The Effe c t.s c..i unialgal andMi xe dDiet s onGr o wth ofHa tc hery- Pr o d u c e d Spat. of the Sea Sca llop ,
Pla~magellanicus (Gme lin)
by
eCa r o l y n A.Gil l is , B.Sc .
A thesis submitted co the Schoolof Gr a d u at e St.udies in partial fulf il lment ofth e
requirement.s for the de g r e e of Master of Sc i e nc e
Department of Biol o g y Me mo r i a l University of Newfoundland
September, 1993
St.John 's Newfoundland
1+1
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ABSTRACT
Sea scallop (~ magc ll an icu s l juvenileshav e beenreare dina hatcheryat the Ocean SciencesCentre, Memo ri altjndver-sit.yofNewf ou nd la nd , for seve raj years and transplantedto scallop farms located in three Newfou ndla nd bays. Th i s stud y was undert ake nto investigatethe effect of diet on growt hof sp atto helpoptimize hatchery production. Four experiments we r e performed to determine wh i c h available algaldiet Is) resulted in greatestgrowth (in terms of shell height, dry weight and organic we i gh t ) of juv e n il ef. magellan i c us rangingin sizefrom 1- 4mm shell he i g h t . Itwa s fo und tha t of the dietstested, a ternary mixeddie t consistingof the three species~
~ , Isochr y s i s aff. ~ (T-Iso)and~
epp. (mue llerior~ ) .fe d in a 1:1:1ratioat a fina l co nc ent ra t i onof50 cells/pI , res u l t e d inthe gre atest growth for juven iles sized2 - 4 mm. Aunialgal~
~diet supp orte das much growthas the terna ry mixed die t in scallopssize d1 - 2mm. The unialgal~ diets did notsupportanyappreciablegrowth. ALGAL 161, a commercia llyavaila ble, spra ydr i ed , hete rotrophicallygrown Tetraselmis~product, was found to be a poorfood for g. mag ellanicu s juv eniles. Gros s gr owt h efficiencie s were dete r mine dfor scal l o ps for the differe n t diets tested. No
i i
si-;;nifi cant dif f erenc e s in gr Ol.,.t heffici e nc i es amo ng tte diets were found.
/>.short - t erm expe r ime n t was done tostu dy the effect of rat i on on fi l t r a t i onand ingest i on rat esfor three size classes of ju ve n ile~maqellaniclls (2 - 4mm,4- 6mm, >6mm). It was foundthat as ration increased, fi l t r atio n ra t e s decreased and ingestion rates increased.
valu e s for filtration and ingestionrates (F) were related to growthrat e (W) using the general allome tricequationFlO
ew".The physi ol ogical rateswere found to be increasing fas te r tha n the growthratesofthe s e young juvenil e sca llo p s withvalues of th e exp onen t (bl ranging from1.47 2.24 .
iii
ACKNOWLEDGEMEN'tS
I would like to thank my supervisor , Dr. Patri ck Dabinett, forhis assistan ce and patience throughou t thi s project , As wel l, I sinc e r e l y expressmy thanksto Bar bara Doo l e y for her invaluableadvice, both regarding the project and as a friend.
The researchfor th i s project was car r i e d outat the Oce a nSci e nces Centre, Memorialuniversi ty, Lo gyBayan d the use of the fac ilityand cooperat i on fro m staff is greatly appreciated . This work was supportedin part by grants to P.Dabinett fromth e Newf o undlandDepartmen t of Fisheries and the Canada/NewfoundlandInshoreFisheriesDevelopment Agreementandby bursari es fromthe Sch o olofGr a d u a t e Studies, Me mor i a l University.
ABSTRACT .••. ACKNOWLEDGEMENTS•. CONTENTS.
LIST OF TABLES. . .. LIST OF FIGURES••.
LIST OF APPENDICES•• INTRODUCTI ON •...
CONTENTS
i i iv
vii
xii
I.~. GENERAL, .••• •• •••••• • 1.2. PARTICLE SELECT I ON AND PARTICLE
RETENTION EFFICIENCY... 3
1.3. EFFECT OF MIXED AND UNIALGAL DIETS.. 5 1. 4. BIOCHEMICAL CONSIDERATIONS. . .. .• •. 9 1.5. ENERGY REQUIREMENTSOF LARVAEANDJUVENILES.. lS
1.5.1. FiltrationRate. 15
1.5.i i • In gestion... 16
1.5.i i i. Efficie n cie9. 18
I. G . OBJ E CTI VE ~ 20
rr MATERIALSAND METHODS •. .... . .. . .. . . .. 22 I1. 1. SPAWNING ADULTS AND REARING LARVAE••.• • •,• • •. 22 11.2. ALGALCULTURE.•••.• •••.•••• •• • • . • ••• .•• ••• • • . 24 II .3• ALGALCUL TURESUSED IN GROWTH EXPERIMENTS.. .. 26 I1.4. ALGALDRY WEIGHT AND ORGANIC
WEIGHT DETERMINATIONS... • • • •, ••• •• ••• . .• . • 27 11. 5. EFFECT OF DIET ON GROWTH••.••• •. .•••.•• •• •••• 27 II.5. L. Experiments 1 and2.. .... . .•.. ...• • .. 28
11.5.11. Experiment 3 29
II.5.iii. Ex pe rime n t 4 30
II.G. GROWTHDETERMINATIONS... . . 31
II.6.i. Shell Height. 31
II.6. i i Dr y Weight.. . .. .. .... 31
I1.6.iii.Organic Weight. 32
11.7. DETERMINATIONOF INGESTIONRATES.•. .•. 32 II. 8. DETERMINATION OF GROSS GROWTH EFFICIENCY..•. 32 I 1.9. EFFECT OF RATION ON FI LTRATION AND
INGESTION RATES .•••.•••.• • • • ••• •.••• • ••••. 33 II.1 0. DETERMINATION OF FILTRATIONAND
INGEST IONRATES.... ... 34 II .11. STAT ISTICALMETHOD.•.••.. . ••.•••• •.•. . . . .. .• 36
II I RESULTS•• • . . . •.• .. .. . . • • ... • .• . . ... .. 38
!II.l. ALGAL SPECIES' CHARACTERISTICS .. ... 38
III.2. GROWTH - EXPERIMENT 1 ... '" .10 III,2.i. She l l He ight.. .. .... . ....'10
III.2.Ii. Dry Weight..• • •. . 42 IIL2 . ii i . Organic We i g h t 42 111. 3. GROWTH - EXPERIMENT 2... -IS III.3. i. Shel l Hei ght .... '15
IlI.3 . i i. D::y We ig h t .. .... . . .. 46
IIL3.iii. Organic Weight.. . ... '16
III.4. GROWTH - EXPERI MENT3. . . . .... 49
IIL 4.i. Shell Height... . . . .. . . .. 51
II I.4. ii . Dry Weight ... .•.•... . . . ...•. • • 53
III.4. i i i . Or g a n i c Weight ... ..••.•. . . . ... . 55
IlI.5. GROWTH - EXPERIMENT4 55 III.5.i. ShellHeight... . ... •.•.. ... . . 51
III. 5 .ii . Dry Weight.. ... . ... . . . ..••• •. .. . . 59
IlL 5.iii.Or g a n icWeight .. .•. . ... . .•. . . ... 59
IU .6 . GROSS GROWTH EFFICIENCIES... . . . . • •• ... . GO 111.1 . EFFECT OF R.l\.TION ONFILTRATIONAND INGES TI ON RATES . IV DIS CUSSION•. 65 . ....eo IV .l. EFFECT OF DIET. . . .. . . . ... . . ... 80
IV.2. GROSSGROWTHEFFICI ENCI ES . . ... 87
IV . 3 . EFFECT OFRATIONONFILTRA TIO NAND INGEST I ON RATES... . . . • • • • . ..•. . ... . ..• .... 89
IV.4. SUMMARy.. . . . ... .. ... .... . . .... . .. . .. 92
IV.5. CONCLUSIONS.... ... ... . . .. 95
REFERENCES ... .•.• . •.•• ••. .. ....•• •.•• • ••... ...•• . 96
APPENDIX.•• •• • • ••. •••• • • • •• •• • ••• • • • • • ••• •• •..• ••••••.• .•112
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LIST OF TABLES
TABLE1: Summaryof experimentalfactors andalgal 28 combinations used in growthexperiments.
TABLE2: General characteristicsof the algal species 38 used in Experiments 1 - 4.
TABLE 3: Literat u revaluesfor algal cell 39 characteristics.
TABLE4: ~~mage llanicus. Summaryof re s ults 41 of Duncan 'smeanstest for di f f e r e nc e s in shell heightsof spat (2 - 4mm) rearedonvarious diets (Ex p eri tlle nt 1).
TABLE 5: Placopectenmagellanicus. SultlJ1lary of results 43 ofDunc a n' s eeana test fordi ff e r e nc e s indry we ight s of spat (2 - 4 mm) rearedonvarious diets(Experiment 1).
TABLE 6: Placopec teomagella nicus. Summaryof results 44 of Duncan'smeans test for differences in organic weight s of spa t (2 - 4mm) rearedon variousdiets (Experime nt 1).
TABLE7: Placopecten~~. Summary of results 47 of Duncan'smeanstest for differences in shellhe i ghts of spat (2- 4 mm) reared on various diets (Experiment 2).
TABLE 8: plaCQpectflDmage]lan icys . Summaryofre s ul t s 48 of Dunca n'smeanstest fordiffe rencesin dry weights of spat (2 - 4 mm) reared on various diets (Exp eriment2).
TABLE9: Placopecte;O~ : i. Summary of results so ofDunc a n ' s meanstest for dif ferencesin orga nicweightsof spa t (2- 41I\lll.) rearedon var i ou sdirJt s (Experiment 2).
TABLE10:~ ~ 1Il.5I~. Summaryor results 52 of Dunca n 'smeanste st tor di ffe rencesin shell he i g ht s of spat(1 - 2 mm) rearedon vario us di e t s (Experiment 3).
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TABLE11: ElA&2~magella n i cu s. Summa ry of results 54 of Duncan 'smeans test fordiffere ncesin dry weights of spa t (1 - 2mm)reared onVAri o us di ets (Ex pe r i me nt J).
TABLE 12: ~mage)lan icus. Summa ryof reeutes 56 of Duncan'smeans test for diff e r enc e s in organ i cweightsof spat(1 - 21l\lll)rear edon variousdi ets(Experiment J).
TABLE13 :~ IIL~. Summary of results 58
ofDunc a n 's means test for differencesin shell heights of spa t (2 - 4mm) rearedon variousdiets (Experiment4).
TABLE14: ~ma ge ll a n i c us. SUllllDary of re s u lts 61 cfnunoen es means test for diffe r enc e s in dry weightsofsp a t (2- 4mID)reared onvarious diets (Experiment4).
TABLE 15: ~magellan icus. Summaryof resu lts 62 of Duncan'smeanstest for diffe rences in organicweightsorspa t (2 - 4mm)reared on various diets (Experiment4).
TAB LE 16:Literatureva l ue s for gross growth 64 effi cienc i e sof laboratory-reared'[u venILe bivalvemolluscs fed algal diets.
TABLE 17 :Mean filtration andLnqestiLonratesfor 71 three size classesof scallops(2-4 ,4-6 and 6+mm) at three food rations(20, 40 and80cells/pi).
TABLE 18 : Literaturevalues for filtrati on ratesof 72 va rio u sbi va l v e spe cies.
TABLE19: Specif icfiltrationand ingestionratesfor 74 threesize classesofsc a llo ps (2- 4, 4-6 and 6+ lIllll) at three foodra tio ns (20 , 40 and 80cells/lJl).
-v i ii -
LI STOF FIG URES
PIGURE1: ~magellanic:us. Hean shellheights 41 (t sd ) for spat (2- 4mm) rearedonvarious algal diets batch fedat50cells/pI (Ex periment 1).
FIGURE 2: ~ m~. Meandryweights 43
(t sd )forspa t (2 - 4mm) re ared onva r ious algal dietsbat c h fedat 50cells/p I (Experiment 1).
FIGURE 3: ~mage l la nisus. Me an orga n ic 44 we ight8(t sd) forspa t (2- 4 1llIlI)rearedon various alga l diets batch fed at50cells//Jl (Experiment1).
FIGURE 4: ~mag e ll a nicus. Me an she ll 47 he ights(1sd) forspa t (2- 41IUll)rearedon vario us alg aldietsbatch fed at 50cells//Jl (Exp e rime nt 2).
FIGURE5: ~mage lla nicus . Me an dry weIghts 48 (±sd) f"r sll8t (2 - 4IIUD.) rea r ed onva ri o us alga l diets batch fedat 50cells /pI (Ex pe ri me nt 2).
FI GURE 6: ~mage JJa nf c:us. Me an organi c 50
we ights (tsd) for spat (2 - 4mm)rear e don vario us algal dietsbatch fed at 50cells//J1 (Expe ri me nt 2).
PIGURE 7: ~magelJ ani c:us. Meanshell 52 heig hts(t sd) for spa t (1- 2u )reared on va riousal g a l dietsbatchfed. at 50cel ls//JI (Experiment 3).
FIGURE8: ~mag e lla nicys. Meandry weig hts 54 (± sd) for spa t (1 - 2mm)reared onva r i ous algal diets batch fedat 50cell s /pI (Experiment3) .
FIGURE9: ~magellanicus. Meanorganic S6
weights (t sd) forspa t (1 - 2mm)reared on variousalgal diets batch fed at 50cells/ loll (Experiment3).
-ix-
FIGURE 10: PlaeopeeteD magel 1anicus. Mean shell 58 heights(±sd) for spat (2- 4 mm)re a r e d on various algaldiets batch fedat 50cells/~l (Experiment 4).
FIGURE11: Placopectenm . ~. Hean dryweights 61 (±sd) tor spat (2- 4 mm) reared on various algaldiets batch fed at 50 ce11s/1-11 (Experiment 4).
FIGURE12 : Pl acopecten magellll niclls . Hean organic 62 we i ghts (±sd) for spat (2- 4 mm) reared on variousalgaldiets batch fed at 50 cells//JI (Experiment 4).
FIGURE13: PlacQpecte nmagellanicus. Gross growth f6 efficiencies (Kl) of spat (2 - 4 mm) reared on various diets batch fed at 50 cells/pI
(Experimen t 1).
PIGURE 14 : placgpectenmage1Janjclls. Gross growth 67 efficie nciesof spat (2 - 4 mm) reared on variousdietsbatchfed at 50 cells//Jl
(Experiment 2).
FIGURE15 : p1acopecte nmagellaDicus. Gross growth 6B eff icienciesof spat (1 - 2 mm) rearedon va r i o u s dietsbatchfed at 50 cells//Jl (Experiment Jl•
FIGURE16: placopectenmage1Jan icus. Gross growt h 69 efficienciesof spat (2 - 4 mm) reared on variousdietsbatchfedat 50 cells//JI (Experiment 4).
FIGURE 17: Plac opecten mage1Ja nicus . Specific 75 filtratio nratefor spat ofva ri o u s stee classeswh e n fed varyingrationsof ami x e d alga l die t.
FIGURE IB: placopeete nmage llanjcus . Specific 76 ingestio n ratefor spat of various size classes when fed varying rationsof a mixed algal diet.
FIGURE 19: plgcopectenmggellanicus. Filtrationrate 7B per animalas a functionofti s su e organic weig h t l\t varyingfood rations.
FIGURE 20 :~magell a n i c us. Inge s tionrate 79 peranimal as a fun ct i on of ti s sue
organic weight at vary i ng foodrati ons.
-xi-
TABLE A-l:
TABLEA-2 :
TABLE A-3 :
TABLE A- 4:
TABLE A-5:
TABJ.EA-6:
TABLE A-7:
TABLE A-8:
TABLE A-9:
FI GUREA- I:
FIGURE A-2:
LIST Of' APPEJrfDICES
Resultsforgr owt h parameters of 112 juven il e Plac opectenmagellanlc\l5 in Experiment1.
Ing e s tionvalues for~ 115 magellani cusjuvenilesduri ngthe courseof Experiment1.
Resultsfor growthparametersof 116 juv e nile P1acQpectenmagel]anicus in Experiment 2.
Ingestion values forP1acope c t e n 120 magellani clls juvenilesduring the cou r s eofExperiment 2.
Results fo r growthparametersof 121 juvenileP1ac ope c t en ma ge 1l 8ni c u5 in Experiment J.
Ingestionvaluesfor~tl.gn 125
magell anjcus juvenil esduringthe course of Exper iment 3.
Resultsfor growth parameter sof 128 juvenile PIaco pe cte n magellanicus in Experiment 4.
Ingestionvaluesfor ~ 132
maaell anicus juv e n iles during the courseof Experiment4.
Summaryof conditionsyielding highest 135 growth ratesof E. magellanicus spa t from experiments 1-4.
~magellanicys. Filtration 136 rate per animal for spa t of varying size cl a s s es when fedvarying rationsof a mixed algal diet.
~mage'lan 1Clls. Ingesti on 137 rate per animal for spatof various size classeswhen fed varying rations of a mh:ed alga l diet.
-xH-
TABLEA-tO: Inorganic dr y weigh ts and orga nicdry we ightsofsca ll o ps foreac h tre atment stud ied.
-x iii-
138
I INTRODUCTI ON I.I. Ge n e ral
The seasca llop, Placgp e c t eo magellaoi cJ!s, is an imp o r t a n t na t i v e commercialmollusc (c l a s s Biv al v ia , family Pectinidae ) , found in the northwes tAtla nt.icfr om the no r th shoreof the Gulf of St. Lawrence toCa pe Hatte r a s, North Carolina. Most scalloplan d i n g s are obtainedfrom th e comme r c ial fisherieson Georges Bank of f the ea s t coa s t of NorthAme r ica. The past ten yearsha v e shown annual ca tc he s fromal lsources rangingfrom about 10,0 00mt to 40 , 000 mt, with landed prices rangingfr o m a highof C$17/ k g inthe early1980sto the present value of C$8 - 10/kg (Couturier, 1990).
In recent yearsthere has been considerableinte r e s t in the aquaculture of thisspecies.Thereare two possible sources of seed (spat or juvenile scallops).The fi r st inv o l v e s collectingspat onartificial substratesdeployed in suitablesitesin th e natura l environment,and thenusing these spat to enhancenatural populations or for aquaculture purposes. The secondinvolvesproductionof hatchery-grown spat, an approachthat is presentlybeing studiedas a possible al ternativeto naturally-producedspat.
Successful re a r ing of F.. magellanic llsla r v a e through metamorphosisin the laboratory has be en limited(Cul l i n e y, 1974; Fournier and Mars o t , 19 8 6 ). Resea rch at the Ocea n SciencesCe nt re inae. John's, Newfoundland ,onha t c h ery
methods for the sea scallop commencedin thelate seventies and by 19 8 7had advancedto produce spatof adequate size to transportand suspendin several aquaculturesites .
Artificialspatproduction con sistsof 2 phases: hatcheryandnu rs e ry. Th e hatcheryphas e involves reari ng the larvaeto post -larvae (up to1 mral. In the nursery phase , spatare rearedunt i l tran s pla n t i ou tothe natural envi r o n me nt (2 - 10 mm} , The nu r s e r y phaseis impo rt a n t in that it minimises lo s s e s duetohandling,preda t i o n, tempe r a t u r eshocks and shortag eoffo od (DePauw, 19 81 ). For both the hatcheryand nurse ry ph a s e s, it is impo r tan t to knowwh i c h alga ldi e t s support growth of larvae, eurvfval throughmetamorphosisandprov i d eop timum growthof the pos t-larv a e andjuve ni l e s.
The successful rearing of bival ve larvae (Loo s a n o f f and Davis, 1963;walne, 1964) has led to studieson their nu tritionalrequiremen ts IUke l e s , 197 0 ) andaspects of their biochemistry (Millarand Scott, 1967;Hol land, 1918; Whyte g,t.li., 198 7 ;Gabbott, l!:l7 61. These studiesin v e s t i g a t e d fa c to rs influencinglarval su c c e s s and vi a b ili t y thro ugh metamczphc e La andsett leme n t. Much of the rese a r c h ava ilable con c e rns oy st e r, clamand mus s e l larvae. Some info rmat ionconcerni ng larvae of
.e.
mage llanicu s is reported incu l l i n ey (1 974) and Mann i ng (1986). NaiduIIll. (19891 presen t an ove rvie wof cultu r e methods for.e .
magellanicusand, Dabinett (1989) has reported successes in hatche ry production and grow-out inNew f o u n d l a n d. Other research on juvenileR. magellanicus has focused on effect of ration (Hollett and Dabinett, 1989). effect of current speed and food concentrationusing a microparticulatediet (xean-gowie IIg.,1989) and the effects of unialgal and mixed dietson growth (Gillisand Dabinett, 1989). However, as the scallop aquacultureindustrygrows, the r e is a need for further research on requirements for larval and juvenile scallops.
1.2 . PARTICLESELECTI ON AND PARTICLERETENTIONEF FIC IENC Y
Scallopshave been described asnon-s i p h on a t e , ciliary suspensionfeeders (Bricelj and Shumway, 1991) and have been termed suspens ivoresrather than filterfeeders (Be nni ng e r and Le Pennec,1991). Suspendedpazt.Lr-Le s are captured on the gillsby a combinat ionof directciliary action, mucus secretionandentrapmen ton the gill fi lame n t s . Due to the dimensionsof the eu-Iatero -frontalcirri on thegill filaments, it is believedthat different speciesof bivalves can retainpar ticlesof different sizeswith differing ef ficiencies. Br i c e l j andShumway (199 1) poi n t outhowmost post-set tlementstagesof suspension feedingbivalves can retain partic lesabov e 3- 4jJ.mwit hlOOt efficiency, with retentioneffici e nc ydecreasing wi t h dec r e asingpar t icle
size. However, the limit foref f e c t i v e retentionof particlesin members of the Pectinidaeisabout 5- 7«m, Winter (197B) fo undthat info ur speciesof bivalves
(~ ~ , ~ ~, CrUSOgtrea ~,
~~) ,particleswere effective lyret aineddownto about 7 p.m in diameter.~~could re ta i n par t icles downto about 2 p.min diameterand par t icles be l ow 1 ,urn were ret a ined toa very limi t ed degreeby all spe cie s . Re c e n t studieswith.f.magellani cu s have re p orted100 \ rete n tio n of particlesdo wn to 3f1m (Cranfordand Grant 1990, D.Deibe landR. Thompson,Oce anSciencesCen tre, Memori al Uni versi t y, pers. com.) .
Haven and Morales-Alamo (1970) fou ndthat th e American oy s t e r, Cr a fls o s t r ea~ (2 years old) exhibit eda sharpincreaseinth e perc entag eof particlesremoved as particlesizein creasedfro m 1~mto between 2 and4~m, with a consistentinflectionpoint at a particlesize of 2 3 p.m, ind ica t ingthatthedist a n c e bet....een adja c ent latero- fron t a l ci liadeterminesthe par t i cl e sizesret ained , and th e absen c e of a mucoussh e e t. Per c e n t ag e remove d le v e l l e d of f at largersi z e s with no incre a s e inef f i cie nc y.
Riisgardttill. (1 98 0 ) found thatjuvenile,M.~ (1 - 4mm long) completelywithheld part icl e sabove 4 ntn, with retent ioneffi c i e nc y gra d u a lly dec r e asi ng below 4sm to 20t for 1 ,u.mpar t i c les. Wil son(l9BO) foundthat while gra z ing
rate s increasewith thesi z e of~edul i s spat, part icle-sizepreference rema ins co n s t a n t .
Va h l (1 97 3 ) has shownthatthe Iceland scall op (~
~), 45.5 - 51.2 mm shell he ight ,eff e ct ivel y retained partic lesdown to 7JIm diameterwith few pa r t i c l e s 1.2 ttmin diameter being retained. This iscon si stent on observationson the queen scallop, chlamys (~ l opercularis (Va h1, 197 2). Sprung (l98 4) su gg e s t e damaximum retentionefficiencyforM. edulisla r v a e of 3.5 JIm diameter, the maximum in ge s ti b l e particlesizebeing 9lim di a me t e r.
1:.3 . EF F ECTOF MIXEDAND UNIALGAL DIETS
For hatchery and nurserypurposes, Lr isimpo r ta nt to know the algaldiet support inggrowth and surviva l of larvae th r ou g h metamorphosisand resultingin optimum growthof the juveniles. Thereare several featuresof the die t which mustbe taken into account when determining whichalgal species wi l l provideth e best food suchas: th e costof production of algae; th e ease withwh i c h the algaeca n be cult uredina lab o r a tor y;the optimum food ration (a l g a l ce l l density) for growth.These features may varyaccording to the bivalve species under consideration.
Some algae support better growt hof bivalves than
others. Wal ne 11~70) studiedth e food value of19ge n e r a of algae , pr e sen t e d as unialgaldiets tojuv e nile bivalves.
Somealgalepeeree resulted in gr eatergrowthde pendingon the spe c i e s of bivalveus ed . Walne (1 970 ) consid e red tha t diff erenc e s in the rigidity of the cell wall or in chemical compos i tio n cannot expl ai n all the res ults obt a i n e d, and he suggested th a t differing food value s maylie inprotein diges t i bi lity as awho leorinso me othe r aspectof pro t ei n chemistry.
As we llas studyi n g the food value s ofun i a l g a l die ts for bivalves, some reeearc be c e ha vealsoco nsidered mixed al g al diets. Wal ne {1974} desc ri bed howdie tscon s istingof mor e than one spe c ies tendtoresult inbetter growth . Epifanio (19 7 9 ) studied th e effectsof 15 diets composedof various mix t u r esof4 spe c iesofalga e on growth of juvenile
Cras s o s trea~and~~. The r e was
littlecor rela t i o nbetwee n gross chemicalcompositionand nutr it i o na l va lueof the diets . 'No n-add it i v e ' growt hwas definedas growthdiffe re nt from that. pr e dic ted by summing the growthre spons e s of theoy st ers fed the ind iv i d ua l componen tsof the diets . The te r m 'synergis tic' wa s applie d to mi x eddietswhic hyie l d grea ter growththan th a t pr o d uc e d by eitherof the dietary components alon e. Obse rved synergismwas sugge s t e d tobedue to micronu t r i e n t s or fa t ty acid compo siti on of the algae.
Romberge r an d Epifanio (1 981) studi e dthe effec t sof th r e e speci es of algae onaes ImiLa c Ion ef f i ciency and growth in juvenileoy s t e r s ,~virg ini ca , fe d si ng leor mixed di et s. In general, it ...as fo u n d that inges t i o n of any two-speciesmixturewas greater thanforany unial galdi et , witha mixeddietconsistin g of Thalassiosira~and Isochrysis galbana resu lt ing in synergistic growt hres po n s e s which cou l d not be expressed in terms of assimil at i on or ing e stio n. It was sugges t e d that thiswas pro ba b l y due to theavaila b ili ty and balan ceof fatty acids or
micronutrie nts suchas vita mins or mi.n e r al s . En ri g h t ~gl. (1986) studiedthe food valueof 16 phytoplankton dietsongr o wt h of juvenileOstrea~
co mp a r e d toa referencediet of IsochrysiRaff. oa Ibane(T- Is o). The highest growthrate swe r e observe dus inga mixed alga l die t co n t a i n i n g fiv e species. In general,the be s t dietshadhighle v e l s of the fatty acids , 22: 6n3 and 20:5n3 and high carbohydrate levels, wh il e prote inleve lsappeared to be less critical. Discrepanciesfrom 1t.ee reeu r e values were at trib utedto su c h differencesas the speciesused, th e strainsandphysiological state of thealgal cells (wh ich is influenced by cu ltureconditionsl, and the ration used.
Davis and Guillard (1958) also determined the valueof te n genera of microorganisms as food for the larvae of oysters (Cr a s s o s tre a ~ )and clams (Me r c e n a r i a
~) . Again,a mixeddiet providedbetter growth than unialgal die t s. The fourbe s t foods foroyster larvae were al l naked flagellates , while clamla rva e were able to utilize several formswithcell wal ls. Toxicity of algal metabolit eswas thought toaffect theva l ue ofan al ga as food, an d a metabolitethat in hibitsgrowth in one species of biva lve ma y promote growthinano t her.
Helm (1977) also found th a t a mixeddietof~
~and~l.m.i§.~resu lted in impr o v ed growt h
in~.f:..!!!.!.l..ila rv ae and provided grea t e r yie lds of spa t.
He concludedthat th e mixeddietprovidesa betterbala nce, such that any deficienc ie sinone speciesar e compensated forbythe otherspecies in the mixture , andsuggestedthat the triglyceridefraction of theal gae mayberesponsible for dif ferencesin thefoodval u e for oyste r larvae.
Ca ry~.io!l.. (1 9 81) found that a mixed di etof
~, MQDOchrysis and ~showeda negat ive
synergisticeffecton growt hinlarvae of the purple-hinge rockscal lopHi.n.n1.t.umultirugasl1s, possibl ydueto ciliate contamination or buildu pof toxic metabol i tes. It was also sh own that so me algae (~ ~ ) resultedin bette rgrowthwhen harvestedinth e stationaryphase,during wh i c h th e cell s contained the lowest carbo h y d r a t e and highest lipi d leve l , whil e oth e r s (Mo n ochr y s ie .l.Y.t.h.u..il su p po r t e d sc a llopgrowth better when har ve s te d inthe
expone n t ial pha se.during wh i c h highlevelsof pro tei n and lip i d wereprod u ced . ~aff.~ IT -I so) produ c e dsim i lar gr owt h whe nharves tedineitherthe exponent ial or th e sta tionaryphase.
The effect. ofye a s t.- a l g a l mixed di et s as foods fo r bivalvesha s al so be e n examined. Ep ifanio (1 979) compared four dietsof varying propor t ions of the yeast~
~and th e diatomThala s siosira~onfour speci e s of b.i.valvemolluscs. I twa s found that Candid.") Y.t.il.i.§.was suitable as a replacemen t fo r up tosot of algae indietsof 3of the 4 species tested. Meangrowthwas not closely related togrosschemicalcomposi tionof thediets oraminoac id composition. Diets hig her inlipi d genera lly yie l d e d gre ate r growth. This isproba b l y du e to lipid quality rat he r than l ip i d quant ity.
I. "• BIOCHKMICALCONSIDERAT IONS
Many researchers have stud iedandcompa r e dalg a l diet s inan att e mpt to determine theop ti mal diet for bivalve s . I t is impo rt a n t tocon s i der why suc hdiet s resu l t in incre a sedgrowth. Altho ugh th e pr e s en c e of a rigidcel l wall mayhaveso me ef f e c t, thi s alone can n o t account for man y of the resultsobtainedand many researchershave looked furt h er toassess the importance ofthe biochemical
co comp o s it ionof thealgae on itsfoodva lue.
In ma ny marine invertebratelarvae, lip id is the maj o r diet ar y reserve mate r i al (Hol l a n d1978). The diffe r e n t stag es of the bivalves'slif e are marked by varyi ng bioche micalcompositions . Holland (1 9 7 8) measuredli p i d reservesand energy metabo lismof benthicmarine inve r tebrates. Marine invertebrateeggs are comp ri s e d primarilyof prot.ein,followedby lipid and then carbohy d r at e . Re s u lts from~~ind i c ate that lipid isthemai n reserveduringthela r v a l stage up to and inc l u d i ngmetamo r p h os i s (Holl a n d ,1978). It appears th at the larvaeutil i s e mainly lip i d st ore s where as theadults st o r e glycogen as an energyreserve.
Holland and Hannant (1974) studied the biochemi cal cha ng es which oc c u r inthe spatofoy s t e r s , Ostrea~, fol l o wi ng met amorphosis. For a few months after settlement , the neutral lip i d conte nt wa s greater th a n the glycogen content. Followingth i s time, glycogenreserveswere ac c umulatedfasterthan neutral lipid so that 3 to 5 mont h- old spat showed greateramountsof glycogen thanne u t r al lipid. Ho lla n d (1 978) associatedthisin c r e a s e in glycogen with an increasein the number ofLe y d i g cells, connective tissuecells scattered throughout the adult oysterand responsible for glycogenstorage. Furthermore,glycogen levels fluctuatedwiththe reproductive cycle, showing an
11
increase in levelsduringthe sp r i ng and earlysumme rpr i o r to gonadri p eni n g, and a decreaseduring th e summer spawning season witha SUbsequent inc r e as e inth e autumn.
Ho lla nd (19 7 8) suggested !:.ha t the po ns Lb Le reasonswhy larvae st oremainlylipidwhe r e as adu l t s store glyc og e n concerns the fact that lipids can onlybe ut i l i zed aerobically, whereas carbohydrates canbe used eit he r ae r obic a l l y or anaerobi cal ly . Glyc og e nsto r a g ema y enable the adul ts to surviveanaerobi cco ndi ti ons, suc has when they are exposedatlowti d e. Another impo r t a n t diffe r en ce may lieinthe li f e s t y l e s, efnc e th e larvae ar e plankt oni c and theadu l ts eeeeLl.e. Inthefree-swimming la r valstage thestore dli pid will alsocontribute more to the buoyancy of the animal.
Whyte~ll. (1 9 8 7) identified lipid and prot e inas major components and carbohydrate (gl uc a n l a minorco mpo n e nt in the eggs of the scallop Patinopecten yessoens is, with total energyle v e l s twi c e those observed in thelar v a... of other scallopsand oysters. The stored glu ean, probably gly c oge n , was depleteddu r i ng ear lylarval developmen t.
During development, scall op larvae utilisedreserves of lipid and protein for approximately20days, after whi ch lipidwas accumulated as the larvae approached metamorphos is, a process requiring sign i f i cantenergy
12
Whyte IIli. (1990) correlated the nutritio nal con d it ionofro c k sc a ll o p (~ ~ l la rva e wit h di e t a ry carbohyJrate. LdpLd,and protein. The gr e ates t energy reserves wereobse rved in larvae feda diet withthe highestle v el of carbohydrates, ratherthan lipid or protein. Furthermore, itwas shownthat while th e levelsof 20:503 or22:603fattyacidsdo not effect thenu t r it i o n a l condit ion ofthe larva e, a cer t.a in threshol dle v el of essential fattyacids mus t be met in order to maintain nutritional levels. Apparently, requirementsfor fa tty acids are moreeasily satisfiedthan for other micronutrients.
Langtonand waldeck (1 98 1 ). st udying Cr a s s o s tre a~ sp a t, foundthateither 20: 503 or 22:603 coul:i fulfi l l the higher n3 polyunsat uratedfatty acidrequirementsfor-th e spat which are una b l eto producelong-c ha i n fatty acids of the n3 family. A dietarydeficiencyof these fattyacids was found to limit growth.
Enright II 21. (1986) studiedthe growt hof juvenile Ostrea edulisEedChaetoceros gracilis of va r y i ng chemical composi tion. This was done by varying the nutrient conditionsof th ecu l t u r e. Cells cultured in completef/2 nutrientmedium (c o n t r o l) and a silicate· limitedmedium had a simi larprotein content, whereas cellsfrom a nitrogen- limited medium contained approximately 60%lesspr o t e i n .
13
Allthr ee cul t u re shad a similar amino-acid composition.
The lipid contentof silicate - limitedcellswa s more than twice that of the control or nitrogen -limitedcells. At the lowest ration levels, the silicat.e- limiteddiet produced the highestgrowthrate. Thiswa s attributedto the fact that sincenoneof the diets pro bablyhadsuf ficie n t caloric content for ma ximumgrowth, the increasedamount of fatty acidscouldbe used to supply energyso the proteinscould be us e d for growth. At the highestfeedingratio n , the control cultu r e re sul te d in the bes t growt h . The control had 3 timesthe 22 : 6n3 conten t ofthe silica te-li miteddiet, whichmay be impo rtan t once thecalor icre quirements are
The hi g h carbohydratecontent of the ni t r o g en-l i mi t e d dietseemsto be of little use if the protein contentis limiting.
Holland (1978) poi n te dautthat bothn3 and n6 polyunsa turated fa t ty acids are importan t for me mb r a n e flu i d ityandessential membra ne functions. The dou b l e bonds formedin then3 fatt yacidsmay be respons ib le for re t a ining membranefluidityatlow andrelat ive lyconsta nt tempe r ature s, su ch as arefo un d inthe marineenvironment.
Other adva n tagesof li p i d as anene rgysource includethe fact tha t itis an effic ientfue l, espe cially fo r organis ms that ma y underg o pe r i odsof food shor t a g e s or cr it i c al de velopmentalchanges in th e i r life historie s. Ld.p Ld sar e
14
also impo rta n t fo rbuoyancy , mai n t e nan c e of body structure and thermal insulation in variousmarine cr c enreme.
Whyte(19 8 7 ) comparedthe chemi ca l composition inbo t h the stati onaryand the exponent ial phasesof growt.h. of th e followingspeciesof algae: (1) Thalassiosirapseudonana, (2 ) Chaet.QcaTos calci t rans , (3) ~ap., (4)
~~, (5) !sochrysisaff.qalba na (T-IsQ)and (6 ) Te t ras e l mi s ~. I twas foundth a t bothspecies of
~at bothstages of growth hadth e highe s t lipid levels. The diatomsexaminedhad higher levelsofmo no- ol i g o s a cc h a ri de s and total carbohydrates than did phytoflagellates. Withthe exception of Tha l assios ira, the phase of gr owt h had little effecton the polysac charide content. The phytoflage llateshad hi gher tot al ni t r oge.n co nce n t r a tion s thar.the diatoms , witha greater concentrationobserved du ri n g the stationaryphase. The con c en t r a t ionof free amino acids,pept.Ldes an damines was similar for most species. Th e algae studiedwererankedas followsinterms of energy from the constit uents: (1 )
~aff. ~ (T - I s o), (2) ~~, (3)
Chaetoceros~, (4 ) I.~, (5)1:. pseudonana and (6 )Ch a e t o c e r o s sp.
is
I.5. ENERGYREQUIREMENTSOFLARVAE AND JUVENILES I.S.L Fi l tra t i o nRate l
Two approachesto the studyof the passageof water throughbival ves ar e: (1 1 direct meth ods, which separateand measure the exhalentflow, and givea ·pump ing rate" ;and
(2) indirect methods,whic h measure..filtration or clearance rates" (the vo l ume ofwa t e r cleared of suspende dparticles per unit time), us ing the rate ofre mo v a l of suspen ded mate rial from aknown volum e of wat e r to estimat e the rate offlow through the mantleca vity (Owen, 1974). Owen (1974 ) also givesfour assumptionsof indirect methods when determiningfilt e r i ng rates:
(11 the reduct ionin the concentrati on of particle s is dueto filtration;
(2) the an imal'spumpingrateis ccnscanc , (3) particle retention is 100 \ effi cient ;
alternatively, a known co nsta n t percentage is re t a i ne d; and
(4) th e test suspension is at alltimes homogenous. Aninverse re lationshi p between filtration rate an d alg a l concentr a t ion (below thre s holdle vel s ) has be e n reportedfor pecti n ids (Pa l me r andWillia ms , 198 0 ; Malo uf andBr i ce l j , 1989). Genera lly, above a thr eshol d conc e ntrationof about60 cell slld (2 mg dryweight/II, the ing es tionrate becomesind ependent of concent r a tion (Palmer
16
an d Wi ll i a ms , 1980) un t i l very highconc e n t r a ti o n s causethe filteringmechanism toblock .
In general, filtrationrates decre asewith incre a sing cell concentrat ions (winter, 1978;Owen, 1974; Sc h ul te , 1975;Foster-Smith, 1975 ) . In terms ofha t c he ry- p r od u c e d bivalves, itis important to determinethe optimum food concentrationwh i ch is charac te r ized bylow filtration ra te s, no production of pseudofaeces (particlesfiltered, bound in mucus and eliminatedbeforein g e s t i on ) and complete inge s ti o n of food particles. This concentration has been te r me d "pseudofaeces-free ce l l density" (Wi n t er. 1978) or
"critical cell density" (Schulte, 1975). If tbe re isno productionof pseudofaeces, the amount of food ingestedis equal to the amount of food filtered.
As algal concentrationinc r eas e s fr om minimum levels, the bi v a l ve filters more algaeper hour, althoughthe water volumepassing throughth e gills decreases (Schult e, 1975).
Sprung (1984) foundthat with mussel larvaein dense food concentrations, there wa s a constant.ingest.ion rat.eover a wide range of foodconcent rationsand a decliningrate with increasing foodco nc e nt r a tio ns. In dilute food
co nc e ntr a t i o n s , there was a constant. filtrationrateand an inc r e a s e of ingest ionrate withinc r e a s i ng food
concentration. Sc hulte (1975) reportedthat filt r a tio n ratesat differe nt concentrationschan ge considerably in
17
short periods of timeand, generally, filtrati on ra t e s de c r e a s e wi thtime at all concentrations.
Winter (1 97 3) also foundvarying fi ltra t i onratesin
~ ~under constant experimental co ndit i onsand algalconcentrations. As wel l, the re s u l t s showed that filtration rates and amount of algaefiltered increasedwith inc r e a sin g bodysize,while filtrationrate pe r mgdry weightof tissuedecreased with increasingbody size. I t was shown that musselsof the same bodysize fil te re d approximatelythe same amountof algaeat highor lo w concentrations, withlower concentrationsbeing count erba l a nc e d by higherfiltration rates.
There is an allometric relationshipbetweencle a r a nc e rate and body size : CR=aWb, whereCR=cle arance rat e (l /h ) and W~ ti s su e dry weight (g) . Va l u e s forthe parameters (a) and(b) are given in Bri cel jand Shumway (1 9 9 1) forarange of pectin ids ; the weight exponentbis variable, ranging from0.58 to0.94 ,with a meanof 0.7, which is within the range reported for other bivalves.
It has been observedth at different bival vespeciesmay ha v e different filtrat ionrates over a period of time. For example, Palmer (1 9 8 0 ) foundth a t filtrationactivity remainedrelativelyconstant over a period of 24 to 33 h for
~ ~t;Qn c e n t r i c u s, although CraS90strea
~exhibitedhigh and low periods of filtration
a.e
act i vity. Itwas sugg e s t e d that this serves to re gu l a te inge s tion rate .
I.5. ii. Inge.tion l
Bayne (1983) re portedthat inge stionrateva r i e d directly withpartic l e conc e n t r a tion until ath r e s ho l d conc e n t r a t i o n was reachedabove which a furtherincreasein co n c e n t r ationdidnot result in an increase in ingestion rate. Th isis referredto as the "fu nctio n a l response" to food. The rate at which cells are ing ested is a functionof clearance ra t e , part icleconcentration,particlesiz e and the rate at whi c h th egut is empt ied .
1.5.i i i. EfficienciesI
Fo r aquacul t urepurposes , it is notonlyimpo r t ant tha t growth bemax im ized.butalsothat the optimumgrowthcan N.
achi eve d withthe leastpossible amount of food. Growth can be descrihedinterm s ofenergy or calori c cont e n t, wet weight or dry weight. Cris p (1971) su g gests th a t growth effici ency ra t i o s sh o u l d bebasedon energy contentrathe r th an we t or dryweight, but if onl ywet or dryweigh t is known, the term "conversionrate " shouldbe us ed .
The literatu re (Sprung , 19 84 ; Jorgensen, 1976 ; B..yne and Ne wel l, 1983;Warren and Davis, 19 67 ; Crisp, 1971;
Morton.19 8 31describes different types of effici encies
19
which describeenergybudgetswithin an organism.
As s i mil a tio n eff iency (AE)refers to the proportionof ingestedfo o d (I ) usedfor growth (G) and res pi r a t i o n (R) (a s s i mi lat e d ) , and ia given by:
AE- {{R+GJ / I] x100
Gross grow t h effi ciency (Kl) refers t.o the proport ionof ingested foodtha t is converted to growth, and is givenby:
Kl '" (G / r] x100
Net growt h ef fi c i e n cy (K2) refersto thepr op o r t i o nof as simi l a t e d foodwhich is convert ed int o growth, and is gi ve nby:
K2..(G / (G + R) 1 x100
sincetheamo unt of food assimUated is always le s s thanthe amountof foodingested, K2 is alwaysgreater th an Kl.
Growthefficienci eshave been determined inte r ms of dry weight, ni trogen, ph o s pho r u s, or ca rbo n con tent (Co r ner and Davis, ~971).
The ter m"mainte nanceration" hasbeenused to describe therate of food co ns umpti on whi c h allowsmaintenance activities t.o be performedwithno increase in biomass
(Crisp, 1971 ). Anotherterm that has been widelyused to desc r ibeenergeticsin mol lu scsis "scope fo r growth", which isdef ined as"t h e difference betwee nthe ene rgyof th e food an anima l consumes andallother energ yutil i satio nsand losses " (War r enand Dav i s,1967) . Thescope for gr owthand
growthefficiencies mayva r ywi ththe species,sizeandag e of the bivalveand with rati on level, temperatureand salin ity (Ba y n e, 1983 ; Bayneand Newell, 1983 ) .
I.6. OBJECT IVES
Ex per ienceatthe pilothat chery at theOS~/MSRLbased on twelve successivespawnings (Dabi n e t t , pers.comm. )has shownthat survivaland growth ofla rva e throughsettlement to postla r v a e of 1 mm in shell height (i . e . the hatchery phase) is predi ctableandofunifo r m rate. Inco n t r a s t , nur s e r y growt hof pest-larvae from 1 mm shel lhe ight onwa r d s has beenun p r e dic t a b l e . Growthrateshave fluctuated indicatingthat conditionssuch asdi et , ration, and physical conditionssuch as temperature and water flowwe r e not ideal.
This study wa s undertakento investigatethe use of unialgal,binary andter n a r y algal mixtures of available algal speciescult u r e d tosupport scallop
(.e.
magellanicus ) growth wi thnursery-sizedanimals (>1 mm shellheight ).These cultures were~ ~,~ (Ta h i t i a n
strain) T-Iso , ~ ~and Chaetoceros
~. Furthe rmore, a commerciallyavailable spray- driedheterotrophically growncultureof Tetraselmis~ knownas ALGAL161was testedfo r efficacy as a die t supplementfor juv e n ile scallops in a hatchery-nursery
21 situation.
The hypothesiste s t edwa s th at , bas e d on stu dies previously ci te d inthisch a pt er , growth ofju v e n ile scallops on the variousalgal diets wou l ddif fer significantlywith the prediction that the ranked or d e r wouldbe ternary, binaryandun i a l g a l diets respective ly.
Conversionefficiency or gross growth ef f iciencyis a useful index whichhas beenus ed in the cult u r eof many speciesenabling hatchery managersto opti mizegrowthand minimize foodco s t s. Afu r t h e r aimof thi s study was to inv est igatetheus e f u lne s s of conversion effic iency ina hat cheryculturing scallopsa po ten t ia l tool to descr ibe th e effectiveness of hatcherypractice.
The effect of fo od rat i o non filtra t i onand ingestio n rates for threesizecla s s e s of juv e n il e£.... magellan i c us spat was investiga tedtocomp l e me n t th e di e tstudies. Information was thereby provided for hatcherymanagement on both the dietand rationto help optimise the growthof spat in nurseryculture.
22
II MATER.IALSAND METHODS
11.1. SPAWNING ADULTSAND REAR:INGLARVAE,POST - LARVAE AND SPAT
Sca llops foruse as broodstock we r e collectedby SCUBA di v e r s from natura l populations in Newfoundlandin late May , threemonths priorto natural spa wning. Malesand femal es wer e put into separate tanks in ambi entseawate rand fed cu lturedalgae, at approxima t e ly2\ drywei g h t of the scallop drywe i g ht perday, for 6 weeks prior to spawning.
At the time of spawning, a male and femalewith ripe gonads we r e chosen. Th e shellsof thechosen scallopswere cleaned, measuredandla bell ed .
The maleand femalewe r e thenplacedin a single plasticta nk (34x~8x 16cm) containing 10 1 seawater filteredto 1 11m and aanitised wi t h INat lSQC. Water was vigorously recirculated over thean ima l s with a pump until spawningcommenced. When t.he male spawned, the water became frothywit h bubbl es. Whe",l the female spawr.ed, the water be c ame pink due to the color of theeg g s. Onceeither of the adult scommencedspawning, the pump wa stur ne d offand theadult swe r e put into cleanfil te red seawaterin separa te containersto complet e the spawningprocess.
Ifthe ferneLe hadnot spawnedbythis point, shewa s inducedto spa wn by reimme rsion inthe origi nalfiltered seawa t er towhic h sp erm hadbeenadde d fora short period of time . Alt ernatively , the pumpwas usedaga into st i mulate spawning.
23
Once the male and femalecompletedspawning inthe separatecontainers, the adultswereremoved from the egg or spermsuspensions . Aco un t of the egg suspensionwas done usingaCou l t e r counter. The requirednumber ofegg s was removedand re-suspendedin a vo lume of filt e r e d seawater. Sperm was addedto this egg suspension to obt aina rat ioof 2 - 10 sperm per egg based on observations using dark field microscopy. 1\higherratio is not recommendedsince this may cause polyspermy, which may result in lysisofthe eggs or abnormal larvae.
The newlyfertilizedeggs were hel dinpyre x dishes(3B x26x5em) un t il the y reached the D-veligerst a g e3days later. Fil teredseawaterwa s put intoeach di sh , tog e t her with the antibiotic ne omy c i n at a concentrationof 0.0 2 5gil as a precautionarymeasureto control bacterial growth.
Ap p r o x i mate l y 70,000 fertilisedeggs wereadd e d per dish (100eg g s / c m2area) ina fin al volumeof 750 mI. The di s he s were covered and maintainedat 15°C.
The larvae wereleft for 3 days unt i lthey reached the O·veligerstage. They were then removedfrom the dishes by filtrationusing a submerged 50nm mesh screen. The lar vae were thenput in a 1000 1tank (lml) at a density of approximately1/ml and fed a diet of Isoch rys i s~, Isochrysisaff.~ IT -Iso ),Chaetoceros~and :r.b.a.l.sut.~pseudonana, mainta inedat a concent ra tionof
24
12 cells/ltl in a ratio of 2:1phytoflage llatesto diatoms.
Wate r in the tankswaschangedei thertwice or threeti me s weeklyandreplacedwi t h fresh filtered seawater. After 10 days, the larvae were counted using a Coulter counter. This procedurewas repeated for approximately 35- 40 days while gradually inr:reasingthe food supplyto 30 cells/ltl, by whi ch time the larvae began to show adhesivetende nc i es .
Corrug~tedplastic sheets were hung in the tanks and left for4- 6we e k s with fresh filte redseawater and food addedda ily by displ ace ment. During this time the larvae underwentmetamorphosi sto post-larvae, set or spat.
After 4- 6we e ks , the spat weregently brushed off the plastic sheetsand placedon either300/lm or 500 /lm mesh screensin a 10001tankcontaining filtered seawater. The wa t e r in the tanks waschangedby displacementdaily andthe tankswere cleaned appr ox i mat ely twice a month. The ration ofalgaefed was increa s e dasrequiredbythe spat mai nt a i ni ngafoodconcentra t ionof 30 cells/pi.
Spatwer e sizegr a ded usingmesh screens to obtain sizes appropriateforthe feedi ngand growt hexpe r iments.
II.:2. ALGAL CULTURE
A culture collectionof variousalgal species was maint ainedat a consta nt temperatureof 15°C. These unialga l cultures (althoughnot axe ni c) we r e maintained in
25
20ml,cul t uretu b e s with '2/ 2 mediaWu il l a r d,1 97 5l, and transferred every month.
Algal culturesfor feeding broodstockandjuv e niles we r e grown in250 1 fibreglass cylinderson F/2 growth mediumund e r continuous ligh t fromcoolwhite andday light fluores c e n t tu b e s at 20°C. Thevolume ha r v e sted eve ry 2 - 3 days was replacedwith fresh filt ere d seawat er and PI?
medium. Cultureswe r e counteddai_y usi ng a mode l Zf Coulter Counter with? 100 ILmaperture or i f ice tube, and the volumeof culture requiredforfeed ingwas cal cul at ed.
Cultures forfeeding larvaewere grownin2 or 41 Erlenmeyerflasks on F/2medium. These cultures wer e ha rve ste d every 2 or 3 days andfr e s hfilt e r e d seawater an d medium weread d e d. Algalcell countsweremade andthe appropriate rationwas fed to the larvae .
Cultures for feeding juvenilesin experimental work were grown in101 glass battery jarsinn o cu l a t ed with 2 1 of algae collec tedfrom4I Erlenmeyer fla sks. Cul tu r es were harvested every1- 2 days and fresh filteredseawater and mediumwe r e added. Al g a l cell countswe r e performed daily using a Coulter Counter to det.ermfno the appropriate culture volume for the ration required .
All seawaterus e d for the culture of algae was passed througha series of fourfil t e r s: a prefi lterdesignedto remove sedimentand large part icles , followedby a series of
26
three Gelman cartr idge fi lte r s of pore siz e s 10 jJrn,3 nm andl~m. The filte redseawater was the n passedthr o ug h a Trojan Te c hn olog i e s Inc.UV 405water steril izer. When the te mp e r a t u r eof the ambientseawaterfell belowaoe, the filtered seawater was he a t e d to 10 - 11" C. Air was bubbled throughthe filteredseawater during heat ing to ensure propermi xi ng and avoidsupersaturation.
I:I. 3• ALGALCUL'I'tJRESUSED IN GROWTH EXPERIMENTS Fourspeciesof algaewere used in eitherunialgal or mixedalgal diets . These species were chosenbecause the y proved to be suc c ess fu l in rearingE.magellanicYs la r va e through metamorphosis . The four species emp l o y e d included
twophy toflagella t es, ~ ~and~aff.
galbanA. (T~ I s o),both hi g h- t e mp e r atur etolera n t species , fromth e Cl a s s Prymnesiophyceae, and twodi a toms , Chaetoceros cal ci t r an s and Chaetoceros ~, both from the ClassBacillariophy ceae. In addition, adr i e d alga!
fo od, knownas "ALGAL 161" (Ce ! l Systems , Cambridge, Eng!and) wa s us e d inExp t. 4. ALGAL 161co n s i s t s of spray driedce ll s of heterotrophically grown Tetuse'mis~ (Cl a s s Prasinophyceael (1 gramof ALGAL 161 is equivalent to Sx 109cells). The necessarymass of ALGAL 161to givethe requirednumber of cells was addedto100- 200 ml fil tered seawater and mixed gently.
27
II.4. ALGAL DRYWEIGHTANDORGANIC WEIGHT DETERMINATIONS Fiveto ten coun ts were pe r f ormed on threeseparate aliquots of each algalcultureto determinecell concentra tion. Measuredvolumesof thealgalculture, in the rangeof 10 -300 ml, depe nd i n goncell concentra tion , were filt ered th r ou g hpr e -we ighe d , ashed Wha t ma n GF/C fil t ers usinggentle vac uum. The cell s in the filtra te were al so count ed, and the to tal number of cellscaugh t onthe filte r wasdete rm i ned . The cells on the filter were rins ed wi t h 5 - 10 ml 3\ ammoniumformate (isotonic wi th seawater) and the fi l te rpapers were pl a c e d in aluminum we i g h i ng pa n s and dried to a consta nt we i ght at SDOCfor 24 hours.
Once algal dryweightswere determined. thefilters and al u minumwe i g h i ng pa nswerepl a c e d in a muffle furnaceat 450°Cfor 5 hoursto completelyoxidise an d remove any organicmaterial. The resulting ino rga ni c weightwas subtractedfrom the dryweight togive th eorga nic we i g ht of theal g a e.
II.5 • THE EFFECT OFDIE TONGROWTH:
Table1gives a summa ryof th e ex perimentalconditions andal gal speciesused ineachofth e fou r expe rimentsto dete r mi n ethe ef f e c t of diet on growthofjuve nil e£....
magellanic u s .
"
Tab le1. Summary of experimental factor s and algal combinationsused in growth experiment s.
Er.perlment
a
Sca.llop shell he:'ght (IMI) St ock ingdensity(II/ll Food rationleells/~ l) Age of scallops (mon t h s) Temperature (OCl
Algaldiet (11 Iso
(2) eh
m
(4) Iso ;Ch Iso / ell
(5) ChiT-I so Ch;T~ Iso
(6) Iao;Ch; Iao iCh ;
T-Iso r- a s e 171
IS'
tao;T-Iso
e .
c.ceaT-Iso 161; 1 8 0 Iso / ell rscrc.car cnrr - rse 'r-aeerc. cai Iso/Ch; i~~~~h;
T- Iso
taol T-lso laa l e . c a l;
T-Iso sta rved st a rve d control control
Iso "'~ ~
ell ..~muelleri
'r-aec..tsachrysis~(T-Iso) 161 ..ALGAL161
C.cal.~~
:n.S.i . Experiments 1 and 2;
Scallopsaged7months (Exp e ri me n t 1) and 11 months (Exper iment 2) were size graded using mesh screensto give a
29
size classof 2. 4 mm shellhei g h t. scallops (200 ) were pu t on each of 3screen s suspended in tanks contai n ing 60 li t r e s of filteredse awa t e r standing in a wet be nchto ma int a i n tempera tureat 10° C. Water wasci r cula ted ove r the sc r e ensusin g ai r : ift s to cre at e <ldownwe l l i ng circulation of water overthe screens. Water was changed 3time s per weekby displacement.
Eachtank was batch-fed algae daily a rationof 50 cells/ILl with the mixeddiets consist ingof equal porti ons of each constituent species based on cell number.The water wasmi x e d thoroughly to resuspend any set tledal galcells prior to samplingfor cell co untsbe causeno control tanks were available.Cell co un t s were madeda ily us i nga Coul t e r Co u n t e r andgr owth (s h e l l height, dry wei ghtand organic we ight ) was mon itoredevery two weeks by subsampl i ng 30 scall op s per screen.
II.5. ii. Experiment3:
Sc all op s aged 3 months were size gradedusin g1 mm and 2mm mesh screens. Thirty-twoexperimental ta n k s were set up infourwaterbathsat 7°±loC. Batchesof 100 0juveniles were placed on screens suspended in the tanks in10 1 filteredse a wa t e r , soth a t the density of animalswas 1 scallopper 10ml filteredseawater. Three replicates we r e set up for each tr eatment, together wi t h controlswith
30
noanimals todet e rm ine the se t t l i ng out of algal cells in the ca l c u lat ionof fi l tration ra tes. Water was ci r c u lat ed, andfoodke pt in suspensio n ,bygentlebubbli ng andan airlift do wnwel le r . Fresh filteredseawater was adde d 3 timesa weekby displacementat a flow ra t e of approximately B1/ min. for5minutes.
The ration of the appropriate diet was batch- fe d daily at ;:;0cell s / Joll wi thmixed diets made up from equal ratiosof eac hcomponentspecies basedon cell number.
Cell countsweredone dailyusi ng aCou l t e r Co un t e r an d growth (sh ell he i g ht. dry weightandorganic weight )was moni toredeverytwoweeks bysubs ampl i ng SOsc a llo p s per tank.
I:I.5. i i i . bp" r iDl8 nth
Juve nilesage d 9 monthswe r e size gr a d e dusing 2mm and 4mmmesh scree n s resul tingin selec t ion of scallops 2 4 mmin length. Thirty-twotanks wereset up inthree water bathsmaintainedat a temperature of 10· t:lo C. wit h three replicat esof eachtr e a t ment, plus con trolswithno animals. 16 0scallo p s we r epl a c e d on screens suspendedinth e tanks cont a ini n g101 fil t ered sea wa ter.The densityof ani ma l s was 1 scallopper 63 ml fi lt e r e d seawat er. Conditions were similarto experiment 3, except that~ ~and Alga l
31
161were testedas dietarycomponents. Growth was monitore d bysubsampling 20 scallopspe r tank every seven days.
II.6 • GROWTH DETERH:INATI ONS : :Il:.Ei . i . Shel l Height:
The required number of scallops were removedfrom the expe r imentalrear i ng tank. Scallops sized2- 4 romwe r e placed in a petri dish and an imag ewas recordedon a photocopier. The sca llopimageson the photocopy we r e then impo rtedinto Jandel Scientific's Sigma Scan program
(veretcn3.90) for measure me nt of shell height. Scallops 1 2mmin size werevideotaped on a JVCERvideotapeusing a wildM420MicroscopeandCohu Mo de l 4615cameraconnectedto a JVC model H4-D 4400 videocassette recorder. Scallopswe r e sized usi ng ima g e ana l ysis (JAVAR program,Version1.20, JandelScientific).
:l:l:.6.i i . DryWeigh t :
Thewholescallopsused for shell height
det ermi nat io n s we r e filtere donto a pre-ashed, pre- we ighed WhatmanGF!C filt e r usinga minimu m volu me of filtered seawater. Th e scallops>wer e thenrinsedwith 5 - 10 ml isoton i c ammoniumformatedriedat 90 ° C (24hours) and weighe d .
32 II.6. i ii. organicWeight.l
Aluminum weighing pans containingfilterpapers and scallopswere placed in a muffle furnace at 450°Cfor 5 hours to removeany organic components. The organic weight was determinedby subtracting the re ma i n i ngin o r ga n i c component fr o m the dryweight.
11.7 . DETERMINATION OF INGESTIONRATES
Since the ration used (5 0cells/p.l) wasassumed tobe below the crit icalcell co n c en tration (Hollett an d Da b inet t, 1989), that is, the concentr at ionabove which pseudofaeces isproduced. the number of ce llsremovedfrom suspension was considered tobe ingestedby the animals. Therefore, the ingest i o n rate lc e ll s/h) was calculatedbydeterminingthe number c·fcells removed from suspensionover a period of time.
Thedryweightor organi cweight of cellsingest.ed can thenbe determi nedby mul tiplying th e numberof cells inge s t ed by the drywe i g ht or organic weightpe r cell of the appr o p r i a t ealgae.
II. 8. DETERMINATIONOFGROSSGROWTH EFFICIENCY Gr oss gr owt h effici ency (Kl) is growthper uni t of inge s t e dration. Becaus e respir a t i o n ra tes wereno t
33
determined, assimilated portion offoo d could notbe determined and net growth efficiencies (K2) were not ca lcu l a t e d. Becausethe algal rationus e d was belowthe level ne c e s s a r y for productionof pseudofaeces , the fo od removedfrom the suspen sionwas equal to the fooding e s t e d by the animals.
Growthwasmeasuredas an increasein organicwei g ht per animal during a specified period of time. The numbe r of cellsingestedby the animals was de termined us i ng the Cou l t e r Counter. The organic weight of ce l l s inge s t e d was ca lcul a t e d bymult i plyingthisnumbe r by theorg a n i c wei ghts of the algal ce ll s as showninTa b l e 2.
II.9. EFFECT OF RATIONON FILTRATION ANDINGEST ION RATES Anexperimentwas done todeterminethe effectsof rat i o non filtrationrates for th r e e size classesof juv en ile.e...magellanicusage d5 mont hs. Th e t.hree size cl a s s e s studied were 2- 4mm shell height, 4 - 6mm shell he i g h t and>6 mm shell height. The re qu i r e d sizes were collectedby gently sievingscallops throughappropriately sized submergedscreens as in previous experiments. The thr e e food rations st ud i e dwe r e20 cells;"ll, 40cellsfl,t.l and 80cellS;"!! '
Thir tytankswe r e setup infourwa t e r baths. Each tankcont ained 10 1 filtered seawater. Water was circulated
34
andfoodkept in suspensionbygent lebubbling and a downweller. Temperatureineach tankwasmai n tai nedat 7·C - 7.SoC. Th~r ewere threereplicatesof eachofthe followingtreatments:
(l) 72 scallops (2) 144 scallops (3 ) 2Se scallops
<4} 20 sca llop s (5140sc a llo ps (6) 80 sca llo ps (7) 12 scallop s (8) 24 sca llops (9) 48 scallops
(2 -4 rom) at a rationof 20cell s/pI (2 -4 mm) at a ration of 40 cellshl1 (2 - 4 mml at a ration of 80 cells/pI (4 - 6 mm) ata ra t i o n of 20 cells/Joll (4- 6 rom) ati!lra t i o n of 40 cells/pI (4 - 6mml at arationof 80cells/p I (;,. 6mm) at a ration of 20 ce llsht l (:>6mm) at araeLonof40 cells/pI (:>6mm) at a ra tion of 80 cel l s / ill and controlsat each cell de nsity with no ani mal s .
Thenumbe r ofscallop swasincreased withincreasing rationinorder for any de creaseinrationtobe observed in theha l f-ho u r measuringtime. The ex pe r ime n t lasted 30 hours. Counts weredoneever-y 30 minu t e s and algae were added tomaintainthe appropriatelevel using a 1:1:1 mixtureofL.. ~. ~ ~and.L.~(T- I s o l based on cell number. Anothe rcount was then done 30 minutes later and the number of cells removedfrom suspension was usedto determ ineingesti on rat e. Cell count s wer e do neusinga model Z~Coulter Counte r with a 100 J.l.rn apert u re orif ice tub e .
Foranimals2 - 4 mm shell he ig ht , orga n icweig h tswe r e determi ne d using the wholeani mal . For those ani ma lssiz ed 4- 6mm and:-6 mm shellhe ight.org anicweights were determine dseparately fo rtiss ue andshe ll s.
35
II.10. DETERMINATION OFFILTRATIOJIANDINGESTIONRATES The ce llsremoved fromsuspen s i on du r inga 30~minute samp ling peri odwer e consi de r e dtobeinges t edby the scallops, as therewa s no product i on ofpseudofa e c e s and settling was neg ligible. Filtration ra tewas determi ned using the followingformula (Co ug hla n , 196 9 ):
m ..M/In*t) log.conco/Conc~
wherem..fi l t e r i ng rate of animal M..volumeof suspen s ion n..number of animals t ..period of time
Cone..andcone,..conce ntra tions ini tia llyand after time t insuspe n s i on
Filtra t i on rat e s we re det e rmi ne d for eac h 30-minute samplingperi odand an aver a g e filtr a t i o n rat e was calculated for the entir e expe r i me nt a lperi od for each experimentaltank (ml/scallop/hr). Dividing th e se filt ration values by th e organicweight of animalsin thetank(mg/ s calloplresulted in fil tration rates per un i t we i g h t of animal (ml /mg/ h r).
Correspondinginges tionrateswere determi nedbymulti p lying these fil trationra te value s by the conc entrationof fo od in the ta nk (c ell s/sca llo pj hrl and specif i c ing es tion rates (ce lls/mg/hr )we r ecalcula t e d.
36 II.11. STATISTICALMETHODS
All statistical analyses.were performed using 5AS (St atistical Analys i s syecee!, deve Lcp.ed by SAS Ins t itut e Inc.,Cary, N.C••USA.
Thegrowthdat afo r th e juv en i l eswas su bj e ctedto atwo- way analys is of va ria n c e with in te r a ction using the catego r ica l varia b les of diet and ti me. Aprobabil ity of greater tha n0.0 5 fortheWtest fo r normal ity (Sha p i r o - Wil ks test) wa s accepte d. Residual plots we re examined for ind e pe nde nc eand con s tantvariance .
In those case s where vari an c e wa s not co nsta nt , a log tr a ns f ormation of thedatawa s pe r f o rmed befor e ana l ys isof va r iance. In ce r t ai ncase s, wher e a log tr a nsformatio n did not result in cons t a ntva r i an ce, outliers were removedor a weigh t e d ana l ys is of variance wa s perf o rmed. Of the twelv e ana lys es perf o rme d . only two did not result in a p value gr e ate r than O.os in thewtestaf tereuchmanipu l ation s . In thesecases,examination of thenorma l ity plot s ind ica tetha t the yare close to no r mal and are symmetrical.
Time 0 points were not included in the analysis si nc e on ly one average initial sample was taken for all the experimental groups.
Duncan's means testswereus edto test fordifference s between dietsat individual samplingti mes .
37
Two no n- p a r a me t r i c tests, Friedman 's method for randomi ze dblocks andthe Krusk a l l-wallistest, we r e used to test for significance in thegrossgrowthefficiency data.
For thest u dy of the physiological rates of filtration and ing estion,log phys iological rat e wa s plottedagainstlog orga nic weig ht and thepl o t inspe c tedfor linearity. Alinear regress i on wa s performed to determine theparametersfor the allometr ic equation relating physiological function IF) to size (o r ga n i c weight) namely: F .. aWb• The values for the constant 'a'andwe i gh t expon ent"b' wer e determined from the linearregre ssion of the log transfo r med va r i a bles namely:
lo g F= loga +b logW (SakalandRohlf, 1969).
38 XII RESULTS
IIL l .CHARAC1'ERI ST:ICS OFALGAL SPE CIES
The cell cha rac teris ticsof th e algalspecie s usedin thi s stud yare given in Table2.
Ta b l e 2. Ge n e ra l charac teristics of thealgalspecie s used in Experiments 1 - 4.
Spec i e s Dry weight Orga ni c weig h t Meancell (p g/c e U ) (pg/celll diamet er
Mean±sd Mea n±sd (~I
~ 21.Btl.0 19.81 0.9 3.8
~ (n
.
10 1cbeecocerea 26.4±2.4 16.l,t1..6 3.S
l!lllOllll.i. (n _71
1·aU. 30.5:t:2. 1 26.1±1.0 S. '
~ (n - 61 T- Iso
Cha e t o ce ro s 14.4±l.B 10.5 i 1. 0
4.'
~ (n • 81
The weights and sizes found inth is aeudyare in gen eral agreement with thosereported in th e literature , whichfor comparisonarepresented inTa b l e 3.
39
Table 3. Literatu re va lue s for algal cell characteristi cs. Species
Isochrys is 23. 5
galbana
Isochrys isaff. 20.1
~(T- I s o)
~galbana 16.1
Isochrysis galbana 24.2
~ a f f. 30.8 galbana (T - I s o) Chaetoceros
~ IsQchrysis afE.
~(T -Iso )
~aft.galbana
(T - I s o)
so u rce
Epif a n io and Ewa r t (19 77\
Urb a n ~.li.
(1 9 8 3) Rombergerand Epifan io (1981) Manning (1 9 86 ) Ma n ni n g (19 8 6 )
Lai n gand Mi l l i can (1986)
20 Laingand
Milli can (19 8 6) 5 x 2. 5 Caryl l .sj..
(1 9 8 1) 5x2.5 Ca r y g,t..el..
(19 8 1)
The algal species used were in the sizera ng e of 3~ 5 JLmdiameter. ~ aff.~ (T- Iso) ha d thehi gh e s t dry and organicweigh tsand thegr e ates t cel l diameter. The Chae t o ce ros ~cells, although not the sme Lte ee , had the least dry and organic weights. The other two species studied, Chaetoceros.!!l!Lillsu.:1and~ ~!!fall between these two exc reeee. In terms of organic weight supplied to theju v e n i l e s , the dietswere ranked as fo llowS
