`
`Vol.173:149—162,1998
`
`MARINE ECOLOGY PROGRESS SERIES
`Mar Ecol ng Ser
`
`d
`Publishe November 1
`
`2
`
`Changes in lipid composition of the Antarctic krill
`Euphausia superba in the Indian sector of the
`Antarctic Ocean: influence of geographical
`location, sexual maturity stage and
`distribution among organs
`
`P. Mayzaud‘, E. Albessard, J. Cuzin-Roudy
`
`Equipe d’Océanographie Biochimique et d'Ecologie. LOBEPM, URA-CNRS 2077. Observatoire Océanologique. BP 28,
`F—06230 Villetranche sur mer, France
`
`ABSTRACT Lipid content and lipid class composition of Euphausr‘a superba were studied at different
`levels for populations and individuals sampled in the Indian sector of the Antarctic Ocean. Strong site-
`to—site variability was recorded which could only partially be related to sex or development stage dif-
`ferences Three groups of stations could be differentiated. Northern stations were characterized by
`‘high lipid—high triglyceride‘ content. western and eastern locations by 'high lipid-high phosphatidyl
`choliiie’ content and southern areas by ’low lipid-high phosphatidyl ethanolamine/glycolipid' content.
`Such variability was likely related to advected populations having spent variable lengths of time in the
`area studied. Lipid content and class among organs were studied in 5 body fractions: abdomen, stom—
`ach. digestive gland. gonad and fat body. In absolute terms, the highest concentrations were observed
`in the ovaries of mature females and the abdomens of the other stages. In relative terms 1% dry weight).
`the digestive gland displayed the highest level, except in mature females. Distribution varied With
`stages, with low triglyceride levels in abdomen tissues of most stages and in the fat body and stomach
`fractions of subadults. High triglyceride levels were recorded in the other fractions for post spawning
`females and males, as well as in the fat body fraction for mature females and in subadult gonads. A
`reverse pattern was observed for
`the relative content of phosphatidyl choline. Phosphatidyl
`ethanolamine showed maXimum values in the abdomen and the gonad. Glycolipid percentages were
`maxmium in the abdomen, suggesting a structural role The roles of the different lipid classes are dis-
`cussed With respect to the function of the organ.
`
`KEY WORDS: Krill - Lipids ~ Spatial heterogeneity . Maturity stage organs
`
`INTRODUCTION
`
`The. role of lipids in Antarctic krill has been the con-
`cern of several papers in relation to reproduction
`(Clark 1980, 1984, Kolokowska 1991, Pond et al. 1995,
`Virtue et al. 1996], energy storage for overwintering
`(Quetin & Ross 1991. Hagen et al. 1996] and trophic
`interactions (Bottino 1974, Reinhardt & Van Vleet
`1986, Virtue et al. 1993a, b). Krill accumulate lipids
`mainly as triacylglycerols during the spring and sum-
`
`' E-mail: mayzaud@ccrv.obs-vlfr fr
`
`© IntenResearch 1998
`
`Resale of full article not permitted
`
`mer when phytoplankton are abundant (Clarke 1984,
`Hagen et al. 1996).
`Neutral lipids are utilized whenever energy levels
`exceed food intake. In krill the 2 major energy utilizing
`events are summer reproduction and winter survival
`under low phytoplankton conditions. Krill store signifi‘
`cant amounts of lipids (Clarke 1984, Hagen et al. 1996),
`although most studies have concluded that the concen-
`trations are not sufficient to meet the energy require—
`ments during the winter, when food supply is low
`{Quetin & Ross 1991, Quetin et al. 1994). However. the
`contribution of lipids to the overall survival strategy of
`
`RIMFROST EXHIBIT 1084
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`

`

`150
`
`Mar Ecol Prog Ser173' 1497162, 1998
`
`krill appears quite significant (Hagen et al. 1996) and,
`during the summer period,
`is channeled mainly into
`reproductive output.
`Krill reproduction takes place over several spawning
`phases
`(Cuzin—Roudy 1987, Quetin & Ross 1991);
`hence. their reproductive output seems directly related
`to the richness of the food supply (Ross & Quetin 1986).
`Yolk accumulation is characterized by high concentra-
`tions of neutral lipids and high levels of triacylglycerols
`in ovaries of mature females (Clarke 1980, Hagen
`1988, Pond et a1. 1995). Males do not seem to accumu-
`late neutral lipids in relation to spermatophore produc-
`tion, but various reports have suggested that
`the
`energy cost of frequent remating may be high, though
`distributed differently over time (Virtue et al, 1996).
`The resulting time integral may represent a significant
`depletion in lipid content, as reported by Pond et al.
`[1995).
`Contrary to the high levels found in large calanotd
`copepods, wax esters are not present in krill lipids, and
`reliance on triacylglycerols as a storage moiety is now
`well established (Clarke 1980, Hagen 1988, Mayzaud
`1997). Reports by Elligsen (1982), Saether et all (1986),
`Hagen (1988), and Hagen et a1. (1996) suggested that
`polar
`lipids,
`and more
`specifically phosphatidyl
`choline (PC). may also serve as storage lipids. The
`involvement
`of
`cell
`structural components as
`an
`energy source has been reported for PC as a source of
`essential polyunsaturated fatty acids in fish for egg and
`larval development [see Fraser et al. 1988). This differ-
`entiation in the actual me of the lipid classes could
`
`provide additional resources during times of increased
`energy demands,
`Our knowledge on the sites of lipid synthesis and
`catabolism in krill is still limited. Lipid composition has
`been given for 3 main body fractions, ie. abdomen,
`digestive gland and mature ovaries (Clarke 1980,
`Saether et al. 1985, Virtue et al. 1993a), with respect to
`neutral lipid accumulation. Little is known on the vari-
`ability of such composition with growth or sexual
`maturity stage. The potential role of the glyco—lipo-
`protein complex present in the fat body described by
`Cuzin-Roudy (1993) remains to be evaluated.
`The objective of the present study was to evaluate,
`for an open-ocean krill population,
`the influence of
`exogenous and endogenous factors in the control of
`lipids during summer. Changes in lipid concentration
`and composition were investigated at 2 different lev~
`els: population and specific organs or body fractions of
`different growth and maturity stages
`
`MATERIAL AND METHODS
`
`Sampling. Euphausia superba were obtained from
`RMT 8 oblique tows made to a depth of 100 or during 2
`cruises of the RV ‘Marion Dufresne' in February 1981
`(FlBEX) and February 1994 (ANTARES 2). Positions of
`sampling stations and cruise track for the first cruise
`are given in Fig. 1. Samples from the second crmse
`were obtained at 2 stations (66“41'5, 61°50‘E and
`63° 00’ S, 70° 20' E). Krill were sorted immediately after
`
`
`
`
`
`Fig. 1. Cruise tracks and stations surveyed as part of FIBEX [February 1981)
`
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`

`Mayzaud et al.: Lipids in Antarctic krill
`
`151
`
` Fig. 2 Euphausia superba. Dissection of krill into 5 body fractions for
`
`the separation of the pnncrpal organs. 1 to 5: Exctsion lines. dg: Diges-
`tive gland; lb: fat body; 0v. ovary; st: stomach
`
`capture, rinsed with distilled water and deep frozen
`(—80°C). They were stored at —70°C under nitrogen
`and transported to France within 4 mo.
`Additional
`samples were collected at different
`depths for particulate chlorophyll and proteins as
`descriptors of the trophic environment. Protocols and
`detailed results can be found in Mayzaud et a1. (1985).
`Stage determination. For population study (FIBEX),
`at each station a subset of 20 individual krill were iden-
`
`tified to development or sexual stage, measured (BL:
`standard length 1; Mauchline 1980), weighed (wet
`weight: WW] and extracted. The rest of the sample was
`weighed and that part was treated as representative of
`the whole population. Female were separated into
`‘maturing’ and ‘post spawn' categories. Maturing fe-
`males were recorded as lIlC (Makarov & Denys 1980).
`For the study of specific stages and organs, frozen
`krill were scored individually for sex, sexual develop-
`ment and maturity (Makarov 8c Denys 1980, Cuzin-
`Roudy & Amsler 1991) and measured for body length
`(BL) while thawing on an ice cold plate under a micro-
`scope. Mature female krill (BL = 44.52 to 58.79 mm)
`with a swollen thorax were staged IIID and SDS 7
`(Fig. 2). Spent female krill (BL : 43.52 to 55.?0 mm]
`had a small ovary and contracted and irregular lobes.
`The thoracic cavity was mainly filled with hemolymph.
`They were scored as SDS 9 rather than MA, in order to
`take into account the ovarian regression which occurs
`normally at the end of the reproductive season (Cuzin—
`Roudy 1987, Cuzin-Roudy & Amsler1991). Among the
`10 male krill dissected (BL : 36.?5 to 60.52 mm), 4 were
`mature and 6 had empty ampullae and were scored
`’post mature'. immature young adults (B1, = 38.35 to
`47.04 mm) will be referred to as subadults.
`Separation of main organs. The krill were dissected
`while thawing to separate either organs or body
`fractions containing a main organ. During the dissec—
`tion the specimens were placed on pre—weighted/
`pre-extracted filter paper (Whatman 42 extracted in
`chloroformzmethanol 2:1), which collected the fluids
`originating from each step. Five fractions were sepa—
`
`rated (Fig. 2): (1) The abdomen fraction was obtained
`from excision lines 1 and 2 and contained mostly mus-
`cle and cuticle from the abdomen and the various
`
`appendages. (2) The anterior fraction containing prin-
`cipally the stomach was next obtained by excision
`line 3. Minor components were, in decreasing order:
`eyes, brain ganglia, and cuticle.
`(3) The digestive
`gland and the digestive tract were next excised as a
`whole from the anterior section of the thorax (excision
`line 4). Whenever the digestive gland was not suffie
`ciently cohesive, recovery was accompanied by leak-
`age of greenish fluid, which was collected on the filter
`paper, extracted and combined.
`(4) The fraction con-
`taining the gonad was obtained from the dorsal part of
`the thorax. In mature females the thoracic cavity was
`overfilled with the swollen ovary and excision along
`line 5 could not be made Without damaging it. The
`resulting leakage of fluid was collected on the filter
`paper, extracted and combined.
`(5) The last fraction
`contained the [at body, the conjunctive tissue which
`fills the ventral part of the thoracic cavity and comes in
`to contact with the ovary (Cuzin»Roudy 1993). A very
`minor component was the nervous tissue and cuticle.
`The wet weight of the 5 organs or body fractions was
`recorded, and additional specimens were dissected the
`same way to obtain wet weight/dry weight ratios. In
`this case dry weight was obtained after oven drying at
`60°C to constant weight.
`Lipid extraction and determination. Entire krill
`were placed frozen on crushed ice and brought to 0°C.
`Size (BL) and fresh weight
`(WW) were measured
`before lipid extraction, according to the method of
`Bligh & Dyer [1959). Either the extracted lipids were
`weighed in tarred vials or their concentration was esti-
`mated according to Barnes & Blackstock (1973) but
`w1th Euphausr‘a superba lipids as standards instead of
`Cholesterol. Both determinations yielded similar val»
`ties. The lipid extracts were then placed under nitr0<
`gen at —70°C until analysis. Body fractions were
`extracted immediately after dissection using the same
`protocol.
`
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`

`152
`
`Mar Ecol Prog Ser173: 149—162,1998
`
`Lipid classes were quantified after chromatographic
`separation coupled with FM) detection on a latroscan
`Mark 111 TH 10 [Ackman 1981). Total lipid extracts
`were applied to chromarods Slll usrng microcapillaries
`(1 pl) and analyzed in duplicate. Neutral lipids were
`separated using a double development procedure with
`the following solvent systems; n~hexane:benzene:for-
`mic acid 80.2005 (by volume) followed by nvhexane:
`diethyletherzformic acid 97:3:05 (v/v). Glycolipids
`were separated according to Hirayama & Morita (1980)
`with chloroformzethyl acetatezacetone:methanolzacetic
`acidzHZO 60:12:15:16:3:3 (v/v), Phospholipids were
`separated with chloroformzmethanoltl-IZO 65:35:4 [v/v).
`Individual calibration of rods was achieved with com-
`
`mercial standards according to Ackman (1981). Using
`the solvent system indicated, all listed lipid classes,
`and in particular free fatty acids. were separated and
`accounted for.
`
`To validate the Iatroscan separation and identifica—
`tion, neutral and polar lipids were further isolated on a
`preparative scale by column Chromatography on silica
`gel {Bio~Sil HA, minus 325 mesh). The neutral lipid
`fraction was eluted with 6 column volumes of chloro-
`form, the acetone mobile compounds were eluted with
`4 volumes of acetone and the phospholipids were
`eluted with 6 volumes of methanol. All operations took
`place under nitrogen. Each fraction collected was fur—
`ther separated by thin-layer chromatography (TLC) on
`pre-coated silica gel plates (Analtech, Uniplate) and
`developed with hexanezdiethyletherzacetic acid 80:20:
`1.5 (v/v)
`for neutral
`lipids, or chloroform:methanol.
`aqueous ammonia 85:30zl
`(v/v) for glycolipids. and
`chloroform:methanol:acetic acid2HgO 25:15:42 (v/v)
`for polar lipids. Lipid classes were visualized using
`dichlorofluorescein and identification was achieved by
`comparison with standard mixtures. Specific detection
`of phospholipids and glycolipids by TLC was made
`with molybdenum blue and diphenylamine reagents
`[Stahl 1969). Each of
`these lipid classes was then
`applied to chromarods SH] and developed as previ-
`ously to confirm both identification and retention times
`of the peaks recorded with total extract.
`No attempt was made to hydrogenate the samples
`(Shantha & Ackman 1990) because no evidence of sub-
`fractionation effects due to the vanety of fatty acids could
`be detected. As recently shown by Miller et al. [1998).
`the use of commercial standards resulted in an underes-
`
`timate of the actual concentration of triglycerides but
`without changes in the shape of the FID response.
`Data analysis. The allornetric relations for the differ-
`ent maturity stages (WW = aBL”) were computed after
`log-log transformation and model I regression (Sokal &
`Rohlf 1981).
`One- and two‘factor variance analyses were per-
`formed for total lipids as well as lipid classes. Multiple
`
`comparisons of means were achieved using the Tukey
`test procedure. Comparison of linear regression equa-
`tions was achieved by covariance analysis. All
`the
`above procedures were carried out according to Sokal
`& Rohlf (1981). Systat 7.0 statistical package was used
`for all bivariate tests (Wilkinson 1996).
`Principal component analysis (PCA) was performed
`after arcsine transformation to normalize percentage
`data. Details on the method and means of interpre-
`tation are given in Mayzaud et al. (1989). To facilitate
`the representation of the factor scores structure, an
`ascending hierarchical clustering method was used
`(Lebart et al. 1995) to group those observations which
`displayed maximum similitude and to produce a num»
`ber of classes best represented in a dendrogram.
`Correspondence analysis
`(Benzecri 1969, Gower
`1987) was performed on a data matrix transformed to
`relative frequencies and scaled so that each row (or
`column] can be viewed as a row (or column) of condi—
`tional probability distribution. Distances between pro-
`files were computed with x’ metrics. This distance
`gives symmetry to the 2 sets of data so that each facto-
`rial axts of the cloud of variables corresponds to a fac-
`torial axis of the cloud of observations. Thus,
`it was
`
`possible to represent simultaneously descriptors and
`observations on the plane defined by the factorial axes.
`Interpretation and representation followed that de-
`scribed above for PCA.
`
`Computation of multivariate tests was made using
`the SPAD 3.0 software (Lebart et al. 1995).
`
`RESULTS
`
`Size, weight and lipid relationships
`
`The size, wet weight and lipid content of the 4 cate-
`gories of krill. collected during the FIBEX cruise are
`summarized in Table 1. Subadults displayed the small-
`est size and weight of the 4 groups, while males and
`females showed similar size ranges (31 to 44 mm) but
`slightly different wet weight distribution. Mean values
`were not statistically different (t-test, p < 005) but
`ranges suggested a trend towards maturing females
`and males being heavier [0.57 to 1.28 mg and 0.4 to
`1.21 mg. respectively) compared to post spawn fer
`males. Lipid content relative to wet weight suggested
`maximum accumulation in maturing females (3.3%).
`Males tended to show minimum lipid content although
`the high variability in this case prevented statistical
`significance.
`The wet weight (WW) relationships with size or lipid
`content were established for each stage present in the
`samples collected,
`i.e. subadults, males and females.
`The log‘log regressrons between size and weight
`
`RIMFROST EXHIBIT 1084
`
`page 0004
`
`

`

`Mayzaud et al: Lipids in Antarctic krill
`
`Table 1. Euphausla superba. Range and mean values of krill size, wet weight and total lipid content [% wet weight) of specific
`maturity stages of individuals collected during FlBEX. n = number of individuals analyzed
`
`
`
`Subadults
`Males
`Metering females
`Post spawn females
`
`n
`
`33
`54
`14
`20
`
`
`Size
`Mean
`Wet weight Mean wet
`Total lipid
`Mean total
`range
`Size
`range
`weight
`range
`lipid
`(mm)
`x SD
`(mg)
`xSD
`(% wet wt)
`x SD
`
`23—35
`31—43
`35—44
`35—42
`
`29.7 r 31
`37.1126
`39.3 1 26
`38.1118
`
`0.20—0.59
`0404.21
`0.57428
`0.53—0.96
`
`0.37 I 012
`074 10.18
`0.93 1 0.22
`0.74 i 0 ll
`
`1.1—4.7
`0.7—5.0
`1.6—4.8
`1.1—4.8
`
`2.9 x 0.9
`2.2109
`3.3 a: 08
`2.8 $1.2
`
`(Fig. 3) appeared to be similar for all 3 stages (slope:
`this = 2.07; intercept: F2120 = 0.26] corresponding to
`an overall regression equation of:
`
`logWW : ~0.08 + 3.12logBL
`[r = 0.967, F1124 : 1779, p < 0.0001)
`
`Relationships between WW and total lipids or phospho~
`lipids (Fig. 3) were all significant (p < 0.008). The re»
`gressions for male individuals showed slopes or inter—
`
`cepts different from the other 2 stages. Males and fe~
`males appeared to accumulate lipids and phospholipids
`at a similar rate (slope: 1-185 2 0.272) but with a lower in-
`tensity (intercept: Furs = 14.25) for a given size in males.
`Triglycerides illustrated a different pattern of changes
`with only 2 significant regressions, those for subadults (n
`: 33, r = 0.673) and females (n = 33, r = 0.556), which
`showed a significant difference in intercept (Free: 5.54)
`but not in slope (131.55: 0.04). In these 2 stages, triglyc-
`
`I
`
`:—
`
`Ill(II
`
`
`
`LogWetweight(g/ind)
`
`
`
`
`
`Logtotallipidcontent(mg/ind)
`
`5‘:8
`
`41.75 '
`
`-l.00
`
`Ea
`
`F\lb.
`
`0.42
`
`
`
`
`
`
`may SubudulLs
`U Mules
`
`A
`tit-males-
`
`'
`l.4l
`
`L——_J
`1.61
`
`'
`
`'
`1.5)
`Log Length (mm)
`
`O
`
`U
`
`9
`
`[I
`
`
`
`
`
`LogPhospholipidconcentration(mg/ind)
`
`
`
`
`
`LogTriglyceridescone.(mg/ind)
`
`1.24 '
`
`0.93 ' 0.61
`
`o
`
`
`
`0.31
`
`0.00 2
`
`mo.” Subudults
`——Cl-—- Malia
`
`—-— Fr-mnlm
`
`
`-0.00
`41.75
`-0.50
`-0.25
`Log Wet weight (glind)
`
`
`
`
`
`
`.075
`050
`41.25
`
`Log Wet weight (glind)
`
`—.—vaalt\<
`—-o—— Males
`.. o-v- Suhudulb
`.
`
`0.10 ~77 ,
`'
`4
`*— -
`4.00
`41.75
`0.25
`0.00
`050
`Log Wet weight (glind)
`
`Fig. 3 Euphausia superba. Log-log regressions, for the 3 ma]or developmental stages, between wet weight and body length and
`between wet weight and total lipid, phospholipids and triglycerides content
`
`RIMFROST EXHIBIT 1084
`
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`
`

`

`154
`
`Mar Ecol Prog Ser173: 149-162, 1998
`
`erides were accumulated at a similar rate but with a
`
`lower intensity in females than in subadults. The lack of
`correlation for the male data set was to a large extent as-
`sociated with an increased variance, with low concen-
`trations in the medium to large size range
`
`Lipid content and spatial heterogeneity
`
`To test the relative influence of geographical loca-
`tion and biological descriptors we conducted an analy—
`sis oi variance, The relative lipid content recorded at
`the population level was significantly related to stage
`distribution and location of sampling but not to size or
`weight (Table 2). The overall mean lipid percentage
`(2.6% wet weight) can be divided into 2 groups of sta—
`tions, irrespective of the sex or stage composition (t-
`test: 7.65. df = 48, p < 0.0001): (1) those north and west
`of the grid (Stns 2, 3, 17, 18, 19, and 23) and those to the
`extreme southeast (Stns 9A and 10) with high concen»
`trations (mean value; 3.1% wet weight), and (2) those
`from the central and southern part of the grid (Stns 5,
`5A, 6, 9, and 11) with low concentrations (mean value:
`19% wet weight).
`Over the spatial grid surveyed during FlBEX, a rela-
`tively complex pattern of lipid classes was observed for
`
`the lipid distribution of
`Table 2. Analysis of variance for
`Euphausia superba as "i. wet weight over the FIBEX grid of
`stations
`
`
`Source of
`variation
`
`Sum of
`squares
`
`df Mean F»ratio Signif-
`squares
`icance
`level
`
`I Main effect
`Station
`Stage
`Covanates
`Size
`Weight
`Resrduals
`
`3633
`16.81
`
`0.28
`0.06
`80.42
`
`Total (corrected)
`
`144.64
`
`3.03
`5.61
`
`0 28
`0.06
`0.76
`
`3 99
`7 39
`
`0 37
`0.07
`
`0.0000
`0.0002
`
`0.553
`0.789
`
`12
`3
`
`1
`1
`106
`
`123
`
`
`
`the different growth and maturity stages. Both struc—
`tural polar lipids and reserve triglycerides displayed
`positive linear relationships with total lipids, suggest»
`ing that part of the polar lipids acted as reserve lipids
`(Fig. 4). Interestingly, the relationship significantly dif-
`fered with the stage considered. A covariance analysis
`of the various regressions indicated that regressions
`for polar lipids showed a trend for increasing slopes
`(p < 0 05), with subadults (r 2 0498) < male (r = 0.579)
`
`Subadults
`
`Mature Females
`
`Polar lipids
`
`Polar lipids
`
`
`
`”Triglycerides
`
`
`
`Polar lipids
`
`
`
`Males
`
` MainClassesofLipids(mg/100gwetweight)
`
`
`
`
`
`Polar lipids
`
`
`
`Fig 4 Euphausza superba. Linear regressions between total lipid content and Lipid classes for sexual and developmental stage
`
`Total Lipids (mg/100g wet weight)
`
`RIMFROST EXHIBIT 1084
`
`page 0006
`
`

`

`Mayzaud ct at: Lipids in Antarctic krill
`
`
`155
`
`AXIS2(22.0%)
`
`PE
`
`5T9 %....S'l‘5A. LTOJ'
`
`-0.4
`
`-02
`
`0.0
`
`0.2
`
`0.4
`
`AXIS i (75.5%)
`
`b
`
`
`h5%
`
`| srts
`ST17
`
`s‘rzg
`STtO
`
`
`
`_
`
`'— srs
`_ifL30% _
`L_
`
`STQA
`T STSA
`
`< mature females (r = 0659) < spent
`females (r : 0.802) and the reverse for
`triglycerides. Within a given stage.
`polar lipid and triglyceride relation-
`ships were significantly different for
`males and spent females (F1105 : 5.76;
`151.36 = 89.95, respectively) but not for
`subadults and mature females (FIN :
`0.07 and F1135: 2.74, respectively).
`The spatial structure was analyzed
`using multivariate
`correspondence
`analysis. The factorial plane defined
`by the first 2 axes accounted for 97.5 %
`of the total variance, The first and sec~
`
`0nd axes were respectively related to
`the richness in triglycerides and phos-
`phatidyl choline (Fig. 5). Grouping of
`stations, based on proximity analysis,
`showed 3 major clusters, which com-
`prised (1) those stations with high pro-
`portions of
`triglycerides in the total
`lipids, located in the northern part of
`the grid (Stri 23 at 62°S; Stns 17 and 19
`at 63°S), (2] those stations with a high
`proportion of phosphatidyl choline in
`total lipids, located at both the western
`and eastern edges of the grid (Stns 2, 3
`and 11], and (3)
`those stations with
`high percentages of structural compo-
`nents(phosphatidylethanolamine,g1y-
`colipids and to a minor extent phos—
`phatidyl choline), in the southern part
`of the grid (Stns 5A, 6, 9 and 9A at
`64°S) (Fig.5).
`
`Distribution of lipids in body
`fractions and organs
`
`
`
`
`
`F) STH
`8T3
`8T2
`
`25%
`
`the
`The respective contribution of
`different fractions and organs to the
`Lipid compos1tion of Euphausia superba
`was studied for different development
`and maturity stages: subadults, males,
`mature females and spent females.
`The mean Sizes of the specimens dis-
`sected and the dry weight of the differ-
`ent fractions are presented in Table 3.
`Size varied over a
`relatively small
`range (42 to 51 mm) and was chosen so
`that variability around mean size remained more or
`less constant. In terms of dry weight,
`if the mature
`females, for which the heaviest fraction was the ovary,
`are not included, the abdomen represented the largest
`body component. The other
`fractions and organs
`
`Fig. 5. Correspondence analysrs of the spatial changes of tota. lipids and lipid
`classes of Euphausia superba collected during the FIBEX cruise (a) Protection of
`biochemical descriptors and stations on the first 2 axes. Descriptors of
`the
`trophic environment (proteins and chlorophyll) projected on the factorial plane
`as supplementary variates, LTOT:
`total
`lipids; PC: phosphatidyl choline;
`PE; phosphatidyl ethanolamine; GLYC‘ glycolipids; TG:
`triglycerides; CHL:
`chlorophyll; PRO: particulate proteins. For station (ST) locations refer to Fig.
`l
`(b) Dendrogram of hierarchical clustering of stations based on the scores on the
`first 5 factorial axes %. percent total variance accounted for by each cluster
`
`showed dry weights ranging from 9.6 to 43.4 mg, with
`no significant differences (p < 0.05). Lipid contents in
`both absolute and relative terms are presented in
`Table 4. with the highest concentrations observed for
`the ovary in mature females and for the abdomen in
`
`RIMFROST EXHIBIT 1084
`
`page 0007
`
`

`

`156
`
`Mar Ecol Prog Ser173zl491162.1998
`
`Table 3. Euphausra superba. Mean (1 SD) size of individuals (mm) and dry weight
`triglyceride levels were recorded in
`(mg) of the diiferent body fractions and organs. n = number of individuals
`abdomen tissues (mean: 28.7 1 2.19..)
`in most stages, and in the fat body and
`stomach fractions of
`the subadults
`
`Subadults
`
`Males
`
`Females
`
`Mean size
`
`41.9 1 3 5
`n = 5
`Body fractions and organs
`Abdomen
`83.2 1 2.4.3
`Digestive gland
`9.6 1 4.3
`Fraction including
`33.6 1 37.2
`23.8 110.5
`Gonads
`304. 118.9
`11 11 6.4
`Fat body
`221 114.4
`101 I 4.8
`Stomach
`
`
`51.3 1 4.8
`n I 9
`
`142 1 58
`20.6 1 7.8
`
`Spent
`47.11 4.4
`n :12
`
`98.8 1 30.2
`20.2 1 11.2
`
`23.9 113.9
`2511112
`19.9 1 9.1
`
`most stages. On a percent dry weight basis, the diges-
`tive gland displayed the largest lipid content in all
`stages except in mature females. The Significance of
`these differences was tested by covariance analyses,
`which confirmed that changes in lipid content were
`related to the above stages (p > 0.002) and organs (p >
`00001), and in absolute terms to size (p > 00001). The
`ovary in mature females 18 very rich and contains about
`60 % of the total lipids, while in the other stages the
`abdomen contributes 30 to 40% to the total lipids, and
`the digestive gland accounts for 20 to 30%. Fat body
`and stomach fractions were in all categories minor con—
`tributors, with respective ranges of 8 to 14% and 4 to
`9% of the total lipids (Table 4).
`Distribution of lipid classes among organs showed
`different patterns with stages (Tables 5 & 6). Low
`
`
`
`Mature
`50.8 I 4.3
`n :10
`
`85.4 1 26.4
`43.4 1 25.2
`
`175.3 1 63.2
`329 1 8.3
`22.8 i 7.8
`
`Maximum percentages were observed
`m the Other ”Mum? tor Spent females
`and males (respective means: 42.2 1
`1.7% and 38.61 1.0%), as well as the
`fat body fractlon m mature females
`and the gonad fraction in subadults,
`As anticipated, phosphatidyl choline
`(PC) displayed a reverse pattern with
`.
`.
`hlgh “”815 m the abdomen Of a,” the
`stages (mean: 47.6 1 1.8%) and in the
`gonad fraction in mature females.
`Minimum percentages occurred in the
`fat body and stomach of males and mature females as
`well as the digestive gland and gonad fraction oi
`subadults. Phosphatidyl ethanolarnine (PE) showed
`maximum values in the abdomen (mean: 6.4 1 0.4 %)
`and minimum percentages in the gonad fraction
`(mean: 2.4 1 0.4 %). Glycolipids (GLY) showed low per»
`centages in the abdomen (mean: 2.4 1 0.4 %), mainly in
`males and post spawn females. intermediate values in
`the digestive gland and gonad fraction (respective
`means: 3.7 1 0.4 % and 3.0 1 0.4%) and maximum val—
`ues in the fat body and stomach fractions (respective
`means: 4.7 1 0.5% and 4.1 1 0.4%). Monoglycerides
`(MAG) showed maximum values in the abdomen of
`most stages. except mature females. The variability in
`monoglycerides was significantly related to both stage
`and organ (ANOVA, p > 0.0003). Lysophosphatidyl
`
`Table 4. Euphausia superba. Absolute (per mg) and relative (% dry weight of each organ] lipid content (1 SD) of the different or»
`gans or tissue fractions and lipid distribution wrthin body components (% total lipid]
`
`
`Subadults
`
`Males
`
`Females
`
`Body tissue and organs
`
`Abdomvn
`
`% total lipid located in tissue
`
`Digestive gland
`
`% total lipid located in tissue
`
`Fraction including
`Gonads
`
`% total lipid located in fraction
`Fat body
`
`‘31} total lipid located in fraction
`Stomach
`
`total lipid located in fraction
`
`mg
`" -L)W
`
`mg
`"oDW
`
`mg
`‘3-1. DW
`
`mg
`"IliDW
`
`mg
`0A) DW
`
`
`
`13.1138
`15.9131
`40811.6
`
`0813.3
`52.7 1 7.8
`21.1111
`
`6913.4
`28317.6
`21411.3
`2.9127
`24719.2
`9.2107
`2411.6
`21.8177
`7.4 105
`
`15216.4
`11.2125
`35511.8
`
`14.1156
`53.11 9 9
`32.9113
`
`4611.7
`26116.7
`10712.3
`5813.4
`22.5141
`13.5119
`3.1113
`20216.6
`7.3 10.6
`
`Spent
`
`13514.6
`13.7128
`30911.3
`
`13616.0
`46.2 111.0
`31.2115
`
`6713.1
`30217.1
`15.21 11
`5.9126
`24.3163
`13.6118
`3.9112
`21.1161
`9,010.9
`
`Mature
`
`11314.2
`13513.9
`13712.3
`
`10.4-15.1
`25.4 110.0
`12712.8
`
`4981160
`31317.6
`60511.8
`7111.8
`21.9146
`8.6107
`3.7115
`16.1140
`4.5 10.4
`
`RIMFROST EXHIBIT 1084
`
`page 0008
`
`

`

`Mayzaud et al.: Lipids in Antarctic krill
`
`157
`
`Table 5. Euphausr‘a superba Lipid classes composition for the different organs and tissue fractions of subadult and male stages.
`LPC. lysophosphatidyi choline; PC: phosphatidyl choline; PE: phosphatidyi ethanolamine; GLY: glycolipids; Chol. cholesterol;
`MAG: monoacylglycerols; TAG: triacylglycerols. Standard deviations are given in parentheses
`
`Body tissue
`and organs
`
`Abdomen
`
`Digestive gland
`
`LPC
`—
`
`57
`(1.0)
`
`1.0
`(0.1)
`
`Fraction including:
`C‘ionads
`3.0
`(2.4)
`6.1
`(1.3)
`5.5
`(2.6)
`
`Fat body
`
`Stomach
`
`Subadults
`
`Chol MAG TA
`GLY
`PE
`»
`("’1‘ total lipids)«~------—~
`»
`
`PC
`
`--
`
`45.3
`(10 0)
`
`32.2
`(8 3)
`
`30.3
`(7.0)
`50.5
`(7.2)
`38.7
`(8.1)
`
`4 9
`(1.8)
`
`3.8
`(1.4)
`
`1.9
`(1.4)
`3.8
`(1.8)
`4.4
`(2.1)
`
`34
`(0.7)
`
`6.5
`(3 5)
`
`2.1
`(1.3)
`4.9
`(5.7)
`4.1
`(2.7)
`
`2.3
`(1.8)
`
`1.8
`(1.5)
`
`3.0
`(19)
`1.5
`(1.1)
`2.3
`(2.1)
`
`
`6.0
`(1.8)
`
`4.8
`(1.4)
`
`6.5
`(3.2)
`1.8
`(3.6)
`2.8
`(2.3)
`
`30.9
`(7.8)
`
`39.6
`(5 3)
`
`51.0
`[6.0)
`29.1
`(5.4)
`35.2
`(16.3)
`
`Males
`Chol MAC TAG
`(SLY
`PE
`»--(% total lipids)
`
`PC
`LPC
`___-.-
`
`1.8
`(0.7)
`
`2.3
`(1.1)
`
`0.9
`(0.8)
`1.6
`(1.2)
`2.6
`(1.8)
`
`552
`(8.9]
`
`43.8
`(11.6)
`
`46.6
`(20 5)
`34.9
`(9.8)
`36.8
`(9.9)
`
`5.7
`[3.6)
`
`2.9
`(1.6)
`
`1.7
`(1.0)
`3.4
`(1.7)
`3 5
`(2 3)
`
`1.9
`(0.3)
`
`2.1
`(1.6)
`
`2.4
`(2.0)
`2.9
`(2.6)
`3.2
`(1.8)
`
`3.2
`(1.3)
`
`0.9
`(0.7)
`
`2.0
`(1.8]
`2.3
`(1.4)
`2.1
`(2.3)
`
`11.4
`(2.4)
`
`6.3
`(2.2)
`
`3.0
`(3.0)
`9 4
`(18)
`5.3
`(3.2)
`
`(117)
`
`201
`(4.0)
`
`37.3
`(12.7)
`
`43.2
`(189)
`44 0
`(181)
`46 3
`
`Table 6. Euphausia superba. Lipid class composition for the different organs and tissue fractions of females at 2 maturity stages.
`Abbreviations as in Table 5. Standard deviations are given in parentheses. tr' trace
`
`PC
`LPC
`——--n
`
`
`
`
`Abdomen
`
`Digestive gland
`
`3.2
`(1.2)
`
`4.1
`(1.8)
`
`Fraction including:
`Gonads
`
`it
`
`Fat body
`
`Stomach
`
`19
`(1.8)
`3.8
`(2.5)
`
`45.2
`(7.9)
`
`37.8
`(8.1)
`
`40.2
`(6.9)
`35.6
`(7.2)
`35.1
`(4.2)
`
`8.0
`(2.4)
`
`4.3
`(2.4)
`
`3.3
`(1.3)
`4.6
`(1.6)
`4.0
`(2.1)
`
`1.2
`(0.7)
`
`2.6
`[0.6)
`
`0.6
`(0.2)
`2.8
`(1.0)
`4.9
`(2.7)
`
`3.3
`(1.2)
`
`1.3
`(1.0)
`
`2.2
`(1.6)
`1.8
`(0.8)
`2.6
`(12)
`
`6 7
`(2.3)
`
`4.0
`(1.9)
`
`4 3
`(3 3)
`6.1
`(1.7)
`5.8
`[3.2)
`
`32.4
`(9.4)
`
`42.6
`(10.2)
`
`49.5
`(7.8)
`43.6
`(11.9)
`42.7
`(5.9)
`
`
`
`
`Body tissue
`Spent females
`Mature females
`
`and organs
`PE
`GLY
`Chol MAG TAG
`PE
`GLY Chol MAG TAG
`PC
`LPC
`(‘f-b total lipids)---~—
`—
`—»—--——————— l "a total lipids) —
`
`4.8
`(2.0)
`
`2.3
`(2.1)
`
`1.9
`(1.8)
`3.5
`(1.6)
`3.9
`(0.9)
`
`42.7
`(10.2)
`
`43.8
`(11.6)
`
`53.9
`(7.8)
`35.1
`(5.5)
`38.0
`(5.7)
`
`7.2
`(1.3)
`
`2.9
`(1.6)
`
`3.4
`(1.6)
`4.6
`(1.5)
`6 0
`(3.2)
`
`3.1
`(1.0)
`
`2.1
`(1.6)
`
`6.3
`(4.4)
`3.3
`(1.7)
`4.3
`(2.5)
`
`5.0
`(15)
`
`0.9
`(0.8)
`
`3.0
`(1.0)
`2.5
`(0.8)
`4.4
`[1.1)
`
`2 7
`(2.3)
`
`6.3
`(2.2)
`
`2.7
`(1.7]
`3.1
`(0 3)
`1.4
`(12)
`
`32.2
`(8.1)
`
`37.3
`(12.8)
`
`26.7
`(8.8)
`44.3
`(5.1)
`39.2
`(9.8)
`
`no
`
`significant
`
`changes
`
`showed
`(LPC)
`choline
`(ANOVA, p < 0.05).
`lipid
`The
`relationship between distribution of
`classes, organs and stages can be more clearly
`described using a multivariate approach,
`i.e. PCA.
`Three factorial axes were needed to explain 83% of
`the total variance. Correlation between axes and lipid
`descriptors showed that
`the first axis was strongly
`related to the opposition between PC and triglycerides
`and accounted for 40.8% of the total inertia (Fig. 6).
`The second and third (not shown) axes accounted for
`22.8% and 19.6 % of the total variance (Fig. 6). They
`were respectively correlated to monoglycerides and
`PE for axis 2 and to glycolipids for axis 3. Hierarchical
`clustering of the organs and stages (Fig 6) suggested 2
`ma)or groupings, each associated with the dominance
`
`of 1 lipid descriptor: [1) high proportion of polar lipids
`(PC and PE) with the abdomen from all stages and the
`various fractions
`from mature females (except
`fat
`body), and most
`fractions
`from subadults (except
`gonad and digestive gland); (2) high proportion of the
`2 acylglycerols (MAC and TAG) with mostly spent
`females and males, with digestive gland and fat b