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ASSIMILATION OF ORGANIC MATTERBY ZOOPLANKTON'RobertJ. ConoverWoodsHole Oceanographicnstitution, oods Hole,Massachusetts 2543

ABSTRACTA methodfordirectmeasurementf percentage f assimilationf organicmatter yzooplanktonhatdoes notrequire hequantitativeecovery f fecesand can be adaptedforuse in the field s well as in laboratorynvestigationss described. f it is assumed hatonlytheorganic raction f the food ngested y a zooplankton nimal s affected y thedigestive rocess, t canbe shown hatpercentage futilizations defined sU'l=f ( E')( ))]X 100,

whereF' is the ash-free ryweight:dryweight atio fraction f organicmatter) n theingested ood, nd E' is the same ratio n a representativeampleof feces.The method ompares avorably,s a measure f assimilation, ithdirectmeasurementof thequantityforganicmatterost from oodduring assagethroughhedigestive ract.

INTRODUCTIONDigestionand assimilationre vital tothetransfer f energyfrom one trophic evelto the next. These processes also influencethedistributionnd composition fparticu-late organic matter at all levels in theoceans. Little is knownabout the forma-tion of particulatedetritus r of its impor-tancein sustaining heanimalscontributingto itsformation.A methodfordeterminingthe fraction f energyremoved by the di-gestiveprocess each timeparticulatematterpasses through n animal s needed.Previous estimates of percentageof as-similation y zooplanktonrange from6 to99% (Conover 1964). Differentspeciesfeedingon different oodsunder differentexperimental onditionsno doubt accountfor ome ofthevariability, utthemethodsused to measure assimilationmay not al-waysgivecomparableresults. The quantityof a radioisotope ccumulatedin the bodyof an animal fed labeled food is not ameasure of assimilationunless it is cor-rected for osses due to recycling. On theotherhand, the directmeasurement f as-similation equires hequantitative ecovery

1Contribution o. 1719 from heWoods HoleOceanographicnstitution. his researchwas sup-ported by National Science FoundationGrantsG-23134and GB-3120.

ofzooplanktonfeces,a processthatcan bedifficultven in laboratory xperimentse-cause pellets are frequently ornopen orreingested by actively feeding animals.Particles of foreign matter and "pseudo-feces," formed from uningested organicmatter oncentrated y feedingmovementsof the copepod's appendages, are difficultto separatefrom ruefeces.The method describedin thispaper re-quires neither he quantitative ecovery ffeces norknowledgeof the amountoffoodeaten if only the percentageassimilated srequired; hence, the method can be usedin the field almost as readily as in thelaboratory.The authorwould like to thankR. R. L.Guillard who supplied the algal culturesand R. F. Vaccaro who made bacterialcounts on the fecal pellets. Technical as-sistancewas providedby ThomasRenshaw,JohnWoodcock, Robin Walden, and Bar-bara Steinberg.

METHODSThe methoddepends on the assumptionthat only the organic component of thefood is significantlyffectedby the diges-tiveprocess. If this assumption s correct,it is necessaryonly to obtain the ratio ofash-free ryweightto dryweight fractionoforganicmatter)for sampleof foodand338

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ASSIMILATION OF ORGANIC MATTER BY ZOOPLANKTON 339a sample of feces to calculate percentageof assimilation,U', usingtheequation

u~ [(1Et)(F'J x 100, (1)where F' and E' are therequiredratiosforthe food and excreta, respectively. Thederivation of this equation is as follows.Food assimilated s definedas that amountof the food ingested which is taken inthroughmembranes fthe digestiveorgansof an animal. Then,percentage f assimila-tion,U', maybe defined s

U'=(I/ )xlOO, (2)where I is the quantityof organicmatteringested, nd N is the quantity xcreted nthefeces. Thus, I is equal to thetotalpar-ticulatematter ngested(F) minus ts ash(Af) and N equal to the total particulatematter xcreted E) minus ts ash (Ae), sothatequation (2) can be rewritten

U'= [(F At) (E e)_ x 100. (3)However,Af s assumedto be equal to Ae;hence, 3) becomes

U' = (F-A-)x 100l (4)F' and E', the ash-freedry weight: dryweightratios for ngestedfood and forthefeces,can now be written

F' = F ' (5)and

El = E (6)Since Af=Ae, equations (5) and (6) canbe rearranged o that

F- Af (7)(1- F')'andE (1-E) (8)

Substitutinghese values forF and E, equa-tion (4) reducesto(I, - E')( P)

Food samples for dryweight determina-tion were filtered onto tared, glass-fiberfilters. Samples and seawaterblankswerewashed with isotonic ammoniumformateto remove adventitious alts and dried at60-70C for at least 12 hr. Blank filtersgenerally howed a smallweight oss or nochange; as there was no evidence ofnitrogen ontamination,ompletesublima-tion of the ammoniumformatemusthaveoccurred. ilters nd sampleswereweighedon a quartz helix microbalance (Micro-chemicalSpecialtiesCo., Berkeley,Califor-nia) or on a Mettler ingle pan microgrambalance. Weight of samplewas determinedby differencerom hefilter are,after or-recting or ny weightchange n theblank.Ash-free ry weightwas sometimesde-termined by differencefrom a secondweighingafter gniting ilters n a mufflefurnace t 450C, but an alternativemethodgave more reproducible esults.When suf-ficient oodmaterialwas available, a thickpad was first deposited on a Millipore'filter f suitableporosity nd washed withammonium formate. A portionwas thencarefullycrapedfrom hefilterwitha dullspatula,dried,weighed, shed,andweighedagain.Fecal pellets were collected in severalways,dependingon thetypeofexperimentand food source. If feedingwas heavy,sufficientmaterial ould be pickedup witha fine-bore queeze bulb pipette n 5 or 10min. When feces were scarce, theywereseparatedfromhefood culture yfiltrationonto a suitableboltingcloth (usually No.25 Nytex). Sometimespellets were col-lected in the fieldwith a suspended trapmade from n 8-inch 20 cm) plasticfunnelfittedwitha shellvial in whichthesampleaccumulated. Feces thus collected werecomparedwitha surface ampleofparticu-latematter akenat the timethetrapswereset out. Alternatively, ooplanktonwerepermittedto feed on natural particulatematter nd feces were collected by pipetteorfiltrations described bove. Field sam-ples werefrozen nd returned o the abora-toryforanalysis.

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340 ROBERT J. CONOVEA

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X 1EE ' ? 1; (v5 cS O co j cS o , i

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t m 0 e > t~~~~~~~~~~ol C CO r-- Na 0 ~~~ ? ~~~ m e t m C b ? O Ho ~~~~~~~~H0

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ASSIMILATION OF ORGANIC MATTER BY ZOOPLANKTON 341Once a sample was collected, t was ex-aminedunderthe dissectingmicroscope na Syracuse stainingdish and any foreignmaterialremoved. If care is taken to keepexperimental lassware freefromdust andotherparticulate ontaminants,his task isgreatlysimplified.The sample was thenswirled gently,causing the pellets to ac-cumulate near the center of the dish. Ex-cesswater was siphoned off eavinga pasteof pellets or, alternatively, arge pelletswere collected nto a bolus and directly e-moved from hewater. In eithercase, pel-letsweretransferreds a lumptothe foldededge of several thicknesses f hard filter

paper (S and S sharkskin)nd were washedby droppingammoniumformateon themwith a capillarytube; each drop was al-lowed to soak intothe paper before ddingthe next. Finally the bolus was transferredto a small tared platinumpan fordrying,weighing, nd ashing. As littleas 20 ,ugofpellets could be used to obtain the neces-saryratiowithan error fusually ess than15% in the finalcalculation of assimilation.In laboratoryexperiments, tage V orfemale Calanus hyperboreus, ither fromthe Gulf of Maine or nearby slope water,were used as experimental nimals. Foodorganismswere chieflybacteria-free ul-tures of marine diatoms and flagellatesfromthe Woods Hole Oceanographic In-stitution ollectionor naturalphytoplank-tonassemblages. The experiments ere runfrom 2 to 24 hr n 1-gallon 4 liter),poly-ethylene ontainerswith small populationsof animals (20-35) in 2 liters of cultureor in pint (0.5 liter) containerswith oneanimal to about 200 ml ofmedium.

EVALUATION OF THE METHODA major fraction fthefood ofherbivor-ous copepods such as Calanus finmarchicusconsists fdiatoms Marshall 1924; Lebour1922). Diatoms have a high ash content,chieflysilica (hydrated SiO2) (Parsons,Stephens, nd Strickland1961). AlthoughLewin (1961) showed that diatom shellsdissolvedfaster t hightemperatures, ighpH, and after reatment ithacid orchelat-ing agents,dissolutionwould probablynot

introduce significanthange n theweightof the ash during the relatively rief pas-sage through he gut of a planktonicherbi-vore.To see if copepods mightcause the dis-solution of silicate from diatom shellscrushed in feeding,three sets of Calanushyperboreus ere each placed in a richcul-ture ofThalassiosirafluviatilis ontainingknownamountofdissolved silicate, nd anadditionalthree ets were placed in filteredseawater of known silicate content. Ap-propriate ontrolswithout nimals were in-cluded. At the end of 46 hr in the dark,therewas no differencen thedissolved ili-cate level (as determined y the methodofMullin and Riley 1955) in the containerswith animals and those without,whetherdiatoms were present or not. Because nu-merous fecal pellets were present in thecontainerswithboth copepods and T. fluvi-atilis,the animalsmust have been feedingactively.On severaloccasions attemptsweremadeto recoverfecal material quantitatively ocompare a directmeasurement f organicmatterassimilatedwith the ratio method.If the fecal pelletswere compact,with lit-tle damage due to recapture, nd if therewas no tendencyforclumpsof uningestedparticulatematter o be formed, greementbetweenthemethodswas goodwithdiatomfood (T. fluviatilis).Anexampleofanotherexperimentwith C. hyperboreus sing thedinoflagellate xuviellasp. as a food sourceis shown in Table 1. The resultsof thisexperiment re particularly ignificant e-cause they suggestthat the ratio methodmay give a reasonable estimate fpercent-age of assimilation ven whenthefood or-ganism does not containa largefraction frelativelynert ilica.

BACTERIA IN THE FECESThe feces of higheranimals are knownto containmanybacteria thatplay an im-portant ole in the digestiveprocess. Noth-ing is knownregarding he intestinal loraof the zooplankton,but large numbersofbacteria could contribute ignificantlyo

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342 ROBERT J. CONOVERTABLE 2. Number f bacteria n fecalpellets roducedby C. hyperboreustage V feeding n differentfoods

No. No.Date Temp. of No. bacteria/ bacteria/mg(1963) Food incubation bacteria/ mgdry wt organcw(C) fecal pellet ofpellet ani wt->K10-6 1-26 Sept Thalassiosiraluviatilis ca. 25 150ca. 25 4009 Oct T. fluviatilis ca. 25 1,20016 Oct T. fluviatilis ca. 25 2,700 4.4 13.5Dunaliella p. ca. 25 4,520 6.4 10.524 Oct T. fluviatilis ca. 25 1,800 2.3 6.45Skeletonemaostatum ca. 25 1,500 2.5 6.25Coscinodiscussp. ca. 25 625 0.9 1.82T. fluviatilis 4 1,200 1.5 4.3S. costatum 4 300 0.5 1.25Coscinodiscusp. 4 375 0.53 1.131 Oct T. fluviatilis ca. 25 250 0.5 1.8T. fluviatilisafter daysat room emperature) ca. 25 3,440S. costatum ca. 25 1,480 2.4 5.7S. costatumafter daysat room emperature) ca. 25 1,590Coscinodiscusp. ca. 25 730 0.65 2.35Coscinodiscusp. (afterdaysat room empera-ture) ca. 25 11,0007 Nov T. fluviatilis ca. 25 580 1.35 4.4521 Nov T. fluviatilis ca. 25 580 0.88 2.23T. fluviatilis ca. 25 370 0.56 1.42T. fluviatilis ca. 25 510 0.775 1.95

the quantity f organicmatter n the fecesor break down some of the unassimilatedorganic matter after defecation. Newell(1965) has shownthatfresh ecesproducedby the deposit-feedingmolluscs Hydrobiaulvae and Macoma balthica wererelativelyhigh n organiccarbon and low innitrogen,but after everaldays of incubation heni-trogen content n the pellets rose and thecarbon level fell slightly.Apparently ac-teria were using fecal carbon as an energysource and utilizingsome source of dis-solved nitrogen oform heir wnbodysub-stance.To obtain an approximatequantitativeestimateofbacterialabundance,C. hyper-boreus feces were ground, uitablydiluted,and plated on seawater agar enrichedwithbeef extract nd bacto-peptone.The countsare summarizednTable 2. Assuming achbacteriumhas a densityof one and a vol-

umeof 1 /3, thehighestoncentrationsb-served,2,700 bacteriaper pelletforThalas-siosirafluviatilisnd 4,520forDunaliella(16 October 1963), would represent asmuchas (13.5 x 106/109)x 100 = 1.35 nd1.05%,respectively,f the totalorganicmat-terpresent n thefeces only fthebacteriacontained no water or ash. It is doubtfulthat ll bacteria nthefeceswerefavored ythemediumused, but even if countswerelow by an order of magnitude, bacteriawould seldom have contributedmore thana fewper cent to the totalorganic contentof thefeces. When pellets were incubatedat 4C (the temperature t which most ofthe assimilation xperimentswere carriedout) thetotal countwas lower thanat 25C,and the ncubation eriod had tobe at leastdoubled beforecolonieswere largeenoughto count. When assimilationexperimentsare run at low temperatures or24 hr or

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ASSIMILATION OF ORGANIC MATTER BY ZOOPLANKTON 343TABLE 3. Assimilationfnatural rganicmatter

LocationDetCruise Date Animals Depth %(N lat) (W long) (in) assimilationGosnold 40 20 Mar 64 42050' 69050' Meganyctiphanes surface 65.0norvegicaCrawford 06 8-9 Apr 64 42?50' 69050' mixed ooplankton 10 81.2Crawford 106 13-14 Apr 64 41055.2' 65014.2' mixed ooplankton surface 32.5Crawford 06 13-14 Apr 64 420261 67?10' mixed ooplankton 100 72.8Crawford 106 15-16 Apr 64 43024' 67048' mixed ooplankton surface 45.9Crawford 106 15-16 Apr 64 43024' 67048' mixed ooplankton surface 3(6.7Crawford 106 15-16 Apr 64 43024' 67048' mixed ooplankton surface 65.2Crawford 106 17-18 Apr 64 420 12' 69039' mixed ooplankton 10 72.6Crawford 06 17-18 Apr 64 42012' 69039' mixed ooplankton 50 79.6Crawford 106 17-18 Apr 64 42012' 69039' mixed ooplankton 100 87.1Gosnold 44 8-9 July 64 42050' 69050' mixed ooplankton surface 75.7Gosnold 44 8-9 July 64 42050' 69?50' mixed ooplankton surface 79.5Gosnold 44 8-9 July 64 42050' 69050' mixed ooplankton 10 74.3Gosnold 44 8-9 July 64 42o5OQ 69050' mixed ooplankton 50 72.7Gosnold 44 8-9 July 64 42050' 69050' mixed ooplankton 100 88.7Gosnold 44 10-11 May 64 41055' 69014' mixed ooplankton surface 38.6Gosnold 44 10-11 May 64 41o55' 69?14' mixed ooplankton 50 78.1Gosnold 44 10-11 May 64 41o55' 69014' mixed ooplankton 100 92.1Gosnold68 14-15 May 65 42011.3' 69039' mixed ooplankton surface 54.1Gosnold 68 13-15 May 65 39050.3' 69033.3' mixed ooplankton surface 40.7Gosnold 68 13-15 May 65 39050.3' 69033.3' C. hyperboreus surface 43.8Gosnold 71 10 May 65 39046' 69044.5' Euchirella rostrata surface 17.7Gosnold71 11-12 May 65 42015.6' 69041.0' mixed ooplankton surface 80.6Gosnold 71 12 May 65 420?17.6E' 69036.7' mixed ooplankton surface 40.0Gosnold 71 12 May 65 42017.6' 69?36.7' mixed ooplankton surface 58.2Gosnold 71 12 May 65 420 16.5' 69042' C. hyperboreus surface 69.5

less, bacteria in the feces probably havelittlequantitativeeffecton the estimationofpercentageof assimilation.ASSIMILATION OF ORGANIC MATTER

IN NATUREAssuming that the ash content of nat-ural particulate organic matter is unaf-fectedby the digestive process, it shouldbe possible to determine he percentageofassimilationfrom a naturalpopulationbythe zooplankton f representative amplesof uneaten organicmatter and feces pro-duced from tare available. Corner 1961)presented videncethatCalanus helgoland-icuspreferentiallyelected theorganicfrac-

tionofthe particulatematternnatural ea-wateroff lymouth, ngland. Ifparticulatematterwas selected on the basis of its or-ganic content, serious errorwould be in-troduced into the calculation of assimila-

tion.On severaloccasions,feceswerefoundto contain a smallerash fraction han theparticulatematteroccurring n the water;but away fromthe coast where the inor-ganicparticulate ractionmightbe affectedby material of terrestrialrigin,fieldesti-mates of assimilationwere usually com-parable to laboratorymeasurementsTable3, cf. Table 1).When traps were used to collect fecesfromdifferent epths,the ash content npellets from deeper water was alwayshigher, ndicating a largerpercentage as-similated therethan in the surfacewater(Table 3). The values derivedfromfecescapturedat 50 or 100 m should be consid-ered maximum stimates, ecause somepar-ticulatematter t thesedepthsprobablyhasalready passed through he intestine f an-other nimal orundergonepartialdegrada-tionbybacteriabefore ngestion nd so has

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344 ROBERT J. CONOVERa higher sh content hanthe surface am-ple used forthe calculations.

DISCUSSIONTo determine ssimilation ccuratelybythe ratiomethoddescribed in this paper,organicmatterand ash must be ingestedby thezooplanktonn the sameproportionsas they occur in the naturalfood. Alterna-tive methodswould also yield inaccurateresults forganicmaterialwas lost from heparticulate ractionwithoutbeingingested;othermethodsgenerally lso requirequan-titative recovery of feces. Beklemishev(1954) and Cushing 1955) observedcope-

pods ingestingthe cell contentsbut notthe testsof large diatoms. Corner (1961)found that C. helgolandicus removed ahigher percentage of particulate organicthanof inorganicmatterfromnaturalsea-water taken at the entrance to PlymouthSound,England. Because the sampling o-cationwas near land, a significant ortionof the uningested norganicmattercouldhave been terrigenous.On theotherhand,C. hyperboreusompletelyngested hetestof a large Coscinodiscusbut probably ostsome of the cell sap duringthe feedingprocess (Conover 1966). About15% ofthecarbonremovedfrom culture f T. fluvia-tilisby the feedingof C. hyperboreuswasreturnedto the medium as dissolved or-ganic matter,presumably some throughleakage from cells damaged during thefeedingprocessand theremainder hroughexcretionby the animals (Hellebust andConover, unpublisheddata).At present, he effect f selective nges-tion of organic matteror the leakage oforganic matter fromcrushed cells on thedeterminationf assimilationby the ratiomethodcannot be assessed. If zooplanktoningesta higherproportion f organic mat-ter than inorganic n their food, as sug-gestedby Corner, he ratio methodwouldgivea deceptivelyow estimate fassimila-tion, while if the reverse were true, assuggestedby the preliminaryxperimentsof Hellebust and Conover, assimilationwould appear too high. However, in the

laboratory he ratio method gave reason-ably consistent stimatesof percentageofassimilation forganicmatter y zooplank-ton,whether hefoodconsistedof the dia-tomT. fluviatilis, ithhighsilica ash con-tent,or the dinoflagellateExuviella, witha relatively ow ash, and resultsobtainedfromnaturalpopulationsagreedwell withthose from controlled laboratoryexperi-ments.Whateverthe error n the ratiomethod,the results btained also comparefavorablywith some earlierestimates rom he litera-ture see Table 1,Conover1964). Becauseonlyorganicmatter ctuallyremovedfromthe feces (ratherthanthe fraction ppear-ing in the tissues of the animals) is con-sidered, there is no confusionusing theratio method whether assimilation orgrowth fficiencys beingmeasured (as inmany experimentsinvolving radioactivetracers). The quantitative ecovery ffeces(a laboriousprocess and subject to error)is usuallynecessary omeasureassimilation.S. M. Haq (personal communication)hasrecently hownthatthe copepod Metridialonga does not consistentlyormfecal pel-lets butdefecates portion f tsundigestedfood in small, unrecognizablefragments.When large quantitiesof feces are to berecovered by filtration r centrifugation,care mustbe taken to keep food culturesuncontaminatedydust, int, nd other ineparticulate matter of atmosphericoriginthatquicklyaccumulateson glassware, ndso on. Cotton plugs, used to keep algalculturesfree frombacterialcontamination,contribute onsiderably o particulatecon-tamination. Althoughprecautions shouldalwaysbe taken to limit he ntroductionfforeign ontaminants,n adequate samplefor determination f assimilationby theratiomethodcan be recoveredby sortingunder a microscopeeven if a mistakehasbeen made. The measurement f the as-similation findividualcompoundsor com-ponentsof organic matter uch as nitrogenorphosphoruss also facilitated yrelatingthem to the ash fraction.The methodhasthe added advantageof simplicitynd lowequipmentcost.

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ASSIMILATION OF ORGANIC MATTER BY ZOOPLANKTON 345REFERENCESBEKLEMISHEV,C. W. 1954. Feedingof severalcommonplanktoncopepods in far easternseas[in Russian].Zool. Zh.,33: 1210-1230.

CONOVER, R. J. 1964. Food relationsnd nutri-tion of zooplankton.Symp. Exptl. MarineEcol. OccasionalPubl.No. 2,p. 81-91. Grad-uate School Oceanog., Univ. R.I., Kingston.. 1966. Feeding on large particlesbyCalanushyperboreus,. 187-194. In HaroldBames [ed.], Some contemporarystudies inmarinecience.GeorgeAllen ndUnwinLtd.,London.CORNER,E. D. S. 1961. On the nutritionndmetabolism f zooplankton. . Preliminaryobservationsn the feedingof the marinecopepod,Calanus helgolandicusClaus). J.MarineBiol. Assoc. U.K., 41: 5-16.CUSHING,D. H. 1955. Productionnd a pelagicfishery. isherynvest.,London,Ser. 2, 18:

1-104.LEBOUR, M. V. 1922. The food of planktonorganisms.J. MarineBiol. Assoc.U.K., 12:644-677.LEWIN, J.C. 1961. The dissolutionf silicafromdiatomwalls. Geochim.Cosmochim.Acta,21: 182-19,8.MARSHALL, S. 1924. The food of Calanus fin-marchicusuring 923. J.MarineBiol.Assoc.U.K.,13: 473-479.MULLIN, J. B., AND J. P. RnIEy. 1955. Thecolorimetric eterminationf silicate withspecial referenceo sea and naturalwaters.Anal. Chim.Acta,12: 162-176.NEWELL, R. 1965. Detritus n the nutritionfmarinemollusks. Proc. Zool. Soc. London,144 (Part 1): 25-45.PARSONS, . R.,K. STEPHENS, AND J.D. H. STRICK-LAND. 1961. On the chemicalcompositionof eleven species of marinephytoplankton.J.FisheriesRes. BoardCan., 18: 1001-1016.