Nutritional Value -of Marine Harpacticoid Copepods as Live Food for Marine Fish Larvae.

BY

Dominic Andre Nanton

Submitted in partial fulfillment of the requirements for the degree of Master of Science

Dalhousie University

Halifax. Nova Scotia

CANADA

May, 1997

O Copyright by Dominic Andre Nanton, 1997

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TABLE OF CONTENTS

TABLE OF CONTENTS .............................................. ...... ..... .. ..... i v LIST OF TABLES, FIGURES AND PLATES v i

0 0 0 ABSTRACT ............................................... ...... ............ . . . .... VI II

ACKNOWLEDGEMENTS ............................................ . ............ .. . ...... i x

Chapter 1 Fatty Acid Composition of Haipacticoid Copepods

and their Nutritional Value for Marine Flsh Larvae

O F m O N COPEpOD UpiDs IMnODUCTION . . .. . . . . . .. .. . . ... . .. . ... . . .. .. . .... .. . .. . . .. . .. . . .. . . .. . . . . . .. . . . . . . . . . . . . 1 5 MATERIALS AND M E M O D S . . ~ . . . . . . . . . . . . . . . . . . ~ ~ m . . . . . . . . . m a l 6

COPEPODCULTURE ...................................................... . ......1 6 ALGAL C U L T U R E . . . . . . . . . . . m . . . . . . a . . ~ . . . . . . o . . . . m 8 LlPlD ANALYSIS .................................... . ................... ........1 9 STATISTICAL ANALYSIS ..................... ........ .. ..... .. .. . . . . 2 3

RESULTS . ............... ..... ........ ............... . ......... .... ...... ......... ....... 2 3 DISCUSSION . . . .. . . .. . . . . . . . . .. . . . . . .. . . . . .. . . . . . . . . .. . . . . . .. . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . O

EFFECT OF DlET ON COPEPOD UPIDS. ............ ... . ... .. . . .. . . ... . .. . ..A O NUTRITIONAL IMPLICATIONS FOR MARINE FISH LARVAE . . . . . ... 4 7

IMRODUCllON . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .. . m l MATERIALS AND MRHODS ................................... ...... . . . . ...... . 3 RESULTS ............................................. . ............................. ........... 5 4 DISCUSSION . e . . . . . . . . . . . . . . . . . . . . . . m . . . . . . . m . . . . . . . 6 O

EFFECT OF TEMPERATURE ON COPEPOO UPIûS.. .. . . .. . . . ... . .. . . . .. .6 O NUTRITIONAL IMPLICATIONS FOR MARINE FISH LARVAE . . . . . . . .6 4

ID COMPOSITION OF qllVlNAtlVE -eFC(ES INTRODUCTION . . . . . .. . . .. . .. . . .. . . . . . .. . .. . . . . . .. . . .. . . . . . .. . . . .. . . . . . . . . . . . .. . . .. . .. . . . . . .6 6 MATERIALS AND METHODS ............. ........... . . .. . . . . . . . . . . . . . . . . . . . . . . . . . .. 6 6

. . . . . ....... ............................................................ . .

DISCUSSION ........................................ 5 LlPlD COMPOSITION OF ALTERNATNE UVE FOOD SPECIES ..... 75 NUTRITIONAL IMPLICATIONS FOR MARINE FlSH LARVAE ........ 77

Chapter 2 Free Amino Acid Composition of Harpacticoid Copepods

and their Nutritional Value for Marine Fish Lawae

.................... ............... ............................... INTRODUCTiûN .. ..i 2 FREE AMIN0 AClD M E T ' L I S M IN COPEPODS ...................... 82 FREE AMIN0 AClD METABOLISM IN MARINE FlSH LARVAE ....... 84 EXPERIMENTAL OBJECTIVES . . . ~ . m . ~ . . . . ~ . . . . m m ~ . ~ . m . . . ~ . . a m 8 6

MATERIALS AND METHODS .............................. ... .......................a... 0 8 RESULTS ....................................... 1 DISCUSSION ....................................... 9 5

Chapter 3 Mars Culture of a Harpacticoid Copepod Tisbe sp .

..... ............................................. INTROOUCTION ................... .. .. 0 1 ............................................................. MATERIALS AND MtlTnODS 102 RESULTS AND DISCUSSION .am.e.mmmmmm.mm..m~~ . . m m . . . m ~ ~ m m m m m m l 0 5

Chapter 4 Preliminary f rials Using a Harpacticoid

Copepod. Thbe sp., as a Dlet for Marine Fish Lawae

.................. ......................... JMRODUCTlON ...................... . . 1 1 ...... ................ MATERIALS AND MEMODS .. .............................. 1 1 4 RESULTS AND DISCUSSION .................... ... .. .... .....................m... 119

GENERAL CONCLUSIONS ........................................................ 2 7 APPENDICES

A . LlPlD ANALYSIS TECHNWES ........................... ... .................... 130 B . ASTkWüWHIN MEASUREMENTS ................... ... .... .... ...... ....A 32 C . FREE AMIN0 AClD IXITWCTION ............................. ..... ............ 135 ....................................... BIBLIOGRAPHY ........................... ... 1 37

UST OF FIGURES

Fig . l a . Fig . 1b . Fig . 2 . Fig . 3 . Fig . 4 . Fig . 5 . Fig . A1 . Fig . A2 . Fig . B I . Fig . 62 .

The n-3 and n-6 families of fatty acids ......................... 4 Alternative pathway to A 4 desaturase ......................... 5 Photographs of the harpacticoid copepods .................... 17 Tisbe sp . and Amonardia sp . Diagram of the nsbe mass culture systern ................ 103 Population counts from the nsbe masa .................... 106 culture system . Photographs of haddock l a ~ a e fed Tisbe .................. ..l 25 Bligh and Dyer lipid extraction method ...................... 130 Methyl ester formation method .................................. 131 ..... Visible absorption spectrum for wild zooplankton 133 astaxanthin at different wavelengths . Astaxanthin standard plot and regression .................. 134 analysis .

vii

ABSTRACT

Live food organisms containing relatively high concentrations of 13-3 highly unsaturated fatty acids (HUFA) are required for the first-feeding of cold-water marine fish lantae. In particular, long- chain fatty acids such as 20:5n-3 and 22:6n-3 are essential. Two species of harpacticoid copepods isolated from the Atlantic Oœan near Halifax, Nova Scotia, Tîsbe sp. and Amonardia sp., demonstrated the ability to elongate and desaturate 18:3n-3 to 20:5n-3 and 22:6n- 3 when fed a diet deficient in these essential fatty acids (EFA). Decreasing th8 culture temperature from 20 to 6OC increased the proportion of long-chain EFA (20:5n-3, 22:6n-3 and 20:4n-6) in the copepods. Both harpacticoid copepod species maintained a 22:6n-3 to 20:5n-3 ratio of greater than two for al1 dietary (the algae Chaetocenrs calcitrans, DunalieMa te rtiolecta , lsochrysis galbana and baker's yeast) and temperature (6, 15 and 20°C) regimens, suggesting their usefulness as a live food for marine fish larvae. For corn parison. trochophores of the common mussel Mytilus edulis and wild zooplankton obtained from St. Andrew's N.B. were examined for use as potential live food and were also found to have a high percentage of long-chah EFAs.

The relative amounts of free amino acids (FAA) in Tisbe rernained f airly consistent despite large differences in the amino acid composition of the diet. The propoition of essential free amino acids was nearly doubled in Tisbe fed 1. galbana (clone T-iso) compared with Tisbe fed yeast.

The harpacticoid copepod Tisbe was mass cultured in a system which produced nearly 10' individuals in a relatively small tank volume of 32 L.

P reliminary feeding trials comparing growth and su wival O f marine fish larvae (American plaice and haddock) fed rotifers or Tisbe were conducted. The plaice and haddock larvae were observed feeding on the harpacticoid copepods. Tisbe, when compared w i th the rotifers, gave superior growth but poorer survival of the haddock larvae.

I would like ta extend thanks and appreciation to al1 the

membem of my supervisory cornmittee, D~s. JmD. Castell, I.A.

McLaren, J.S. Craigie, SBJB Iverson, R a Ackman and my external

examiner, Dr. C.C. Parrish, for their advice and comments regarding

the research.

Special thanks to Dr. J.D. Castell foi his daily guidance and

encouragement, to Dr. I.A. McLaren for organizing the finances, and to

Dr. J.S. Craigie for the amino acid analysis of the copepod samples.

1 would like to thank L. Boston foi showing me the l ip id

analysis techniques and for her helpful advice. Statistical

assistance was kindly provided by DrB R Rodgers. Appreciation goes

to Dr. S. Johnson and Dr. R. Huys for harpacticoid copepod

identification. Thanks must also be extended to Dr. C. Monisson and

J. Martel1 for the sectioning and preparation of the lawal haddock

slides. I am grateful to the Aquarium staff, F. Rahey, P. Fraser and B.

Bassett, for their help in the setting-up and maintenance of the

culture systerns.

I would also like to thank my family for their support and

encouragement.

This work was funded in part by a research grant from Atlantic

Fisheries Adjustment Program (Dept. Fisheries and Oceans, Canada), the New Finfish Aquaculture Species Prograrn (CanadiadNew

Brunswick Cooperative Agreement on Economic Diversification and

Aquaculture Development for Nontraditional Species), and the

Patrick Lett Fund (Dalhousie University).

Chapter 1

Fatty Acld Comporit ion of Harpact icoid

Copepods and Implicatlonr on their

Nutritional Value for Marine Firh Larvae

GENERAL INTRODUCTION

OVEMON

Aquacuîture is the most significant growth component of the

Canadian fisheries industry. Salmon culture in the Maritimes now

represents an estimated 100 plus million dollar industry (McGeachy

et al., 1996). The world wide growth of salmonid culture has

resulted in a depression of th8 market value, and consequently

interest in the culture of other valuable marine fish species such as

halibut Uippoglossus hippoglossus, haddoock Melanogrammus

aeglefinus, flounder Pseudopleuronectes americanus and cod Gad-

morhua. lnterest in marine fish culture has dramatically increased

with the recent failures in the commercial fishery for northem cod

and other important fish stocks. There hm been some success in

rearing hali but, cod, sea bream Spanrs aurata, turbot Scophthalmus

maximus and other fish species in Noway, France, the United

Kingdom, Spain, Japan, Canada and other countries eround the world.

The main bottleneck for fry production in the species

mentioned above and other cold-water marine fish is associated

with larval first feeding. First feeding occum when the endogenous

energy resewes in the lama's yolk sac are depleted and the larva

must begin to feed exogenously. Lawal mortalities are generally

greatest at this stage. The high mortality is, in part, due to the lack

of nutritionall y adequate live food organisms. Fish l a ~ a e req u i re

live food organisms which have relatively high concentrations of the

long-chain, n-3 highly unsaturated (HUFA; M double bonds) or

essential fatty acids (EFA) such as 20:5n-3 (EPA; eicosapentaenoic

acid) and 22:6n-3 (DM; docosahexaenoic acid). These are essen tial

because the lawae do not have th8 necessary A-6, A95 and putative

6-4 desaturases to synthesize these fatty acids (FA) from shorter

chain n-3 fatty acids (Fig. 1). The bnne shrimp Artemia sp. and the

rotifer Brachionus plicatifis, which are widely used as live food f O r

cfustacean and fish lanral culture, have limited value in the culture

of cold-water marine fish larvae because of their low long-chain

EFA levels. Marine copepods, which are the principal cornponents of

the natural diet of many marine fish lawae, have higher amounts of

the long-chain EFA which makes them an attractive alternative l i ve

food source for the first feeding of cold-water manne fish larvae

under hatchery conditions.

My first objective was to evaluate the nutritional value of

indigenous marine copepods as live food for use in the commercial

Essential in Diet

Fig. la. The n-3 and n-6 families of fatty acids. The slanted and vertical arrows represent elongation and desaturation reactions, respectively. An alternative pathway to the putative 6-4 desaturase is depicted in Fig. 1 b.

Fig. Ib. Alternative pathway for the biosynthesis of 22:6n-3. The slanted arrows represent elongation or retroconversion. The vertical arrows represent desaturation reactions. The pathway al t e rnative to A-4 desaturase was discovered in a radio-isotopic study of rat liver microsornes by Voss et al. (1 991).

culture of locally important marine fish larvae such as halibut, cod.

flounder, and haddock. This was to be achieved by establishing

cultures of the native harpacticoid copepods, Tisbe sp. and

Amonardia sp. The effects of algae and yeast in diets, as well as

those of temperature on the essential fatty acid composition of the

copepod populations were then to be detemiined.

FArrY AClD REQUIREMENTS OF MARINE flSH L A M

Most marine fish larvae have an essential requirement for the

n-3 long chain HUFAs, 20:5n-3 and 22:6n-3 (reviews: Greene and

Selivonchick, 1987; Watanabe, 1982). Cowey et al. (1976) were

among the first to examine the fatty acid requirements of marine

fish. Turbot Scophthalmus maximus juveniles were fed isocal O ric

diets containing differing amounts of the n-3 series of fatty acids.

They demonstrated that weight gain and protein efficiency fat i O

were highest in the turbot when polyunsaturated fatty acids (PUFA)

of the n-3 family were present in the diet. Scott and Middleton

(1979) examined the EFA requirements of turbot larvae, and

observed that the larvae had better sunrival and growth, when fed

rotif e ts which contained elevated concentrations of 20:Sn-3 and

22:6n-3 in their lipids. The ratio of DHA to €PA also significantly

affects the suwival of marine fish. lncreasing the ratio of DHA t o

EPA from 0.1 to 0.5 in the diet of turbot markedly decreased the

mortalities (Bell et al., 1985). There is other evidence for the need

of a high DHA to €PA ratio in the diet of marine fish lawae. For

example, the yolk of wild marine fish eggs contains a DHA to EPA

ratio of about 2.0 (Parrish et al., 1999, and this high ratio has been

observed in the polar lipids of copepods, the natural prey of marine

fish lawae (Kattner et al., 1981).

Tocher et al. (1 989) incorporated various radio-labelled PüFA

into cultured turbot fin cells to detemine which specif ic

desaturation and elongation reactions were taking place. The ce1 ls

exhibited A-6 desaturase activity converting 18:Zn-6 to 18:3n-6 and

18:3n-3 to 18:4n-3, however, they could not convert the supplied

18:3n-3 into 2031-3 indicating either a lack of 6 5 desaturase or of

the C-18 to C-20 elongase enzymes. Tocher (1993) found that the

A-5 desaturase was also lacking in the turbot brain astroglial cells.

The enzyme activity required to biosynthesize 22:6n-3 was also low.

When 20:5n-3 was the sole PUFA incorporated into turbot astroglial

cells, the elongation reaction of 2Mn-3 to 22:5n-3 predominated

over the reaction converting 22:5n-3 to 22:6n-3. This may explain

why turbot and other cold-water marine fish have sudi a large

dietary requirement for 22:6n-3 (Bell et al., 1985).

Arachidonic acid (AA; 20:4n-6) is another fatty acid which may

be considered essential fo i marine fish. Castell et al. (1 994)

discovered that, when turbot S. maximus juveniles which had

previously ben fed a diet with high proportions of 22:6n-3 but

deficient in 20:4n-6, were fed a diet containing 20:4n-6 as the sole

HUFA, they expenenced higher growth and suwival than those

supplied 22:6n-3 as the sole HUFA Arachidonic acid is the major

fatty acid precursor of several important eicosanoids including

prostaglandins, leukotrienes and hydroxytrienoic acids. Bell et al.

(1 995) discovered that increasing the amounts of 20~411-6 i ncreased

the levels of prostaglandin (POE and PGF) in the tissue homogenates

of the juvenile turbot.

The live food organisms traditionally used for warm-water

fish culture, the brine shrimp ARemia salina and rotifer Brachionus

plicatilis, are not as nutritionally valuable as copepods for col d-

water marine fish larvae. Brine shrimp cannot synthesize or

incorporate significant arnounts of the 22:6n-3 fatty acid which i s

crucial for the survival of most marine fish larvae (Watanabe et al.,

1978). Davis and Olla (1 992) fed lamal walleye pollock Theragra

chalcogramma a diet of Artemia sp. only, and found reduced g rowth

and suMval. However, when wild-caught copepods were added t O

the diet, growth and suwival of the lawae improved. Rotifers can

synthesize highly unsaturated fatty acids (HUFA) to a lirnited extent,

but to supply sufficient amounts of HUFA to marine fish lanrae, the

rotifers must be fed EFA-rich food (Lubzens et al., 1985). Kitajima

et al. (1980 a,b) demonstrated that ayu Plecoglossus altlvelis and

red sea brearn Pagnrs major lawae cultured with rotifers which

were fed bakers' yeast supplemented with cuttlefish liver oïl (high

in EFA) had superior growth than those fed a diet of unenriched

rotifers.

The value of the trocophores of the mussel MytiIus edulis, as

an alternate live feed for marine fish larvae, was evaluated by

Howell (1973). They did not support growth in lawal plaice

Pleuronectes platessa and sole Solea solea. However, if a diet O f

trocophore lanrae is followed by rotifers, the diet can support the

development of lemon sole to the point when newly-hatched Artemia

nauplii c m be used by the lawae for further growth t o

metamorphosis (Howell, lQ7l).

The future of marine fish lawal culture may lie wi th

fomiulated micro-particulate feeds. Micro-particulate diets WOU Id

eliminate the need to culture algae for consumption by live food

organisms thus simplifying larval culture. They could also eliminate

the need for live food if microcapsules could be fed directly to the

marine fish Ianrae. Up to the present, such diets have been

unsuccessful as food for manne fish lanrae. This is thought to be

due primarily to the deficiency of appropriate digestive enzymes i n

the incomplete digestive system of the earliest larval stages

(Walford, 1991). Munilla-Moran (1 990) proposed that exogenous

enzymes supplied by live food organisrns such as copepods play an

essential role in their own digestion in the gut of the marine f ish

larvae.

COPEPOD UPlD METABOUSM

Watanabe et al., (1 978) suggested that it is the copepods' high

HUFA levels that make them nutiitionally valuable for marine fish.

It is well established that manne calanoid copepods, parti CU l arl y

those from northem latitudes. are generally rich in lipid which i s

made up prirnarily of wax esters. In calanoid copepods the lipids can

mach 80% of the dry weight with the wax esters making up to 90%

of the total lipid (Sargent and Henderson. 1986). The marine wax

esters are contained in oil sacs which nm parallel to the gut and

consist primarily of long-chain fatty alcohols (usuaily saturated O r

monounsaturated) esterif ied to fatty acids (often pol yunsaturated) . Copepods accumulate wax esters after phytoplankton blooms (spring,

early fall) and we them as fuel resewes during prolonged periods

(usually winter) of food shortage (Sargent and Henderson, 1 986).

The long chain EFAs for cold-water marine fish, 20:5n-3 and 22:6n-

3, are prominent in the lipids of these copepods (Ackrnan et al..

1974; Sargent et al., 1977; Kattner et al., 1981; Nonbin et al.,

1990). For calanoid copepods the large amounts of n-3 HUFA are

probably incorporated directly from their phytoplankton diet. Prahl

et al. (1 984) demonstrated the selective absorption of n-3 HUFA i n

calanoids by feeding the green alga, Dunaiieiia primolecta, t O

Calanus helgolandicus and observing a drop in the n-3 HUFA

composition of the food as it passed through the gut to f o n faecal

pellets. Stattnip and Jensen (1 990) demonstrated that, because O f

the inability of Acartia tonsa to elongate and desaturate 18:3n-3 t O

longer chain HUFA, this calanoid shows reduced growth and egg

production when fed Dunaliella tertiolecta, an afgae deficient i n

20511-3 and 22:6n-3. These findings were confirmed with a series

of studies looking specifically at the fatty acid content of the diet

and relating it to egg production in the calanoids Acartia

(Jonasdottir, 1994; Jonasdottir and Kiarboe, 1996) and Temora

longicomis (Jonasdottir et al., 1995). The fatty acid requirements

were for high n-3 to n-6 and high 22:6n-3 to 20:5n-3 ratios. The

fatty acid 22:6n-3 was also positively correlated with egg

production for both species.

Harpacticoid copepods, which are benthic with some

exceptions, live in an environment with a more stable food supply

than calanoid species. They are known to feed opportunistically ,

efficiently utilizing various food sources such as vegetables (Kahan,

1 979), polychaete tissues (Guidi, l984), bacteria (Rieper, 1 Q78),

unicellular algae, detritus (Ustach, 1 QW), dried mussel powder,

yeast and macroalgae (Miliou and Moraitou-Apostolopoulou, 1 99 1 b) . Therefore, they do not need to accumulate wax esters to the same

extent as calanoids, and their lipid composition is typicalty around

10% of their dry weight (Miliou et al., 1992). Norsker and Stprttrup

(1 994) discovered that a harpacticoid copepod Tisbe holothuriae

does have the ability to elongate and desaturate the shorter chain n -

3 HUFAs from Duneliella tertiolecta to produce relatively large

amounts of the essential far( adds 20:5n-3 and 22:6n-3. When fed

D. tertiolecta, egg production of the harpacticoid was not

significantly decreased by the lack of long-chah €FA in the diet

when compared with Rhodomonas baltica, an alga with a relatively

high content of long-chain EFAs.

Poikilothermic animals, including copepods, tend to increase

their unsaturated to saturated fatty acid ratios as tem peratu res

decrease. The increase in this ratio is an adaptation thought t o

allow membrane fluidity to be maintained at lower temperatures

(Hazel and Williams, 1990). Farkas (1979) noted a large increase i n

the PUFA in the phospholipids of freshwater copepods as the

temperature decreased from approximately 20 to 1O0C. The largest

proportion of this increase in PUFA consisted of the EFAs, 22:6n-3

and 20:Sn-3. The levels of total PUFA including 20:5n-3 and 22:6n-3

were also obsewed to increase in the lipids of various species of

marine calanoid copepods in the North Sea during the colder winte r

months (Kattner et al., 1981).

OBJECTIVES

The nutritional or EFA value of indigenous mafine harpact i coid

copepod species as lhre food for the commercial culture of

important marine fish larvae (Le. halibut, floundei, cod, and

haddock) was evafuated by:

a) establishing cultures of native harpacticoid copepods;

b) determining the effects of diet (algae and yeast) on the fatty acid

composition of two species of cultured harpacticoid copepods (Tlsbe

and Amonardia) ;

c) detemining the effects of temperature on the fatty acid

composition of the cultured harpacticoid copepods (Tisbe and

Amonardia) fed the same algal diet;

d) comparing th8 fatty acid compositions of the two harpacticoid

copepod species (Tisbe and Amonardia);

e) cornparing the essential fatty acid composition of the

harpacticoid copepods with other potential native live food

organisrns for marine fish larvae. Wild zooplankton (made up almost

entirel y of calanoid copepods) and 24-h-old f ertilized eggs and

trochophores of M ' u s edulis were the other live foods evaluated.

Effect of Diet on Copepod Lipids

INlRUMlCTlON

One objective of this study was to determine if the

harpacticoid copepods are able to elongate and desaturate shorter

chain n-6 and n-3 fatty acids such as 182n-6 18:3n-3 into the long-

chain EFAs 20:4n-6. 20:5n-3 and 22:6n-3. This ability can be

detennined by feeding the copepods diets which have only trace

amounts of these EFAs (Le., the alga, Dunalieilla tertiolecta. and

bakers' yeast) and then detemining the copepods' fatty acid

compositions. Norsker and Stattrup (1 994) discovered that the

harpacticoid copepod. Tisbe holothuriae. does have the ability t O

elongate and desaturate the shorter chain n-3 HUFAs in Dunaliella

tertiolecta to produce relatively large amounts of the essential

fatty acids 20:5n-3 and 22:6n-3. lt was, therefore, expected that

our indigenous harpacticoid copepods (Tisbe sp. and Amonardia sp.)

would also posess the sarne desaturase and elongase enzymes needed

to produce 20:5n-3 and 22:6n-3 from the shorter chah n-3 f at ty

acids such as 18:3n-3.

The other main objective was to examine which diets gave

higher levels of the n-3 and n-6 essential fatty acids and high DHA

to EPA ratios in the copepod lipids. These parameters are crucial

for the survival and growth of cold-water marine fish larvae

(Watanabe, 1982). The copepod diets were chosen on the basis o f

their large differences in long-chain EFA composition. lt was

expected that, because of the harpacticoid copepods' elongating and

desaturating abillties, they would maintain relatively high levels O f

20:4n-6, 205n-3 and 22:6n-3, and a high DHA to EPA ratio even when

fed long-chain €FA deficient diets.

MATERlALS AND MElHWS

Copepod Culture

The harpacticoid copepods, Tisbe sp. and Amonardia sp. (Fig.

2), were captured and isolated from the plankton in Halifax Harbor,

Nova Scotia, Canada in Febniary 1994. They were cultured for over

10 generations on a rnixed algal diet at 20°C (generation time was

approx. 2 weeks). The calanoid species, Acartia hudsonica and

Eurytemora herdmani, were also captured in the same tow, but due

to low production rates and poor survival the numbers necessary for

lipid analysis were not obtained. The harpacticoid copepods we re

raised in 6-L cylindrical plexiglass (McDonald) jars containing

seawater (32 ppt salinity) which had been filtered through 10 prn

Fig. 2. Photographs of the two marine harpacticoid copepods evaluated as potential live food organisms for marine fish larvae. A. Female Tisbe sp. with nauplii (anow) hatching from egg sac. B. 10% formalin preserved femele Amonsrdie sp. with egg sac. Scale bars = 100 Pm.

pore size cartridges and UV treated. Oxygen saturation and wate r

circulation were maintained with air flowing through airstones at

the bottom of each cylinder. Temperatures were maintained a t

2WI0C. The seawater was changed approximately twice per week

by collecting the copepods on a 40 pm mesh and transferring them to

jars of fresh, filtered seawater. Three replicate cylinders of

copepods were used in the feeding trials and the algae and yeast

were fed to the copepods in excess (ca. 1 mg dry weight/L of sea

water) each time the water was replaced. The copepods were

cultured in this manner for approximately 4 weeks pn'or to l ipid

analysis.

Algal Culture

Pure strains of the algae lsochrpis galbana (UK isolate),

Chaetoceros calcitrans and DunaIieIIa tertioiecta were purchased

frorn the Provasoli-Guillard Center for Culture of Marine

Phytoplankton (West Boothbay Harbor, ME). ErJenmeyer flasks (2 5 0-

m l ) containing 150 mL of autoclaved sea water and f/2 medium

(Fritz Algae Food, Leland, MS) developed by Guillaid and Ryther

(1 962) were then aseptically inoculated with the pure strain of

algae and stoppered with cotton wool to allow for gas exchange.

The cultures were maintained at 18OC for 3-5 d before being

transferred to 1 1-L carboys. The 1 1 -L carboys contained 10 L O f

autoclaved seawater. 2 mL of Fritz Algae Food f/2 Solution A and 2

mL of Solution B were then added. The carboys were maintained a t

18°C and bubbled vigorously with air. Carbon dioxide was added at a

flow rate of 0.5 Umin. The algae were hawested while in the log

phase at 6-8 d (approx. 5 million cells/mL) for feeding to the

copepods.

Lipid Analysis

Samples of the algae used in the feeding trials were separated

from the culture medium by centrifugation. The algae were

harvested in the log phase and equal volumes were poured into 2 5 0 -

mL plastic flasks. These flasks were placed in a 5.75 in. maximum

radius rotor and spun at 5000 rpm (g force = 334) for 20 min using a

Serval RC28 centrifuge. The seawater was decanted. The algal

pellet was then scraped out of the plastic flask and analyzed f O r

lipids and dry weight.

Adult copepods were collected on 200 pm mesh nylon screens

and transferred to jars containing fresh 10 Pm filtered, UV-treated

sea water. They were then stanred for 24 h to allow for the

clearance of dietary algae present in the gut and rescreened t o

remove any accumulated waste products. More than 200 animals per

replicate were isolated in this manner for iipid analysis.

Subsequently, the copepods were suction-filtered on Whatman no. 1

filter pape? and rinsed with distilied water. The copepods plus 1 0

mL of chlorofonn:methanol (2:l v/v) were then homogenized in a 20-

mL glass culture tube using a polytron (8n'nkman Instruments).

Total lipids for the diets and copepods were extracted using a

modified method based on that of Bligh and Dyer (1959). For details

of method see Appendix A; Fig. Al. If, after removal of the

chloroform, the total amount of lipid was too small (4 mg) for an

accurate weight measurernent, a known amount of an interna1

standard fatty acid (23:O) was incorporated in the sample t o

estimate lipid weijht. Methyl ester derivatives of the total f ip id

were prepared by adding 2 mL 7% BF3 in methanol to the lipid sample

in 15-mL glass tubes with teflon-lined screw caps and heating t O

1 OO°C. After cooling, the methyl esters were recovered by adding 5

mL anhydrous sodium sulfate and extracting twice with 4 mL

hexane. The hexane was evaporated and the methyl esters were

purifieci by thin layer chromatography. For details of method see

Appendix A; Fig. A2.

The fatty acid methyl esters were t k n quantified using a

Varian 3400 mode1 gas liquid chromatograph (GU) equipped with a

hydrogen Rame ionization detector (FID) and a polar column

(Omegawax 320 flexible fused Mica capillary column; 20 m i n

length x 0.32 mm ID). The injection temperature in the GU= went

from 100 to 250°C in 0.51 min and was held at 250°C for 1 min so

that the sample was fully vaporized. The det8~tor temperature was

300°C, and the oven temperature was programmed as follows: the

initial colurnn temperature was 160°C increasing to 240°C at 3.S°C/

min for 22.85 min and then held for an additional 12 min at 240°C t o

clear any residual material from the column. Total nin time was

34.85 min. The pressure of the helium carrier gas was set at 82

kPa; gas flow through the column was 3.0 mumin. An inboard data

handling system detemined retention times and integrated the

areas under each methyl ester peak. Individual peaks were

tentatively identified by compaiing their retention times w i t h

known standard fatty acid methyl esters, typical of marine oils,

which were previously identified in an Ornegawax Column Test Mix

(Supelco, Bellefonte. PA).

Identification of some peakl not present in the reference

sample was f acilitated through the use of hydrogenation. The peak

that iepresented an unsaturated fatty acid methyl ester w i l l

disappear after hydrogenation and the equivalent chain lengt h

saturated fatty acid methyl ester will increase by an amount equal

to the previous unsaturated fatty acid methyl ester. Thus, we can

determine the carbon chain-length for the unknown unsaturated

fatty acid methyl ester. The methyl ester sample to be hydrogenated

was placed in a 250-mL Erlenmeyer flask with 20 mL methanol and

approximately 0.5 mg of platinum oxide catalyst. The flask was

flushed with hydrogen gas two or three times stoppered and left t O

stir (magnetic stirrer) for 1 h. The plaünum dioxide was removed

from the sample by suction-filtering the methanol through Whatman

no. 1 filter paper and rinsing with with 20 mL hexane. The sample

was then evaporated to dryness under nitrogen and the same volume

of hexane used for the unhydrogenated sample was added for dilution

prior to injection onto the GLC (Christie, 1982).

Statistical Analysis

Signif icant differences in the lipid compositions of the

materials examined were evaluated by analysis of variance (ANOVA)

using Systat 5.1 for personai cornputers (Wilkinson, 1990). Tukey's

HSD (Highly Significant Difference) test which is a post-hoc

paiwise comparison test was used at the pS0.05 level to compare

diff erences between means. The fatty acid compositions we re

calculated as a percent of total lipid. Data represented as

percentages are not normally distributed (the tails of percentage

data distributions are shortened due to the finite O and 100% l i m i ts)

which violates the assumptions for ANOVA. The data were therefore

normalized or transfomied using the arcsine of the square root o f

the proportion (arcsine X ln) before ANOVA was applied.

RESULTS

Analyses for the ash weight and lipid percent of dry weight of

the three algae and one yeast diet are presented in Table 1. The

diatorn Chaetoceros calcitrans had the highest ash weight due to the

presence of its siliceous shell. The ash content of the flagellates,

Dunaliella tertiole cta and lsochrysis galbana, were similar a t

around 10% dry weight. lsochrysis galbana had the highest lipid

Table 1. Ash weight and total llpid content of copepod diets.

Dunafiella tertiolecta 8.95 * 2.15 25.67 f 3.09

Chaetoceros caIcitrans 24.30 * 2.33 21 -67 f 4.24

Isochrysis galbana 10.65 1 .O0 28.41 f 0.99

Ba kers' yeast 5.12 î 0.85 2.19 i 0.14

Values are mean (n-3) f standard deviation on a dry matter basis.

The algae was washed with ammonium formate pikr to ashing.

content, followed in decreasing order by D. tertiolecta and C

calcitrans. Both the ash and lipid contents of the bakers' yeast

were very low.

The three species of aigae in this study were chosen because

of the large differences in their EFA compositions (Table 2). Lipids

of I. galbana had significantly more 22:6n-3 than the other diets

(about 1/4 of the total FA) and a small amount of 20:5n3.

Conversely, Cm calcitrans contained significantly more 20: 5n-3

(about 1/3 of the total FA) compared to the other diets and only a

small amount of 22:6n-3. 0. tertiolecta had only trace amounts of

both 20:5n-3 and 22:6n-3. However, Dm tertiolecta lipid had

significantl y higher amounts of the 18-carbon fatty acids, 1 8:2n-6

(almost 10%) and 18:3n-3 (almost 50%). The content of 20:4n-6 was

present at low levelç in al1 diets (<0.3%), but was significantly

higher in C. calcitrans than in any of the othei diets. These extreme

differences in the EFA levels allowed a cornparison of the effects of

dietary €FA on the fatty acid composition of the harpacticoid

copepods.

The bakers' yeast diet (Table 2) had small percentage of both

20:Sn-3 (not significantly different from the levels in I. galbana)

Table 2. Fatty acid compositions of the algae (C cal= Chetoceros calcitrans, D tert- Dunaliella tertiolecta and I gal= lsochrysis galbana) cultured at 18OC with f/2 media and of bakers' yeast used in the copepod feeding trials. Diffeient letters indicate sig ni f ican t differences at pc0.05 level.

Fatty acid C d 0 tert lad Yeast

mm 7.7 f l.7r 9.4 f 6.h 10.0 f 4.8r 23.7 f 0.66 33.8 f 2.3r 11 .O i 2.5b 23.5 & 3.lc 89.8 I 1.2d

C PUfA 57.1 f 1-68 76.1 f 5-36 65.1 & 6.6. 5.8 I 0.7~ C ri-3 37.4 f 3-98 57.8 * 3-86 51.6 & 6.5b 4.6 f 0 . 7 ~ C n-6 5.3 f 0.4. 15.1 * 5-76 11.8 f 0.9b 0.8 f 0 . 1 ~ DHAEEPA 0.1 f 0.0. 2.0 I 1-46 12.2 f 3 . 1 ~ 1.1 I 0.4a C UNK 0.6 f 0.4 1.6 f 1.3 0.2 f 0.1 t r

Results are expressed as 45 of total fatty acids and represent means and standard deviations of 3 repficates. Unknowns el% not included in table. Abbreviations; nd= not detected, tr= trace (<0.05%), 7 Me= 7 methyf, SA'= saturates, MûNQ monounsaturates. PUFk polyunsaturates, UNK= unknowns, DHAt docosahaaenoic acid, E P h eicosapentaenoic acid.

and 22:6n-3 (not significantly different from the levels in C

calcitrans). The fatty acid composition of bakers' yeast d i f f e red

markedly when compared with the algae. The levels of saturated and

monounsaturated fatty acids were at least double that of the dosest

algae. The fatty acids, l6:l n-7 (20.4%), 18:l n-7 (27.3%) and 1 8: 1 n-9

(17.9%) were the major contributors to the high levels of

monounsaturates.

The fatty acid composition of the harpacticoid copepod, Tisbe

sp., fed these four organisms are presented in Table 3a. The yeast

was fed to the copepod at a different time of year than the algae

(Jan., 95 W. Aug., 94, respectively), so that other factors, such as

season, may have affected the fatty acid composition of the

copepods when compan'ng the algaf and yeast diets.

In Tisbe, the relative amounts of the 18 carbon fatty acid,

1 8:3n-3, incorporated by the copepod corresponded with the amount

in the algal diet. 18:3n-3 was highest in D. tertiolecta (45.0%) and

the copepods fed with this diet. It was much lower in copepods fed

with C. calcitrans and I. galbana. This was not the case with 1 8:2n-

6. Tisbe fed with I. galbana incorporated slightly higher amounts

of 18:2n-6 than Tisbe fed with D. tertiolecta, evee though

Table 3a. Fatty acid composition of Tisbe sp. cultured at 20°C and fed algal (C cal= Chaetocem calcitrans, I gai= Isochrysî" galbana and D tert= Dunaliella tertiolecta ) and bakers' yeast diets. Differences in lettering indicate signîficant differences at the pc0.05 level.

l4:O l 4 : l n-7 l 4 : l n-5 l5:O DMA16 l6:O l6: l n-7 7-Me16 l 6 : l n-5 16:2n-6 16:2n-4 l7:O 16:3n-3 16:4n13 16:4n-1 DMA 18 l8:O 18:ln-9 l 8 : l n-7 l 8 : l n-5 18:2n-9 18:2n-6 18:Zn-4 18:3ri-6 18:3n-4 18:3n-3 18:4n-3 20:o 20:1 n-11 20:1 n-9 20:1 n-7 UNK 20:2n-6 20:3n-6 20:4n-6 20:3n-3 20:4n-3 20:Sn-3

22:o 6.9 * 2.9. 1.9 0.460 3.8 * O h 1.6 * 0.56~ 22:ln-11 0.3 * 0.1 0.3 * 0.1 0.2 * 0.1 0.5 * 0.4 22:In-9 0.4 I 0.1 0.3 * 0.1 0.3 * 0.1 0.2 * 0.0 22:ln-7 0.1 f 0.2 0.3 * 0.1 0.2 * 0.1 r d 21:Sn-3 0.2 f 0.2 0.2 * 0.1 0.6 * 0.2 nd 22:4n-6 0.1 f 0.1 0.7 * 0.7 0.4 * 0.5 nd 22:Sn-6 0.5 * 0.5 1.2 k 0.2 1.4 * 0.7 0.9 * 0.3 22:4n-3 0.2 t 0.3 0.1 î 0.0 0.3 * 0.4 0.2 * 0.1 22:5n-3 1.0 î 0.4 0.4 0.2 0.5 * 0.1 1.1 * 0.5 24:O 3.8 * 0.6. 1.5 0.2b 2.5 * 0.60 rid 22:6n-3 21.4 * 1.9.b 12.4 2.98 22.9 * 9.8.b 28.4 * 6.b 24:ln-11 6.6 * 4.1. 0.2 * 0.lbc 1.6 1.2.0 1.2 * 0.4bc 24:1 n-9 1.3 * 0.1. 0.6 0.lb 0.6 * 0 . lb nd 26 :O 4.0 * 2.3 2.7 * 1.1 3.7 * 0.5 0.9 * 0.3 C a . 40.3 * 3.3.6 43.7 6.8. 33.0 * 5.6.b 28.0 2 3.26 PW 18.0 3.2 18.3 * 1.2 13.8 * 2.7 20.7 * 3.7 z WFA 36.2 * 1.4.6 35.4 * 5.4. 45.7 * 9.- 50.1 * 3-06 C n-3 31.6 * 2.0.b 25.9 * 5.5. 32.1 * 11.0.6 43.3 3-76 2 n-6 4.1 * 1-38 8.3 & 0.7d 11.7 * 3.46 6.3 1.6.0 D M P A 2.6 * 0.3 2.0 * 0.1 3.3 * 0.5 3.1 1.3 C UNK 5.5 * 1.0. 2.7 1.h~ 5.4 * 2-24 1.2 * 0.46~

Results are expressed as the 96 of total fatty soi& and r q c r e n t means and standard deviations of 3 replicates. Unhomu ~ 1 % not inciuded in taôle. Abbraviations: n& not detected, tr= trace (<0.05%). 7 Me= 7 methyl, D M k dimethyl anteisu, SAT= saturates. M C N b monounsaturates, PUFA.: polyunratuiates. UNlG unknowns, DCUI doco98her~.noic acid, EPAr eicosapentaenoic acid.

Table 3b. Percent change in the fatty acid composition of Tisbe sp. cultured at 20°C as compared with the fatty acid composition of the algal (C cab Chaetoceros calcitrans, O tert= Dunaliella tertiolecta and I gal= I s o & ~ s galbana) and bakenr' yeast diets. The minus (-) symbol indicates the fatty acid is found in the algae but not detected in Tisbe sp. The plus (+) symbol indicates the fatty acid i s found in the ~0p8p0d but is not deteded in the algae.

Fatty acid C d D tert l ml veast

Results are expressed as 96 change in total fatty ad& from the diet to the q m p d and represent the change in the mean of 3 repficates. Abbreviations; 7 Me= 7 methyl, SAT= saturates, MON- rnonounsrrturatm, PUFk polyunsaturatcw.

Dm tertiolecta contained a higher proportion of this fatty acid. The

lipid from copepods fed al1 three algal spedes contained

substantially lower percentages of 18:3n-3 than was present in the

lipids from those algal species. The largest reduction in 18:3n-3

from the diet (ca. 45%) to the copepod (ce. 4%) was seen in the

copepods fed D. tertiolecta. This represented a 91% decrease of

18:3n-3 (Table 3b).

The propodon of 22:6n-3 was higher in the lipids of Tisbe fed

1. galbana and calcitrans but not significantly different (pS0.05)

from the levels in the copepods fed 0. tertiolecta. There were

sig nif icantl y higher concentrations of 22:6n-3 in these two algae,

especially 1. galbana, than in D. tertiolecta which produced SI i g h t l y

lower levels in the copepods. The amount of 20:5n-3 was

significantly higher in the copepods fed with C. calcitrans than in

those fed with D. tertiolecta. The level of 20:Sn-3 in the copepods

fed with 1. galbana was not significantly different from the other

two algal diets. The DHA to €PA ratio was much higher (2.6) in the

copepod fed with C. calcitrans than in C. calcitrans itself, wheie

this ratio was extremely low (0.05). This was due. to both a large

reduction in the proportion of 20:5n-3 (about a 75% decrease; Table

3b) in copepod lipid compared with that in the Cm calcitrans diet, and

a corresponding 10-fold increase (Table 3b) in th8 proportion of

22:6n-3 in the copepod. The lipids of D. tertiolecta-fed copepods

contained relatively large amounts of 20:Sn-3 and 22:6n-3, even

though only trace amounts of these EFAs were present in that alga,

and this accounted for a 45-fold and a 40-fold increase,

respectively (Table ab). In copepods fed with lm galbana, there was a

2-fold increase in the proportion of 20:5n-3, but a slight (10%)

decrease in the proportion of 22:6n-3. Tisbe fed the bakers' yeast

had extremely high amounts of both 20:5n-3 and especially 22:6n-3

in their lipids (Table 3a).

It was apparent from differences in the EFA content between

each of the diets and the copepod, that Tisbe managed to maintain a

consistently high DHA to €PA ratio despite the extreme diff erences

in the arnounts of these fatty acids in the diet. This ratio ranged

between 2.0 and 3.3 and there were no significant differences

(ps0.05) in this ratio between the ~0p8pods fed any of the diets.

The levels of 20:4n-6 were relatively high (~1.0%) for Tisbe

fed each of the diets. The copepods fed with Dm tertiolecta

contained 1.5% of 20:4n-6 in thelr lipids, even though this fatty acid

was below detectable levels in this alga.

Two of the algae (D. tertiolecta and I. galbana) were also f ed

to another species of harpacticoid copepod, Amonardia sp. (Table

4a). This copepud had low levels of 20:5n-3 (4%). when fed w i t h

either D. tertiolecta or 1. galbana. The levels of 22:6n-3 (>13%)

were much higher than those of 20:Sn-3 (Table 4a). Amonardia fed

with 1. galbana increased the percentage of 20:5n-3 in their l ipids

slightly, and those fed with D. tertiolecta dramatically increased

(>4-fold) the percentage of 20:Sn-3 in their Iipids compared to the

diet (Table 4b). The ~0p8pods fed with 0. tertiolecta also had a ve ry

high, almost 300-fold, increase in 22:6n-3 compared to the diet. As

was the case in Tisbe, the percentage of 22:6n-3 in the copepods fed

with 1. galbana was slightly reduced compared to the diet (Table

4b).

Unlike Tisbe sp.. Amonardia sp. did not maintain a constant

DHA to EPA ratio and there was a significant difference in th is

ratio between the copepods fed the two algal diets: 24.0 for those

fed 0. tertiolecta compared with 6.6 for those fed 1. galbana. This

was due prirnarily to the low level of 20:Sn-3 in the copepods fed O.

Table 4a. Fatty add composition of Amonardia sp. culhired at 20°C and fed the algae lsochrysis galbana (1 gaî) and Dunaliella tertiolecta (O tert). The asterisk indicates significant diff erences at the p<0.05 level.

Fat& acid l cral 0 tert ug LipW copepod 0.51 f 0.12 0.31 f 0.04 14:O 1.4 f 0.4 0.3 i 0.3 14:1 n-9 0.4 f 0.2 0.2 f 0.2 14:ln-7' 1.3 f 0.8 0.1 f 0.2 15:O' 0.4 f 0.1 0.9 i 0.2 16:O 32.9 f 3.8 47.9 f 6.5 16:l n-7' 3.3 f 0.4 0.4 f 0.1 7-Me 16 0.6 f 0.2 nd 16:1 n-5 0.9 f 0.5 0.2 f 0.2 16:2n-6 0.4 f 0.2 nd 16:2n-4' 0.9 f 0.2 1.7 i 0.3 17:O 2.0 f 0.4 2.5 f 0.6 16:3n-4 0.4 f 0.4 0.3 f 0.3 16:3n-3 0.2 f 0.1 0.2 f 0.2 l6:4n-3' 1.8 f 0.7 0.7 f 0.1 16:4n-1' 0.7 f 0.1 1.8 f 0.2 18:O' 7.5 f 1.2 3.9 i 0.6 18:1 n-9' 4.0 k 0.3 2.6 i 0.1 18:l n-7' 2.3 f 0.1 1.5 f 0.0 18:1 n-5 0.4 f 0.2 0.2 î 0.2 18:2n-9 0.4 f 0.3 nd 18:2n-6 1.0 f 0.1 0.3 î 0.2 18:2n-4' 0.3 f: 0.0 1.2 f 0.4 18:3n-6 1.1 f 0.7 0.4 f 0.2 18:3n-4 0.8 f 1.0 nd 18:3n-3' 1.1 f 0.1 2.3 f 0.7 t8:4n-3 0.8 f 0. 7 nd 18:4n-1 0.7 f 0.8 1.6 f 0.6 20:o 1.1 * 0.1 1.1 i 0.1 20:1 n-9 0.7 k 0.5 nd 20: 1 n-7 0.1 k 0.0 nd 20: 1 n-5 nd 2.7 * 0.4 20:2n-6' 0.1 f 0.0 1.4 f 0.4 20:3n-6' 1.2 f 0.3 0.3 I 0.1 20:4n-6 0.3 f 0.1 0.1 f 0.1 20:3tt-3 t r 0.2 I 0.3 20:4n-3' 0.6 I 0.2 1.2 i 0.3 20:5n-3' 2.7 i 0.8 0.8 f 0.1 UNK 0.3 * 0.6 0.4 k 0.0 22:O 2.0 f 0.3 1.8 f 0.3 22: 1 n-1 1 0.5 f 0.1 0.1 f 0.2 22: 1 n-9 0.2 f 0.1 nd

26:09 0.7 f 0.2 1.7 f: 0.8 C mT 52.1 f 5.8 60.1 f 6.2 C MONO' 14.5 f 1.4 9.4 f 0.6 C PüFA 34. 6 f 4.6 30.0 f 6.0 C n-3 26.0 4.1 19-9 + 4.7 C n-6 4-4 f 0.5 3.6 f 0.8 DHA/EPA* 6.6 * 3.0 24.0 * 3.8 C UNK" 1.4 f 0.8 0.6 f 0.2

Results are erqrressed as 96 of total futty a& and repnisent means and standard deviations of 3 replicates. Unknowns 4% not iriduded in taMe. Abbreviations; n& not detected, tr= trace (<O.OS%), ?-Me= 7-methyl, SATI saturates, MONOI monounsaturates, PUFR polyunsaturates, UNK= unknowns, DHAt docosahexaenoic acid, €PA= eicosapentaenoic acid.

Table 4b. Percent difference in the fatty add composition of Amonardia sp. cultured at 20% as compared with the fa€ty acid composRion of the algai (D tert- DunaIfella tertiolecta and I gai= lsochrysis galbana) diets. The minus ( 0 ) symbol indicates the fat t y acid is found in the algae but not detected in Amonardia sp. The plus (+) symbol indiates the fatty acid is found in the copepod but is not detected in the algae.

Fatty acid I ad D tert 14:O - 1 2 371 14:1 n-9 229 - 6 1 14:1 n-7 889 - 4 6 14:l n-5 - - 15:O 807 1 4 0 4 16:O 352 4 7 3 16:ln-9 - + 16:l n-7 4 8 -03 7-Met 6 - 7 0

16:l n-5 4967 - 8 7 16:Zn-6 1941 0

16:2n-4 - 8 1928 17:O 3283 525 16:3n-4 496 548 16:3n-3 262 - 8 9 16:4n-3 25827 - 9 4 16:4n-1 1 IO7 13321 18:O 1185 1 089 18:ln-9 -63 16 18:ln-7 7 6 2 f 8 : l n-5 685 + 18:2n-9 + - 18:2n-6 - 7 9 - 9 7 18:2n-4 + 6 3 1 3 18:3n-6 1126 - 9 0 18:3n-4 1873 + 18:3n-3 - 9 1 - 9 5 18:4n-3 - 9 2 - 18:4n-1 + + 20:o + + 20:1 n-11 - - 20:1 n-9 2757 - 20:1 n-7 + - 20: 1 n-5 + + 20:2n-6 - 7 8 2237 20:3n-6 592 21 8 7 20:4n-6 - 1 0 + 20:3n-3 - 298 20:4n-3 188 4456

Results are expressed es 96 change in total fatty adde from the diet to the cupfmd and represent the change in the mean of 3 replicates. Abbreviatkns; 7-Me= 7-methyl, SAfr saturates, MON- monounsaturates, PUFA= polyunsaturates.

tertiolecta, as the difference in the percentage of 22:6n-3 in the

copepod lipid fed the two diets was not significant.

The levels of 20:4n-6 were very low in Amonardia, 4).3% and

not significantly different (pS0.05) for those fed either alga.

DISCUSSION

Effects of Diet on Copepod Lipids

Dunaliella tertiolecta (CMorophyceae) contained only t race

arnounts (<O.S%) of the EFAs 20:Sn-3, 22:6n-3 and 20:4n-6. This

suggests that this alga contains low levels of active A-6 and A -5

desaturases, which are necessary to convert 1 8:3n-3 into 20: 5n-3

and 22:6n-3 and I8:2n6 into 20:4n-6. Chaetocmos calcitrans

(Bacillario ph yceae) contains relative1 y large amounts of the €FA

20:5n-3 and little 22:6n-3, which suggests that this algae contains

low levels of active enzymes necessary for 22:6n-3 synthesis.

lsochrysis galbana (Prymnesiophyceae) contained large amounts of

22:6n-3 and very little 20:5n-3, which is consistent with the

presence of either an active A 4 desaturase or the Schoenberg shunt,

which catalyzes the transfer of 20:Sn-3 on through to the 22:6n-3

end-product (Fig. 1 b). These fatty acid compositions are typical f O r

these classes of algae (Volkman et al., 1989). The bakers' yeast has

a large amount of A-9 desaturase activity as is evidenced by the

high level of 18:ln-9, 16:ln-7 and 18:ln-7 (a 16:ln-7 elongation

produd). These three A-9-desaturated fatty acids make up 65.5% O f

the lipid. The yeast contains low levels of th8 other desaturase

enzymes as is evidenced by its low amount of total PUFA (5.8%).

These species were chosen as diets because of their large

differences in €FA composition. The diets Mgh in 20:Sn-3 (C.

calcitrans) and 22:6n-3 (1. galbana) allowed us to examine the level

of incorporation and modification of these EFAs from the diet to the

copepod. The diets deficient in long-chain EFA (D. tertiolecta and

yeast) allow us to evaluate the desaturating and chain-elongating

abilities of the harpacticoid copepod.

Most crustaceans thus far used as live food organisms in cold-

water marine fish culture have no or only a limited ability t o

bioconvert shorter chain n-3 PUFA into the longer chain EFA, 20:Sn-3

and 22:6n-3 (Lubzens et al., 1985; Howell and Tzoumas, 1991). Both

harpacticoid copepods, Tisbe sp. and Amonarclia sp., that we

studied are able to synthesize significant amounts of 20:5n-3 and

22:6n-3, when fed Dunaliella tertiolecta, which contains only trace

amounts (<0.5% of lipid) of these EFAs. This confirms the work done

by Norsker and Stettrup (1994) on a European strain of Tisbe

holothuriae. They fed 0. tertiolecta to this harpacticoid and found

that il also could convert n-3 PUFAs from the algae into 20:5n-3 and

22:6n-3. This was further evidenced in their egg production study

with 1. holothuriae. There was no significant drop in the copepods'

egg production, when fed the €FA-poor Dm tertiolecte versus the

relatively €FA-rich aigae Rhodomonas baltica.

The harpacticoids, when fed D. tertiolecta, converted mainly

18:3n-3 (45.0% of lipid) into 20:Sn-3 and 22:6n9. This was

evidenced by a large decrease in 18:3n-3 from the amounts present

in the alga relative to the amount in both Tisbe and Amonardia

when fed this species. Along with this decrease, there was a big

increase for both copepods in the amounts of 20:5n-3 and 22:6n-3

relative to that in 0. tertlolecta. These copepods have the ability t O

convert 18:3n-3 into substantial quantities of 20:5n-3 and 22:6n-3.

Therefore, to be capable of this conversion the copepod must contain

suff icient amounts of the C 18-to-C20 and C20-to-C22 elongase, as

well as A-6, A-5 and putative A 4 desaturases (Fig. 1).

Tisbe, when fed C. calcitrans which is rich in 20:5n-3,

reduced its proportion by about 75% in its lipids. There was also a

concurrent 10-fold increase in aie percentage of 22:6n-3 in Tisbe

fed with calcitrans. This is consistent with the hypothesis t hat

the copepod uses C20-to-C22 elongase to convert 20:5n-3 to 22:5n-

3, and either A-4 desaturase or the Schoenberg shunt to convert

22:5n-3 to 22:6n-3. The disappearance of 20:Sn-3 may also be due,

in part, to oxidative catabolism. However, 20:Sn-3 constitutes over

90% of the total n-3 fatty adds in C. calcltram, and an n -3

precursor is needed to produce the levels of 22:8n-3 seen in the

copepods fed with C. calcitmns.

The copepods (Tisbe and Amonardia), fed an alga rich in 22: 6n-

3 (1. galbana) decreased the proportion of this EFA in its lipids when

compared with that in the alga. There was a slight decrease in Tisbe

and a somewhat larger decrease in Amonardia. As previously

mentioned, Tisbe when fed a diet rich in 20:Sn-3 (C. calcitrans)

reduced the proportion of this EFA in its lipid when compared with

the alga. When the copepods were f8d with the EFA-pooi a

tertiolecta and yeast, the proportions of 20:5n-3 were increased

dramatically in its lipid, relative to that in the diet (ca. 45-fold for

D. tertiolecta fed to Tisbe: ca. 5-fold for D. tertiolecta fed t o

Amonardia; ca. 6-fold for yeast fed to Tisbe). A similar increase was

obsewed for 22:6n-3 (40-fold for 0. tertioIecta fed to Tisbe; 4 5 - fold for Dm tertiolecta fed to Amonardia; almost 15-fold for yeast

fed to Tisbe). Because of these changes, the DHA to EPA ratio

remained high ( ~ 2 ) for boai harpacticoid copepod species for a l l

diets, despite the huge variations in the EFA composition of their

diets. In Tisbe, this ratio also remained remaikably constant.

There was also no significant difference in the DHA to EPA ratio

arnong the copepods fed any of the four diets. If the diet contained

quantities greater than a certain threshold for either 20:5n-3 or

22:6n-3, the harpacticoid copepods reduced the percentage of these

in its lipids. If the amount was below this threshold then the levels

were increased so that a consistently high DHA to EPA ratio was

maintained (Fig. 1 ).

The lipids of Tisbe fed with yeast contained th8 highest levels

of long-chah EFAs of any treatment group, despite the very l ow

levels of EFAs in the yeast itself (496 of both 20:5n-3 and 22:6n-3).

This can be partially explained by th8 copepods' low total I ipid

content. Tisbe fed with yeast had the lowest average lipid content

(0.20 pg/copepod) of any treatment group. Therefore, the i r

proportion of membrane phospholipids would necessarily be greater

and their storage lipids (triglycerides) less. The membrane lipids

are generally high in the EFAs, and their proportionate increase

would partially account for the high €FA levels of Tisbe fed wit h

yeast. The overall low lipid content of the copepod could be

accounted for by the low lipid content of the yeast (2.2% of the dry

W.). However, the yeast feeding trials were performed at a

different tirne of year from the algal feeding trials, so that seasonal

effects may also have played a role in lipid composition differences.

The other long-chain €FA, 20:4n6, was present at highei

levels in the harpacticoid Tisbe (A% for al1 the diets with no

significant differences among them) and at very low quantities in

Amonardia (<0.3% for those fed either diet). More evidence for A - 6

and A-5 desaturases in the harpacticoids can be seen in Tisbe fed

with D. tertiolecta. No 20:4n-6 was detected in D. tertiolecta, yet i t

was found in Tisbe fed this alga. For both harpacticoid species fed

with D. tertiolecta, there was a decrease in the levels of th8

ptecursor fatty acid 18:2n-6 (by 65% for Tisbe and by 97% f O r

Amonardia) and for Tisbe a concurrent increase in 20:4n-6 to 1.5%.

In Amonardie fed with O. tertiolecta the levels of 20:4n-6 were

very low (~0.5%). Most of the 18:2n-6 in these copepods was

elongated to 20:2n6 (>20-fold increase) and not desaturated to

18:3n-6 (the next step in the conversion to 20:4n6; Fig. l), which

was actually decreased in Amonadia by 90%.

Some general trends were seen in Tisbe fed al1 of the diets.

The levels of 18:O were increased, compared with the diet, for the

copepods fed al1 of the diets. 77368 may preferentially synthesize

this fatty acid de novo, as oppoged to 16:O. Th8 monounsaturate,

16:i n-7, decreased in the copepod lipid compared with the levels i n

the diet for the copepode fed al1 diets. There was a concurrent

increase in I8 : lnJ for the copepods fed al1 algal diets. suggesting

that the copepod rnay be elongating 16:l n-7 in the diet to 18:1 n-7.

Both 20:l n-7 and 22:l n-7 were also increased in Tisbe fed al1 diets

which indicating that 18:ln-7 may be further elongated to these

fatty acids in the copepod. However, 20:ln-7 and 22:l n-7 were

present in much lower quantities in the copepod lipid, compared

with 18:ln-7. In yeast. 18:ln-7 was present at extrernely high

levels, therefore, there was decrease in its level in the copepods fed

with yeast.

Amonardia, increased the total arnount of saturated f a t ty

acids, including 16:O and 18:0, relative to that in the dietary lipid.

Unlike Tisbe, Amonardia wntained a large amount of 16:0, which

was greater than the amount of 18:O. This suggests that there i s

less elongation of 16:O to 18:0, or that there is preferential de novo

synthesis of l6:O in Amonardia. As was the case in Tisbe, 16:O was

present at a higher level in the aie copepod fed with Dm tertiolecta

(48%) than in those fed with I. galbana (33%). However, th is

difference was not significant. The fatty acids 18:ln-9, 16:ln-7 and

18: 1 n-7 (a l6:l n-7 elongation produd) were al1 significantly higher

in Amonardia fed with I. galbana, indicating A-9 desaturase activity

was greater in this copepod. Those copepods fed Dm tertiolecta may

be channeling their energy into making the desaturases needed t o

convert 18:3n-3 into the EFAs (20:5n-3 and 22:6n-3), while l i rn iti ng

the synthesis or activity of the A-9 desaturase.

Nutritionai Implications for Marine Fish Larvae

In mafine fish larvae, 22:6n-3 has more €FA value than 20:5n-

3. This has been demonstrated in several species of manne fis h

larvae in feeding studies using the brine shrimp, which cannot

synthesize or incorporate high levels of 22:6n-3. Howell and

Tszournas (1991) fed brin8 shrimp from Brazil and Utah to larval

sole (Solea solea). The Brazilian strain was low in 22:6n-3, but had

significantly more than the Utah strain. The larvae had significantly

higher growth rates when fed the Brazillan strain, but there was not

enough of the 22:6n-3 to promote good survival. The ratio of EPA t o

DHA was also show to significantly affect the survival of marine

fish. Changing the ratio of €PA to DHA from 13.8 to 2.2 in the diet of

turbot markedly decreased mortalities (Bell et al., 1985). The

increased importance of 22:6n-3 compared . with 205n-3 as an EFA

for marine fish larvae was reviewed by Watanabe, 1993 f o r

yellowtail Serjola quinqueradiata. striped jack Pseudocaranx dentex.

striped knifejaw Oplegnathus fasciatus, red sea bream Pagms ma jar

and f lounder Paralichthys olivaceus.

Tisbe, because of its high desaturase activity, is able t o

produce large amounts of 22:6n-3 and maintain a consistently hig h

DHA to EPA ratio (2.0 to 3.4), despite the long-chah EFA

composition of the diet. Amonardie also had a very high DHA to EPA

ratio (23.4) when fed a long-chain EFA deficient diet. This

extrernely high ratio was due, in part, to very low levels of 20:5n-3.

These harpacticoids have a great potential as live food, because they

do not need to be fed an EFA-rich diet, but maintain high DHA to EPA

ratios within their lipids even when cultured on low cost and

maintenance foods such as yeast.

For both harpacticoids, higher 22:6n-3 values were achieved

when they were fed a long-chain €FA-rich diet. The exception was

Tisbe fed on yeast, which had lower total lipid levels. The long-

chain, EFA-rich algae, lsochrysis galbana and Chaetoceros

calcitrans, gave higher levels of 22:6n-3 in Tisbe, when compared

with Dunaliella tertlolecta ('Table 3a). Arnonardia also contained

higher levels of 22:6n-3 when fed with 1. galbena as oppoged to LX

tertiolecta (Table 4a). Therefore, feeding the harpacticoids w i t h

diets n'cher in long-chah €FA increases the amounts of 22:6n-3 in

the harpacticoid lipids, which can be made available to marine f i s h

larvae.

Arachidonic acid (20:4n-6) is also essential for marine fish.

Linares and Henderson (1 99 1 ) discovered that radiolabelled 20:4n-6

is incorporated into turôot phosphatidylinositol at a very specific

and high level. They suggested that, because of this specific

incorporation of 20:4n-6 into phosphatidylinositol, and because O f

its role as a precursor for prostaglandin synthesis, 20:4n-6 i s

probably essential for marine fish. Marine fish have very low levels

of A95 desaturase activity. Therefore, they cannot convert 18:2n-6

into sufficient amounts of 20:4n-6 and so require preforrned 20:4n-6

in their diet (Mourente and Tocher, 1993). Although marine f ish

require 20:4n-6, the demand is not as great as that for 22:6n-3 as

indicated by the higher whole-body retention of dietary 22:6n-3

compared to 20:4n6 in turbot juveniles (castell et al., 1994).

The harpacticoid Tisbe sp. contained greater than 1% 20:4n-6

with al! diets tested, including the alga, D. tertiolecta, which

contained only trace amounts of this EFA (Table 3a). Amonardla

produced lower levels of 20:4n-6 (4.3%) than Tisbe, producing

instead, increased proportions of 20:2n-6 and 20:3n-6, when fed the

algal diets (Table 4a). Tisbe, in terms of its arachidonic acid

composition, thus appears to be best suited for manne fish iarval

culture, when grown on various diets.

EFFECT OF TEMPERATURE ON COPEPOD LlPlDS

I M O N

Acute changes in environmental temperatures may have

deleterious effects on the structure and function of cellular

membranes of crustaceans as well as other poikilothermic animals.

Changes in temperature impact both the permeability of cel l

membranes and the activity of integral membrane proteins,

presumably via changes in the physical characteristics of the l ip i d

bilayer. It is generally assumed that, for a given set of membrane

constituents, there is an optimal range of ternperatures wi t h in

which suitable molecular interactions (either lipid-lipid or l i p i d - protein) occur for proper membrane structure and function. and that

this range is above the critical phase transition (gel to liquid-

crystalline) temperature of the bilayer (Pniitt, 1990).

This maintenance by poikilothemic animals of their membrane

lipids at a standard fluidity above the phase transition temperature

is known as "homeoviscous adaptation' and was first described f o r

Escherichia coli by Sinensky (1974). Poikilotherms tend t O

increase the ratio of unsaturated to saturated fatty acids of thei r

phospholipids in cell and organelle membranes as tempe rature

decreases (Chapelle, 1 078; Farkas, 1 979). Lower tem peratu res

increase the viscosity of the cell membrane by reducing the thermal

energy and causing greater cohesiveness of the macromolecules

which comprise the membrane. lncreased desaturation of membrane

phospholipids at lower temperatures increases the disorder of the

macromolecules in the membrane so that a standard fluidity i s

maintained.

Rainbow trout (Salmo gairdneri) hepatocytes had higher levels

of n-3 PUFA, in particular 22:6n-3 and 20:5n-3 as well as higher

levels of 20:4n-6 when acclirnated at 5OC rather than 20°C (Sellner

and Hazel, 1982). lncreases in 20:4n=6, 20:Sn-3, 22:6n-3 and to ta1

n-3 PUFA at lower temperatures have also been obsewed i n

crustaceans (Harrison, 199 1 ). Among copepods, large increases i n

2Mn-3 and 22:6n-3 fatty acids were also obsewed in freshwater

species as tempe rature decreased (Farkas, 1 979).

The objective of rny present study was to detemine i f

decreasing the rearing temperature will significantly increase the

level of EFAs (20:4n-6, 20:Sn-3 and 22:6n-3) in the harpacticoid

copepods, Tisbe and Amonardia, thereby making them more valuable

as a live food for marine fish larvae. In the copepod feeding t r ia ls

with various diets, a large potential for desaturation of fatty acids

was observed. In particular, harpacticoids had a great ability t o

convert 18:3n-3 into 20:5n-3 and 22:6n-3 and 18:2n-6 into 20:4n-6.

One would expect that, as an adaptation to the lower temperatures,

the copepods would further increase their already large amounts of

EFAs to maintain a standard membrane fluidity.

MATERIALS AND MEiHODG

The harpacticoid cupepods nsbe sp. and Amonardia sp. were

raised in 6-L cylindrical plexiglass jais containing seawater (32 ppt

salinity) which had been filtered through 10 prn pore site cartridges

and UV treated. Oxyg-en saturation and water circulation were

maintained with air flowing through eirstones at the bottom of each

cylinder. They were maintained for approximately 4 weeks at 3

temperatures 20, 15 and 6OC (i 1 OC) while being fed the same alga,

lsochrysis galbana. The medium was changed approximately t w i c e

per week by collecting the copepods on a 40 pm mesh and

transferring them to jars of fresh, filtered sea water. Three

replicate cylinders of copepoâs were used in the study and the alga

was fed to the copepods in excess (ca. 1 mg dry weight IL of sea

water) each time the water was replaced.

Lipid and statistical analyses were perfoned using the same

methods descilbed for the feeding trials (pp. 19-29).

RESULTS

There was a direct relationship between the percentage of

saturated fatty acids and temperature. As the temperature of the

environment decreased from 20 to 15 and e0C, the copepod,

Amonardia sp., significantly decreased the levels of saturated f a t t y

acids from about 52% to 32% in its lipid (Table 5). This difference

in the levels of saturated fatty acids was due principally to the

lower content of 16:0, which decreased by about half as the

temperature decreased from 20 to 15 and 6OC. The percentage o f

total PUFA increased at 6OC compared to 15 and 20°C. The PUFA

were at their lowest levels at 15OC. None of these differences i n

the PUFA at the various temperatures were significant (pS0.05).

The EFA 20:5n-3 in Amonardia increased significantly at 6°C

compared with 20°C, but there was a decrease in this PUFA at W C .

The pattern was similar for 22:6n-3, which showed a slight increase

at 6°C compared with 20°C and a significant decrease at 15OC

compared with 6 and 20°C. There were no significant differences in

the DHA to EPA ratio for the three ternperatures. As expected, the

Table 5. Fatty acid composition of the harpacticoid copepod, Amonardia sp., fed the aîgae Isochvsis galbana cultured at three temperatures. Different lettering indicates significant differences at the pe0.05 level.

l4:O 14:ln-9 14:ln-7 l 4 : l n-5 l5:O DMA 16 l6:O 16:ln-7 7-Mû 16 16:ln-5 16:2n-6 16:2n-4 17:O 16:3n-4 16:3n-3 16:4n-3 16:4n-1 l8:O t8: ln-9 l 8 : l n-7 l 8 : l n-5 18:2n-9 18:2n-6 18:2n-4 18:3n-6 t8:3n-4 18:3n-3 18:4n-3 18:4n-1 20:o 20: 1 n-11 20:1 n-9 20:1 n-7 20:Zn-6 20:3n-6 20:4n-6 UNK 20:3n-3 20:4n-3

CMoNo 14.5 k 1.4b 32.0 i 9.9r 22.9 I 1.Oab C PUFA 34.6 f 4.6 29.6 f 8 A 41.8 f 3.9 x n-3 26.0 î 4.0ab 18.0 i 5.78 31.0 I 4.3b C n-6 4.4 * 0.56 11.6 * 4.58 10.5 I 1.0a D HA/ E PA 6.6 f 3.0 7.2 f 0-4 3.4 f 0.6 C UNK 1.4 * 0.8 2.7 i 0.6 3.1 f 0.7

Results are expressed as % of total fatty ecids and represent means and standard deviations of 3 replicates. Unhomu, 4% not induded in taMe. Abbreviations; nd- not detected, tr= trace (4.05%). 7-Met 7- rnethyl, D M k dimethyl anteiso, SAT= satutates, MCWQ rnonounsatumt8~, PUFA= poîyunsaturates, ü h k unknowns, DHA= docogahexaenoic acid, €PA+ eicosapentaenoic acid.

content of 20:4n-6 in the lipid increased as the temperature

decreased. The diierence between 20 and 6OC was significant.

The effects of temperature on the fatty acid composition of

Tisbe sp. were evaluated (Table 6). One replicate sample out of

three for Tisbe sp. cultured et 6OC was contaminated, so that

statistical cornparisons were only made between 20 and lS°C. The

amounts of PUFA increased only slightly at 6OC compared with 20°C,

but there was a significant decrease in the PUFA at 15OC compared

with 20°C. Th8 latter was a similar temperature response to t hat

shown by Amonardia. The saturated fatty acids rernained nearly

constant at about 30% for the three temperatures. The levels of

20:5n-3 were about 9% at 20°C and decreased substantially to 4%

at 15OC and 6OC (significantly so at 15°C). The amounts of 22:6n-3

increased at 6OC compared with 20°C (no significance att r i b uted) . However, there was a slight drop in the level of 22:6n-3 at lS°C,

which is anothei temperature response similar to that of Amonardia.

There was a steady increase in the DHA to EPA ratio as the

temperature decreased. As in Amonardia sp., 20:4n-6 reached i t s

highest levels in Tisbe at the lowest temperature tested.

Table 6. Fatty acid composition of the harpacticoid copepod, Tlsbe sp., fed the algae lsochryss galbana cultured at three temperatures. The asterisk symbol indicates significant differences at the pe0.05 level for 20 and lS°C.

Çaîty Acid 20°C 15OC 6°C 14:O 0.7 I 0.3 nd 0.1 14:ln-9 0.2 * 0.3 nd t r 14:l n-7 0.3 f 0.3 nd t r 14:1 n-5 0.1 f 0.2 nd O. 1 1 5:0 0.5 f 0.2 nd 0.2 DMA 16 0.1 f 0.1 nd 0 -2 DMA 16 0.1 f 0.2 0.1 16:O' 19.5 f 5.4 9.9 * 2.6 14.1 16:1 n-7 7.0 f 6.5 0.9 f 1.2 0.2 16:ln-5 0.4 0.2 0.3 î 0.1 0.3 16:2n-6 0.2 f 0.1 nd nd 16:2n-4 0.4 f 0.2 0.9 f 0.5 0.1 17:O 2.8 i 0.9 1.6 f 0.5 1 .O 16:3n-4 0.5 i 0.2 nd 0.3 16:ln-3 0.5 I 0.5 0.2 f 0.2 0.4 16:4n-1 1.1 f 1.1 1.7 f 1.0 0.6 18:O 8.1 f 1.8 11.0 1.9 7.4 18:ln-9 3.9 f 0.8 10.3 f 5.1 1.8 18:ln-7' 2.8 f 0.7 5.5 î 1 .3 1.5 18:1 n-5 0.4 i 0.3 0.6 f 0.4 0.1 18:2n-6 1.7 f 0.2 1.2 f 0.8 0.5 18:Zn-4" 0.3 i 0.2 2.5 f 1.3 0-3 18:3n-6 0.1 I 0.1 0.2 f 0.1 O. 1 18:3n-3 0.8 f 0.1 0.6 f 0.2 0.4 18:4n-3 0.6 f 0.2 0.4 f 0.1 0.3 18:4n-1' 0.2 i 0.2 2.7 f 1.7 0.4 20:0* 0.4 f 0.2 1.1 f 0.2 2.1 20:1 n-11 0.4 f 0.1 nd nd 20: 1 n-9 0.9 0.7 1.4 f 0.4 0.3 20:l n-7* 0.3 f 0.1 0-8 î 0.2 0 .5 20:l n-5 0.1 f 0.2 0.5 f 0.9 nd 20:2n-9 0.3 f 0.4 0.4 f 0.4 0.6 20:2n-6' 0.2 f 0.0 2.6 f 1.6 0.3 20:3n-6 0.8 I: 0.4 0.9 f 0.3 3.0 20:4n-6 0.7 î 0.1 0.5 f 0.3 2.1 20:3n-3 0.1 f 0.0 nd 0 -4 20:4n-3 0.5 f 0.2 0.3 f 0.1 0 .6 UNK nd 2.5 f 1.5 nd 20:Sn-3' 9.3 f 0.5 2.2 * 0.5 2.5 22:O' 1.2 f 0.5 3.6 f 1.4 5.0 22:1 n-11 0.1 f 0.1 0.3 f 0.1 t r 22:1 n-9 0.2 f 0.3 0.2 i 0.2 0.4

22:Sn-6 1.3 f 0.5 1.6 0.3 1 .1 22:ln-3' 0.3 i 0.5 2.4 f 1.3 1.8 22:Sn-3 0.3 f 0.3 nd 0.6 24: 1 n-11 0.6 f 0.4 1.5 f 0.6 2.6 22:6n-3 26.9 f 9.4 20.5 f 1.6 38.4 24: 1 n-9 0.4 f 0.3 nd 1.3 26:O' 0.4 f 0.2 1.4 f 0.3 1.7 C SAT' 33.7 f 2.2 28.6 f 1.5 31.8 C m 18.1 f 7.9 22.4 f 7.8 9.5 C PüFA 47.2 f 9.8 42.0 f 7.1 56.0 C n-3 39.5 f 10.5 26.5 f 1.9 45.5 C n-6' 5.0 i 0.7 7.3 f 1.2 8.3 D HNEPA* 2.9 f 1.0 9.9 I 3.4 15.7 C UNK* t r 5.5 f 2.9 0 -2

Results are qressed as % of total fatty adds and represent means and standard deviations of 3 replicates except for 6OC which is the mean of 2 replicattw. Unknomis ~ 1 % not included in table. Abbreviations; nd= not detected, tr= trace (<O.OS%), SAT= saturates, MONOc monounsatutates, PUFA= polyunsaturates, UNlG unlaiomis, D W dooosahexaenoic acid, €PA= eicosapentaenoic acid.

0lSCUSSl0N

Effects of Temperature on Copepod Lipids

As the temperature decreased from 20 to 6OC, the percentage

of saturated fatty acids in Amonardia decreased from about one half

to one third. In Tisbe, the saturated fatty A acids were slightly, but

significantly higher at 20°C than et lS°C. but slightly higher again

at 6OC. This suggests that there was either more desaturase

activity, or less de norio synthesis of the saturated fatty acids a t

the lower ternperatures, particularly in Amonardia. There were

slightly higher levels of PUFA in Amonardia at 6OC than at 20°C, and

in Tisbe at 6OC than at 20°C (significance not attributed). At the

intermediate temperature of lS°C, the PUFAs were at their lowest

level in Arnonardia and in Tisbe. The monounsaturated fatty acids

were at their highest levels at 15OC in both Amonardia and Tisbe.

perhaps making up for the lower levels of PUFAs at th is

temperature. Thus, the total amount of PUFAs were highest in both

copepods at 6OC relative to 15 and 20°C. In Amonardia at 6OC, th is

increase in the level of PUFAs was at the expense of the saturated

fatty acids. Thus, these copepods seem to be undergoing

homeoviscous adaptation at 6OC to maintain a constant membrane

fluidity at this lower temperature.

The EFAs also exhibited distinct temperature responses i n

Amonanlia and Tisbe. The EFAs, 20:5n-3 and 22:6n-3, showed the

sarne general trend. In both harpactlcoids, except for 20:5n-3 in

Tisbe, the EFA occurred at higher levels at 20°C, decreased at 15°C

and then increased at 6OC. From 15 to B°C, which aie within the

range that the copepod would naturally encounter in the collection

site at Halifax Harbor, the levels of 22:6n-3 and 20:5n-3

dramatically increased with decreasing temperature for both

harpacticoids. This increase in 20:5n-3 and 22:6n-3 was also

observed for f reshwater copepods at lower temperatures (Farkas,

1979). There are some possible explanations for the increase in n-3

EFA a? 20°C compared to 15OC. The most probable explanation f O r

the increase in n-3 EFAs at 20°C, is that the phospholipid levels

increased over the neutral lipids as a proportion of the total lipid.

The stress of the abnormally high temperature of 20°C cornbined

with a higher metabolic rate could release depot lipid reserves

(Miliou and Moraitou-Apostopoulou, 1991 c; Pollero et al., 1 99 1 ).

There were no significant differences in the total lipid per copepod

for Amonardia at the three temperatures. At this highest

temperature, a higher proportion of females undergoing I i pi d

analysis may contain egg sacs, so that the total lipid per individual

copepod remains fairly constant, but more of it may be phospholipid.

There may also be selective catabolism of the monounsaturates,

such as 18:ln-9, to meet the higher energy demands at the higher

tem peratures. In Amonardia 1 8: 1 n-9 decreased 5-fold and in Tisbe

almost 3-fold as the temperature increased from 15 to 20°C (both of

these decreases are significant at pSO.10). This large decrease in

the monounsaturates, especially in Amonardia, would help to make

the n-3 EFAs more prominent in the lipid. Some or al1 of these

factors might explain the increase in n-3 EFAs from 15 to 20°C.

The EFA, 20:4n-6, generally followed the expected trend f O r

PUFAs, increasing steadily as the temperature decreased from 2 0

to 15 to 6OC in Amonardia and, in Tisbe, remaining nearly constant

at 20 and 15°C while increasing dramatically at 6OC. The increase

in this PUFA at the lower temperatures may be another example of

homeoviscous adaptation in the copepods.

For both harpacticoid copepods, the saturated fatty acid, 1 6:O,

was twice as high at 20°C compared with 15OC. although it remained

neai constant between 15 and 6°C. The high levels of this saturated

fatty acid may help maintain cell membranes at the proper liquid-

crystalline state at 20°C. The level of 16:O at 20°C is probably

achieved for the most part by de novo synthesis. The drop i n

monounsaturates frorn 15 to 20°C might indicate that they are the

primary energy source at 20°C, freeing up 16:O to be incorporated

into the phospholipid bilayer.

The main explanation given above for the increased levels of n-

3 PUFA at the highest temperature of 20°C was that the neutral

lipids were being bumed off du8 to increased metabolic rate or

stress, thereby decreasing the levels of saturated fatty acids and

increasing the phospholipid charader of the copepods' lipids. The

explanation for the increased levels of n-3 PUFA at the lowest

temperature of 6OC was that these decreased the viscosity o f

membranes by increasing the level of unsaturated fatty acids in the

phospholipid bilayer. To test these hypotheses, fatty acid analyses

would have to be performed on the neutral and phospholipid fractions

of the copepod lipids to detemine if the changes with respect t o

temperature in fatty acid composition occur in the membrane or the

depot lipids. If the total phospholipid was further divided into

classes. an increase in the levels of phosphatidylethanolamine a t

lower temperatures might be predicted. Phosphatidylethanolamine

in poikilothermic animals tends to be the most unsaturated of the

lipid classes, and increases in this phospholipid have been observed

in cold-acclimated f ish (Hazel, 1 979) and crustaceans (Chapelle,

1986). Evaluating the fatty acid composition of the various l ipid

classes in th8 copepods is n0t essential for the purpose of marine

fish larval nutrition. Suwival and growth in first-feeding marine

fish lantae c m be related to the EFA composition of the total l i p i d

in the live food (Watanabe, 1993).

Nutritional Implications for Marine Fish Larvae

The lowest temperature (6OC) gave the highest amounts o f

22:6n-3 for both harpacticoids. However, there is a trade-off in

decreasing the culture temperature. Loweiing the temperatu re

lowers the biomass of copepods available from a mass culture

system by decreasing egg production and growth rate and by

increasing generation times (Miliou and Moraitou-Apostolopoulou,

1991a). The nutritional value of the individual copepods for the

marine fish larvae will be increased, but there will be fewer

copepods for feeding to th8 larvae. The copepods had very high

levels of 22:6n-3 and high DHA to EPA ratios at 2Q°C, so that the

EFA value of the food would not be limiting for marine fish larvae

fed copepodg cultured at high temperatures. Therefore, it i s

piobably not necessary to lowei temperature to increase the

harpacticoid copepods' nutritional value.

As the temperature decreased from 20 to 6OC, the amounts of

arachidonic acid (20:4n-6) increased in both harpacticoids over 4 - fold for Amonardia and 3-fold for Tisbe (Tables 5 and 6). The

biggest advantage in ternis of increasing the EFA composition of the

live food with lowered temperature, lies with increasing the

amounts of arachidonic acid. However, requirement of marine fis h

lawae for 20:4n-6 is relatively low when compared to that for the

n-3 fatty acids 20:5n-3 and 22:6n-3, as is evidenced by the l ow

total amount of 20:4n-6 in the laivae (Castell et al., 1994).

LlPlD COMPOSITION OF ALTERNATlVE LlVE FOOD SPECIES

I r n W O N

The lipid compositions of some other native potential live food

organisms were exarnined in addition to those of the harpacticoid

copepods, Tisbe and Amonardia. Wild zooplankton (consisti ng

primarily of calanoid copepods), useâ in lanral marine fish feeding

trials at the St. Andrews Biologicai Station, and the trochophores of

a local mussel species. M'lus edulis, were the alternative l ive

food species evaluated. Cornparisons were then made with the

harpacticoid copepods, to determine which live foods had the most

appropriate EFA compositions for marine fish lanrae. It was

expected that th8 harpacticoid copepods would have the highest

levels of 22:6n-3 and the highest DHA to €PA ratios because of their

extrernely high desaturase activity, thus meking them the most

attractive live food for marine fish larvae, in ternis of fatty acid

composition. The wild zooplankton tend to inhabit environments rich

in n-3 EFAs (20:5n-3 and 22:6n-3) and incorporate high levels of

these EFAs in their lipid. The DHA to EPA ratio in the lipids of most

north Atlantic calanoid copepod species is usually around 1. although

this number cm Vary widely with respect to species and

environmental parameters (Kattner et al., 1981). Shellfish tend t O

have higher levels of 20:5n-3 and DHA to €PA ratios of less than 1

(Kluytmans et al., 1985). The trochophores of M. eduM may not have

as high an €FA value as the copepods as Iive food for marine f is h

larvae, because of this low DHA to €PA ratio.

MATERlALS AND METHODS

Freshly collected local (Nova Scotian) mussels were held in

buckets at ambient seawater temperature (6OC). They were then

isolated in trays containing filtered (10 pm), UV-treated sea water

and brought up to room temperature (lQ°C) to trigger a spawning.

The spetm and eggs of an individual male and fernale mussel were

cornbined in a 20-L bucket and after 24 h the fertilized eggs and

trochophores were captured on 40 jm screen and suction f i l te red ,

using a Buchner funnel, on Whatman no. 1 filter paper. Fatty acid

compositions were detemined for three replicates in the same

manner as for the copepods (p. 19).

The lipid composition of wild zooplankton taken f rom

Passamaquoddy Bay, Na B., in a tow (mesh site = 84 pm) on 27 J uly ,

1995 was also analyzed. The sample consisted of freeze-dried

calanoid copepods (Eurytemora sp. and Acarlia sp.) which were used

as live food for marine fish larvae at St. Andrews Biological Station.

Three replicate samples were weighed and anaiyzed for lipids using

the methods described for copepods (p. 19).

Differences in the relative amount of EFAs in the harpacticoid

copepods, zooplankton and mussel trochophore larvae were compared

using Tukey's HSD pairwise cornparison test.

RESULTS

The fatty acid composition of 24 h-old fertilized eggs and

trochophores of the mussel Mytilus edulis were determined (Table

7). The trochophores contained relatively large amounts of the €FA

20:5n-3 and 22:6n-3, but had a low DHA to €PA ratio (0.37). They

also contained 1.3% of the €FA 20:4n-6. The 16 carbon fatty acids

16:0 (18.5%) and 16:ln-7 (10.6%) comptised the greatest portions of

the saturated and monunsaturated lipid compositions, respectively.

The total PUFA content of the trochophores was extremely high

(alrnost half of the total FA), and approximately equal to the sum of

the saturates and monounsaturates combined. It was predominantly

made up of the n-3 family of fatty acids.

The lipid composition of wild zooplanMon captured i n

Passamaquoddy Bay is shown in Table 8. The percentage of total

Table 7. The fatty acid composition of 24 hour old fertilized eggs and trochophores of the blue mussel, Mytilus eduk

FaW acÏd Percentaae 14:O 1 .O I 0.9 15:O 0.1 f 0.1 16:O 18.5 f 1.0 16:ln-7 10.6 f 0.5 .

7-Me 16 0.1 f 0.0 16:l n-5 0.5 f 0.0 16:Zn-6 0.4 f 0.1 16:2n-4 0.5 f 0.1 17:O 0.3 f 0.0 16:3n-4 0.2 f 0.2 16311-3 0.4 f 0.1 16:4n-3 0.1 f 0.0 16:4n-1 4.8 f 0.6 18:O 2.7 f 0.4 1811 n-9 1.5 f 2.0 18:l n-7 3-1 f 0.6 18:1 n-5 0.2 f 0.0 18:Zn-6 1.2 f 0.2 18:2n-4 0.7 f 0.2 18:3n-6 0-1 f 0.0 18:3n-4 0.3 f 0.0 18:3n-3 1 -6 f 0.3 18:4n-3 2-7 f 0.5 18:4n-1 0.4 f 0.1 20 :O o. 1 f 0.0 20:1 n-11 0-7 f 0.6 20:l n-9 1.9 f 0.6 20:1 n-7 1.6 f 0.3 UNK 1.4 f 0.1 20:2n-6 0-6 f 0.0 20:3n-6 O. 1 f 0.0 20:4n-6 1.3 f O -8 20:3n-3 0. 1 f 0.0 20:4n-3 0.5 I 0.0 20:5n-3 24.0 f 3-5 22:o 0.2 * 0.0 22:1 11-31 O. 1 f 0.0 22:1 n-9 0.3 k 0-2 22:1 n-7 0.4 f 0.3 21 :5n-3 0.9 I O. 1 22:4n-6 0.2 f 0.0 22:5n-6 O. 1 f 0.0 22:4n-3 O. 1 f 0.0 22:5n-3 1.3 f 0.1 24:O O. 1 f 0.0

22:6n-3 8.8 f 1 ,O 24: 1 n-1 1 0.2 f O. 1 C m 23.5 I 1.2 CMoNo 20.6 f 2.2 C PUFA 46.7 f 4.1 C n-3 40.6 f 5.1 C n-6 4.0 f 0.7 D W A 0.4 i 0.0 C UNK 3.8 f 1.8

Resulfs are expraned as 96 of total fuüy adds and represent means and standard deviations of 3 mplicates. Unknowns 4% not induded in table, Abbreviations; ncb not detected, tr= trace (~0.05%). ?-Me= 7-methyl, DMA= dimeîhyl anteiw, SAT= saturates, monounsaturates, PUFA= polyunsaturates, UNlb unknowns, D H k docosahexaenoic acid, €PA= eicosapentaenoic acid.

Table 8. The lipid composition of wild zooplankton taken f rom Passamaquoddy Bay, N. B. in a tow (mesh size = 84 pm ) on 27 July, 1995.

Fatty acid Percentage % Lipidl dry wt 8 -20 * 0.08 % Astaxanthinl lbid wt, 0.62 f 0.08 14:O 0.4 l 4 : l n-9 14: 1 n-7 l 4 : l n-5 l5:O l6:O l 6 : l n-7 7-Mû16:O l 6 : l n-5 16:2n-6 16:2n-4 17:O 16:3n-4 16:3n-3 16:4n-3 16:4n-1 DMA 18:O 18:O 18:ln-9 18:ln-7 l 8 : l n-5 18:2n-9 18:2n-6 18:2n-4 18:3n-6 18:3n-4 18:3n-3 18:4n-3 18:4n-1 20:o 20: 1 n-11 20: 1 n-9 20:1 n-7 20:1 n-5 20:2n-9 20:Zn-6 20:3n-6 20:4n-6 20:3n-3 20:4n-3 20:5n-3

22:o 0.2 f O -2 22:1 n-11 0.1 f 0.0 22:1 n-9 0.5 f 0.0 22:1 n-7 0.1 f 0.0 21 :Sn-3 0.2 f 0.1 22:4n-6 0.1 f 0.1 22:s n-6 0.2 f 0.0 22:4n-3 0.2 f 0.1 22:5n-3 0.5 f 0.2 24:l n-11 0.2 f 0 .2 22:6n-3 16.6 f 2.9 24:1 n-9 4.1 f 0.7 26:O 0.4 f 0.2 CmT 46.2 f 1.6 CMaw 12.8 f O -7 C PUFA 35.3 f 4.7 C n-3 31.5 f 4.7 C ri-6 2.9 f 0 .2 D M P A 1.8 f 0 .2 t UNK 5.7 f 5-7

Results are expressed as % of total fatty ad& and reprewnt means and standard deviations of 3 replicates. Unknowns t1% not induded in taMe, Abbreviations; nd= not detecteâ, tr= trace (<O.OS%), 74- 7-methyl, D M k dimethyl anteisa, SAT= saturates, monounsaturates, PUFA= poiyunsatutates, UNb unknowns, DHAt docosahexaenaic acid, EPAs eicosapentaenoic acid.

lipid was low for calanoid copepods at 8.2% of the dry weight. The

zooplankton sample was freeze-dried for storage which may cause a

lower lipid yield when extracted (Dunstan et al., 1093). The

carotenoid pigment astaxanthin made up 0.6% of the lipid weight

(Appendix 6). The EFAs, 20:5n-3 and 22:6n-3, were present in

relatively high arnounts. The DHA to €PA ratio was also hlgh at 1.8.

The €FA, 20:4n-6, was present in very low quantities. The

zooplankton contained large amounts of saturates (almost half O f

the total FA), somewhat less polyunsaturates and much less

monounsaturates. Most of the saturated fatty acid was 16:O.

The fatty acid compositions of the four live food species (the

two harpacticoids, the wild zooplankton, and the 24 h old M. edulis

trochophores) are compared in Table 9. The 18-carbon fatty acids

18:2n-6 (precursor for 20:4n-6) and 18:3n-3 (precursor for 20:Sn-3

and 22:6n-3) were relatively highest in Amonardia. The percentage

of 20:4n-6 was highest in the trochophores and lowest in the w i I d

zooplankton. Significantly larger quantities of 20:Sn-3 occured i n

mussel larvae, followed by the wild zooplanldon, with the

harpacticoids containing comparatively the lowest percentages.

Tisbe, however, contained the highest percentage of 22:6n-3, not

Table 9. Fatty add composition of potential live foods for marine fish larval culture. Different letter superscripts indicate significant differences at pe0.05 level.

Fatty acid riabe ' Amondia wild wopl-kton3 M. 18:2n-6 1.2 * 0.84 6.4 * 3.56 1-5 i 0.û 1.2 * 0.2.

1. Tisbe sp. fed lsochrysis galbana cultured at 1 SOC. 2. Amonardia sp. fed Isochrysis flaibana culturd at 15%. 3. Wild zooplankton capturd fiom Pamcuguoddy Bay, N. B. in July. 1895. 4. 24 h old Mytilus eduiis eggs and trochophores cultured at 18OC.

Results are wressed as % of total fatly acids and represent means and standard deviations of 3 replicates. Abbreviations; SAT= saturates, MONOc monounsaturates, PUFh polyunsaturates, D b docosahexaenoic acid. EPA= ricosapentaenoic acid.

significantly greater than the wild zooplankton, but significantly

more than was present in Amonardla and M. edulis. Not suprisingly,

the harpacticoids had significantly higher DHA to EPA ratios when

compared wïth the other live food specïes. The M. edulis larvae had

a lower DHA to EPA ratio than the wild zooplankton (0.37 vs. 1.77).

DlSCUSSiûN

Lipid Composition of Alternative Live Food Species

The larvae of M edulis had a significantly higher level of

20:Sn-3 when compared to the copepod species. The lower levels of

20:5n-3 and higher levels of 22:6n-3 in the copepods could be an

adaptation to their more motile lifestyle when compared to that of

M. edulis (suggestion by Dr. R. G. Ackman). The fatty acid 22:6n-3 i s

present at high levels in the nervous systems of motile animals. The

functioning of the nervous systern is based on the activity of ion

channels and of receptors situated within the lipid bilayer. The

proper functioning of these proteins depends on a bilayer wit h

certain physical properties (i.e., fluidity and permeability) which the

n-3 PUFA confer, in particular 22:6n-3 (Dumont et al., 1992). The

mussel trochophore larvae were the only potential live food species

we studied which had a DHA to EPA ratio of less than 1. This fa tt y

acid composition is typical of other marine bivalve larvae (Marty e t

al., 1992). Unlike the copepods. M edulis larvae do not use

desaturase and elongase enzymes to convert most of their 20:5n-3

into 22:6n-3.

Both 77sh and Amonardia had significantly highei DHA to EPA

ratios compared to the wild zooplankton. The latter (consist ing

almost entirely of Acartia sp. and Eurytemora sp.) captured i n

Passamaquoddy Bay during the month of July, contained very low

lipid levels (about 896). These copepods do not contain an oil sac,

thus they do not accumulate the large lipid reserves of some other

calanoid species. The phyîoplankton concentrations in July also tend

to be low, so that the wild zooplankton will not have a high energy

intake, or b8 able to accumulate high levels of lipid (Sargent and

Henderson, 1 986).

The lipid compositions of the two harpacticoid copepods

differed considerably. Tlsbe seems to have a greater desaturating

capacity than Amonardia. When both species were fed I. galbana a t

1 5OC, Tisbe produced almost twice as much of the n-3 EFAs, 20:5n-

3 and 22:6n-3. Amonardia, however, contained signif icantl y more

18C precursor fatty acid 18:3nB (Table 9). This suggests that Tisbe

contains more of the desaturases necesssary for this conversion

( g . 1 ) . Amonardia also had a signif icantly higher level of 1 8:2n-6,

but the levels of 20:4n-6 were not significantly different from

those of Tisbe, a further indication of less active A-6 and A - 5

desaturase. Also, the overall levels of PUFA were much higher i n

Tisbe, compared to Amonardia, whereas, Amonanfia contained much

higher levels of the saturates and monounsaturates.

Nutritional Implications for Manne Fish Lawae

The nutritional value of the wild zooplankton for marine f ish

larvae will Vary considerably with season. Our zooplankton sample

taken in midsummer was lipid poor (about 8% dry wt.). Amounts of

lipid reach their lowest levels in July for North Atlantic calanoid

copepods. Also, the fatty acid composition (particularly of the

neutral lipids) would be quite different, immediately after or during

a phytoplankton bloom (Kattner et al., 1981 ; Norrbin et al., 1 990).

The wild zooplankton have a much higher caloric and nutritional

value for mafine fish larvae dun'ng or directly after the spring or

fall ph ytoplankton bfoorns, when some calanoid species accumulate

wax esters, which can make up to 70% of their dry weight (Sargent

and Henderson, 1986). The advantage of using cultured animals

versus wild moplankton is that the cultured animals can be

manipulated to give a consistently high lipid and EFA composition.

The large arnounts of both long-drain EFAs, 20:5n-3 and 22:6n-

3 and their high D M to EPA ratio indicate that these calanoid

copepoûs have high EFA value for marine fish lawae. This high EFA

value is one reason why field-captured zooplankton aie widely and

successfully used for the culture of cold-water manne fish larvae

(aga Kuhlman et al., 1981 ; Boxsspen et al., 1990; Shields, 1992).

The value of trochophores of the mussel, Mytilus eduiis, as an

alternate [ive feed for marine fish lawae was evaluated by Howell

(1973) and was found not to support growth in lawal plaice,

Pleuronectes platessa, and lemon sole, Solea solea, when used as

the only food. However, when the trochophores were offered in

combination with rotifers to the lemon sole l a ~ a e at the early

first-feeding stages, a survival rate of 32% and an average growth

increase from 5.8 to 8.1 mm length for the first 30 days post-hatch

was obtained. Ch days 12 and 20 post-hatch, al1 the first-feeding

larvae contained only trochophores in their guts. This demonstrates

their preference for trochophores over rotifers during the early

first-feeding stage (Howell, 1971).

The advantage of using mussel lawae as live food is the ease

of culture. Simply raise the culture temperatures of adult male and

fernale mussels and they will spawn. Combine the spem and eggs of

the mussels and within 24 h trochophore larvae are produced.

Trochophores may not have as high an €FA value as copepods

for manne fish larvae because of their lower DHA to €PA ratio.

However, the trochophores do contain much higher levels of the

long-chain EFAs, 20:5n-3 and 22:8n-3, than do the traditional l ive

food organisms, bfine shrimp and rotifers. The rnwsel trochophores

also contain a higher proportion of the EFA 20:4n-6 than do the other

alternative live food organisms analyzed.

Live foods, which contain either the most 22:6n-3 or the

higher ratio of 22:6n-3 to 20:Sn-3 tend to have the highest EFA value

for cold-water, marine fish larvae (Bell et al., 1985; Watanabe,

1993). The two harpacticoid copepod species studied had the

highest ratios of DHA to EPA, when cotnpared with wild zooplankton

or mussel lawae. Tisbe hed the highest percentage of 22:6n-3 of al1

the live foods that we examined, and it contained much more of th is

EFA than the traditional live food organisms such as the brine

shrimp and rotifers. Tisbe is able to produce large amounts o f

22:6n-3 and maintain consistently high DHA to EPA ratios (2.0 t O

3.4) despite the €FA composition of its diet, due to its high levels O f

desaturase activity . This 22:6n-3 producing capability toget h er

with its ability to be mass produceci at high densities make the

harpacticoid Tlsbe, and to a lesser extent Amonardia, very

attractive potential live food organisms for marine fish l arval

culture.

Chapter 2

Free Amino Acid Composition of Harpacticoid

Copepodr and lmplicationr on their

Nutritional Value for Marine Fish Larvae

Introduction

Free Amino Acid Metabolism in Copepods

Marine invertebrates, crustaceans especially, accumulate large

reserves of free amim acids (concentrations around 300-500 mM

are typical) for use as osmolytes to help maintain their intracellular

osmotic stasis relative to that of their seawater medium (Pierce,

1981; Fyhn et al., 1993). Theiefore, changing the salinity of the

seawater medium will effect a diange in the free amino acid pool.

With exposure to reduced salinities, the free amino acid levels are

reduced in both calanoid (Farrner and Reeve, 1978; Fyhn et al., 1 993)

and harpacticoid (Goolish and Burton, 1 989) copepods. The f re s h l y-

caught calanoid copepod AcaHia tonsa starved during sa l i n i t y

acclimation did not increase its free amino acid pool at salinities

above 100% seawater, perhaps du8 to a lack of nutritional reserves

needed for the extra synthesis of amino acids at the higher

salinities (Farmer and Reeve, 1 978). The calanoid Calanus

finnmarchicus, however, did increase its free amino acids as

salinity increased from 15 to 45 ppt (Fyhn et al., 1993). Jeffries

and Alzara (1969) also obsewed an increase in the total amount o f

free arnino acids as salinity increased, when comparing calanoid

copepod species which live in f reshwater environments wit h t hose

from estuarine and marine environments. The harpacticoid copepod,

Tigriopus, inhabits splash-pools of the intertidal zone and

experiences a wide range of salinities, being active in water from O

to 80 ppt salinity. Tisbe sp. occupies the sublittoral zone or t ide

pools that are regularly inundated by seawater and so experîences a

narrower range of salinity in its environment. It is only active f rom

30 to 45 ppt salinity (Finney, 1979), therefore, one might expect a

larger free amino acid pool in the euryhaline Timpus when

compared to Tisbe, particularly when acclimated to higher

salinities. Tigriopus californicus was the subject of a number o f

studies on free amino acids versus salinity (Burton, 1986; Burton

and Feldman, 1982; Goolish and Burton, 1989). The non-essential

amino acids proline, glycine and alanine, in decreasing order of

concentration are the dominant contributors to the free amino acid

pool when Tm californicus is put under hyperosmotic stress. These

high free amino acid levels drop rapidly (within 5 h proline dropped

40%), when the copepods are transfened from water of 68 to 34 ppt

salinity (Burton and Feldman, 1982). Therefore, the copepods must

be ingested within a short period of time after exposure to the

hyperosmotic medium. if the marine fish larvae are to benefit

maximally from the increased level of non-essential free amino

acids of the copepods.

The dominant contributor to the free amino acid pool differs

between the harpacticoids and calanoids which have been acclimated

to 100% seawater. Glycine dominates the free amino acid pool in the

marine calanoids (Fyhn et al., 1993; Famer and Reeve, 1978),

whereas proline dominates the free amino acid pool in the

harpacticoid copepod T 'opus (Burton and Feldman, 1 982).

Crustaceans, when injected with radiolabelled acetate, were

shown to lack the enzymes needed to synthesize the same 10 amino

acids which are essential for marine fish larvae (Cowey and Forster,

1971). Therefore, any increase in these 10 essential amino acids as

a response to either salinity or diet is due to increased

incorporation from the diet and not de novo synthesis.

Free Amino Acid Metabolism in Manne Fish Larvae

Manne fish larvae also require certain amino acids. Halver

(1 957a, b) discovered that chinook salenon Oncorhynchus fsha wytscha

required 10 essential amino acids (phenylalanine, tryptophan,

histidine, arginine, lysine, valine, isoleucine, leucine, threonine and

methionine). These amino acids were omitted from test diets and

growth responses were compared with those of complete diets. No

significant growth was obtained on diets devoid of any one of these

amino acids. The marine f latf ish plaice Pleuronectes platessa end

sole Solea solea were ako unable to synthesize these amino acids

from radiolabelled glucose. Thus, these amino acids are also thought

to be essential for marine fish (Cowey et al., 1970). nierefore. a

higher level of essential free amino adds will increase the

nutritional value of the live food for manne fish larvae.

Free amino acids have a special role in the eggs and early

larvae of marine fish. Marine fish eggs and yolk-sac l ame contain

much higher amounts of free amino acids compared to the adolt

tissues. The large pool of free amino acids is contained almost

exclusively in the yolk-sac cornpartment of the fish lawae. The free

amino acid pool is depleted during development and reaches l ow

levels at first feeding (Rsnnestad and Fyhn, 1993). As this free

amino acid level decreases with development, there is a concomitant

increase in ammonia excietion and oxygen consurnption, suggesting

that the free amino acids are being aerobically catabolized as a

source of energy (Fyhn and Sergistad, 1987; Finn and Fyhn, 1993).

First-feeding mafine fish lanrae have a relatively undifferentiat ed

digestive system and low proteolytic capability (Hjelmeland et al..

1993; Pedersen et al., 1987). Dietary proteins will, for the most

part, be unuseable as an energy source by the larvae at this stage.

Also their supply of free amino acids from th8 yolk sac will have

been largely depleted. Thus, at the initiation of exogenous feeding,

the marine fish lawae may require large amounts of free amino

acids. As noted above, marine zooplankton typical l y accum ulate

large amounts of free amino acids which play a dominant role in

rnaintaining the osmotic balance of their intracellular fluids. Fyhn

(1989) suggested that, in choosing a live food organism, the free

amino acid pool of the prey should match the free amino acid pool

consumed by the fish embryo during endogenous feeding.

Experimental Objectives

The purpose of this study was to determine if the diet fed t O

the harpacticoid copepod, Tisbe sp., affects the size and

composition of the free amino acid pool in the copepod. The t wo

diets used in the copepod feeding trial, the alga lsochrpis galbana

(clone T-iso) and bakersg yeast, were chosen because of their large

differences in amino acid composition (Chau et al., 1967; Ren et al.,

1993). It is expected that the proportions of nonessential free

amino acids in the copepod fed the two diets should remain f ai rl y

uniform with nonessential free amino acids such as glycine, tau rine,

proline and alanine dominating. However, the increased amount of

essential amino acids available in the alga 1. galbana (T-iso)

compared with yeast, should increase the level of essential free

amino acids in the copepods fed T h D An increase in the size of the

free amino acid pool might increase growth in fish lawae R the free

arnino acids remain an important source of fuel and precursors f O r

protein synthesis after yolk sac absorption (Fyhn et al., 1993). Large

variations in the total free amino acid pool, foi the copepods fed the

two diets should be expected, considering the dynamic nature of

these metabolites (Stnrttrup et al., 1995). A higher level of

essential free arnino acids in the copepods will also increase the

nutritional value of th8 live food (Cowey et al., 1970).

The free amino acid composition of wild zooplankton (calanoid

copepods) from a tow in Passamaquoddy Bay, Ne B. (27 July, 1995)

was also compared with that of the harpacticoid copepod Tisbe. The

free amino acid compositions should be similar for the two copepod

88

classes with the non-essential amino adds glycine, proline and

alanine dominating (Farrner and Reeve, 1978).

The salinity tolerance of Tisbe sp. was also tested. It i s

expected that this copepod will be stenohaline as it lives in an

environment with a low variability in salinity (Finney, 1979).

Because of this stenohalinity Tisbe will not have the same potential

as Tigropius for increasing its free amino acid pool, and therefore

energy value, for marine fish lawae when subjected to h yperosmotic

stress (Burton and Feldman. 1982).

Materials and Methods

The free amino acid composition of an alga, lsochrysis galbana

(clone T-iso), a bakers' yeast diet, and of Ti- sp. fed these diets,

was evaluated. The alga, T-iso, was cultured and centiifuged using

the sarne methods described earlier for lipid analyses of aigae (p.

19). T-iso, was grown at 18°C on f/2 medium (Guillard and Ryther,

1962) and harvested by centrifugation while in th8 log phase at 8 d.

The copepods were cultured for over one generation on the yeest and

algal diets using the same culture setup as described for the lipid

study (p. 16). The copepods were screened through a 200 pm mesh

and subsequently transferred to jars containing fresh 1 0-prn

filtered, UV-treated sea water. They were then stanred for 24 h t o

allow for the clearance of dietary algae or yeast present in the gut

and rescreened to remove any accumulated waste products. The

copepods were then counted, suction filtered through Whatman no. 1

filter paper and rinsed with seawater.

Free amino acids were extracted from the copepods and t hei r

diets using boiling 80% ethanol. Two replicate samples of the

copepods containing approximately 500 individuals per sample we re

analyted. Three replicate samples of the diets consisting of an

average of 84.0 mg dry weight of bakers' yeast and 86.1 mg dry

weight of T-iso were used for free amino acid analysis. The dietary

and copepod samples were immersed in the boiling ethanol and

homogenized in 20-mL glass culture tubes using a polytron

(Brinkman Instruments). The homogenized samples were t hen

filtered through Whatman no. 1 filter paper and rinsed with excess

boiling ethanol. The ethanol extract from each sample was then used

for free amino acid analysis.

An emount of standard norleucine was added to the extract so

that area percent could be converted to pmoles of free amino acid

units. The standard norleucine peak area was approximately 10930%

of the largest peak area. The amount of norleudne to be added for a

given amount of sample was detemined by either the estimated dry

weight of the sample. or by preliminary analysis with various

dilutions of the sample. The extract was then evaporated to dryness

in a rotary flash evaporator (Haakebuchler Instruments). Samples

were redissolved in a citrate buffer (pH 2.2) and injected on the

automatic amino acid analyzer (Bedunan 119 CL), which was based

on the system developed by Moore et al. (1958). The presence of

glutamine in the yeast samples interfered with quantification of the

threonine and serine peaks. Therefore half of the yeast sample was

made to 2 M with trifluoroacetic acid and hydrolyzed at 1 OO°C f O i

1 5 min (sufficient to quantitatively destroy glutamine), after w h i ch

it was evaporated to dryness and dissolved in citrate buffer (pH 2.2)

for injection on the amino acid analyzer.

A cornparison between two free amino acid extraction

rnethods- the aqueous phase of the Bligh and Dyei (1 959) versus the

boiling 80% ethanolic extraction method- was conducted using a

hornogenized wild zooplankton sample (Appendix C). The 80% ethanol

method gave significantly higher extractions for 75% of the amino

acids detected in the zooplankton. Thus, the 80% ethanol method

was chosen over that of Bligh and Dyet (1959), for the extraction o f

free amino acid from the copepod samples.

The salinity tolerance of Tisbe was also tested using

different amounts of lnstant* O-' (Aquarium Systems) dissolved

in distilled water. Approxirnately 20 adult copepods were e~posed

to different salinities (20, 30, 40 and 54 ppt.) for 5 hours. The

copepods were then examined under a dissecting microscope (20X

magnificat ion) to determine percent survival . Results

The free amino acid composition of bakers' yeast and

Isoch~sis galbana (clone 1-iso) differed considerably (Table 1 O) .

The alga had a total free amino acid concentration greater than 3.5

times that of the yeast. The dominant free amino acids in the T-iso

sample were gamma aminobutyric acid (ca. 39 pmol/g dry W.) and

alanine (ca. 19 pmollg). Proline, taurine and arginine were the other

major free amino acids in T-iso (ca. 11 prnoVg). The dominant free

amino acid in the yeast was glutamic acid (ca. 25 pmoVg diy wt.).

Alanine (ca. 5 pmollg), aspartic acid (ca. 3 pmol/g) and proline (ca.

3 pmol/g) were the next largest contributors to the free amino acid

pool in the yeast.

The mol % free amino acid composition of the copepods was

very similar (Table 1 1). among diets (T-iso W. yeast) and copepods

(calanoid vs. harpacticoid). The wild zooplanîcton sample was made

up almost entirely of the calanoid spedes, Eurytemora sp. and

Acartia sp. Glycine was the dominant free amino acid in al1

copepod samples. It was highest in th8 wild zooplankton (43.1%),

followed by nsbe f8d yeast (38.0%) and T-iso (33.5%). In the w i l d

zooplankton, glycine was foiîowed in decreasing order, by taurine,

alanine, proline and arginine. In nsbe fed with yeast, glycine was

followed in decreasing order by alanine, taurine. arginine and

proline. In Tisbe fed with T-iso, glycine was followed by arginine,

taurine and alanine. Proline was present at a comparatively l o w

level in Tisbe fed with T-iso. Thus, the three copepod samples had

fairly similar patterns with respect to mol % free amino acid

composition. The nonessential free amino acid glycine dominated,

followed in lesser arnounts by alanine, taurine and proline (Tisbe

fed T-iso excepted). The essential free amino acids were present i n

much smaller amounts in the copepods, when compared with the

nonessential free amino acids. Arginine was the only essentiel free

amino acid which exceeded 5% of the total free amino acids. Tisbe

Table 1 O. Free amino acid composition of the alga Isochrysis gaibana (clone T-iso) and bakers' yeast used in copepod feeding trials. Results are expressed as pmol/ g dry wt. and represent means and standard deviations of three replicates.

Amino acid Yeast T-iso taurine nd 11.18 f 1.85 aspartic acid threonine* serine glutamic acid proline glycine alanine cysteine valine methionine* leucine* isoleucine* tyrosine phenylalanine* GABA histidine* lysine* arginine* Total EFAA % Total NEFAA % Total FAA

Abbreviations: nt#= mt deticted, GA&= gamma amino butyric acid. FAA= free amino acids, EFAA= essential free amim acids. NEFM= nonessential free mino acids. denotes individual essential free amino acids.

Table 11. Free amino acid composition of Tisbe sp. fed the alga Isochrysis galbana (clone T-iso) and bakers' yeast compared with a homogenized, freeze-dried sample of wild zooplankton isolated f ro m the plankton of Passamaquoddy Bay, N. B. in July, 1995. Results are expressed as moi % and represent th8 means of two replicates f O r Tisbe and three replicates for the wild zooplankton samples.

Tisbe fed Amino acid Yeast T-iso Wild zooplktn8 taurine 13.05 11.37 15.70 aspartic acid threonine* serine glutamic acid proline glycine alanine cysteine valine* methionine* leucine* isoleucine* tyrosine phenylalanine* GABA histidine' lysinet arginine* Total EFAA % Total NEFAA % Total FAA (pmol/ind.)

Abbreviations; mi= not detected. wild zooplktn= m'id zoaplankton, gamma amino butyric acid, FAA= free amino acida, €FM= essentiai free amim acids. NBAA- nonessentiai free amino acids, denotes individual €FM.

a. absolute data (pmol F M g dry W.) given for wild zocplankbn in Appendk C (p. 137)

fed T-iso contained th8 largest fraction of essential free amino

acids, with levels of the basic amino acids arginine, lysine and

histidine approximately double those in the other copepod samples.

The free arnino acid level per copepod was approximately

twice as high in Tisbe fed yeast as in Tisbe fed T-iso. The level of

total free amino acids in the homogenized, freeze-dried wi ld

zooplankton was expressed as pmoV g dry wt. and was not d i rectl y

corn pared.

The salinity tolerance of Tisbe tested using Instant Oceana

(Aquarium Systems) and was found to be fairly stenohaline. The

survival was 1ûô% at 30 and 40 ppt. after 5 h exposure. At 20 ppt,

survival was 7ï%, but the copepods were quite inactive and at 50

ppt, survival was only 29% after 5 h.

Discussion

The large differences in the free amino acid composition

between the yeast and alga used for th8 copepod feeding t r ia ls

should be expected considering that the orgmisms are classified i n

different kingdoms and inhabit diff erent environments. T-iso has a

much higher total free amino acid content than the yeast. The role

of free amino acids as osmotic effectors in marine algae was f i r s t

demonstrated for the red aiga Porptyn'dium purpureum (Gilles and

Pequeux, 1968). The total free amino acids increased 4-fold when

this alga was moved from fresh to sea water culture. Presumably,

T-iso also uses free amino acids to help maintain an osmotic

balance in its marine environment.

The high level of glutamic acid, which malces up approximately

50% of the total free amino acids, is typical for yeast (Ren et al.,

1 993). The alga T-iso (Isochrysis galbana ) has a large percentage

of free gamma eminobutyric acid (about 20%). There appears to be

no published data on the free amino acid composition of T-iso.

Gamma aminobutyric acid does not play a role in the structural

proteins (Enright et aL, 1986) but is the dominant free amino acid i n

T-iso. This indicates that its major role in the algae could be as an

osmotic effector.

In general, the harpacticoid copepod Tisbe demonstrated a

similar free amino acid pattem when fed the yeast and T-iso diets,

despite the very different free amino acid compositions of the diet.

The wild zooplankton also demonstrated a very similar pattern t O

Tisbe. The total amount of free amino acids per individual Tisbe

was about twice as great in those fed on yeast as aiose fed on T-iso.

Such variations in total free amino acids were also seen in the

rotifer Brachionus plicatilis fed various algal diets (Stmrttrup et al.,

1995). and might be expected considering the transitory nature of

the free amino acid pool.

Glycine was the dominant free amino acid in the copepod

samples including those from the wild zooplankton. The free glycine

levels of Tisbe more closely resembled those of the calanoid

copepods (wild zooplankton sample), than those of the i n t e rt idal

harpacticoid copepod Tigropius califomicus examined by Burton and

Feldman (1982). Th8 proline concentration was 5 times higher than

glycine in T. californ~cus, and was the primary free amino acid used

by this euryhaline copepod to osmoregulate in extremely variable

salinities. It appears that the non-essential free amino acid

composition of marine copepods is more dependent on their f u nct ion

in osmoregulation than on their classification (harpacticoid vs.

calanoid). Free amino acids of euryhaline copepods are dominated by

proline. whereas stenohaline copepods store large amounts of free

glycine. The stenohaline copepods with dominant glycine in the free

amino acid pool include calanoids such as Acartia tonsa (Famer and

Reeve, 1978) and Calanus finmarchicus (Cowey and Corner. 1 963),

as well as our harpacticoid copepod Tisbe . The percentage of essential free amino acids in 77sbe fed the

alga T-iso was almost twice that of nsbe fed yeast. This may

partially reflect the higher total amount of free amino acids

available in T-iso, which also has a much higher percentage of f ree

essential amino acids approximately double that in the yeast.

Copepods have high enzyme levels and contain large amounts of

protease (Munilla-Moran et el., 1990; Mayzaud et al., 1992).

Therefore, the proteins in the yeast and T-iso can also contribute to

the free amino acid pool in Tisbe . Like the copepods, marine flatfish l a ~ a e aie unable to

synthesize the 10 essential amino acids and must, theref ore,

incorporate them from their diet (Cowey et al., 1970). Tisbe fed

with the alga T-iso had a higher percentage of essential free amino

acids than either the Tisbe fed with yeast, or the wild zooplanidon,

and thus might provide better nutrition fo i the fish lawae. Marine

fish larvae tend to have simple digestive systerns and l irnited

protein catabolizing ability. This is demonstrated in the marine fish

larvae Chrysophrys major, Plecoglossus aitiveiis and L ates

calcarifer, which can ingest protein-membrane microcapsules, but

are unable to digest the walls of these capsules (Kanazawa et al.,

1982; Walford et al., 1991). As noted above, the proteins in l ive

food may be partially digestible because the fish lanrae use the

exogenous enzymes of the live food foi digestion. Walford et al.

(1991) demonstrated that adding rotifers to a microencapsulated

diet caused increased digestion of the protein membranes of the

microcapsules, suggesting that the fish larvae are using the

exogenous proteases of the rotifers to aid in digestion of the protein

membrane of the microcapsules.

Amino acid composition of proteins in crustaceans fed

different diets tends to remain fairly constant (Frolov et al., 1991;

Tamani et al., 1993). Therefore, assuming a constant amino acid

composition foi the proteins, Tisbe fed with T-iso rather than yeast

would appeai to be the better food for marine fish larvae because its

free essential amino acid content is double that of Tisbe fed w i t h

yeast.

Chaptei 3

Mars Culture of the Harpacticoid Copepod, T l s b sp.

Introduction

Harpadicoid copepods have been mass cultured in vaiious

laboratories for use as an alternative live food source. The marine

harpacticoid copepods, Tigropius jatponicus (Fukusho, 1 98 O),

Amphiascoides atopus (Sun and Fleeger, 1995) and Tisbe sp. (Kahan

et el., 1982; Uhlig, 1984) are among those that have been cultuied on

a large scale.

The harpacticoid copepod genus Tisbe is epibenthic and l ives

in the subtidai zone. Tisbe is therefore more suited to production

with a more constant supply of fresh seawater. A funning water

system shows decisive advantages over a closed system. Zhang and

Uhlig (1993) found that lawal mortality is about 20% lower in

running water systems, the time of larval development is about two

days shorter and thete is a high rate of naopliar production. Uhlig

(1981) also demonstrated that an increase in the production of Tisbe

was correlated more to the available substrate or base area in the

container, than to seawater volume.

U hlig (1 084) gave five reasons why harpacticoid copepods,

such as Tisbe, are more suitable than calanoids in mass culture as

live food for marine fish larvae. Of particular importance are: 1 )

their tolerance of a wide range of environmental conditions, 2) their

ability to utilize many different food sources, 3) their high

reproductive capacity. 4) their relatively short life cycles, and 5)

their ability to produce high population densities in appropriate

culture systems.

The purpose of my study was to create an appropriate mass

culture system for Tisbe, and evaluate the copepod population

present in the system over time. The copepod populations in the

mass culture system were evaluated for two diets: a commercial

larval diet, Microfeast L-1 O@, and the alga lsochrysis galbana. Given

its ability to thrive on a van'ety of food sources (Miliou and

Moraitou-Apostopoulou, 1991 b; U hlig, 1 Q8l), high densities of Tisbe

were expected in this system for both diets.

Materials and Methods

The mass culture system used in this study (Fig. 3). contained

32 L of seawater (20°C) in a plastic culture tank. The water was

successively filtered through 5 pm and 0.25 pm pore size cartridge

f Mers (model #150071; Atlantic Purification Systems Ltd.,

Dartmouth, N. S.) end approximately 15-20 L was added every 2-3 d.

Fig. 3. Diagram of the harpacticoid copepod mas$ culture system (not to scale). The system indudes A) an airlift made of 1 .5" diameter A M pipe containing an airstone and B) a plastic ~upperware~ container which houses an airstone and is connected to the outlet with a 100 pm screen glued over a 6x18 an hole cut in t O

the lid. This culture system was placed in a 100 L tank (not depicted) with continuously flowing seawater heated to 20°C.

A constant flow rate was not used, because the 100 pm screened

outlet became obstnided in the tank containing the oily ~ i c r o f e a s t ~

diet, causing the tank to overflow within 12 hm An airlift pump kept

the water in the tanks well aerated, and provided a means of

bringing copepods from the bottom of the tank to the surface f O r

collection. A plastic ~upperware@ container (24x12 cm) was affixed

to the outlet. The ~upperware* lid had an 18x6 cm hole cut out and a

100 pm screen glued over the opening. The 100 pm screen was large

enough to permit good water flow through the outlet pipe, but it did

allow th8 escape of some of the copepod nauplii, although the adults

and copepodites were ietained. An airstone was used to maintain

water circulation in the ~upperware' container, thereby keeping the

mesh free from obstruction. The culture system was placed in a 100

L tank, with a continuous flow of 20°C seawater to maintain a

constant water temperature.

The harpacticoid copepod Tisbe which we had been culturing in

srnall, 6 L cylindrical jars for biochemical analyses, was introduced

to our mass culture system. Two replicate culture systems were

used for each of the two dietary treatments. The copepods were fed

twice per week with either 0.5 g of Microfeast L-IO* lawal diet or

3 L (appiox. 5 million cells/mL) of log phase lsochrysis galbana

(clone T-iso).

Enurneration of copepod populations in the mass culture tanks

were perfomed at various time intewals. The copepods were

transferred from the mass culture tanks into 20 L buckets using

0.25 pm filtered sea water. They were then collected on a 40 pm

screen to decrease the water volume and transferred to a 2 L

graduated cylinder. They were then thoroughly mixed and a known

volume was removed.. The majority of adult copepods were

separated from the copepodites and nauplii, by seiving through a 200

pm screen. After a number of sirnilar dilutions, copepods of the two

size fractions were evenly mixed in a 20 mL graduated cylinder, and

0.5 mL drops were micropipetted onto a petri dish for counting using

a dissecting microscope (20X magnification). Five drops were

counted to determine an average. Dilution factors were used to

calculate the number of copepods in the original culture.

Rerults and Diacurrion

The copepods fed the Microfeast L-IO@ larval diet were first

- . . O 5 10 15

time (days)

m

2000 - 10 20

timr (dayr)

Fig. 4. Time courses for Tisbe growth in the mass culture system. Counts were performed on two sire classes, those retained on a 200 ym mesh (mainly adults) and those that passed through (copepodites and nauplii). The effects of two diets A) Microfeast L-1 O' larval diet (log scale) and 6) the alga lsochrysis galbana (clone T-iso) were determined for two ieplicates of Tisbe (Tanks A end B).

counted after approximately 3 weeks of culture (Fig. 4). Over a

period of 12 days, the adults (~200 pm size class) increased on

average from about 22,000 to 32,000 indviduals. During this same

time period, the juveniles and nauplii (QOO pm size class)

increased on average from about l89,OOO to 829,000 individuals.

The copepods in the mass culture tanks fed T-iso were counted f ro m

the first day, when 384 adult copepods wefe introduced to each of

the two tanks (Fig. 4). Growth of the populations were very si mil ar

in the replicate tanks fed 1-iso. Over the first 6 d, the adult size

ciass decreased from 384 to an average of about 225 individuals

(41.4% mortality). Meanwhile, the juveniles and nauplii increased

from O to an average of about 2,500 individuals. The adult

populations thus produced an average of about 1.4 nauplii par

individual per day in this mass culture system over the first 6 d.

After 16 d, the adult population (>ZOO pm) reached about 7,000 as a

result of the maturation of the first generation of nauplii. There

was a large difference in the population growth of the 9 0 0 pm size

class between tanks from 6 to 16 d. In Tank A, the population

increased from 2,500 to alrnost 11,000 and in Tank B. from 2,500 to

6,000 individuals.

The expefiment was terminated du8 to contamination by

other harpacticoid copepod species in the tanks. Thus. we were not

able to compare the carrying capacities of the mass culture tanks

containing Tisbe fed the two diets. To prevent contamination i n

future studies, it is recommended that the copepods be cultured i n

an isolated area to prevent untreated se% water from getting into

the tanks. The copepod populations fed T-iso had not reached the

levels of those fed Microfeast. The mass culture of Tisbe fed w i t h

T-iso demonstrated the short time span needed to obtain a f a i rl y

large population of copepods. After 16 d the total copepod

population (both site classes) had reached a level of alrnost 20,000

copepods per tank. The terminal population of the Microf east-fed

animais in Tank A approached 1 million of the d l 0 pm class and

32.000 adults in a relatively small tank volume of 32 L. This

indicates that Tisbe has the capacity necessary to produce the

numbers needed for larval fish culture. The original purpose of the

experirnent was to detemine the carrying capacity of this system

for Tisbe fed both diets. Unforhinately, the copepod population was

still growing in al1 tanks at the time of contamination. Once the

canying capacity of the system is determined, a study of harvesting

rates for the copepod population could be carried out. This study

should simulate the exploitation levels needed for the culture of

marine fish larvae. Different proportions of the population would be

taken out at various time intervals to determine the ability of the

population to retum to previous levels (Ohno et al., 1990).

The copepod population could be further increased if the

available substrate area in the tank was increased. Uhlig (1 98 1 )

dernonstiated that the mass production of Tisbe is essentially

related to the available substrate area, and less to the available

water volume. A series of black removable plates placed in the tank

might be an advantageous addition to the m a s culture system.

Harpacticoid copepods are negatively phototactic and will be

attracted to the surface of the black plates. These plates could then

be rernoved and the copepods washed off to be fed to the fish larvae.

Chapter 4

Preliminary Trials Uring a Harpacticoid

Copepod, Tlrbe rp., as a Diet for Marine Fish Larvae

Introduction

Calanoid (Last, 1978; McLaren and Avendano, 1995)

harpacticoid (Jindasa et al., 1 991 ; McCall, 1 992) copepodites

nauplii constitute the principal food of many mafine fish lawae.

culture of most cold-water marine fish species requires

and

and

The

the

provision of live prey for a variable period .from the onset of larval

feeding. When the marine Rsh larvae are 0ffer8d C0p8pOd~ and other

zooplankton @.gag rotifers Brachionus plicatilis) in their diets, the

fish larvae tend to select the copepods (Kuhlmann, Quantz, and Witt,

1981; van der Meeren, 1991). Meng and Orsi (1991) demonstrated

that larval striped bass in the San Joaquin Estuary select native

over introduced species of copepods.

Growth of marine fish larvae fed on a copepod diet alone has

been demonstrated for both calanoids and harpacticoids. for

example, the harpacticoid Tisbe sp. has been used in preliminary

studies as a live food for laival saury Scomberesox saurus

(Brownell, 1983) and sea bream Spanrs aurata (Kahan et al., 1 98 1 ) .

Euterpina a cutiforons was fed to larval mahimahi Cotyphaena

hippurus (Kraul et al., 1991). Tigropius japonicus was fed to the

112

mud dab Limanda ydrohamae (Fukusho et el., 1979) and the black

sea bream Mylio macrocephaius (Lee et al., 1981).

The calanoid copepoâs have ais0 been used as live food for

manne fish larvae. Euryiemora affinis has been wed as the sole

food foi striped b a s larvae Morone saxtiIis (Chesney, 1989; Tsai Ce,

1991), and for turbot larvae Scophthalmus maximus (Kuhlmann e t

al., 1981 ; Witt et al., 1984). Wild zooplankton, presumed to be made

up almost entirely of calanoid copepods, have been used as food fo r

hali but larvae Hippoglossus hnippglossus (Boxaspen et al., 1 990 ;

Shields, 1992).

The main advantages in using harpacticoid rather than calanoid

copepods are the ability of the former to be mass cultured and thei r

high €FA (20:Sn-3 and 22:6n-3) composition which is independent o f

the long-chah €FA composition of the diet. A further advantage o f

using harpacticoids in marine fish culture is that those which are

not eaten are able to find nourishment in the fish tanks by feeding on

detritus or the rapidly developing biofilm, including live bacteria on

the tank surfaces. Thus, they maintain their nutritional value while

helping to keep the tank ctean, both important factors in the

successful rean'ng of marine fish larvae (Stnrttrup, 1 993).

A disadvantage to using harpacticoids for marine fish culture

is their inability to remain suspended in the water column.

However, Kahan et al. (1 981) devised a floating tray culture system

for Tisbe, in which a basket with a 80-100 pin mesh bottom f loats

on the surface of a larval marine fish rearing tank. This allowed

newly hatched nauplii to fall through the mesh, thereby making the

copepods more available in the water column as a live food for the

fish lawae.

A preliminary, small-scale study was perfoned w i t h

American plaice Hippoglossoides platessoides and haddock

Melanogrammus aeglefinus larvae feeding on a sole diet of either

rotifers Brachionus plicatilis or the harpacticoid copepod Tisbe sp.

The main objective was to detemine if the marine fish larvae w i l l

grow and survive, on a diet of Tisbe alone. The lanrae should show

increased growth and suwival, due to the high EFA value of Tisbe

cornpared with rotifers. However, this benefit might not be realized

by the possibility that Tisbe may be less available than the rot i f ers

to the fish lanrae in the water column. As an aside. a f i rst-f eeding

haddock larvae fed with Tisbe was longditudinally thin-sectioned,

to determine if there were copepods in its gut.

Materials and Yethods

Eggs f rom one naturally ripe American plaice Hippoglossoides

platessoides were stfipped on March 29, 1996, and placed in dry

plastic containers floated on ambient temperature sea water (Z°C).

The sperm from an adult male was then added to the container and

mixed with the eggs. Fertilization occured over a perlod of 5 min.

Filtered (5 pm), ambient temperature (2OC) sea water was then

added to the fertilized eggs and they were tiansferred to floating

trays with a 200 pm mesh bottom in a flow-through ambient sea

water (2OC) tank. The trays were checked at regular intervals and

unfertilized or undeveloped doudy eggs were removed to prevent

disease or anaerobic conditions. Hatching took place approximatel y

14 d after fertilization. Initial lengths of the larvae were measured

using slide calipers before they were placed in larval culture

containers. The containers consisted of 15 cm (6 in.) diameter

white PVC pipe 15 cm in height with a 40 pm mesh screen inserted

5 cm from the bottom (total volume 1.9 L). These were placed i n

flow-through ambient seawater tanks at 2.0°C (range 1.2-2.6°C).

The l a ~ a e were cultured in green water with approximately 250 mL

(a. 5 million cells/ml) of the alga, lsochtysis galbana (clone T -

iso), added daily. To allow for optimal aeration, a stream of air was

directed over the surface. Two arches (2 X 3 cm) were cut out of the

bottom of each container to allow for better water flow and

exchange through the mesh bottom. Only one container with 20

plaice larvae was used for feeding with the copepod (Tisbe) diet and

four containers with 50 plaice larvae each were used for feeding

with the rotifer Brachionus plicatilis. This irnbalance was due t O

the fact that our mass copepod culture system was just being

established and there were insuffident copepods for feeding a large

number of fish larvae. The main objective was to determine whether

the l a ~ a e would grow and survive fed on a sole diet of Tisbe. The

rotifers were offered at a concentration of approximately 10

animals/mL. The concentration of Tisbe was approximatel y 0.9

copepodlmL in the >200 pm size fraction (mainly adults) and 3.5

copepods/mL in the 4 0 0 pm size fraction (copepodites and nauplii).

Tisbe was cultured in 6 4 cylindrical jars and fed bakers'

yeast using the procedures described for the copepods produced f O r

lipid analysis (p. 16).

The rotifers were fed 3 g of Microfeast L-1 O" lawal diet

daily. The rotifers were cultured at 20°C in 100 L of filtered (5

pm), 20

with an

replaced

ppt seawater contained in a 500-L plastic cylindrical

airstone. Approximateiy two thirds of the seawater

116

tank

was

each week. The rotifer population averaged about 30,000

individuals per L.

A second group of plaice larvae (hatched 6 June, 1996) was

cultured in 27 an (11 in.) diameter Mack buckets (total volume 7.8

L) with two 10 X 10 cm square holes cut in the sides. These holes

were covered with 40 mesh to allow water flow through the

container yet retain the live food. These buckets were placed in the

flow-through ambient seawater tanks (at average 6.1 O C ; range 5.4-

6.8OC). The larvae were cultured in green water with approximately

250 mL (ca. 5 million cells/mL) of the alga, I. galbana (clone T-iso),

being added daily. Thirty plaice larvae were cultured in each of three

buckets and fed rotifers at approximateiy 10 rotifers/mL. Nine days

after hatching, DHA-enriched brine shnmp (1-2 d post hatch) were

also fed in excess to the larvae. Unfortunately, most of these larvae

died about 20 d post hatch due to water-flow problems. However,

sixty larve8 of this same batch were cultured in floating trays w i t h

a 40 pm mesh bottom and were unaffected by these water-flow

problems. These plaice larvae were fed in the same manner (rotifers

and brine shrimp) as those cultured in the black buckets. The tim ing

of metamorphosis and the dimensions of these plaice larvae at

metamorphosis were recorded.

The bn'ne shrimp wem cultured using the following procedure.

Ten mL of brine shiimp cysts (8iomarine Inc., Hawthorne, CA) were

added to a 6-1 cylindrical jar (with a bottom drain), containing 4 L

of 20°C, filtered (5 pm) seawater. Most of the brin8 shn'mp hatched

after 48 h. The newly-hatched brine shrimp were then sepaiated

from the dead cysts which rose to the top of the container. This was

done in a dark room, by holding a light near the bottom of the

container. The newly-hatched brine shrimp were attracted to the

light as the water was drained from the bottom, and caught on a 40

pm screen. lhey were then transferred to another 6 L cylindrical

jar, containing an airstone and approximately 0.25 g of DHA-rich

~e lco ' (Artemia Systems N.V., Ghent, Belgium) emulsified in 4 L of

sea water. The brine shrimp were enfiched for 24 h, and rescreened

through a 40 pm mesh to remove excess oil prior to feeding to the

fish larvae.

Fertilized eggs of haddock Melanogrammus âeglefinus, obtained

from Dr. Ken Waiwood (St. Andrews Biological Research Station, N

B.) were placed in floating trays and treated similarly to those o f

the American plaice. The eggs were fertilized April 5, 1996 and

hatched approximately 10 d later. The newly-hatched larvae were

measured and separated into two culture containers (average 2.0°C;

range 1.2-2.6OC) with the same setup as used for the plaice lafvae.

Thirty larvae were placed in each container and fed either the

rotifers or copepods (Tisbe). The concentration of Tïsbe was

approximately 0.7 copepod/mL in the a200 pm size fraction (mainly

adults) and 2.7 copepodlmL in the 4 0 0 Fm size fraction

(copepodites and nauplii). The rotifers were fed at a concent ration

of approximately 10 animals/ml. The rotifers were cultured on

Microfeast L-10" larval diet while Tisbe was grown on bakers'

yeast . A first-feeding haddock larva (17 d post hatch) was taken from

each dietary treatment (Tisbe and rotifer) and fixed in 1%

glutaraldehyde and 4% fonnaldehyde and thin-sectioned, so that the

gut content could be microscopically examined. The fixed specimens

were dehydrated in methanol and embedded in historesin blocks

which were sectioned et 2 pn and stained with toluidine blue. After

staining , the sections were dried with compressed

chlorodifluoromethane (MG. Chemicals, Surrey, B. C.) instead O f

solvents. Sections were photographed with a Zeiss photomicroscope.

Reaults and Discussion

The plaice larvae fed with copepods (Tisbe) had significantly

higher growth (psO.05) compared to those fed with rotifers, when

assessed after 6 d (Table 12). This may have b e n due more to the

lower density of copepod-fed larvae present in their containers than

to diet. Suwivai of the copepod-fed plaice larvae (65%) was very

similar to that of the rotifer-fed plaice l a m e with the highest

survival (68%) 12 days after hatching. Their was a large variation in

the suwival of plaice larvae fed rotifers (Table 12). Suwival of

plaice larvae declined precipitously 2-3 weeks after hatching. This

mortality could have been due to a number of factors: 1 )

deteriorating water quality, particulaily in the tanks with Tisbe as

food. because we were unable to completely separate the benthic

copepods frorn the detritus in the copepod culture container before

feeding them to the fish larvae; 2) inappropriate fish lawae culture

containers (containers with dark walls and conical bottoms would be

preferable); and 3) rotifers may have been too small as live food f O r

the two week old l a~ae . Even so, the lame were obsewed to be

Table 12. Survival and growth measurements of American plaice (HippogIossoides platessoides) lame ieared at 2OC and f ed harpacticoid oopepods (Tisbe sp.) or rotifers (Brachionus plicatilis). The copepod was fed bakenr' yeast and the rotifer was fed Microf easte L-10 Larval Diet. The asterisk symbol indicates significant ciifferences at the pe0.05 level.

Copepod-feda Rotifer-fedb Survivat% Survival% (n=5û)

Time (days) Length (mm) (W02 Length (mm) Median Range O 4.21 f 0.31 1 O0 4.35 f 0.38 1 O0 100-1 00 6 6.32 f 0.43' - 5.56 f 0.58 - 12 O 65 - 24 68-1 O 15 9 25 9 12 48-6 23 O O 7.61 f 0.72 4 14-0

a Survival in phice lanrae fed with appods foi 1 container containing an initial population of 20 larvae.

b. Survival in plaice larvae fed with rotifen is the median aid range for 4 containers containing an initial population of 50 lawae each.

feeding on Tisbe and we were able to obtain relatively good growth

and sundval of the lanrae for two weeks after hatching. considering

that the culture conditions were less than optimal.

The plaice larvae, which were cultured in the second

experiment in three black buckets, had extremely good growth and

suwival during the first three weeks after hatching. The results

were as follows: day 1 post hatch (suwival 1 W%, length 5.37i0.21

mm), day 6 (suwival 1 00%. length 6.1 3I0.38 mm), day 13 (survival

87.8fl.9%, length 7.5if0.10) and day 20 (survival 78.9I5.1%, length

8.4810.38). These length measurements were taken f rom a sample

of five larvae. All larvae in the black buckets died shortly after 2 0

d due to water-flow problems. The increased early growth and

survival of these plaice larvae raised in the black buckets, compared

to those in the white PVC pipe could have been due to a number of

factors: 1) a higher percentage of the eggs fiom this batch were

transparent floaters and the hatching success was higher; 2) the

density of the larvae in the black buckets was much lower; 3) the

black buckets provide better contrast for the larvae to see and

capture their live food prey; and 4) DHA-enriched brin8 shrimp were

added to the diet 9 d post hatch.

Approximately 60 plaice lame of the same batch as those in

the black buckets, were reared on the sarne rotifer and brine shrirnp

diets in floating trays with a 40 pm mesh bottom. These lawae

were unaffected by the water flow problems. 01 these larvae, 2 0

were raised through metamorphosis. Metamorphosis was defined by

complete eye migration, fin ray and scale development. At this

point, they started to feed on aie pelleted marine fish lawal diet

developed by Dr. J. D. Castell. Metamorphosis occured approxirn atel y

85-90 days post hatch. The newly metamorphosed larvae were

25.59I2.74 mm long and 11.39I0.41 mm wide (n=5). The

temperature rose from 6 to g0C over this time period. To my

knowledge, there are no published studies of Amen'can plaice larvae

being raised through metamorphosis.

Haddock l a ~ a e were also cültured with Tisbe and rotifers

(Table 13). The haddock lawae demonstrated better growth but

poorer survival when fed the copepods compared to the rotif ers.

Unlike the plaice larvae, the containers with haddock larvae fed the

two diets had similar numbers of lawae suggesting that the better

growth was due more to diet than stocking density. Feeding behavior

of the haddock on the copepod lanrae was also obsented. The biggest

Table 13. Sunrival and growth measurements of haddock (Melanogremmus aeglefinus) larvae reared at 2OC and fed harpacticoid copepods ( Tisbe sp.) or rotifers (Brachionus plica tilis) . The copepod was fed bakers' yeast and the rotifer was fed Microfeaste L-IO Lawal Oiet.

copepod-fed rotifer-fed s u n r ~ v a ~ sunrival96

time (days) length (mm) (ndo) lerigth (mm) b 3 0 ) O 3-17 * 0.41 1 O0 3.47 * 0.41 100 8.5 5.36 f 0.78. 57 4.57 f 0.15 93

18.5 5.89 f 0.61" 23 5.00 * 0.41 57 21.5 - 1 O - 10 25.5 - 3 - 7

Significant differences at P O . 1 O.

" Significant differences at p40.05.

drop in suivival was durlng the first two weeks post hatch for the

copepod-fed larvae and after approximately 2.5 weeks for the

rotifer-fed larvae. The reasons are probably similar to those

proposed for the plaice larvae.

Proof that the first-feeding haddock larvae ingested Tisbe can

be seen in photographs of a thin-sectioned lama (Fig. 5). This

haddock larva contained a partially digested adult or l ate r-stage

copepodite (over 200 pm ir; length) in the stomach lumen, a smaller

early copepodite (100-200 pm in length) in the intestinal lumen and

possibly a third welkdigested copepod in the most posterior section

of the intestinal lumen. A haddock larva taken from the tank with

rotifers as food showed nothing in the gut lumen.

These preliminary larval feeding trials demonstrated that the

haddock and plaice lawae will ingest the harpacticoid copepod Tisbe

and will grow on this diet over the first two weeks after hatching.

The culture conditions were not optimal and work needs to be done

on a larger scale with better culture parameters to determine

whether rotifers or Tisbe is superior as a live food diet. Tisbe

spent most of ils time on the walls and bottom of the culture

container, and so was not as accessible to the larvae as were the

Fig . 5. Thin section of a first-feeding haddock (Melanogrammus aeglefinus) larva (17 d post hatch) fed the harpacticoid copepod (Tisbe sp.) and reared at 2°C. A. Whole rnount of the larva with arrows depicting the gut lumen containing the partial1 y-digested copepod (bar= 500 pm). B. Magnified view of the gut content of the lama (bar= 1 00 pm).

rotifers which swim freely in th8 water column. This too, may be a

reason for the drop in suwival of the larvae approximately two

weeks after hatching.

In summary, the increased growth of the marine fish larvae

fed with Tisbe compared with rotifers, could be due to both the

increased EFA vdue of th8 copepod. and the decreased densities O f

l a ~ a e in the tanks that were fed Tisbe. The decreased suwival o f

the fish larvae in the copepod-fed haddock larvae could be due to the

inaccessbility of the lhre food to the lawae in the water column. To

make Tisbe more accessible as a live food foi the fish larvae, a

floating tray system similar to the one designed by Kahan et al.

(1981) might be used. Because of their detritivorous, benthic

nature, Tisbe may better serve as a tank cleaner and EFA-rich, live

food supplement to rotifers and bn'ne shn'rnp, rather than as the sole

live food diet (Stattrup et al., 1995). Further studies comparing

growth and suwlval of fish larvae on a diet of Tisbe, rotifers and

brine shrimp, versus rotifers and brine shrimp alone, should be

performed to determine the value of Tisbe as a live food

supplement.

GENERAL CONCLUSIONS

The greatest obstacle in the culture of cold-water marine fis h

is the provision of a nutritionally suitable live food for the larvae a t

f irst-feeding . Harpadcoid copepods ( Tlsbe sp. in particular) are

prornising candidates as potential live food foi marine fish larvae.

They are easy to culture, can be grown at high densities and have the

necessary nutritional composition, in ternis of EFA, for marine fish.

Pemaps the rnost important requirement for marine fish larvae is a

diet with a high DHA to EPA ratio. The harpadcoids maintained this

high ratio despite a diet low in EFA or a high culture temperature.

Tisbe also contained >1% of the €FA arachidonic acid in its lipid at

20°C, irrespective of the diet's EFA composition.

Unlike fatty acid composition, the effects of free amino acid

composition of the live food on growth and survivai in manne fis h

larvae is largely unknown. Free amino acids are used primarily as an

energy source in the developing larvae. The harpacticoid copepods'

free amino acid pattern is fairly consenrative for the major

components, irrespective of the free amino acid composition of the

diet, with glycine contributing over 30% of the free arnino acids

followed by alanine, taurine and arginine. The essential free arnino

128

acids in 7ïsbe fed T-iso (30%) were airnoet double that of Tisbe fed

yeast (17%). The levels of total free amino adds per copepod was

highly variable.

The harpacticoid copepod Tnsbe demonstrated the necessafy

production parameters for mass culture, and could give the numbers

needed for marine fish lanral culture. ln a 32 L container nearly 1 O6

copepods were produced. This system could easily be modified t o

increase this production significantly by increasing the surface area

available to the copepod.

Survival of the marine fish larvae was poor when fed only on

the harpacticoid copepod Tisbe. This could have been due to several

factors including inappropriate culture containers and the

unavailability of the copepod in the watei column. Tsbe may better

serve as a tank cleaner and EFA-rich live food supplement to

rotifers and brine shrimp, rather than as the sole live food (Stettrup

et al., 1995). Further studies should be performed to confimi this.

Presently there is much local interest in using the

harpacticoid copepod Tisbe sp. as a live food organism for marine

fish culture. Research on the nutritional composition of Tisbe and

related studies are critical if marine finfish culture is to progress

in the Atlantic provinces. To date, replacement of live food has not

k e n accomplished for any of the marine finfish species considered

important for cultivation (Holt, 1993). Thus EFA-rich, live food

organisms such as Tisbe are, at this point the only viable

alternative for getting the larvae past first-feeding stage en route

to metamorphosis.

Appendlx A - Lipid Analysis Techniques

comôine sample with 10 mL methanol + 5 m l chloroform

homogenize for 1 min in polytron

suction filter through Whatman no. 1 filter paper rinse sample tube and filtrate with 5 mL chlorofom

add 9 mC distilled water to give 0:10:10 solvent ratio

dry chlorofom and lipid discard top aqueous with anhydrous sodium sulfate methanol layer*

filter through glass wool in a Pasteur pipette to test tube

evaporate chloroform under nitrogen

transfer to preweighed via1 and obtain total lipid weight

Fig. Al . Diagram of the Bligh and Dyer (1 959) method for total l ip id extraction. The asterisk symbol indicates where the sample w as extracted for amino acids.

Obtain total lipid using Bligh and Dyer (1959) method. Add 2 mL 7% boron trifluoride in methanol and 0.5 mL of toluene per 1 mg of lipid. Boil for 30 min at 100°C or ovemight at 50°C in pressure-tested 15 mL teflon-lined screw cap culture tubes. Cool tubes and add 10 mL of distilled water. Extract the top hexane layer mice with 2 to 3 mL of hexane. Discard the water layer. Dry the hexane with anhydrous sodium sulfate. Filter through Pasteur pipette containing gless wool. Evaporate hexane under nitrogen till dry. Add 30pL of chloroform to 1 mg of sample. Apply a 1 cm streak 2.5 cm from bottom of a precoated silica gel thin layer chromatographie (TLC) plate. 3 or 4 sarnples can be added to a 20 X 20 cm TLC plate.

10)Also add 15 pL of a reference methyl ester sample on the side of the plate 2.5 cm from the bottom.

1 1) Develop the plate in hexane: diethyl ether: acetic acid (90: 10:l V/V/V) for 45 min.

12)Allow the solvent to evaporate and spray the section of the plate containing the reference rnethyl esters with the visualking agent 0.1 % 2',7'-dichlorofluorescein in methanol while covenng the experimental sarnples.

13)Under ultraviolet light the reference methyl esters appear as two averlapping spots or bands. The upper band containing the more saturated and the lower containing the more unsaturated methyl esters. Mark the portion of the plate where your methyl esters would be and scrape the silica off the plate into a test tube.

14)Add ca. 7 mL of chloroform to methyl esters and silica in the test tube. Filter through a Pasteur pipette containing glass wool to remove the silica.

1 5) Evaporate to dryness under nitrogen and iedissolve in hexane prïor to analysis of fatty acid methyl esters on the GLC.

Fig. A2. Method for obtaining methyl esters from total lipids for analysis on GLC.

Appendix B - Astuanthin Analysis

The wild zooplankton captured in a plankîon tow (84 pm mesh)

on 27 July, 1995 was analyzed for both lipid (8.2% of dry wt.) and a

carotenoid pigment tentatively identified as astaxanthin (0.6% o f

total lipid). The chlorofom layer of the Bligh and Dyer (1959) lipid

extraction contained al1 of the astaxanthin pigment. The peak

absorbance of the pigment found in the zooplankton occuned at

approximately 460 nm wavelength (Fig. BI), typical of the esterified

astaxanthin pigments dissolved in hexane (467 nm; Czeczuga. 1971).

The absorbance of the pigment at 467 nm was related to the we i g h t

of astaxanthin through the use of known amounts of standard

astaxanthin sample. The positive linear relationship is shown i n

Fig. 82.

300 400 500 600

wavelength (nm)

Fig. BI . Absorbante spectnim of wild zooplankton astaxanthin measured in hexane.

.O0 0.01 0.02 0.03

Astaxanthin (mg/mL)

Squared Multiple R = i .ow

Linear Regression Equation

Absorbance = 0.070 X Astaxanthin (pg/ml)

Fig. B2. Standard plot and iegression analysis of astaxanthin versus absorbante at 467 nm.

Appendix C - Free Amino Acid Extraction

A freeze-dried sample of wild zooplankton was obtained f rom

the plankton of Passamaquoddy Bay, N. B. in July, 1995, using an 8 4

pm mesh plankton net. There appeared to be some leaching of f ree

amino acids in this sample, pehaps due to thawing in the f reeze-

drying process. This was evidenced by large variances in the

zooplankton f ree amino add compositions. This zooplankton sam ple

was then homogenized, so that two extraction techniques could be

compared; the water phase of th8 Bligh and Dyer (1959) method

versus the boiling 80% ethanol method. The boiling 80% ethanol

method gave significantly higher extractions (p<O.OS) for 75% of the

free amino acids detected in the zooplankton (Table Cl). Migration

of some of the free amino acids from the water phase into the

chloroforrn layer in the Bligh and Dyer method might explain the

lower free amino acid extraction values obtained with this method.

Thus, the Bligh and Dyer method, aMhough advantageous in ternis o f

allowing one to analyze both lipid and free amino acid compositions

with a single sample, significantly underestimates the free amino

acid content. Therefore, it should not be used for the purpose of

free amino acid extraction.

Table Cl . A cornparison of two amino acid extraction techniques, the Bligh and Dyer aqueous phase extraction and the boiling 80% ethanol methods. Amino acid composition of a homogenized, freeze- dried sample of wild zooplankton isolated from the plankton of Passamaquoddy Bay, N. B. in July, 1995. The asterisk symbol indicates significant differences at the p<O.OS level.

Amino acid Bliah and mer h m d g diy wt.) ûô% Ethaml (umdlg dry wt.) taurine* 66.67 * 4-06 82.10 i 2.39 aspartic acide 1.43 f 0.08 3.67 f 0.73 threonine* 4.91 f 0.20 5-78 i 0.12 serine' glutamic acid* prolinee glycine* alanine* valine methionine leucine* isoleucine* tyrosine* phenylalanine histidine lysine' ammonia 3.83 I 0.99 arginine* 28.20 f 0.63

See Appendix A (p. 130) for more information on extraction techniques.

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