid metabolism andthermoregulatory adaptations, can affect the composition anddistribution of lipids in the blubber. These factors must beconsidered when evaluating the data from studies employingmarine mammal blubber, e.g. in analysis of foraging patternsand diet composition. It should be emphasized that the depth-specific differences in fatty acid composition of the blubber alsoprovide the opportunity to investigate in more detail theendogenous factors mentioned above (i.e. variable lipidmetabolism and thermoregulatory adaptations). For instance,the insulation capacity of superficial blubber may be related tothe fatty acid and lipid composition, or the nutritional status ofindividuals may be evaluated on the basis of biochemicalvariations in the deep blubber. The biochemical and functionallayering of the blubber also probably affects the distribution andmetabolism of lipophilic compounds, such as lipid solublevitamins and persistent organic pollutants (POP) in the blubber(Debier et al., 2002, 2003). Vitamin A has been employed inhealth assessments of seals, and POP concentrations of blubberare frequently used in environmental biomonitoring (Käkelä etal., 1997; Aguilar et al., 2002; Mos et al., 2007). Previous studieshave documented that lipophilic compounds are nothomogenously distributed throughout the blubber layer andthat 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 compoundsin the blubber would significantly add to our understanding ofthe metabolic fate and deposition patterns of these compoundsin marine mammals and help in reducing methodological datavariation due to different blubber sampling procedures.

58

This study demonstrated that the blubber of small-sized phocidseals is biochemically layered, and that the pattern of layering issimilar among the studied seals irrespective of the differences inthe blubber fatty acid composition. All the studied species wereclosely related, and the fatty acids were generally stronglystratified throughout the blubber. One factor that probably has astrong impact on the stratification pattern of fatty acids inblubber is thermoregulatory needs. The studied species areamong the smallest pinnipeds and also inhabit a coldenvironment. Small sized pinnipeds are susceptible to greaterheat loss because of their larger surface-to-volume ratio, and thismay be one reason for the strong stratification of fatty acids inthe blubber. The blubber of walruses (Odobenus rosmarusrosmarus) was found to be less stratified, most likely because oftheir 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 bydepth-specific and selective metabolism, and if the deep blubberis metabolically the most active layer, the seasonal changes inenergy balance (associated with reproduction and molting)probably induce stratification and layering of fatty acids inblubber.

Diet influences the blubber fatty acid composition, and thusthe interspecific differences in the fatty acid composition of sealblubber were pronounced. Marine food webs are rich in longchain monounsaturated fatty acids (>C18 MUFA), and theabundance of these fatty acids strengthened the fatty acidstratification in the Arctic ringed seal blubber. Although thedegree of fatty acid stratification was less pronounced in thefreshwater seals, the detailed stratification patterns among thestudied seal species were similar. In particular, the fatty acidcomposition of the superficial blubber seemed to be relativelystable, while the fatty acid composition in the deeper blubberlayers is likely to be influenced more by the diet. The thicknessof the superficial blubber was estimated to be about 1.5 cm in allthe studied species, regardless of total blubber thickness.

59

The composition of the membrane lipids (specifically theSM/PC ratio) suggests that metabolic activity may be lowest inthe superficial blubber. If the superficial blubber expresseslower lipolytic activity, it may help to sustain blubber forinsulation during negative energy balance. Biochemically thelayer of postulated high metabolic activity next to the musclewas most recognizable in seals with thin blubber, particularly inpups (possibly due to the switch from nursing to independentfeeding, and/or the post-weaning fast). This suggests that recentchanges in the nutritional status and diet composition could beevaluated on the basis of significant compositional changes inthe deep blubber (deepest centimeter next to the muscle). Themiddle blubber could be categorized as a storage layer thatexpands and contracts according to the nutritional status.Similar functional layering has been documented for bottlenosedolphins; the blubber was morphologically stratified into threelayers, which may have variable lipid dynamics (Montie et al.,2008). The exact depths and degree of layering probably varydepending on the species and environmental conditions, whichwarrants detailed biochemical studies of adipose tissue in avariety of species from different environments.

The novel findings on the variability in membrane lipidcomposition throughout the blubber and the links with depth-specific differences in lipid metabolism should be studiedfurther, as this would provide valuable information on lipidmetabolism in marine mammal blubber. Comprehensiveknowledge on the function and dynamics of the blubber layer isessential not only for physiological and ecophysiological studieson marine mammals, but also for ecological studies utilizing theblubber tissue.

61

5 References

Aguilar A, Borrell A & Reijnders P J H (2002) Geographical andtemporal variation in levels of organochlorine contaminantsin marine mammals. Marine Environmental Research 53: 425-452.

Al-Makdissy N, Younsi M, Pierre S, Ziegler O & Donner M(2003) Sphingomyelin/cholesterol ratio: an importantdeterminant of glucose transport mediated by GLUT-1 in3T3-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 PrinsKarls Forland, Svalbard, assessed via scat and fatty acidanalyses. Canadian Journal of Zoology 82: 1230-1245.

Anonymous (2002) Physical constants of organic compounds. InLide 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 JapaneseConference on the Biochemistry of Lipids.http://lipidbank.jp/.

Bartlett E M & Lewis D H (1970) Spectrophotometricdetermination of phosphate esters in the presence andabsence of orthophosphate. Analytical Biochemistry 36: 159–167.

Bastiani M & Parton R G (2010) Caveolae at a glance. Journal ofCell Science 123: 3831-3836.

Beck G G & Smith T G (1995) Distribution of blubber innorthwest Atlantic harp seal, Phoca groenlandica. CanadianJournal of Zoology 73:1991-1998.

Beck C A, Iverson S J, Bowen W D & Blanchard W (2007) Sexdifferences in grey seal diet reflect seasonal variation inforaging behavior and reproductive expenditure: evidencefrom quantitative fatty acid signature analysis. Journal ofAnimal Ecology 76: 490-502.

62

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 southernelephant seals (Mirounga leonina): implications for dietanalysis. 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 theforaging ecology of southern elephant seals (Miroungaleonina) using blubber fatty acids. Proceedings of the RoyalSociety of London B 270: 1283-1292.

Brockerhoff H (1966) Fatty acid distribution patterns of animaldepot fats. Comparative Biochemistry and Physiology 19: 1-12.

Brockerhoff H, Hoyle R J & Huang P C (1966) Positionaldistribution 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 ofsphingolipid- and cholesterol-rich membrane rafts. TheJournal of Biological Chemistry 275: 17221-17224.

Bryden M M (1978) Arteriovenous anastomoses in the skin ofseals. III The harp seal Pagophilus groenlandicus and thehooded 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 atthe low picomole level by nano-electrospray ionisationtandem mass spectrometry. Proceedings of the NationalAcademy of Sciences of the United States of America 94: 2339–2344.

Budge S M, Cooper M H & Iverson S J (2004) Demonstration ofthe deposition and modification of dietary fatty acids inpinniped blubber using radiolabelled precursors.Physiological and Biochemical Zoology 77: 682-687.

63

Budge S M, Iverson S J & Koopman H N (2006) Studying trophicecology in marine ecosystems using fatty acids: a primer onanalysis 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: Acomparison with morphological analysis, using Arctocephalusseals fed a known diet. Journal of Experimental Marine Biologyand 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 triacylglycerolsynthesis and their regulation. Progress in Lipid Research43:134-176.

Cook H W & McMaster C R (2002) Fatty acid desaturation andchain 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. PolarBiology 32: 1137-1145.

Costa D P & Ortiz C L (1982) Blood chemistry homeostasisduring 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, LeBoulengé E, Larondelle Y & Thomé J-P (2003) Dynamics ofPCB transfer from mother to pup during lactation in UK greyseals Halichoerus grypus: differences in PCB profile betweencompartments of transfer and changes during lactationperiod. Marine Ecology Progress Series 247: 249-256.

64

Dowhan W & Bogdanov M (2002) Functional roles of lipids inmembranes. In Vance D E & Vance J E (Eds) Biochemistry oflipids, lipoproteins and membranes (4th Edn). Elsevier, TheNetherlands, pp. 1-35.

Duffin K L & Henion J D (1991) Electrospray and tandem massspectrometric characterizartion of acylglycerol mixtures thatare dissolved in nonpolar solvents. Analytical Chemistry 63:1781-1788.

Dunkin R C, McLellan W A, Blum J E & Pabst D A (2005) Theontogenetic changes in the thermal properties of blubberfrom Atlantic bottlenose dolphin Tursiops truncatus. Journal ofExperimental 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 speciesof diatom (Bacillariophyceae). Phytochemistry 35: 155-161.

Fahy E, Subramaniam S, Murphy R C, Nishijima M, Raetz C RH, Shimizu T, Spener F, van Meer G, Wakelam M J O &Dennis E A (2009) Update of the LIPID MAPS comprehensiveclassification system for lipids. Journal of Lipid Research 50: S9–S14.

Fayolle C, Leray C, Ohlmann P, Gutbier G, Cazenave J-P, GachetC & Groscolas R (2000) Lipid composition of blood plateletsand erythrocytes of southern elephant seal (Mirounga leonina)and antarctic fur seal (Arctocephalus gazelle). ComparativeBiochemistry 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 differencesin energetic requirements and use of lipids and proteinstores. Physiological and Biochemical Zoology 78: 491-504.

Flowers M T & Ntambi J M (2008) Role of stearoyl-coenzyme Adesaturase in regulating lipid metabolism. Current Opinion inLipidology 19: 248-256.

Folch J M, Lees M & Sloane-Stanley G H (1957) A simplemethod for the isolation and purification of total lipides fromanimal 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

65

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) Procedurefor 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 onfatty acid composition of blubber fat of seals as determinedby biopsy: a multivariate approach. Marine Biology 110: 59-64.

Grahl-Nielsen O, Andersen M, Derocher A E, Lydersen C, WiigO & Kovacs K M (2003) Fatty acid composition of the adiposetissue of polar bears and of their prey: ringed seals, beardedseals 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 acidcomposition of the blubber of the Baikal seal, Phoca sibirica,and its marine relative, the ringed seal, Phoca hispida. MarineEcology 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 reflecttheir 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 LakeBaikal. Aquatic Biology 13: 1-10.

Gray R, Canfield P & Rogers T (2006) Histology of selectedtissues of leopard seal and implications for functionaladaptations 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 lipidomedata. 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

66

porpoise (Phocoena phocoena). Journal of Morphology 261: 105-117.

Han X & Gross R W (2001) Quantitative analysis and molecularspecies fingerprinting of triacylglyceride molecular speciesdirectly from lipid extracts of biological samples byelectrospray ionization tandem mass spectrometry. AnalyticalBiochemistry 295: 88-100.

Han X & Gross R W (2005) Shotgun lipidomics: electrosprayionization mass spectrometric analysis and quantitation ofcellular lipidomes directly from crude extracts of biologicalsamples. Mass Spectrometry Reviews 24: 367-412.

Hart J S & Irving L (1959) The energetics of harbour seals in airand 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 andReproduction 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 humansusing stable isotopes. The Journal of Clinical Investigation 87:1841-1852.

Henderson J R, Kalogeropoulos N & Alexis M N (1994) The lipidcomposition of selected tissues from a Mediterranean monkseal, Monachus monachus. Lipids 29: 577-582.

Henderson J R & Tocher D R (1987) The lipid composition andbiochemistry of freshwater fish. Progress in Lipid Research26:281-347.

Herman D P, Matkin C O, Ylitalo G M, Durban J W, Hanson MB, Dahlheim M E, Straley J M, Wade P R, Tilbury K L, BoyerR H, Pearce R W & Krahn M M (2008) Assessing agedistributions of killer whale Orcinus orca populations fromthe composition of endogenous fatty acids in their outerblubber 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 MM (2009) Age determination of humpback whales Megaptera

67

novaeangliae through blubber fatty acid compositions ofbiopsy 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 andtissues of captive seals: Implications for dietaryreconstruction involving marine mammals. Canadian Journalof Fisheries and Aquatic Sciences 53: 528-533.

Hsieh S L & Kuo C-M (2005) Stearoyl-CoA desaturaseexpression and fatty acid composition in milkfish (Chanoschanos) and grass carp (Ctenopharyngodon idella) during coldacclimation. Comparative Biochemistry and Physiology B 141: 95-101.

Hsu F-F, Bohrer A & Turk J (1998) Electrospray ionizationtandem mass spectrometric analysis of sulfatide.Determination of fragmentation patterns and characterizationof molecular species expressed in brain and in pancreaticislets. Biochimica et Biophysica Acta 1392: 202–216.

Ikonen E & Vainio S (2005) Lipid microdomains and insulinresistance: Is there a connection? Science STKE 268:pe3.

Irving L & Hart J S (1957) The metabolism and insulation ofseals as bare-skinned mammals in cold water. CanadianJournal 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 signaturesreveal fine scale structure of foraging distribution of harborseals and their prey in Prince William Sound, Alaska. MarineEcology Progress Series 151: 255-271.

Iverson S J, Field C, Bowen W D & Blanchard W (2004)Quantitative fatty acid signature analysis: a new method ofestimating predator diets. Ecological Monographs 74: 211-235.

Jakobsson A, Westerberg R & Jacobsson A (2006) Fatty acidelongases in mammals: their regulation and roles inmetabolism. Progress in Lipid Research 45: 237-249.

Jump D B (2001) The biochemistry of n-3 polyunsaturated fattyacids. The Journal of Biological Chemistry 277: 8755-8758.

68

Ju S-J, Kucklick J R, Kozlova T & Harvey H R (1997) Lipidaccumulation and fatty acid composition during maturationof three pelagic fish species in Lake Baikal. Journal of GreatLakes Research 23: 241-253.

Kazuharu T & Reue K (2009) Biochemistry, physiology, andgenetics of GPAT, AGPAT, and lipin enzymes in triglyceridesynthesis. American Journal of Physiology - Endocrinology andMetabolism 296: E1195-1209.

Kleivane L, Severinsen T, Lydersen C, Berg V & Skaare J U(2004) Total blubber burden of organochlorine pollutants inphocid seals: methods and suggested standardization. Scienceof the Total Environment 320: 109-119.

Koopman H N, Iverson S J & Gaskin D E (1996) Stratificationand age-related differences in blubber fatty acids of the maleharbour 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 andmorphology associated with starvation in the harborporpoise (Phocoena phocoena): evidence for regionaldifferences in blubber structure and function. Physiologicaland Biochemical Zoology 75: 498-512.

Koopman H N, Iverson S J & Read A J (2003) High concentrationof isovaleric acid in the fats of odontocetes: variation andpatterns of accumulation in blubber vs. stability in the melon.Journal of Comparative Physiology B 173: 247-261.

Koopman H N (2007) Phylogenetic, ecological, and ontogeneticfactors influencing the biochemical structure of the blubber ofodontocetes. Marine Biology 151: 277-291.

Kotronen A, Seppänen-Laakso T, Westerbacka J, Kiviluoto T,Arola J, Ruskeepää A-L, Yki-Järvinen H & Oreši M (2010)Comparison of lipid and fatty acid composition of the liver,subcutaneous and intra-abdominal adipose tissue, andserum. Obesity 18: 937-944.

Kozlova T A & Khotimchenko S V (2000) Lipids and fatty acidsof two pelagic cottoid fishes (Comephorus spp.) endemic to

69

Lake Baikal. Comparative Biochemistry and Physiology B 126:477-485.

Kunnasranta M, Hyvärinen H, Sipilä T & Koskela, J T (1999) Thediet of the Saimaa ringed seal Phoca hispida saimensis. ActaTheriologica 44: 443–450.

Käkelä R, Hyvärinen H & Vainiotalo P (1993) Fatty acidcomposition 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) fromthe Baltic. Comparative Biochemistry and Physiology B 105: 553-565.

Käkelä R, Ackman R G & Hyvärinen H (1995) Very long-chainpolyunsaturated fatty acids in the blubber of ringed seals(Phoca hispida sp) from Lake Saimaa, Lake Ladoga, the Balticsea and Spitsbergen. Lipids 30: 725-731.

Käkelä R & Hyvärinen H (1996) Site-specific fatty acidcomposition in adipose tissue of several northern aquatic andterrestrial mammals. Comparative Biochemistry and PhysiologyB 115: 501-514.

Käkelä R, Hyvärinen H & Käkelä A (1997) Vitamins A1 (retinol),A2 (3,4-didehydroretinol) and E ( -tocopherol) in the liverand blubber of lacustrine and marine ringed seals (Phocahispida sp). Comparative Biochemistry and Physiology B 116: 27-33.

Lee R F, Hagen W & Kattner G (2006) Lipid storage in marinezooplankton. 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 membranesphingomyelin increases insulin sensitivity. Molecular andCellular Biology 31: 4205-4218.

Litchfield C, Greenberg A J, Caldwell D K, Caldwell M C, SiposJ C & Ackman R G (1975) Comparative lipid patterns inacoustical and non-acoustical fatty tissues of dolphins,porpoises and toothed whales. Comparative Biochemistry andPhysiology B 50: 591-597.

70

Lowry L L, Frost K J & Burns J J (1980) Variability in the diet ofringed seals, Phoca hispida, in Alaska. Canadian Journal ofFisheries and Aquatic Sciences 37: 2254-2261.

Lydersen C & Gjertz I (1987) Population parameters of ringedseals (Phoca hispida Schreber, 1775) in the Svalbard area.Canadian Journal of Zoology 65: 1021-1027.

Lydersen C & Kovacs K M (1999) Behaviour and energetics ofice-breeding, North Atlantic phocid seals during the lactationperiod. 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 monitoringpollution burdens in phocid seals. Science of the TotalEnvironment 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 wildbottlenose dolphins (Tursiops truncatus). Journal of ComparativePhysiology B 178: 529-543.

Mellish J-M, Horning M & York A E (2007) Seasonal and spatialblubber depth changes in captive harbor seals (Phoca vitulina)and Steller’s sea lion (Eumetopias jubatus). Journal ofMammalogy 88: 408-414.

Miyazaki M & Ntambi J M (2003) Role of stearoyl-coenzyme Adesaturase in lipid metabolism. Prostaglandins, Leukotrienesand Essential Fatty Acids 68: 113-121.

Molyneux G S & Bryden M M (1975) Arteriovenousanastomoses in the skin of the Weddell seal, Leptonychotesweddelli. 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 (Tursiopstruncatus) from the Southeastern United States: influence ofgeographic location, age class, and reproductive state. Journalof Morphology 269: 496-511.

Morris R J (1984) The endemic faunae of Lake Baikal: Theirgeneral biochemistry and detailed lipid composition.Proceedings of the Royal Society of London B 222: 51-78.

71

Mos L, Tabuchi M, Dangerfield N, Jeffries S J, Koop B F & RossP S (2007) Contaminant-associated disruption of vitamin Aand its receptor (retinoic acid receptor ) in free-rangingharbour seals (Phoca vitulina). Aquatic Toxicology 81: 319-328.

Mu H & Høy C-E (2004) The digestion of dietarytriacylglycerols. Progress in Lipid Research 43: 105-133.

Muje P, Ågren J J, Lindqvist O V & Hänninen O (1989) Fattyacid composition of vendace (Coregonus albula L.) muscle andits plankton feed. Comparative Biochemistry and Physiology B92: 75-79.

Nakamura M T & Nara T Y (2004) Structure, function, anddietary 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 chemicalmarkers in freshwater ecosystems. In Arts M T & Wainman BC (Eds) Lipids in freshwater ecosystems. Springer, New York,pp. 21- 44.

Nelson G J (1970) The lipid composition of the blood of marinemammals – I. Young elephant seals, Mirounga angustirostisand harp seals, Pagophilus groenlandicus. ComparativeBiochemistry 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 resourcepartitioning between adult and juvenile southern elephantseals. 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 (Miroungaangustirostris) pups during the postweaning fast: size doesmatter. 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 nearlyweaned pup, yearling, and adult Weddell seals (Leptonychotesweddelli). Physiological and Biochemical Zoology 81: 868-879.

Ntambi J M & Miyazaki M (2004) Regulation of stearoyl-CoAdesaturases and role in metabolism. Progress in Lipid Research43: 91-104.

72

Olsen E & Grahl-Nielsen O (2003) Blubber fatty acids of minkewhale: stratification, population identification and relation todiet. Marine Biology 142: 13-24.

Palo J U & Väinölä R (2006) The enigma of the landlocked Baikaland Caspian seals addressed through phylogeny of phocinemitochondrial sequences. Biological Journal of the LinneanSociety 88: 61-72.

Parrish C C, Myher J J, Kuksis A & Angel A (1997) Lipidstructure of rat adipocyte plasma membranes followingdietary lard and fish oil. Biochimica et Biophysica Acta 1323:253-262.

Parry D A (1949) The structure of whale blubber, and discussionof its thermal properties. Quarterly Journal of MicroscopicalScience 90: 13-25.

Perona J S & Ruiz-Gutiérrez V (1999) Characterization of thetriacylglycerol molecular species of fish oil by reversed-phasehigh performance liquid chromatography. Journal of LiquidChromatography 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 intriacylglycerol deposition in rat adipose tissue. British Journalof Nutrition 84: 765-774.

Pierce G J & Boyle P R (1991) A review of methods for dietanalysis in piscivorous marine mammals. Oceanography andMarine 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: exploringadipocyte 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 thedistribution and composition of adipose tissue in wild arcticfoxes (Alopes lagopus) on Svalbard. Journal of Zoology 236: 593-610.

Pond C M, Mattacks C A, Colby R H & Ramsay M A (1992) Theanatomy, chemical composition, and metabolism of adipose

73

tissue in wild polar bears (Ursus maritimus). Canadian Journalof Zoology 70: 326-341.

Quinn G P & Keough M J (2002) Experimental Design and DataAnalysis. Cambridge University Press, United Kingdom.

Raclot T & Groscolas R (1995) Selective mobilization of adiposetissue fatty acids during energy depletion in the rat. Journal ofLipid Research 36:2164-2173

Raclot T (2003) Selective mobilization of fatty acids from adiposetissue triacylglycerols. Progress in Lipid Research 42: 257-288.

Ramsay M A, Mattacks C A & Pond C M (1992) Seasonal andsex differences in the structure and chemical composition ofadipose tissue in wild polar bears (Ursus maritimus). Journal ofZoology 228: 533-544.

Reddy J N & Hashimoto T (2001) Peroxisomal -oxidation andperoxisome proliferator-activated receptor : an adaptivemetabolic system. Annual Review of Nutrition 21:193-230.

Rice D W (1998) Marine Mammals of the World. Systematics andDistribution. Society for Marine Mammalogy SpecialPublication 4: 1-231.

Rosen D A S & Renouf D (1997) Seasonal changes in blubberdistribution in Atlantic harbour seals: indications ofthermodynamic considerations. Marine Mammal Science 13:229-240.

Ryg M, Smith T G & Øritsland N A (1988) Thermal significanceof the topographical distribution of blubber in ringed seals(Phoca hispida). Canadian Journal of Fisheries and AquaticSciences 45: 985-992.

Ryg M, Smith T G & Øritsland N A (1990a) Estimating theblubber content of phocid seals. Canadian Journal of Fisheriesand Aquatic Sciences 47: 1223-1227.

Ryg M, Smith T G & Øritsland N A (1990b). Seasonal changes inbody mass and body composition of ringed seals (Phocahispida) on Svalbard. Canadian Journal of Zoology 68: 470-475.

Sargent J R, Gatten R R & Henderson R J (1981) Marine waxesters. Pure and Applied Chemistry 53: 867-871.

Schey H M (2005) Div, grad, curl and all that: an informal text onvector calculus (4th edn). W W Norton, New York.

74

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 MarineMammal Commission (NAMMCO) Scientific Publications,No 1. pp. 83-99.

Sjögren P, Sierra-Johnson J, Gertow K, Rosell M, Vessby B, deFaire U, Hamsten A, Hellenius M-L & Fisher R M (2008) Fattyacid desaturases in human adipose tissue: relationshipbetween gene expression, desaturation indexes and insulinresistance. Diabetologia 51: 328-335.

Skoglund E G, Lydersen C, Grahl-Nielsen O, Haug T & KovacsK M (2010) Fatty acid composition of the blubber and dermisof adult male Atlantic walruses (Odobenus rosmarusrosmarus) in Svalbard, and their potential prey. MarineBiology 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 ofmale southern elephant seals, Mirounga leonina, duringmoulting fast. Australian Journal of Zoology 40: 235-243.

Slotte J P (1999) Sphingomyelin–cholesterol interactions inbiological and model membranes. Chemistry and Physics ofLipids 102: 13-27.

Smith R J, Hobson K A, Koopman H N & Lavigne D M (1996)Distinguishing between populations of fresh- and salt-waterharbour seals (Phoca vitulina) using stable-isotope ratios andfatty acid profiles. Canadian Journal of Fisheries and AquaticSciences 53: 272-279.

Sprecher H, Luthria D L, Mohammed B S & Baykousheva S P(1995) Reevaluation of the pathways for the biosynthesis ofpolyunsaturated fatty acids. Journal of Lipid Research 36: 2471-2477.

Struntz D J, McLellan W A, Dillaman R M, Blum J E, Kucklick JR & Pabst D A (2004) Blubber development in bottlenosedolphins (Tursiops truncatus). Journal of Morphology 259: 7-20.

75

Suzuki S, Ishikawa S, Arihara K & Itoh M (2008) Molecularspecies-specific differences in the composition oftriacylglycerols of mouse adipose tissue and diet. NutritionResearch 28: 258-262.

Thiemann G W, Iverson S J & Stirling I (2007) Variability in theblubber 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 inblubber fatty acid composition among marine mammals inthe Canadian Arctic. Marine Mammal Science 24: 91-111.

Thomas J, Pastukhov V, Elsner R & Petrov E (1982) Phocasibirica. Mammalian Species 188:1-6.

Thompson Jr G A (1996) Lipids and membrane function in greenalgae. Biochimica et Biophysica Acta 1302:17-45.

Thordarson G, Vikingsson G A & Hersteinsson P (2007)Seasonal variation in body condition of adult male hoodedseals (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 carpby transcriptional and posttranslational mechanisms. Science271: 815-818.

Trueman R J, Tiku P E, Caddick M X & Cossins A R. (2000)Thermal thresholds of lipid restructuring and 9 –desaturaseexpression in the liver of carp (Cyprinus carpio L.). Journal ofExperimental Biology 203: 641-650.

Tucker S, Bowen W D, Iverson S J & Stenson G B (2009) Intrinsicand extrinsic sources of variation in the diets of harp andhooded seals revealed by fatty acid profiles. Canadian Journalof Zoology 87: 139-151.

Vainio S, Bykov I, Hermansson M, Jokitalo E, Somerharju P &Ikonen E (2005) Defective insulin receptor activation andaltered lipid rafts in Niemann-Pick type C diseasehepatocytes. 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

76

(2001) Synthesis and function of 3-phosphorylated inositollipids. Annual Review of Biochemistry 70: 535-602.

van Meer G, Voelker D R & Feigenson G W (2008) Membranelipids: where they are and how they behave. Nature ReviewsMolecular Cell Biology 9: 112–124.

Ventura M (2006) Linking biochemical and elementalcomposition in freshwater and marine crustaceanzooplankton. Marine Ecology Progress Series 327:233-246.

Walton M & Pomeroy P (2003) Use of blubber fatty acid profilesto detect inter-annual variation in the diet of grey sealsHalichoerus 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 fromblubber biopsies of sperm whales Physeter macrocephaluslocated around the Azores. Journal of the Marine BiologicalAssociation 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 verticalstratification of blubber fatty acids alters diet predictions forlactating Weddell seals. Journal of Experimental Marine Biologyand Ecology 352: 103-113.

Wheatley K E, Nichols P D, Hindell M A, Harcourt R G &Bradshaw C J T (2008) Differential mobilization of blubberfatty acids in lactating Weddell seals: Evidence for selectiveuse. Physiological and Biochemical Zoology 81: 651-662.

Weslawski J M, Ryg M, Smith T G & Øritsland N A (1994) Dietof ringed seals (Phoca hispida) in a fjord of west Svalbard.Arctic 47: 109-114.

Worgall T S (2007) Sphingolipids: major regulators of lipidmetabolism. Current Opinion in Clinical Nutrition and MetabolicCare10: 149-155.

Yoshii K, Melnik N G, Timoshkin O A, Bondarenko N A,Anoshko P N, Yoshioka T & Wada E (1999) Stable isotopeanalyses of the pelagic food web in Lake Baikal. Limnologyand Oceanography 44: 502-511.

Zeghari N, Vidal H, Younsi M, Ziegler O, Drouin P & Donner M(2000a) Adipocyte membrane phospholipid and PPAR-

77

expression in obese women: relationship tohyperinsulinemia. American Journal of Physiology -Endocrinology and Metabolism 279: E736-E743.

Zeghari N, Younsi M, Meyer L, Donner M, Drouin P & ZieglerO (2000b) Adipocyte and erythrocyte plasma membranephospholipid composition and hyperinsulinemia: a study innondiabetic and diabetic obese women. International Journal ofObesity 24: 1600-1607.

Örtegren U, Karlsson M, Blazic N, Blomqvist M, Nystrom F H,Gustavsson J, Fredman P & Strålfors P (2004) Lipids andglycosphingolipids in caveolae and surrounding plasmamembrane of primary rat adipocytes. European Journal ofBiochemistry 271: 2028-2036.

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

Publications of the University of Eastern Finland

Dissertations in Forestry and Natural Sciences

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 stratificationpatterns and verticallayering of lipidsin seal blubber– source of ecophysiological information

Fatty acids are among the most ana-

lyzed components in marine mammal

blubber, and increasingly employed

in ecological and physiological stud-

ies. However, the interpretation of

the results from these analyses is not

straightforward, as the fatty acids

and other lipids are stratified in the

blubber. This thesis documents the

detailed stratification patterns of

lipids in blubber, discusses the physi-

ological and ecological implications

of the biochemical stratification of

blubber and provides a suggestion for

standardized sampling procedure.

disser

tation

s | No

65 | Ur

sula S

tra

nd

berg

| Deta

iled stra

tifica

tion

pa

tterns a

nd

vertical la

yering of lip

ids in

seal b

lub

ber

Ursula StrandbergDetailed stratificationpatterns and vertical

layering of lipids in seal blubber

– source of ecophysiological

information