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EFFECTSOFPHOTOPERIOD AND LIGHTINTENSITYON THESURVIVAL, GROW THAND FEEDING BEHAVIOU R OF LARVALSTRIPEDWOLFFlS H

(Anarhichas lupus)

BY

Kelly J.Morcl©

ATHESIS SUBMITTED TOTHE SCHOOLOFGRA DUAT ESTUDIESTNPARTIA L FULFILMENTOF THE REQUIREMENTS FORTHEDEGREEOFMASTEROF

SCIENCE (AQUACULTU RE)

MEMORlALUNIVERSITYOF NEWFOUN DLAND

AUGUST2002

ST.JOHN'S NEWFOUr\ DLAND

Abstract

Thisthesi~investigated theimpact oftwo environmentalfactorson the performanceoflarval striped wolffish (Anarhichaslupus).Specifically,Idescribethe impact of photoperiodandlight intensityonthe growth,survival, andfeeding behaviour oflarvalwolffish.

In the photoperiod experiment,larval wolffishwere subjec ted tophotoperiods consistingof 12 hoursIightll2hours dark,18 hourslight/6 hourslight,or 24 hours continuouslight.Results showed thataphotoperiodof18U 6Dyielded thebest survival and growthafter 50 days.Providing24hourslight,acommon technique in larvicultu re, did not offer anyadvantagein terms of survivalor growthcompared to thel8L treatment.

Thehigher performance results seenfor the18Ltreatmentis attributed to thesimilarity in photoperiod ofthenaturalenvironmentforthe species.

Theinvestigation into the effects of lightintensity onthe survival and growth of larval wolffish compared intensities of 10, 40,160, 320, 750,and1200lux.Forallvalues tested, survival andgrowthincreased with increasinglightintensity.Alightintensityin the range of750lux-1200luxproduced survival rates ofapproximately92.0%byday 50.

Theeffects ofl ightintensity(320 lux, 750 lux, 1200 lux)on thefeedingand activityof larvae werealso investigated.Thefrequencyof feedingincreased with increasinglightintensity.Theimpactoflight intensitywas most significant duringdays 30-40,aperiod corresponding tothe switch fromendogenou sto exogenous feedingin larval wolffish.Duringthisperiod,thelarvalin thehighestlightintensity treatment

(1200 lux) had significantlygreater frequenciesoffeeding comparedtothelowestlight intensitytreatment (320 lux). By the endofth e study(day 50),there was nodifference observedbetweentreatmentsin terms of successfulorunsuccessfulforaging.

Fortheproductionofl arval wolffisha photoperiodofatleast18Lin conjunction with 1200lux is recommendedformaximum growthandsurvival uptoat least day50 post-hatch.

iii

Acknowledgements

Without doubtor hesitation,the firstperson I haveto thank for thecompletionof this thesisismy supervisor, Dr.JoeBrown.Throughoutthe years hehasprovidedme withsubtle,yetverypositive,support,guidance, and encouragement. More so,henever oncelosthis humour ordoubtedthecompletion of thisproject,despitethe multitude of delaysranging fromchildbirth, toajob,to malaria! Thank you Joe,Iwill beforever gratefulfor your patience.

Withequal and heartfelt gratitude,Ihaveto thank myfamily(especially Mom) for nevergiving upon mewith regards to seeingthisthesis completed.Myparentshave providedmewithcountless support(financial, encouragement, and evenbabysitting) and countlessreminders "t hat it would allbefinishedoneday".Thankyoufromthebottom of myheart.

To anyonewho providedfundingforthisresearch,graduatesupport, orbursaries forconference travel opportunities,(CCFI,graduatestudies, OSC,School ofGraduate Studies)Iam verythankful for all the support givento me.

Duringmyworkon wolffish,I had thepleasuretohaveworked witha dynamic group ofresearchers.To LauraHalfyard,Dena Wiseman,and Sherra (Tullock) Fam.

Thank youfor makingthis journeyofdiscoverya fun one.Thankyouas wellforall the sharedinformation,ideas,and mostimportantly, sharing inthedream thatthis species will onedaybea successdueto efforts suchas our own. Gratitude must alsobe extendedto John WatkinsandBarryHicksforinitiallypursuing wolffishforculture

iv

purposes,and forproviding the facilities and eggsllarvae whichallowedthisresearch to be conducted.Inaddition, Iwishto thankDr. JoanneMorgan (DFO),Dr. Sue Clearwater, and Dr. Jay Parsons for anyassistancethey providedtome duringthe writing ofthis thesis.

Appreciation mustalso beextended to the"workshopboys"attheOcean Sciences Centre (Danny,Dami an. Jimmy,Geny,andTeny) for all yourassistance,and humour.

We allknow thatwithout you gentlemen,most research conductedattheOSC would be less successful.Thankyou as well,to allmy lab mates(Donna ,Karen,Martha, Craig, Lisa) for any and all help they providedduring this thesis.

Last but not least,Iwishto thankDanny BoyceandPuvanendran(Puvey) for really teachingme the basics ofaquaculture.They maderesearchfunand pract ical. I am forever grateful10 them for what theyhavetaughtme.

I wouldalsolike 10 thank KeithRideout, for all hislo ve and supportand for helpingme with this work....whenever needed. The same extendsto Miranda Pryor, for herundying friendship,and encouragement.

I dedicatethis thesis to my daughterCatherine.Altho ughsheis the reasonthis thesiswasdelayed,more important ly,sheismy source ofinspiration, and the reason I want to strive to be better...hencethe completionof this thesisl!

TableOrCoe tea ts

Abstract .ii

Ackno wled gemen ts IV

TableofContents vi

List of Tables. Listof Figu res.

.. viii

.. xi

Chap ter1. General Introdu ction-Cu lturing New Spec ies 1

Chapter2.Technical Review ofWolffishCulture 5

2.1Overview 5

2.2Brood stock and Reprod uct ion 6

2.3 EggIncubation 10

2.4LarvalStage 12

2.5Juv eni le/On-Growing 16

2.6Concl us ion 19

Chap ter3.TheEffect ofPhotop eriod onLarvalWolffishGrowthand Survival 20

3.1Introduc tion 20

3.2Materi alsand Methods 23

3.2.1Egg Collection andLarvalSelection 23

3.2.2PhotoperiodProtocol 25

3.2.3MeasurementsandAnalys is 26

3.3Results 29

vi

3.4 Discussion .41 Chapter 4.The Effectof Light Intensityon Larval Wolffi shGrowthandSurvival .48

4.1Introduction....

4.2 Materialsand Methods...

... ... ....48

. .51

4.2.1GeneralMethodology for BothLight IntensityExperimen ts .51

4.2.2ExperimentI Methodology 51

4.2.3Experiment 2Methodology... .. .53

43Results 54

4.3.1Experimentl... .54

4.3.2 Experiment2 67

4.4 Discussion 79

Chapter5. TheEffectof Light Intensityon theLarvalWolffishFeedingBehaviour 86

5.1Introduction 86

5.2Materialsand Methods 90

53Results.... . 92

5.3.1 Modal Action Patterns-Frequency 92

5.3.2FeedingActivities-Frequency 101

5.3.3ActionPattern-Frequen cyand Duration 106

5.4 Discussion... ....113

Chapter 6.Conclusion 118

References 119

vii

Listof Tables

TableI:ResultsofTwoWay Analysisof Variance(ANOVA)onthe mean percent survival, mean wet weight,and mean standard length oflarval wolffish for

thephotoperiod experiment 32

Table 2:Results ofTukey' sStudentizedRangeTeston the meanpercent survival, meanwetweight,and meanstandard length oflarvalwolffish, for each sampledayseparately,for thephotoperiodexperiment... 33 Table3:Tukey'sgroupingsforthe mean percent survival,mean wetweight,and

mean standardlength for thephotoperiodexperiment.. : 34 Table4:Results of Two Way AnalysisofVariance (ANOVA)on the specific

growthrates(% wetweight!day,%standard length! day)oflarval

wolffishforthephotoperiodexperiment .. .38

Table5: ResultsofTuke y'sStudentized RangeTeston thespecific growth rates (%wet weight/day,%standardlength!day) oflarval wclffish,foreach sampling dayseparately,forthe photoperiodexperiment. 39 Table 6:Tukey'sgroupingsforthe specificgrowth rates(%wetweight!day,%

standardlength!day)for thephotoperiod experiment .40 Table7:ResultsofTwoWay Analysisof Variance (ANOVA) on the meanpercent

survival,mean wetweight,and mean standardlength oflarval wolffish

for Light IntensityExperimentI 56

Table 8:Resultsof Tukey'sStudentized Range Test on themean percentsurvival, meanwet weight,and mean standardlength oflarvalwolffish, foreach

sample day for Light Intensity ExperimentI .57

Table9:Tukey'sgroupingsfor themean percentsurvival,mean wetweight,and mean standardlengthfor Light IntensityExperiment I .58 Table 10: ResultsofTwo WayAnalysisof Variance(ANOVA) onthespecific

growthrates ('Yowetweight!day,%standard length!day) oflarval wolffish forLight Intensity ExperimentI... .. 64 TableII :Results ofTukey'sStudentized RangeTest on the specific growth

rates(%wet weight!day,%standardlength!day) oflarval wolffish, viii

for eachsampleday forLightIntensityExperimentI... . 65 Table 12:Tukey'sgroupings for thespecific growth rates(%wetweigh t!day,

%standardlength! day)forLight Intensity Experiment I (significan ce level p<O.05,means with thesameletter are notsignificantly different,

p<O.05) 66

Table 13:ResultsofTwo Way Analysisof Variance(ANQVA) on the mean percentsurvival,meanwetweight,and meanstandard lengthof larval wolffishforLight IntensityExperiment 2 69 Table14:Results ofTukey'sStudentizedRangeTest on themean percent survival,

mean wet weight,and mean standard length oflarval wolffishforeach

sample dayfor Light Intensi tyExperiment2 70

Table15:Tukey'sgroupingsforthe mean percent survival, mean wet weight and meanstandard lengthforLightIntensityExperiment2 71 Table16:Resultsof Two WayAnalysisof Variance (ANOVA)on thespecific

growth rates(%wet weight!day,%standard length'day) of larval

wolffish forLight Intensity Experiment2 76

Table 17:ResultsofTukey'sStudentizedRangeTest on the specific growth rates(%wet weight/ day,%standardlength' day)oflarvalwolffish,

foreach sampledayforLightIntensity Experiment2 77

Table 18:Tukey 's gro upings for thespecificgrowthrates(%wet weight!day,

%standard lengt h!day)for Light Intc:nsityExperiment2 78 Table19:OperationalDescriptions ofthe Modal ActionPatterns (MAPs) for

Striped Wolffish Larvae 91

Table20: OperationalDefmitionof theBehaviouralActionPattern for

StripedWolffish Larvae 91

Table21:Results ofTwo WayAnalysisofVariance (ANOVA) on the frequency (number of occurrencesper minute)of the MAPs(modal actionpatterns:

(orient-fixate,lunge-bit e,captures,miss)forlarval wolfishfeedingunder

varying lightintensities 94

ix

Table22: Results ofTukey'sStudentizedRangeTeston thefrequency (number ofoccurrencesper minute)of the MAPs(orient-fixat e,lunge-bite, captures,miss)foreachobservationdayfor larvalwolffish feeding

under varying light intensities. . 95

Table 23:Tukey'sgroupingsfor the meanfrequencyofthe MAPs[orient-fixate , lunge-bite,captures,miss)forlarvalwolffish feeding under varying

light intensities... ...96

Table 24:ResultsofTwo Way Analysis ofVariance (ANOV A)onthe frequency (number of occurrences perminute)ofthefeeding activities(foraging activity,successfulforaging,unsuccessfulforaging,feeding effort) forlarval wolffishfeedingundervarying light intensities.... . 107 Table 25:ResultsofT ukey'sStudentizedRangeTeston the frequency

(numberof occurrencesperminute)ofthe feedingactivities (foraging activity,successfulforaging,unsucc essfulforaging,feedingeffort) for each observationday for larvalwolffish feedingunder varyinglight

inteneities.; . 108

Table26:Tukey' sgroupingsfor the mean frequencyof the feeding activities (foraging,successful foraging,unsuccessful foraging,feeding effort)for larvalwolffish feedingunder varying light lnrensities.; ...109 Table27:ResultsofTwoWayAnalysisof Variance(ANOVA)on the frequency

(number ofoccurrence sper minute)and theduration (seconds)of swimm ing activityfor larval wolffishfeedingunder varying light

intensities 111

Table28:ResultsofTukey's StudentizedRangeTeston the frequency(number occurren cesperminute) and duration(seconds) of swimmingactivity foreach observation day forlarval wolffish feedingunder varying

light intensities I 11

Table 29: Tukey'sgroupings forthemean frequency(occurrencesperminute) and duration (seconds)of swimmingactivityforlarval wolffish

feedingundervarying light intensities 112

List ofFigure s

Figure I:Geographiclocationsof theegg mass collectionsite(A) andresearch facilities(B and C) for experimentalprotocols 24 Figure 2:Mean percent survival(%)oflarval wclffishovertime (days)for

photoperiod experiment.... .. 30

Figure 3:Mean wet weight (g) oflarvalwolffish overtime (days) for

photoperiod experiment... . 31

Figure 4:Mean standard length (mm)of larval wolffish over time (days) for

photoperiodexperiment 36

Figure5:Themean wetweight specificgrowthrate (fig.a) and the mean standardlength specific growth rate(fig. b) over time(days) forlarval

for the photoperiodexperiment. .37

Figure 6:Mean percent survival(%)oflarvalwolffishovertime (days) for

Light Intensity Experiment1 55

Figure7:Mean wetweight (g) oflarval wolffishover time(days) for Light

IntensityExperimentI 60

Figure8:Mean standard length (mm)ofiarvaI wolffishovertime (days)for

LightIntensity ExperimentI 62

Figure 9: Mean wet weight specificgrowthrate(fig. a) andmean standard length specificgrowthrate(fig. b)ofIarval wolffishovertime (days)

for Light Intensity Experiment1 63

Figure 10:Meanpercent survival ('Yo) ofIarvalwolffishover time (days) for

LightIntensity Experiment 2. .. 68

Figure 11:Mean wet weight(g) ofiarvaI wolffish over time (days) for Light

Intensity Experiment2 73

Figure12:Meanstandardlength (mm) oflarval wolffishover time (days)

for LightIntensity Experiment 2... . 74

xi

Figure13:Mean wet weightspecificgrowthrate(fig.a)andthe mean standard length specificgrowthrate (fig.b) ofl arvalwolffish over time (days) for LightIntensityExperiment2... . 75 Figure14:The frequency(numberof occurrences per minute) of orient/fixate

over time (days) for larvalwolffish feeding undervaryinglight

intensities.; . 93

Figure 15:Thefrequency (numberof occurrencesperminute) oflunge-bite over time(days)for larvalwolffishfeedingunder varying

lightintensities... . 98

Figure16:The frequency (number of occurrences per minute) ofcapture over time(days)for thelarval wolffishfeeding undervarying

lightintensities.... 99

Figure17:The frequency (numberofoccurrences per minute)ofmiss over time(days)forlarval wolffishfeedingunder varying lightintensities 100 Figure 18:The frequency(numberofoccurrencesper minute) offoragingactivity

over time (days)forlarvalwolffishfeedingundervarying light

intensities 102

Figure19:Themean percent(%)frequency ofsuccessful foraging over time (days)for larval wolffish feeding under varying light intensities 103 Figure20:Themeanpercent(%)frequencyofunsuccessfulforagingover

time(days)forthe larval wolffish feedingundervarying light

inrensiues.... . 104

Figure21:Themean percent(%)frequencyof feedingeffort over time(days) forlarvalwolffishfeedingundervarying lightintensities 105 Figure22:Theduration(seconds;fig.a)and frequency(occurrencesperminute;

fig.b)of swimmingactivity over time (days) for larval wolffishfeeding

under varyinglight intensities . 110

xii

Chapte rI: Genera l lnlro duclion-Cullu rin g New Species

Market value,cost of production,and the quality of fannedfish have been identified as decisivefactors in assessinganew species' potential for aquaculture development (filseth,1990;Tilsethel al.,1992).In Atlantic Canada biological suitabilitymustbe consid ered an equally important factoras anycandidatespecies investigated for productionmust be abletotoleratesub-zero«O"C)temperatures for sign ificant portions of the year (Brownetal.,1992; 1995a). Factorsdealingwithpolitics, economic s,infrastructure,pollutionand diseaseare also crucial to new species' developmentand productionsuccess (Hem pel,1993).Consequently,the developmentof a newspecieswill onlybelucrativeif the animal'sbiologyissuited to thehabitatwhere it willbe grown and if cooperativegovernment/research,andindustry alliancesexists.

In general,there isusually an incentiveor motivatingfactor drivingthe developmentor expansion ofthe aquacultureindustry.In1990,worldaquaculture production reachedapproximately15 million metrictonnes(mt),by1998 thisexpanded to approximately 30 .8 million metrictonnes(FAO statistics,2001). By theyear 2025,it isestimatedthat62.4millionmetrictonnes(mt) of aquaculture: productsareexpected to beproduced.Since theworldcapturefisheries will remainstableat about100 million metrictonnes(mt),increasedmarket demand will have tobefilled through aquacultu re production(Hempel,199 3).

Traditionally,theincen tiveforthe aquaculture ind ustryhas notbeentosupply or meet theworlddemand forseafoodprodu cts.Instead,thenorthernandtemperateregions

havefocussed theirattentiononhigh value finfish products.Althoughthe Northern European aquacultureindustry has beendominatedbysalmonids,severalfactors have prompted research into culturing alternatecold watermarine species.Factorssuch asthe fluctuationsin the Atlantic salmon(Salmo salar) marketcaused by excessive world supplyand decreasingvalue in the internationalmarket(Strandet al.,1995)and decreasedlandings in the traditionalcommercialfisherieshaveserved as a strongimpetus forexpandingcold waterproduction intootherspecies.Similar ly,the imposed moratorium onthe eastcoast Canadian groundfish fishery,whichresultedinadrastic shortageofonce readily availabl efish,alsoprompted extensiv einvestigati onsintocold wateraquaculture.

In an effort to deal with the situations mentioned above,theNorthernEuropean andAtlantic Canadian aquaculture industriesidentified severalfinfish specie s,namely halibut(Hippogl ossushippoglossus),haddock(Melanogramm us aeglefi nus ),cod(Gadus morhua),and two speciesofAtlantic wolffish,the striped wolffish(Anarhichas lupus) and thespotted wolfish(Anarhichasminor)as potentialcandidatesfor coldwater development{Tilseth,1990;Brownet01.•1995a; Stefanussenet al.,1993).Since all these speciesoccurnaturallyin the cold waters around AtlanticCanada and Northern Europe,theirsuitab ilitytothe existingenvirorunentlhabitatwas notconsideredan impedimentto production.

Researchinto allaspects of aquacultureproductionstages (broodstock, egg, larval,juvenile/grew-out)is consideredcrucialfor the successof theindustry . Although

literature may be available for each productionstage, caution shouldbeexercised when drawin gdirectcomparisons between wild and cultured fish as factors dealing with density,stress,feedingand environmentalconditionsare often inconsistent(Blaxter, I975a) andtherefore nor directlyapplicable.When comparing wild to rearedfish, Blaxter(1975a)noted that variabilityinbehaviour(aggression,feeding),morphology (abnonnalfins and heads,pigmentation),physiology (egg size,qualityand fecundity) and bioch emistry (fat, water,and ash contentin the body)does occur despite thefact that growth rates and conditionfactors of culturedfish exceed those of the wildpopulation . Whereinconsistenciesoccur,it is eviden tthat problemspecificresearchisessential for resolvingproduction impediments.

Norway'ssuccess in culturingalternatecoldwaterspecies is dueinpart to the strong cooperativeeffortamong scientists,governmentandindustry.Canadian industry has takentheleading roleintheselectionof newspeciesfor cultureandhas fostered relationships withscientistsfromprovincialandfederal governmentsas wellas universities inorder to develop research activities.Scientists fromboth countriesagree whenconsidering a new speciesforproduction,research anddevelopment must occur at eachstageof productionin order for producers to incorporatescienceinto the rearing protocols.

Insummary,stripedwolffishis consideredanidealspec iesfor aquaculture production in the coldwatersof Newfoundlanddue to its suitabilitytoa coldwater environm ent.However,littleresearchhas been conductedondetermining the specific

environmentalrequirements for thevarious developmentstages ofthis species. In particular.havingenvironmental protocolswhich are easilytransferred intoproduction protocolsremains an obstacle.Therole oflight (photoperiodand light intensity)in the cultureoftbis species is one keyresearch area which remainslargelyunaddressed .Based upon thislack of informatio n, thefocus ofmythesisis 10 determinethephotoperiod and lightintensityrequirement s of thelarval stage andtodetenninethe effect oflight intensityonthefeeding behaviourofiarvaJwolffish.In thefollo win g chapteratechnical review, includingaccomplishme ntsand obstacles to culturingthis specieswill be presented foreachstageofprod uction.

Chapter Two: Technical Review ofWolffisbCulture

z.rOvervtew

Theconcept ofdeveloping wolffish asanaquac ulturespecies originated in Norway and Russiaduring the1980's asfluctuationsin thesalmon aquacultureindustry createdgreate r initiativestoculture alternativespecies.Todate,the vast majorityof researc hand developmentforthis species occursprimarilyinNorway but research interest hasalso spread to Scotland andAtlanticCanada.

Wolffish aremembers ofthe familyAnarhichadidae,with atleast threespecies Jivingoff Canada'sAtlanticcoast (ScottandScott,1988).Thespecies includethe striped (Anarhichaslupus).spotted(A.minor) and thenorthern(A. denticulatus)wolffish.

Altho ugh both the spotted and stripedspecies are considered aquaculture candidates,the spotted wolffish hasbecometheprimaryfocusofaquaculture research.The solitary and reclusivehabitofthefishin combination with lowcommercial landings (usuallyreported asaby-catch) createunpredictablemarketavailabili ty,asituation conduciveto aquaculture development .Furthermore.thefilletscfwolffish areofexcellent quality.

comprised offinn, whiteflesh.Filletproductscan be smoked.pickled .ordriedand even theliver.bileand roe can beutilized .The skincanbetanned intoafine leather (Butt.

1993;Moksness and Pavlov.1996).and antifreezeproteinsin theblood canbeextracted and utilizedin thebiotechnologyindustry,medical.andfood indus tries(Wiseman.1997;

Brown,1998).Consequently. the profitability associated with total utilization of the animal highlight sthe culturing potentialfor thisspecies.

The natural ecologyofwolffish, including habitat preferences(Baruskov, 1959;

BeeseandKandler,1969;Albikovskaya,1982a;Kinget aI.,1989;Pavlovand Novikov, 1993),distribution andmigrations(Powles, 1967;Jonsson,1982;Templeman,1984a;

Keatset aI.,1986a;Ortovaet al.,1990),morp hology(Barsukov,1959;Jonsson, 1982;

Templeman, 1984b;Scott andScott,1988) and feeding (Albikovs kaya, 1982b,1983;

Templeman, 1985;Keats etaI.,1986b)have been previously studied.Forthepurposesof this chapter,a review ofthe biologyand ecologyofthe animal will notbepresented.

Instead, an overviewof each stage of aquacultureproduction(reproduction,egg development,larval, and juvenileand on-growing)willbepresented.A summationof the scientificandindustrialbreakthroughswhichhavepermi tted the culturingof this species,as wellas some of the obstacleswhichstili remain for full scaleproduction will alsobediscussed.

:Z.:ZBroodstock and Reproduction

Establishinga wolffishbroodstock whichcouldproduce eggs andspenn, and subsequently larvae,on a consistent andreliablebasishas plaguedaquaculturists since first efforts were madeto culturewolffish.Unlike manyother fishspecies,wolffish (in particularfemalewolffish)donot spawnreadilyincaptivity.Infact,the only docum entedcases of naturalspawningin captivitywerereport ed for fishmaintained ina rearing facilityfor many years(Ringeet al.,1987;RingeandLorentsen,1987).To dale, artificialreproductionby insemination is theprimary techniq ueused to obtainand fertilizegametes.

The first published report for successfully fertilizing wolffisheggs under artificial condi tions and successfull yraisingthe larva, was by PavlovandNovikov (1986).In additionto success fulfertilization,this experiment provided someinitialinformationon spermphysiology.In1991,asecond successfu lartificialspawning and fertilization from wild-caught broodstock was reported (pavlov and Radzkihovskaya,1991).Duringthis time,researchers also started to observe thereproductivebehaviourofwolffish.

Kvalsund (1990) was oneof the first to suggest thatwolffi sh was aninterna l fertilizerbased upon the followingobservations:I)males produced a verysmall volume ofmilt (approximately1.2ml);2) thetime during whicheggs are extruded (15-20 minutes) is inadequatefor fertilizationofeggs withsuch asmall quantityof sperm; 3) the urogenital papilla whichdevelopsin males mayfunction as a copulatoryorgan;4)there is periodicclose contactofthe femaleand maleprior to egg ovulation.Observa tionson the spawningbehaviourofwolffishbyJohannessonet al.(1993)also supportedthe hypothesis of internalfertilization.

AdditionalresearchconductedbyPavlov (1994a; 1994b) and by Pavlovand Moksness (1994a)further confirmedthatwolffish were indeed internal fertilizers.In water, the ability of eggs tobefertilized decreased,but withoutwater,andinovarian fluid,the capacityfor fertilizationincreased to approximate lysix hours.A contactperiod ofat least two hourswas determined necessary to ensurehigh fertilizationsuccess (90- 95%).Ithas alsobeen determinedthat atemperature ofO"Callowed sperm to remain fertileforup to tenhours.After thistime,sperm viabilitydecreased.

Subsequent research conductedbyPavlovandMoksness(1994b;1995;1996a)

resulted in rapid improvementsand refinementsinartificialinsemination,and yielded a tremendousamountofinfonnationonegg and spenn quality.Theseadvancementswere quickly followedby a repeat maturation and spawningof captivebroodstock(Pavlovand Moksness,19%b)andanunders tandi ngofthe bio logica l,physiologi cal,and envirorunentalfactors which affect the qualityof gametes andthe successof artificia l reproductivetechnology(Pavlovand Moksness,1994a;1996a; 1996b; Pavlovet aI., 199 7).

As aresultof this researchtwo methods of artificialfertilizat ion(internaland external)were developed(Pavlov, 1994 a;1996a;I996b;PavlovetaI.,1997).Interna l fertiliza tionis accomplis hedby introducingmiltinto theoviduct of the female.

Approximately10·25 mlof sperm solution is injectedwitha syringeintothe genital openinginto the middleof the ovary.Afterinseminatio n,femalesare kept inhol ding tanks for fourto six hours,after which theyare strippedof their eggs. A timeframe of fourto six hoursis used because after sixto eighthou rs,the femalereleases her eggs into the waterand the eggsadheretogether.

Externalfert ilization consistsof stripping ripe femalesand males and mixingthe gametes(eggs and ovarianfluid withmilt). Thegametes areplaced in a cylinderwith the sperm dilutant (Ringer'ssolution)and the cylinderisinvertedup to 20 timesduringthe first hour (fewerinversionsthereafter).Gametesaremaintained at a temperatureof -4.7"Cforfourtosix hours(a sufficien tenough timeto ensure approx imately100"10 fertilization).Fertilizedeggs arethen removed and distributedover thebottomof special trays toprevent sticking .Nodifferencesinthequality ofeggs weredeterminedfor either

the internal or external fertilizationtechniques.To ensure good quality of gametesand successfulhigh fertilization rates:I) the concentrationof sperm shouldexceed I.Oxl0⁶ perml;2)onefemaleshouldhe fertilized with the spermof severalmales; 3) use of the cylindrica lchamberforinsemination enablesthe concentrationof sperm tobe maximized;4) Ringer'ssolutionis recommended as a dilutantinsteadof seawater becauseit enablesspermto live longerin the ovarian fluidandprevents egg swelling;4) eggs and spermshouldbein contactforatleast2hours to ensurehigh fertilization success ;6)activa tionand fertilizationof eggs byspermatozoa andtheinitial development of eggs shouldoccur in the ovarian fluid,however, eggs shouldbereleased into seawater before the beginningof cleavageto ensure subsequentnormal development;7) to prevent fertilizedeggs from stickingtogetherinclumps,eggs should be placedin static seawater (five to six hours) andthen separatingindividually onaspecialtray beforeflushing the remainin g ovarian fluidfrom the egg surface (Pav lov,1994b;PavlovandMoksness, 1994a;Pavlovand Moksness, 1996a;I996b;Pavlovet aI.,1997).

Theimportan ce of environmentalinfluenceson thesuccessfulspawningand fertilizationofwolffishwas presented by PavlovandMoksness(1994301996a) and Pavlovet al. (1997).At temperatures below1000C egg fertility is increasedandis accompaniedby fewerresorbed eggs.Normalegg ripening infema lesrequiresthat broodstockbekept at a temperat urebelow1000efor atleastfourmonthsbefore ovulation.

Forspermatozoa,whichare activated inseminal plasma, sperm are abletoremain motile forup to tendays, provided they are kept at temperatur es closeto O°C.Motility,and viability decrease significantlyasthe temperatureincreases .

Morerecentresearchon temperature effectson broodstock haveindicatedthatthe temperatures experiencedbythebroodstockduringbreedingseason affectsboththefinal maturation,thetiming of ovulation,and egg quality(Tveiten and Johnsen, 1999;Tveiten et aI.,1999;2001).

Photoperiodhad no effecton spawningmales (Pavlovet al.,1997). Photoperiod affected femalematurationtime and resulted in a protractedperiod ofeggmaturation, therebyconfirmingan endogenousrhythmin the controlof reproduction(Pavlovand Mcksness,1994a).Thereisalso evidence that the intensityof light mayaffect spawning andegg quality.In a technicalreport byMoksnessandPavlov(1996),theystated that stronglight intensitiescausedprematurereleaseof eggs oflower quality. Unfortunately, noexperimentswereconducted on theeffectsof lightintensity on spawning andgamete quality.

Other variablessuch as rearing femalebroodstoekin the presenceof males,and dietquality andcomposition fed to the broodstockare believedto be importantfactors in obtaininghigh quality viable eggs year-round(pavlovand Moksness,1994a).

2.3EggIncubation

For wolffish,theperiodof eggincubationfrom fertilizationtohatch,is approximately1000degree days.Eggmasseswhich havebeenincubated under ambient watertemperaturesexperiencednormal egghatchingand subsequentlarvaldevelopment, despitenegativeambient water temperatures.Pavlovand Moksness(1995)attempted to determinetheincubationtemperatureswhich could shorten embryonicdevelopment

10

while ensuring normal ontogeny.The resultsindicated that successful incubation ofeggs was possibleat temperaturesbetween5-11°Cbut thehighestincubationoccurred at lower temperatures(between 5-7"C).A temperatureof 9°Cseems tobethe upper limitfor normal development.Beyond this temperature,many fin rays(particularlypre-caudal parts of dorsal and anal fins) were absent.

The results suggesta temperatureregimefor minimizing wolffish egg incubation time could be:incubateat 7"C from eggfertilizationto the morula stage(2 days);

incubateat 11°Cfrom this stage to 50%vascularizationof theyolk sac (30 days);

incubateatT'Cfrom the latter stageto formationofrays and caudal and pectoral fins(57 days);HOCfrom the latter stage to hatching(104 days).

Pavlovand Moksness(1993) stated that bacterialiving on the surface ofwolffish eggs maycause low gas exchange,gradualdestructionof egg membranes,premature hatchingof embryos,and high mortality.Theyrecommendedtreatingeggs with a gluteraldehyde bath.Currentpractice{Falk-Petersenetal., 1999)is to use a gluteraldehydetreatmentof ISOparts per million twicea month to deal with the growth of microorganismsoneggs. The authors state that premature hatchinghas been a problem in individualegg bathes althoughthey do not fullyunderstandthereasons for the premature hatching.

Breakingthe egg massesinto smaller portions,and incubating thesepiecesin Heath TrayTIllunits (standard egg incubationequipment usedfor salmonids)was a good method for egg incubationin Newfoundland.Theunits consisted of severaltrays,and hadahighwater flowwhich cascadeddown through the trays.High water flow,along

II

withan airhoseforeachtray, andregularcleaning of the systems resultedin high proportionsof nonnallarvae,with littleeggmortality,andlittlebacteria linfectio n (Halfyard,1995,pees. comm.;Watkins,1995pers.comm.;Wiseman, 199 7).

2.4LarvalStage

At hatching,wolffishlarvae areapproximatel y20mm long ,they have large pigmentedeyes,pigmented skin, developedfins,approximately50 teeth,andlittletono yolksac (Barsukov,1959;Moksnessand Pavlov,1996). This contrasts withthe majority of marinefinfishlarvae, whichgenerally hatchat 2· 5rom in length,withlittleorno pigmentation,poorlydevelopedsensory systems (e.g.pooreyesand vision),andhavea hugeyolk sac.Mostof thesefishlarvaelive off their huge yolk sacuntil a functional mouth and digestive system developsand thelarvae areableto switchfrom endogenou s to exogeno usfeeding.Generally theselarvae,undergoan energeticall ydemanding metamorphosis,and survival beyond this stage is verylow (Blaxter,1981).Larval wolffish however,arecapableof eating within daysafter hatchingand donot go through adistinctiveand stressfulmetamorphosis.

Ingenera l, marine larvaeare usuallystan-fed on small, wildorcultured plankton (e.g.,roufers),and are successive lyweaned ontolarger,culturedzooplankton(e.g., Anemia)beforeweaningonto commercial pellets.Theadvanced"juvenile-like" stage of larval wolffish,aswe ll as a reviewofthe dietsfromwild-caught larvae{Falk-Petersen et al.,1990 ) suggested larvae arecapableofeating largepreyitemssoon afterhatching, therebyeliminating theneed for smaller live-fooditems.

12

Preliminary research on first-feeding in larval wolffish indicated that high survival waspossibleiflarv ae were fed a dietof wild zooplankton and that other diets such as Anemia.fish products. or commercial pellets compromised survival during this period (Ringe et al.,1987).Ringe et al.(1987) found newlyhatched wolffish fed diets of wild zooplankton had a survival rate of97%,at day 120 post-hatch, compared to larvae fed on a preparedcodroe diet which had 0%survival,at day 50 post-hatch. Diets of dry pellets (varying moisture levels) alone,orin combination with Anemia,failed to achieve the survival rates obtained by Ringe et at. (1987).Inthe initial study (Moksness etal.; 1989) higher survival rateswere obtained when larvae were fed Artemia in combination with the dry pelletsversusdry pelletsalone,implying Anemia provided some additional benefit to first-feeding larvaethandry pellets alone could not provide.

Additional studies conducted by Blanchard (1994) and Wiseman (1997) using only Artemia as the diet for first-feedingwolffish larvae,revealed that regardless of prey density,survival rates were much lower for wolffish fed Artemia than for wolffish fed wild zooplankton (Ringe et al.;1987).Wiseman (1997) conducted experiments where larvae were fed varying densities of enriched Anemia (100 perIL, 900 preylL) in combination witha commercialdietfeedtoexcess. At the end of the study (9 weeks post-hatch),larvae fed prey at high densities (9001L anddryfood) had a final survivalof 94.3%. Results from the behavioural analysis revealedthat larvae fed significantly more on Anemiaduring thecriticalperiod(up to 5 weeks post hatch) (Wiseman,1997).

However, byweeks6-7 thelarvae fedequally on Anemia and dry feed and by week 7, the larva preferred dry food,having weaned themselves offAnemia(Brown et al.,1997).

13

The wolffish's ability to "self-wean"in the presence of suitable diets, can be considered a benefit withrespect to potential commercialproduction.

More recent studies conducted by Hendry and Halfyard (1998) comparedthe growth and survivalrates of larvae fed three different diets.Results indicate thesurvival of the larvae was greatest withenrichedAnemia/drydiet(>90%), followed by unenriched Anemia/drydiet (>80%),and finallythe dry pellet only(76%).Theseresults support the theory that the presenceoflive foodduring the first feeding stage promotestheinstinctive predatoryresponse whiletheAnemia(particularly,the enriched fonn) offers some nutritional contribution10the larvae (Brownet ai.,1995b;Hendry andHalfyard,1998).

In Norway, a studyconductedbyStrandet al.(1995) demonstratedit was possible to start-feed larvae and obtain high survivalby using only a commercialdiet.In thisreport,larvae were fed two commercial diets (diet A: floating pellet, diet B:sinking pellet) for 60 days post-hatching.Attheend ofthe study, bothgrowthand survivalwere higher among larvae fed dietA (finalsurvival82%).The authors postulated that a floatingdietstimulateda higher start-feedingincidencedue to larvae more readily attackingthe floating diet.Start-feeding larval wolffishsolely on commercialpelletsis thepreferred and most commonlyused techniquein Norway.

One constantnoted among aJlthe feeding studies was the timeframe during which significantmortalities occurred (Moksnesset al. 1989(days 20-49); Blanchard, 1994 (days 27-36);Strandetal., 1995 (days 22-40);Wiseman, 1997(days 21·35).Day 20-40 post-hatch therefore representa critical periodfor larval wolffishgrowth and survival. This time period corresponds tothe total absorptionof the yolksac and a switch

14

from endogenousto exogenousfeeding. High mortalitiesobservedat this timewere attributedto a failureof larvae to initiatefeeding and anunsuccessful switch to the exogenousfood provided (Strand et al., 1995;Wiseman,1997).

In additionto ensuring optimaldietaryrequirements. factorssuch as optimal environmentalandrearing conditionsalsoaffectthe growthand survivalof a species.

For example,temperature isknown to be an importantvariable in rearinglarval fish as it canaffect incubationtime, size at hatch,yolkutilizationefficiency,growth,feedingrates, time to metamorphosis,behaviour,swimmingspeed,digestion rate, gut evacuation,and metabolicdemand (Blaxter,1988). For mostspecies, growthratetendstoincrease with increasing temperaturesuntil theoptimalgrowth rate isreached and beyond this "optimal temperature" growth rate decreases (Jobling,1983).

Variousstudies haveindicated thatwolffish are capable of toleratinga widerange of watertemperatures(1.0-13.7'C) during rearing (Stefanussen etal.,1993;Ringe et al., 1987;and Moksness,1994). A studybyMoksness(1994)onthe growth ratesof striped andspotted wolffishrecommendedatemperature ofbetween7-9"C.Itwas recommended that rearingtemperatures not exceed

urc,

especiallyforthe spottedwolffish,which appeartohavea lower optimaltemperature ofthetwo species.

Temperaturestudiesconducted by Wiseman (1997)onlarval wolffishshowed thatforthe first 6 weeks post hatch,temperatures shouldbebetween4· goC.Temperature affected survivaloflarval wolffishup to6 weeks post-hatch butnot after this period.

Theseresultsindicatedthatfishreared inlower than optimal rearing temperatures may havebeen unsuccessfulintheirtransitionfrom endogenous to exogenous feeding and the

is

inadequaterearing temperatures prevented the larvae from feeding at a level thatmet their metabolic demands.

2.5Juvenil e/On-Growing

Forproductionpurposes. oneof the first studies on the feedingbehaviourof juvenilewolffish was conductedby Ortovaetal. (1989).This studyrevealedthatthe frequency of foodingestiondepends on watertemperatureswherejuvenilesheld at high temperatures(6-10"C)feed daily.However,atlow temperatures«()'2^GC)the intervals between ingestion of foodareincreased10 2-3days.

Dietcompositionand theprocessing techniqueusedto formulatethe commercial dietsarecrucialtomaximizingjuvenileproduction.Accordingto Stefanussenet al, (1993)inorderto achieve a high growth rate inwolffish, thefeedcomposition should have a highproteinconcentration(>5Q&1o)and a lowcarbohydratecontent(<20%).High fat contentin the feedresults inhigher fat contentin the filletandanenlargedliver.

whereas thewater contentinthefeed didnOIaffec tthe growthrate.Moksnessetal, (1995)compared moist (squid diets), regular fish mealdiets,and lowtemperature(LT) processeddrypellets and foundthere were nodifferencesingrowth,feed conversion, proteinefficiencyrate,orproductive proteinvaluesbetweenthemoist and LT diets.

However.theLThadbetter resultsinall parameterswhencomparedtotheregulardiet.

Withrespect10rations,Ortovaet al. (1989)found that foradult wolffish dailyfeeding rations weremaximalat9-1O"Cand thatO- I"Cwasclosetothe critical temperature for feeding.

16

higher densities by using re-sorting of fish to preventstarvationand mortalities ofthe smallestfish.Moksnessand Pavlov (1996)reported that yearlingsand adults have been successfully maintain ed without mortalities at stockingdensities of 100 kglm²•Fam (1997)determined50gIL to be sufficient,whileincreased densities lowered feed conversion ratios and lower densitiesaffected the proteinefficiency ratio.

Determiningthe optimal environmentalrequirements is essential foranystage of prod uction. For wolffish juveniles,little isknown about their light requirements.Pavlov (1995)determinedthainewly-hatchedlarvae reactpositivelytolight but duringthe course of ontogenesisthey become increasinglydemersal andthe role of light and vision inthe search for food decreases.Itwas alsonotedthatin winter and autumn, juveniles periodicaJlydiscontinue feedingandthat growthratesdecreased implyinga seasonal rhythmpossibly linked tothe decreasingphotoperiod at thistime.Moksness andPavlov (1996)noted thatthe positivereactionof juvenilestolight disappearsinfishgreater than 1 g in weight andlongerthan 50-60mm. They alsosuggested that continuouslightwas important duringthe pelagicphaseofwolffishandthatthe strongreactionsoflarvaeand juveniles tolight could beused as a management1001for their behavio ur.

Feedingfrequency studiesforjuvenilewolffishdeterminedthatfeedingrates varied from everyday forsmallerwolffish 10 every other day for larger fish(Stemarrson and Moksness,1996).Studies conductedby Ortovaet at.(1989)and Fam (1997) confirmthaifeeding of large juveni les need onlyoccur every second day.

\8

2.6 Conclusion

In summary.muchof therearing technologyfor this species has been determined.

The role of light (photoperiodandlight intensity) consistentlyremains an area which needs to beaddressed for each stage of production.Inthe followingchapterstheroleof light onlarvalproduction is examined.Theobjectives of mystudyare:1) to detennine the role of photoperiod on growth and survival oflarval wolffish;2) to determine therole oflight intensity on the growth, survival.and feeding behaviour oflarval wolffish; and3) torecommend light protocols which can be incorporated intohatcheryculture technology.

19

Chapter 3: TheEffect ofPbotoperiod on Larv alWolffisb Growtb and Survival

3.1Introduction

Wolffish have been identifiedasastrong candidatefor cold wateraquaculturedue inpart to an inconsistentmarketsupplyand its potential biologicalsuitabi lityto cold- waterrearing.Relativeease inlarval and juvenile production, good qualityflesh(fillet), and small landedquantities from thecommercialfisheries havehighlighted the wolffish's appealas an aquaculturecandidate. Research on the cultureof striped (Anarhichaslupus) and spottedwolffish (Anarhichasminor)ispresentlyunderwayin Newfoundland, Quebec,Norway,and Scotland.

Manyof the culture techniques dealingwithreproduction (pavlov,1994a,b;

PavlovandMoksness,1994a;1994b;1996a;1996b),eggincubation(pavlovand Moksness,1993;1994a;1995). temperature requirements (Pavlovand Moksness, 1994a,b; 1995;I996a,b;andWiseman 1997 ),dietand feedingprotocols (Moksness et al.,1989;Ortovaet01.,1989;Stefanussenet al.,1993;Moksness, 1994;Pavlov and Moksness,1994a;Moksnesset01.,1995; Pavlov, 1995;Strandetal.,1995;Wiseman, 1997),stocking densities (pavlov,1995), andgrowthrates (Stefanussen et aI.,1993;

Moksness,1994;Moksnesset01.,1995;Pavlov,1995),have beendetermined for this species.

Despite the tremendousamountofresearch conductedon wolffish,the roleof photoperiodin wolffish cultureremains largelyunknown. For broodstock, it isknown

20

thatunder natural conditions spawn ingisapparently synchronized bythe decreasein day length duringthesummer and autumn months(Moksnessand Pavlov,1996).During laboratorytrials Moksnessand Pavlov(1996) experimentally altered thephotoperiod of theirbroodstoek (from18L:6D to6V18D)and observeda failureoffish to spawn inover 50010ofthe femalessubjected tothealteredphotoperiod .Theseresultsindicated that the light cycle plays aroleinthe detenn ination ofwolffishspawningtimeand suggeststhe possibility for themanagementoffinalmaturat ionby seasona lity(day length).Despite the limitedinformation on theeffectsofphotoperiod on wolffish,the effects of photoperiodonother fish species havebeenextensively studied.

Severa lauthors have shown that photoperiodhasa significant effect on the biologyand behaviouroffish.Photoperiodand light intensitymayaffectgrowthand survival via a number of physiologicalpathw ays.Forexample, Fuchs (1978)stated light stimuli affects sensory receptors infish (eyes,pinealgland),and induceschangesin their physiology.Photoperiod and light may also exert directeffects onthebehaviourof an organism,not necessarily linked to any endogenous rhythm (Richusand Winn,1979).

Suchbehavioursincludethe activityoffish (Schwassmann, 1971;Britzand Piennar , 1992) ,reproduction(gonadmaturation,gameteproduction/fecundi ty,delayingor synchronicity of the spawn ingseasons; Baggerman ,1980;Ridha andCruz,2000; Loiret aI.,2001;Rodriguezetal.,2OCH ),physiology(e.g.thyroxinelevels;Noeske andSpieler, 1983).feedin gbehaviour(Schwassmann,1971;Tandler and Helps,1985).responsesto visualstimuli, diurnalrhythm, andvertical migration,(Blaxter,1966;1968a,b;1973;

1975b ;Rahmannet al.,1979).

21

The response of fish to light isnot only species specific butvaries with the stage of development.The responses to lightlevels maybeconsiste ntthrougho utthe developme ntstages or it mayvary.Forexamp le,yolk-sac larva e of Atlant ichalibut (Hippoglossus hippoglossus)develop abnonnally in the presenceoflight(Bolla and Hol mefjord ,19 88), whereas juven ile-ad ult stage halibutexposed to continuo us light (24 ligh t)hadhigher growth ratescomparedto those raised undershorterphotoperiodsof8 hours lightl16 hours darkness (Simenseneral.;2000).Variation inligh t requiremen ts within a species can alsobeseen foryellowtailflounder.Experime ntsconductedon yellowtai llarvaetPleuronectesferrugineus) demonstrated thathighergrowth and survival rates were obtained under continuouslight (Puvanendran,I999b;unpu blished data ) whereas for the juveni lestage(same spawningbatch as thelarvae described above) a photoperiodof 12hourslightproduced comparab legrowth and survivalratesto those raisedunder continuous light (P urchaseandBro wn, 1997).

The natureand extentoftheeffect of increased phot operiod on growth and survival of a species canvary greatlydespitesimilaritiesin developm ent, habitat, morphologyandphysiolo gy (Bar lowetai.,1995). Asanexample, TandlerandHelps (1985) demonstrated that forthefirst 12days, larvalgiltheadsea bream(Sparus aurora) survivebetterunder 24hours lightversus12hours light. However,Dowd andHoude (1980)showedthat13 hours of light resultedin the bestgrowthandsurvivaloflarval sea bream(Archosargusrhomboidalis ) upto day16 post-hatch.Forlarva e ofyellowtail flound er(Pleuronecres f errugi neus) continuouslight resulted inimpro ved growth, survivaland specific growthrates (Puvan endran,1999b pers.comm.)however,in

22

summer flounder (Paralichthys dematus),continuouslightoffersno benefittogrowthor survival (Huber et ai,1999). ConsequentJy,selectingan optimalphotoperiod for maximumgrowth and survival,for anynew speciesshould be based onexperimentation.

No studies have beenconductedonlarval wolffishconcerningtheeffects oflight onthe survivalor growth oflarvae.Duringlarva l studies,photoperiods ranging from16 hoursto24 hours lighthavebeen used withoutexplanation(Moksnesset al.,1989;

Moksness, 1990; Strandet aI.,1995;Moksnessand Pavlov, 19%;Wiseman,1997).The objectiveof thisstudywas todetermine theeffect of photoperiodonthegrowth and survivaloflarval wolffish.Thehypothesis thatincreased photoperiodwill resultin increasedgrowth and survivalofwolffish larvae wastested.

3.2Materials aad Metbods 3.2.1EggCollection and LarvalSelection

During October 1994 ,wolffish egg masseswere collected bySCUBA diversin Bauline,ConceptionBay, Newfound land.Eggmassesweretransport edto the WesleyvilleMarineFinfishHatchery,in Wesleyv ille,Newfoundland (Fig.I).Atthe hatchery,egg masses werebroken intosingle layers of eggsandincubat ed in up-welling vertical trayincubatorsat adensity of approxima tely1000 eggs/trayuntil hatching.

Ambientseawaterwasusedduring incubatio n.Topreventbacterial infection, allegg masses weredisinfected twiceweeklyusing aglutera ldehyde-seawa terbath (Salvesen andVadstein, 1995).Disinfectionended whenthelarvae started hatching.

At 0-12 hours post-hatch, larvaefromthree egg masses were randomlyselected 23

u-

N

A'Eggmass collectionsite,Baulin e C; n"ption Bay , Finfish Hatchery, B'WesleyvilleManneI

W"l< yviJIo Momon"

c.

Oc~ Science~~:ntre, University, Logy

Ilection site (A) and research hie locationsofth~ e:t~:~;;ols.

Figure

~~~I~:1B

^{andC)forexpen m}

24

from the incubationtraysand transferredto theOceanSciences Centre.Memorial University.LogyBay,Newfoundland,for experimentation.

Fish were randomlydistributedin theexperimental tanksat 6-14 hours post-hatch andacclimatized for an additional24 hours prior tostarting the experiment.Theinitial stockingdensity for the tankswas5fishfL(Pavlov,1995)withequalproportionsofthe threeeggmasses stockedineach tank.Mortalitiesobservedduring this time were removed and replaced withnewfish.DayI repres entsthe dayon whichtheexperimen ts started(28-40hours post-hatch).

3.2.2 PhotoperiodProtocol

Threephotoperiods:12 hourslightl12hours dark (12L;8:00 a.m.·8:00p.m.),18 hoursIighl/6hoursdark (I8L;8:00 a.m.-2:00a.m.),and 24hours light (24L ) were selectedforexperi mentation. To avoidpossiblelight interference,eachphotoperiodtrial was conductedina separatelight-controlledroom.

A lightintensityof750luxwasused for all treatmentsandwas achievedby placingincand escentbulbsapproximatel yI metre abovethe testtanks. A20 minute twilight period (180lux) was activatedbefore and afterthe mainligh ts were turnedon/o ff inorder to avoidlight-shockingthelarvae(Morkand Gulbrandsen,1994).AlIlighlS werecontrolled electronicallywith a timer.Light intensities(recordedinwrits of lux) were measured at the water 'ssurfaceusinga SPER Scientifi cLightMeter.

Nine(3replicatetanksper treatment)flow-through,rectangular,glass aquaria (45 cm x 30 cmx30 em,30 L) wereusedas experimentaltanks.Thefour walls(sides)of

25

the glass tanks were covered withblack plastic to controloutside disturbances and limit mirrorreflections within thetanks (Pearce,1991;Barlow et al.,1995;Naas ecal.;1996).

The mean ambientsea water temperaturewas 6.O"C (range:l.g-12.0"C). Waterflows were adjusted dailyto maintainatemperaturebetween 6-8°C (Wiseman, 1997).

Each treatmentreceivedthreedaily feedingsof Artemiafranciscananauplii (1000 preylL) anda commercialdrypellet fedtoexcess (Wiseman, 1997),duringlighthours (10:00a.m.,2:00p.m.,6:00p.m.).Artemiadecapsulation andenrichment werein accordancewith AnemiaSystems standardmanual (Sorge1ooset al.,1986). A fourth (dryfood only)supplementalfeedingwas given toalltreatments at 5:30a.m.which coincidedwiththe darkhours ofthe12L and 1BLtreatments.Drypellets wereobserved to sinkto thebottom of thetanks shortlyafterintroduction. A previous weaning study conductedon the feedingbehaviouroflarval wolffish demonstrated thattheycanfeed on dry food onthe bottomof the tanks (Wiseman,1997),thereby ensuringthat fish ineach treatmenthadan opportunity to feed onthe fourth supplemental ration.Thephotoperiod experimentwas terminatedon day 50.

3.2.3Measurementsand Analysis

~

Alltanks were siphoneddaily,priortofirst feeding,toremove excess feed and feces.Mortalitiesobservedduringcleaningwereremovedandrecorded.

26

GrowthMeasurements

Initial (day I) size measurementswereconductedon a sub-sampleoflarvae from eachofthe threeegg masses.Thirty fish (10 fromeachegg mass)weremeasured for standard length andwet weight. Inorderto avoid introducingpotentiallystressed or moribund fishinto a treatment,none of thefish usedin theinitial measurementswere placedintheexperimental tanks. Subsequentgrowthmeasurements wereperformedon sub-samplesof fish(10 larvae)randomlychosen from each experimentaltank.

Measurementswererecordedevery tendays untiltheexperiment wasterminated, Protocolswereas follows:

Wet weight

Wet weights were recorded foreach oflhe10 larva in the sub-sample.Fish were removed fromtheexperimentaltankswith a dip-netandexcesssea waterwas removed fromeach larvaby gently towel dryingthefish.Fishwere placedinapre-weighed,petri dish whichwas filledwith sea water.AtoploadingMettler PC4400 scientificbalance was usedto record measurements to thenearesthundredth(0.01) ofagram(g).

Standardlength

Todetermine standard length,individual fish were transferredfrOm theweighing dish to ameasuring dish.The measuringdish was a modified petri dish equippedwitha flexible,plastic, holding chamber(used toenclose thelarvaeand preventitfrom swimming),andametricrulerfor measuring thelengths ofthefish.Basedupon

27

preliminary trails with this apparatus,wolffishlarvae ceasedswimmingwhen surrounded by the "chamber"and remained stationary until it was removed.The apparatus didnot injure any fishduring sampling.Standard lengths wererecorded in millimetresto the nearest 0.5millimetres (rom).

Specific Growth Rates

Specificgrowth ratesweredetermined for alltreatmentsbased upon an equation by Buckley et al.(1997):

SGR={(Iog.Sl-log.S I)/Tl-T11+100,where SI=initial fish measurement (wet weightor standardlength);

Sl=final fish measurement (wet weightor standard length);

T1

=

initial time;and

Specificgrowth rates werecalculated at ten dayintervalsfor the duration of the experiment.

StatisticalAnalysis

Alldata were first tested for nonnality.Datawhich werenotnonn ally distributedwere transformed (survival data:arcsin transformed,and growth data:log transformed) 10 meetthe assumptions of the test. A two-way ANOVA (SAS version 6.1, Cary,NC) was used for all comparisons.

Where significantday-treatment interactions occurred,a Tukey's Testfor 28

Multiple Comparisonswas perfonned among treatmentmeansfor each sample day.

Levelof significancewas set at0<=0.05.

3.3Results Survival

Photoperiod had asignificant effect on thesurvivalof larval wolffish (Table I).

Although a significantdifferencein survivalwas seen among treatmentsforday 10 (Table 2) themortalities recorded duringthis timeweredirectlyattributed to siphoning errors and nottreatment effect. Statisticallysignificantdifferences in survivalwereseen among treatments between days30-50oftheexperiment.According tothe Tukey's results (Tables 2,3) for days30·50 inclusive,both the18L andthe24L treatmentshad a significantly highersurvival ratethan the12L treatment (Fig.2).

Althoughno statisticallysignificantdifferenceinsurvivalwas recorded between the18L andthe24L treatments throughout the entire experiment,significantmortalities commenced on day30for the 12Ltreatment and continueduntiltheexperimentwas terminated on day50.Finalsurvivalrates for each treatmentwereas follows:18L (66.89"1o);24L (58.67%);and 2L (34.22%).

Wet Weight

Photoperiod had a significanteffect onthe overallweightofJarval wolffish (Table' I,Fig.3).No significant differences in wet weight were observedamongtreatments for day10(Table 2).Although statisticallysignificantdifferenceswere observed on day20,

29

100

80

e

60

"ij

·E

>

:>

'" ;:

₄₀

"

"'" ~ _-e-

_I2l

20 ___18L

---A---24L

10 20 30 40 50

Time (days)

Figure 2: Mean percent survival (%) of larval wolffish over time (days) for photoperiod experiment (vertical bars represent standard error, n=30 for each data point).

30

0.40

0.35

0.30

:§

_0.25

.J:

_OJ) 'il

'"

^0.20

~

0.15

0.10

0.05

0 10 20 30 40 50

Time (days)

Figure 3: Mean wet weight (g) of larval wolffish over time (day s) forphotoperiod experiment (vertical bar s represent standard error. 0=30 for each data point).

31

Table):Resultsof Two Way AnalysisofVariance(ANOV A) on themeanpercentsurvival,mean wetweight.and mean standard lengthof larv alwolffishforthephotoperiodexperiment (signifi cancelevel p<0.05.- denotesa signi ficant difference).

EXPERIMENT SOURCE DF F·VALUES [I-VAL UES

Treatment 2 32.56 0.0001-

MeanPercentSurvival Day 4 128.48 0.000)-

Treatment-Day 8 6.03 0.0001-

- -- -

Treatment 2 52.72 0.0001-

MeanWetWeight Day 4 272.81 0.0001-

Treatment-Day 6 6.12 0,0001-

- - - -

Treatment 2 22.85 0.000'-

MeanStandard Length Day 4 462.66 0.000'-

Treatment-Day 8 3.31 0.00 72-

32

Table2:Resultsof Tukey's StudentizedRange Test onthemeanpercent survival.meanwetweight, and mean standa rd lengthoflarvalwelfflsh,foreachsampleday separately. forthephotoperiodexperiment(significancelevel p<0.05, • denotes significanldifference).

VARIABLETESTED DAY DF F·VALUES p.VALUES

10 2 7.00 0.0270·

20 2 0.3 1 0.7450

MeanPercent Survival 30 2 15.25 0.0044·

40 2 16.5g 0.0036·

SO 2 14.96 0.0047·

IU 2 3.90 0.0822

20 2 6.46 0.0319·

Mean Wet Weight 30 2 20.44 0.00 21·

40 2 150.59 0.0001·

SO 2 12.58 0.0071·

10 2 1.65 0.2690

20 2 4.07 0.0764

Mean Standard Length 30 2 12.51 0.0072·

40 2 2.61 0.1521

SO 2 11.76 0.0084·

33

Table3:Tukey'sgroupings for the mean percent survival, mean wet weight. and mean standardlength for the photoperiodexperiment(Note:means withthe sameleiter, foreach age,arenot significantlydifferent.p<O.05).

Tukey'sgroupings (means) foreach sampleday Time

(days) Treatment Survival(%) Wet Weight(g) Std .Length (nun)

\0 12U 120 100.00"

o.u s

20.07^E

18U 60 99.78^AB 0.13^c 20.28^E

24UOO 99.11⁸

o. ro -

20.53^E

20 12UI 20 93.78"

o.u >

21.05^E

18U 60 97.11" 0.12^c 21.5S^E

24UOO 95.S7" 0.\0" 20.92^E

30 12U1 20 48.67^B 0.11⁰ 22.25^F

18U 60 93.78" O.1Sc 24.22^E

241)00 84.00-' O.1Sc 24.00^E

40 12UI20 38.oo^B 0.14^D 25.11£

181)60 76.89" 0.23^c 27.88^E

241)00 7l.11" 0.22^e 26.82£

50 12U1 20 34.2i' 0.25° 29.01^F

18U6D 66.89" 0.36^c 32.48^E

241)00 S8.67" 0.3S^c 31.85£

34

the Tukey' s groupings(Table3)show the actualdifferencein thewet weightswas minimal(12L(O.ll g);18L(0.12g);24L(0.lag).Althoughno significant differences in wet weight occurred between the18Land 24Ltreatments during days 20-50, significant differences in mean wetweight occurred between these treatments andthe12ltreatment duringdays 30-50.Consistently,the 12l treatment hada significantlylower wetweight thaneither the18l or 241treatments.

StandardLength

Photoperiodhadasignificanteffect on the standardlength of larval wolffishfor days30and 50 of the experiment (Table2).For each ofthese samplingperiods, the differences in standard length between the18L and 24L treatmentswerenot significant butwere significantlygreaterthanthe standard lengths for12l treatment (Fig. 4, Table 3).No significantdifferences wereobserved amongtreatmentsfor days10, 20,or40.

Specific GrowthRate (SGR;%wet weight/day)

ResultsfromtheANOVA (Table4)showa significant interaction between day andtreatment.However.dayeffect,notphotoperiodtreatmenthas the most significant influenceon the wetweight-SGR.Although,negativeSGR's were seen forboththe12l and 18ltreatments onday 20 (indicatingaloss in weightfor the fish;Fig.5), day 30 was the onlytime periodinwhich asignificantdifference occurred among treatments(Table 5).larvae sampled from the12Ltreatmenthada lowergrowthrate (0.18%wet weight/day) thaneitherthe18l (1.90%)orthe24L(3.39%)treatm ent. Therewere no

35

40.0

37.5

35.0

32.5

E .§.

30.0 .c

Oh

_27.5

...., e

~

^25.0

-e

§

_22.5

'"

20.0

17.5

15.0

10 20 30 40 50

Time (days)

Figur e 4: Mean standard length (mm) oflarval wolffish over time (day s) for photoperiod experiment (verticalbarsrepre sentstandard error , 0=30 for each data point).

36

~ ~

~ ~

~.~

~ ~

" g _0."- ~

on -1

2.0

~

S

- "

^1.5

"':;' -"1ii>

. o"E ^{0-" "}

^1.0

~~

'[~

^0.5

C/}e

0.0

~'2UI2D a.

18U6D 24L1OD

I ^{: R fl} n

---

10 20 30 40 SO

~12U12D b.

18U6D 24UOD

In~ I

10 20 30 40 SO

Time(days)

Figure5:Themeanwetweightspecificgrowth rate(fig. a)and the mean standard length specific growthrate(fig.b) over time (days)forlarval wolffishfor the photoperiod experiment (0=30foreach datapoint).

37

Table4:Results of TwoWayAnalysis ofVariance(ANOVA)onthe specificgrowthrates('I_wetweighVday.%standard length/day)oflarval wolffish forthephotoperiodexperiment(significancelevel p<O.05.-denotessignificant difference).

VARIABLE SOURCE DF F·VALUES p·VALUES

SpecificGrowthRate Treatment 2 2.48 0.1005

(%wetweight/day) Day 4 32.83 0.000' -

Treatment- Day 8 3.27 0.008S-

Specific Growth Rate Treatment 2 3.97 0.0295-

(%standardlength/day) Day 4 28.17 0.000. -

Treatment-Day 8 2.84 0.0180-

38

Table5:ResultsofTukey'sStudentizedRangeTestonthe specificgrowth ratesW.wet weight/d ay,and%standard length/day)oflarvalwotflish,for eachsamplingday separately,forthephotoperiodexperiment(significancelevel p<O.05,• denotessignificant difference).

VARI ABLETESTED DAY DF F-VALU ES p.VALUES

10 2 4.02 0.0779

SpecificGrowthRate 20 2 1.13 0.3836

('Yowetweight/day) 30 2 16.83 0.0035-

40 2 1.03 0.4137

'0 2 0.89 0.4572

10 2 1.82 0.2406

Speci ficGrowthRate 20 2 2.89 0.1322

(%standardlength/day) 30 2 18.92 0.0026-

40 2 1.06 0.4038

'0 2 0.54 0.6101

39

Table6:Tukey's groupings forthe specificgrowth rates(%wet weight/day,

%standardlength/day )forthe photoperiod experiment(Note:means withthe sameletter,foreach age,arenot significantly different, p<O.05).

Tukey'sgroupings(means) foreachsampleday Time

(days) Treatment %WetWeightIDay %StandardLengthIDay

10 12UI2 D 2.29" 0.92"

18U6D 3.57" 1.03"

24UOD 1.26" 1.14'"

20 12UI2D _0.86" 0.48'"

lSU 6D .0.22'" 0.61'"

24UOD 0.81'" 0.20'"

30 12U12D O.ISo 0.55°

18U60 1.90" 1.16"

24UOD 3.39" U7"

40 12U12D 3.28-'" J.21^A

18U 6D 4.23" 1.4 1^A

24UOD 3.68" 1.11"

50 12Ul 2D 5.52" 1.44"

18U6D 4.64" 1.53"

24UOD 4.56" 1.72"

40

statisticallysignificant differencesin wet weight-SGRbetweenthe ISL and 24L treatments(Table6). Fordays 40 and 50, the SGR of the12L treatmentwas statistically similarto the othertreatmentsindicatinggrowth for thistreatmentwasnot com promised beyondday 30.

SpecificGrowthRare(I/ostandardlength/day)

Results for thestandardlength-SGR'sshowsimilar trends asthe wet weight- SGR's(Fig 5.,Table4).Day3D,wasthe only day onwhichsignificantdifferenceswere found amongtreatments.Differencesinthe SGR between the 18L (1.16%)and24 L (1.37%)treatments werenol significantbutboth had significan tlyhigher SGR'sthan the I2L(0.55%) treatment(Tables5,6).

3.4 Discussion

Theresults ofthis experiment demonstratedthat photoperi od had an effect on larvalwolff ishsurvivaland growth.During the first 20 days.the effectsof photoperiod on theseparameters.were not significant.Althougha slightstatist icaldifferencedoes occur forbothsurvivalandweigh t during thisperiod.thechangesin growth and survival were minimaland were directlyattributableto hwnan error.Duringcleaning several larvaewerekilled when theywere accidentlysiphoned .

Photo period effect onlarval survivalandgrowth wasmost evident during days 20-40post-ha tching.Duringthisperiod,the trend forbothsurvival and growth was statisticall ysignificant and consistent.Althoughno significant differe nces were recorded

41

between the18land24ltreatments, both treatme ntsdiddiffer signific antly fromthe 12ltreatment. Themortalityrateof larvaein the12ltreatmentnearlydoubled thatof the18ltreatmentduringthis periodindicatingthatthe12Lphotoperiodhad adeleterious effecton larval wolffishsurvival from day 20 onwards . However,beyondday40the changes in survival andgrowth becomelesspronouncedimplyingthatthe photoperiod requirementchanged asthelarvaeapproached thejuvenilephase.

The trend ingrowth rates (meanwet weight ,meanstandard length)wassimilarto those for survival ,asthe 18Land24Ltreatments producedthe longe st and heaviest larvaewhilethe12Ltreatmentproduced thesmallestfish.Consistent withsurviv al, growth forthe12lphotoperiod showedthe largest statisticaldifferencesbeyondday 20 post-hatch.

Theresults suggestthat days 20-40 post-hatch are a criticalperiod forensuring larvalwolffish growth andsurvival.Itisapparentthata signi ficant biological phenomenon occurred duringthisperiod and was reflected in thevariations ordifferences in survivalrates, growthrates(lengthand weight)and specificgrowthrates for thethree treatments.Duringthis criticalperiod, thereducedsurvivaland growth ratesofthe12L treatmentindicatethat the reducedphotoperiodis inadequateforensuring maximum survivalorgrowthofl arval wolffishduring this time.Several studiescondu ctedon larvalwolffish suggest thatthecriticalbiologicalphenomenonwhich occurs duringdays 20-40,isthe switchfrom endogenou stoexogenous feedingand that thisfeeding transition may coincide withweightlosses and/orsignificantmortalities(Blanchard, 1994; Stran detai.,1995; Wiseman, 1997).

42

Despiteaweight loss(negative specific growthrates) occurrin ginboththe12L and 18L treatmentson day20.the 18Ltreatment(day 30) was able to attain a much higherSGR than the12L(approximately 10 timeshigher) suggest ing thattheadditional6 hoursof photoperiodcontribut edto theimproved survival and growth during this transitionto exogenousfeeding.

Negativegrowthratesduringthetransitionalfeeding stage have been reportedin previouswolffishstudies(Strandelal.•1995;Wiseman ,1997)and in alarval rabbitfish Siganusguttanus study (Durayand Kohne,1988).Studies conducted on thestarvation of larval wolffishdetermined themortalityrateforunfed larvae commencedaroundthe third week (dayztpost-hatcb)with100%mortality occurringbyweek five(day 35 post- hatch;Wiseman,1997).Several authorssuggest thathighmortalities duringthis period maybe caused byeithera reduced feedingopportunity (resulting in a deficiencyin nutrientsand energy)or an inappropriatefeeding adaptation(Moksness et al.,1989;

Blanchard,1994;Strandelal.•1995;and Wiseman, 1997).

Itseemsthat18L and 24L photoperiods inthis studyprovidedalongerfeeding opportunitycomparedtothe12Ldueto thegreaterdurationinwhich thecommercial diet was presentinthe rearingtanks.Personalobservations revealed that wolffish do not appearto feed activelyduring periods of darkness and larvae remainstational)'onthe bottom of the tanks duringthis period.Initiallyit was perceivedthatsincelive food was onlypresentedduring thelighthours andthat flushingrates forthe tanks weresimilarfor alltreatments that all larvae had an equalperiod of time to forage.However.the dry food whichsanktothe bottomof thetanks.was notsubjectedtothesameflushing rate as the

43