degree in

EFFECTS OFSMALL-SCALETURBULENCE ON MICROZOOPLANKTON PREDATOR-PREYINTERAcnONS

by

MARCIANNAPTAK, B.Sc.

A thesissubmittedtotheSchoolof Qrad uateStudies inpanialfulfillme ntof me requirementsfor thedegreeof

Master of Science

DepartmentofBiology OceanSciences Centre Memorial UniversityofNewfoundland

April 1998

SLlohn's Newfoundland

ABSTRACT

Thebiologic:aIand chemicalprocesseswhich affect bacterioplanktongrowthand mortality havebeenwellstudied.Littleisknownaboutphysicalcontrolsonmiaobial systems.Turbulentmixingcan influencetherates of preyencounterandgrazing characteristicsofsmallmetazoangrazers,ho wev er models suggest thatsmell-scale turbulence (Kolmogorovlength scale) shouldnot haveaneffect on microorgani smsless than 10micrometersRecentstudies suggestthat.despit e theory,turbulent mixingcan influencemicroflagellates. Thisstudyhasexaminedtheinteractionsbetweentemperature (rt',5"',I<t,IS"C)andturbulentmixingonthegrowthandtrophodynamics between the heterotrophicmicro8agellateParaphysomonas impeiforataanditsbacterialprey,Vibrio splendidus.It wasfoundthatgrowth rates ofP.imperjOl'alaatS toIS*Cwere1.05to2 foldhigher underturbulent comparedto staticconditions.However.as the temperature decreased fromIS to O"C.ingesti onand clearance ratesincreased10 fold.but no significantdifferencewasfound betweentheturbulentandstaticconditions.Itisbelieved thattheincreasedgrowthratesinthef1ageUatesat the warmertemperatur es inthe turbulent oonditionwereduetoincreased encounter ratesbetweenthemiaoflageUateand bacteria."Thehigheringestionandclearance ratesatthecoldertemperaturesarebelieved tobedue tothe increaseintheviscosityof seawater,allo wingflagellatestomovea greater volumeafwateracro ssitsboundary layer.Theseresu lts suggest thatgrowthand ingestionrates determineddurin g static incubations ofinsitusamples underISoCfrom previousl ypublished studiesmaybeover orunderestimatedsinceturbulence iscontinuous innature.

Table of Contents Ah>md..

Listo fTablc:s...

Listof Figures...

Listcf'Notations .

Acknowledgements...

Chapter1...

1.1Background..•.

1.2 ThesisObjectives...

. ...•..•...• . ...•.• .. ....vi .. .... ... ..vii

Chapter2•TheEffects of Turbulence on ProtistanGrazersSmallerthan the KolmogorovLengthScale.inTheory and Applied... . 10 2.I.I.lntroducti on...

2.1.2 .Objectives.. . 2.2.Th<o<y

2.].MaterialsandMethods..

2.].1.Ex:perimentai Apparttus.. .

.... .. ..10

. 1]

15 ....19

...19

2.3.2.Quantification ofTurbulcnce 19

2.3.3 . Maintenanceof Cultures..

2.3.4.F1ageUateGrowthand Ingestion of Bacteria...

2.3.5. SampleAnalysis ..

...20

. 21

.. 22

2.3.6.Calculations 22

2.4.Results. .. 25

:<:.:>.~"' . . 27

2.5.1. Theoreticalve:rsusMeasuredKineticEnergy... . . 27 2.5.2. BioticPanmeten....

2.5 .3. Conclusion... .

28 ..31

Chapter 3-The EffectsofSmall-Scale Turbul ence and Low SeawaterTem peratureS OQthe Growthand Gnuingofthe HeterotrophicMiaotlage1late.

Paraplrysomonasimpf!rjorata... . 44

3.1.1. Introduction 3.1.2.Objectives.. . 3.2.Materialsand Methods...

3.2 .1. ExperimentalApparatus...

3.2.2. QuantificationofTurbulence...

3.2.3.Maintenanceof Culrures.. ..

.... .. ...44

. 46

. 47

. 47

. 47

48 3.2.4.Flagellate Growthand Ingesti on of Bacteria....

3.2.5.SampleAnaIysU...

3.2.6.Calculations.•.

3_3.Resul ts

. 48

49 50

... .. ...52

3.4.Discussion. .. 55

3.4.1. Biotic Parameten, Static versus TwbulentConditions... . 55 3.4 .2. Biotic Parameten.,Tem peratureDependena:.. .... . 58

iv

3.4.3. Conclusion... .

Chapter 4

4.1.1.Sununary•..

4.1.2.FutureResearch...

LiteratureCited .

. 61

.... ... . .. ... ... ..73 ...73

. 74

. TI

List of Tables

Table 2.1 VlSCOSities of seawater coUectedfromLogyBay, Newfoundland(32%0),calculatedfromasetofequatio ns listed inAppendixAfromJwnaesetai.(1993).. . . 41 Table 2.2 Thegrowthratesof the heterotrophicflagellateParaphysomonas

impe rfOl'alaand the bacteriaVibriospf~ndidus(±:9O"/o)foreach

experiment atIS-C... . 42

Table 2.3 Tbeingestionandclearancerates of the heterotrophicflagellate Paraphysomonas imperforataforeachexperimentatIS-C.Mean bacterialabundancewaslOSto10'cellsmfl... .. 43 Tab le3.1 1bemeangrowthrates oftheheterotro phicflagellate

ParaphysomonasimperforaJaandthebacteriaVibriosp/~ndidltl

(±9O%)fo.-_ experiment 67

Table3.2 Theclearance,ingestionandcommunitygrazingrates ofallfour experimental temperatures .Meanbacterialabundances available were10⁵to10'cells mr^l•Each rate isa mean ofIto4

experiments.... ... ...68

Table3.3 QIOvaluesfor thegrowthoftheheterotrophic flagell at e Paraphysomonas imperforatafedon themarinebacteri umVibrio spl~ndidusoverthetemperature rangeof0to IS·C 69 Table3.4 QIOvalues for thegrowthof the marinebacteriumV;briosp/~ndidus

everthetemperaturerangeof0toIS-C 70

Table 3.S Comparisonofgrowth.ingestion andclearancerates ofP.imperforala from otherpublishedresu lts.Resultsofthisstudy are in bold 71 Table3.6 Compariso nofmeangrazing rates on bacteria by heterotrophic

microflagellatesintheoceanfromother publishedresult s.Results

of this study areinbold 72

vi

ListofFigures

FigureI.1 Schematicdrawing of theheterotrophicmicroflagelJate Paraphysomonas imperjorato.fromEcceIston-ParryandLeadbeater

(1994).... . . 10

Figure2.1 A)Preyparticle encountering predatorthroughBrownianmotion and8)Preyparticleencounteringpredatorbydirectinterception (Vogel1994 ).Arrowsrepresentthestreamlineof the 8uid's

Bow 33

Figure2.2 Experimental apparatus consistingof3static and3stirred (turbulent) incubation tanks.Turbulencecreatedby vertically

oscillatingplundersconsistingofa plasticwandanda perforatedPVC plate.A) Still-photoofexperimentalsystem

andB)schematic drawing ofsystem... . 34

Figure2.3 Laboratocyset-upofthelaser Dopplervdocitimeter.Laser beamissplitinto4 beamsandpositionedjustbelowthePVC plunger.A)Schematicdrawingofexperimentalset-up and

B)still--photoo fset-u p... . 35

Figure2.4 Thechangeinabundance(±SE)of the heterotrophicflagellate Paraphysomonas imperjorata(opensymbols)and bacteriaVibrio splendidus(filledsymbols)of ExperimentAatIS·Cin turbulent

(circles) and static(triangles)conditions.. . . 36 Figure 2.5 Thechangeinabundance(±SE)of theheterotrophicflagellate

ParophysomonasimperjoraJa(opensymbols)andbacteria Vibrio spkndidus(fiJIedsymbols)ofExperimentBatIS·Cinnubulent

(circles)andstatic (triangles )conditions. ... ..37 Figure2.6 Thechangein abundance(±SE)oftheheterotrophic flagellate

Parophysomonasimpe tfora ta(opensymbols)andbacteriaVibrio splendidus(filledsymbols)of ExperimentC attS· C in turbulent

(circles) andstatic(triangles) conditions.. . . ...38 Figure 2.7 Changeinbacterialabundance(±SE)ina grazer-free environment

ofExperimentAatatenpearureof !SOCinturbul ent(circles)

and static(triangles)conditions 39

vii

Figur e2.8 Changeinbaaerialabundance(±SE)inagrazer-freeenvironment ofExperimentBat a temperatureoflS-cinturbulent(circles) andstatic(triangles) conditions... . 40 Figure3.1 Thechangeinabundance (±SE)of the heterotrophicflag ellate

ParapIrysomonasimperforota(opensymbols)andbacteriaVibrio spI~ndidus(filledsymbols) ofanexperimentat IS-Cin turbulent

(circles)andstatic(triangles)conditions 63

Figure3.2 'Theeffea of tempcntureon the mean growthratesof ParapIrysomonasimperforata(open symbols)andthemarine

bacteriumVibriospI~ndidus(tiDedsymbols)in turbulent (cirdes ) and static(triangles)conditions... . 64 Figure3.3 Thechangeinabundance(±SE) oftheheterotrophic flagellate

Paraphysomonas imperforata(opensymbols) and bacteriaVibrio spfendidus(filledsymbols)of anexperiment at1000einturbulent

(circles)andstatic (triangles)conditions... . . . 6S Figure3.4 'Theeffect of ternperarure on themean ingestion ratesof

ParapIrysomonas imperforata(opensymbols)in turbulent (circles) and static(triangles) conditions.. . . 66

viii

LISTOF NOTAnONS

Dynamic:viscosity,Pas0#'specific:growthrate, divisionsh-^I Densityofsea wat er{ lS"C,32%0), 1.024gan')

Kinematicviscosity, mIs·1

Kolmogorovlength microscaJe ofturbulence.nun Twbulent kinetic energy dissip ationrate,W kg"

So Smallest turbulenteddysize.mm D Diffusio ncoefficient,an¹s·¹

A Prcda.tor c:dl radius,~

Ke Boltzmann' sconstant, 1.38 x IO-DJ1("1 TIC TemperarurcindegreesK elvin (213.15+ 15°C"K)

Ro Prey cell radius,urn Wavenum berk-21t

if

a.y.a Standard deviationsof particlevelocit ies

n' Sumofa'. a',.anda',

T Period of one oscillation.1 sec

Acknowled gmen ts

I would liketo thankaneoonnou.sm.unbc2"ofpeoplef~helping methroughthis projecttheselast couple ofyean.Fimand foremost,.tomy supervisor.Dr.Richud RivIcin,woonotonlypuc:IUsconfidencein meand invitedmetohis labovertwoandhalf yearsago. buthas providedmewith a wealthofknowledgeinthemicrobialworld. Sincerethanksto PaulMatthews, forwithouthishelpon a dayto day basisIwouldhave truly been lost,andmost likelyhave takenanother yearto finishthisproject.Thanksto Diane Moon ey and HeatherBussey forloaning someof theirvaluabletimeto assist me in bacterialand flagellatecounts.Muchthanksto Zanna Chase,a student of Dr.NeilPrice atMcGillUniversity, for supplyingmewiththat cutelittleflagellate.Paraplrysomona imperfora ta.. Mygratitude toSteve Sooley for the actualconstruct ionof my experiment al apparatuS;he took.mydrawingsand made them areality.Thanks torunMillanoflhe InstituteforMarineDynamics forthe useof thatfantastic machine called alaserDoppler vdocitimeter-thechance tocontrol a 300mWargon-ionlaserwasquitetherush.Thanks toDr.Charles Shirkeyfor takin gthatbJge amountof dati.obtainedfrom that fantastic Ia.serandhelping me processitforhumanconsumptio n.Again, addanotheryeartothis projectifhehadnotcomeand rescue me from the perils ofsignalprocessing.Abig thank you toDr.John Gowwho not only providededitorial adviceon thissomewhat massive docum ent,butalso forsupplying me with theinitial cultu res ofVibrio sp/endidusso my poorlittle flag ellates woul dno t starve.Thanks to Dr.Don Deibel forhis guidance, patienceandeditorial advice on thisthesis.A small thank you toHo ng Chen who

providedmewithsome veryvaJuabIestatisticaladvice.Allowme to extend mygratitude to my wonderfulanddear familyandfriendswho havecheeredme onthese lastcoupleof years.Many blessedthankyou'! to mymoth er for notonly allowin gher littl egirltomo ve toanother country awayfromher,but also forcontinuallyprovidingsomuch emotional supporteachandt:VerYday.Andlastbuthardlyever least, Iwouldliketoshowan immenseamountof appreciationto my Wber, my phantom committee member.who contributedagreatdeal of inputonthephysicsofmyresearch. Mostimportantly,I am etemally gratefu lbecausebe has put50much faithinme,not only ashisdaughter,but as a truescientist.

CHAPTER.

1.1. Background

Themarinemicrobialtrophic levelsprocess aconsiderable portionof dissolvedand particulatecarbonintheworld's ocean.Themarinebacteriaare responsibleforthe rernincralWd:ion ofdissolvedorganicmaterialemanatingfromphytoplanktonand heterotrophicgnzcn(DavisandSiebwth1984,Jumarsdol1989andref:citedwithin).

Bact erial production alone.has beenreportedto constinne 20 to60% of primary productioninthe ocean(Whitcel a/.1991.DuddowandCarlson1992).However.

bactcria-controUedcarbon fluxesintheocean can behighlyvariableand range from zero to morethan100"....of 1ocal primary production(pomeroyttlat.1991,Hochand Kitchman1993.AzametaI.1994).Eventhoughphotosynthesis limitsthe amountof atmosphericCQzabsorbedintotheocean'ssurface layer, itisthe function of themarine bacteria andmicrobialfood web to export thecarbonfromthe surface layer to themixed layerofthe ocean.No neth e less,the energyconsumed bythemarin ebacteriamustbe transferredtothenexttrophiclevel bymeansof predationbyprot o zoansandother microzooplankton(e.g.pelagictuniwes;Kingetal.1982, Urbanelal.199 2, Deibeland Lee199 3).

TheconcepIofthemicrobialfoodwebwasrecentlymodifiedby Legendre and R.tssou1zadegan(1995) tointrod ucetheideaof amultivorou sfoodweb where mesozooplankton omnivory or herbivory, and micro zooplanktonherbivoryor bactiverory co-exist.For example., heterotrophi cmicroflage llatesare majorbactiverous grazers whichplaya substantial rolein thecydingofbiogeniccuban(Fenchd1982a,b,eandd,

I

SherrandSherr1983).Flagellatescan repackagebaGterialbiomassinto particles eccessbteto mesozooplankton,andhence recycle carbonbacktothemiaobial loop (CaronetaL1985andref.cited within).Tofullyunderstandthefactonthatregulatethe dynamicsandflowofthebiogenic carboninthemarine microbialfoodweb.it isnecessary to determine howphysical factors, suchas smalIscaleturbulence and low seawater temperatures. influencetheinteractionsbetweenthese trophic levels. Turbulentmotion that hasbeendissipated toa micro-scale can increasetherelativevelocity of a rciCfOZOOpLanktonanditsbacterialprey.andpossi~yincrease theirencounter rates Increasedencounter rates usuallyresultinincreased grazingrates, and hence the cycling ofcarboninthe rciaobialfood webcouldoccurata fast er rate

MicroOageUatesexperiencelifeatlow Reynold'Snumbers where.atleast in theory, inertia has littleeffect.The Reynold's numberis adimensionless indexwhich relatesthe dragofa solidthrougha viscousfluid; the higherthevisco sity.thelow erthe Reynold's number (Reynolds1883).Theforcesexerted onthemicroorganismto assistinits movementareforcesactingonitsbodyatthatmomentandthatmoment alon e (Purcell 1971). AmicroOageUateswimmingthrough seawaterisanalogoustoahuman attemptingtoswimthroughapOOof molasses.Hence,whenthe micro flagellale stopsits propulsion throughthefluid, the animal's movement immediatelyceases.Withsuchlow Reynold'snumbersto consider,small-scale turbulence can possiblybenefit a microOagellate becausetheincreased water velocitiescreat ed bythe turbulenteddiescan increasetheanimal'srdativevelocity throughthe fluid.

Small-scale turbulenceistheresultofthc dissipation oflacgerturbulent eddies createdbystrongdisruptionsto theWlller'Ssurface.suchashigh windsaeatingwaves.

Anysmallscaleoceanicmotion (smallerdissipated eddies)relies OnJyontherateat which itis providedwithenergyby•largerscale motion(large turbulen teddies) and onthe kinematicvisco sityof theseawater (Termekes 1972 ).Thesmallestturbulentscaleis det enninedbytheKolmo go rovlength scale.This theoreticalmicro- scale existswhere viscousdragbegins todomina teandviscositystartsto smooth out turbulent water Ouauations (Tennelces1972,Vogel1987.MannandLazier 1989).Therefore. the equation forthe Kolmogorovlengthscale(ttl isgovernedby thekinematic viscosity(\I)or themolecular diffusivity of momentum. andthekinetic energy dissipation me (e)which can IWIgeintheoceanfrom10')to10-¹⁰Wkg-^I(Osborn 1978. Oakey and EDict1982.

Yamazaki and Osborn 1988):

(I) ^q=

(V')'

^-

_e "

To further emphasizethatsmall scale motionisdominatedbyviscousforces.the Reyno ld'snumberwillequal-I-when'1and0 (Duidvelocity) are combined:

(2)

!l!!.=1

v

emphasizingthatthedissipationofthe viscositycan modifY itse lfto the amo unt of energy suppliedbytheturbulenteddies(Tenne kes1972). Atscales smal le r than the Ko tmog c rc v scale., flowisclassifiedaslaminar(or-smooth-)andmicroorganismswill onlyexperiencethe Iarninarshearcreatedbythedissipaled eddies (Shimera etaI.1995).

Tutbulemmotiooisa complicated concept that hasbeeninvestigated for yeanby physici ...and""""""'oceanognphe<s (seeNdIcin1992 ).QuantilYingturl>uJence""'Y scaleisdifficultsince itmustbedeterminedthrough a set oftheoreticalequations Modem technologypermitsthe measurement of turb ulencc atthelargerscales,butsmall scale turbulenceisimpossible tomeasuredirectly.Furtherm o re,inmost casesturbulence andplankton havebeen studied independentlyinnatural systems, particularlydue tothe difficultiesinconlroUingthephysicalparameten (AJcarueraL1988,Sanfo rd 1997).

lberefore,anumber oftheoreticaJ.equations and standardsexistfor micro--scaIefluid motionwhichalloriginatefromKolmogorov'sUniversal Equilibrium Theory (1941).This physicaleffect onabiologicalsysIcmhasbeeninvestigated conventionallybya number of researchers.Purcdl (1978)wasthefirsttoderivea model characterizingthe effect of stirringonapredator's

«

50urndiameter )absorption of preyparticles based onatheory developedby Smoluchowski(1916).Purcell had det ermi nedthatparticles(ororganisms ) lessthanafewmicronsinsizewouldnotbeaffectedby vigorous stirring, butthose swimmingmiaoorganjsms greaterthan5J.Utlinsizecouldenhancetheabsorption of prey particles.Latier andMann(1989)furtherexplored Purcell'sideas,focusing theirtheo ryon the diffusive boundary layeraroundamicroorganism.Theirmodelsuggeststhat turbuIcnccdoesDOtaffect sphcrical organismslessthan100 J.Utlindiameter.Lazierand Mann (19 89) believethedifference fromtheirresults andotherspublishedresultsarose from the misuse oftheKolmogoro vlength scale,under esti matin gthelength of the smallest turbulenteddybya facloroften. RothschildandOsborn(1988)created a

turbulenceinducedpredator -preyencountermoddwhichsuggeststhatzooplankton feeding

rates.

asweDas other plankton rates suchasthe optimal foragingtheoryand nutrient exchange in oligotrophic systems. may be underestimated by failuretoconsider turbulentmotion. KisrboeandSaiz(199 5) funher expanded Rothschild and Osborn's modelbecause theybelievedthe resultsdidnot applytolargerpred ato rs, such as copepods.They enhanced the equations by adding behavioralcomponents to include charaaeristicssuchasrandomwalkpatternsandambushpredation.

The generalargumentinthefieldofsmallscaleturbulenceisthatonlymeso-sized organisms(>SO~)canbenefitfrom turbulence,andeventhoughthere hasbeena considerable amount ofresearchontheeffectsofnubuJence oncopepods(SaizIttal.

1992, MalTaseIttal.1990, Costelloet al.1990 )and fishlarvae (MacKenzieandLeggett 199 1,Mue lbenIttal.1994 , SundbyandFossum1990)studies of smallerorganisms.such as ciliatesandmicroflageUates..have beenneglect ed.Kierboe andSaiz (1995)had reportedthat turbulenceistobeconsidered -unimportantforverylarge predatorsandfor mostverysmallpredators-.butpossiblyeffectiveto meso-sizepredato rswhich filllctio n aroundtheKo!mogorovlengthscale.HillIttaL(1992 )concludedtheirstudywiththe same hypothesisinwhich"the encounter rate of particles similarinsizeto the Kolmogorovscaleiscontroll ed by turbulent eddying motions.- However,thisisonly theory,andalltheory must be testedandapplied.Asurveyof the literaturehaspresented onlyfour articles on theeffectsof turbulentmotionson microorganismssmallerthan10 JUtI.Peters and Gross (1994)reportedthe grazing ratesof ParaphysomcmasimPf!rforata feeding on rrwine bacteriaintheGulfof Mexico during stagnan t andturbulent water

S

conditions.They found nodiffCl"CDCtin theingestion ruesbetweenstaticand turbulence conditions.butresultsshowed • changeinOagel1ateabundanceandcd1 size.Ahigher flagellateabundancepredominatedundO'"theturbulentcondition.however.cellsize appeared smaller thanthose foundunderthestaticcondition.ShimetarlaJ.(199 5) subjected planktivoroussuspensi onfeederstolaminarshear fields justbelowthe Kolmo gor ovlengthscale.TheyfoundthattheshearcreatedbysmaIlscale turbu lencehad DOsignificant effectontheflagellateand ciliatespecies(Paraplrysomoncusp.,2 chrysomonads,Diaphanoecagrandis.Favella$p.and anunidentifiedheterotrich).excep t forthecboanoOagellateMonostgosp.Thisled toa hypothesisthatturbulenceeffectsmay bespecies-specific,havingastrongerinfluenceonl'IOM\OtiIeorganismsorweak swimmers such as foraminiferansandradiolari ans.Petersetai:(1996)investigatedthe effectof turbu1e ntmixingonP.imperjora tdsingest ion rates.Micro flagellates were exposed to12to24hourturbulentperiod s overarange of turbulentintensities.They foundno significantdifference(p<0.05)in theingest ion rates among turbulentinte nsities and sugg estedthattwbulence maynothaveaneffectontheorganismsunlesstheyare exposed toturbulencefor morethan12h.Theeffect ofturbulenceon bacterial productionwasreponedbyMoseneder andHemdI(1995)_They found thatturbulence increasedbacterialproductionwhenphyto plankt o nwaspresent,but not when phyt oplanktonwasabsent. Sinceindividual bacte rialcellsaremuch smallerthanthe Kolmogo rov scale (LoganandKirchman 1991 ).turbu lence should. intheory.not influencetheratesof producti o nunlesstheyareinlarger aggregates, such as anached to dumpsof phytoplankton ceUs.Therefore,Moeseneder and Hemdl(1995) suggestthat

6

turbulencemayonlyalter abacteriopLanktoncornrwnityst:rlJCIWeandnot affectiu actual rates ofproduction.

Forthe researchproposedherein.theheterotrophicmicroflagellate Paraphysomonasimperfoeuawasthechosenanimalbecauseitis.ubiquit ou s microflagellatc,easytoculture andgrows overawiderangeoftemperatures.Itis sphericalinshape,coveredwithsiliceousSPines.withtwoflagella,oneshortandone long, whichassistinlocomotion (Figure1.1) (Fenchd1982a ).Thelonger of thetwo flagellahasabila1era1iU'TaYof beterokonttubularhairs., wbereastheshorteris smooth (EccJcslorHJanyandleadbeater1994).Itisacolorless microflagel1ate.andforittofeed, abacteriumoralgalceDmustcomeindirectcontactwiththeflagellate'sventral furrow wheretheprey isthenphagocytized(Fencbel1982a).Caronet af.(1986)did an extensive studyon theeffectofincreascdtemperaturesonthephysiologicalpro pertie sof growthandingestionratesand cyclingof carbonandnitrogenbyP.imperforata.They foundthatincreasingtemperature from14to26·Cresultedinincreasedgrowthand grazing ratesofthemiaoflagellalc.However,therewas no relationshipbetween incnascdtemperamre andgrossgrowthefficiency.ChoiandPetm (1992)measuredthe feedingratesoftwo coldoceanstrainsofP.imfWrforataat a temperaturerangeof-1.8 10 2O"C.Theirresultsagreedwiththoseof Caron et al.(1986)inwhichlhere was an increaseinfeeding rates withtheincreaseintemperatures.However, theresults ofChoi andPeters (1992)also showedan increasein thegross growthefficiencyofP.

imperfor ata. Mostresearch concerninggrazingandgrowthrates ofmicrof1agellates havebeenconductedathigher temperatures (-20'"C),leaving agreat needformore

7

infonnationonhowjcwe-seawater tcmpcn.tures an affectthefeeding andgrowthofP.

i~iforalQ·

1.1.Thesis ObjKtives

No two experimentalresults or models absolutelyagreeupon howsmaUscale tul'buIenceaffectsmicroorganisms andtheirencounterprobabilities.Inadditio n.nostudy hasyeccombined the effects of turbulencewithdecreasing seawater temperaturesto consider the consequences of the increased viscosityofthe8uidonmicrozooplankt onina turbulentenvironm ent.Nonetheless.many studiesdo haveone underlyingidea.andthat is. ifturbulenccdoespro ve tohave an effecton thephysio logicalratescfmicrco rg anisms.

thenallpreviouslypublished ratesassessed understagnant incubaUolI5maybeunderor overestimated.Hence.the purpose of this research istodeterminewhetherturbulence influencesthegrowthand grazing rateofParaphysomonas imperfoanaatseawater temperatures belowI

s-t:.

Itis hypothesized thatsmallscale turbulencewill increasethe growthandgrazingofP.imperforataataUtemperat uresbyaugmen ting theenco u nter ratesbetweenpredat orand prey

Withinthisthesis.,Chapters2and3accomplishthefo Uowingobjectives:Cha pter 2 discusses theorydeveloped todiscern whether turbulence impactstheheterotrophic microflagellatc.P.imperforolaandits contactwithitsbacterial preybymodifyingthe equationsofPurcell(l977).The theoryis then applied todeterminegrowth, ingestion and clearance rates ofP.imperjorala,in a turbulent enviro nment.Chapter2also quantifiestheturbulence or cak:ulatestheenergydissipatedinthe experimental environmenI.Chapter3investigatesandreports the combined effects of small-scale

turbulenceandseawatertempcratun:sbelowIS-Conthegrowth.ingestionandclearance rates ofP.impt!rfOl'OIa.Chapter4simplyconcludes thisthesiswith.varietyof future directivesaimedatthis fieldof research.

II

lOum

II

Figu re1.1:Schematicdrawi ngof Paraphysamona imperforarafrom Eccelston-Parryand Leadbeater ( 1994).

10

CHAPTER.

The EffectsofTurbuIence00Protistan Grazers Smallerthan the KolmogorovLength Scale,inTheoryandAppli e d.

1.1.1.lntrodUcnOD

Itiswidely believedthatthemicrobialfoodwebisadominantpath wa y for energyflow(Azamet al.1983).Heterotrophicmicro tlage llat es are known tobeprim ary predatonofbaeteria(SherrandSherr 1984,Fenchel1986) and areconsideredtobea aiticaltrophictinkinthemicrobial foodweb(Aumetal:1983.CboiandPeters1992, Fuhrman 1992 ).Paraphysomona.Jimperformais.ubiquitousheterotro phic microflagellatc.Itcan apidly increaseitspopulationwhen conditions are optimal and consume avariety ofsizes(0.5lJ.II1.^Jto 200~1andtypesofprey(bacteria andalgal cells)(FencheI 1982a,ChoiandPeters 1992,Eccleston-Parry andLeadbea ter1994).

Until recen tly,all reportedgrowth.inge stion and clearance ratesof lhisflage lla te.and other microb ialgroups,have beendetenninedinnon-turbulentcondition s.In many cases.microorganisms thatwerecollected fromthefield were obtainedfrom turbulent waters,and growthandgrazing rateswereascertained during staticincubation conditions.

Thedissipationof turbulentkineticenergyfrom the largetosmallsize scalesis an inherentcharacteristic of the ocean. Turbulence bes beenknownto contributetothe formation ofmarineaggregates(Kisrboe1993)anddispers ionof planlctonpopulatio ns (Lasker1975.Haryetal.1990).Rothschildand Osborn(1988) suggest edthatsmall scale turbulence couldalso significantlyinfluencepreda tor-preyinteractionsandthe flow

II

ofenergywithinandbetweenmarinefoodwebs. Several studies on the effects of turbulcnce00copepods andfish larvaeproposed that contactand feedingrates maybe serious ly biased when excludingthecontributio nof sma ll scale turbu lencetothese rates (Marasseetal.1990, Costelloetal.1990.MacKenzieandLeggett 1991,Sundbyand Fossum 1990 ).Inconsiderationofthese studies,a questionariseswhether-turbulence couldhavethesameeffect on smaller organisms(10)JJllor less)andconsequ e ntly,what impactitcould have onthemarine microbialfood web.

Only recentlyhavetheeffects of turbulenceonaflagell ate'singestionrates been examined (petersandGross 1994,Peters etoJ.1996 ).Thefast ofthesetwo papers investigat ed ingestionratesoffluorescentl ylabeledbacteriabyParaphysomonas impeTjorata.Petersand Gross (1994)foundthat therewasanincrease intheabu ndance ofP.impeTjoratQunder a turbulent environment,butcellsize haddecreasedovertime Theirresultsalso showed ingestionrateswereslightlyhigher under turbulence,bulthe rateswere not significantlydifferentthanthose of the static condition .Theseaut ho rs proposedthat theturbulence causesa changeinbehavioral responsewhich resul tedin incr ea sed grazing,furthersuggest ingthatthisresponseis similar tothatreportedin experimentsusingcalanoi dcopepods(Saizand Alcaraz 1992a,Saiz etaJ.(992 ).Peters etaL(1996)expandedonthisin studiesof theingest io n rates ofP.impe rjOl'OlD.butthis timeconcentrated ontheeffectof differentturbulentintensities(O.OS,O.IS,IScm²

s')

Theirresultsshowed that theflagellates influencedonlyby "highturbulentlevels "(IS cm^2s·l)fortwelvehours were moreabundantthan the non-turb ulentconditio n.

12

Shimcta (1993)postulatedthatmicroorganis ms would encounter prey atahigher ratebecauseoflaminar shear created bythe smallestturbulenteddies which arejust below the Kolmo go rovlengthscale.They tested theirhypot hesisinorderto determ ineif shearrateaffected clearancerates of. variety cf'plankrivcrcussuspensionfeeders,such asflagellatesandciliates (ShimetaetaL1995).Theirresultssuggestedthatthe effectsof shear seemedtobespecies-specific.For example,ParapIrysomonassp.andthe cboanoOagellateMOtX2Sigasp.werebothfedF1.Bofsimilarconcentrations(10'mr^l). The clearancerates det ermi ned fromtheParaphysomonassp.exposedto thelami nar shearfields werenotsignificantly differe nt fromtheclearance ratesof thosein the static condition. In contrast,MOIlOSigasp.•whichissimilar insizetoParaphysomanassp.•

did show. significantdifferencewit h . muchgreaterclearance ratewhenexposedtothe laminarshearfield.

2.1.1.Objmins

Theobjectiveofthis studyistoquantify the effects of smallscale turbulenceon the growt h and inge stio nrat es ofthemicroflage llateParaphysomonasimperfarata feedingon bacterialprey.Equations ofPurccIi(1978)are first discussedand then applied to micro-sized(5to6j.U1l)grazers.Thentotest Purcelrstheory,experiments weredoneto evaluate theeffects of turbulence uponfeed ingonthe psye hrotro phic mari nebacteria, Vibriosplendidus,byP.imperfcxala.Theseexperimentstestthe hypo th esis that micr otlagellat e growt h andits ingestion of bacteria willbe greater under turbul entthanstagnantconditions.lfturbulence enhancestherateofgrowthof micro het erotrophsandtheir grazing of marinebacteriathen itislikelythatpublished

13

ratesofgrowthandingestion determined uDderstaticincubation conditionsmaybeunder ocoverestimated.

14

:u.

THEORY

Thesizeoftbe smallestnubulent eddiesistypically referencedto the

Kolmogorovlengthscale.TJ.This isascalewhichisdet erminedbythekinematic viscosi tyofa fluidandthe velocityshear(o rotherwiseknownasthe kineticenergy dissipationrate). Kinematicviscosi tydiffersfrom dynamic viscosity(j..L)inthat the latteristhefrict ion of a fluid.Itisa measure of. fluid's resistanceto shear when afluid isinmotionandcanbedefinedas:

(I)

when:F(Newtons) is theforceactingonone fluid layer orbodymovingacro sstheother, z(meters)isthedistancebetweenthetwobodies. U(ms^l)isthevelocity resultingfrom theforceacting uponthelayer,andS(m^l)isthe area ofthefluidvelocit y or body (Vogel 1994).Thedimen sion of dynamicvisco sityisaPascalsecond (pas)inSIunits Kinematic viscosity(v)is thensimplyaratioofthe dynamicviscosity andthe fluid's dens ity(p):

(2)

v =1!

p

TheSIunitdimension for kinematicviscosityis mJs·^l.Thisratio isdefinedasthe measurem ent ofthe-ability ofmolecu lactransport to eliminatethenon-uniformitiesof fluidvelocit y " (Batchelor 1967, Vogel1994 ).Table 2.1 list sthe dynamicand kinema tic visco sitiescalculated for seawaterchancterizedforLogyBay , Newfoundl andatfour experimentaltemp eratures.

IS

TheKolmogorovlengthscale relatesthekinematic viscositytothe energy dissipationme (e)ofaturbulenteddy,It isdefined as'

(3) q= -

(V ')'"

e

whcre£can rangefrom 10-3toICr^lOWkg·¹or-IOta 10'"a nIsec·)(Osborn 1978.Oakey and EUiot 1982,yamazaki andOsbom 1988)( 1W kg-I-10" anIs"1.By using this range ereandassuming visapproximately 10'" m^I,I.then1IcanvuybetweenIto6

According to Lazier and Mann(1989).thesizeof thesmallestturbulenteddy(5..

mm)hasbeenroutinely underestimatedin thepast~ysomefactora,and ther efore:

(4) Ss=a q=a

( £' v' )"

Thevalueof2xhasbeenoptedfor-QbyLazierandMann(1989)because22tisoften usedinthedefinition of the wavenu mberkformathematical convenience. It is relativelyunimportantwhat the absolutevalue isforu,justaslongasitisclearwhatis the percentofshearenergythatisused bythesmallest turbulent eddy.Whena-2lt,the Kolmogoro vlength scalewouldrange from6 to 37 mm andthesmallest turbulenteddy wouldcontainapproximatelyJ% ofthemaximumshear energy oftheoriginalturbulent flow.Consequently,anyturbulent motion which spanslessthan. fewmillimeters will diminishtolinearshear.

Forsuspendedparticles (e.g.predaJ:orsandprey)to encounter oneanother,they mustmove at differentrelativevelocities.This can occur by eitherswimmingorsinking

'6

atdifferent velocities.generationof a feedingcurrent,orfluidmotion bringingparticles into contact. Aquesti on arises as tohowapreyparticleis absorbedbya predator.Does the predator experience direct interceptionor-diffus io nalormotilepanicle depo sition - (Vogd1994)'?FordirectintCl"CCption.,apRyparticleIID.Istpasswithinthe radius ofthe predator's cellandbestreamlined direaJ.y tothe predator,a techniquetypicalforamotile predatorandnon-motile prey.For diffusionaldeposition,a predatorcapturesa prey particleas aresultoftheprey'sBrownianmotion.orotherwisestatedastheprey's random motion(Figure2.1).For thepurposeof thisstudy,wewill assume the latter situatioo. sinceVibriospl~ndidtuis. motilebacterium.Twootherassumpt ionswhich must be made formathematical convenienceare:(I)alltheparticles inquest ionare perfect sphericalbodiesand(2) twbulenceis isotrop icatthesmal lscale.

Ifwe areto assumethai.turbulentmotion increasesthe ccnteceofpreda to rand preyparticles,thenthenext questiontoaskisbowmucb-stirring-orturbulent energyis requiredtodoublethecontaet rateofpreyparticles.Purcell(1918) deri ved anequation whichinvo lvesthediffusioncoefficient(0,eml s"l) ofa preyparticle, the dyna mic viscosity(J1, W s2e m-J)ofafluid andthepredator'scellradius(A, J.U11) to dete rmi nethe -stirring .powerdensityfor doublingtherateof absorpti on-

«s>...

Wem"J):

(5)

"Thediffusioncoefficientwuascertained withthe Einstein-Smoluchovski equatio n:

(6)

.7

whereItaisBoltmwut's constanl (1.38XIcr^DJIdegreeK),TI:is thetemperature in Kelvin(I

s-c '""

288K), Jlis thedynami cviscosityofseawater-(at1S'"C and )2%0, Jl=l.l x10"W,tan",andItoistheradius ofthepreyparticle(inour

ease.

O.S JlII1-the radius of.Ium bacterium).

Purcelltested theequationbyfirst usinga predatorradiusof IiJlI1which is absorb ing.particlewitha0of10·'cmls·l.Thisresultsin.stirring power of 0.5W cm.J,a very'powerfuland impractical valueforapredator of this size.He then appliedthe same situationto.pmiator of10um radiuswhich resultsin astirringpower of5x lcr'W em-)whichismuc h more realistic and obtainab le.Let us now examine bow much energy itwouldtaketo doublethe absorption rateof •predator withthe radius on.5to3.0um(simi larinsizeto the micrcflage llateP.imperjOl'Qla),a 0oD.8)(

10-9cm2s-\anducrt.!J(10-9Ws2e m·)(for 15°C,32%0salinity):

<S> _500(1.1x1O-~Ws^Jcm^lX 3.8x10-'cm^JS-I}J - (25(0lOx10~cm)'

(1) (79x1O-~Wscm-^I)

<S>_ 39 ro81x I O-ucm"

This range issimilartoPurceJl's estimatio nof. 10iJlI1predatorand therefo re a reasonable value toattain.This, in fact,means that theturbulent ener gy createdat 2X10-9Wcm-Jto9x to·IOW em')can be effect ive andincrea se the rateofdiffusio nal depos ition of. bacteriu m to a microflageIJateofSto 6iJlI1insize.

18

1.3. MATERIALS AND METHODS

1.3.1.uperim~.taJap para t us

Theexperimentalappantus consistedof tht ee insull.led plexiglass conlainers (46 em x 20 em) containingtwo smallerplexiglassincu bationcontainers(IIcm x II em x 14 em heigh t ),Thethreetankswere connected to .temperatu re controlled recirculati ngwaterbath (NeslabRTE-210 ).Ofthcsix smallerincubatio nchambers, threearcthecontrol(i.e,non-twbulent) conditionandthreechambers aretheturbulent conditton(Figure 2.2).

Tbeturbulencewascreatedbyverti caJlyoscillat ing plungersconsistingofa plasticwandandaperforated polyv inylchloride (pVC)plate(platesizewas9.5cm², perforationdiameterwas2mm,-93%solidity).The plateswerecentered in each chamberwith2.5 mmcleara nce between theinsidewaIls ofthetankandtheedgeofthe plates.The vertical amplitude oftheplungermo tion was 3 em, travellin g in theupper portion of the water column,andosc illating atarateof IHz±0.3Hz. (Figure2.3) 1.3.2.Quanlilia-t wDofturbulenc~

Turbulencewasmeasured with.1. 2-ax.is 300mW argo n-ion laser Doppler ve10citimeter(DuteeElectronics)and processedwitha Flow VelocityAnemom eter (DanteeElectronics).Vertical and horizontalcompo nen ts of waterveloci ties were measuredat different positionswithinthewatercolumnusingacomputercontrolled traversemec hani sm.Latexfluorescentbeads (Ij.UTI).similar insizetothe bacterial prey, wereusedas tracerpanicles inthewater column.Each velocity compo nentwas measuredat a samplingrateof -120Hzwitha bandwidthof 0.12MHzand-2nun fringe

19

spacing(0-35).0a1awereooUc:eted at random locations withinthetankfor an accwoolation of 10,000sample velocities or a maximumof10minutespersite.

Thestandarddeviationswere calculatedfor eachU~(velocitycomponentinthe x- direction)andV,(velocity componentin they-d irect ion).Athirdstandarddeviation for the velocities in thez-directjcn wasassumed to besimilar toU~andVy.The energy per volume., orthekineticenergydissipationrate,e(Wkg·^I).was determinedfirst byfinding thesumofthestandarddeviations ofthethree velocity compone nts:

(8)

Thissumwasappliedtoanenergyfornulato determinethe energy pervolume(m'1:

(9)

wherep isthe density ofseawlt erat 15°C(1.02 36 g cm·^J) Then to equateWattskg"

into the formula, (9) is dividedbyp and T.the period ofone oscillationof the plunger(1sec;Trin on1988):

(10)

2.3.3.Maintenanceof Cultures

Cultur es oftheheterotrophic miacflagella teParaphysomonasimpeiforalQwere maintainedin 70mlglassculturetubesat15°C.Stock cultu resweretransferredinto SO ml offresh medium every 3 to 4 weeks.The medium used was sterile.0.2urn filtered seawaterenriched with Imlof10"1eyeast extract (Difeo )and1mlof 0.2%proteo se peptone (Difco).A sterile rice grainwasadded to each cultur e tube.Vibriosplendidus

20

cultureswen:maintainedonagarplates foc upto onemonth.Twostepswererequired forthepr-epantionof.stock culture to be used foranexperiment.FU'St, 25mlof deionizedwater(01) wasenrich ed with0.9]5gofpreparednutri entmix(Marin e Brot h 2216.DUco )andlUtoclaved in100mIErlenm yer nasks.A swab off'amarine brothagar platewastransferredtotheaqueous mediumTheflaskwasthen placedon a reciprocating sbUerca.12 batroom temperature.This timewassufficient forV.

splendidus to reachexponentialgrowtILThenelttday.1mIaftheculturewas dilutedto 800mIofsterile, 0.2IJDIfilteredseawater. and replaced ontheshaker for6 to12 hours. Thisworkin g stock culture was thendivided amongthesix incu batio nchamberst-1] 0 ml each).dilutedtoI.]Lwith sterile 0.2IJDIfiltered seawater.andallowedtoacclim ate to the experimentaJtemperatures before the addition ofP.imperfonzta.

2.3.4.Fb.gdlateGrowthandIngest toDorBacte ria

Fourorthesixincu bat io ntanks(two staticandtwo twbulent)containingthe diluted bacterial cultureswere inoculated with30ml of theflag ellate cultureandallowed anunstirred acclimationperiod of ca.12hbeforetheinitia l(P=O)samp ling. The two remaining incubationtanks(one staticandone turbulent}weregrazer-freecontro lsto assesstheeffectofturbulenceon bacterialgrowth. Sampleswere taken at6to12 h interval s foc 72 h intervals. All samples(5mI)forenumerating bacteriaandflage llate abundances wereimmediate lypreservedwithImIof gluteraldehyde (5% final concentration),storedat room tem peratureinglass20mlliqui dscintillatio nvials,and processed within 3to 5 daysoftheircollect ion.

2\

u .s.

Samp leAaalysis

&aerialabundancewas determinedwith the acridineorange direct count method (Hobbieetal.197 7).f\o~tohighbacterial abundances, samplesweredilutedupto20 foldwith0.2}.l1nfiltered seawater.The diluted subsamples were collected ontoapre- stainedblack 0.2J.U1lPorttics polycarbonat:e filter and posr-stainedwithacridine orange.

TwofiltCf'Sperslide were placed onsmeareddropsofCargilleTypeA immersionoilon aglassmicroscope slide.Eachfilterwastoppedwithanotherdropofimmersionoiland thecoverslip was gentlypressedonsothatthe oilwasevenly dispersedoverthe filter.

Bacteriawere counted usingaBH2~RFCOlympusepitluo rescencemicroscope undera magnificationof 1000x.Amercurylamp (100W)wasused toemitblue excitation (BP440. DM455.AFC+Y475) .Atleast 300cdlson each filter were counted by randomlyselecti ngfields

Flagellateabundancesweredeterminedbydilutin g2-m1a1iquouto5ml (with0.2 umfilteredseawater)andfilteri ng the entireSmlonto1.0 J.UTl Poredcs pre-stainedblack poIycarbonatefilters.Each filterwas post-stainedwith 0.2ml of acrid ine orange. Filters werepreparedasdesaibedabove.Flagellate abundanceswereenumerated by counting 10randomfield s oneachfilter usingepifluoresce nce microscopy.

2.3.6.eakulations

Eachbacterial and flagellate abundance from the microscop ic counts were calculated byusing the followingequation:

(II )

N .[ _~ A ' ] .1000

V 22

where Nisthe average number cfcellscountedperfilter ,A2isthe area or filteroccupied bythestainedsample(mm2),XistheDl1IIlbc£ofsquares countedin theoculargrid(100 squares, Imm2),and Visthe volumeof the filteredundilutedsample

Foreachexperimentaltank,celldivisionrates(JL.b-I) orflagellatesand bacteria were determinedfrom the linear regressio nofthenatuea1l ogarithm(1n)of abund ances plotted against time. Fo reach grazed tank,eachplot ofbact erial abundance wasdivided intothreeintervals, representinglag, growth,anddeplet ed cells (negat ive slope ). in which linearregressionwasapplied individually.Thenet grazing mortalit y,or apparent growthrate (AGR, b^ol).of bacteriainthegrazingchamberswasalso determinedwith linear regression oftheplott eddaaLines ofbesl fit foreachinterval wereconsidered to bethose that maximizedthe

r'

andminimizedthe standarderror ofeachdivision rate.

Thegrowth(J.l.)andAGR were then appliedtoFrost's equations(1972)to equate mean bacterial concen trat ion([C),cells mI'I),flagellate clearance (CR, nl flagell a le-lh-^I)and ingestion(IR,cellsflage llal e'lh-l).

(12)

(13)

(14)

[C]=

C, (pe ':~~~;: -t,J

CR=

A~R

IR

=

CR• [C]

whereClisthe concentrationofbecteria in thenon-grazertanksat the beginni ng(tl)of thetimeinterv al, t2istheendof thetimeinterval,and N isthe concentratio nof flage llates mrlatthatspecified timeinterval.Ingestionrates were normalized by

23

dividingIRwiththe mean flasellatepopulation for • given timeinterval.Studentpaired t-tc:st.sor two-wayANOVAwith integrationwen: usedforstatisticalanalyses ofeach experiment.Eachrate(growth,gruingandingestion) was tested forsigni ficant differe nces betweenexperime nt replicationsandfo r the staticand turbulen t conditions

24

2.4.RESULTS

ThreegrazingexperimentswereanalyzedfortheresultspresentedinTables2.2 and2.3. Inallexperiments.the specificgrowth rate ofP_imperfoeatawassignificantly higher (p-=O.OOOI.ANOVA)in the twbulentthanthestatic condition.Flagellate populationsreacheda maximumbetween30 to48houn(ca.5I(10'cdls mfl)inthe turbulentincubationtankwhereas the populationsinthestaticincu batio ntanksreached maximum abundance at-60boon(ca.3I(10'cells

ml";

Figures2.4.2.5,2.6).The meangrowthrateofP.imperfOl'CllQwas2.35±0.670¹and1.01±O.4d-I in the three turbule ntandstatictanks,respect ively (Table2.2)

Bact eri algrowthinthe grazer-freetanks wu not significantlydifferent(p>O.05, Student t.test)betweenthe turbulentandstaticconditions.The meangrowth rateofV.

splrndiduswas0.89±0.540¹and0.65±0.77olintheturbulenttanksandstatictanks.

respectively.Itshouldbenotedthatalthoughgrowthinthetwoconditio ns was not significant lydiffere nt, the finalbacterial abundancesafter 60hou rstended tobe approximately 1.5 foldhigherinthe turbulentcondition(Figure2.7).

'ThecommunitygrazingofthebacteriabyP.impt!rjoraIQwassigni fica ntly (pcO.OOl. ANOVA)greaterintheturbulentthanstatictanks(Table2.3).Forthethree experiments, baaerialgrowthexceeded grazingmortali ty inthefirst24to 30hfor both conditions.After 30h,bacterialabundances declined intheturbulenttanks.Howev er, bacterial abundance sof thestatic tanks showed no obviousgrazingmonality forthenext 6 to12h,thencontinuedfor the remainderofthe incubation with a low grazing mortality

25

(Figures2.4,2.5,2.6).FmaJbaacria.labundanceswerealways greaterinthestaticthan theturbulentconditions.ExperimentAshowedadecreasein bacterial numbersafter 30b.inthear...ulCii%.cor'.d.~ion.whereasinthe static:tank, a noticeable decrease didnot occur until&fie!"48hours(Figure 2.4).Inexperiments BandC.bacteri al abundances were diminishedtozerosoon after 36hours (Figures 2.5and2.6);this correspo ndedwith thepeakabundancesofP.impel'jorata.

Ingestionandclearance rates variedamongtheexperimentsandaresummarized inTab le 2.3. Inspite ofthe variability,the mean ingestionrates between the two conditions are almostequalwherethe statictankssho wed 4.2cellsflagellale-^Ih-^Iandthe tuJbulent tanks showed 4.1cellsflage llate-Ih-I.Themean clearance rateswereslightly higher inthestatictanks(1.04x

I<r'

mIflage llate" hOI)than theturbu lent tanks (7.28 x 10-¹mlflagellate·¹h·^I).

Cellvolumes were not routinely measured throughout the experiments.However.

there wasanoticeable decrease inthecellvolume of theflagellates in theturbulent conditions.Averageflagellatecell volumes duringthefll"St24 to30hours wereCL20 JUD^J•withsome cells 250 IJlO^J(-loutor 20cells countedbada cell radius4 to51JlO).

Towardsthe endof tile experiment, though, mean cellvolumeswere ca..7 JUTl^J.

26

1.5. DISCUSSION

1.5.1.Theoretical venumeasuredlUBuKeDergy

Theenergy dissipation rateestimatedforthreerandomloca tions inthestirred tankwasapproximatel y USl(10-'W kg'l.Thekineticenergydissipation rates measuredin theopen ocean range fro m 10-3to10-10Wkg,1(Osborn 1978, Oakeyand Elliot 1982, Yamazaki.andOsborn 198 8).50the measuredrate observedin this studyis simil arto that foundinnature. Inregardsto the amount of energy requiredto"doa ble the absorptionrate"(Purcdl1918)of aSto6 JUl'lpredator.10"Wkg"ismore than enough energyto enhance thecaptureofprey particles.

Inmost

cases.

experimentsthatare basedontheorytend to produce resuhsmore idealthan whatwouldbe found in nature.lbis experimentalset-upmayhavegenerated diss ipat ionratessimilar to those foun d inthe open

ocean.

but it providedconstant. steady stirring.Environmentalturbulencecan be characterizedas intermittentand unpred ict able.Furthermore.the equations used to determinethe energyneeded to double theabsorptionrate ofapredator assumedthe preyparticleandpredato rtobespherical. V.splendidMsisarod shapedbaaerium. and eventhoughP,impe rfrxalaisspherical.it iscoveredinsiliceousspines(Fenchel1986a).Theimplications ofthe equationare slightlyquestionable . Thespherica lshapewasassumed onlyto maJc:ethemathematics easier.Hence.itwouldbeamathematical challengetodetermin ehowa rod-shaped particle, oranynon-sph erical body, is consideredinto a similar equat ionforthediffusion coefficient.

27

2.S.1.Bioticpuamtttn

Purcell's (1978)firstexampleof<5>.... whichwasexplored earlier.showed that abacteriumwouldrequirean enormousamountof energy toincreasetherate of nutrient uptake.[fit is assumed that nutrient uptakecontrolsbacterial production.then theresults of this study areconsistent with thoseofMosenederand Hemdl (1994).Thebacterial production(BP)wasmeasuredin seawatersamples after agitati onfor24boonandin noo-agitatedcontrols. Inthesampleswhichcontainedonlybacteria,BPwassimilarin stagnantandrurbulent conditions.This is consistent withtheresultspresentedinthis study.Therewasnosignificant difference inthebacterial growthbetweentheturbulent andstaticgrazer-freeconditions. Since a singlebacterium ismuch smallerthanthe Kolmogoro v scale, itexperiences only lam inarshearandmust depend uponitsown motilityto encounterandabsorbitsnutri ent s. The higherbacterial abundance at the end of the experiment in theturbulent condi tion wouldthenbea resultof abetter homogeneous mix of nutrients withinthetank,already an assumedcharacteristic of turbulence(see Section2.2).

It isevidentthatturbulence didhave a clear effect onthegrowthand grazing of themicroOagellate.Growthrueswerez2foldhigher intheturbulent(2.3

cr

^l)than staticconditions(1.1

cr

^l;Table2.3).Consequently,with higher Oagellateabundances.

thebacteriaweredepletedfrom theturbulentsystemmore rapidlythanthose ofthestaric.

Theseresults sugg est that flagellategrazing doeshave contro l of the bacterial populatio n inthepresen ce of turbulence.

28

Theingestionrates ofthemiaoflagellate were variableunderthetwo conditions. lngestiolt rateswerehigherin the6Tst6 to24h ofallthree turbulent experiments.

however.tberewas a massiveincreaseinflagellateDUmber'sandthe~ofprey particlesingest edperflagellate decreasedto themeanrateslistedinTable 2.3.The opposite occurred forthe static condition. For thefirst6to 24 h flagellate grazingUK!

ingestionbadv«ylittlecontrolon the bacterial abundance.After36hours.whengrazing mortalitysurpassedbacterialgrowth,flagellatesofthe static conditions stillingested the samenumberof«lish,1 as those flagell atesofthe turbulent conditions.These results are simi lar totheresults ofPetersandGross(1994)and Peterset aL(1996). Peters and Gross(1994)found that turbulenceeffective lyincreased the grazing ofP.imperforara, buttheingest ion ratesinthetwoexperimental conditionswerenot significantlydifferent.

PetersetaL(1996) foundthaJonly atveryhighle velsof turbulenee (IS emls'l)didthere appeartobedifferencesinthe flagellate abundances,feedingrates on fluorescently label ed bacteriaandgrossgrowthefficiency.They annbutedthe loweringestionrates of the turbulentcondition tothe decrease in cellsizeasthe flagella tepopulationincreased.

According to FencheJ(1982b),miaotlagellateswill continue to divide at lowfood concentnrions.,buttherewillbelittleincrease inthe totalbiomass,suggesting that these protozoa may resort to a survival techniquewhenconfrontedwithlittle or no food availa ble,orperiodsofstarvation.Cell division withoutgrowthmay have occurred in this experime nt because of the flag ellat eabundan ce outnumbering the bacterial abundance inthe turbulentcond itionsatthe endoftheexperime nts.Inadd ition,there

29

wasanoticeablesizedifference intheflagellate cellsattheend oftheturbulent inaJbatioDS.

Shimetaetat(1995)also reportednopromi nenteffect ofapplicdlaminarshear 00theclearance ratesofP.impetjOlYlta.TheyusedCouettetankstoprodu celaminar shear fields.Cou cttetanksusearotating platterandspindletogentlyspinthewater withinthetank.Aspreviou slydiscussed.laminarshear is characteristically found just belowtheKolmogorovturbulentlengthwhereviscosityisdramatically smoothing the smallest turbulenteddies.Shimetaetat(1995)did,however.observeasigni ficant increasein theclearanceratesofMonosiga sp.,andsuggested thattheeffectof twbulenceisspeciesspecific.Monosiga$p.isa choanoflagellateandistypicall ythe samesize(5 to 6urndiam eter ).or smallerthanP.imperforatQ.How turbulence effect s thefeedingrateofone micrcfle gellat e,but notanother,mayreflectthe differencein the mode of feed ingandhow preyparticlesarecaptured.Fenchel(1982&)describes Monosi ga ashaving acollarwith pseudopodiaarisingfromthebaseofthecellar.Prey particlesonlyneedtocome intocontactwiththepseudo podiatothen be carried downto the posteriorendoftheorganism.P.imperjoratrJ.ontheotherhand.musthavea prey particle come intodirectcontactwithitsventralfurrowinorder forittcbephagocytized.

Thepseudopodia ofMonosigamayprovidea largerswiaceareathantheventralfurrow ofP.impt!rfOl'ata.Alarger surface area couldbetranslatedtobencr chan ce sat encounte r.andtherefore a higheringestionor clearance rete.

Higher encou nter rates caused bythe fluidmotion surroundinganorganismdoes n01necessariJy resullinhigheringest ionrates,aswasprevious ly reportedinTable2_3

30

Thequestion stillremains:Whydo increasedwater movementscrea t ed bynlpidstirring or twbu1aJce influencethegrazingofmiCl"OClf"gallisms?ManymicronageUates..

includingP.imperfora ta,have been observedtohavea rando m walkpattern (Berg19 82, Peters 1994).Hence"it couIdbe arguedthatincreasedwater fluctuation swoul d only reinforce therandompatternsof the swimmingorganism..Mitwasdiscussed in Section 2.2, equatio05 (5)through(7) showed thatweakstirring would notenhance encounter rateswithprey particlesanymorethansimplediffusion.However. higherturbulent energies willallowsmallergrazersto~outrun-diffusionby movingfaster andcovering larger distances (Purcell 1917).Purcellhad also suggestedthat perhaps turbul enceis oaly leading microocganisms to~~pastures- wherehigherfoodcencen trarions exist.Ithas beenreponed thatsmall-scale turbulencemayaetuaJlypromoteplankton patchi ne ss(Kierbce1993) orparticl e concentratio nrather thanunifo rm distribution (Squirts and YamazaJci 1995).Hence.small-scale tw'buIencemayallow smallgrazers a betterchanceforswvival bytransportingthemtonutrientrichareas withinthe water colu mn.

2.UCODd ustoDJ

Thisresearch showsthatturbulence can augmentthe comm unitygrowthand grazing of the microflagellatespecies.P.impe rforata.This further suggeststhatgrowth and ingestion rates ofsomeprotistangrazersthathavebeendetermined under static incu bat ions,maybe under-estimatedcompared with the rateswhich occurinnature, whereturbulentconditions exist.Resultsfrom various other researchersdo notyet agree asto whysmall-scalenubulenceeffects microorganismsandbyhowmuch. Therefore

JI

moreresearchinthisfieldisneededfor a better understanding ofthis phenomenonand howitimpactsuponthetrophicinteractionsofthe microbial food web.

32

A B

Figure2.1:a) Prey particle encountering predatorthroughBrownian motionand b)prey particle encountering predat or by direct interception.(Vogel1994) Arrowsreprese nt the streamline of thefluid'sflow.

)J

DeMotor

plungers

~ ,:==--J III

A

-, ~ J

Coolantlines out

Coolantline in

\

Figure 2.2 Experimentalapparatusconsistingof3 staticand 3 stirred(turbulent) incubation tanks.Turbulence createdby verticallyoscillating plungers consisting of a plastic wandand a perforatedPVC plate.A) Still-photoof experimenta l system.B)Schematicdrawing of system.

...CO""4'utereonrro lkd tra"er..,mccham, rn

lkarn Spliner

Fig2.3Laboratoryset-upof thelaserDoppler vclocitimeter.Laser beam issplit into4 beams andpositionedjustbelowthePVC plunger.A)Schematicdrawing of experimental set-up.B) Still-photoof set-up.

35

A

8

BA~TERIA

- - , Static

r ;

^{f-- _ _ ....}

Tuftlulent

t-

T

10'

10'

-'E '8

^10'

10'

10'

a

12 24 36

hou rs

FLAGELlATES

48 60

Figure2.4:Thechangeinabundance:(±SE)ofthebeterotrophic Oagellate Paraphysomonosimperforata(open symbols)andbacteriaVibrio splendidus (filled symbols)of ExperimentA at15°Cinturbulent (circles)andstatic (triangles) conditions.

36

10'

-'E

^10'

~

B

10'

10"

0 12 24 36

hoors

FLAGELLATES

48 60

Figure2.5:ThechangeinabuOOance(±SEl oftheheterotrophicflagellate ParaphysomonasimfNrforoco(opensymbols)andbacteriaVibriospIendidus(filled symbols)ofExperiment8atISOCinturbulent(circles)andstatic (biangles) conditions.

37

10'

10'

60 48 36 24 12

10'-l---~---_---~--~---1

o

hours

Figure2.6:Thechangeinabundance(±SE) ofthc heterotrophic flagellate Parophyso"'onas;",pe rjora ta(open symbols)andthe bacteriaVibrio $plendicius(filled symbols) of Expcriment Cat150CintutbWent(cades)andstatic(triangles)conditions.

"

~ _--L~

~Il ·

- .

Turbulent Static

60 36 48

24 12

10'+---~--~-~_-~--~---'

o

hou..

Figure2..7:Changeinbacterial abundance(±SE)inagrazer-free envircnmeerof ExperimenI.AatateroperaneeofIS'Cin theturbuknt(triangles)anistatic(circ les) conditions.

"

10', -- - - - -- - - - -- - -- - ----,

10'

E

'8

10' Turbulent

Static

60 48 36 24 12

10'+---~---~---~---~--~

o

hours

Figure2.8:Changein bacterialabundance(±SE) in a grazer-free environmentof Experiment B atatemperatureof 15"Cin the turbulent(triangles)andstatic(circles) conditions.

40

Table2.1:Viscositiesofseawater collectedfrom LogyBay,Newfoundland (32%a), calculatedfromasetofequationslistedinAppendix A fromJuman~Ial,(1993).

Temperature ("C)

10 15

Dynamicviscosity (Pas)

1.83X10'}

1.56X10'}

1.33X10'}

1.11^X10'}

"

Kinematicviscosity (m^2s·^l)

1.78

x

10~

1.53x10~

1.30x10~

1.09X10..6

Table 2.2:The growth ratesoflheheterotrophicflagellateParapirys0m0na3;mperforata andthebacteriaYibriosplendidus(±~")foreach experimentatISDC.

Experiment Bacterial growth Flagellate growth (divisions d·^l) (divisions d·^l)

Experiment A

Static O.24±O.12 L10±0A3

Turbulent O.77±O50 355±L30'

ExperimentB

Static 1.06±0.60 O.86±O.24

Turbulent O.98±O.34 1.67±O.31'

ExperimentC

Static 1.06±O.60 1.03±O.26

Turbulent O.98±O.34 2.04±O.46'

Gran dMean

Static O.74±O.39 1.01±O.31

Turbulent _{O.77±O.29 2.35±O52}

• denotes a significantdifference(p<0.05,ANOVA)betweentbestaticandturbulent conditions.

"

Table2,3:Theingestionandclearance ri tesofthehetefOlrofhic fll geUltePorophywmo"asiMfNiforatafor eachexperimentItIS·C,Meanbaeteri.l.bundancewas10 to 10'cells mr',

Experiment Ingestion rate

Flagellate

Total bacteria

(bacteria clearance rate consumed

flagellate '

hoI)

(n l flagellate

hoI) (ml"h^{O' )}

Experiment A

Static

1.12 0.26 1. 09

x

10'

Turbulent 1.37 0.43 2.83x 10'

~ Experiment B

Static

7.03 1.8 8.72

x

10'

Turbulent

7.52 1.2 9.71

x

10 '

ExperimentC

Static

4.34 0.46 5.10

x

10 '

Turbulent 3.46 0.54 1.27

x

10 '

CHAPTERJ

TheEffectsof Small-Scale Turbulence andLowSeawater Temperatureson the Growth andGrazingofthe Heterotrophic Microflagellate,Paraphysom onas imperforata

3.1.1.latrod udio n

Itiswidelyacceptedthatheterotrophicbacteria arethefoundationofthemarine microbialfoodweb(Pomeroy1974,Azameta/.1983).Bacteriatakeupandrecycle dissolved organi c manerandothernutrientsintheocean whic hareproduced byprotistan and metazoangrazers.Moreover,bacteria aretoo smalltobeeffic iently ingest edand utilizedbymostmetazoangrazers(Fortieretai:1994 ).Hence.itisthe becteriv ercc s protists.suchas flag ellatesandciliates, whichinco rpo rat etheenergy of thatorganic matter intothemarine food web by repackaginglbesmal l cells into particleswhich can beefficientlyingest ed bylargerzooplankton(Goldmanet al.1985.Gokhnaneral.

1987).AJthoughmarine bacteria havethe potential forrapidgrowt h,the abundance s remain relativelyconstantthroughtime withina rangeor O.5to 3" 10' cellsml·^l (Anderse nandFenchel1985.DucklowandCarlson 1992),Itisbelieved that hecerotropbicmicroflagell atesare themajor grazers whichconuo lbacterialabu ndan ce (SherrandSherr 1984.Porterel01.1985,Fenchel 1987).Sherretal.(1986) suggested thatmiaoflagellat esare capable ofconsuming~.4Ofmore ofmebacterialbiomass.and Fenchel(1982d) calculatedthat 10to 10%of thewatercolumnis cleared of'bacteriaeach daybymicroflagellates.Withsuch alargegrating potential. itis notsurprisi ng that both

pbototrophicand beterotrophic protozoadominate the plankton communityinthe euphoticzone(GoldmanandCaron 1985).

Manymodelsforthemarinemiaobialfoodwebare qualitative mode lswhich require that biologicaland physicalprocesses beparameterizedand constrainedto the grazing impacton bacteria.Thisis essential to understandthefunctionalityof the microbialsystem (Wright 1988.).Manyphysicalprocessesinfluencetheconditionsthat cootrolimportantbiologicalprocesses,suchISgrowth,grazing. and panicledistri bution.

Clearly,bio~gicalprocessesc:annotbeconsidered in isolationof the physicaland chemicalenvironment (MmnandLazier 1990).Physical

processes.

suchas nubulence andseawatertemperatures.canpotentially impactthe pathwaysoftnnsferof microb ial production tolargerconsumers (e.g.mesozooplankton).Inthepastdecad e., several studies suggested that small-scaleturbulencecould influencethe encounterrate sofa planktongrazerand its prey(RothschildandOsborn 1988.MacKenzieand Leggett1991.

Kierboe andSaiz 1995).This theory has been examined numerous timeswith predat ors such as copepod.s (Marraseetal:1990 )and fish larvae(DoweraaL1996),but very few studies have investigatedtheimpactoftutbulenceonheterotro phic microflage llares Moreover,(0dare,studiesexaminingtheeffect ofsmall-scaleturbulence on microorganisms., including miaoflagellates.,have beencarriedoutItseawater temperatures above ISoC.Itseemsthatstudiesontemperature dependent bact erial and heterotrophicprocesses have been under-representedfrom cold orpolar environments (Rivkinetal.1996).Consideringthatmorethan70010ofthe oceanisalwaysbelowSoC.

and90%oftheocean issea.sonallybelow SoC(Baross andMorita1978.Levitus 1982.

"

RivIcineta!1996). itisnecessarytoassesstheeffectsof small-sca.le turbulence and cold seawat ertemperatureson thegrazingofbacteria byheterotrophic microflagellates.

3.1.1. Objectives

This chapt er examinestheeffectof twbulenceonthetemperaturedepend ent growthof the heterotroph icmicrofl agellate.Paraphysomonas imperforato.and the grazing on itsbacterialprey.Vibrio splendidus,atISoC,IOoc.SoCandO"c.The experimeotswere designedto encompass the seasonal temperaturevariabilityof Logy Bay,Newfoundland. eastern Canada..Itishypothesized that microflagellategrowthand iuingestionofbacteriawillbehigherin the turbulentcondition at all four experimental tempentures.

3.2. MATERIALS AND METHODS 3.2.1.Experimratalappantus

Theexperim e ntalappara tus consist edofthree insulated plexi glass containers (46em x20em) containi ngtwo smaller plexigl as sincubat io n containe rs(11cmxIIcm x14em height).Tbe threetanks were connectedtoarempera ture controlled rrcircu.Iating waterbath(NeslabRTE-2 (0).OfthesixsmaJler incu bationchambers, threeare thecontrol(i.e.noIPtuJbulent) oonditionandthreechambersarethetu:rtlulent condition (Figure2.2).

Theturbule ncewas creat ed by vert ical lyoscillating plungers consisti ngofa plast icwand andaperforatedpolyvinylchlori de(PVC)plate(plate sizewas9.Scm'. peri'orationdiameterwas 2nun,-93%solidity).Theplateswere centeredin each chamberwith2.5mmclearance betweentheinside walls of the tank:andthe edgeof the plates.Theverti cal amplitu deofthe plungermotionwas3em,travelling inthe upper portionofthewater column,andoscillating atarateof I Hz±0.3Hz. (see Figure 2.3) 3.2.2.QuantifiatioD ofturbulence

Turbulence wasmeasuredwitha2-axis300 mWargon-ionlaser Doppler velocitimeter (Dantec: Electronics)andprocessedwitha Flow Veloc ityAnemom eter (Dantec Electronics).Verticalandhorizonul componentsofwater velocitieswere measuredatdiffere ntpositio nswithinthe water column usingacomputercontrolled traversemechanism.Latexfluo rescentbeads(I~).similar in sizetothebacterial prey.

wereusedastracerparticlesinthe water colu mn.Eachvelocitycompone ntwas measuredata samplingrateof -120Hzwithlbandwidth of 0.12MHzand -2mmfringe

"

spacing (0-35).DalawerecollectedItrandomIcxationswithinthetankforan acc:umu!atioooC10.000 samplevelocities or a maximumoC 10 minutespersite.See Section%.3.1Corcompletedetailsonthedataanalysis ofthelaser do ppl er anemometer.

3.1.J. Maintenance ofeulturn

CulturesofParaphysomonasimpeiforatawere maintai nedin10mlglassculture tubes at allfourtemperatures.Stock cultureswere transferredinto 50mlof fresh mediumevef)'3to 4weeks.Themediumusedwas sterile,0.2J.lIDfilteredseawater enrichedwith Im1oClO% yeastextract (Difco)andIm1of 0.2%proteose peptone (Difco).Asterilericegrnn was added toeach culture tube. Vibrio splendiduJcultures weremaintainedon agarplates for upto one month. Twostepswere requiredforthe preparationofastockcultur e 10beusedforanexperiment.First,25mlofdeionized water(01) was enrichedwith0.935g ofpreparednutrient mix (Mari neBroth 2216.

Difco)and autoela ved in 100ml Erlenmy er flask s.A swaboff a marine broth agarplate wastransferredtotheaqueousmed ium.Theflaskwasthen placedon a reciproca.ting shakerCI..12 baI.room temperature.This was sufficienttime forV.splendidustoreach exponentialgrowth.. Thenextday.Imlof the culturewasdilutedto 800mIofsterile.

0.2um filtCl"td seawater.andreplacedonthe shakerfor 6 to 12 hours.This working stockcuJturewasthendivided among the sixincubar.ionchambent-130mleach).

diluted to1.3Lwithsterile, 0.2J.U1Ifiltered seawa ter.and allowedto acclimateto the experimentaltemperaturesbefor ethe additionofP.imperforata.

..

3.2....FlagdlateGrowth aDdlagestiodofButeN

FourofthesixiDO.lbat iontanks(two staticandtwo turbulent)containing the dilutedbacteri al eulrureswere inoculated with30mlofthe flagell ate cultu re andwere maintainedinstaticcondit io nforca.12hbeforetheinitial (t-O)sampling to allow for acclimation. Thetwo remainingincu bationtanks(onestaticandone turbulent) were grazer-freecontroI.s to assesstheeffea of turbulence onbacterial growth.Theduration oftheflageUategtO'Mhexperimenuwasdependentuponeachtemperature. Forthe ISoCandIO"C experiments.samplesweretakenat6 to 12 hinterv alsforthree days.For the SoCand O°Cexperimen ts,sampleswerecollectedevery 24 hfor thefirst 72h,and at every12hforthe next 72h(with the exceptionofone SoCreplicat ionwheresampling wassimilarto those of me highertemperatur es).All samples(SmI)for emu nen.ting bacteriaand flagellateabundanceswereimmed iatelypreservedwith 1mIof g1uteraldehyde(5'Y.finalconcentration),storedat room temperatu rein glass 20mlliquid scintillationvials,andprocessed with in3toSdays oftheircollection.

3.2.!. Sa m ple Adalylis

BacteriaJabundancewasdetenni ned withtheacridineorangedirectcountmethod (Hobbie etaI.1971).DuetohighbaaeriaJabundances.sampleswere dilutedupto-20 foldwith0.2urn filtered seawater.Thediluted subsampleswerecollectedonto apre- stainedblack0.2umPceeo cspolycarbo nate filterandpost-stai ned withacridi ne orange.

Twofiltersperslide wereplaced on smeared drops of CargilleTypeA immer sion oilon a glassmicroscope slide.Each filterwastoppedwith anotherdrop ofimmenion oil and the covenlipwas gentlypressed on so thatthe oilwasevenlydis persed overthe filter.