GREEN GONADS

COMPARISON OF NUTRIENT COMPOSITION OF GONADS AND COELOMIC FLUID OF GREEN SEA URCHIN STRONGYLOCENTROTUS DROEBACHIENSIS

CHANDRIKA LIYANA-PATHIRANA,' FEREIDOON ^SHAHIDI,'

^"*

AND ALAN WHITTICK'

^Department of

^Biology,

-Department of Biochemistry. Memorial

University

of Newfoundland.

St.

John's,

Newfoundland, AlB 3X9, Canada

ABSTRACT

Thecompositional characteristics ofsea urchin gonadsand coeloniic lluid from Stnmgylocemnitus droehuchiensis harvestedin the coastsofNewfoundland andthereafterrearedinan aquaculture facilityand fedonaLaminariadiet for a3-week period,wereassessed. Evaluationswereperformed onthe basisof proximate composition,lipidclass distribution,fattyacidcompo-

sition, total andfreeamino^acid composition,andcontents ofnucleic acids andcarotenoids. Noticeable changesexistedbetween proximate composition of^seaurchingonadsand coelomic fluid.Moisture contentwas 74.7+0.04and96.5

±

0.03%ingonadsand coelomicfluid,respectively.Gonadscontained very high^levelsoflipids,proteins,andcarbohydrates; whereas,thesewerepresentat verylowlevelsinthecoelomicfluid.Majornonpolarlipidclassesweretriacylglycerols(TAG),freefattyacids(FFA).andsterols(ST) while

dommant

polarlipidclasseswerephosphatidylcholine (PC), phosphatidylethanolamine^(PE).sphingomyelin/ lysophosphatidyl- choline(SM/LPC).and phosphatidylserine^/phosphatidylinositol(PS/PlIinboththegonadsandthe coelomicfluid. Majorsaturated fatty acids(SPA) were 14;0and 16:0;whereas. 20:ln-15 wasthe main monounsaturated fatty acid

(MUFA)

present. Furthermore, 20:5n-3 (eicosapentaenoicacid,

EPA)

wasthedominantpolyunsaturatedfattyacid

(PUFA)

inthegonadsandthecoelomicfluid.The

totalaminoacid

(TAA)

andfreeaminoacid

(FAA)

profiles were dominatedbyglycine.Thetotal

FAA

^contentwas

much

higherin thegonadsthaninthecoelomicfluid.In addition, the^totalcarotenoid content ofseaurchingonads wasapproximately6.4timesgreater thanthatof coelomicfluid.Hence,mostofthecarotenoidswereconcentratedinthegonadal^tissue.Echininoneand fucoxanthinwere the dominant carotenoids in the gonads and the coelomic fluid, respectively. The content of deoxyribonucleic acid

(DNA)

and ribonucleic acid

(RNA)

was

much

higherinthegonadthaninthecoelomicfluid,thus indicating greaterbiomassandprotein synthetic activityintheformertissue.Thepresentstudydemonstratesthatsea urchingonadshave

much

in

common

withseaurchin coelomic fluidon a qualitative^basis. However,therewere markedquantital;ve differencesbetweenthe twotissues.

KEY WORDS:

aminoacidcomposition,carotenoids. fatty acidcomposition, lipid class distribution, nucleic acids,Slmngyhcen- trolusdroehachiensis

INTRODUCTION

Sea

urchinsbelongto the marineinvertebrate

phylum

Echino-

dermata

or spiny-skinned animals.

These

relatively small echino-

derms

have spherical bodies enclosed in a hard shell or "test"

completely covered with

numerous

sharp spines.

Sea

urchins are

omnivorous

animalsthat live

on

theoceanfloor, feeding

on

small crustaceans, fish offal, but mainly

seaweed

(Smith 198(5). Thus, the eating quality of sea urchin

gonads

is dictated, to a certain degree,

by

the quality of kelp

consumed. Laminaria

kelps are the preferredsource of feedforsea urchins.

Kramer

and

Nordin

(1979) reportedthatthegreenseaurchinStrongyloceiitniliisdroebachien- sis produces high-quality

gonads when

the availability of fresh kelpis adequate.

The

ediblegreensea urchinS. droehachiensis ^is abundantly distributed in the

North

Atlantic, Arctic,

and

North Pacific Oceans, but this species is currently exploited to a

much

lesserextentinthe

Northwest

Atlantic

and

intheNortheastPacific, and NortheastAtlantic

(Walker &

Lesser 1998). Furthermore,S.

droehachiensisisa target species for the

development

of

commer-

cial echiniculture

(Hagen

1996).

The

edible portions of the sea urchin

body

areits reproductive organs;ovaries,

and

testes.

Gonad

yield

from

seaurchin

may

vary with the time

and

the site ofharvest

and

generally ranges

from 8-20%

ofthetotal

body

mass.

When

seaurchinsareprocessed^for gonads,theinitial stepistobreaktheshell

and open

it sothatthe five

gonad

sacs areexposed.

The

cracked^shells arethen allowed to drain for several minutes to dispose of coelomic fluid. Thus, duringextractionofseaurchingonads,large

amounts

of coelomic

fluidare obtained.

So

far,there are

no

effective

means

of usingsea urchin coelomic fluid in a useful manner. Furthennore,

no

infor- mation isavailable

on

the nutrientcompositionof seaurchin coe- lomic fluid. In fact,

knowledge

of nutrient composition

may

be useful todetermine

whether

seaurchin coelomicfluidcouldserve as a potential source ofa flavoring in fabricated seafood.

The

objective ofthis study

was

to assess the nutrient

compo-

sition ofsea urchin

coelomic

fluid as

compared

with that ofthe gonads. Thus, proximate composition,^lipidclass distribution,fatty acid composition,

amino

acidcomposition,

and

contents ofcarot- enoids and nucleic acids of

gonads and

coelomic fluid

were

de- termined. This

may

leadtopotential

commercial

utilizationofthe processing byproducts

from

sea urchins,

which would

otherwise be discarded.

MATERIALS AND METHODS

Materials

*Corresponding author. Tel.: (709) 737-8552; Fax: (709) 737-4000;

E-mail: fshahidi@mun.ca

One hundred

twenty-five seaurchins

were

procured

from

the

Sea

Urchin Research Facility

(SURF)

at Bonavista Bay,

New-

foundland

and

subsequentlytransportedin

aquarium

coolers toour laboratoryat

Memorial

University of

Newfoundland.

Urchins

were

captured

from

thewild (June 2000) and raisedin

raceways

feeding

on

a

Laminaria

diet. Urchins

were

harvested for analysis after three

weeks

of feeding

on

a purelyalgal diet. Live urchins

were

stored at

4°C

before the extraction of tissues.

The gonads and

coelomic fluid of sea urchins

were

separated after breaking the shell, using a specially devised sea urchin cracking tool. After extraction,sea urchin

gonads were homogenized

for 2

min

usinga cooled

Waring

^blender

(Dynamics

Corporation,

New

Hartford, CT),

and

coelomicfluid

was

usedfor analysis as^{itis.In this}study.

861

seaurchin

male and

female

gonads were

pooled togetherforanaly- sis.

The

tissues(both

gonads and coelomic

fluid)

were

flushedwith liquidnitrogen

and

storedat

-20°C

untilusedforfurtheranalyses.

All chemicals used

were

obtained

from

either Fisher Scientific (Fair

Lawn.

NJ) or

Sigma Chemical

Co. (St. Louis.

MO). The

solvents

were

of

ACS-,

pesticide-, or

HPLC-grade.

Determination of Proximate Composition

Moisture

and

ash contents ofsea urchin tissues

were

deter-

mined

accordingtothestandard

AOAC

⁽1990) procedures.

Crude

protein content

was

obtained by Kjeldhal

method (AOAC

1990).

and

total lipids

were

extracted

and

quantified by the Bligh and

Dyer

⁽1959) procedure.

Carbohydrate

contentofeach

sample was

determined by difference.

AnalysisofLipid Classes by latrnscan

Instrumentation

The

crude lipidsobtained

from

Bligh

and Dyer

(1959) extrac- tion

were chromatographed on

silicagel-coated

Chromarods

^-SIII

and

then analyzed using an latroscan

MK-5

(latroscanLaborato- ries Inc.,

Tokyo)

analyzer

equipped

withaflame ionizationdetec- tor(FID)connected to a

computer

loaded with

TSCAN

software (Scientific Products

and Equipment. Concord. ON)

for datahan- dling.

A hydrogen

flowrateof160 niL per

min and

anairflow rate of2.000

mL

per

min were

usedinoperatingtheFID.

The

scanning speed of rods

was 30

secperrod.

Preparationof

Chromarods

The Chromarods were soaked

inconcentratednitricacidover- night followed

by

thorough

washing

withdistilled water

and

ac- etone.

The Chromarods were

thenimpregnated by dippingina

3%

(w/v) boric acid solution for five minutes to

improve

separation.

Finally, the cleaned

Chromarods were scanned

twice to burnany remainingimpurities.

Standards

and

Calibration

A

stock solution ofeach ofthe nonpolar lipids; namely, free fatty acid

(FFA;

oleic acid), cholesterol ester (CE). cholesterol

(CHOL), monoacylglycerol (MAG;

monoolein). diacylglycerol

(DAG;

diolein).

and

triacylglycerol

(TAG;

triolein),and the polar lipids;

namely,

phosphatidylcholine (PC), phosphatidylethanol-

amine

(PE). phosphatidylinositol (PI), phosphatidylserine (PS), lysophosphatidylcholine

(LPC). lysophosphatidylethanolamine

(LPE).cardiolipin(CL).

and sphingomyelni (SM) was

prepared by dissolvingeach in a chloroform/methanol (2:1. v/v) solution

and

stored at

-20°C. A

range ofdilutions ofthe stock solution,

from

0.1 to 10|j.g per(xL.

was

preparedforuse as

working

standards.

Each compound was developed

individually

and

run

on

the latro-

scan-FID

todetermineitspurity

and Rf

value.For each

compound

peak, area

was

plotted against a seriesof

known

ct)ncentrations to obtainthe calibration curve.

latroscan

(TLC-FID)

Analysis ofSea UrchinLipids

The

total lipids extracted

were

dissolved in

chloroform/

methanol

(2:1, v/v) toobtainaconcentrationof¹ p.g lipidper

mL.

A

^I ji,L aliquot of

sample was

spotted

on

silica gel-coated

Chro- marods

^-

S

III

and

conditionedin ahumidity

chamber

containing saturated

CaCl,

for

20

min.

The Chromarods were

then

developed

in

two

solvent systems.

The

solvent system hexane/diethyl ether/

acetic acid (80:20:2. v/v/v)

was

^u.sed for separation ofnonpolar lipids (Christie 1982).

Following

theirdevelopment,

Chromarods were

dried at 1

10°C

for three minutes

and scanned

completely to reveal nonpolarlipids. Forpolarlipids,followingthe

same

proce- dure

and

drying, the

Chromarods were

scannedpartiallytoapoint just

beyond

the

MAG peak

to

bum

the nonpolar lipids.

These

partially

scanned Chromarods were developed

ina

second

solvent system of chloroform/methanol/water(80:35:2, v/v/v) for thesepa- ration ofpolar lipidclasses (Christie 1982) followed

by

dryingat

110°C

for three minutes. Finally, the

Chromarods were

scanned completely to reveal polar lipids; the identity ofeach

peak was

determined

by comparison

with a

chromatogram

of standards ac- quired concurrently withthesamples.

The

determination of weight percentages of individual lipid classes

was

achieved using the standardcurves obtained foreachauthentic standard.

Analysis ofFattyAcid Composition of^Lipids

Fatty acid composition of lipids

was determined

using gas

chromatography (GC)

asdescribed

by Wanasundara and

Shahidi (1997). Fatty acid methyl esters

(FAMEs)

of total lipids of sea urchin

gonads and coelomic

fluid

were

prepared

by

transmethyl- atingapproximately 10 to

20 mg

ofeachlipid

sample

in2

mL

of freshly prepared transmethylating reagent

[6%

(v/v) sulfuric acid in 99.9

moK/f HPLC-grade methanol

containing 15

mg

of t-

butylhydroquinone

(TBHQ)]

at

65°C

for 15 h in a6

mL

^Teflon-

lined

screw-capped

conical vials. After incubation, the mixture

was

cooled,

and

1

mL

ofdistilledwater

was added

toit.This

was

followed by extracting the

FAMEs

three times with 1.5

mL

pes- ticide-grade hexane.

A few

crystalsof

TBHQ were added

toeach

sample

before extraction with hexane.

The hexane

layers

were removed and combined

in acleantest tube followed

by washing

twice with 1.5

mL

ofdistilled water

by

vortexing.

The aqueous

layer

was

discardedafterthefirstwash, whilethe

hexane

layer

was removed and

placed in a

GC

^{vial following} the second wash.

Hexane was

evaporated

under

astreamof nitrogenina

fume

hood.

The

dried

FAMEs were

thendissolvedin 1

mL

ofcarbondisulfide

and

used for

GC

analysis.

FAMEs were

separated using a gas

chromatograph

(Hewlett-Packard

5890

SeriesII,Hewlett-Packard.

Mississuaga,

ON) equipped

with a fused silica capillary

column (SUPELCOWAX-IO. 0.25-mm

diameter.

30-m

length. 0.25-p,m film thickness;

Supeico Canada

Ltd.. Oakville,

ON). The sample was

injected intothe

GC

analyzer using aHewlett-Packard

7673

autoinjector(Hewlett-Packard.Toronto.

ON). The

temperature of the

oven was programmed

at

220°C

^for 10.25

min

followed by

ramping

to

240°C

at

20°C

per min.

where

it

was

held for nine minutes.

Helium

at a flow rateof2

mL

per

min was

used asthe carrier gas.

The FAMEs were

identified by

comparing

their reten- tiontimes with those ofauthenticstandard mixtures

(GLC

^- 461.

Nu-Check-Prep) and

literature values (Takagi etal. 1980.

Takagi

etal. 1986).

The

relative content offatty acidsin the

sample was

determined using the peakareasoffatty acids.

CarotenoidPigments

Extractionand Determination ofTotal

and

IndividualCarotenoids Carotenoids

from

each ^tissue

were

extracted threetimes witha total of

50 mL

^{of acetone for}

^two

^minutes.

The homogenized

samples were

centrifuged

(lEC

Centra

MP4

Centrifuge. Interna- tional

Equipment

Co..

Needham

Heights.

MA)

^at

4000 x

_gfor five minutes.

The

supernatant

was

subsequently filtered through a

Whatman

No. ¹ filterpaper.Carotenoid

pigments

inacetone

were

then transferred to

40

niL of n-hexane ^{in a}

250-mL

separatory funnel.

One hundred

niilhlitersofa 0.57c

sodium

chloride solution

were added

tothemixtureto

maximize

the transferofcarotenoids.

The

hexane layer

was

then transferred into a

50-mL

volumetric flask and

made up

to volume.

The

absorption spectrum

was

then recorded

(400-600 nm)

using a Spectronic spectrophotometer (Spectronic Genesis. Toronto.

ON

^I.

The

total and individual ca- rotenoid contents

were determined by

the

method

of

McBeth

(1972).

The

totalcontentof carotenoidspresent per100 g of^tissue

was

calculated using the followingequation.

mg

Carotenoid per 100 g ^tissue

= (A

x

V

x 10')/e x

W)

where,

A ⁼

absorbanceat\„„,;

V ⁼

total

volume

ofthe

sample

(mL); e

= _molar

extinction coefficient,

and W ⁼

weight ofthe tissue (g).

Because

thecrudeextractsusuallycontaineda varietyof carotenoids an averagecoefficient of2,500

was

used in the cal- culations.

The

total

pigment

extracted

was

separated into individual ca- rotenoids

by means

of thin-layer

chromatography (TLC). The

crude carotenoids

were

separated

by

preparative

TLC on

silicagel

G

(20

X 20 cm, 230

(jim, Aldrich

Chemical

Co., Inc.,

Milwaukee, WI)

using acetone/n-hexane (3:7, v/v) as the developing solvent.

Characterization of Fractions

Cochromatography on TLC

providedthe ultimatetestfor iden- tification

when

authentic samples

were

available for

comparison

with

unknown

^pigments.

The unknown

fraction

and

the authentic sample

were

spotted

on

either side in an equally proportionated mixture ofthe

two

pigments

on

silicagel

G

plates(20x

20 cm. 250

fjLm, Aldrich

Chemical

Co., Inc.,

Milwaukee,

WI):

unknown

frac- tions

were

consideredtobeidentical tothe authentic

sample

ifthe

two

did not separate

upon

subsequent

development

ofthe plate.

When

authentic samples

were

unavailable, the type of carotenoid ineach fraction

was

tentatively identifiedaccording toitsabsorp- tion

maximum

in n-hexane, ethanol,

and

chloroform

(Goodwin

1955, Krinsky

& Goldsmith

1960,

Fox & Hopkins

1966, Britton 1995).

Determinationof^Total

Amino

Acids

The amino

acidcomposition ofseaurchin

gonads

and coelomic fluid

was

determined according to the procedure described by Blackburn (1968).

Samples were

lyophili/.edandthenhydrolyzed for

24

h at 1

10°C

with

6M

HCl. Hydrochloricacid in the hydro- lyzate

was removed

under

vacuum, and

the dried

sample was

reconstituted with a lithium citrate buffer (0.2

M, pH

2.2) for analysis.

The amino

acidsinthehydrolyzate

were

separated, iden- tified

and

quantified using a

Beckman

121

MB amino

acid analyzer

(Beckman

Instruments Inc., Palo Alto,

CA).

Sulfur- containing

amino

acids

were

determined

by

oxidizingthesamples with pertbrmicacidbefore theirhydrolysis ina

6M HCI

solution (Blackburn 1968).Cysteine and methionine

were measured

ascys- teic acid

and

methionine sulphone, respectively.

To

determine tryptophan,

samples were hydrolyzed

in

3M

mercaptoethane- sulfonicacidat1

10°C

for

22

hundernitrogen

and

thenneutralized with lithiumhydroxideandadjustedto

pH

2.2(Penkeet al. 1974).

Determination of Free

Amino

Acids

Samples

(10 g)

were

extracted with

20 mL

of a

6%

(v/v) perchloric acid

(PCA)

solution

by homogenization

^usinga Poly-

tron

homogenizer (Brinkmann

Instruments, Rexdale,

ON)

at 10,000

rpm

for

two

minutes in an ice bath.

The homogenized

samples

were

then incubated in anice bath for

30

min.This

was

followed

by

centrifugation

(lEC

Centra

MP4

Centrifuge, Interna- tional

Equipment

Co.,

Needham

Heights,

MA)

^at2,000 x g^{for 15} min.

The

residue

was

re-extractedwith another

20 mL

of

6% PCA.

The

supematants

were combined

andfilteredthrough a

Whatman

No. 4filterpaper.

The pH

of thefiltrate

was

adjusted to7.0using a

33% KOH

^(w/v) solution. Precipitatesofpotassiumperchlorate

were removed

by centrifugation at

2000

x g for 10 min.

The

supernatant

was

then acidified to

pH

2.2 usinga 10

M ^HCI

^solu-

tion, and the

volume

of the extract

was

brought to

30 mL

^with

distilled water.

Three

millilitersoflithiumcitrate buffer

(pH

2.2,

0.3M) were added

toI

mL

ofthe extract,andthe resultant solution

was

analyzed using a

Beckman

121

MB amino

acid analyzer

(Beckman

Instruments, Inc., PaloAlto,

CA)

for individual

amino

acids.

DeterminationofNucleic Acids

The DNA and RNA

contstituentsof

gonads and

coelomicfluid ofsea urchins

were

extractedaccordingtothe

method

of

Schmidt and Thannhauser

⁽1945)asmodified by

Munro

and Fleck(1969).

Five

grams

of each

sample were homogenized

in 80

mL

ice-cold deionized water usinga Polytron

homogenizer (Brinkman

Instru- ments,Rexdale,

ON)

at 10.000 rpm. Five millilitersofthe

homo-

genate

were

allowed to stand for ten minutes in ice

and

then centrifuged

(lEC

Centra

MP4

Centrifuge, International

Equipment

Co.,

Needham

Heights,

MA)

at

2000

x _g for ten minutes.

The

residue

was

subsequently

washed

with 2.5

mL

ofice-cold 0.2

M

PCA and

centrifuged at 2,000

x g

^{for ten} minutes followed

by

digestionofthe residuein

4 mL

ofa 0.3

M KOH

^for

one hour

at

37°C

inawaterbath.

The

resultant solution

was

coolediniceand

mixed

with 2.5

mL

of 1.2

M PCA and

allowed to stand for ten minutes,

which

finallyresultedinthecoagulationof^proteins.

The

mixture

was

centrifuged at 2,000

x

_g for ten minutes,

and

the supernatant

was

recovered (Extract No.l).

The

precipitate

was

then

washed

twice with 2.5

mL

of a 0.2

M PCA

solution

and

centrifuged at 2,000

x

_g for five minutes.

The

supernatant

was combined

^with extract No. ¹

and

10 niL ofa 0.6

M PCA were added

tothe mixture.This

was

used for

RNA

determinationafter diluting it

up

to 100 niL with ^distilled water.

The

residue

was

dissolvedin 17

mL

^ofa 0.3

M KOH

solution at37

C and

diluted tothe

50 mL mark

inavolumetric flaskwithdistilledwater.

The

contentof

DNA

in the

samples was

estimated

by

determiningthe deoxyribose content in the extract using the indoleprocedure of Ceriotti(1952), while

RNA ^was

^determined by recordingthe ab- sorbance of the nucleotide extracts at

260 nm

using a Hewlett-

Packard

diode array spectrophotometer (Hewlett-Packard,

Model 8452A,

Hewlett-Packard [Canada]Ltd.,Mississauga,

ON).

Protein interference at this

wavelength was

eliminated

by employing

a correction factorof 0.001 absorbance unit foreach I (xg per

mL

proteinconcentration inthe extracts.

The

protein concentrationot the extracts

was measured

using the Folin-phenol procedure ^ot

Lowry

etal.(1951).

Bovine serum albumin (BSA) was

usedas a standard. Calf

thymus DNA

(containing

82%

single stranded

DNA)

andcalfliver

RNA (96%

purity)

were

usedasthestandards for

DNA

and

RNA

determinations,respectively.

StatisticalAnalysis

Each

experiment

was

replicated threetimesand

mean

values

±

standarddeviations reported foreach sample. Forstatisticalanaly-

ses,

mean

values oftheexperimental data

were

subjectedto one

way

analysisof variance

(ANOVA)

using

GraphPAD

Instat Ver- sion 1.0. Significance

was determined

at

5%

probabilitylevel.

RESULTS

I'roximaleCoiiiposilidii

Proximate composition ofseaurchin

gonads

and

coelomic

fluid is

shown

in Table ^I.

The

moisture

and

ash contents ofsea urchin

coelomic

fluid

were much

higher thanthoseofthegonads.

On

the other hand, the levels of protein, lipid,

and

carbohydrate in the

coelomic

fluid

were much

lower than those in the

gonads on

a fresh weight basis.

Lipid ClassDistrihiilion

The

nonpolar

and

polar lipid classesof

gonads and coelomic

fluidofseaurchin S. droebachiensisare

shown

inTable2.

Major

nonpolar lipid classes

were TAG. FFA. and

ST; whereas,

main

polar lipids classes

were PC.

PE.

SM/LPC. and

PS/PI in both

gonads and coelomic

fluid. Triucylglycerols constituted the

main

energy reserve inboth tissues,contributing

more

than

63%

to the total nonpolar lipids.

On

the other hand,

PC was

the

dominant

polar lipid, accounting for

more

than

60%

in both

gonads and coelomic

fluid.

The

polarlipidclasses

SM and LPC

as well as

PS and

PI did not

show

aclear

chromatographic

separation

from

each other during latroscan analysis.

FattyAcid Composition

Fatty acidcomposition ofsea urchin

gonads

and

coelomic

fluid ispresentedinTable3.Qualitatively, the fattyacid compositions

were

the

same

in both tissues, while there

were

significant (P

<

0.05) quantitative variations. In both tissues. 14:0

and

16:0

were

the

main

saturated fatty acids. In addition. 18:0

and

20:0

were

present in considerably high levels.

The

fatty acid 20: In-15

was

the

dominant MUFA

in both gonadal

and coelomic

fluid lipids.

Furthermore, 16:ln-7. l6:ln-9. 18:ln-7,20:ln-7, 20;ln-9,

and

22:

In-II

were

detected in noticeable amounts.

Among PUFA,

20:

5n-3 contributedthe highestproportiontothetotalfatty acidcon- tent in both gonadal

and coelomic

fluid lipids,

CarotenoidPigments

The

total carotenoid content,

on

a dry weight basis, of sea urchin

gonads and coelomic

fluid

was

23.2

±

0.04

and

3.7

±

0.1

mg

per g^tissue, respectively.

Crude pigments from gonads and

coe-

TABLE

1.

Proximate cumpositionof sea urchingonadsand coelomicfluidafter feedingurchinsonaLaminariadiet forthreeweeks.

Constituent

TABLE

3.

Fatly acidcomposilimi(Ht'i(;lit '7fI oftotal lipidsseaurchingonads andcoeloniic fluid afterfeeding theurchins with iMininariadietfor

three weeks.

TABLE

4.

Total

amino

acidcontent Img/gprotein!of sea urchingonads

and

coelomicfluid afterfeedingurchinsonaLaminariadietfor

threeweeks.

FattyAcid

TABLE

S.

Free

amino

acidconttnlin/ft.dryweight) of sea urchin gonads

and

coelomicfluid afterfeeding urchinson Laminariadietfor

three weeks.

characteristics ofsea urchins' coelomic tliiid. In our study,

when

urchins

were

fed

on

u Lainimiria diet,

gonads

contained 74.7

±

0.04%

moisture,

which was

significantly(f

<

0.05)lower thanthat ofthecoelomic tluid.

IIIthe present study, the relativecontent of

ST

inthe

gonads was

significantly(P

<

0.03)higher than that in thecoelomic fluid.

Vaskovsky and

Kostetsky (1969) have

performed TLC on

polar lipidsof sea urchin ^5. iiiuhis

and

S. intennediiis.

The

polarlipid fraction

was

separatedinto five

components

of

which PC,

PE, and

SM

constituted the

major

polarlipidclasses present.Furthermore, lipidextractsofdifferentorgans ofthe

same

animal

had

a similar qualitative polar lipid

composition (Vaskovsky &

Kostetsky 1969). Inthisstudy,both

gonads and

coelomicfluid

showed

quali- tativesimilaritiesintheirpolarlipidfractions.Thus,

PC,

PE,

SM/

LPC, and

PS/PI constituted the polarlipids ofS. droebachiensis

gonads and

coelomic fluid, and

PC was dominant

inbothtissues, witha contributionof

more

than65'^/c tothe totalcontentofpolar lipids.Similarly, Floretoetal.(1996) demonstratedthatseaurchin Tiipneiistes gratila fed

on

a

seaweed

diet had

PC and PE

as the majorlipid constituents,

and PC

contributeda greaterproportion than PE.

FattyAcid Composition

The

fattyacidsoftotal lipidsof sea urchin

gonads

and coelomic fluid

were

typically similartothoseofothermarinespecies witha

dominance

of 16:0

and

20:5n-3

(Wanasundara

1996). Although.

22;6n-3isa typicalfatty acidin marinelipids,itcontributed only

1.4

±

0.1

and

0.6

±

0.1

%

^tothetotalfattyacidsinthelipidsofsea urchin

gonads and coelomic

fluid, respectively. Holland (1978) reported thatthe

predominance

of 20:5n-3 and 22:6n-3in typical marinefatty acidsisaresultof low- temperature adaptation.This helps in the maintenance ofcell

membrane

fluidity inorganisms living in thecold environments.

Considerabledata are available

on

thefattyacidcomposition of seaurchins (Takagietal. 1980.

Kaneniwa

and Takagi 1986).

The

fatty acid 16:0

was

the

major SFA

in the sea urchin 5. droe- bachiensis harvested

from

Herring

Cove, Nova

Scotia (Takagi et al. 1980). Fujinoetal. (1970) analyzed fatty acidcomposition of sea urchins Anthocidaris crassispina. S. piilclwninuis. S. fraii- ciscanus. S. intermedins,

and Echinus

esculentus. In all these samples, 16:0

was

the

prominent SFA

followed by 14:0.

The

^fatty acid 18:0

was

foundtooccurinconsiderable amounts. Similarly, in the present study, the

predominant SFA were

16:0

and

14:0in the lipidsof both

gonads

and

coelomic

fluidofS. droebachiensis.

Among MUFA

20: In-¹5

was

present up to

11%

in the total fatty acids of urchins (Takagiet al. 1980).

Ackman and Hooper

(1973) reportedthatsuch marine animals asperiwinkle {Littorine littorea).

moon

snail (Liinata triseriata).

and sand shrimp (Crangon

septemspinosu.s) contain 20:ln-l5, but at

much

lower levelsof

up

to

0.2%

ofthetotal fattyacids.

However,

this hasnot

been commonly

reportedasbeingtypicalof marine lipids.Inour study, 20:ln-15

was

also the

major MUFA

in both tissues ana- lyzed.

On

the otherhand, seaweeds,the naturaldietofsea urchins,

have

not

been

reported to

contain 20:ln-15 (Ackman &

McLachlan

1977);hence, theformation of 20:ln-l5in sea urchin tissues

may

bebiosynthetic inorigin,becausethis

was

notdepen- dent

on

the diet.

The

occurrence of such unusual ."i-olefinic fatty acids as 18:ln-13, 20:ln-15, 20:2A5,11, 20:2A5,13, 20:3A5,11,14.

and

20:3A5, 11,14,17 has

been

noticeable in lipids ofsea urchins ac- countingfor as

much

as

6-2

1

%

ofthe fatty acids oftotal lipids (Takagiet al. 1980.

Kaneniwa & Takagi

1986). Inthis study, the

amount

of 5-olefinic acids found inthe lipidsof both

gonads

and

coelomicfluid

was

intherange of

7-10%. The

presenceof5-ole- finic fatty acids has

been

reported in 12 species of Echinoidea collected inJapan (Takagietal.l986); thus,theyserve a

common

andcharacteristic feature ofsea urchinlipids.

The amount

of eicosapenlaenoicacid(20:5n-3)

was

quitehigh inseaurchinlipids(Takagietal. 1980). Pohl

and

Zurheide (1979) reportedthaturchinsthat

consumed

Laniinaria

had

ahigh content of 16:4n-3, 18:4n-3, 20:4n-6.and20:3n-3. Similariy. sea urchinS.

droebachiensisinour study

consumed

Laniinaria foronlyathree

week

period,

and

theirgonadaland coelomicfluidlipidscontained quitehigh levelsofthese fatty acids. Thus,the fattyacid profiles ofseaurchintissues

somewhat

reflect thatoftheirdietsas

was

also observed by Floreto et al. (1996).

However,

certain fatty acids, suchas 16:4n-3,20:4n-6, 20:5n-3,

and

20:ln-l I,

which

constitute the

major

fatty acids ofsea urchin tissues,

were

not detected in their diets; therefore, suggesting that sea urchins are capable of synthesizing

them from

lower fatty acid precursors. Similarly, in the presentstudy 16:4n-3,20:ln-l1, 20:4n-6,and 20:5n-3,

among

others,

may have been formed by

chain elongation ofprecursors.

In general, the sea urchin fatty acids; namely, 16:4n-3, 20:4n-6,

and

20:5n-3,

may

possibly confer

some

structural function and, hence, are purposely synthesized by the animal (Floreto et al.

1996).

Carotenoid Pigments

In the sea urchin .S'. dnfcbacliiensis. carotenoids

were

mainly concentrated in the gonadal tissue.

Hence,

the total content of carotenoidsinthe

gonads was

about6.3times

more

thanthatofthe coelomic fluid.

However,

the content of carotenoids in different tissues

may

vary with the reproductive stage ofurchins. Hence, during gametogenesis

most

ofthecarotenoidsinother tissues

may

be transferred into gonads, consequently increasing their carot- enoid content (Griffiths

&

Perrott 1976).

Echininone

and

fucoxanthin

were

characterized as the

major

carotenoidspresent in the

gonads and

the coelomic fluid,respec- tively. In addition, (J-carotene

was

identified in both tissues.

Echininone was found

tobethe

main pigment

witha lesser

amount

ofP-carotene in the

gonads

ofS. piirpiiraliis (Griffiths 1966),S.

droebachiensis^(Griffiths

&

PeiTOtt 1976) and Tripnenstesgratila (Shinaetal. 1978).

Tsushima

etal.(1995) found^that(J-echininone and p-carotene

were

the

major

carotenoidsinthe

gonads

of19 out of

20

sea urchin species

examined. Meanwhile,

the

major

carot- enoids of

brown

algae, the natural preferred diet ofsea urchins, consist ofp-carotene, violaxanthin,

and

fucoxanthin

(Matsuno &

Hirao 1989).Furthermore, thereisbioconversion of p-carotene^to P-echininone^via P-isocryptoxanthin in sea urchins;

which

takes place mainly in the gut wall,

and

the resultant p-echininone is

incorporatedintothe

gonads (Tsushima

etal. 1993).

Kawakami

et al.(1998)

showed

thatfucoxanthin,the major carotenoidin

brown

algae, did not accumulate in the gonads. In fact, in the present study

on

5. droebachien.sis. fucoxanthin did notoccur inthe go- nads.

On

the contrary,coelomicfluid

had

fucoxanthinasits

major

carotenoid.

Amino

Acid Composition

Although

marineinvertebrates characteristically containahigh intracellularconcentration of

FAA,

the composition of the

FAA

pool

may

vary

among

species (Gilles 1979). In the present study, glycine

was

the

dominant amino

acid in both

TAA and FAA

profiles inboth sea urchin

gonads and

cocloniic fluid.

Komata

el al.(1962) reportedthatglycine

was dominant

ni the

gonads

ot"sea urchin S. piilclwnimiis.

and

itscontent ranged

from 35—41%

of total

FAA. Lee and Haard

(1982) reportedthatglycineconstituted

18-60%

ofthe

FAA

inthe

gonads

ofseaurchinS. droebachiensis.

The gonads and coelomic

fluidofseaurchin.S'. droebachiensis in this study contained 1

1.9-14.6%

glycine in the

TAA

profile, re- spectively.

However,

glycine

was

not the

dominant amino

acidin the

gonads

of the sea urchin Paracentrotus lividiis. although it

contributedaconsiderable

amount

tothe

TAA

pool(Cruz-Garcia et al. 2000).

Other

than glycine, alanine, arginine. glutamic acid, lysine,

and

methionine are considered important for taste,

even though some

of

them were

present in small quantities (Lee

&

Haard

1982).

These amino

acids

were

present in considerable

amounts

inboth

gonads and coelomic

fluidofsea urchins in this study.

It has been found that different combinations of taste-active

components

(substances that influence the taste of

any

food) as well as their relative

amounts

are of

paramount

importance in producingthe characteristic flavorof each seafood

(Puke

1994).In general, glutamine

and

glycine,

which were

present in higher

amounts

inthe

gonads

than

coelomic

fluid,are

known

tobe^taste- activein sea urchins

and

otherseafoods, regardlessoftheirquan-

tity.

Sea

urchin

gonads seemed

to be sweeter

when

little or

no

glutamine

was

present,

and

alanine

was

foundⁱⁿconsiderably high levels. Alanine is a taste-active

component

in sea urchin tissues, contributingnoticeably toboth

TAA and FAA

contents. Further- more, valine

and

methionine are

known

tobetaste-activeonly in

seaurchins;whereas, arginine

was

also taste-activeinsea urchins because of its high content (Fuke 1994).

Both

methionine and arginine

were

presentata higher proportion inthe

coelomic

fluid than in the gonads. Similarly, the contents ofaspartic acid,histi- dine.

and

especially proline

were much

higher in the

coelomic

fluidthan thoseinthegonads.Thus,

amino

acidsplay a

major

role inthetasteofseaurchingonads.Inourstudy,various

amino

acids contributed differently to both the

TAA and FAA

of sea urchin

gonads

and

coelonnc

fluid.

ContentsofNucleicAcids

Ingeneral, quantitative analysisofnucleic acidprovidesarela- tively simple

means

of estimating recent

growth

rate ofsea ur- chins.

The

processes ofcellular

growth and

division require the synthesis ofnucleic acids

and

proteins.

The

fact that

RNA

^is a precursor to protein synthesis led to its use as an indicator of

growth

rate

(Church &

Robertson 1966).

The

primary function of

RNA

^involves protein synthesis; whereas.

DNA

is the primary

carrier of genetic information.

Because

the majority of cellular

DNA

^is

chromosomal,

the quantity of

DNA

^{percell isquasicon-}

stantinsoinatictissues;the tissue

DNA

concentrationreflectscell

numbers

(Sulkinetal. 1975;

Bulow

1987).Therefore.

DNA

con- tenthasusually

been

usedasan index ofcell

numbers

or

biomass

(Regnault

& ^Luquet

^{1974). In this study, the}

DNA

content inthe

gonads was

approximately fourtimes higherthan that in thecoe- lomic fluid.

Although

the

gonad

isa tissuewith ahigher

biomass

as

compared

with

coelomic

tluid, the lattercontains mostly coe- lomic fluid with a lower biomass.

On

the other hand, the

RNA/

DNA

ratiohas

been

usedas an estimate of

growth

fora varietyof invertebrates (Sulkinet al. 1975).Thus, the

RNA/DNA

ratioisan indexofprotein synthetic activity percell

and

reflects the protein synthesizing capacity forestimatingrecent //;situproteinincrea,se

(Bulow

1987,

Hovenkamp &

Witte 1991^). In fact,correlationbe-

tween RNA

concentration or

RNA/DNA

ratio

and growth

ratehas been observed for a

wide

variety of

organisms

(Sutcliffe 1970).

Furthermore,thegonadal

RNA

^content

^was

about5.4timeshigher thanthat in the

coelomic

tluid,thus demonstrating higherprotein synthetic activity in the gonads. In general,

gonad

is the site of gametogenesis,

which

involves

much

protein synthesis. Further- more,the

RNA/DNA

ratio

was much

lower in

coelomic

tluidthan that ingonads,indicating greater protein synthetic activitypercell in the gonads. This is an indication that

gonad

is a tissue with greater//; sitit protein

growth

as

compared

^with

coelomic

fluid.

CONCLUSIONS

The

pi'esent study demonstrated ^that sea urchin gonadal

and coelomic

tluid tissues

had many common

compositional charac- teristics.

Most

oftheparametersanalyzed didnot

show

qualitative diffeiences; whereas, there

were

quantitative differences. In fact,

gonads

ofsea urchins are a site ofnutrient storage in addition to being the reproductive organs.

The

accumulation ofnutrient re- serves contributes to the

growth and development

ofthe

commer-

ciallyimportant seaurchingonads.

Although

.seaurchin

coelomic

fluid has not yet

been

exploited commercially, evaluation of its

composition

may

lead to itspotential use as a flavoring source.

ACKNOWLEDGMENTS

The

author(C. L.-P.) gratefully

acknowledges

theassistanceof the

Canadian

International

Development Agency (CIDA)

through a Mai'ine Science Scholarship.

Thanks

are also extended to Mr.

Keith Collins at the

Sea

Urchin Research Facility

(SURF)

at Bonavista

Bay, Newfoundland

^for providing sea uichin

samples

for the study.

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PItysiol. 4<iB:l.'i.3- J65.

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R. G.

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Agalsuma. Y. 1998. Aquaculture of the sea urchin (Strongylocentroiiis nudus)transplantedfromcorallineflats in Hokkaido. Japan.J.Slwll- fish Res. I7:1541-LM7.

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