Grazing of particle-associated bacteria-an elimination of the non-viable fraction

h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Environmental

Microbiology

Grazing

of

particle-associated

bacteria—an

elimination

of

the

non-viable

fraction

Maria-Judith

Gonsalves

,

Sheryl

Oliveira

Fernandes,

Madasamy

Lakshmi

Priya,

Ponnapakkam

Adikesavan

LokaBharathi

CSIR-NationalInstituteofOceanography,BiologicalOceanographyDivision,DonaPaula,Goa,India

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Articlehistory:

Received5February2015 Accepted7April2016

Availableonline9November2016 AssociateEditor:WelingtonLuizde Araújo Keywords: Bacteria Ciliates Grazing Particle Protists Viable

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Quantificationofbacteriabeinggrazedbymicrozooplanktonisgainingimportancesince theyserveasenergysubsidiesforhighertrophiclevelswhichconsequentlyinfluencefish production.Hence,grazingpressureonviableandnon-viablefractionoffreeand particle-associatedbacteriainatropicalestuarycontrolledmainlybyprotistgrazerswasestimated usingtheseawaterdilutiontechnique.Invitroincubationsoveraperiodof42hshowedthat attheendof24h,growthcoefficient(k)ofparticle-associatedbacteriawas9timeshigher at0.546thanthatoffreeforms.Further,‘k’valueofviablecellsonparticleswasdoublethat offreeformsat0.016and0.007,respectively.Whilebacteriaassociatedwithparticleswere grazed(coefficientofremoval(g)=0.564),thefreeformswererelativelylessgrazed indicat-ingthatparticle-associatedbacteriawereexposedtograzersinthesewaters.Amongthe viableandnon-viableforms,‘g’ofnon-viablefraction(particle-associatedbacteria=0.615, Free=0.0086)wasmuchgreaterthantheviablefraction(particle-associatedbacteria=0.056, Free=0.068).Thus,grazingonviablecellswasrelativelylowinboththefreeandattached states.Theseobservationssuggestthatnon-viableformsofparticle-associatedbacteria weremorepronetograzingandwereweededoutleavingtheviablecellstoreplenishthe bacterialstandingstock.Particlecolonizationcould thusbea temporaryrefugeforthe “persistentvariants”wheretheviablefractionmultiplyandreleasetheirprogeny.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Introduction

Bacteria are capable of consuming more than 50% of pri-maryproductionintheformofdissolvedorganic matter.1,2

Thus, they play an important role as energetic subsidies for higher trophic levels. In recent years, studies focusing ongrazingofbacteriabybacteriophagousmicrofaunahave

∗ _{Correspondingauthorat:Aqua-geomicrobiologyLaboratory,CSIR-NationalInstituteofOceanography,DonaPaula,Goa403004,India.}

E-mail:mjudith@nio.org(M.Gonsalves).

gainedimportance.Predationbymicrozooplanktoninaquatic habitatsisknowntodecreasebacterialabundanceand stimu-latemineralizationofnutrients.3_{Theprocesscouldinfluence}

the morphological structure, taxonomic composition4 _and

physiological status of bacterial communities.5 _The

graz-ers(microzooplankton)consistofholoplanktonic(protozoa, ciliates, flagellates, copepod nauplii, etc.) and meroplank-tonic organisms (larval stages of benthic invertebrates:

http://dx.doi.org/10.1016/j.bjm.2016.10.009

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

trochophores,veligers,etc.).Protozoansareknowntoplaya majorecologicalroleintheaquaticenvironmentduetotheir effectivenessinconsumingawiderangeofpreysizeclasses andtypes.6_{Theyarecapableofconsumingotherprotozoa,}7

phytoplankton8andbacteria.9–11Theirgrazingabilitydepends on the concentration ofbacteria and digestion capacity of thegrazer.Someubiquitouslydistributedprotistslikeciliates occasionallyformamajorcomponentofthe microzooplank-toncommunity12,13_{andhaveakeypositioninplanktonfood}

websas majorconsumersofpico- and nano-plankton.14,15

Availability and concentration of the prey size and type, attachedvs.unattachedcellsandviablevs.non-viablecells aresomeofthefactorsaffectingciliategrazingrates.

Among the planktonic bacterial community, particle-associatedbacteria(PAB)havereceivedmoreemphasisthan free-living formsbecause of their relatively easier accessi-bility to filter feeders16,17 _{and their abilityto improve the}

nutritionalqualityoftheparticles.18_{ThePABcanbedirectly}

grazedbylargermetazoans,bypassingconsumptionby proto-zoangrazersandshort-circuitingthemicrobialloop.16_Reports

onpelagicbacterialcommunityhavedemonstratedthatitis unwarrantedtoclubparticle-attachedorunattached bacte-riaasasingleunit.19_{Tounderstandgrazingpreferenceson}

thebacterialcommunityinatropicalestuarydominatedby protists,weconductedaseawaterdilutionexperimentto mea-sure the coefficients of increase ‘k’ and grazing ‘g’.20 _The

followingquerieswereputforth:(1)dograzersexhibit selec-tivepreferenceforfreeor particle-associatedbacteria (PAB) and (2) which physiological state (viable or non-viable) of freeor particle-associated bacteria ofthe bacterioplankton willbeeliminatedbythegrazers.Thoughpreviousworkhas highlighted the importanceofPAB as food forgrazerslike microzooplankton16,17,21 _{for thefirst time wehave}

demon-stratedelevatedgrazingpressureonnon-viablecellsofPAB and conservationoftheir viableformsforstock replenish-ment.

Materials

and

methods

Studyareaandsampling

ANiskinsamplerwasusedtocollect watersamplesat3m depthinDonaPaulaBay(15◦27N,73◦48E;Fig.S1)whichis locatedattheterminusofZuariestuaryinwestcoastofIndia. Thissemi-enclosedbayisundisturbedbyseaportandriverine traffic.Detailsontheclimaticconditions,annualvariationin hydrographicparametersandbioticvariablesinthestudyarea havebeendescribed elsewhere.22 _{Thetemperature,salinity}

andpHofseawaterduringsamplingwere24◦C,21psuand 6.3,respectively.

Grazingstudies

The dilution technique23 _{was used for grazing studies in}

the present work. Thetechnique has been widely used to reduce the number of predators by creating a gradient.24

Themethodhasalsobeenusedtoestimategrowthratesof phytoplankton/bacterioplankton25,26 _{aswellasgrazing}

pres-sureexertedbymicrozooplanktonontheseforms.26,27_Weset

upincubationswithwholeanddilutedseawatertodetermine bacterialabundance(totalbacterialcells,PABandfree-living). A schematic representation of the experimental design is showninFig.S2.

Forpreparingparticle-freewater(usedfordilutingwhole seawater),thefiltrationassemblywaswashedwith50%HCl andrinsedwithMilli-Qwater.Twolitresofseawaterwasthen filteredthrougha0.22␮mfilter(pressure<100mmHg).Thus, toprepare‘diluted’samplesforincubation,untreated‘whole’ samplewaterwasaddedtoparticle-freewaterintheratio1:4. ThePABwereconsideredasthosethatwouldberetained ona3␮mpore-sizefilter(Fig.S2).Bacteriathatpassedthrough thesefilterswereconsideredasfree-livingbacteria.An esti-mateofthetotalandfree-livingbacterialpopulationinthe wholeanddilutedseawaterwascarriedout.Theabundance of PAB wasderived bysubtracting the free-living from the totalbacterialpopulation.Controlsweremaintainedforbottle effectandthesewereusedtosubtractfromtheexperimental values.Dilutedandwholewatersampleswereincubatedfor up to42hat27±2◦Cunderstaticconditions. Sub-samples were removedfromthewholeanddilutedseawaterbottles at6hintervalsfortheestimationofTC(totalbacterialcells), thetotal,viableandnon-viablefractionofPABandfree-living forms.Fortheenumerationoftotalbacterialcells(TC),a5mL aliquotofwatersamplewasfixedusing250␮Lofbuffered for-malin(2%finalconcentration)asdescribedbyHobbieetal.28

For enumerationofviablecells(VC),5mLofseawater sam-pleswereamendedwith0.001%finalconcentrationofyeast extract and 0.0016%final concentration of antibiotic cock-tail solutioncontainingpiromedic,pipemedicandnalidixic acid whichwereinthe ratioof1:1:1.29 _Sampleswere

incu-batedstaticallyindarkfor6h.Attheendoftheincubation, thealiquotswerefixedwith2%ofbufferedformalin.ForTC andVC,1mLofsub-samplewasfilteredovera0.2␮mblack Isoporepolycarbonatefilterpaper(MilliporeCorp.,MA,USA) andstainedwithacridineorange(finalconcentration0.01%, w/v). The samples were incubated for 2min and then fil-tered.Bacterialcellsretainedonthefilterpaperswerecounted using Nikon 50i epifluorescencemicroscope equippedwith a 100× oilimmersion objective. Viable cells refer to those whichenlargeusingthedirectviablecount(DVC)procedure.30

Theseenlargedcellscompriseofamixtureofcellswhichhave increasedinsizeandactivelydividingcells.Thus,inthisstudy, onlycellsthatenlargeorreplicatearecountedasviablewhile thenon-viableformsareconsideredastheoneswhichdidnot enlarge/replicate.Bacterialabundancehasbeenexpressedas cellsL−1.

Thespecificrateofincreaseinbacterialcells‘k’and mor-tality/decrease ‘g’ of bacterioplankton were estimated.The constantskandgarecoefficientsofpopulationincreaseand grazingmortality,respectively.23_{Theseconstantsalsoreferto}

increase andremovalnotnecessarilylinkedtogrowth and death.Therefore,itwouldmeananincreaseinonepooli.e., particleassociationbycolonization,anddecreaseinanother pool i.e.,free-livingforms.Boththesecoefficientsmayvary withtimeofdaywithoutaffectingourcomparisonsofincrease in bacterialnumbers in thedilution over a fixedperiodof incubation. Thus, the abundance and grazing mortalityof bacterioplanktonwasinferredfromobservedchangesin pop-ulation.

Whole seawater 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 42 36 30 24 18 12 6 0 Abundance (x 10 9 cells L -1)

TC PAB Free-living bacteria

Diluted seawater 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 42 36 30 24 18 12 6 0 Time (h) Abundance (x 10 9 cells L -1)

A

B

Fig.1–Abundanceoftotal,particle-associatedand free-livingbacteriainwhole(A)anddiluted(B)seawater.

Microzooplanktondensityanddiversity

Duetolowdensityofplanktoninthestudyarea,10Lof sea-waterwasobtainedfortheenumerationofmicrozooplankton densityanddiversity.Thewatersamplewasfilteredthrough a200␮mmeshtoexcludemesozooplankton.Further,this fil-teredwaterwasslowlypassedthroughanetof20␮mmesh size.Theseawaterwasthenconcentratedto1Lbysiphoning outexcesswaterwiththehelpofaTygontubing(Saint-Gobain PerformancePlasticsCorporation,USA).Thedippedendofthe tubingwascoveredwith20␮mmesh.Thereafter,thesample was preserved in 1%acid Lugol’s solution and left to set-tlefor24hand furtherconcentrated to10mLbysiphoning outtheexcess water.Onemillilitreofconcentrated sample wastransferredintoaSedgwick-Rafterchambertoenumerate microzooplanktonabundance.Countingwasdoneusing an invertedphasemicroscopefittedwithawhipplegrid.About 30–40fields representing150–200individualswerecounted. Themicrozooplanktonwereidentifiedtogenusand/orspecies level.TheirdensityhasbeenexpressedasindividualsL−1.

Results

Grazingofparticle-associatedandfree-livingbacteria

Theabundanceofbacterialcellsinwholeseawaterincreased steadilyover timeand peaked atthe end of36hto reach 1.16±0.06×1010_cellsL−1_{(Fig.1A).Inthedilutedsample,peak}

incellabundancewasobservedat24hofincubationandwas marginallyhigherat1.51±0.09×1010_cellsL−1_(Fig.1B).

Table1–Coefficientsofincreaseandremovalof

bacterioplankton. Bacterial fraction Coefficientof increase(k) Coefficientof removal(g) k−g PABTC 0.546 0.564 −0.018 PABVC 0.016 −0.056 0.072 PABNVC 0.528 0.615 −0.087 FreeTC 0.064 −0.077 0.141 FreeVC 0.0066 −0.068 0.0746 Free NVC 0.074 0.0086 0.0654

Note: PAB, particle-associated bacteria; TC, total bacterialcells; VC, viable bacterial cells; NVC, non-viable cells. Coefficient of increaseincludesactivelyreplicating/enlargedcellsandnet col-onization/exchangefromthefreetotheparticle-associatedpool andviceversa. Thecoefficient of increaseof non-viableforms was higher than that of viable ones as these include non-replicating/non-enlargedcells.

Likewise,thepeakinPABabundanceinwholeanddiluted

seawater was observed at the end of 36 and 24h,

respec-tively. ThemaximumabundanceofPABinwholeseawater

was8.10±0.73×109_cellsL−1_(Fig.1A).

The viable PAB abundance was not affected both in whole(R2_{=0.6937,p<0.05,n=5;Fig.2B)anddilutedsamples}

(R2_{=0.2204).Indilutedseawater,PABabundancewashigher}

byoneorderi.e.,1.20±0.04×1010_cellsL−1_(Fig.1B).

Thenon-viablefraction increasedover time(R2_=0.6845,

p<0.05,n=5;Fig.2C)withakvalueof0.528(Table1)in com-parison tothe viableforms(k=0.016).However,removalof non-viablePABbygrazerswasfargreater(g=0.615;Table1) than thatofviablecells (Table1). Free-livingcells were far lesserinabundancecomparedtoPABandwereslowgrowers asobservedfromthelowkvalues(Table1).Their numbers in whole seawater increased from 0.81±0.07×109_cellsL−1

at the beginning of the incubation period to a maximum of6.25±0.66×109_cellsL−1 _{atthe end of30h(Fig.1A).The}

increaseinabundanceoffree-livingnon-viablecellswasseen tobestronglydependentontheincubationtime(R2=0.7725, p<0.05,n=5;Fig.2F).Indilutedseawater,asteadyincreasein free-livingbacterialcellswasobservedupto36hwherethey numbered about 3.34±0.22×109_cellsL−1 _{(Fig. 1B). Increase}

in the abundance of viable free-living cells over time was marginally higher (R2_{=0.3756, n=5; Fig. 2E) than the}

non-viablefraction(R2_{=0.3653,n=5;Fig.2F).Removaloffree-living}

formsbygrazerswasfoundtobenegligible(Table1).

Microzooplanktondensityanddiversity

Leprotintinnus nordquisti and Stenosemellasp. dominated the

microzooplanktoncommunityin theDonaPaula bayatan abundanceof25and24%ofthetotal,respectively(Fig.S3). OtherimportantmicrozooplanktonincludedFavellasp.(12%),

Dictyocysta sp. (8%), Codonellopsis sp. (8%) and Tintinnopsis

uryguayensis(5%).

Discussion

Bacteriaare relevantmembers oftheplanktonic foodweb, both in terms ofbiomass and production share.However,

y=0.1323x+0.986 R2_=0.622 y=0.0847x+0.468 R2=0.6238 0.0 1.0 2.0 3.0 4.0 5.0 6.0 24 18 12 6 0 Free-living_TC (x 10 cells L 9 -1) y=0.0737x + 0.128 R2=0.4477 y=0.0183x + 0.196 R2_=0.3756 0.0 0.5 1.0 1.5 2.0 2.5 3.0 24 18 12 6 0 Free-living_VC (x 10 9 cells L -1) y=0.0693x + 0.572 R2_=0.7725 y=0.0598x + 0.208 R2=0.3653 0.0 0.5 1.0 1.5 2.0 2.5 3.0 24 18 12 6 0 Time (h) Free-living_NVC (x 10 cells L 9 -1) y=–0.0087x + 2866 R2 =0.017 y=0.426x – 1.46 R2=0.7307 0.0 2.0 4.0 6.0 8.0 10.0 12.0 24 18 12 6 0 PAB_TC (x 10 cells L 9 -1)

Whole seawater Diluted seawater y=0.0602x + 0.116 R2=0.6937 y=0.0255x + 0.258 R2_=0.2204 0.0 0.4 0.8 1.2 1.6 2.0 24 18 12 6 0 Time (h) PAB_VC (x 10 9 cells L -1) y=–0.0777x + 3.16 R2=0.3449 y=0.4242x – 1688 R2=0.6845 0.0 2.0 4.0 6.0 8.0 10.0 12.0 24 18 12 6 0 Time (h) PAB_NVC (x 10 9 cells L -1)

A

B

C

D

E

F

Fig.2–Regressionanalysisoftotal(A),viable(B)andnon-viable(C)particle-associatedbacteria(PAB)inwhole()and diluted()seawater.Regressionplotsfortotal,viableandnon-viablefree-livingbacteriaareshownin(D),(E)and(F), respectively.Linearregressionlineforwholeseawatersampleshasbeendepictedusingasolidlinewhereasthedashed linedisfordilutedseawatersamples.TheR2_{valuesdenotedinthefigureshavebeenconvertedtothecorrelationcoefficient}

(r).Subsequently,thelevelofsignificance(p)foratwo-tailedtestwasinferredfromastandardtable(WheaterandCook, 2000)43_{andwasacceptedatp<0.05.}

bacterialmortality/lossoccurslargelyduetoprotistgrazing31

whereas,viral lysis accounts for ∼10–20%of the loss.32 _In

aquaticsystems,PABareknowntoconstituteabout60%ofthe totalbacterialcounts.Inthepresentstudy,microscopic exam-inationrevealedthatPABformedupto76%oftheTC(Fig.1B)in wholeseawaterwhereas,thefree-livingbacterialcellsvaried between26and82%(Fig.1C).Ourattempttodemonstratethe effectsofgrazingonparticle-attachedandfreebacterial pop-ulationinnaturalsamplesusingdilutionmethod,estimated anincreaseandremovalcoefficients ofPAB andfree-living bacterialcells.Thisapproachminimizedthegrazingpressure onbacterialcellsasevidentfromapeakintheirabundance within24hofsampleincubationascomparedtothe undi-lutedsamplewherethepeakwasat36h(Fig.1B).Basedonthe gvalues(Table1),thegrazingpressureonPABwasfargreater thanonfree-livingcells.Inaquaticsystems,bacteriaattached to detritalparticles serve as an importantfood source for zooplankton33_{e.g.,ciliateswhichtransfersignificantamount}

ofbacterialproductiontohighertrophiclevels.34_Atthetime

of sampling in the Dona Paula bay, tintinnid ciliates viz.,

L. nordquisti and Stenosemella sp. were dominant (Fig. S3).

There was a considerable bacterial load in the study area whichimpliesthatthebacterivorousciliatesdidnotfaceany limitation forfood. Itmay benoted that theexperimental approachusedinthepresentstudydidnotdirectlymeasure grazing.Therefore,allreferencestograzingareinferredfrom indirect observations. We intend to study the impacts of grazingonbacteriabyciliatesinourfutureresearch.

An increase of viable cells in both diluted and whole water samples (Fig. 2B and E) was observed. The increase wasmoreprominentinthelatterwithanegativecoefficient of removal suggesting avoidance/escape. More viable cells perhapsget added fromthe freepool besidesthe increase bymultiplicationonparticles.Incontrast,thecoefficientof increase forPABNVCwas marginallylesserthan theirrate ofremoval(Table1)indicatingtheireliminationassoonas theyaccumulate.Thisinturnleavestheviablecellsrelatively intact.

Not only do the free viable cells but also the particle-associatedviableonesescapegrazing.Weobservednegative grazing coefficients for viable cells of PAB and free-living bacteria.Attachedbacterialcommunitiesareknowntorelease progenyintothesurroundingwater.35_{Consequently,ageneral}

exchangeofattachedandfreebacteria36 _{intheviablestate}

couldbeexpected.AsthecoefficientofincreaseofPABwas morethan2×higherthanthatoffree-livingforms(Table1), itisapparentthatfreecellsattachandmultiplyonparticles. Theadherenceofbacteriatodetritalparticlesandabilityto rapidly reproduce isan importantresponse forsurvival in thepelagicmarineenvironment.37Itispossiblethat bacte-rialcellsfirstlosetheirviabilityandwhentheaccumulation reachesathreshold,grazingeliminatesthem.Fromour exper-iments,itcanbenotedthatthepercentageofnon-viablePAB andfree-livingcellsisrelativelymorethantheVC(Fig.2).For example,attheendof6hofincubation(Fig.2B),the viabil-ityofPABinwholeanddilutedseawateris<21%oftheTC; whereas,the NVCcanconstitutealmost90–99%.Whenthe NVCreachessuchathreshold,thelownumbersofremaining viablecellsi.e.,the persistentvariantsdetachintothe free pooltostartanewcycleonfreshparticles.De-colonization hasbeen suggestedasa meansofavoiding sinking.38 _Itis

alsoameansofquittingtheless labile,over-agedparticles fortherelativelymorelabilefresherparticles.Previous stud-ieshaveshownapositivecorrelationbetweenabundanceand productionoffreeandattachedbacteriaindicatingthat pro-ductionbyattachedbacteria supplies‘new’ freebacteria to thepelagicmicrobialcommunity.39_{Thoughlargefree-living}

activecellshavebeenshowntobepronetograzing,40_someof

theseformscouldescapebeinggrazedtemporarilyby divid-ingorcolonizingondetritalparticles.Itissuggestedthatfresh particulateorganicmatterwouldnotattractgrazerstillthey are sufficientlyagedwhilethe mostcomplexpolymersget convertedtosimpleronesbybacterialaction.Bacterial asso-ciationwithparticlescanbeactiveanddynamic.41_Ourstudy

demonstratesthat grazingpressurecancontrolthe physio-logicalstateofthebacterialcommunitybesidesotherknown factorslikegenotypeandphenotypiccomposition.42

Conclusion

ThisstudydemonstratesthatPABaresignificantlygrazedand thenon-viablefractionismorepronetoremovalbygrazers like tintinnid ciliateswhich are abundantin tropical estu-aries.Theviablebacterialforms(persistentvariants)which aregenerallymuchlowerinabundanceinboththeattached andfree-stateescapegettinggrazed.Therefore,itissuggested thataccumulationofthenon-viablecellsonparticlesupto athresholdcouldsignaldirectly(1)theremainingviableor “persistentvariants”todetachor(2)indicateparticle matu-ritytograzers.Incontrasttothegeneralcontentionthatactive cellsaremorepronetobeinggrazedupon,itnowseemsmore apparentthatthenon-viablecells generallygeteliminated, leavingtheviablelessdisturbedtoreplenishthestock.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

ThispaperwassupportedbygrantsfromtheOfficeofNaval Research, Washington,D.C.,USA.Theauthors arethankful totheDirector,NIOforthefacilities.Theauthorsthankthe twoanonymousreviewersfortheirvaluablecomments.This isNIOcontributionno.5885.

Appendix

A.

Supplementary

data

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,atdoi:10.1016/j.bjm.2016.10.009.

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