Effectof algal cell density,dietary compos ition.,growth phaseand macronutrient concen tratio nongrowthandsurvi valof giantscallopPlacope cten magellanieus
(Gmelia,1791)larvaeandspatina commerc ial hatchery.
by
<0CatherineMaxine Ryan,SSc.
Athesis submitted to the Schoolof Graduate Studiesinpartialfulfillmentof the
requirements for thedegree of Masterof Science(Aquaculture)
SchoolofGrad uate Studies Aquac ultureProgram Memori al University of Newfoundlan d
October1999
St.John's Newfoundland
Abstract
This studywas aimed atoptimizing thegrowth and survivalofPtacopecten mage ltamcu s larva eandspat bymanipulation ofthe hatcheryalgaldiet with respect to cell density,speci es composition.phase ofharvest., andmacro nu trien t concentrationof the algalgrowthmedium.Algae were culturedin a chemcstee systeminwhichvariationsin celldensityand growthratecouldbe controlled.
Thisstudy indicatedthatanalgalcelldensity of 40 cellsh llresulted in mo st efficient useof thealgal ration.This ration wasthe n fedto spatin subsequentgrowth trials. Spat werestained withcalcein(0.15gILseawate r)priorto experimentation.for a duratio n of72hours,tointrod ucefluores cent marker ban dswhichcould beusedto measurenewshell growth inthesetrials.
Attemptstoimpro vethestandardhat chery dietby the additionof algalspec ies showedthatTetrase lmtssliecica, ChaeloceroscalcitransandChroomonas salinawer e not beneficialas a supplement toa mixedalgaldiet forPtacope ctenlarvaeandspat.
Scalloplarvae were found togrowbetterwhenfedlogari thmi cphase algaeor algaecultu redund erhighmacron utri entconcent ration.suggesti ngahigherrequirem en t forprot einduring early development. Spatwere foundtogrowbetteroverall when fed statio nary phasealga e oralgae cultur edunderlo wmacro nutrientconceruraticn,which were foundtobe highinlipidand carbohydratecontentimpo rtan t formetamorphosisand pcsttarval (sp at)development. Spatshell growth and total lipid and carbohydra te ofthe diet were positively correlated.
Theresu lts ofthe study also indicatedthatIbatassiostro~issjlogiiwas beneficialin a mixed algaldietfor giant scallo p spat. and thatspatselectivelyingest significan t ly more1halassiosiraweissflogiiby volume incompariso n toaspecies of similar celt size,Tetrase /mis suecica
This study ind ica teswhereimpro vements can be madeto traditi onally used mixed algal dietsfedtolarv aeand spat ina commercialhatch ery givinghigher growth and survival priortotransfertothenatu ralenvironmentatnursery orgrow-outsites
Acknowledgeme nts
Iwould liketoexpressmygreatappreciationtomy supervisor. Dr.Patrick Dabinen, forhisfriendship.help.guidance and patiencethroughoutevery aspect ofthis project.A5well,I expressmythanks to Dr.JayParsons,Dr.Ray Thompson, Dr. Chris Parrish,and JeannetteWellsfo r theiradvice, and theOcean SciencesCentre,Memori al University. foruseof the facilitytoconduct lipid analysis.
Therest of theresearchfor this projectwascarried out atthe sea scallop hatchery locatedin Belleoram, Fortune Bay. Newfoundland.Isincerely expressmy thanksfor the useofthe facility and cooperati on,friendship and help from thestaff;namely,Jennifer Caines. Newman Rose,Steward May, Abigail Crock er,and Kevin Crocker.
This workwassupportedinpart bygrants from theCanadian Centrefor Fisheries Innovationand the Aquaculture Compo nentof the CanadaINcwfoundland Economic RenewalAgreement, andbyfellowships fromthe Schoolof Graduate Studies, Memorial University,allof whicharegratefully acknowledg ed.
iii
Contents
Abstract... . ji
Acknowle dgeme nts... . .iii
Contents .... ... ....iv
List oftables... . vii
List of ligures.... . viii
. 2
.. 1
. 1
.. 3
. 5
. 6
..__8
. .10
. 10
. 11
Introductioo...
l.lGenera1....
12 Algalculture...
1.3 Algal cell density...
1.4Alg aldiet ary composi tio n....
l.5Algalgrowthphase...
1.6Algalmacronutrie ntconcentration...
1.7Selectionofalgalspeciesbyfilter-feedingbivalves ...
L8Calceinstaining...
'1.9Objectives....
MaterialsaDd Metbods.. ... 13
2.1Study sit e... . 13
2.2Alg al culture... . 13
2.3Algaldryweight... .. 15
2.4 Larval andspatexperimen tal rearingtanks,; .. 15
2.5Spatstainingwith calcein... . 16
2_6Duration of growth trials.... . 17
2.7 Algalcell densi tyandspatgrowth.. . 17
2.8Attemptedimprovementof stan dardhatch.erydiet by add ition
ofalgalspecies... . 18
2.8. 1Chaeloceros calcitransinlarval diet s... . 19 2.8.2Chae toceroscalcitransinspat diets... . 19 2.8.3 TetrasetmtssuectcaandChroomonassalinainspat diets 19
2.9Algalgrowthphase... . 20
2.9.1Larvae... . 20
2.9.2Spat... . 20
2.10Algalmacronutrientconcen tration... . 21
2.10.1Larval diets ofalgae harvested from continuous cultureathigh andlowmaaonutrient concentrations... ... 21
iv
2.10 .2Spat dietsof algae harvested from contin uous cultureat high
and low macronutrient concentrations.... 22
2.10.3Larval diets ofalgaeharvested fromcontinuou sculture at highandlo w macronutricntconcentrations andhighand low
tem peratureregimes... .,.,
2.11 Tetraselmtssuecica,Tnatassiostraweissflogiiand T-lso fed tospat
singlyandinco mbinatio n... . 23
2.12Lipidanalysis ofalgae... . 24
2.13 Carbohydrateanalysis ofalgae... . 25
2.14Statistical analysis... . 25
Resull'.... . -27
3.1Spat stainingwithcalcein... . 27
3.2Algal cell densityand spatgrowth... . 27
3.3Attempted improvementofstandard hatcherydiet byaddition
of algal species... . 27
3.3.1Chaetoceros ealeitrans in larval and spat diets... . 27 3.32Tetra setmts suectcaandChroomonas salinainspat diet s 31
3.4 Algal growth phase... . 3I
3A-l Larvae... . 31
3.4.2 Spat... . . 31
3.5Algal macr onutri entco ncentra tio n... . .32
3.5.1 Larval diets of algae harvested from continuouscultureathigh andlowmacronutrient concentrations... . .32 3.5.2Spatdietsof algae harvested fromcontinuo usculture athigh
andlowmacronutrient con centra tions... . .38 3.5.3 Larval diets ofalgaeharvest ed from continuous cultureat
highand low macronutrientconcent ratio ns and highand low
temperatureregimes... . 38
3.6 Tetrasetmts suecica,Thalassiosiraweissflogiiand T-Isofedto spat
singly and in combination... . .39
3.7Lipid analysisofalgae... . .. _50
3.7.1 Larval diets of algae harvested from continuous culture at high and low macronutrientconcentrati o nsandhighandlow
temperatureregimes... . 50
3.7.2 Spat dietsofalgae harvested fromcontinuo usculture at high
and low macronutrien tconcentrations... . 50
3.7.3 Tetraselmissuecica,Thalassiosira wetssflogit and T-Isofedtospat
singly andin combination... . 50
H Carbohydrateanalysis of algae... . 51
3.8.1 Spat diets of algae harvested fromcontinuo uscultureathigh and lowmacronutrie ntconcentrations... ...51 3.8.2Tetrasetmtssueciea.Tha1assiosiraweissjlogiiandT-Is o fedto spat
singlyandin combination. . 51
... . ... .... ...66
. 66
.__67 Discus sion...
4.1Spatstaining with calcein...
4.2Algal celldensity and spatgrowt1L.
4.3Attempted improv ement of standard hatchery dietbyaddition
of algal species 69
4.4Algalgrowth phase..; . 70
4.5Algalmacronutrientconcentration 73
4.6Tetrase/mis suecica , Tha iassiosira wetssfiogiiand T-Iso fedto spat
singly andincombinatio n 76
4.7Growthratesof scallo plarvaeand spat... . 84
Litera tureCited...
vi
...88
Listof Tables
Table 1:Algalculturesused for-feeding giantscalloplarvaeandspat 15 Tab le2:Concentrationof lipidclasses in algal species culturedatlow and high
macronutrient conce ntr ations andtemperatur eregi mes and fedto
Ptacopec tenspat... . 54
Table3:Pears on correlationcoefficients:larval shellheight andsp atshellgrowt h rate
vs. lipid class compositionof thediets 56
Table4:Concentration oflipidclassesinalg al speciesculturedat low andhigh macronutrientconcentratio ns and fedtoPlacopectenspat _ 58 Table5:Concentrationoflipid classes inalgalspecies cultu redatlowmacronutri ent
conc entra ti onsand fedtoPlacopec tenspatsingly andincomb ination 60 Table6:Concentr atio noftot al lipid andtotal carbo hydrate in micro algaeculturedat
highandlowmacronutrientconcentrations andfedtoPlacopecte nspat 63 Table7 Concentrationoftotallipid and total carbohydra teinmicroalgae cultured at
lowmacronutrient conce ntr ati ons andfed toPlacopec ten spatsingly and in
combination 64
Table 8 Compari son ofconcentrati ons oftotallipidandtotalcarbohydrate foundin
microal gaeunder different culturing regimes.. . 79
vii
Listof Figu r es
Figure1:stainingof scallop spat(0.5mm-2 mm)withcafcein,•.•••••.•.. ..•.••.•...••....28 Figu re2:Growth of scallop spat fedadiet ofisochrysisgalban a,T-lso,Chroomo nas
salina.Tetrasetmtssuecica ,Thalassiosirapseudonana andOaetoceros
muelleriatvarious ceUdensities... . 29
Figur e3:Clearanc erateofPlacopecten spat feda dietoflsochrysisgalbana,T-Is o, Chroomonas sa/ina, Tetrase/missuectca,Ihatassiosira pseudonanaand Chaetoceros muelleri atvario uscelldensities.,. . .30 Figure4:Ingestio nrateofPlacopecten spat feda diet ofIsochrysi s galbana,T'-lso,
Chroomonassalina,Tetrase/missuectca,Thalassiosirapseudonana and Chaetoceros muelleri at variouscelldensities. ... .30 Figure5:Theeffectof addingChaetoceroscalatranstoa standarddiet(Chaetoceros
ceratosporum,Chaet oceros muel/eri,1halassiosira pseudonana,/sochrysis galbana and T-Iso)on growth ofPtocopectenlarvae.... . .33 Figure 6.The effect ofaddingCnaetocerosoatcttranstoa standard diet(Chaetoceros
ceratospo rnm, Chaetocerosmuel/eri ,Thalassiosira pseudonana,/sochrys is galbaha and T-Isc )ongrowth ofPlacopeetenspat .33 Figure 7.Theeffectof addingTetraselmis snecicaandChroomonassalinato a
standarddiet(Chaetocer osceratospontm, Chae toceros muelleri, Thalassiosirapsendonana.Isocbryasgalbanaand T.150)ongrowth
ofP/acope ctenspat.;. . 34
Figure8:Growth of scalloplarvaeon diets ofmixed algaeharvest edfromcultures maintainedinlo g andnear stationaryphase... . 35 Figure9a-d:Growth ofscallop spat on dietsof mixed algaeharvestedfromcultures
maintainedinlog and near stationary pha se 36
Figure 10 Clearancerate ofscallop spatgrown on diets of mixed algaeharvested from cultures maintainedinlog andnear stationaryphase 37 FigureII:Effect ofdietsof algae culturedunder conditionsof highand low
macron utrientconcentrationsongrowthofPtacopeaenlarvae... . .40 Figure12 Effect ofdiet s ofalgaeculturedunder conditions of high and lo w
vi i i
macronutrie ntconcentrationsongrowthofPtocopectenspat .41 Figure 13 Clearance rate ofspat fed diets ofalgaecuhurcdunder cooditionsof high andlow
macronutrient concentrations... . ..41
Figure 14:Effectof diets of alga ecultured underconditio nsof high and low macronutrient concentrations and high and lowtem perature regimes
ongrowthofPlocopectenIarvae.. . 42
FigureIS Effect offeedingT -Iso,TetrasetmtsslIeci cn and ThalassiosirolI'eissjTog;;
singlyandincorrtination ongrowthofP/accpectenspaL... ...44 Figure16 Clearance rate ofP lacopectenspatwhenfed T-Iso,Tetrasetmissuecica
and ThalassiosiraweissfIogiisingly and in combination... . 44 Figu re17 <a).Coulter Multisizerplot s showingtheinitialnumber of cellsin a50150
ratiodietofT- lso/ ThalassiosiraweissjIogii and after4 daysina
feedingtrial.. . .45
Figure17(b) CoulterMultisizerplotsshowingtheinitial volumeofcellsina50150 ratiodiet of T-Iso/Iha/ass;osira weissjI og Uand after 4 daysina
feedingtrial . .46
Figure18 Number ofalga1 cells of each speciesinthe50150ratioofT~[so/Terrascfmis suecicadieting est ed byPlocopeeten spat per hour.Data from Multisizer
plots 47
Figure19:Numberof algal cellsofeachspeciesin the50150ratioofT-Iso!
ThaJassiosira weissf10giidietingested byPlocopectenspat per hour.
Data fromMuhlsizer plots... . .47
Figure20:Vol ume of algalcells ofeach species in theS0I50ratio ofT-lsoITerraselmis suee/eadiet ingested byPlacopeetenspatperhour.Data fromMultisizer
plots .48
Figure 21;Volumc of algal cellsof each speciesinthe 50150ratioofT-lsoI Thalassios ira weissjI ogi idietingested byPlacopectenspatperho ur.
Dat afro mMultisizerplots.... . .48
Figure22:Ingesti on rates ofPlacopeetenspat fed 50/50rati oT_lsofTetrasel mls suecicaandT·lsoIThalassiosiraweissfIog;;diets overtheduration
of the feedin gtrial... . .49
ix
Figure 23:Lipid class compositi onof algaldiets gro wnatlo w and highmacronut rient
concentrations and temperature regimes ... . 53
Figure24:Lipidclass compo sitionofalg aldietsgrownatlo w and highmacro nutrient
concentrations 57
Figure25:Lipidclasscomposition ofdiet s consistingof T-Iso,Tetrasetmts snecica andTbatassiosra weissjlogii fed to Placopectenspat... . 59 Figure26 Totallipid(withou thydrocarb on)ingest ed perhou r from each alg alspecies
ofthe T-isortetmsetmtssuectcadiet byPlacope cten spat.... ...61 Figu re27 Totallipid(witho ut hydrocarbon) ingestedper hour from each algal species
of theT-l501Thalassiosiraweissflogii diet by Plocopectenspat 61 Figu r e28:Tot alcarboh yd rate inalgaecultu red at low andhigh rnacr onutrien t
concentration and fed toPlacopecten spat . . 62
Figure29:Total carboh ydra t ecfT'-lso,Tetrasetmts snecicaandThatassiosira weissf/ogiifedtoPfacopeeten spat singly and in combination 62 Figure 30 Total carboh ydra teingestedbyPfacope ctenspat per hourfromeach algal
species ofthe50/ 50 ratioofT-l501Tetraselmissuecica diet...•...•... ... •.65 Figure31:Total carboh ydrate ingest ed byPtacopectenspat per hour from each alg al
speciesof the50/50ratio ofT-IsoiThalassiosirawetssftogitdiet 65
Cha pter1 Introdu ction
W. !i<Iw:lII
The giantscallop or sea scallop(Placopect enmagellanicusGmelin,1791) ranges from the nonhshoreof the Gulf of St.LawrencetoCape Hatteras, NorthCarolina (posgay,1957). Itis abenthic,subtidal, activesuspensionfeeder ingestingphyto plankt on, small zooplankton,spores,and detrital particles (Shumwayet at,1987)
Inthe giantscallop, the sexesareseparate,gametes are releasedinto the surrounding water;and fertilization occursext~y(Levitan, 1991).Fertilized egg s undergo developmentinto plankt oni c Dcshapedveligerlarvae approximately 4daysafter spawning.Whenlarvae reach about2201A-ttl,approximately28 days afterspawning, they develop pairedeyespots and a foot (pediveligerstage),settle on the bottom, undergo metamorphosis (spat stage)(approximately35 daysafter spawning),andattach toa varietyofsubstra tes (CuUiney,1974).
Placopectenmagellanicus once supportedavaluablefisheryin eastern Canada and theno rtheastern United States. andstilldoes in certain regions.Between1976and 1987 giant scallop harvests representedabout30% of theworld production ofallscallop species.butove rfishing has depletedsomenaturalstocksinrecent years (Naidu,1991).
This has stimulated renewedintere stingiant scallop aquaculture .
Placopectenhas anumber of featu res that make it an exceUentcandidate for cultureinAtlantic Canada (Dadsw eUand Parsons,199 1).Scallops ofcommercial size (80-100mm) containalargeamount ofmeat whichcommands ahighmarketprice (Wildishetat.•1988).The species is tolerantof the low wintertemperaturesfoundin Atlantic Canada,and itishardy whenhandled.Scallop culturecan be divided into three stages (Claerebo udtetet.,1994 ):a breeding or hatchery stage, whereadultsare conditio nedand spawned and larvae raisedtomaturity,a nursery or intermediate stage where juveniles areraised to anoutplantingsize,and a growout stage wherejuveniles are
grown to commercial size.Numerous studies have examined methods of improving intermediate and grow-out techniques (Dadswell et al.,1988;Parsons and Dadswell, 1992;Couturier et al.,1995). but spat (juvenile scallop) supply remains critical to the economic viability and success of a scallop aquaculture orenhancement venture.The major problem facing the industry today is unpredictabilityin the availability of spat (Helm, 1994).Ifspat couldbe reliablygrownin a hatchery, a reliable source of seed for the farmers would be ensured, eliminating the seed supplyproblem When hatcheries are used to producejuvenile bivalves,maximum survival of maturelarvae must berealizedto make bivalve culture economically viable.Lowsurvival oflarvae and juveniles results in very high costs of hatchery operations.Inorder to maximize production, it is necessary to study the biology of larval and juvenile scallops,and determine the optimum conditions for their growth and survival.
The production of unicellular algae is a very important step in the rearing of marine bivalves on both laboratory and commercial scales. A variety of batch and continuous culture systems has been used.Batch culture can be defined as a culture of constant volume confinedwithina vessel with no replacement of culture medium (Eppley, 1991) The great advantage of batch culture is its simplicity; its main disadvantage is that conditions are constantly changing in the culture vessel.Growth rate changes with time. as does the physical and chemical environment of the cells. It is possible to stabilize the cells' chemical environment by using cage cultures.whereby cells are grown inside dialysis bags exposed to a reservoir of medium.Nutrients diffuse into the bags and exudates diffuse out (parrish and Wangersky, 1990).Theuse of cage cultures does not, however,overcome the problem of varying growth rates and an increasing degree of self-shading by the algae;
this can be done only by using continuous culture methods.
Continuous cultures can be defined as constant volumecultures provided with a continuous inflow of new mediumand a corresponding outflow ofculture. Two types ofcontinuous cultures can be identified:turbidostat and chemostat.The standing stock
ofcells isregulatedin turbidostat cultures,whilethe flow rate of new medium is regulated in chemostat cultures (Eppley,1991) . Co ntinuous cultures are particularlyattractivein laboratory work and mass culturework becausetheyprovidea continuou smeansof samplingor harvesting and because they are amenable to automation(Wangerskyetat., 1989) .
The algal productionunitat the Belleoram Sea Scallop Hatchery,locatedin Fo nune Bay,Newfo undland, obtainedfrom SeasalterSheUfisheries isa large chemostat in which manyalgal culturevessels are suppliedcontinuo us ly with nutrientsandcontinuo usly harvested at the same rateas the nutrient supply.Therate of nutrientdelivery is controlledbya valvewhich regulatesthepressureinthe nutrient deliveryline.When the cell densityofalgaeinthe culturevessels is constant,thegrowthrate ofthe algae is determined by the pressure ofthe nutrient line.Manipulationof the concentration softhe majorchemical nutrients supplied to the culture vessels changes the cell density in the vessels,with an increase in theconcentra tionof limiting nutrient leading toan increase in cell density (Dahinett et al.,1998).Adjustments in the rateof nutrient delivery tothe algal chemostat culture system andconcentra tio n of nutrients maygivebetter growth and survivalofPlacopeotenlarvae and spat.
U Algal
te"
dens ityAlgalcell density affects theclearancerate,ingestion rateand absorptionefficiency ofPlacopectenmagetlantcus,and thus growthand survival .Growthof bivalvesis a function of suspens io n feeding,which can be determined from themeasurement of clearance rate(the volumeof water swept freeof particles per unit time per animal).
Essentially,ciliated gillsgeneratewater currents, filter particles from the water,trap these particlesinmucus,and transport them tothelabial palpsfor further sortingandeither ingestionor rejection as pseudofacces (particleswhich are removedfromthe water but are notingested)(Thompson and Bayne,1972).
Two gen eral approaches to themeasurementof clearance ratehave been identified,adirectand an indirect method.Thedirectmethod has beenused wherethe wat er from theanimal' sexhalantsipho ncanbe sampled directly,andcan beusedto determinepumpin grate .Theindirectmethod measurestheremoval ofpaniclesperunit timefrom a known volumeofwater whichcontains theanimal (s) .This is the more commonl yused method. Theapparatusused tomeasureclearancemaybeasimplestatic systemcompo sedofa cont ainercontaining the animal (s)and suspens io n ofcultured algae, in which thede cr ease inparticle density is measu r edovertime,ora flow-t hrough system where wate r with a constant algal densityflowsthrough achambercontaining the animal(s),andclearance ismeasured by detennining the particleconcentrati on inthe inflowingandoutflowin gwater(lbompsonand Bayne,1972 ).
Thegeneralrelationshipbetween algal densityand particleconcentrati o nisthat clearance ratedecreaseswithincreasing algal density.Manybivalveshavetheability to regu latefeedingwithina range of particle densitiesinorderto obtain aconstant ing est ion ration (Thompson and Bayne,19 72;Winter ,197 3;Palmer andWilliams,1980; Sei der eret al.,1984).Fooduptake ina dense algal culture islimited by thecapacityof the gutand the digestibili ty of thealgae(McMahon and Rigler,196 5) . Atlow algal densities, food uptakebyPlacope ctenmagellamcusis char acteriz edby aconstant clearancerate and ingestionrate increaseswithincreasing algal densiti es(Manning,1986).Inaquacultur eit isimportantto determine the maintenanceand optimumrationsforagiven species.
Maintenancerationistheamount oH o od whichsuppo rts basal metabolismbutresul tsin zerogrowth.Optimumrationisthatwhich maximizesgrossgrowth efficien cy.The algal ration fed tolarvae and juvenilesshou ldbesufficient tocover maintenanc e costs,but should not exceedthe optimum ration because of thehighcost ofgrowingthealgae (Manning,198 6).
Hollett(l98 9)investig atedtheeffect of ration(alg al cell density) on thegrowthof juvenilePlacopectenmagellanicustodetermine the most efficient use of algal cultures to promoteoptimumgrowthina hatchery.The diet consistedofa mixtureof three cultured algaeina 1:1:1ratio basedon cell count.The mixed algaldiet was batchfeddaily to
provideinitial mean cell densitiesof12. 22.45.68.and 87cells/ul.Clearance and ingestionrates weremonitored.Theresults indicated thatclearance ratewas inversely relatedto algal celldensity.Ingestionrate ina-eased at lowcelldensityto a maximumat 22cellsl~l.Between cell densitiesof22 and 45cellsl~1,.ingestion rate remained constant.
Further increasesin algal celldensitycauseda decrease in ingestion rate.The algal cell density of45ceOsl~1resulted in superiorgrowth andmost efficient use ofthe algal ration.
Ut Algal djet,ryc;o roPOsitio n
For hatchery purposes.it is important toknow the algal dietsupportinggrowth and survival oflarvae through metamorphosis, andresultinginoptimumgrowthofthe juveniles.Anumber ofstudies haveexaminedthenutri tive valueof avariety of phytoplankto nspecies formolluscs.Fromthe severalthousands ofphytoplankt on species existinginnature, only40(De Pauw,1981).distributedover eight taxonomic classes, havebeentested as food for juvenilebivalves belon gingto the genera Ostreo, Crassostr ea, Mercenarta,MytilllS.andVenerup is.Fromthe resultsof these studiesitcan be concludedthat thereare considerab ledifferencesinfood value betweendifferentalgal speciesused as food forjuvenile bivalves(DePauw,1981).Only16species out of40 suppo rtedexcellent growth in juveniles:Dtcrarertaino mata,lsochrysisgalbana, Pavlovalutheri.PseudoisochrysisparadoJUJ,Tetroselmissuecica,T.chui,T.maculata, T.letrahel e,T.inconspicua;Chaetoceros calcitrans,C.curvisetus.C.simplex, Bellerocheapolymorpha,B.spinifera,Skeleto~macostatu m,and Thalassiosira pseudonana. Eighteenhadvery poor nutritivevalue and somewereeven toxic.In general,representativesof theChlorophyceae,Cyanophyceae,Dincphyeeae,and Xantho phyceaewereless suitableas food for bivalves because ofathick cell wallorthe productionoftoxicmetaboli tes (DavisandGuillard,1958;Guillard, 1958;Ukeles, 1971.
1980).00thecontrary,small chrysomonads, havingnothick ceUwallandproducingno toxicmetabolites, weregood food as, were crypto monads,green flagellates, anddiatoms.
However,even relatedspecieswithinthe sameclass,family,orgenus,may be nutritionally
good or poor.Fwthennore, not all the bivalves considered reactedinthe sameway towards given algal species;for example,Dunaliella tertiolectawas a poor food source for O.edulis and M mercenaria,butgood for C.gigas (De Pauw,198 1).Algal diets that promote rapidgrowthof the bivalve generaexaminedare not, however.necessarily amongthe best scallop diets, so information on nutritionalrequirements of these genera cannot be applied directlyto scallops.
During the last three decades. considerableeffortshave been made todetermine thevalueof mixed dietsof monospecific algaeversus that ofsingle algal diets as food for juvenile bivalves.The results showed that with suitablealgal species,far better growth of juveniles is obtained with mixtures oftwo or more species than with either species alone (Helmet al.,1973;DupuyetaI.,1977;Ukeles et al.•1984;Lucas etal.,1986;Gillis.
1993).
Inaddition to consideringwhether or not an algal species possessesa thickcell wall orproducestoxic metabolites. several other properties must be taken into account when detennining which algal specieswrillprovide thebestfood, including:(I) cost of growing the algae (2) ease with whichthe algae can be cultured in a hatcheryand (3) biochemical compositionof the algae (wikfcrs etal., 1984;Enright etal.,1986;Gillis, 1993).
1.5 Alg.!growt h pha s e
Many researchers havecompared algal dietsinan attempt to determine the optimal diet for bivalves. Itis importantto consider why some diets resultinincreasedgrowth.
One enigma is the relationship between the food value of analgaand its chemical composition (Epifanio.1979). Phytoplanktoncan vary intheir nutritionalvalue to a variety ofplanktivorous animals.During early experimentationinaquaculture,variationin nutri tional value of phytoplankton did not appear tobe relatedtotheir general biochemical composition(walne,1963).These earlyfindings suggested that palatabilityand digestibility were significant factorsindetermining the nutritional value of phytoplankton.
However. the availability of more sophisticated analytical techniques providedsuperior
toolsfor investigatingthe biochemical compositionof phytop1anlcton,e.g.,the Chromar od -Iatr o sca.n (TLCIFID) system used for lipid class measurements (Ackman, 1981),and the analysisof thecomposition of individual fatty acidshave shown thatthe biochemicalcompo sitionof the algae is highlysignifi cantinaffecting molluscan growth.
Phytoplanktonas food must supplyboth energy andcsscmia.lrartri e ms.Although littleisknownofspecificnutrient requirementsforbivalves(Langdonand Waldock, 1981),lipid.,carbohydrat e,andprotein remain the majordietarysources cf'energyfor growth and development.Inmost marine invertebratelarvaelipidisthe majorreserve material(Hollan d,1978),whereasadultsuseglycogenastheir majorenergy source (Giese,1969).Duringthepelagic stage, neuttallipidisaccumula tedby larv aeandreaches apeak just before metam orphosis.Most bival velarvae areunableto feed onparticulate matter during metamorpho sisandsettl em ent,asaresult ofthelarvalfeedingorg an(the velum) beingcast off,and theadult feedingorgan(thegill)not yet being functional (Hickman andGruffydd,1971 ).Lipidreservesare rapidly dep letedduring thisperiodto meetthe costs of metabolismand morp ho genesis(Hotlandand Hannant,19 74 ).
Therefore, thecorrectdiet isrequirednot onlytoensure good growthof pelagiclarvae, butto allow them toaccumulate enoughenergyreservesto sustain themthrough metamorphosis.
Theconditions under whichphyt o plankt o n growdetermine their biochemical composition andthel"ebyaffect theirenergycont ent and nutrientvalue (Whyte,1987). One factor influencingalgalbiochemicalcompositionisthegrowthphase ofalga e harvestedfOTfood.Inthe logarithmicphase, synthesisof metabolites andcell divisionis rapidandceunumben increase inageometric progression,whileinthestationaryphase cell numbers remain constantand their viabilityis maintainedby storedenergyreserve s.
Studies onchanges in biochemicalcompositionofphytopl ankton during the different growthphasesarenot very common,and publisheddatausual lyrefer toasinglephase of the culture (Femandez-R eirizetat.,1989).
Brown et al.(1996)investigated the effect of harvest stage (logarithmicand stationaryphase s) on the biochemicalcomposition of the diatomTnakusiosra pseudonana, and found that with theonset of stationary phase; carbohydrate andto a lesser extent lipid conte ntincreased, and proteindecreased,whereas cells in logarithmic phase contained the most protein.nuswasa result of metabolism being shuntedfrom protein synthesis (cell division) duringthe logarithmic phaseto energy (carbon) storage during the stationary phase.
Otherstudies of thenutritivevalue of phytoplanktonspecies during different growth phases have shown thathighlev els of carbohydrate., obtainedfrom algaeharv est ed duringstationary phase,produce the best growth injuvenileoysters(Ostrea edlllis) (Enright et al., 1986). High dietary protein., obtainedfrom algae harvested during logarithmicPhase.prod uces optimumgrowthinjuvenilemussels(MytiIlIstrossuhts ) (Kreegerand Langdon. 1993).F1aakand Epifanio (1978) also demonstrated thatalgae in thestationary phase containedmore carbohydrateand resulted in oysterswith more glycogen;algae intheexponential phase containedmore protein butsuppo rted le ss growth.
1...6. AlgI'marronlltricgtconcent ration
Biochemical studies on microalgae arealsorequired to determine how organic composition may be affected by the concentrationsof dissolvedmacronutrients in the surrou nding med ium . Changes in the chemicalcompositionof algal speciesculturedat different macronutrie nt(nitrate,pho sphat e and silicate)concentrationshave longbeen observed(Ketchumand Redfield, 1949; Spocbr and Milner,194 9).Most such studies have shownrepeat able trendswhenthe nitrogen sourceis manipulated:nitrogen deficiency resul tsinanincrease in carbohydrat eanddecreasein protem.whereas nitrogen enrichmentresultsincellsrichinprotein and low in carbohydrate (Thomas etal.,1984; Wikfors,1986).
Harri son et al. (1990),measured changes in prot ein,carbohydrate,.andlipid of three algal species(Isochrysisgaibana.Chaeroceroscakntrans,andThalassiosira
p5e1/dona"a)(harvested duringmid-logarithmi cgrowth phase) as growthbecam elimit ed bynitro g en(N) or silico n (Si).UnderNstarvation (2 days)%lipid remained relatively constant,while%carbo hydrat e increased and%protein decreasedinallthree speci es, compared withcellsgrowing undernonutrie nt limitat ion.Under Si starvatio n (6 hour s) therewasno change in lipid,proteinor carbohydrate. No changes in lipidwereobserv ed, probably because cellcompo siti on was determined after only6hours of Sistarvatio n. An increasein lipidsdue toSi limitationinCye/o fellacryp tica andChaeloceros muellerihas beenobserved byseveral workers(Vau lotetal.,1987 ;Roessler,1988).
Wtkfors et al.(1984) analysedtwoalgal flagellates(Duna/iellatertiotecta and Tetrase/mis macu/ata)fortotal carbohydrate,protein. and lipid.The two algalspecies werecultu red in three differentgrowthmedia and harvested in stationary phase.When cultured in the enriched-nutrient reduced seawater mediumX,bothspecies contained more carbohyd rateand less protein thanthey did when cultured inE (stan dar d fonnulation/enrichedseawatergrowthmedium).The third medium(NIP)(allmedium components decreased except nitrateand phosphate)produced algaewith decreased carbohydrateand increased protein when compared with E.The total lipid content ofD.
temotectawas significantlyless than that ofT.macu/ataregardlessof culture medium.
Tetrase /mis maculata was a consistently better food source than D. tertio /ecta, indicating a relationshipbetween algal lipid content and oystergrowth.Growth ofoystersfed algae cultured in medium X was higherthan growthof oystersfed algae culturedinEorNIP, suggesting a nutritional requirement for more carbohydrate than protein.
Several authors have pointed outthe importance of lipid (Dunstan et 81.,199 3 ; Parrish et el., 1998)and carbohydrate(Enright et al.,1986)forgrowth of mollusclarvae and spat,and Brown eraI.(1997)reported thattheaminoacid compositionof the 40 species of micro algae examined during their study was strikingly similar,suggestingthat protein quality was also similar.The protein quality of micro algaealso remainshighunder different culture conditions (Brown etaI.,1993).Forthese reasons the protein contentof
10 the microaIgae was notdetermined during thepresentstudy andonlylipid and carbohydrateanalyseswen:performed.
Theimportanceof pro viding larvaeandbivalve spatwitha 5Uitablcalgaldietinthe hatc heryisveryclear.theywill growfasterandIIIOCeefficiently.reachahigher quality, andperformbetterwhentransferredto the natunl.1environment.
L1 Sdrctjonof,Ig"'Pednbyfiltcr·(ttdin,bjnly n
Thechoice ofalga!speciesisparti allydepend ent on theparticularmorphological and hydrodynamic characteristi csof the etenidia ofa givenbivalvespecies.whichcan leadto different filtering and particleretentio n capabilitiesbased on size, electrical charg e, and flowrate s(Rubenstein andKoehl,1977;Jargensen,1983;LaBarber a. 1984;Riisgard, 1988)
Experimentsconducted by Lesser etaI.(1991)withPlacopec ren magellanicus, usingdifferentsiz esof phytoplanktonatdensitiesthatdidnot producepseudofaeces, revealed thattheselecti o nof algaewas notbasedonsizealone.Thetotalnumber of cells clearedwasnot significantlydifferent betweenanyof' theexperiments.Particleselection inthesecases was apparen tly basedODcharacteristicsotherthansize,orbypreingestive sorting ofaI gacbyjuvenile scallop s.This could reflecttheabilityofjuveoiIcscallops to regulatetheirfeedingratesinorder toobtainaconstant ration.If maintaininga constant ratio nispartiallyregulatedbyfactorsother thansize.othermechani sms must be invoked toexplainthe results.ThesemechanismsmightincJudechemosensorycues,the physiologicalstateand biochemical composition oftbe algalcellsdeterminingwhichcells arecleared.andingested(Lesserera1.,1991).
1.1 C.lr cjo3t1joi n'
Chemical tagging oforg anismstopro vide identification,time,orgrowt hmarks has beenusedsuccessfullyinmanystudie s(eg.Bevelander,1965).The chemicalcalcein P,6-D ihydroxy-2,4-bis-[N,N'-di(carboxymet hyl)-amino methylfluoran),Cx.HuN 10 lS.is one such markerwhichhasreceived relativelylittle attention andwhichoffersseveral
II benefits overothers.Calcein was designed as an indicatorfor calcium (Diehl and Ellingboe,1956 ).It can be incorporated into the skeletons ofawide varietyof taxa, providinga fluorescent mark which canbeused for identification, for studiesof calcification.and as a benchmarkfrom which.to measure growthsince marking.It fluoresces bright yellow-greenunder blueorultraviolet light.shows very little toxicity.
and has a good shelflifeinso lu tio n.Applied through bath or injection,itappearstobe pennanentlyincorporated into any calcified structure laid down while the chemical is present,while not modifyingthe growthrateof marked individuals.Asa marker,calcein resembles tetracycline,but fluoresces more brightly,is less toxic,ismoreeasilyabsorbed inabath, and has a much. longer sh.elflife in solution (Rowleyand Mackinnon, 1995).It has been used previously to mark fish otoliths(Wilson etal.,1987) and mammalske let o ns (Suzuki and Matthews,1966),andis presently being used to studysea-urchingrowth and to mark juvenilerazorclams(SiliqllQspp.) in a studyof transplant mortality and growth, and tomark scallop larvae (Crocker.1998).
1.2 ll.bi«:ti=
Experience at the sea scallop hatcheryinBelleoram sinceitopenedin 199 5 hasshown that survival and growthofl arvae prior to settlement ispredictable and consistent.Incontrast survival andgrowthoflarvae through settlementto 1 nun in shell heigh t (spat) has been unpredictable. Growth and survival rates have fluctuated, indicating that physicalconditions and conditionssuch as diet and ration have not been optim al.
This study was undenaken toinvestigatethe effect of manipulation of the algal diet with respect tocell density.species composition, growth stageof harvest, and macronutrientconcentration on growth of sea scalloplarvae andspat.Basedon studies previouslycited in this chapter,thehypo theses tested were that:
I) calcein staining ofPlacopecten spat wouldprovide a fluorescent mark which could be used as a benchmark from which to measure new sh.eUgrowthsince staining
12 2) different algal densitiesofa standard diet fed toPtacopeaenspat would resultin different growth rates of spat.
3)there are differences in food value among different algal species.
4) Placopecten larvae and spat fed algal diets harvested during the stationary phase show faster growth than those fedalgaldiets harvested during logarithmic phase.
S)the biochemical composition of algae depends on the macronutrienr (nitrate!
phosphate/silicate) concentration in the chemostat medium.Macronutrient deficiency results in an increase in absolute carbohydrate content and/or lipid in algal cells, whereas macronutrient enrichment results inalgal cells low in carbohydrate and/or lipid.
Placopecten larvae and spat fed macronutrient deficient diets show higher growth than those fed macronutrientenriched diets.
6) Placopecten spat preferentially select onealgalspecies over another when fed two speciesinequal proportionbased on cell count or cellsize.
II Cbapter2
Mat eri a ls andMethods
Research was conducted at a commercial sea scallop hatcherylocatedin Bellecram, Fortu neBay,Newfoundland.
Stock culturesof variousalgalspecies(Table1) were illuminated underconstant lightfrom fluorescenttubes at about2O"C.The cultures were maintainedinglass test tubes containingseawaterenrichedwith conunerciallyavailablef/2 medium (Guillard, 1975),and weresubculturedeverytwo weeks under asepticconditions.Algal cultures used for feeding larvae and spat wereinoculat ed first into100mLvolumesand then to2 L volumesin 4 L flasks.These cultureswereused toinoculate 500L polyethylenebags, which comprised the algal production unit chemostatatthe hatchery,designedbyand purchased from Seasalter Shellfisheries.England.Each500L culture bag wassupplied witha continuouslypumped inflowofpast eu rised medium and continuouslyharvest edat the same ratethrough an overflow tube.Therat e of medium (nutrient)deliveryor harvest wascontrolledbya valvewhich regulatedthe pressurein thenutrient delive ryline.When the cell density of algaeintheculture bagswasconstan t, the growth rate of the algae couldbe determinedby thenutrient line pressure (Eppley,1991)
Theeffect of algae harvested from differentgrowthphases on the growth of scallop larvae andspatwas investigated byadjusting the nutrientline pressure(5.2 psi) to contr olthe harvestrate, and by adding a secondnutrient delivery line tothose cultures maintained in logarithmicphaseat high growthrates.Dividing thevo lumeofalgae (diatoms and flagellates) harvested per day bythevolumeofthecultu rebags providedan estimation of instantaneousgrowth rate(u).Growthrate indivisio nsper day (d) was obtained bydividingI.lbythe naturallo g (10)of2 (Eppley,1991).Thegrowth rates
14 (divisions/day )wereapproximately 0.70 forlogarithmic phase diatoms, 0.52 for logarithmicphase flagellates.and0.14 for near-stationaryphase diatomsand flagellates.
The concen trationsaCthe major chemical macronutrients(nitrate, phosphateand silicate) supplied to theculturebagswell:varied to modifYthe lipid andcarbohydra te concentnIrionaf tbealgaein order toinvestigate theeffect ofalgal macronutrient concentrationon the subsequentgrowthofscallop larvae and spatwhichwere fed these cultures.Repletecultures were supplied macronutrientconcentrati o ns0£ 883~nitr ate, 36.3~Mphosphate,and 107~silicate(Guillard.197 5)basedonf12formulation.
Deplete cultureswere suppliedconcentra tions0£294~Mnitrate.12J.lMphosp hate,and 107 J.lM silicatebydecr easingthenitrateand phosphat e concentratio ns ofthe fI2med ium by113.Diatoms werefound tobe silicate and phosphate limited in both repleteand deplete cultures,whileflag ellate swerelimited onlybyphosphatein deplete cultures.
Nitrate was notlimitingineitherrepleteor deplete cultures for diat omsand flag ellat es (5.
Andrews.BSe.He ns.Tbesis,MemorialUniversityofNewfo undland, pers.com.). To investigate temperature effectson the quality of algaefedtoscallo p larvae,the temperatureofthemacronu trientcultures was variedbyadjustingthe coldwaterflo win beat exchangersattachedto eacheclture.
Dailyestimatesofculturecell densities were used todeterminethe amount requiredforfeedingIarvacandspat.AModelZrCoulterCounter witha 100~maperture tube was used fo ralgalcellcounting.
15 TableI.Alg al cultu re s used forfeed inggiant scalloplarv ae and spat.
Type Algal Species Celllength~m) Source
flagellate lsochrys is(clo ne T·150) 3-6 CCMP
flag ellate /soch rysisgalbal/o 5-6 CCMP
flage llate Tetrase tmissuectca 10-15 SeasalterSheUfisheries
flagellate Pavlova Iutheri 3-6 CCMP
flagellate Chroo monas salina 6-9 CCMP
diatom Chaetoceroscalcitrans 3-7 CCMP
diatom Chae toce rosoeratosporum 3-1 6 Seasalter Shellfisheries diatom Cha etoc eros muelleri 4-9 Seasalte rShellfisheries
diatom Thalassiosirapseudonana 4-6 CCMP
(clone 3H)
diatom Thaiass iostrawetssflogii lO-15 CCMP
CCMP""Provasoli-Gui llardCenter forCultureof Marine Phytoplankton,Maine,USA
U Alga l drywr jght
Algaldry weightdetenninationswere performed as follow s:twocounts were performed on three separateaIiquots of eachalgalculture todeterminecell density.
Subsarnples (100mL)ofthealgalcultureswerefilteredthrough pre-weighed,pre- combusted WhatmanGF/Cfiltersusinggentlevacuu m.Thecells on the filterwere rinsed with distilled water and thefilterswereplaced in aluminumweighingpans,driedat70°C for 24 hou rs,cooled in a desiccatorandreweighed.
U I ary aland'Pa t "penm enta!rraring ta n k s
Three types of experimental tanks were used throughoutthisresearch.Plastic cylindricaltanks (20L) containing16L of seawater(salinity 30-3 1 ppt, temperature15°C)
16 were used for small-scalespat experimentation. The tanks werepanlysubmergedina water bath fortemperaturecontrol. Air was bubbled into the tanks to maintain oxygen levels and circulate thealgalcells.Spat were removed temporarily every 2 or 3 days while the tanks werecleaned,refilledand algae added.
Conical fibreglass tanks (250L)filledwith200 L of seawater were used for intermediate-scalelarv al and spat experimentation. Air was bubbledinto the tanksto maintain oxygen levels and algal distribution Larvae and spat were removedtemporarily twice a week while thetanks were cleaned,refilled and food added. Spat were held in downweUers hooked ontothe sidesof the conical tanks. Downwellers were constructed from16 Lplastic cylindrical tankswiththe bottoms cut out and replaced with 650Jim meshscreens.Anairliftwas placed on the side of each downwe11erto recirculatewater to the spat at a flowrate of1.2Umin.
Fibreglass hatchery productio ntanks (7000L) filledwith6000 L of seawater were used for large-scale larval experimentation.Airwas gentlybubb ledinto the tanks to provide gentle circulation.Larvae were removed temporarily twice a week while the tanks were cleaned, and refilledand foodadded.
1.S. Spa tstajn jng wjth ra'rri n
Scallopspat shellswere stainedwithcalcein to introduce fluorescent marker bands which could be used tomeasure new shellgrowth in subsequentgrowth trials.One hundred spat,ranging in shellheight from 0.5ffi;ffi~2.0mm,were placedineach of two 10 L plastic buckets containing either 6.6L ofcalcein solution or 6.6L of seawater, partly submergedina waterbath as a temperature control.Airwas bubbled into the buckets to maintain oxygen levels and algal distribution.Calcein solutionwas prepared by dissolving 0.1 5 g calceinllitre seawater, then adjusting the pH with NaOH to equal that of ambient seawater (pH-8.10)(Crocker,1998) . Thecalcein,inpowdered form, was obtained from Sigma Chemical (LotI23H0594). The spat were batch fed approximately 50 cells/ul ofa standard diet, consisting of equal numbers ofccUsofsix unicellular algal
17 species, namely,Thalassiosira pseudonana,Chaetocerosmuelleri,Chaeroceros calcitrans,Pavlova lutheri, lsochrysis galbana,and T-Iso.
Scallop spat wereleft to stain for 24 hours, when 2S were removed from the calcein bath and examined byepifluo rescence microscopy.Fluorescent banding was weak, so the spatwere put back inthe calcein bath and left for an additional 24 hours.
Examination after 48 hours of immersion illustratedthat banding was notsufficiently strong, so the spat were onceagain replacedinthecalcein bath and left for another24 hou rs.Bandingwasstrong after 72 hours of immersion, so thespat were removed from both the cajcein and seawaterbaths, and mortality determined using low po werlight microscopy.This methodwasthen used to stain spat in subsequentgrowth trials.
U Du n tjoQofgro wt h ,rj,l$
All subsequent growth trials were conductedfor a minimum of 12 days.At the endof the trials shell heigh t (from the hinge to the periphery afthesheD)was measured by low power light microscopy for atle ast)0larvae.New shellgrowth (fromthe fluorescentband to the peripheryofthe shell)was measured byepifluorescence microscopy for atleast 20 spat.Mortality was assessed in some growth trials bylow power light microscopy.
U Algal rell dens ity ao dspa ' growth
The effect of algal celldensity ongrowthof spat was investigated to determine the optimum density at which tofeedspat in subsequentgrowt htrials Scallop spat rangingin shell heightfrom 0.3rom-1.7rom were calcein stained, and 20 0were placedineachof ten 20 L cylindrical tanks.Each tank contained a different celldensityof a standard diet (2 controltanks/no cultured food added.twotanks ofl0cells/J.lL.two of20 ceUs/J,lL,two of 40 cells/ilL,and two of80 cellslJ.lL).The standard diet consisted of equal numbers of cells.of six unicellularalgal species, namelyIsochrysts galbana, T -Isc,Chroomonas salina.Tetrase/mis suecica,Tha/assiosira pseudonana.andCha etoceros muelleri.The
18 expc:rirnem.ranfor14days,duringwhichthe algalcelldensities wererestored totheir initialvaluesiftbeydropped below20%of thatvalue beforethe water was changed.
Clearanceratesweredetermined forspatinalltanks dailyfrom cellcounts and the equationof Coughlan(1969):
F;«M*t""•InC./CJ-(M*t-l•In
C :te. '» •
0-1 wneee:F-clearance rate (mlJhIanimal),M-volumeof water withsuspend edalgae(mL ),C.- initialparticleconcentratio n(censl~L).
c,=
concentrationattimet(ceUs!J,lL) ,t-time (hours),C.'=
initialconcentra tionincontrolchamber(ceUsf~L).C:""concentration at timetincontrolchamber (cellsl,uL),and n-nu~of animalsper tank.Ingestionratewasdetermined asthe decreas einthenumber of cellswithtime per scallop.Thefollowingequation was used (Hollen. 1989):
IR-(Cl-CJV •r' •n-Iwhere::
IR""ing estion rate(eeUslsca1loplh our ).C;"initialcell concentra tion(cel1s1~L).Cr-final cellconcentratio n (cellsI,uL),V..volumeof seawater(mL),t'"time(hou rs),andn- numberof scallops
Fivecontroltanks (I foreachalgalcelldensity) weresetupfor Wee days,under thesame conditions as the10treatment tanks,excepttheycontainedno spat.Clearance rateswere monitoredforthecontroltankstodet ermineiftherewassettlingoutofaIgac:.
L.I Attemptedjm p rp yrm eg t orstandudb.tcberydielby.d dj tj o " oral, .1
-
The effectof speciescomposition ofalgaldietsonlarvaJ andspatgrowthwas investigatedby performingsever algrowthtrials in which algal species(Chaetoceros calcitrans,Te1Twelnris~ecica,andChroonwnassa/ina)were addedto thestandard hatc herydietofSspeciesofalgae(Cha etocerosceratospoeum, Chaelocerosmue//erl, Thalassiosirapseudonana,/soch rysisgalbana,and T-Iso)tolookforpossible improv ementsin thediet.19 z.J...l Chge'oqrofcqlctrraminla rva ldiC1s
Larvaeweresuspendedin10Lof seawaterbygemle agitation with a plastic plunger.Onemillilitrealiquot s\VCR:removed and the larvaekilled with ethanol and countedbyinvertedmicro sco py.Approximately2million scalloplarvaewereplacedin eachof two7000L hatcherytanks and batch fed2dietsinlogarithmicphase.One diet consistedofequallkImbers ofcdlsof 5uniceUuIatalgalspecies. namelyChaetoceros cerotosporum.Chtwloceros muelleri,Tha/assiosira p$elldQnana.lsochrysis gal bana and T'-Iso,while theot her dietconsisted of the same5algal speciesinaddition toChaelOCf!F"O$
catcarans.Larvae werefed16ceUslJ,tLatthe beginning of theexperiment and thefood densitywasincreased as thelarvae grew,toafinaldensity afJO ceUslJ,tL.
ll..2. ChaetocerotcqlcjtrwlSjnspatdiet s
Scallopspatranginginshell height from0.5nun.0.75nun were stainedwith caleein, and200placedineach ef'four 20Lcylindrical tanks(2 replicatesof each diet).
Spat were batch fed 40 cdlslJ,tLof2 logarithmic phase diets.Onediet consisted ofequal numbers ofcells of5unicellularalgal species.namelyChaetocero s ceratosporum, Chaeroceros"""elleri ,ThaJassiosirQpseudonona. lsochrysi sga/bonaandT-Iso while the otherdiet consisted aCme same 5algalspecies in additiontoChaetoceros culc;traJU,.
Scallopspatranginginsizefrom 1.7~-2.0nunwerestained withcalcein,and 200placed into eachof eight 20 L cylindrical tanks.Eachtankcontained the same algal cell density (40 ceUslfLL).butdifferentalgaldietary compos itions.Four differentdiets wereexamined.namelya standard diet.astandard+Tetrase lm is suectca+Chroomonas salina diet,a standard+Tetrasetmtsmedeadiet, and a standard+Chroomonassalina diet (2 replicat es ofeachdiet).The standard diet consistedof 4 unice llularalgal species, namelyChae toceros muell eri,Thalassiosirapseudonana./sochrysis galbana,andT-Iso.
Chae toceroscenuosporumwas excluded fromthe standard diet because the cultu re
20 crashed.The diets consisted ofequal numbers cfceusof theunicellularalgalspecies listedfor each treatment.
:.2 Algal growth phaU
Theeffect of algae harvested from logaritlunicand near-stationarygrowt h phases on the subsequentgrowthof scalloplarvae and spat was investigated.
Fourexperiments were conducted to examine theeffect of algae harvestedfrom differentgrowth phaseson growth of scalloplarvae.Inthe first experiment approximately 3.5million larvae wereplacedineachof two 700 0L hatchery tank s and batch. feda standard diet of algae harvested fromlogarithmicandnear-stationary growth phases.The dietconsisted ofequalnumbersof cellsof7unicellular algalspecies,namely Chaetoceros ceratosporum,Chaetocerosmuelleri,Ihalassiosirapseudonana ,fsochrysis galhana.T-Iso,Chaeloceros calcitrans,andPavlova lutheri.Larvae werefed16 cellsl,uLat the beginning of the experiment and the algal celldensity wasincr eased to a densityof30cellsl,uLasthe larvae grew.
The second experiment followed the same protocolasthe first, except that approximately3.9millionlarvaewere placedin eachof 2tanks.
Thethird and fourth experiments followedthe same protocolasthe first two, except that approximate ly3 million larv aewereplaced in each of 2 tanks.Incontrast to thefirst2 experiments,the larvae were fed only6unicellularalgal species.with Cha etoceros calcitrans being excludedfrom the diet.
u.z _
Fourexperiments were conductedto investigate the effect ofalgaeharvested from different growth phases ongrowthofcelcein-stainedscallopspat.Inthe firstexperiment, 400 spat ranging in shellheigh t from 0.3mm-O.5mmand1.4 nun-I.7 nunwere placed in each of eight 20L cylindricaltanks.Eachtankcontained40 ceUsI,u.of a standard
21 diet harvestedfromlog arithmi c and near-stationaryphasegrowth.Spat in4tanks were batchfedalgae harvest ed during the logarithmic phase. while spatintheother 4tanks were fed algaeharvested duringthe near-stationaryphase.There were 2tanks containing spatof each size range foreachharvest phase.The standard dietconsistedofequal numbersofcells offOUTunicellularalgalspecies,namely,Cba etoce ros muelleri, Thalassiosirapseudonana,lsochrysisgalbaJlQ.and T-Iso.Clearan cerateswere determineddailyfrom cell countsusing aCoulterCount er and theequationof Coughlan (1969).
Thesecond experimentfollowed the same protocolas thefirst.withthe excepti on thatspatranged in shell heightfrom0.5mm-O.75mm and 0.75mm-1.4
nun.
and300 spat were placed in each 0£8 tanks.The diet in thisexperim ent consisted 0£6 unicellular algalspecies. namelyThalassiosira pseudonana,Oaaocerosmuelleri,Chaetoceros cerasosponon,Pav lava lutheri,lsochrysis ga/bana .and T -Iso.Thethird experimen tfollowed the same protocolas the secondwith the exception that200spat wereplacedin each of8 tanks.
Inthe fourthexperiment only onesize range was examined (1.4 nun -1.7nun), and there were no replicatetanks. Two hundred spat wereplaced in eachof 2 tanks.and fed the same diets as spat in the first experiment.Clearan ce rateswere not monitored.
~ Alga lmIC ronutrient rnn ren t ra tjQ D
Theeffect ofalgaegrownunder conditionsof high and low macronutrient concentrationon the subsequent growth of scallop larvae and spat wasinvestigated.
2....l.Q.l Iaryal djetsnfalgae harvestedfromcnotioJln!!5culoueathjghandlow macronutrient ocmceotrarioos
Approximately200,000larvae wereplaced in each of twelve 250 L conicaltanks.
Larvae werebatch fed fourlogarithmic phase dietsharvested fromalgaegrownunder highandlow macronutri entconcentrations (J replicatesof each treatment),namelya
22 1000/0low macronutrie nt diet, a1000/0highmacronutrientdiet,a50/50 ratio lowlhigh.
macr o nutrientdiet,anda 80120 ratiolowlhigh macronutrientdiet.The diets werefedto larvaeataninitialalgalcell density of 16 ceUsf",L,andthe densitywas increased as the larvae grew,up toadensity of30cellslJlL.The dietsconsisted ofequal numbers ofcells of4unicellularalgalspeci es, namelylsochrysis gatbana,T-lsc,Oaetocerosmuelleri.
andIhatassiosira pseudonana.
2...l.Q.2.Spat diet s walg nl':harvestedfromcontinuou scultureat highand lo w waCTQOJltrjemconcentrations
Approxima tely3300calceln-stainedspat,ranginginshellheightfrom 0.75mm- 1.4 mrn,wereplacedineachof9 downwellers hungin250 L conicaltanks. Flow rates were adjustedto1.2 Umin foreachdo wnw eller.Spat were batch fed threelogarit hmic phase dietsharvested from algaegrown underhighandlow macronutrientconcentr ations (3replicatesof eachtreatment), namelya 100"10low macronutrientdiet,a100"10high macronutrien t diet, and a50150ratio oflowlbigh macronutrientdiet, at an algal cell density of40 cells/1-tL.The diets consisted of equal numbers of cells of diatoms to flag ellate s of3unicell ular algal species,namelyT-Is o ,Chaetoceros muelleri,and Thalassiosiraweissflogii,Controls with no spat were set up for eachtreatmentatthe beginning of the experiment.Two samplesof eachalg al speciesgrown underhighand lowmacro nutri ent concentrationswere tak en at the end of the experimentforlipid and carbohydrateanalysis.Clearancerates were calculateddailyusing previouslydescribed metho ds .
2.J.Q.lIarvaldiet s oralg ae ha ry eSedfromcontin uousC!!lhm:athigh andlo w macronytrievtcon centr ati o ns andhigh andlo w temperamreregimes
The effect of algaegrownunde r conditionsofhighandlow macronutrient concentrationsandhigh andlow temperatureregimes onthesubsequentgrowthoflarvae was investigated.Approximat ely200, 000 larvae were placedineach of twelve250L conical tanks.Larvae werebatchfed four log arit hmic phase diet s grownunderconditions
2J ofhighandlow macronutrientconcentrations and highandlow temperatureregimes(3 replica tes of eachtreatment),namelyaS0I50ratio cflow macromrtriem-hightem perature /high macronutriem-high temperaturediet.aSOISOratio aflo w macromnrient-high temperarurelbighmaaonutrient-Iow temperaturediet, aSOISOratioafl ow macronutrient -low temperanu-elhi ghma.cronutrient- high temperature diet, and a50150ratio afl ow macronutrient-lowtemperature/high macronutrient-&owtemperaturediet.. The dietswere fedataninitial algalcelldensi tyof16 cdISlJ,olLand the densitywasincreasedas the larvae grew,up toadensity00 0cellsl~.Thediets consistedof equal numbers ofcells of diatomsto flage llatesofJunicellularalgalspecies,namely T-Iso,Chaetoceros muelleri, and Thalassiosirapseudonana.
Thetemperatu reof the algal cultu rebagswasrecordedtwiceweekly.One sample of eachalgalspeciesgrownunder conditionsof high andlo w macronutrien t concentra tions and highandlo wtempera ture regimeswastakenat theendof the experimentforlipid analysis.
.Lll TetnndmqSHm,."n qlq,y iosicqlfri.nIloriiIDdT_hg(rdto'PItsjn gly andjn combin at iop
Based onrecent hatcheryexperience.whereimproved growth ofspatwas obtainedbythe addition of Thalassiosira _issjlogiitothe standard diet,agrowthtrial wasconducted withT~traselmi$ $U~cica,1hakusiosira_iMf/ogii,andT-Isofed tospat singlyandin combinationto determinethe effect onsubsequentgrowthofspatand seek evidencefor possible selectivity.Spat ranging insheDheightfrom0.75mm -1.4mm werecalcein- stainedand150 spat wereplaced in eachof15(3replicates of5treatments) 4 L plasticbuckets each containing 4Lofseawaterandairstones,and partly submerged inawater bath.The5 treatmentsconsisted. of spat being batch fed 40 cellsf,uLof logarithmi cphasealgaegrownunder conditionsoflow macronutrientconcentration.The five dietsconsistedofa100"/0Ti-Isodiet,a 100%Tetraselmis suectcadiet,a100%
Thalassiosira weissflogiidiet,a SO/50ratio ofT·lsoITetraselmi$suectcadiet,anda 50/50
24 ratio T·lso/11Ialassiosiraweissflogii diet, based on ceUcount.Acont rol bucket foreach treatmentwas set up containing no spat.Algal cell densities were calculateddailyusinga Coulter MultisizerII todetermine clearanceand ingestionrates and seekevidence for selectivity,Le.the preferential selection of an algal species from mixeddiet s.Plots obtainedusing a CoulterMultisizer II provided informationpertainingto cell densities of eac h algal species in the SO/50ratio diets.Byoverlaying plotsfrom each successive time interval and subtracting algal cell densities while allowingfor settlingof the algalcellsin the contr olbucket over the same interval,clear ancerate,ingesti on rateandselectivity (number and volumeof each species selected) were determined.Clearance ratewas calculated using the equation of Coughlan (1969) and ingestionrate using the equation of Hollett (1989)
Samplesor each algal species were taken at the end ef the experimentfor lipid and carbohydrate analysis(2 samples of eachspecies).Total lipid and total carbohydrate actuallyingested from each algal species of the 50150ratiodiets byspatperhour were obtained bymultiplying the number of cellsof each species ingested bythe correspon ding amount oflipid and carbohydrate per cell.
z.u
Lipidanalysis Qr.IneExtraction and lipid analytical procedures were based on Parrish (1987),and conducted at the Ocean Sciences Centre, Logy Bay.Newfoundland.Algae were sampled from 100mLvolumes of each culturebyfilteringonto precombusted 47nun diameter glass-fibre filters (Whatman OF/C).The filters were stored in chlcrofbrm at_20°C under nitrogeninglass vials until analysed.Totallipids were extracted with a mixture of chlorofonn and methanol(2:1 v/v) .Lipid classeswere measured by analysingan aliquot of the total lipidextrac t on Chromarod S-ill silica rods with an Iatroscan MK illTH-IO TLe -FIDanalyser.Thesolvent syst em used for the lipid separation was hexane-diethyl ether-acetic acid (60/17/0.5,v/v /v)which resolves triacylglycerolsand free fatty acids from the common neutral lipid classes.Peaks were identified by comparisonwith chromatogramsof standards.
25 Lll Carbohydrate analys isofalgae
Dissol vedcarbohydratewasmeasured using the phenol-sulphuri cacidmeth od (Stricklandand Parsons,1972).Alg aeweresampledfrom100mLvolu mesof each cultu rebyfiltering onto47mmdiameter glass-fibre filters(Whatman OF/C) .The filters werestoredat-2 00Cinglass vials until analysed.Five millilitresof boiling distilledwater were added to each vial.Filterswere groundwitha glassrod.The rodwas rinsed witht mLboiling distilledwater.Vials werecentrifuged fo r 2 minut esat 500rpm. Onemillilitr e ofsample wasremov ed from eachvialand dilute dto 50mLdistilledwaterin an Erlenmeyerflask. Twomillilitres of sample were removedfrom eachflask and placedin anadditionalErleruneyer flask. Two millilitres of phen o l reagent(25 gofpheno l dissol vedin500 mL ofdistilledwater)wereaddedto each flaskcontainingthe2mL samplesusing an auto maticpipette.Tenmillilitr es ofsulphuri c acid reagent(2.5g of hyd razinesulphatedissolvedin500mLconcentr atedsulphuricacid)were addedrapidly to eachflaskusing apipett e withthetip cut off.Theflask s were cooled byallowin g to standforone hou r atroom temperature .Theabsorbancesof the solutionsweremeasu red ina spectrophoto meter using a I em cell,at485nm.Theconcentrat ionof unknown carbohydratewasdet ermin ed graphically using astan dard curve generatedby plottingD- gluco sestandar dsolutionsagainstcorresponding absorbances .
~ SI.tistir.1.n.lysis
Variability between replicatetanksofthe sametreat mentandbetw eendifferen t treatments foreac hexperimentwasanalysed usingne st ed one-wayAnava foUowe dby Tukey'sanalysisifa significantdifferenceexistedbetw een treatme nts.
Stu de ntt-testswere conductedfOTthe larv alexperiments carried out in7000 L hatch ery productio n tank s.No replicatetanks were useddu ring theseexperiment s.A t- test was conductedfor thealgal growth pbase spatexperimentwhereonly onesize range wasavailablewithno replication.Stude ntt-testswere also conductedon the proportio n ofeach species ing estedbynumberandvolume,du ring theselectivityexperiment.
26 The criterion for statistical significance in all analyses was pcO.OS. Correlatio ncoefficients of lipid class composition of the diet and shell growth rate and total carbohydrate and shellgrowth rate were obtained using Pearson correlation analysis.The criterion for statistical significance was ps0.01.
All analyses were performed using the SPSS statistical package.
27 Cbaptoer3
Results
U Sp a tda jp in g with CJll'cjn
Seventy-twohou rs were require dfor-immersion of spat in calcein solution to obtainastrong fluorescent band (Figure1). Sanvivelof the calcein stainedspat and control spat was 99% and 100"/0respectively.
U Alga!gildensity Mod 'PIt growth
Nestedone-wayanalysis of varianceshowedthat growth ratesofspat didnot differsignificantly atdiffer entalgalcell densities(FlU ) '"3.824, P>0.05) (Figure 2).
Clearancerates of spat differed significantlyat differentalgalcelldensities(Nested Anova, F(..~=45.500 ,Pc0.05),withreplicate tanks atthe samecell density not differing significantly(Nested Ancva,F(5.llIO)""0.155,P:>0.05).Data from replicateswerepooled and Tukey's analysis showedthat clearan ceratewas higher at 0cellsf~than atthe other cell concentrations(Figure3).
Ingestionratesof spat differed significamtlywhenspat werefed different algal cell densities(Nested Anova,F(4.5)=68.281,P<0.05)withreplicatetanks at the same cell density notdiffering significantly (Nested AnOYa, F/M'lO)""0.367,P>0.05).Data from replicates werepooled andTukey'sanalysis showedthatingestionrate increased as the algalcell densi tyincrea sed (Figure 4).
M Attemptedim p ro ve m e n t orn_nd,ntb,ttheIY dietby additiono(,I,,!
.l.l..l Chal!tocrrmca/citramin la M! andsp'at die ts
Neithe r larval(t-t est,p>0.05)no r spat;(NestedAno va,F(I ,2)=17.430 , P>0.05) growthwer e significantlyaffected by dietsincladingand excluding thealg al species
100
I
2880
j
n=25
]
60.~
"$. 40
20
o
24h 48h
Duration ofstaining 72h
lIIIIIIIDweak lZZZZlintermed iate cz:z:2lstrong
PigureI.Slainingofscallop spat(0.5 mm - 2 nun sheUheight)withcalccin.
10 20 40 80
29
Algalconcentration (cells/mic rolitre)
Figure 2. Growth ofPlacopecten spat fed a diet of Isochrysis ga/bana,Tclso, Chroomonas salina, Tetraselmtssuecica, Thalassiosirapseudonana and Chaetoceros muelleriat various celldensities. [Mean+SE, n=3D]
Common letterdenotes no significant differenceatp>0.05.
10 20 40 Algal density (ullslm icrolilre )
Figure3. Clearancerate of Placopecrenspat feda dietof Thalassiastrapseudonana, Chaetocerosmuelleri, Tetraselmissuecica. Chroomonossalina, /sochrysis galbana and T-Isoat various cellden sities.
Conunon letterdenotesno significantdifferenceat p>0.05.[Mean+SEtn=20]
10 20 40 80
Algalwnccntnllion(ccllslmicrolitre)
Figure4.Ingestion rateof Placope ctenspatfeda diet ofThalassiosirapseudonana, Chaetoceros muelleri , Tetrasel missuectca, Chroomonas salina,Isochrysis go/bana andT-Isc at various celldensities.
Common letterdenotes no significan tdifferenceatp>0.05. [Mean+SE. n=20]
30
31 Chaetoceros calcitrans(Figures 5 and 6) Survival of spatdidnot significantly differ between the twodiet sineachcase.
1.1..2. Tetraselmir'i7lecicaandChroomwwsmUng in spat diets
Nested one-way analysis of variance showed that growth rates of spat did not significantlydiffer when fedfour different diets(FCU1 =0.090.P>0.05 )(Figure 7).
Survival of spat did not significantly differ between the fourdiets examined,and ranged from 98% to 100%.
3d Algalgrowthpha!5c l.ti 1 . =
Inthree outof'fburexperiments conducted,larv a e fedlo garithmic phase algae were significantly larger at the end afthe experimentsthan thosefed near-stationaryphase algae [t-test, p<0.0 5)(Figure 8).
ll1. SJ>at
Resultsof the first experiment showed that for thesizerange 0.3mm, 0.5 nun, growth rates of spat differed significantly between replicate tanks (Nested Anova,F(;'.I S<ll= 4.650,P<O.OS).Dat a from replicateswere not pooledand Tukey'sanalysis showed that spat fed logarithmic phase algaegrewfaster than those fednear -st atio nary phase algae, withgro wth rates differing significantlybetween replicates 1of both diets(p<0.05).For thesizeran ge 1.4mm -1.7mm,growth ratesdid not differ significantly between diets (Nested Anova, F(I,2)-7.209,P>0.05) or between replica tetanks of the same diet (Nested Ancva,F(2.I~ ""0.069,P>0.05)(Figure9a).
Results of the second experimentshowedthatfor the size range 0.5mm -0.75mm, growthrates did not significantly differbetw een diets (Nested Anove, F(l,2)= 0.426, P>
0.05)or between rep licatetanks of the samediet(Nested Anova, Fa-I%)=1.855,P>
0.05).Forthe size range 0.75nun-1.4mm,growthrates did differ significantly between
32
rep licatetanksof the same diet (NestedAno va,F(1.IKI-24.SSO.p<O.OS).Data from replicates werenot pooledandTukey'sanalysis shewed that spat fednear- statio naryphase algaegrewsignificantly fasterthanthosefedlogarithmicphase algae(p<O.OS)(Figure 9b).
Resultsof thethird~showedthatfor the size range 0.5mm -0.75mm, growthrates differed significantlybetween replicatetanks cfthe same diet(Nested Anova.
Fr:.ll1f;) '"62.580,P<0.05) .Datafro mreplicat es were notpooled andTukey'sanalysis showed thatspatfedlo gari thmicphase algae grew faster thanthosefednear-station ary phasealgae,withbothre plicat es of lhe near-stati o nary dietbeingsignificantlydifferent from the secondreplicateofthelogarithmicdiet.Forthesizerange0.75 nun-1.4nun, growth ratesdidnotsignifican tly differ betweenreplicatetanksafthesamediet (Nested Anova,Fa. l96l'"0.690,P>0.05),but diddiffer significantlybetween diets,withspat fed logarithmic phasealga e growing fast er thanthosefednear- statio nary phasealgae(Nested Anova,F(U) - 283.570,P<0.05) {Figure9c).Thefourth experiment illustrated thatspat fedlogarithmicphasealgaegrew significantlyfaster than thosefednear-sta tionaryphase a1gae (olle sizerange1.4 mm-1.7mm) (t-test,p<0. 05) (Figure9d).
Clearance rates of spatdid notsignificantlydiffer betweendietsor between replicate tanks of the samediet. foreach sizerange,during all threeexperimentsexamined (NestedAnaYa,p>0.05)(Figure10).Swvival didnotsignificantlydiffer between diets.
foreach size range,duringallfour experiments.Survivalrang ed from97% to I()()-I..The different size rangesexaminedduringthreeof the experiments were not comparedto one another.
U AIp'I muroputrirntCOQcrntntjon
.l..S...l brval diets of alga eha rv e st ed fromcontinu o uscu ltu re athighandlo w
macron"trirntconr.cntOltjons
Nestedone-way analysisof varianceshowedthat finallarv al shell heightsdiffered significantlywhen larvae werefed fourdifferent diets(FIU l-46.620,P<0.05)and that shellheights oflarvaeinreplicatetanksfedthe same dietdid not significantlydiffer
'" -j
l3S 1
' 30
1
125 120
'"
33
...Wlthout C"""'I1C~TOS calc"",,,, 110 ...w·,lhClttH'«OfW c: Ic"'altl '05
4 6 e10121416 18 20 22 24 26 Ageoflarvae (daysl
Fig ure 5.TheeffectofaddingChaetacerascatcuransto astandard diet(Choetoceros cerasosporum: Chae toceros muelleri.Thalass iosirapseudanana,Isochrysis golbana and I-Iso) ongrowthofPlacopectenlarvae.
Comm on letter denotes no signi ficantdifferenceatp>0.05.[Mean+1-SE, n>501
with ChaeuX'rros WilholJlChaefOceras
calcilr QfIS calci,I'ons
Diet
Figure 6.Theeffec t of addingChaetooeroscoicitransto a standard diet(Chaetoceros ceratosporum , Chaetoceros muelleri,Thalassiosirapseudonana,Isa chrystsgo/bana and T·rso) on growth ofPlacopectenspat.
Comm on letterdenotesno significant differenceatp>0.05.[Mean+SE, n=50]
