`
`LIPID COMPOSITION OF TWO SPECIES OF ANTARCTIC
`KRILL: EUPHA USIA SUPERBA AND E. CR YSTALLOROPHIAS
`
`Department of Biochemisz and Biophysies, Texas A & M University, College Station, Texas 77843,
`U.S.A.
`
`NESTOR R. BornNo
`
`(Received 21 February 1974)
`
`Abstract—l. The lipids of two Antarctic euphausiids were characterized.
`2. In Euphausia superba complex lipids were the major lipid class followed by triglycerides.
`3. In E. crystallorophias the complex lipids were also the major lipid class, but the second major
`constituent was waxes.
`4. The complex lipids of both euphausiids consisted mostly of phosphatidylcholine with smaller
`amounts of phosphatidylethanolamine and lysophosphatidylcholine. The phospholipids of E.
`crystallorophias were less unsaturated than those of E. superba.
`5. The waxes of E. crystallorophias were mostly esters of oleic (84 %) and palmitolcic (10 ”/o) acids
`with n—tetradecanol (69 7.) and n-hexadecanol (28 %).
`
`INTRODUCTION
`
`THE WORD “krill" in Norwegian means “whale
`food”, and it is with this meaning that the term is
`generally used,
`involving the various crustaceans
`(mostly euphausiids, but also amphipods and
`decapods) which whales eat
`(Nemoto,
`1970).
`However, when one refers to Antarctic krill, one
`generally means Euphausia superba, which is the
`most abundant and far better known Species of krill
`in the Antarctic Oceans. E. superba is usually
`found in open, turbulent waters at the confluences
`of oceanic streams (Ivanov, 1970). But E. superba is
`rarely found in the colder waters in close proximity
`to the ice where the smaller E. crystalorophias seems
`to predominate (Knox, 1970). Both E. superba
`and E.
`crystallorophias are considered phyto-
`plankton fwders (Mauchline & Fisher, 1969 for
`E. superba; Andriashev, 1968 and Knox, 1970 for
`E. crystallorophias). Both euphausiids are consumed
`by whales,
`seals,
`fish, penguins and petrels
`(Andriashev, 1968; Knox, 1970) thus occupying a
`central position in various food chains in open
`waters and in waters close to or under the ice.
`
`During cruise 51 of the USNS Eltanin to the Ross
`Sea, January—February 1972, I was able to collect
`specimens of E. superba in stations 8, 9 and 11
`(Fig.
`l) and specimens of E. crystallorophias in
`practically all of the stations located along the Ross
`Ice Shelf (stations 11—18). Since the lipids of E.
`superba have been studied only superficially and
`those of E. crystallorophias have not been studied
`at all, I decided to scrutinize them in more detail.
`The results are the subject of the prescnt report. A
`preliminary presentation of these results has been
`made (Bottino, 1973).
`
`
`180°
`
`
`Lytltelton
`Depart Lytnlten
`January 17 1972
`
`
`
`
`
`iii- .55 5.
`4,
`.13]
`RossSea
`
`station
`Ross
`ice shelf
`
`
`
`
`MATERIALS AND METHODS
`
`Euphausiids were collected with a l m mid-water
`trawl atdepthsfromOto 300m. Onceonboardthes‘hip
`the samples were rapidly sorted by hand and extracted
`with the chloroform-menthanol (2:1, v/v) mixture of
`Petition for Inter Partes Review
`479
`Of US. Patent 8,278,351
`Exhibit
`
`000001
`
`ENZYMOTEC - 1007
`
`
`
`480
`
`Nae-ma R. Borrmo
`
`Folch et a1. (1957). Portions of 5-10 mg lipids were
`transesterified with methanol in the presence of boron
`trifluoride (American Oil Chemists’ Society, 1970).
`The fatty acid methyl esters thus formed were separated
`from wax alcohols by thin-layer chromatography (TLC)
`on silica gel without binder (Adsorbosil-S, Applied
`Science Co., State College, Pa.) using a mixture of
`benzene—ethyl acetate (95 : 5, v/v) as developing solvent.
`Fatty acid compositions were determined by gas-liquid
`chromatography on a 6 ftxfi in. column of siliconized
`polyethyleneglycol succinate (DGSS-X, Applied Science)
`10% (w/w) on Gas Chrom P 100—120 mesh (Johns-
`Manville, Denver, Colo.) at 170°C. A dual flame Model
`605 Beckman gas chromatograph (Beckman Co.,
`Fullerton, Calif.) was used connected to an Infotronic
`digital integrator (Columbia Scientific Industries, Austin,
`Texas). Final results were calculated on a Hewlett-
`Packard Desk Programmable Calculator Model 7810A.
`Results were expressed as weight per cent. Fatty acids
`were identified by co-chromatography with known
`standards and by plotting relative retention times vs.
`chain length before and after hydrogenation. For
`quantitative fractionation of lipid classes 20—40mg of
`lipids were separated by the method of Freeman &
`West (1966) modified as follows: (a) Silica gel without
`binder (Adsorbosil-S, Applied Science) was used instead
`of silica gel-G.
`(b) Acetic acid was eliminated from the
`solvent mixture No.
`1
`to simplify the drying between
`
`Table 1. Fatty acids of Antarctic krill'
`
`E. superba
`
`E. crystallorophias
`
`Fatty acid
`
`Station 8 Station 11 Station 13 Station 16
`
`12:0
`14:0
`15:0 brT
`15:0
`16:0
`18:0
`22:0
`
`14:1 (n— ?)
`15:1 (n— ?)
`16:1 (n — 7)
`17:1 (rt—8)
`18:1(n—9)
`20:1 (n — 9)
`18:2 (n — 6)
`18:2 (n—3)
`20:2 (n — 3)
`18:3 (n— 6)
`18:3 (n—3)
`20:3 (n— 3)
`18:4 (n—3)
`20:4 (n— 6)
`20:4 (n —— 3)
`22:4 (n — 6)
`20:5 (II—3)
`22:5 (n— 3)
`22:6 01— 3)
`
`0-3
`14-9
`0-3
`05
`21-2
`0-7
`0-1
`
`0-3
`tr
`9-0
`0-7
`18-2
`0-6
`0-3
`2-6
`
`0-3
`1-1
`0-6
`2-2
`0-5
`0-5
`0-2
`16-0
`0-3
`8-6
`
`weight "/0
`0-2
`2-3
`01
`0-2
`13-8
`1-2
`
`8-4
`0-4
`47-5
`0-2
`
`3-3
`0-1
`0-2
`0-9
`0-5
`1-5
`
`11-8
`0-1
`7-3
`
`0-2
`14-3
`0-2
`0-2
`24-7
`1-4
`0-1
`
`0-2
`01
`8-9
`0-3
`21-7
`0-9
`0-1
`2-0
`
`0-2
`1-0
`0-5
`3-3
`0-4
`0-2
`0-3
`11-4
`0-1
`7-3
`
`tr
`2-4
`
`0-1
`14-
`1-3
`
`10-8
`0-4
`45-2
`0-5
`
`2-7
`
`0-3
`09
`
`0-9
`0-7
`0-1
`
`13-4
`
`5-5
`
`"‘ Data from Bottino (1974).
`1' br, Branched-chain fatty acids.
`
`(c) Quantitation of the spots was done
`developments.
`by gravimetry and not by colorimetry of dichromate
`reduction. The gravimetric estimation required the use of
`the following procedure: Once separated by TLC and
`located with dichlorofluorescein spray, the spots were
`scraped all the plates, extracted six times with 4 ml (each)
`of a mixture of chloroform—methanol—acetic acid-water
`(50 : 39 : 1 : 10, v/v) (Arvidson, 1968). The extracts were
`filtered through fine pore sintered glass funnels into
`test-tubes, then 8 ml of 4 M ammonium hydroxide were
`added and the mixture shaken and centrifuged. The
`resulting upper phase, which contained the DCF, was
`diswrded and to the lower phase was added 8ml of
`50% (v/v) methanol
`in water. After the addition of
`anhydrous sodium sulfate, the liquid was filtered through
`a fine pore sintered glass funnel containing a layer of
`anhydrous sodium sulfate. The filtrate was then evapor-
`ated under vacuum and the residue was weighed. Using
`known amounts of total lipids in each determination, it
`was possible to estimate the recovery. Any result showing
`less than 80 per cent recovery was discarded and the
`fractionation repeated. The fatty acids of the lipid classes
`were converted into their methyl esters and studied by
`GLC as indicated above. In the case of the wax fraction
`the methylation procedure using boron trifluoride
`catalyst was found inefficient and was replaced by a
`1-hr reflux with a 2% solution of sulfuric acid in
`methanol. The alcohol components of the waxes were
`studied by GLC without any previous treatment. The
`major component of the alcohol mixture was also
`isolated by preparative GLC and examined with an
`LKB-9000 mass spectrometer. The spectrum was iden-
`tical to that of authentic l-n-tetradecanol. Other alcohols
`were identified by their GLC behavior in comparison
`with known standards and by plotting relative retention
`times vs. chain length.
`
`RESULTS AND DISCUSSION
`
`Table 1 shows the fatty acid compositions of the
`unfractionated lipids of E. superba and E. crystal-
`lorophias. The fatty acid patterns of the samples of
`E. superba were quite similar to each other and to
`that previously reported for another sample of the
`same species.
`In fact, differences among samples
`were only slightly higher than those found between
`duplicate analyses of the same sample (Bottino,
`1974). Similarly,
`the fatty acid patterns of six
`samples of E. crystallorophias difiered very little
`from each other (Bottino, 1974). Table 1 shows one
`example of this. However,
`there are noticeable
`quantitative differences between the fatty acid
`compositions of the two species. For example, the
`level of oleic acid is about twice as high in E.
`crystallorophias as in E. superba, while the reverse
`is true for the content of the saturated acids.
`
`Previous studies from this laboratory (Bottino,
`unpublished) have demonstrated that complex lipids
`constitute 50 per cent or more of the lipids of
`most Antarctic plants and animals other
`than
`mammals. The data in Table 2 show that complex
`lipids are also the major lipid class in the two
`euphausiids of the present study, comprising between
`42 and 58 per cent of the total. In both organisms,
`
`000002
`
`
`
`Lipids of Antarctic krill
`
`481
`
`Table 2. The lipids of Antarctic krill
`
`E. superba
`
`E. crystalloraphias
`
`Station 8 (1)*
`
`Station 11 (2)
`
`Station 13 (4)
`
`Station 16 (2)
`
`Waxes
`Steroid esters
`Triglycerides
`Diglycerides
`Complex lipids
`PCT
`PET
`Lyso PC
`PGT
`Unknown§
`
`—
`——
`8
`17
`54
`
`21
`
`—
`—
`36 i 6
`4 :L- 5
`58 i 14
`48
`8
`1
`1
`2¢22
`
`weight %
`44 i 101
`2 i 3
`—
`——
`53 i 8
`46
`6
`1
`
`-—
`1:2
`
`20 i 1
`27 i 9
`—
`4 i 1
`42 i 8
`
`7:1
`
`" Number of determinations in parentheses.
`1' PC, Phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglyoerol.
`it Weight per cent plus or minus the standard deviation.
`§ R, between those of triglycerides and diglyeerides. The recovered amount of this fraction was too
`small for further characterization.
`
`Table 3. Composition of the waxes of E. crystallorophias
`
`Fatty acids
`
`Wax alcohols
`
`Station 13
`
`Station 16
`
`Station 13
`
`Station 16
`
`Waxes
`
`Waxes
`
`Steroid esters
`
`tr
`72-5
`
`25-3
`22‘
`
`01
`tr
`69-2
`
`28-0
`22“
`0-5
`
`tr
`
`tr
`
`0-1
`tr
`tr
`05
`
`01
`0-6
`0-2
`
`1-3
`11-2
`0-3
`83-1
`0-4
`
`2-2
`
`0-3
`0-1
`
`06
`
`08
`03
`
`0-6
`
`0-1
`8-8
`0-3
`84-
`0-6
`
`3-1
`
`0-4
`
`8:0
`10:0
`12:0
`13:0
`14:0
`15:0 br
`15:0
`16:0
`18:0
`20:0
`22:0
`
`11:1 (n— ‘3)
`12:1 01- ?)
`13:1 (n— '1)
`14:1 (n— ?)
`15:1 (n— ‘2)
`16:1 (ll—7)
`17:1 (fl—8)
`18:1 (n-9)
`20:1 ("-9)
`
`18:2 (n—3)
`
`18:3 (n— 3)
`20:3 (71—3)
`
`18:4 (n— 3)
`
`20:5 (ll-3)
`
`22:6 (71-3)
`
`weight "/0
`14
`0-6
`0-4
`0-1
`8-3
`
`0-3
`14-0
`2-4
`
`01
`0-3
`0-8
`03
`11-0
`14-0
`11
`22-1
`
`19
`
`09
`0-3
`
`1-5
`
`16-5
`
`1-4
`
`Unknown
`03
`
`
`‘ 18:0+17:1.
`
`000003
`
`
`
`482
`
`NESTOR R. Bonn-:0
`
`Table 4. Fatty acids of the complex lipids of Antarctic krill
`
`E. superba——Station 11
`
`E. crystallorophias—Station 13
`
`CL’“
`PC
`PE
`Lyso PC
`CL
`PC
`PE
`Lyso PC
`
`
`9:0
`10:0
`11:0
`12:0
`13:0
`14:0
`15:0
`16:0 br
`16:0
`17:0
`18:0
`20:0
`22:0
`
`tr
`
`tr
`30
`07
`
`0-1
`0-3
`01
`tr
`0-1
`3-4
`1-1
`
`0-2
`0-5
`0-2
`0-2
`0-2
`2-6
`1-5
`
`25-1
`
`25-9
`
`18-8
`
`1-2
`
`0-3
`
`0-1
`
`tr
`
`1-1
`
`0-2
`
`0-1
`
`tr
`
`2-8
`0-1
`13-
`0-9
`
`1-7
`0-2
`
`0-1
`
`0]
`tr
`0-1
`1-3
`
`21-8
`0-3
`
`1-8
`
`1-3
`4-2
`8-3
`5-9
`1-6
`32-6
`1-4
`4-1
`
`0-1
`
`2-3
`
`2-8
`1-6
`
`4-2
`tr
`12-3
`0-2
`
`Weight %
`0-1
`
`tr
`2-9
`0-9
`
`tr
`tr
`
`tr
`.4-2
`1-0
`
`29-1
`
`29-8
`
`1-0
`
`tr
`
`0-1
`
`5-3
`0-2
`21-2
`
`1-0
`0-4
`0-1
`
`6-9
`0-2
`21-0
`0-1
`
`06
`27
`0-6
`1-7
`6-2
`11-1
`5-2
`
`37-8
`
`1-9
`1-8
`2-0
`
`3-9
`2-2
`2-5
`tr
`
`2-4
`
`0-1
`1-4
`0-1
`1-4
`1-6
`4-6
`1-2
`
`26-2
`tr
`2-9
`
`0-2
`
`2-0
`
`31-0
`
`11:1(n— ?)
`12:1 (n— ?)
`13:1 (n— ?)
`14:1 (n— ?)
`15:1 (n— ?)
`16:1 (n— 7)
`17:1 (n— 8)
`18:1 (n—9)
`20:1 (ll—9)
`22:1 (n—9)
`
`18:2 ("—3)
`21 :2 (n— 6)
`
`18:3 (n— 6)
`18:3 (n— 3)
`20:3 01— 6)
`20:3 (n— 3)
`21:3 (n—3)
`22:3 (n— 3)
`
`18:4 (rt—3)
`20:4 (rt—6)
`20:4 (n — 3)
`
`20:5 01— 3)
`22:5 01— 3)
`22:6 (n— 3)
`
`2-9
`0-1
`14-2
`0-8
`
`2-3
`
`0-2
`1-6
`0-1
`1-0
`tr
`0-5
`
`3-8
`1-5
`
`25-3
`0-1
`15-2
`
`2-4
`
`1-4
`
`0-4
`1-8
`0-1
`0-7
`3-8
`0-5
`
`4-7
`1-5
`0-4
`
`22-5
`
`11-8
`
`0-8
`01
`1-2
`4-5
`0-1
`
`0-6
`0-2
`0-2
`
`20-3
`
`20-8
`
`1-1
`01
`
`0-2
`
`tr
`1-6
`
`1-2
`1-9
`0-2
`
`6-5
`
`2-5
`
`2-1
`
`0-1
`1-2
`
`1-4
`1-1
`3-6
`
`1-7
`0-1
`
`2-5
`
`1-3
`
`0-3
`
`0-3
`
`1-5
`
`0-3
`8-4
`
`2-3
`0-3
`
`19-3
`
`14-8
`
`8-6
`
`5-2
`
`15-1
`
`10-3
`
`13-0
`tr
`0-5
`
`0-7
`
`0-4
`
`tr
`0-2
`02
`
`2-4
`
`tr
`
`71-6
`39-5
`36-5
`34-0
`61-3
`26-1
`32-3
`30-3
`Saturated
`24-5
`33-2
`28-2
`26-8
`23-4
`23-7
`17-1
`18-1
`Monounsaturated
`Polyunsaturated
`51-6
`50-6
`50-2
`15-3
`39-2
`35-3
`27-3
`3-9
`
`
`‘ CL, Complex lipids.
`PC, phosphatidylcholine; PE, phosphatidylethanolamine.
`
`the major complex lipid was phosphatidylcholine
`(PC), 49 per cent in E. superba and 46 per cent in
`E. crystallorophias. Phosphatidylethanolamine (PE)
`and lysophosphatidylcholine were also found in
`both euphausiids but in much lower concentrations.
`Unexpected large amounts of waxes were found in
`E. crystallorophias while none was found in E.
`superba.
`In the specimens of E. crystallorophias
`from station 16, slightly more than half of the waxes
`
`were steroid esters! The waxes of each sample of '
`E. crystallorophias were isolated by TLC and the
`fatty acids and alcohols were examined indepen-
`dently by GLC. Both samples had very similar
`compositions, as shown by the data in Table 3. Two
`
`* For the sake of simplicity in the following discussion
`I shall call waxes the esters of long—chain aliphatic
`alcohols only.
`
`000004
`
`
`
`Lipids of Antarctic krill
`
`483
`
`con-
`acids, oleic and palmitoleic,
`monoenoic
`stituted about 94 per cent of the fatty acids of these
`waxes. 0f the four alcohol components, about 70
`per cent was tetradecanol and 28 per cent was hexa-
`decanol. Thus the waxes of E. crystallarophias are
`less unsaturated than the copepod waxes examined
`by lee et al. (1971a, b) and by Benson er a1. (1972).
`The waxes of the present study resemble many other
`waxes of marine animals in their high content of
`oleic acid (Nevenzel, 1970) but differ from the rest in
`having tetradecanol as their major alcohol com-
`ponent.
`In most other marine waxes so far in-
`vestigated, hexadecanol is the predominant alcohol.
`The fatty acids of the steroid esters of E. crystal-
`larophias (Table 3) contain polyunsaturated fatty
`acids while those of the aliphatic waxes do not.
`Table 4 shows the fatty acids of the major phos-
`pholipids of E. superba and E. crystallorophias.
`About half of the fatty acids in E. superba PC and
`PE were polyunsaturated, mostly 20:5 and 22:6.
`Only about one-third of the fatty acids of the PC
`and PB of E. crystallorophias were polyunsaturated.
`Again, 20:5 and 22:6 were the predominant poly-
`unsaturated acids.
`
`In both euphausiids the major lipid class was
`complex lipids (about 20 per cent of the total)
`consisting mostly of phosphatidylcholine with
`smaller amounts of phosphatidylethanolamine and
`lysophosphatidylcholine. The two species difi‘er,
`however, in their neutral lipids, those of E. superba
`being triglycerides and diglyceridss,
`those of E.
`crystallorophias being waxes,
`including steroid
`esters. The alcohols of the waxes were identified by
`mass spectrometry and gas—liquid chromatography,
`before and after hydrogenation, and found to be
`69% n-tetradecanol and 28% n-hexadecanol. Wax
`fatty acids were about 84% 18:1 (n—9) and 10%
`16:1 (n—7). The high levels of waxes in E. crystal-
`Iorophias may be related to their year-round very
`cold environment.
`
`Acknowledgements—I wish to thank Dr. Karl Dahm
`for the mass spectrographic analyses and Mrs. Claudia
`Wiltrout for her technical assistance. These studies
`were supported by a grant from the National Science
`Foundation (GV—30413).
`
`REFERENCES
`
`With few exceptions, the experimental evidence
`indicates that the lipids of aquatic animals living at
`lower temperatures tend to be more polyunsaturated
`than those of aquatic animals living at higher
`temperatures (Johnson & Roots, 1964; Knipprath
`& Mead, 1966; Kemp & Smith, 1970). In the case of
`the lipids of E. superba and E. crystallorophias, a
`peculiar situation exists. Although it
`is true that
`the total lipids of E. crystallorophias, which lives
`in colder waters, are more unsaturated than those of
`E. superba,
`it is due to a higher level of mono-
`unsaturated acids. In fact, the phospholipids of E.
`crystallorophias are less unsaturated than those of
`E. superba (Table 4).
`It is possible that the monoenoic waxes of E.
`crystallorophias may have a role in the adaptation
`of these animals to their extremely cold environ-
`ment. Benson et a1. (1972) have found that cold
`water copepods accumulate waxes. According to
`their views,
`the energy stored as waxes may be
`liberated slowly during periods in which food is
`scarce or not available. These terms certainly apply
`to E. crystallorophias since this euphausid not only
`lives in the cold waters near the iCe but also under the
`ice, almost all year around.
`
`AMERICAN OIL CHEMlsrs’ SOCIETY (1970) Oflicial and
`Tentative Methods, 3rd edn. Method Ce-2«66. Am. Oil
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`000005
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`
`crustacea;
`Index—Waxes; Antarctic
`Key Word
`Antarctic zooplankton;
`food chains; marine phos-
`pholipids; fatty acids; triglycerides; steroid esters.
`
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