Tracking and Growt h of Larvae of the Giant Sca llop.

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ABeU.lHovweU1aformaIioD Compeay 300NonhZedtRo.l,. NmArtuMl"l~l3t6USA

31317' (-4100 I0OI:521-0600

Tracking and Growt h of Larvae of the Giant Sca llop.

Placopecten magellan icus (Gmelin.179\) on a Scallop Farm in No tre Dame Bay. Newfo und land

by Kevin George Crocker

A thesissubmittedto theSchoolof GraduateStudies inpartial fulfillmem oftherequirements

forthedee reeof MasterofScience-IAquaculture )

School of GraduateStudie sAquaculture Program MemorialUniversityof Newfoundland

March\998

Newfoundland

ABSTRACT

CultureofthegiantscallopbeganinNewfoundlandin IQ86after researc h indicated asupplyofwild spatwas possible. Thefailureofwild seedproduction in sufficient quantitieshadnegativeconsequences. andsince19QIwildscallopspatfall has beenverylow IIwasproposedthat thereleaseofmillionsofDcveligerlarvae. raisedby hatchery techniques.on asitemayincreaseannualspatfalland collection.Enhancement wasatte mptedin199 3 ona scallopfann inNewfoundlandbut no increase inspat collectionwasrealized This studywasinitiatedtoinvestigate thepossible fate ofthese releasedlarvae

Three-da y-oldscallop vetigerswere stainedwithcaIcein(a non-tox icfluorescent dye l by immersion in a sclunonof150mg/L calcein for 16 hat densities of 40larvaeJmL.

Highmortalitieswereobserved atdensitiesof~50larvaeJmL Satisfactory fluorescence wasobservedinpreserved samples which subsequenllyretainedthestain forat leasttWO

Two batc hesofcalceinstainedscalloplarvaewerereleased onashellfish fann locatedin Charles Arm.Notre Dame Bay.Newfoundlandduringthesummersof19Q4 and IQQ5lipan recaptureof'theselarvaebyplanktonlOWS.growth rates of3 57 and385 urrvdinIQ~.and972 and2.5211m/d.in 19';5.were observed. Differences inthe growth ratesvaried seasonally and annually(19Q.l&1995).and were related towater ternperarureand food concentrationtchlorophyll-<,) Size frequency distribution.current meter data.andcurrent directio nal mapscreat ed bydnft boule drogue surveysshowed evidence ofpossibleentrainmentof larvae ....jthinCharlesArm.Aswell.evidencewas also reportedsuggesting that bivalvelarvae werealsotransportedoutofthesyste m

Larvalabundance andshellheightdistributionvariedwithtidalstate.Higher numbersandlarger sizebivalve veligers wereobservedduring themid 10lateflood and ebb tidesMean sizeof bivalvelarvaeat four stationssampled on thesite differed

sig rnficamly over thetidalcycle. Variationinlarvalabundancewasalsogreater during neaptidesas comparedto spring tides Diel differe ncesinlarvalabundancewerealso observed Thishas importantimplications foranysamplingregimedirectedto measuring size andnumbers ofbivalvelarvae over time,for example.larval monitor ingprograms used in theaquaculture industry

Anenhancementprojec tdesignedtoincreasethe subsequent spa tfall ofgiam scallopveligersispossiblebUIshouldbeverv..intensive.Hund reds of millionsof greater than ::OOlJrn scallop veligers mayhavetobe released withinthesite toresult in an observed increase in spatfalland to counte ractlos ses duetonaturalmortalityand net outwardtransportfrom thesystem

i i i

ACKNOWLEDGMENTS

Iwould liketoacknowled ~ethe specialinterestandccnmbooonsof the owner of ThimbleBayFarms (CharlesArrru,TeJT~'-\fills \'yuncondit ional useof hisfacdmes al thefarm was instrumen talinthe accomplishmentof thisthesi s I alsothank himand his family(wifeHeathe r andson Marl.:I.relativesand fnendsfor their kindness.friendship and hospitalityduringmy twosummerswith them

IthankCalhyRyan.TonyClemensandbrother-in-lawCraig\1arshaiI for volunteering their timeto helpin differentaspects oftheda tacollectio n andfieldwork

Iextendackno wled gme ntand thanks[0Dr Brad de Young.Jack Foleyand\lark McDonaldfor contributingtheir limeandfor theplacementofcurrent meters within Charles Arm during199 5

IalsothankDr,Cynthia McK enziefo rthe instructionanduse ofrhe epifluorcsce ncemicro scope.Dr.DonDeibelfor discussio ns onplanktonsampling methods.OrJayParson sandCyrCouturiertorthclr discussio nsonaspects of gianl scallop developmentandculture.and DrRayThompsonandC~Tfortheircnnque of the thCSls

Iespeciallyacknowl edgethe exemplaryguidance of my supervisor.Dr.Pa l Dabinett His knowledge and enthusiasmfo r aquacultur eandmor e specificallyscallop culturehasmade this projec tajoy 10 participatein His att itude.thou g htfulness.canng and friendship withhis students shouldbe an example forany supervisor

And finally.aspecia lthankstomy wife.Abigail. and myparent s IGeorge and Elsie jfortheirunre lentingreminders10 finish this thesis.fortheirendun ng suppo rt and patience. and for their assistanceinthe most demanding parts of the field study

Fund ing from theDepa rtment ofFisheries.Go vernmentofNewfoundlandand Labr ado rviaSeabrigh tCorpo ration.Memoria lLmversu ycfNe wfoundland isgratefully ackn owledged

TABLEOFCO NTENTS ABST RACT.

ACKNOWLEDGMEI" S...

TABLEOFCO:\.ENTS . LIST OF TABLES...

LIST OF FIGURES....

. .ii

. iv

. v

... ...viii

. x

INTRODUCTIOS... . 1

Biology... . 1

Scallop aquacultureindustry:Ney,.fo undland... .. . .2

Larvaldispersal

.5

Markandrecapturetechniques... . 8

Larval tracking. . 8

Projectobjccnves.; .. . 10

CHAPTER 1: Developm entand testingof calceinforpractical use withPlocopectenmagelkm icuslarv ae...

1.1 Introduction...

. 14

. 15

1.2

Objective... . 17

1.3

Methods... . 18

Study

site.. .

18

Experimentaldesign... . 18

Spawning&Dcvel igerproduction 19

Calcein preparation.... . 20

Dataanalysis... . 20

1.4 Results .

Mortality. Fluorescence...

v

... 21 ...21

... . 24

1.5Di.scussion....

Monality...

Fluorescenc e...

. 16

... ...26

. 18

CHA PTE R 2: Relea se and recepte re ofC111«instai ned Placopt!cten.mJlgdlanicllSnligenatCha r les Arm,

Notre Dam eBay,New foundla nd... . 29

2.1 Introd uctio n...

Objectives...

...30

... ...31

2.3 Methods.... . 32

StudySile... . 32

Staining... ...32

ReleasctriaJ1994... . 37

Releasc tria ll995... . 37

Reca pturetrial1994... . 38

Recapturetnall995... . 38

CTD (Seabird)casts.... . 39

Microscopicexammation.. . .40

2.4 2.5

Results...

Discus sion....

...40

. 50

CHA PTE R3: Abundanceand dist ributioner blvafveIan:ae

inCha rlesArm.1'\ewfouo dla nd... .. 56

3.1 Introd uction... . 57

3.2 Objectives... . 59

vi

3.3 Methods __

Study site...

Experime ntaldesign...

Microsc opic examina tion...

Dataana lysis... .

Deplo ymentofdriftbouledrogues...

Currentmeters....

3.4 Results...

3.5 Discussion....

CHAPT E R 4: Tidalinn ueoce on quantitat ive sampling of bivalveveligenwitbinCha r lesAnn....

4.1 Introd uc tion...

4.2 Objective...

4.3 Methods .

Sampling

Physica ldata (currentmeters )... . Abundance and netflux. . Sizi ng....

Data analysis....

4.4 Results.... 4.5 Discussion...

Sl'~I ~lA RY .

LITER"Tl'RECIT ED...

vi i

... ... .60

. 60

. 60

. 60

. 61

. 62

. 62

. 63

. 79

. 90

...91

. 92

. 93

...93

. 94

... ...94

. 95

. 95

...96

. .105

...•...111

. 116

Tablela.

Tablelb.

Table 2a:

Table 2b:

Table3

:

Table 4:

LIST OF TA

BLES

Summary of stained giant scallop larvae found over the sampling season of

1994(

from release trial 1).

Totalin sampleis

an

estimate, where indicated

byan

asterisk("). based onstained larvae foundin asubsarn ple

ofa tow, otherwise, the entire sample was searched . Average

size is

for veligers found at all three (release, middle and inlet) stations for each

sample date.

NA

= not applicable. 46

Summary of stained giant scallop larvae found over the sampling season of 1994( from release trial 2).

Tota lin sampleisanestimate.

where indicated

by

an asterisk

(*), based on stained larvae found in a subsample

ofa tow, otherwise

.the enti re

sample was searched. Average

size is for

veligers found at all three (re lease, middle and inlet) stations for each

sample date.

NA-

not applicable. 47

Summary of

stainedgiantsca llop

veligers found over the sampling season of

199 5(from release

trial 1), Total in sample is an estimate , where indica ted by an asterisk ("), based on stained larvae found in a

subsample

ora

tow,

otherwise,

the entire

sample was searched

.

Average size is for veligers found at all three

(release.

middle and inlet

)stations for each

sample date.

NA-

no' applicable. 48

Summary of stained giant scallop veligers found ove

r

the sampling season of 1995 (from release tria12). Total in sample is an estimate. where indicated by an asterisk (.). based on stained larvae found in a subsamp

le

ofa tow. otherwise, the entire sample "as searched

. Averagesize is

for veligers found at all three

(release , middle andinlet) stations

for each

sample date.

NA-

not applicable. 49

Summary of growt hrates (IJmJd)of

bivalvelarvae inCharles Arm

during

the

1995 sampling season for the first spawning episode around August 12, 1995. Values are estimates based on a chan

ge

in mean coh

ort

size. 68

Summary of growth rates

(um/d)

of bivalve larvae in

CharlesArm

during the 1995 sampling season for the second

spawning episode

around August 28,1995. Values are estimates based on a change in mean cohort

size.

69

vii i

Table5.

Table 6:

Summary of currentspeeddata obtai nedfrom the current meters placed within CharlesArm from June l3-October10,1995.Valuesare in ern's.

78

Summa tionof

flood

and ebbphasesbynumbers of bivalvelarvaeJL during a25 htidal regime experiment in CharlesArm on August 19-20,1995 and August27-28,1995.(+)means transportinto Charles Arm. (-) means transport out of Charles Arm,(%)percentage of bivalve larvae from flood.

which Jack in the ebb tideand remain in Charles Ann. 104

t

x

FigureI:

Figure1:

Figure3:

LIST OF FIGl'RES

Mortali ty of 1-day-o ldgiant scallop veligerssta inedwithcalceinat vario usconcentra tions for14hat-250vel igervrn l, (TrialI).Values represent mean+i.standarderrorof3replicates. .,., Mortalityof1-day-oldgtantscallopvehgers stainedWithcalceinat various concentrationsfor24hat-40veligerszrnl,ITnaI2).Values represent mean-'.standard errorof 3 replicates. !3 RelativefluorescenceofJ-day-o ldgiantscallopveligersstainedwith calcem forup to24h.andafter 14and 48hrecovery infiltered seawater.

Fluoresc enc e of 0-undetectabl e.I-fair.2:satisfa ctory.3 .: good and 4

-exce lle nt. 25

Figure4a: Locationof studysite at Charles Arm.NotreDameBay.

Newfo undland. 33

Figure4b: Overview ofCharlcs Ann showing-release"."middle" and"inlet"

stations .current meter locations,and-25 hour-stationfor tidal influences.

Box A-Mouth of Charles Armand B-Endof Ann. J4 Figure 4c: Overviewofthe-Mouth- of Char lesAnnshowinglocation ofheadropes.

co llectorbags ( C ) forscallops.and max imum depths.The depths have been correctedtoshowwater heigh tsduringlowestlow tide.Stippled areasindicate areaswhichare lessthan3m deepor arcshoals whichbreak

the surface duringlowtide. 35

Figure 4d: Overview of the-End-of Charles Ann showing loca nc n ofheadropes.

collectorbags<C)for scallops. and maximwn depths.The depths have been corrected to show water heights during toweselow tide.Stippled areasindica te areas \\.fuch are lessthan 3m deep or are shoals which break

the surface duringlowtide 36

Figure5: Growth rates of releasedcalceinsta ine d3-day-o ldscallop vel igers during 1994 <a) and1995 (b). Valuesaremean sizes(+i.standa rd deviati on )for eachsample date.Dottedlinesrep resentthe 95% confidence

intervals 41

Figure 6:

Figure 7:

Figure8:

Contoursof temperature(DC) during199-tand 1995 in CharlesAnn at the samplingstations(release

=

A&B,middle=C&

D,

and inlet

=

E&F).

43 Contours of chlorophyll-o(l'gIL)during 1994and 1995 in Charles Arm at thesamplingstations (release

=

A&B,middle

=

C&D,and inlet

=

E&

~

«

Averagenumber of bivalve larvae perlitre collected from the three samplingstations(releas e, middle and inlet) in Charles Annduring the twomonth sampling periodin 1995.Valuesare meansoftriplicate sampleswithvertical barsrepresenting standard deviation. 64 Figure9: Shellheightfrequency distribut ions of bivalveveligers foundat thethree

samplestationsinCharlesAnn throughout thesampling period in 1995.

(SE

=

standard error,n=numberoflarvaemeasured ).Arrowsindicate

where cohortsareassumed to be separate. 65

Figure lOa: Surface currentmap ofthe end of CharlesArm.Arrowsindicate the directionofthecurrent drogues.R

=

release areaof the droguesat 06:45 on July 6,1995 (low tide).Times indicatedwere:I

=

10:15,2

=

13:00,3

=15:00andall drogues ashoreat15:50.Circles encompassthe area ofthe drogues at thetimes surveyed.Winds

=

lighttomoderate southwest.

High tide@13:52andLowtide@20:12. 71

Figure lOb: Surfacecurrent map ofthe endof CharlesAnn.Arrowsindicatethe direct ionofthe current drogues.R

=

release area ofthedrogues at 14: 05 on July 20,1995(Hightide).Times indicatedwere:I~15:10,2~16:24, 3~17:35, 4~18:15,5 -21:40,6=12:45(July21),7=13:45,8=14:50 and9

=

17:35.Circlesencompassthe areaof the droguesat the times surveyed.Winds=nowind.Low tide@20:27,High @02:32,Low@

08:37andHigh@15:02. 72

Figure10c: Surface current map of theendof CharlesAnn.Arrowsindicatethe directionofthe current drogues. R

=

release areaofthe droguesat 07:00 onSeptembe r30,1995 (lowtide ).Timesindicated were:I

=

07:18,2

=

08:00,3=10:00and11:30 thedrogues were ashore.Circles encompass the areaof thedrogues at the timessurveyed.Winds⁼verylight southwestswitching to mild-moderate southeast after 08:00survey.High

tide@II:57. 73

xi

Fi~IOd Surfacecurrent mapof theInletof CharlesAnn.Arrowsindicatethe direct ionofthecurrentdrogues.R'"release areaofthe droguesat12:50 onJuly18.1995 tHightide).Timesindicatedwere:I '"13:25.2=14:15.

3-14:50•.J...15:20, 5"15:35and6'"16:00.Circlesencompassthe area ofthc drogues atthetimessurveyed. Winds'"none to very light

southerly.Low tide @ 18:32. 7.J

Figure 10e Surfacecurrent map ofthe Inletof CharlesAnn.Arrowsindicat e thedirectionoftheCUTTeIItdrogues.R'"release area ofthe droguesat 14:30onAugust10,199 5(Lowtide).Times indicatedwere:I

=14:40,2 ...15:10,3=16:55,and4'"17:40.Circles encompassthearea of thedrogues at thetimes surveyed Winds-noneto verylight

northwestwind.Hightide @20:17. 75

FigureII' Temperatureprofil esin Charles Ann. Datacollectedfrom the current metersoverthe sampling period.Site 1-inletstation,site 2sand2d...

~2 5hour" station,site 3 and4~middle sta tionandsite 5..end ofArm

(releasesta tion). 77

Figure 12: (AIAvera genumber of bivalvelarvae per litre from the2S·h station at CharlesArmonAugust 19 and 27,1995_Verticalbars=standard deviatio n.High andLowrepresents timeof tida lhighandlow (B)Depthrelated pressure(dEar)fromCM2dover the sampling

period 97

Figure 13 Shellheight frequencydistttbutionsof bivalvelarvaefromCharles Ann on August 19,1995,collected from the "25 hour"station.(SE'"standard error,n,.numberoflarvae measured).Timeof day is indicatedinthe

rightcomerof each panel. 98

Figure 14: Summaryof bivalvelarvaeabundanceover a 25·h period in CharlesAnn on(A) August 12·13and(B) August25-26,199 5 at the release,middle

andinletstations 100

Figure15 Shell height frequency distributi ons of biva lvelarv aefromCharlesAnn onAugus t 12-13,1995at the release.middleandinletsta tions.(SE'"

standarderror,n-number of larvaemeasured) 101 Figure16 Shell he ight frequency distributionsof bivalvelarvae fromCharlesAnn

on August25·26, 1995at therelease,middle and inlet sta tions.(SE- sta ndard error,n-numberoflarvaemeasured) 102

xLi,

L"'ITRODUcnOS

8m/OIlY

Sea orgiantscallops. Placopectenmag ellamc us{Grnehn,1791).occuron the AtJanticcoast of NorthAmerica.ranging from Cape Hatteras.NorthCaro lina. to Labrador IPosgay.1951).Most wildbedsoccur atdepthsof 10m to100m in the central and nonheropartsof thespeciesdistribution.Inthe southernpanof their range.the majority of giantscallops arefound below50m.presumab ly because of theirinability to tolerat ewater temperatures above 20°C.The giantscallopisabenthic.subtida l.active suspens ion-feeder ingestingphytoplankton.smallzcoptenkro n,sporesanddetri tal particles(Shumwayet a/.•1987).Itcan liveupto and exceeding20yrwith growth rates dependingonlocalenvironmentalconditions

The sexes are generallyseparate.and canbeidentifiedbygonadcolor.with males beingcream-c olored andfemalesbeingbrigh torange.These are easily distinguishable whengonadsareripe.Hermaphrodi tesare found occasionally.Naid u(19 70)reponeda 1.3"-.occurrence ofhennaphrodi tisminscallopsof the Pon au Pan area in Newfoundland

Scallops are highlyfecundwithfemalesprod ucmg over one hundred millioneggs andmalesse vera l billionsperm(c.Couturie r,pers.comm.).Spawni ng isnormally synchronouswith in alocalityandgametes releasedinto the surroundi ng water (Parsons.

1994).Thelik eli hood of fertilizationsuccessisvery low inthis species as itis forother broadcast spawnerste.g.,mussels).Mostpopulations displaya singlc annual spawni ng

period extendin g over one ortwomon ths between JulyandOctobe r.altho ughthere are reports of semiannual spawning (DuPa ulero l..1989~DadswellandParsons.1991:

Andrews.199 2).

Fertilizedeggs (65.70 IJm)de velopinabout3dat15~Cintoplankt onicD-shaped (straight- hinged)velig erlarvae whichbegin to laydown a calcareous(ca lciumcarbonate, she ll(Couturie ret01.,199 5).Thevehgers spendfo urto six weeksinthe water column (C ulliney,1974)wheretheyfeed mostlyon phytop{ankto n.Whenlarv aereach about210 umthc:ydeveloppairedeyespotsandafoot(pedivehgerstagelandeventuallysettle on the bottom(whenappropnatecuesare present»,wherethey attachto a vanety of substratesandundergometamorphosis.Once settledtheyremainattachedbybyssal threads.altho ugh theyarecapableof detac hment andrapidswim ming untiltheyan:

approxi mately15mmin len gth(Man uelandDadswell.199 1)

Scallop aquaculturemdustry,\iewI QlDld fanJ

Large-scalescallop(Pattn()~cten.wuQf!rz.JSls )aquaculturehasbeen practiced in Japanforfonyyears(Ve nti lla., 198 2 ).Annual producnonexceedi ng100 000memc tonnes (mt)of wholeanimal and exports totheUSmark et exceeding10 000mt peryear havemade theJapan esea majorco mpeti torwithgiantscallops. Sca llops makeup 20^G__o of world moll uscproductio n.yieldi ngapproximately500 000mtperyear.Total Canadi an aquacultureproduction(shellfishand finfish)islessthan0.3% ofthe world

production.whichreflectsthe:slowdevelopmentoftechniques for rearingthegran t scallo p(Dads wellandParsons.199 1).

ReportsfromtheNewfound landAquacu ltureIndustry Associatio n indica te that in 1995 . giant scallop culturewas1%ofNewfound land'stotalaquacultural productionwrth J2tonnes produced at avalueors66 945 whic hwas14°/.of the shellfishvalue.This was anincrease of4000,..from1993.when only3 tonneswereproduced.Giantscallopsnow makeup3%of shellfish productionw'ithbluemusselsaccountingforthe other9~

Thetimfield-culturetrials with sea scallops wen: initiatedinthe tare1960'sby researchersal MemorialUnivers ity's Marine SciencesResearchLaboratory(MS RL I (Couturie ret oJ.•1995).Atthatumeatte mptsweremade tocollectjuvenile scallops (spa t) on arti fic ialsubstra tes at varioussites alon gthe coast of Newfoundla nd and Labrador(C outurier.1990).Anexpe rime nta l scallopspat collection programbeganin

\971as a response todwindlin gnatural stoc ks.Thiswasthe tim ofits kind in North America(Naiduetal,19891.Bythelate1970'sthebmlogicaJfeasibility of seascallop culturehadbeendemonstrated but seedsupp ly remained unreliable,Couturier etal., 19951_Earlycommercialworkwasdonein Newfoundland in19 86 (NalduandCahill.

1986)whe n commercialfarms were established at Picc adi lly.PortauPon Bayand CharlesArm,NotreDameBay.Anotherfarmwasestablished alPool'sCove. Fortune Bayin \987.

PJacopecten mageltar ucushas anumberoffea turesthatmake itanexcellent candida tefor cultureinAtlantic Cana da(Caddy,19 89;Dadswe tl.1989).for examplethe

largenatura lpopulationsin manyregion swhich couldprovidethenecessaryspat.One of the cornerstonesofthesecurityof existingenterpri sesandfutureexpansionand diversification in theshellfishsectoristhe reliability ofseed supply(Helm.199 5).andat present inNewfoundlan dthe bivalvecultureindustryreliesonwild-caughtseed-stoc k.

Even thoughthegiant scallophasbeengrowncommerciallyinsuspendedcultureinthis province forthepastdecade (Dab inctt andCouturier.1994 1..thereare snll onlyafew activegrowers.andthis factmaybeattributab le tounreliable seed supply.Both researchers and farmers have identifiedthat either the lackof spat orlackof knowledge of spatsettlement isa limitingfactorfor aquaculture cperancns.Atallevents.the greatest biologicalconstraintisnowseedsupply{Dabmett andCouturier.1994).

AlthoughPtacopectenmagellarucushasbeengrowncommerciallyIn Newfoundland forthepast decad e.itwasnot until1989thattheprovinc ialDepartmentof Fisheries andthe Ponau PortEcono mic Developm ent Associationbegan a program to monitor biological.chemicalandphysicalparametersat the Piccadi llyspatcollection site (Andre ws. 1992).Itwasobserved that spewrung,indica ted bya drop inthe gonadosomaticindex,usually occurred inlate Augustwnhhighestlarvalconcentrations (85urn shellheight) recorded in thefirst weekofSeptember.By early October larvae weregreaterthan 200 urn inshellheightThesedates varywithinabout twoweek s inter- annually.Duringthisstudy.Ponau Port in 199 1 had a recordyear forspe tfall,when the meanyield\1,115 6 379spa t per collector I> 20million seedfrom 6000 collectors).Yie lds thencontinuedtodrop eac hyear withdensities 5·10timeslower in 199 2 (mean yield of I

sn

percollector) andevenloweryields in 1993and1994. with1994havingarecordlow withfewerthan300 000 spatrecoveredfrom 6000collectorsfDabinenandCouturier.

1994).Thecausesforthedeclineareunclear.buta coolin g[rendintheenvironment and/oroverfi shingofthebroodstockpopulations havebeenpropo sed(Dabinettand Couturier,1994).Inspiteof themonitoringprogram.spat collectionattheotherfa nns hasnO(beensuccessfulnordependable,indicati ngtheneedforsomeothermethod to increaseandtor ensurespatcollection.

Larval dispel"SQl

Priortotheearly 1970's. researchon thegiantscallopwas confinedtotheadult stage.butsince that timetherehavebeendescriptionsoflarval development(Cul liney, 197-11.larn l diSbibutions fTre mb layandSinclai r.19 88;Tremblayaal..199 31and settlingbeha..iour (Parso nsetoJ.•19931.This research on larvaldispersio nisofthe utmostimportancewhencons idering scallop recruitment.

literaturerelevanttothedistribution and dispenal ofbivalve veligerswasdivided intothreegroupsbyMann119861.Thefirstof these groupstreats larvaeaspurelypassive particles forwhichdispersalisdeterminedbythephysicalelements.The second. andby far thelargestgroup,consists of fieldobservationsofhorizontalandlorvertical distributionoftheveligerlarvae.Behaviouraldatachara cterizethethirdgroup.Dataare obtainedfrom laboratory experimentsmeasuringrates ofswimm ing velocityand

thresholdlimits to environmental cues,through the manipulationof astimulussuchas tem peratu re.light. salinity(Wpressure whileallothervanablesare mamtamedata constantlevel

Problemsconcern ing thedispersio nof the larvae ofbent hic manneInvertebrat es have been addressed from anumberof different perspectivesand atavari ety ofspatial and temporal scales{Schelte ma, 1986).Someofthcse Include wind -induceddistribution (HudonandFradett e,1993).theretaricnshipbetween feedingandvertical distribution (Rabyet ol.•1994).verticaldismbutioninrelationtowatercolumn stratifica tion (Tremb layandSinclai r.1990&).seasonaland depth chantcterisrics {Robins-Troegerand Dredge. 1993).infl uence oftides(Levin, 1986).driftin g (Tremblayer ul.•19( 3 )andbroad sca le distri but io ninrelat io ntophysicaloceanography (Tre mblayandSinclair.1992) . These studies reportedlarv al dispersalranging fro ma fewcenti m eters to thousandsof kilometersacrosswideexpanses ofocean. Temporal interval s over which dispersalhas beenreported rangefrom afewrmnutes,to over a tidalcycle.to months.Distributi on data areusual lycollected and reported in conjW\Ction withvari ousblo log u:alle .g..

chlorophylland phytoplankton cellconcentrations)and phYSlcaJ(e.g.,temperature.

salinity.cknsity )functions.lnferenceisthen made ofme cause orcausesof theobserved distributions froma synthesisofalldata.

Themaximumpotentialdistan c ethat alarva canbedispe rse d andthe likelihood that it SuM" CStosettleme nt arerelated to(a ) the length of itsplankton iclife.IhJthe rate and directionof the currents that transportit(Sche ltem a, 1986)and c) itsbehaviourin

relationto environmental co ndition s (Gallageret01..1996;Manuelet at,1996).Two principalcharacteristics of the neritic environmentthat have cansignifICantlymodifythe dura tionof planktonicdevelopmentare temperatureandthe'q uality'andquantityof food ti.e..food abundance)(Couturieret or.1995)."Thesecharacteristicshavemainlybeen studiedinthelabo ratory whilevery fewstudiescorre la tingthesecharacterswithlarval development underfie ld conditions havebeenattem pted.

Adeq uate knowledge oflarva l behaviourduringplankt oni c lifeandinvest igation oflarval dispersalrequire suffi c ie ntly detailedinformation oncirc ulation patterns Curre nts and eddy diffusio naffectthedistribut ionoflarvae.Currents

n.e..

advect io n) transportlarva ehorizontall y inthe directionofflow whileeddydiffusionis therandom dispersionof suspended particles thatresultsfromturbulent flow.Thehorizonta l com ponentofeddydiffusioncanbe measured along a two-dimensionalsea surfacebythe change in mean distancebetweenfreelydriftingobjects.Conseq uently.eddydiffusion acco unts forho rizon ta l dispers ion(spati al displacement )of planktoruc larvae....-rth respect toone another.1bedispersalof larvae.as wellasthe varianceindwationofplanktcme dev e lopm ent, determ ines how manylarvae of the samecohon will remaintogetherinthe plan kto n andsubsequentlysettle together in one place(Strathmann,1974)

.

" farkandr~CQptur~ lechnlqllf!~

Nielse n( 1992) reviewedmethods for markingfishandshe llfish.Markingof animalsprovide sthreebroadcategoriesof informatio n:I)tolabel individualanima ls for specificstudy; 2)toidenti fyanimals as theymoveandmrermin glewith others:and3) to provide amethod for populati onstudy.The datacollected fromthesestudies can lhenbe usedfor stock contributionanalysis.population abundance estimates.ageand growth studiesanddistribution and movementstudies.Disaibutionand movementstudies have becomein~ingl yimportantin theshe llfishaquaculture sector.

Larval trucking

ThereateseveraldifficultiesInstudyinglarvaldispersalinthe mari ne environment.Theseincl ude:extreme dilutio nof larvae,parncularlyIf breedi ng:periods ateextended; IU1predietablenmmgofeggor larvalrelease:and smallsize and Inconspicuouscoloring oflarv ae whichlimitthe petenna lfor directlytracking their movements(Will isandOliver.1990 ).Asa result there are very fewinstan cesin which invertebrat e larvaehavebeentrackeddirectly inthe field.Most afthestudies. as mention ed ear lier.combineinfo nnat ionabo utlarvaldistri butio ns in the fieldwith knowledge of larvalsources.behaviour. recruitment patterns.andrelevant hydrod ynami c properties.Often. hydrodynamicmodelsateconstructed orsimulationsoflarvaltransport

are performedto explore probablelarva l trajectori es(Levin.1990 ).Direct rnckinghas alsobeenattem pted usingaeriaJ surveysand satelliteimagery (Willis andOliver.1990 ).

and haveprovidedanimportantperspectivefor identifyingthephysicalprocessesand meteor ologicaland hydrodynami calconditionsthat affectlarva l disper sa l.

Adifferentapproachto thestudy oflarvalmove men tsin thewater column is to re lease and recover la belled larvae.Theearliestreponedattempts to markinvert ebrate larv aeweremadeover fourdecadesagowhenLoosanoffandDavis(1947)successfully stainedoyster(C/tU$o.JtreoWrgln/CO)eggs.trochophoresandveligersbyrmmersicn in neutralred.OtherstainswereaJso testedbuttheseeitherkilledeggsandlarvae or stained [00Iighlly.Since that timeothers like HiduandHan ks(196 8)markedtheshellsoflarv aJ hard-shellclams(M ercenana mercenartavandposllarval hard-she ll.soft-shell(.<\.~a arenanojandcoot clams(.'vfultn/alat erahsvwith alizarinsodium monosulfonate by immersion of activelygrowingindividuals inthe stainfor 2-7d.Manziand Donnelly C1971Jfurtherdevelopedtec hni q ues forstaining large numbersofC',vlrgln/euandJl mercenanolarvaewith alizarin redandneutralred.In mostcasesthesechem ica lswere not usedInthe trackingof stained animalsbutratherforthe speci ficstainingof selective panssothatother research couldbe undertaken.Nile Red has beenusedto sta in intra ce llularlipid droplets.for detecting neutrallipiddeposits intissue sections.

permittingstudy ofmeta bolic pathwaysandlipidreserves (Fowler andGreenspan.1985:

Gree nspanetul..1985:Castelland Mann. 1994).andto monitorphysio logical conditioning (Jackson.1993).

Despuetheavailabi lity ofappropn atestains.therearc very few reportsthat documenttheUSC'ofthesetechniques(0markshellfish fordispersal studies inthefield Onlyone:successfulmark.release,and recaptureIn.t ll Ustudy ofstained larvalmovemen ts inmarine invertebrat eshasbeen described.Millar(196 1)releasedEuropean oyster larvae sta inedwith neutralredattwopointsin aSco ttish loch.He successf ullyrecaptured103 larvae12 to18h later.ever 1mile (2.2krn}fromthereleasesite. Mark,release and recapture methods suchasthese areamon gthe most direcl. means of studyingdispersal.

but are rarely attempted. This project represents onesuch attempt

ProjectObJeclrves

The larvalphaseof the giantscallop isimportant to the aquacultur e industry because highretentio norsurviva lof larvaehasbeen linked to successfulrecruitment to fishablesizesinscallops(Cad dy,1989).Dickie (1955) suggested thatyears ofpoor recruitmentin scallops were due to advection of larvae from favourable settlement areas Atrialenhancement experimentto increase recruitmentwas previouslyperformed by researchers ata mussel/scallopaquaculturesite in CharlesArm,Newfoundlandwhereby millions of3-day-oldveligerlarvae were producedusinghatcherymethodsandrelease d onsite.This didnotresultin increased spat collection 2months later (Dabinenand Couturier,1994 ).The experime nt raisedtwo important questions:1)werethe released larva e retained withinthesite?;and 2) was the methodusedfor spatcollection

appropriate?

Thisprojectwas designedtoinvestigated the retention ofreleasedgiant scallop larvae within an inletinNotre Dame Bay.Newfoundland.Incorporati on of larval markingwithanon-toxic.fluorescentdye (calcein)andtracking (re lease and recapture)•

....'illbe usedto determineif releasedP.mage llamc usla rvae are entrainedwithi nthesue.

Altho ughthe stainingmethod wasada pted fromRowleyandMackinno n(1995 ), speci fic procedures for1'.mag.ellum cus hadfirsttobedeveloped.Furtherm ore.the efficacy ofthissta inonnewlyhatc hedDcvehgershad tobedeterm ined and the procedur e adapted forfieldconditions.

The objectiv es ofthis studywere:

Todescribeaprotocolfor staining scalloplarvae with thefluorescent stain

celcem.

which bindsirrevers iblyto calcium asitisdepos itedintheshell

, Todeterminetheeffic acy oftltis sta ining procedure on newlydevelopedD- veligers.

To co nd uct mar k,release andrecapture trialswithstainedscallo p larvae in Charles Ann,NF.

To determi negrowthrates of calcein-stamed scalloplarv ae undernatural conditions with temperatureand chlorophyll-ameasuremen ts as variablesof the

"naturalconditions".

S. To studythespatialandtempora ldistributionsofstained scallop and WIldbivalve larv ae withinCharlesArm.

6. To measurecurrent speed and direction withinCharlesArmusing mooredcurrent meters,andsurfacedriftpanemsusing bottle droguesand presentthese data in relationto larvaldistnbution.

7. To determi ne the abundanceandsize frequencydistribu tion of bivalvelarvaeat three sampling sitesovertime to giveanindication ofspa wningepisodesandwild growthrates inCharlesArm.

To estimate theeffects of tidalcycles onlarvaldistribut ionbysampling:the distribution andtransportof mesozooptankton throughout tidalcycles inCharles Ann

9. To synthesizetheresults of the markandrelease studiesWlthwildbivalvelarvae distnbutions to examine the possib lefateof artificiallyproduced scalloplarvae releasedinCharlesArmandevaluate this methodasastrategyfor enhanceme nt andrecruitment forscallop aquaculture.

CHAPTER I

Developmeot.ad les tio g ofalttiofor pra ct ial UK

",ilbPJtlcop«t~nmJlgdJtI,,;cM$larn e

1.1IntrodllctioD

Severalmarkingmethods involvechelationorreplacement ofcalci umduring calciumcarbonate deposition.The antibiotictetracyclineproducesafluorescentband.

visibleunder UVlightandhas beenusedbyseveralinvestigato rs tomark avarietyof different species offish.e.g.•eggs andotolithsoflacva layutPtecogtossusultlVeh~) (Tsukamoto. 1985).and otoliths oflarval spot(LelO.'iiram u.~;cant h uru.'ii )and pinfish [Lagodonrhomboides v(Hettle r,1984).Atleastonesuccessful mark-recapture experimenthasbeencond uctedwith tetracycline-labeledayuwhereb y eggsandlarvae wen:releasedintoa river andrecovered. I to 6dlater.inorder10validatethepresence of dailygrowthincrementsinlarv al otoliths(Tsukamol0 andKajihara.,1987).Calcei n\WS

proposed byWilsonetai,(1987)asa saferreplacement for tetracyclineinmarking fish otoliths.Tetracyclineissometimesdetrimental to the health of fish. causing reduced.

activityandcessation of feeding.A laterstUdybyMonaghan(199 3)suppo rtedthe superiority of calceintote tracyclineasachemica l marker in swn mer Flounder (Parallchthys dentatus):unlike tetracycline.calceinbands were moreintenseandthe caleeinstaining procedure wasless toxictothefish.Inany studyusingchemicalmar kers ilisimportantthatneither themarkingprocessnor the chemical itsel faffects the animal inanyway.The chemica l.therefore,mustbenon-toxicand remaindetectab le onthe animalforthedurationofthestudy.

Calcein(3.6-Dihydroxy-2.e-bis- IN.N'-diecarbomethyl)-amino-methylIfluoran )

:6

binds withalka lineearth metals,andalthoughthe processhas norbeen studiedit appears thai calceinispermane ntlyincorporated into calcareoustissuewhenpresentduring calcificatio n(RowleyandMack.innon.1995).Calcein is readilyabsorbed by tissues . bindingpermanen tlytonewlydepositedcalci um.showinglittletoxicityandfluorescing brightgreenwhenirradiatedwith blue light,thus mak ingitvery attractivefor use in marking. identificationandgrowthstudies. These featureshave allowedcalcemto be employedin mark.ingbones andscalesof fish(WilsonetaJ..1987)and in othercalcified structures ofinverteb rate taxasuchasbrachiopods.bryozoans.gastropods,cephalopods.

echmcids.cphicroids,asteroids,holothuroids.pclycha etes.crustaceansand bivalves (RowleyandMackinno n, 199 5).Fluorescent dyes also offer anumber ofadvan tages including:highaffinitytove rylow concentrations of the substanceto be stained; absence of distributionalerrorcharacteristics in absorbance measurements.andthe possibility for InSll llstudies(CastellandMann,1994).Calcein, therefore.haspotentialfor markinga varietyof calcified larval structures,including mo lluscshe lls. echinodennplatesand skeletalelements.and.inthisstudy.the newly-fo nningshell ofthe larvae of the giant scallop

Giant scallopsreachthe Dcveligerstage 3 to4d afterfertilizatio n(Couturieret al.•

1995).Atage 0 to 3 d, giantscallop larvaearetecnh orrcphtc.Secretionsby the she ll gland andmantleepithelium begintheassembly of calcium carbonatecrystalsandan organicmatrixto fonntheprod issoconch I larvalshell(Crenshaw.1980;Jablonskiand Lutz.1980 )_Itis possibletoinducemature giantscallopsto spa wn, fertilizethe eggs and

maintainthe developinglarvae for upto4dunder hatchery conditions.At theonset of theforma tion ofthelarvalshell, Devetigerscanbe immersed in abathofcalcein which then becom es inco rpo ratedin the newshe llgrowth.Once theveligers aremar kedthey are ready10 bereleased intothe naturalenvironment.

Thefundamentalassumptionof chemica lmarkin gisthatneitherthe marking processnortheintrod uced chemical changes the behavio ur,physiology,or bioch e mistry of theanimal(Nielsen.1992). High concentrationsof,orlon g exposure 10.any chemica l can behannful (Tsukamoto.1985).so every proposed che mical markerandmarking process shouldbecarefull yevalua tedItisalsorequiredthatchemical marks remain ide nti fia bleonthemarked animalsthrougho utthe desiredinterv al.ArJ.experimentwas performed10 testtheefficacyofthesta ining proce ssand thetoxicityof thestain.

1.2Objettivc

Todescribea staining protocolforthefluorescentstain.calcein,for use with gianl scallop larvae,andtotest the efficacy ofthisstainingprocedurewithne wlydeveloped D- veligers

Adu ltgiantscallopswere obtainedfrom a com mercialshellfishfarm (Thimble BayFarms)locatedalCharles Arm.NotreDame Bay.NewfoundlandonJuly 20.1994 (tri alI)andonJuly24.1994(trial 2).These scallops served as naturally co nditio ned broodstockforthepurposesofthiSstudy.Fieldlaboearones were establishedin a processing plantandcabinlocated atthe site

Espenmentaldesign

Two trialswere undertake ntoInvestigatethe efficacy cf'rhestai n- Triplicate samples of larvaef250 permL·malland 40permL-m a l 2)wereexposed to:5 concentrations ofthestain plus a control(no stain,1011mfilteredseawaterI. for:5 exposure times of10.100min. 8,16and 24 httnal1)and2.8.and24 nltria l:!l.

followe dby recoveryperiodsin 10urnfiltered seawater fo r24 and48h(m al I)and 24h (trialZ) atIS"C.Thecalceinconce ntra tionswereSOO.200.100,10.Img/L and0mgIL forthecontrolThe cercem,in powderedform.was obta inedfromSigmaChemical(Lo t Il.3HOS94 )

Spawnlfl g and~w"gerproductIon

Larvae were obtainedbyinducing adult giant scallops tospa wn.The adultswere takenfrompearlnetshanging at about3m depthon the fann siteandbrought back to the proce ssingplant.wheretheywereplacedin a1 m'Xacncs PVC tubcontai ning 10urn filtered seawater.Scallopswere inducedto spawn bythe method of Desrosiers andDube (1993).but on a smallerscale.At the onsetofspawning, individualmale and female scallops were placed in5·Lplasticbucketsfilledwith10 pm filtered seawater and allowed to completespawni ng.Fert ilizatio nwasaccomp lished byaddi ng sperm to the egg suspension with a pipett e until aratio ofapproximately5to10 spermpereggwas obtained.This was determi nedbydarkfield microsco py.Eggs wereleft fora few hours andmonitoredusing aninverted microscopeto confirmthat cell division wastaking place.About15millionfertilized eggs were thentransferred10eachof 6 pre<leaned., covered.Im^JXacties PVC tub filledwith filtered seawaterand left for36h.Larvae were thenmonitored to confirmdevelopme ntinto straight hingeDeveliger larvae.When needed. Dcveligersfromthetubswerecoll ect edon50j,lmscreensandwashedinto5-L plasticbucketsfilled with filteredwater.

Catcempreparation

The calcein stock solutionwasprepared using themethod of Rowleyand Mackinno n(1995) bydissolving0.5gof calcei nin onelitre of 10urnfiltered seawaterin a glass jar.Sodiumhydroxide(2.3mLfrom a stock solutionof40gil). andconcentrated Hel (if necessary), was addedtobringthepH close to that of ambientseawater (pH- 8. IO)

AliquolS(2ml.)ofaD-veligersuspension were placedin30mLtissueculture flaskswith calcein stocksolutionand/or filtered sea-water(final volumeof30mL)to give final concentrationsof 0,I,10,100,200and500 mg calceinper litre

A 2mLsample was taken fromeachof the flasks at appropriatetime intervals and larval survivalevaluatedwithaninvertedmicroscope.Sampleswere preserved in4'!'o forma linfor estimating abundance.survivaland mark intens ity.Fluorescencewas determinedon an arbitrary 5 pointscale ranging from 0 (no fluorescence)to 4 (bright fluorescence).Preserved stainedlarvae wereexaminedon a monthlybasis to check for loss offluoresce nce.

After24 h immersion,larv ae inthe stainingsolutionswere removedusing a SO um screen.Thelarva e wererinsedwithclean10 urn filteredseawater to removeany

21

residual calceinsolution. then returnedto thetissue culture flaskswith30 mLof filtered seawater(trialI,highdensity) or tobuckets filledwith 8 L offiheredseawate r(trial 2, low density).

SingleFactorAnalysisof Varianc e (ANOVA)was performed to compare morta lities inallconcentrationgroups,forthe differenttimeperiods sampled.withineach trial.

1.4Results

Mortality

In the first trial mortalit yremainedbelow10%for allconcentrations(Figure 1) duringthestainingperiodwiththe exceptionofthe 500mg calcein/L concentratio n.

whichhad 15%and 16% mortality at immersio nlimesof16 hand24h.respectively.

There was asignificantdifferencebetweenthe500mg calceinILandall other concentrat ions,incl uding the contro lat the 16hsample (ANOVA.F1' .17) ""4.84 1.

P<0.05) .No differencein mortalitywas observedamonggroupsat anyofthe other immer siontimesduringthe stainingperiod(ANOVA.p>O.05). Anincrease in the morta lity of larvaewas observed after the larvaewere removed from the stain(recovery

100

22

-e- 0mgcalceinll

»>

-. -1 mg calcein/L -.A.-10mgcalceinIL

-...- 100mgcalceinlL

•

80

_{-. - 200mgcalceinlL}

-e-500mgcalceinll

•

• ..

/

~ 60 _/

(ij^t::0

$ 1

::;;

;F- 40

20 T T ^:I

T -V

..

^~

v?

^~

..

0

0 10 20 30 40 50 60 70 80

f-

$lIIorll.. gPenod

~ IE Time (Hours) '--- ^~

Figure 1 Mortality or z-dev-otd giant scallop veligers stained with calceinat various concentrations for24 h at-250 veligervml,(Trial I) Valuesrepresent mean+1-standard error of3 replicates

30 -r-;:========:;-- -- - -i

23

20

-e-.0mgcaJeeino'l ___ 1mgcaJceinIL ---..-10mgca~rVL ...- 100 mg caJceinll --+-200 mgc.lcelnIL _ 500 mgcalceinIL

10

o 10 20 30 40 50 60

f--- --.- -+----.----l

TIme(Hours)

Figure 2Mortality of2-<lay-old gianl scallop,,~i!lenstained at variouswithalcein al various COlICe!Itrati0ll5for24hat-40veligerslmL(Trial2),Valun represenl mean-f·

standarderrorof3replicates

"

period).After24 h recovery.alltreatments includingthe control groupsexhibited greater than 45%mortality.After 48 h recovery.mortalitieswere-9 5-1.atall concentrations withtheexceptionaCme200mg/l,(mo rta lity-75.,...

Inthesecondtrial.no mortalitiesover20%wereobse rved fo r anyaCme sta ining conce ntratio ns or thecontrolduringthe staining orrecovery periods (Figure 2).No mortalitiesgreater than 4%wereobserved inthe 100and200 mgcalceuvl, concentratio ns even during therecoveryperiod.Therewasnosignificantdiffe re nce inpercent mo naJi ty foreachconcentra tiongroupandthe control(ANQVA.p>O.OS)during the staining period.Mortalitiesincreasedoverthestudyperiodfo r the controlgroup (0 mg calceinIL) (ANOVA.F'l.Il=91.06.p<O.OSl.and10mg calceinIL(ANOV A,Fa..,-'23.65.p<O.051 but nosignifican tincreasein percentmonalitywas evidentfor the 100 .200andSOOmg calceirvl.groups(ANOVA..p>O.OSl.Microscopicobservationduringtherecovery period revealedthatprotozoans were prevalentinthe controlgroup whilelarvalsamples prt"iouslybathed in calcein wert protozoanfree.

"'Juo,e.~cence

Fluoresc e ncewasobservedin larvae afteras littleas 8himmers ionin the stain (Figure3).butwasfaint and visibleonlyin thehighest concentrationof stain(500 mg calceinIL).After16himmersion.larv ae inboth the SOOand200 mg calc einzl,

25

D

0mg calceinIL

o

¹mg calceinIL

o

¹⁰mg calceinll

D

100mgcalceinll

o

^{200mgcalceinll}

CJl

500mgcalceinll

Q)

4

0c:

s '"

~

0

3

"

u:

Q)

.2:

iii

Qi

a: 2

30

,L -

o 10 20 40 50 60 70 80

I--

S'*''''''''llP-.od - ,. .

11-<.----

RIKJ:I... ryP."Od ~

Time (hours)

Figure 3 Relative fluorescence of j-dey-oldgiantscallopveligers stainedwith calceinfor up 1024 h,andafier24 and 48 h recovery infiltered seawater

Fluorescenceof0-undetectable,1=fair,2·satisfactory.3-good and eeexceltenr

26

conce ntrationswerestainedsatis facto rilyand a faintfluorescen cewasobserved inthe 100mg calceinILconcentra tion.After24h.the200 rng calcem/l,samplesshowed excellentfluorescence.Fluorescenc e was undetectable inthecontro lortheImg calceinILconce ntratio nafter24 him mersio nNodepletionoffluoresc enc ewasobserved duringthereco veryperiodinanyof thestainedvehg ers. Preservedlarvaehave retained theirfluorescencewiththe same intensityaftertwoyears.

loSDiscussion

Mortaluy

Althoughhighmeasurementsofpercentmortalitywereobse rvedinmallin all calceintreatments theydid not differ fromthe controltreatment.Incontrast.duringtrial 2thecontrolgroup hadthehighestmortality observed(Figure2).Somefac tor.otherthan the calce inbath.must berespo nsible for the increasein mortalityof the controls.The differe ncein percentmorta lityobservedbetweenthetwo trials (FiguresI and 2), especiallyduring the recoveryperiod.suggests two possibilities.

Firstly,thedifferencemaybeattributabletoth.elarvaldensityinthetissue culture l1ask s.TrialIusedaveryhigh.density of-250 veligervml,whileduringDial2 density wasreducedbyalmos t soelo to40veligers/mLThehighe rdensitiesmeanthatlesstime

istakenfor metabolicwaste to reac hcrit icallevelsandforoxygen10be depletedThe extremely high mortah nes(>40%)observed duringtherecovery period oftrial I (Figure IIwereunsatisfactory,andwouldrenderthe staining procedure impract icalformarkin g thelargenumbers of gian tscallop larva eneededfor a fieldexperiment. Lowering the densityof thevehgersin

w

flask.. for trial2.would reduce stress anddecreasedpercent mortalitywouldbeexpec ted Notonly was thedensitylowe rduringthestainingperiod.

but once removedfrom thesta in these veligers wereplaced in8 Lof water.thereby greatlyreduc ingthedensity (6veligers/ml. )during therecoveryperiod. Largenumbers of larvaecould possibly be supported byusinglargerincubation vessels(bucketsortanks], i.e..alower density.andpossibly with aeration.

Secondly,different batches of scallop vehgers wereusedin the trials These batchesofveligersmay be of a different geneticmakeup,related possibly tothe genetics ofthe broodstock or tothe ripenessof thegametes(Cou turier,1986)andmay accou nt for the differencesobservedinpercent mortalitybetween the twoexperimentaltrials

Large numbersofprotozoanswere observedinthecontrol group(trial 2) after24h post-stai n.whereasnone were observedinthe samplesbathedin calcein.This findi ng isof someinterest and may benoteworth y for furtherstudy.Itmaybepossib le thatcalcem has someeffec t on protozoan s or bacteria suchas reducin gtheirabundance.

Fluorescence wasnotevident untilatleas t8hafterImmersi on.andwasfa intat thistime(FigureJ).Stro ngerfluorescencewasobservedat16hforthe200 and500 mg calce in /l. samples.but thegreat es tfluorescence wasseeninthe200mg calcem/L sample after24h.

Giantscallopveligerscouldbesatisfactorilymarkedthroughimmers ion inthis stainfora minimumof16hataconcentrationbetween100-200mg calceinIL.Altho ugh better fluorescencewasobservedafter a longe rimmersion time(Figur eJ).it is more convenientto use 16h because scallops canbeplacedinthe stainin theeveningandused thefollowingmorni ng.Furtherm ore.the sho rte rthelengthof timethattheve lige rs are handled.the lesslikelythatmonalitieswill occur.Afluoresc entmarkerneedonlybe detectablethusa minimwn amountisrequired. Using-ISOmg calceinll.. wouldbe suffic ientfor fluoresc enceingiant scallopvelige rs andwould makemo re efficientuse of thecalcem, Aconcentrationof 12S mg calceiwl,was previcustyusedbyWilsonaat.

(1987) to mark otoliths of larvalandjuvenile ftshalthough. Rowley and Mackinnon 1199S ) used a calcem solutionof SOOmg/l, to stainbivalvesas well asothe rtaxa.The resultsofthisexperimentind icat e tha t only1/3that coecenrranon needbe usedforgiant scalloplarvae buthigher concentra tion smaybenee ded for othertaxa.

Celcem isusefulin itsabilityto markgia ntscallop larvae.Not onlyisit no n-toxic 10 thelarva e. italso providesabright,permanent fluoresc entmark.thathasalo ng lifein preservedsamples.

CHA PT ER2

Reka se and recapture or eelee re stain edliantscallopveli2en at Cba rfu Arm, !lriotnDameBay,Sewfou ndla nd.

30 1.1(_trod ud ion

Earlier enhancementstudies at CharlesAnn, Notre DameBay.during which millio nsof j-day-cldgian tscalloplarvae were released. didnot result inincreased spat collection(Dabmett andCo uturie r. 1994).There aresevera l possib le expla nations for the lackofobscrvedincrease inspatyield.Firstly,the veligers producedbyhatc hery techniques mayhavebeen moribundandthereforedid notsurvivetoset. Secondly.the released larvaemayhave been ttansponed out ofthcsitelOsetelsewher e.Thirdly.the predationlevelmayhave beensuffi cien tlyhighthatthenumber of releasedlarvae that survivedto SCI wastoolow tomak e a significant difference.Fourthly,too few larvaemay have been re leased CharlesAnnholdsapproximate ly10mill ionnr'of water.so releasing onlyafewmillion larvae would probablynotresul t in a highpercen tageincrease ofspat found onthe collectorsas compared to previous years.Finally.theveligers may have beenretained inthesitebut the collec tio nmethod or place mentof collectorswithi n thesitemayhavebeeninappropriatetoprovideany substrate for the releasedlarvae.

Before stainingtechniquesweredeveloped. stud ies on the movementsand distributionof larvaehadtodealwithunknownpopula tions.Since astainingmethod for oyster JaMIewithneutral redwasdevelopedbyLoosanofTandDavis(194 7).manystai ns have been used success fu llyforavarietyofinverteb ratelarvae.for acomprehensive reviewseeLevin(1990).Despitethe availability of appr opriate stai ns therearevery few studies inwhichinve rtebratelarvae have beentracked direct lyin the field.Mosttracking studieshave combinedinfo rmationon larvaldistributionswith larva l sources.behaviour.

recruitment patternsand relevant hydrodynamic properties (Levi n, 1990 ).

Mark.releaseand recapture methodsareamongthemostdirectmeans of studying larv a l dispersal .They are rarely attemptedand Millar(1961 )ISthe onlyreponed success ful attempt

1.2Objectives

The stainingprotocol sdescribed inCha pter Iwereusedto stai n batchesofscall op larvae prior10releaseata single point within a sheltered sitefor recapture studies.The purpose of thismark. releaseand recaptureexperi men t was:1)todetermin ethespat ial distribution oftheveligers which maybe movedby swimm ing. windtransport or current/w ater movement;2) toestablish if the veligersare beingentrai nedwithinChar les Arm;and3)toemploystai nedveligers,upon reca ptured.tomeasuretheirgrowthrates under insitu conditions.Temperatureandchlorophyll-adatacollec ted byaCTDmeter willbeused as measuresofinsnuco nditio ns.

32 2.JMethods

Studysite

The study area isloca ted at CharlesAnn.Notre Dame: Bay (Figure 4a and b).

This is a commercial mussel(Mytilus edulis)and giant scallop fann.The site is located in a sheltered narrow inletwitha shallow sill (5 m) at the mouth.The inlet extends fornearly 2 kilometers and has buoyed head ropes anchored shore to shore across thesite.The farm occupies nearly 100 hectares and holds about 10 millionm' of waterwitha maximum depthof20 m(Figure 4candd)

Stammg

Batches ofPtocopeaenmagelkmtcusvetigers were produced using standard hatchery techniques and stained usingthe protocols developed in Chapter 1.i.e, immersion in150mg calceinIL for 16h.

Figure 4a Location ofstudysite atCharlesAnn. NotreDame Bay.

Newfo undland.

33

B

34

Figure 4b: Overview of Charles Annshowi ng"release", "middle"and"inlet"stations, current meter locations,and"25 hour"station for tidal influences Box A:::

Mouth of Charles Arm and B=End of arm

35

A

Figure4c: Overview of the"Mouth"of CharlesArmshowi ng locationof headropes.

collector bags ( C )for scallops,and maximumdepths.The depthshave been corrected to show water heightsduringlowestlowtide.Stippled areasindicate areaswhicharelessthan3m dee p or are shoals whichbreak the surface duringlow tide.

36

Figure 4d: Overview of the"End" of Charles Ann showing location of head ropes.

collector bags ( C ) for scallops.and maximum depths.The depths have been corrected to show water heights during lowest low tide.Stippled areas indicate areas which are less than 3m deep or are shoals which break the surface during low tide.

37 Release trIal/994

Stained larvae were filteredfromthe calccinbath withaSOumscreen.Larvae werewashedwith 10urnfiltered sea-waterand placed in16- L bucketsfilledwith11.5 L filteredwater(-175veligerslmL)untilthetimeof rc leasc (within 2 h).Larvae were releasedonlyathightide.The buckets of stainedlarvae were transported to therelease pointinthefarm'sharv est boat and emptied gentlyovertheside oftheboatbytiltingthe partly submergedbucket in the water.Eighteen million(t.8Im^l)stained vehgers were releasedin the firsttrialonjuly27 at11:41.withan observed 17.25millionlarvae viable.

On August19 at 08:00.a second batch 0£21million larv ae (2.l /m^l)wasreleased.

Mot1alitywasobserved tobe30% and approximately15 million larvae were viable.

Reiease trial/995

Stainedlarvae(15 millionand 27 million) wererelease d on two occasions (August 12 and 25.respectivel y)at the same release point asin1994.

36 Recapt.~mczll994

Recapture ofmarkedvehgerswasattemptedwithhorizontalplanktontowsat two depths (ImandSmlat three differe nt stations(releas e.midd leandinlet).Towswere cond ucted1.14.21,and72hafter thefirstrelease onJuly27witha30 emdiam ete r64- um-rnes hnet. Subsequentto wswere completedonAugust6.11.19.23 . 24and26th.

September8and October4(10.21.23.27. 28.30.4)and69 d after release).Thesecond release of stainedlarvae occurred3wklater on August19.Towscompletedafte rJuly29 were performedwitha50 em diameter 64-prn-meshDelPlankton sampleswere preserved inS%buff eredfonnali n untilmicro scop icexam inationforstainedlarvae.

Towing timesvariedfromIto 3 min, depend in g ontheobse rvedconce ntrationof planktonin thewater.Towi ngtimeswerereducedonoccasion topreven t theplankt onnet from clogging.AGeneralOceanics..Inc.2030R Standard Flowmeterwasused to quantify thevolume ofwater filtered during eachtow.

Recaptun trial/995

Recaptureof stainedveligers wasaccom plis hedusing obliquetows.donein triplicate.atthe samethree stati ons des ignated in 1994.Towswere completedwith the same 50 em diame ter64 gmplanktonnet and pcrfo nn ed 2.4.8,16,and24 hafte r release and then weekly.The first releasedatewasAugust12 andsubsequentto\\''5were

3.

performedonAugust 13.19,25.26.28,September7,21and30th (1.7,13.14.16,26.40 and49 d later)withthe second releaseoflarvaeonAugust25.The obliquetowsatthe releaseandmiddle stations were70mlong. dropping I mindepthforevery10 m of horizontalmovement.Distanceanddepthconsideration allowed a50 m obliquetowat the inlet stationonly.To aid inthereplication aftowsandto ensure thattheboat remainedonthesamplingstation.fluorescentlypalmedJavexbottles were attachedto a fixedheadropc10mepart, Samplingbeganatoneendof thisheadrope.Sampleswere pre-filteredthrough a

sao

urn screen to remove any1a..fi'eTplanktonic animalsand then preservedin5%formalin untile:wninedforstainedlarvae.

CTD (Seabird)casts

Watercolwnnstructurewasexaminedwitha conduetivity,lemperanue,depth m~{cm.manufacnaedbySeabirdElectronicstec.,WashingtOnUSA).equippedwith an fluorometer tomeas ure chlorophyll-aconcentration Tempearureand t:hlorophyl1-a contour plotswerepreparedwithSurfer(Wi n 32)5Oftware.Versio n 6.01 (Golde n SoftwareInc.Colorado.USA, 1995).

'0 Af,CTOSCOpte~mlflallOn

Preserved sampleswerefiltered ontoaSOurn screen and re-suspended in2.5mLof 2JImfilteredseawater.Twenty fiveJmLsubsam pleswere examinedwithaZeiss AxiovenInvertedMicroscope withastandard bluefilter set (Zeiss4877 09),Chroma tic BeamSplitte r510,Barrier Filter BP 515- 565 andExciter fillerrange450490run.When stained sca llop vehge rs were found.she ll heightandlength weremeasure dwitha calibratedeyepiece.To sho rte nsearch time. samplescollect edwithin a weekofrelease weresizegradedtolessthanandgreaterthan186um(mtexmeshscreenmeas ured on the diagonal).Onlythose sampleswithlarvaesmaJlerthan186 JIm were examined.Samples collected after thefirst.week but not laterthan2 wk afterrelease wereseparatedimoless thanand greaterthan295 JImwithonlythose sampleswithlarvae smallerthan 29511m examined.

2.4 Results

Larvaewere-60-80IJ.m inshellheight uponrel easein 1994.Larvae remained v.ithinthe site for over 40 d in 1994withmaximwn shell heigh ts of240 and270urn observedfor theJuly27and Augus t19.1994 releases,respective ly.Gro wt h I11tc SoD.S3 and3.85 j.1mJdwere observedforthe 1stand2nd release s.respectively(F igureSa).

Larvaewere70 urninshe llhe igh t uponrelease in1995.A maxim umshell heigh t

350

-r;:======::::::;---=-i

300 ^tsireeese

(July27,1994) 2nd release(AugusI 19,1994)

250 200

150 100 50

~

0 + --,-- ,----,- ---,-- ---,-- -,--- , ----,--1

I •

1st release (August 10.199 5)

I

• 2nd release(August28. 1995)

300

350 ,-;::= = = = = = ::::;-- - - ,

250 200

150 100 50

O +----,- -r---,- ,..---,- ,---,- r ----j

200 210 220 230 240 250 260 270 280 290

July August September

I

October JulianDay

Figure 5.Growthratesofreleasedealcein stained3-day-old scallopveligers during1994 fa)and 1995(bj,VaJues an:meansieesC+t.standard deviation)foreach sample-date Donedlincsrepresenlthe95% confidenceinlervaJs

"

of-240gmandagrowthrateof9.npmldwereobservedwithin3witfor thelim.release onAugust 10.1995.Larvae ofthe second release remainedwithinCharlesArmforever 30 dandhadan observed growth ratc of2.52pmld (Figure Sb).

In 1994,surfacetemperaturepeaked durin g late Julywith18°Cobserved at all threesamplingstations.Te mpe ratures remainedgreaterthanISoCuntil earlySeptember atthe release andinlet stationsbut remained above 16·Catthe middlestatio n until la te Septem ber.Temperatures greaterthan J4-IS·Cwereobservedtoadepth0(7matall sampling stationsfrom lateJulytolateSeptemberin1994(figure6).

In 199 5.surf ace temperaturereacheditsmaximum duringearlytomid-August with14.18and17-C observed at therelease.middleandinlet statio ns. respectively.The temperaturedeclinedrapidly to12°C (Se ptem berandOctobe r ).Ata depth0( 7m, temperaturesabove12·Cwere notobserveduntil earlyOctober atthe releasestation.late Septemberatthemiddl e station andearlyAugustat theinlet station.

Temperatures greaterthanISoCwereobservednear the surface from early Julyto lateSeptember during 1994.whereas temperaturesabove(S·Cwererecordedonly from late July10late Augustin 1995.

In1994.thefirst indicationof a phytoplanktonbloomappearedin late Julyatthe releasestatio n.peakingat 10 IJ.gchlorophyllaJ1..(Figure1).Atthemiddlestatio n.the chlorop hyll-amaxim um remain ed around8IlgILbutwaspresent atdepthsof greater than 10m.Anotherbloom occurred inearlySeptemberwithan observed concentration of II IJ.g/Lchlorophyll-a at14 m.

43

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44

In 1995,aphytoplankton bloomoccurredatthe release station in themiddleof June(Figure 1).ChlorophylkJconcentrattonreached a maximwn of71Ig.1.withthe peak.

between6 and 8 m depth.Asecond bloomoccurred during early Augustwithchlorophyll- aconcentra tionsreach ing 13 P8:1..(IImdepth)atthe releasesteuon and 10I.IgrLat the middlestation.Tbechlorophyll~concentrationatthemiddlestationremainedhighover thestudyperiodwithva lues greater than 10I.Ig1l.observed at depthsgreaterthan 8m.

Stained scallopswererecaptured at allthree samplingstationsduring1994 (Tables laandlb]and 1995 (Tab les2aand2b)andrangedfrom 70 to 27011minshell height.

Table1a: Sum maryofstainedgiantscallopveligers foundoverthe samplingseasonof 1994(fro mrelease trial11.Totalin sample isanesnmate.whereindicatedbyan astensk(.)'based onstamedlarvaefoundinasubsam ple ofa tow.otherwise.the entire sample was searched. Average sizeis forveligers foundatall three (Release.MiddleandInlet)stations foreach semple date.SAanotapplicab le

Oalo SiteFound Number Tota l Range A,,,,,", Stan dard

Retrieved Found m (11m) Sizeuc m Devia tion

Sample

July 27 Release 1 m

, s

60-80 68.8 8.2

Middle 5m 8 8

July28 ReleaseI m 2 70" 70 70 0

August6 MiddleI m

,

8'" 105·160 125 18

Inlet5 m 2 34·

Augusl1 7 Release5 m 1 I 155 -165 158.8 3.5

InletIm

•

^68"

Inlet5m 3 SO"

August19 Release Im 1 1 145·160 149 8 lOA

Middl e5m

" ^rs

lnIetlm

,

102·

Inlet Sm

•

^153·

August23 Release_ReleaseMiddle 5 m₅Im_m

• • , • • _s

^{130-170 145.6 .7}

Inlet lm 3 51"

Inlet5m

,

102·

August.N Release1m I 17" 140-160 152.5 '.8

Middle5 m

,

_34·

lnIet lm

,

102·

lnlet Sm I 34"

Se ptember8 Release1m 1 17" 240 240 NA

Table Ib Summary ofstained giant scallopve ligers found overthe samplingseason of 1994 (fromrelease trial2).Total insample isan estimate.whereindicatedbyan asterisk(.),based onstainedlarvae foundin asubsampleofa low . otherwise.the entiresample was searched.Averagesizeis forveligersfoundatallthree (Rel ease.MiddleandInlet) stations foreachsample date.NA-notapplicable

Date SiteFound Number Tota l Range Average Standard

Retrieved Found tn (p m) Size (p m) Devta tion

Sample

August19 Release 1 m 2 2 85-100 92 106

August2J Releasc:5m 2

,

_{95-120 107 lOA}

ReleaseI m 2

,

September8 MiddJe 5m 1 17' 140- 175 158.7 16.5

Inlet5 m 3 3

October 4 Inletl m

,

'7' 270 270 NA

·,

Table2a: Summary of stainedgiant scallopvehgeesfound over the sampling season of I99S(fromrelease tria l I).Totalinsampleis anestimate.whereindicatedby an asterisk(-I.basedonstainedla rva efoundinasubsample ofalOW.otherwise,the entiresamplewassearched.Average size isforvelig ersfoundatall three (Release.MiddleandInlet ) statio ns foreachsamp ledate.NA-nOIapplicable

Da te Site Foun d Number Tota l Range Average Standard

Retrie...ed Found on (~ m) Sizeturn] Deviation

Sample

August12 Release '0 '0 70-90 797 63

Middl e 6 6

Inlet 2 2

August13 Middle 2 2 70-9'

n.,

94

Inlet 4

•

August 19 Inlet

, , _'0

80 NA

August25 Release

, ,

230 230 NA

August 26 Inlet I I

u s

^{185 NA}

49

Tablelb' Summary of stainedgian t scallopveligersfoundoverthe sampling seasonof 1995(fromreleasetrial 2).Tota l in sampleisanestimate.whereind ica ted by an asterisk (.).basedonstainedlarva e found

In.

subsamplc oratow,otherwi se.the ennre samplewassearched.Avera ge size isforveligersfound at allthree (Release. MiddleandInlet)stations for each sampledate.NA-notapplicable

Date Site Found Number Total lUnge Average Standard

Retrieved Found in _(~m' Size (f1m) Deviation

Sample

Augus 125 Release 1 1 75 75 NA

August 26 Release 1 1 85-90 87.5 35

August28 Middle 3 3 75-80 76.3 2.5

Inlet 1 1

September7 Release 1 1 80.95 88.3 7.6

Inlet 1 1

September21 Middle 1 1 140-1 55 150 8.7

Inlet 1 1