Effects of zooplankton herbivory on biomarker proxy records. Kliti (3rice, 1 Wim C.M. Klein Breteler, Stefan Schouten, Vincent Grossi, 2. Jan W. de Leeuw, and ...
PALEOCEANOG•HY,
VOL. 13, NO. 6, PAGES 686-693, DECEMBER 1998
Effectsof zooplanktonherbivory on biomarker proxy records Kliti (3rice,1Wim C.M. KleinBreteler,StefanSchouten, VincentGrossi, 2 JanW. de Leeuw,andJaapS. SinningheDamst• NetherlandsInstitutefor SeaResearch(NIOZ), Den Burg,Netherlands
Abstract. The stablecarbonisotopiccompositions of cholesterol, generallythe mostdominantsterolin the copepod
Temora, bearsthe513C"signature" of itsdietaryprecursor sterolwhenfedonIsochrysis galbanaandRhodomonas sp. The513C of cholesterol in thefaecalpellets released fromTemora longicornis fedonRhodomonas sp.is identical to the • 13C of thesterols in thediet,indicating thatnosignificant carbon isotopic fractionation effects occurwhenthecopepod modifieseukaryoticprecursorsterolsto cholesterol.Furthermore,the ratio of long-chainalkenonesand their stable carbonisotopiccompositions in I. galbanawereidenticalto thoseegested in faecalmaterial.Thuszooplankton herbivory doesnot invalidatetheuseof thesealkenones asa proxyfor seasurfacetemperature andpCO2. 1. Introduction
Long-chain C37 alkenones [e.g., de Leeuw et al., 1980; Volkmanet al., 1980a, b; Rechkaand Maxwell, 1988a, b; Thiel et
al., 1997] biosynthesized by certainhaptophytes,for example, Emiliana huxleyi [e.g., Marlowe et al., 1990], have been used widely asa proxyof seasurfacetemperature (SST) [e.g.,Brassell et al., 1986; Prahl and Wakeham,1987; Eglinton et al., 1992; Prahl et al., 1995; Chapmanet al., 1996], on the basisof the
distributions of isomers (e.g.,uk'37whichis the ratio of (C37:2
phytoplankton to sedimentaryorganicmatter [Suess,1980], althoughtherestill can be an indirectinputvia detritalmaterial from zooplanktoncomprisingtheir faecal pellets and body remainsupondeath[Srnetacek, 1980;Corneret al., 1986;Killops and Killops, 1993]. In this respect,marinecrustaceans, suchas copepods (mesozooplankton) abundantlypresentin our oceans today[Hardy,1970],play a majorrole in the pelagicfoodweb, formingthefirstvital link in the foodchain.As such,theyform a link betweenproduction anddeposition of biolipidsfromprimary
producersand result in structuralmodificationsand, in turn, alkenones)/(C37:2 + C57:3 alkenones)[Prahl et al., 1988]). The compositional differencesof biomarkercomponents presentin individualstablecarbonisotopiccompositionsof alkenones,in surfacesediments[e.g., Volkrnanet al., 1980c; Corner et al., combinationwith other data, have provided an estimate of 19861. ancientpCO2 levels [dasperand Hayes, 1990; Freeman and Copepods, like mostothercrustaceans, are not capableof de Hayes, 1992; Jasperet al., 1994]. As such,thesecompounds novo cholesterol(sterol I) synthesisso they must obtain it haveprovidedvaluabletoolsfor paleoceanographers in SST and directlyfromtheirdietor indirectly by dealkylating ingested C28 pCO2reconstructions. Furthermore, additionalinformationabout and C29sterols[Goad, 1981]. Besidessterols,modificationof the origins of other biomarkersin sedimentsbased on their other phytoplanktonlipids in general by zooplanktonis individual1Stsc values[e.g.,Hayeset al., 1990;Freemanet al., importantasthereis someselectivitytowardthe components that 1990] has aided geochemists in reconstructing ancient are digested[e.g., Volkrnanet al., 1980c; Tanoueet al., 1982; environments of deposition[e.g.,Freemanet al., 1990;Hayeset Prahl et al., 1984;Harveyet al., 1987]. al., 1990; Kohnen et al., 1992; Collister et al., 1992, 1994; In termsof feedingrelationships the stablecarbonisotopic Schoutenet al., 1997; Grice et al., 1996, 1997]. In this respect compositionof the whole body tissueof a consumershowsa the •Sl•Cof steroids(sterolsand steranes) havebeenusedas a systematic increase in l•C relativeto itsdietwhichis estimated to stable carbon isotopic reference point for phytoplankton be-0.5%o-1% o (within analyticaluncertainties) per trophiclevel populationsin past environments with the assumption that the [De Niro and Epstein, 1978]. It is thereforeimportant to stablecarbonisotopicfractionationeffects associatedwith the determine whether such an enrichment in 13Cof a consumer is a biosynthesis of sterolsaswell ascell sizes/cellgeometry[Poppet generalfeaturefor individualconstituents suchas algal lipids al., 1998] andvariablegrowthrates[e.g., Goerickeet al., 1994; whichare consumed, retained,and/oregestedby zooplankton. Lawset al., 1995]of the eukaryoticcommunitymaybe averaged What carbonisotopicfractionationeffectsdoes zooplankton outfor sedimentsamplescoveringtensto hundredsof years. suchassteroIswhichcan Grazing of phytoplankton by herbivorous organisms herbivoryhaveon ingestedcomponents be further modified? Furthermore, does it invalidate the use of (zooplankton) can greatly reduce the direct contribution of othercomponents suchas alkenonesas a proxy of sea surface temperature (SST) and pCO27 As studieson carbonisotopic t Now at Petroleum Environmental OrganicGeochemistry Centre, fractionations of individualbiologicalconstituents withinpelagic Schoolof Applied Chemistry,Curtin Universityof Technology,Perth, Western Australia, Australia.
2 Laboratoired'Oc6anographie et de Biog6ochimie, Facult6de Luminy, Marseille,France.
Copyright 1998 by the AmericanGeophysicalUnion. Papernumber98PA01871. 0883-8305/98/98PA-01871
$12.00
686
food webs have been limited, we have carried out a series of
continuous algalculturefeedingexperiments usingthe copepod Ternoralongicornis(Mfiller) representative of mesozooplankton to assess the effectof zooplanktonherbivoryon the stablecarbon isotopiccomposition of eukaryoticmarkers.Isochrysis galbana andRhodomonas sp.werechosen for thefeedingexperiments as theyhavebeenshownto be readilyingested by copepods [e.g.,
GRICE ET AL.: ZOOPLANKTON
HERBIVORY
EFFECTS ON BIOMARKER
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687
Harris, 1996]andlackcholesterol. In addition,I. galbanawas
washing withdoubledistilledwaterandcentrifugation to remove
especiallychosenas it is known to biosynthesizelong-chain alkenones[e.g.,Pattersonet al., 1994], was easilycultured,and was readily availablefor the experiment,thoughit shouldbe noted that I. galbana is not an open-oceanhaptophyte.The
any floating debris. The remaining undesirabledebris was
distributions and •513C valuesof sterols(and alkenonesfor I. galbana) in the food sourceare comparedwith those in the copepodsand in the pellets egestedfrom the copepodon the corresponding diet.
removedwith a Pasteurpipetteundera dissecting microscope. An aliquot of the algae from the overflow was also collected typically every 2 days and centrifuged.At the end of the experimentthe copepodbodieswere retainedfor analysis.All sampleswerefreeze-driedovernightand kept at -20øCto avoid bacterialdegradation [HonjoandRoman,1978].
2.2. ControlledMesocosmFeedingExperimentWith IsotopicallyAlternating Food
2. Experimental Procedure 2.1. Controlled MesocosmFeeding Experiments
Two independent grazingexperiments (Figure 1) were carried out,the first oneusinga haptophyte(I. galbana) as the principal food source and the second one using a cryptophyte (Rhodomonassp.) as the principal food source. Both algal cultureswere grown continuouslyat 15øCwith a constantair supplyin an F/2 medium [Guillard, 1975] with nitrate as the limitingnutrientanda 16:8 light (L)/dark (D) regime.A dilution rate of 0.16 per day was used in order to maintaina constant
In an additionalexperimentthe 813C of the food was manipulatedto trace the flow of carbonthroughthe refined ecosystem. Two continuousI. galbana cultureswere used.One culturewas only fed with air; the other was suppliedwith a mixture of air and isotopicallylight CO2. The stablecarbon isotopic compositionof the second culture was allowed to
stabilizefor a periodof 2 weeksprior to the experiment.An aliquotof the algal culturewas extracted,and the •513C of the sterols and alkenones were determined by isotope ratio
carbonisotopicfractionation (•2Crelativeto •3C)of the algae monitoring-gas chromatography/massspectrometry (irmrelativeto 15•3C of theinorganiccarbonsourceandhenceconstant GC/MS) at regularintervalsuntil constant•513Cvalueswere 15•3Cvaluesof the lipids in each alga. This was checkedby observed.However,when the experimentcommenced, the •513C monitoring the15•Cof thelipidsandresidual cellmaterialof the of the lipids in the algae fluctuatedsomewhat(see below) continuousculture of L galbana reportedby Schoutenet al. perhaps because of somevariationin theflowrateof theCO2gas [19981. Mixed stage V copepoditeand adults of T. longicornis obtainedfrom a breedingstock[Klein Breteleret al., 1990] were adaptedto the algalfoodsourcefor a periodof 4 daysprior to the experiment. The feedingexperiments werecarriedout in the dark at 15øCin 22 L of doublyfiltered(2 gm) seawatertypicallyfor a periodof up to 1 week (Figure 1). In orderto avoidcoprophagy [Nealet al., 1986],pelletswereseparated fromthecopepods with a 200 gm nylon meshsuspended 2 cm abovethe bottomof the
tank.Foodsupplywasregulated (5 mL h4- 300 gg C L d4) via a peristalticpumpfrom the overflowof the cultures.During the experimentsthe food quality was checkedregularly under a microscope.Typically, every 2 days the copepods(-50), a quarterof the seawater(and algal food), and the 200 gm nylon
stream.
The mesocosmtank setupwas identicalto that used in the aboveexperiments.The copepodswere fed with I. galbana at a
concentration of-300 gg C L-I. Copepods wereadapted to the first(standard) I. galbanaculturefor a periodof 4 daysprior to the experiment.The copepodswere fed with this culture for another4 days.ThereafterthecarbonisotopicallylightI. galbana culturewas fed to the copepodsfor 6 days,after which the food was switchedback to the first I. galbana culturefor a further4 days.Throughout the experiment,every2 daysthe faecalpellets, a numberof copepods(-50), and an aliquot of the I. galbana food were collectedfor analysis.For most samples(but not all
mesh were transferred into a new tank of filtered seawater. The
because of the low relativeamountsof material),•513C valuesof steroIsand alkenonesin the extractswere determinedby irmGC/MS.The•513C valuesof theresidualcellmaterials afterlipid
faecalpelletswere collectedtypically every 2 days under dim light by sequentialfiltration(200, 50, and 20 gm nylon mesh),
extractionhave been observedelsewhere[Grice, unpublished results].
air
f/2medium
I
5 ml h-•from overflow of
pumpcontinuous culture
/ 7,1&• 22 Ltank (filtered seawater) '/ O I faecal pellets
Figure 1. Schematic diagramshowingthebasicsetupof themesocosm feedingexperiment.
688
GRICE ET AL.- ZOOPLANKTON HERBIVORY EFFECTS ON BIOMARKER PROXY RECORDS lOO
2.3. Analysis of Lipids
Rhodomonassp.
Detailsof lipid analysis(extractionandanalyticaltechniques) aredescribed by Schoutenet al. [1998]. 60
3. Results 3.1.
and Discussion
SteroIs
The major sterol biosynthesizedby I. galbana is 24-
methylcholesta-5,22E-dien-313-ol (sterolII, C28:2 ASa•;Figures2a and 3). The copepodT. longicornisfed on this algaecontains three main sterols (Figures 2b and Figure 3), namely I,
cholesterol; III, chotesta-5,24-dien-313-ol (C•7:•AS'•4); andII, the
Isochrysisgalbana
ao•
dietarysterolanda low relativeabundance of IV, cholesta-5,22-
dien-313-ol (C27:• AS'•).It is interesting to notethedominance of cholesterol(I) and the low relative abundanceof the dietary sterol(II) in the copepod.The steroldistributionin the faecal pellets(Figure2c) resembles the I. galbanaprofile,althoughit contains in addition a low relative abundance of cholesterol.
Rhodomonas sp.biosynthesizes two steroIs,C28:2 h5'22 (II) and C27:2 h5'24 (III) (Figure3). Temorafed on thisalgae,like thatfed
algae
copepod
faecal pellets
on I. galbana,containa high relativeamountof cholesterol (I)
and C27:2 h5'24sterol(III; Figure3) and a very low relative abundance of C27:2 A5'22 (IV). Again,thesteroldistributions in the
SteroIs
Alkenones
I
I
(a) Figure 3. Bar plots showing the relative percent of sterols: I, cholesterol'II, C282 A5'22; and III, C272 ATM,in algae,copepods, and faecalpelletswith Rhodomonas sp.andIsochyrsisgalbana.
HO '• •
39 faecalpellets(Figure 3) resemblethe algal diet, althoughthe
(b)
C37
(c)
C38
IIJ,l• c39 relative retention time
••.
_
dietaryC27:2 AS'24 sterol(III) is presentin muchlowerrelative abundance (percent)thanin the food source,andin contrastis the mostdominantsterolin the copepod. On the basisof the resultsof the two feedingexperiments the generalcompositionof sterolsin the copepodbody seemsto be independentof the sterol compositionof the food. Previous feedingexperimentsby Volkmanet al. [1980c], Prahl et al. [1984], andHarvey et al. [1987] showedthat whenthe copepod Calanushelgolandicus wasfed herbivorously on cholesterol-free diets of Hymenomonascarterae, Dunaliella primolecta, and Scrippsiellatrochoideait alsoproduceda substantialamountof cholesterol in its bodyandin eachcasereleasedfaecalpelletsin whichthis componentwas alsopresent.Thus, the distributionof sterolsin the copepodC. helgolandicus (previousstudies)and T. longicornis(this study), and the pellets egestedfrom them is consistent with the hypothesisthat the copepoddealkylatesthe C2aprecursorsterolto cholesterol as it is unableto biosynthesize this componentde novo [Goad, 1981]. Furthermore,Vollonanet al. [1980c]andPrahl et al. [1984], founda significantamountof
Figure 2. Partial gaschromatograms of lipid extractsof (a) Isochrysis galbana,(b) Temoralongicornisfed on a diet of Isochrysisgalbana for 1 week,and(c) faecalpelletsreleasedfrom Temoralongicornisfed on a diet of Isochrysisgalbana for 1 week. The sterolsare I, cholesterol;II,
the C27:2 AS'24 sterolin the copepodbody,whichwas actually
C282A5'22; andIII, C27:2 hTM.
discussed below.
lacking in the original diet (see the I. galbana experiment mentionedabove). Possible reasonsfor this observationare
GRICE ET AL.: ZOOPLANKTON HERBIVORY EFFECTSON BIOMARKER PROXY RECORDS
689
Table1. StableCarboniS0t6pi•Cømp0sitions •5'3C + 0 of C37andC38Alken0nes •d SteroIs in Isochrysis galbana, Copepod, andPellets Released FromTernora lon•icornis Fedona DietofIsochry, sisgalbana Day
Algae C37
Algae C38
Algae Sterol II
Copepod SterolsII and III
2 4 6 8
-18.0(0.3)3 -17.8(0.4)3 -17.3(0.1)2 -17.2(0.4)2
-17.0(0.4)2 -18.4(0.3)2 -16.8(0.2)2 -16.9(0.3)2
-20.1(0.3)2 -19.8(0.1)2 -19.2(0.1)2 -19.1(0.1)2
...... ....... ....... -18.82
Copepod Sterol I
Pellets C37
Pellets C38
-19.4(0.4)2
n.d. 17.3(0.1)2 16.6(0.5)2 -16.8(0.3)2
n.d. -17.8(0.6)2 -17.8(0.1)2 -16.7(0.1)2
• Superscript numbersindicatethe numberof analyses; numbersin parentheses indicatec• standarddeviation4; n.d. meansnot determined.Carbonisotopiccompositionsare given in per mil. There is a lack of data on the metabolism of cholesterol in sterols,whichare--19.6%0 (average)for I. galbanaand -18.8%0 sp. Furthermore, the /5•3Cof cholesterol (copepods, but it is possiblethat eukaryoticsterolsare converted for Rhodornonas into cholesterol in amountsexceedingthe metabolicrequirements 19.2%o)in the pelletsreleasedfrom Ternorafed on Rhodornonas sp. is identical(within analyticaluncertainty) to /5•3Cof the by the copepod.Thusthe excesscholesterolis not absorbedand could be either broken down to acetate and/or released from the sterols(-18.8%o)in the diet (Table 2). Theseresultssuggestthat in the copepodbodiesandin the pelletsegestedfrom gut into the faecalpellets.In a similarway the excessdietary cholesterol sterolnot requiredis alsoegested.A contributionof cholesterol the copepodare derivedfrom their corresponding dietary sterols in the faecalmaterialcould arise from the releaseof epithelial which is corroboratedby data obtainedfrom the alternating tissuelining the copepodgut becauseof rupturingduring the feeding experimentusing two culturesof the same alga with digestiveprocess[Harvey et al., 1987, and referencestherein]. differentstablecarbonisotopiccompositions(Figure 4a). The The reasonsgiven abovemay explain why cholesteroland the stablecarbonisotopiccompositions of the lipids in the two I. dietary sterolare observedin the faecal pellets.However, the galbana culturesare 20%o-30%o different (Figure 4a). For the extentto which the excesscholesteroland the dietary sterolare first4 daysthe•5•3C of thesterols (in thecopepods) areidentical egestedwill dependupon the nutritionalstatusof the copepod to thosein the I. galbanacultureon which the copepods have (i.e., presentfood conditions),metabolicneedsof the copepod, beenfed. Whenthe food supplyis changedto the isotopically and its feedingand digestiveenzymeactivities.Harvey et al. lightI. galbana,the•5•3C valuesof the sterolsin T. longicornis [1987] found that highestassimilationefficienciesfor sterols shift towardthoseof the sterolof the isotopicallylight algae, were at a lower food level than at increased food levels; the althoughthey do not reach(in the given time) the samevalue. This is, in part, an effect of dilution with the former culture of relative abundanceof sterolsdid not changesignificantlywith the level of the samediet, indicatingthatthe copepodonly retains which one quarter of the volume was retained to keep the whatit requiresfor its ownmetabolicneeds. copepods well fed.In addition,the•5•3C of sterols probablyalso On the basisof previouswork and this studyit is clear that takea longertime to reachthe light algal signatureas a resultof cholesterolis requiredby marine crustaceanssuch as pelagic the presenceof isotopicallyheavy sterolsin the copepodsfrom copepods. It alsoseemslikely that cholesterolis formedthrough thefirstphaseof the experiment.However,sincecholesterolis an modificationsof other sterolswhen cholesterolis lacking in a important componentof all cell membranesand the steroid precursorof manybioactivemoleculessuchas steroidhormones copepod'sdiet. Additionalconfirmationof this view comesfrom [Teshirnaand Kanazawa, 1971] the immediatedietaryintake of the stable carbon isotopic compositionsof sterols in the mesocosm experiments. The stablecarbonisotopiccompositions sterolsis continuallyrequiredby the copepodfor its survival of cholesterolin Temora are -19.4%0 (Table 1) and -19.1%0 [Harrison, 1990, and referencestherein] and leads to a shift (Table 2) fed on I. galbanaand Rhodomonas sp., respectively. toward light values.The mobilizationof previouslyconsumed biosynthesis Interestingly,these values are identical (i.e., within analytical sterolreservesmay alsoplay a role in cholesterol uncertainty) to the •5•3Cvaluesof the corresponding dietary [Harrison, 1990 and referencestherein], but the observed isotopic shift indicates that this is not important in our experiment.In summary,the stablecarbonisotopiccomposition of cholesterol bearsthe •5•3Csignature of its dietaryprecursor Table2. StableCarbonIsotopicCompositions •5•3C + sterol,indicatingthat possiblecarbonisotopicfractionations that o of Sterolsin Rhodornonas sp.,Copepod,andPellets may occur through modifications of the dietary sterols are Releasedfrom TernoralongicornisFed on a Diet of negligible. Rhodomonas sp The stablecarbonisotopiccompositionof the two partially Values coelutingsterols(i.e., II and III; ~-18.8%o)in Temorafed on a diet of !. galbana(Table 1) is similarto cholesterol (-19.4%o), 13 AlgaeSterolII andIII 18.82 alt__hoBgh so__me mi__no_r. variationsin their individual •5 C values Copepod SterolII andIll • •19:32 may existgiventhata 1%odifferenceis apparentbe•een the two CopepodSterolI -19.1(0.0)2 coelutingsterolsandthe averageof the dietarysterol(--19.6%o). PelletsSterolI -19.2(0.0)2
• Superscript numbers indicatethenumbe r of analYSeS; numbers in parentheses indicatec•standard deviation4. n.d. means not determined.Carbon isotopic compositionsare givenin per mil.
These. •results .tentatively' suggest •that•the•C2712 •ATM sterolis slightlymore enrichedin •3C than the othersterols,but this differenceis relativelysmallgiventhe analyticalerror.The stable carbon isotopic compositionsof the two partially coeluting
690
GRICE ET AL.: ZOOPLANKTON
HERBIVORY
EFFECTS ON BIOMARKER
PROXY RECORDS
(a) SteroIs
'*'C282A 5,22
-16
r• cholesterol
I-IC282• 5,22 and C272/• 5,24
-24
-32 -40
-48
I (b) Alkenones
%o
0 C37 mC3•
-16 _
Day
algae
•..... Isochrysis culture 1
faecal pellets I
Isochrysis culture 2
copepod
(airandlight CO2)
bodies
Figure4. Stablecarbonisotopiccompositions of lipidsin algae,Temoralongicornis, andfaecalpellets,in an experiment fed alternately withIsochrysis galbanaof differentisotopiccomposition: (a) steroIsand(b) alkenones.
steroIs(-19.3%o) in TernoraandtheC2s:2 A5'2•sterol(-20.0%o)in the pellets released from this copepod fed on a diet of
Rhodornonas sp.alsoindicatethatthe C•7:2 A5'24 sterolmaybe slightlymoreenriched in •3C,butthisdifference is againwithin the analyticalerror.Evenso,cholesterolin boththe faecalpellets HO
andTernora haveidentical fi•3Cvaluesto theaverage fi•3Cvalue of thetwo dietarysterols(-18.8%o,Table 2). On the basisof these data it seems likely that cholesterolis formed through demethylation of the eukaryoticsterolII (Figure5; i.e., at the C24 positionleadingto a doublebond at A24positionand a
.•/',•methylation
relativelyminor amountof the sterolwith the doublebond at the
A22 position)followedby reductionof the doublebond at A24/A22as proposed in earlierstudies [e.g.,Goadetal., 1981]. HO 3.2. Alkenones
Theprincipalalkenones biosynthesized by I. galbanaareC37alken-2-onesand C3s-alken-3-ones with 2-4 degrees of unsaturation (Figure2a) [cf. Pattersonet al., 1994]. Unlike the sterols,the alkenonesare completelyabsentin Ternorafed on a diet of I. galbana (Figure 2b), althoughthey are presentin substantial amountsin the faecal pelletsegested(Figure 2c). Theseresultsarein agreement with previousfeedingexperiments carriedout on E. huxleyi[Volkmanet al., 1980a] where the copepodC. helgolandicus assimilated only a smallproportion of alkenones;most of the alkenonesin the original food were
HO
IV
III
'• reduction HO
I
Figure 5. Steroltransformations in a copepod.
GRICEET AL.:ZOOPLANKTON HERBIVORYEFFECTSON BIOMARKERPROXYRECORDS 0.15
691
pellets •own•Thester0I is,however, more enriched in •3Cthanthe
algae 0.1
Uk'37
fatty acids,probablybecauseof the differentfractionationeffects occurringin the biosynthesisof isopentyl diphosphatefrom pyruvateandglyceraldehyde [e.g.Rohmeret al., 1996].
The •513C valuesof the alkenones fromthe alternating feeding experimentshow that when the food supplyis changedto the
0.05
isotopically lightI. galbana,the •513C of the alkenones in the pelletstakes ---6days to reach almostthe same stablecarbon I I I [ isotopiccomposition asthat of the alkenonesin the light culture 0 2 4 6 8 10 (Figure4b). After 6 days,whenthe food sourceis switchedback Day to the first culture,the alkenonesin the pelletstake---4 daysto to the •513C of the alkenones in the algae,whereasthe Figure6. uk'37 indices (withstandard deviation) in Isochrysis galbana acclimate andpelletsreleased fromTemoralongicornis fedona dietof Isochrysis sterolsin the copepodstook even longer (see above). This is galbana(grownata temperature of~ 15øC)fora periodof 1 week. probablyrelatedto the fact that the copepoddoesnot appearto havea metabolicneedfor alkenones,alkenonesbeingcontinually egested by thecopepodin thefaecalmaterial(Figure4b).
egested in thefaecalmaterial.Generally, thedistribution of these components appears to be largelyunaltered by foodprocessing Implications in, for example,the gutsof the musselMytilusedulis[Rowland 4. Paleoceanographic and Volkman,1982] and the anchovyfish [Wakehamet al., From our resultsit is clear that modificationsof eukaryotic 1984].
The uk'37indicesof the alkenones in I. galbanaandin the faecalpelletsegested from Temorafeedingon I. galbanawere
sterolswithin the gutsof copepodsdo not have any significant
6arbonisotopic-fracfi•-nation effects.Sterols,not only tho•e-
presentin the algaebut thosealsopresentin the copepodandits determined overa periodof 1 week(Figure6). In eachcasethe faecalpellets,canconstitute a substantial partof the steroidpool uk'37 wascalculated fromtheGC peakareasof theC•7:2 andC•7:3 of particulateorganiccarbon(POC). Furthermore,the stable
alkenones [cf. Prahl et al., 1988].The uk'•7doesnot differ significantly in thealgaeandthepellets(Figure6). Someminor fluctuations in the uk'37of the algaeare apparent duringthe experiment, but thesevariationsare also represented in the
alkenones of thefaecalpellets(Figure6 andTable3). The stablecarbonisotopiccompositionof the C37and alkenones biosynthesized by I. galbanarangebetween-16.8%o and -18.0%othroughoutthe experiment(Table 1). The average fil3Cvaluesof theC37andC38alkenones areidentical(giventhe
carbonisotopiccompositions of the sterolsstill bearthe •5•3C signatureof the precursorsterols.However,the stablecarbon isotopic compositions of heterotrophic organisms that biosynthesize their biological constituentsde novo may be significantlydifferent from their food source.For example, -15
(a)
analyticaluncertainties) to eachother(Table 1 andFigure7). The isotopiccompositions of the C37and C3• alkenones in the faecalmaterialegested fromTemoracompare verywell with the corresponding fi•3Cvaluesof thealkenones in I. galbana(Table 1 and Figure 7), suggesting that the stablecarbonisotopic compositions of alkenones remainconstant whenpassed through
food
-lg
-23
-•5
(b)
0
the gut of a copepod. The 813Cof the alkenones are more enriched in 13Cthanthesterolsby-•2%o(Table1) [cf. Schouten et al., 1998],probablybecause of thefractionations associated with theirbiosynthesis. Schouten et al. [1998] reportedthe alkenones
in I. galbanato be•4%o-5% omoreenriched in •C thantheC• fattyacid,indicating thata differentpoolof acetylcoenzyme A (fromthatusedfor fatty acids)is usedto biosynthesize the and C3• alkenones, thoughthe locationof biosynthesis is not
0
•
faecal
-19
(5•C
pellets
-2•
%o
-•5
(c) •
-•9
bodies
Table3. uk'37 Indices inIsochrysis galbanaandPellets ReleasedFrom TemoralongicornisFed on a Diet of
Isochrysis $albana
Day
0
Algae
Pellets
2
0.07(0.01)
0.09(< 0.01)
4
0.11 (0.02)
n.d.
8
0.10(
