`
`LIPID COMPOSITION OF TWO SPECIES OF ANTARCTIC
`KRILL: EUPHAUSIA SUPERBA AND E. CRYSTALLOROPHIAS
`
`NESTOR R. BoTTINO
`Department of Biochemistry and Biophysics, Texas A & M University, College Station, Texas 77843,
`U.S.A.
`
`(Received 21 February 1974)
`
`Abstract-I. The lipids of two Antarctic euphausiids were characteriz.ed.
`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.
`crysta/lorophias were less unsaturated than those of E. superba.
`S. The waxes of E. crysta/lorophias were mostly esters of oleic (84%) and palmitoleic (10%) acids
`with n-tetradecanol (69%) and n-hexadecanol (28%).
`
`INTRODUCI'ION
`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 ampbipods 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(cid:173)
`plankton feeders (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. 1) 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 present report. A
`preliminary presentation of these results has been
`made (Bottino, 1973).
`
`Deport Lyttelton
`January f7 1972
`
`45
`
`os
`
`Fig. 1
`
`MATERIALS AND METIIODS
`
`Euphausiids were collected with a 1 m mid-water
`trawl at depths from 0 to 300 m. Once on board the ship
`the samples were rapidly sorted by band and extracted
`with the chloroform-menthanol (2: 1, v/v) mixture of
`
`479
`
`RIMFROST EXHIBIT 1038 page 0001
`
`
`
`480
`
`NESTOR R. BoTnNo
`
`Folch et al. (1957). Portions of 5-lOmg 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-5, 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 ft x t in. column of siliconi2:ed
`polyethyleneglycol succinate (DGSS-X, Applied Science)
`10% (w/w) on Gas Chrom P 100-120 mesh (Johns(cid:173)
`Manville, Denver, Colo.) at 170°C. A dual :flame Model
`GC-5 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(cid:173)
`Packard Desk Programmable Calculator Model 7810A.
`Results were expressed as weight per cent. Fatty acids
`were
`identified by C<H:hromatography with known
`standards and by plotting relative retention times vs.
`chain length before and after hydrogenation. For
`quantitative fractionation of lipid classes 20-40 mg of
`lipids were separated by the method of Freeman &
`West (1966) modified as follows: (a) Silica gel without
`binder (Adsorbosil-5, 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. crystal/orophias
`
`Fatty acid Station 8 Station 11 Station 13 Station 16
`
`developments. (c) Quantitation of the spots was done
`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 off 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
`discarded and to the lower phase was added 8 ml 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(cid:173)
`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(cid:173)
`tical to that of authentic 1-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(cid:173)
`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 differed 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,
`
`RIMFROST EXHIBIT 1038 page 0002
`
`0·3
`14'9
`0·3
`0·5
`21·2
`0·7
`0·1
`0·3
`tr
`9·0
`0·7
`18·2
`0·6
`0·3
`2·6
`
`weight%
`0·2
`0·2
`14·3
`2-3
`0·2
`0·1
`0·2
`0·2
`24·7
`13'8
`1-4
`1 ·2
`0·1
`0·2
`0·1
`8·9
`0·3
`21'7
`0·9
`0·1
`2·0
`
`8·4
`0·4
`47·5
`0·2
`
`tr
`2-4
`
`0·1
`14'8
`M
`
`10·8
`0·4
`45·2
`0·5
`
`2·7
`
`0·3
`0·9
`
`0·9
`0·7
`0·1
`
`13-4
`
`5·5
`
`3-3
`O·l
`0·2
`0·9
`0·5
`M
`
`11-8
`0·1
`n
`
`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 (n-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 (n-3)
`22:5 (n-3)
`22:6 (n-3)
`
`0·3
`H
`0·6
`2-2
`0·5
`0·5
`0·2
`16·0
`0·3
`8·6
`
`0·2
`l·O
`0·5
`3-3
`0·4
`0·2
`0·3
`11-4
`0·1
`n
`• Data from Bottino (1974).
`t br, Branched-chain fatty acids.
`
`
`
`Lipids of Antarctic krill
`
`481
`
`Table 2. The lipids of Antarctic krill
`
`E. superba
`
`E. crystallorophias
`
`Station 8 (l)• 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
`
`weight%
`44± lOt
`2±3
`
`53±8
`46
`6
`1
`
`1±2
`
`36±6
`4±5
`58±14
`48
`8
`1
`1
`2±22
`
`20±1
`27±9
`
`4±1
`42±8
`
`7±1
`
`• Number of determinations in parentheses.
`t PC, Phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol.
`t Weight per cent plus or minus the standard deviation.
`§ R1 between those of triglycerides and diglycerides. The recovered amount of this fraction was too
`small for further characterization.
`
`Table 3. Composition of the waxes of E. crystal/orophias
`
`Fatty acids
`
`Wax alcohols
`
`Station 13
`
`Station 16
`
`Station 13
`
`Station 16
`
`Waxes
`
`Waxes
`
`Steroid esters
`
`tr
`72·5
`
`25'3
`2·2·
`
`0·1
`tr
`69·2
`
`28·0
`2·2·
`0·5
`
`tr
`
`tr
`
`0·1
`tr
`tr
`0·5
`
`0·1
`0·6
`0·2
`
`1·3
`11'2
`0·3
`SH
`0·4
`2'2
`
`0·3
`0·1
`
`0·5
`
`0·8
`0·3
`
`0·6
`
`0·1
`8·8
`0·3
`84'1
`0·6
`H
`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- ?)
`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 (n-9)
`18:2 (n-3)
`18:3 (n-3)
`20:3 (n-3)
`18:4(n-3}
`20:5 (n-3}
`22:6(n-3}
`
`Unknown
`
`weight%
`1'4
`0·6
`0·4
`O·l
`8·3
`
`0·3
`14·0
`2-4
`
`0·1
`0·3
`0·8
`0·3
`11-0
`14·0
`M
`22'1
`
`1·9
`0·9
`0·3
`
`1·5
`16·5
`1'4
`0·3
`
`• 18:0+ 17:1.
`
`RIMFROST EXHIBIT 1038 page 0003
`
`
`
`482
`
`NESTOR R. BoTnNo
`
`Table 4. Fatty acids of the complex lipids of Antarctic krill
`
`E. superba-Station 11
`
`E. crysta/lorophias-Station 13
`
`CL*
`
`PC
`
`PE Lyso PC
`
`CL
`
`PC
`
`PE Lyso PC
`
`Weight%
`0·1
`
`1-8
`
`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
`3·0
`0·7
`
`0·1
`0·3
`O·l
`tr
`O·l
`3-4
`H
`
`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
`
`H
`
`0·2
`
`O·l
`
`tr
`
`2·8
`0·1
`13-2
`0·9
`
`1-7
`0·2
`
`0·1
`
`0·1
`tr
`O·l
`1-3
`
`21-8
`0·3
`
`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
`
`tr
`tr
`
`tr
`.4·2
`1·0
`
`tr
`2-9
`0·9
`
`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
`
`0·6
`2-7
`0·6
`1-7
`6·2
`11'1
`5·2
`
`37·8
`
`1·9
`1·8
`2·0
`
`3·9
`n
`2·5
`tr
`
`2-4
`
`O·l
`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 (n-9)
`22:1 (n-9)
`
`18:2 (n-3)
`21:2 (n-6)
`
`18:3 (n-6)
`18:3 (n-3)
`20:3 (n-6)
`20:3 (n-3)
`21:3 (n-3)
`22:3 (n-3)
`
`18:4 (n-3)
`20:4 (n-6)
`20:4 (n-3)
`
`20:5 (n-3)
`22:5 (n-3)
`22:6 (n-3)
`Saturated
`Monounsaturated
`Polyunsaturated
`
`2·9
`0·1
`14·2
`0·8
`
`2·3
`
`0·2
`1·6
`0·1
`l·O
`tr
`0·5
`
`3·8
`1-5
`
`25-3
`0·1
`15·2
`
`30·3
`18·1
`51·6
`
`2-4
`
`1-4
`
`0·4
`1·8
`O·l
`0·7
`3-8
`0·5
`
`4·7
`1·5
`0·4
`
`22·5
`
`11·8
`
`32·3
`17-1
`50·6
`
`0·8
`0·1
`1-2
`4·5
`0·1
`
`0·6
`0·2
`0·2
`
`20·3
`
`20·8
`
`26·1
`23·7
`50·2
`
`H
`0·1
`
`0·2
`
`tr
`1·6
`
`1·2
`1·9
`0·2
`
`6·5
`
`2·5
`
`61·3
`23-4
`15-3
`
`2·1
`
`2·5
`
`1-3
`
`O·l
`1·2
`
`1-4
`1-1
`3·6
`
`1-7
`O·l
`
`19·3
`
`8·6
`
`34·0
`26·8
`39·2
`
`1-5
`
`0·3
`8·4
`
`2-3
`0·3
`
`14·8
`
`5·2
`
`36·5
`28·2
`35-3
`
`0·3
`
`0·3
`
`15·1
`
`10·3
`
`39·5
`33·2
`27·3
`
`13·0
`tr
`0·5
`
`0·7
`
`0·4
`
`tr
`0·2
`0·2
`
`2-4
`
`tr
`71·6
`24·5
`3·9
`
`• CL, Complex lipids.
`PC, phosphatidylcholine; PE, phosphatidylethanolamine.
`
`the major complex lipid was phosphatidylcholine were steroid esters.• The waxes of each sample of ·
`(PC), 49 per cent in E. superba and 46 per cent in E. crysta/lorophias were isolated by TLC and the
`E. crystallorophias. Phosphatidylethanolamine (PE)
`fatty acids and alcohols were examined indepen-
`and lysophosphatidylcholine were also found in
`dently by GLC. Both samples had very similar
`both euphausiids but in much lower concentrations.
`compositions, as shown by the data in Table 3. Two
`Unexpected large amounts of waxes were found in
`E. crystal/orophias while none was found in E.
`In the specimens of E. crystal/orophias
`superba.
`from station 16, slightly more than half of the waxes
`
`• For the sake of simplicity in the following discussion
`I shall call waxes the esters of long-chain aliphatic
`alcohols only.
`
`RIMFROST EXHIBIT 1038 page 0004
`
`
`
`Lipids of Antarctic krill
`
`483
`
`monoenoic acids, oleic and palmitoleic, con(cid:173)
`stituted about 94 per cent of the fatty acids of these
`waxes. Of the four alcohol components, about 70
`per cent was tetradccanol and 28 per cent was hexa(cid:173)
`decanol. Thus the waxes of E. crystallorophias are
`less unsaturated than the copepod waxes examined
`by Lee et al. (1971a, b) and by Benson et al. (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(cid:173)
`ponent. In most other marine waxes so far in(cid:173)
`vestigated, hexadecanol is the predominant alcohol.
`The fatty acids of the steroid esters of E. crystal(cid:173)
`lorophias (Table 3) contain polyunsaturated fatty
`acids while those of the aliphatic waxes do not.
`Table 4 shows the fatty acids of the major phos(cid:173)
`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 PE of E. crystallorophias were polyunsaturated.
`Again, 20:5 and 22:6 were the predominant poly(cid:173)
`unsaturated acids.
`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. crysta/lorophias, a
`peculiar situation exists. Although it is true that
`the total lipids of E. crystal/orophias, which lives
`in colder waters, are more unsaturated than those of
`E. superba, it is due to a higher level of mono(cid:173)
`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(cid:173)
`ment. Benson et al. (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.
`
`SUMMARY
`
`The lipids of specimens of E. superba and E.
`crystallorophias caught in various locations of the
`Ross Sea, Antarctica, were studied. The fatty acid
`composition of E. superba differed quantitatively
`from that of E. crystallorophias, the former con(cid:173)
`taining more saturated acids, the latter containing
`more monoenoic 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 differ,
`however, in their neutral lipids, those of E. superba
`being triglycerides and diglycerides, 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(cid:173)
`lorophias may be related to their year-round very
`cold environment.
`
`Acknowledgements-I wish to thank Dr. Karl Dabtn
`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
`
`AMmuCAN OIL CeEMisrs' SocmrY (1970) Official and
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`RIMFROST EXHIBIT 1038 page 0005
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`484
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`NESTOR R. BornNo
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`LEER. F., NEVENZEL J. C. & PAFFENHOPER G. A. (197la)
`Importance of wax esters and other lipids in the
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`
`Index-Waxes; Antarctic crustacea;
`Key Word
`Antarctic zooplankton; food chains; marine phos(cid:173)
`pholipids; fatty acids; triglycerides; steroid esters.
`
`RIMFROST EXHIBIT 1038 page 0006
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