Siedel 1984 Aquacultural-Engineering

7/28/2019 Siedel 1984 Aquacultural-Engineering

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Aquacu#ural Engineering 3 I 1984) 30 3-3 16

R ap i d D i f f e r e n t i a t i on o f A r t em i a sp p . P op u l a t i on s b yT h i n L a y e r I s o e le c t ri c F o c u s i n g

C.R. Siedel and K .L. Sim psonDep artment of F oo d Science and N utr i tion, Universi ty o f Rhod e Island.

Kingston, Rh ode Island 02 88 I. USA

A B S T R A C TA r t e m i a spp. o f various geographic origins, ),earl), co llec tion lots, dev elop -m ental s tages , and a b#tary geographic m ixn tre were analyzed b y th inlaver polya crylam ide get isoelectr ic tbcusing ([EF). IE F o f geographicalArtemia samples y ie lded un ique pro f i le s for each popu la t ion in the pHrange em ployed . Par thenogene t ic popu la t ions o f Artemia e x h ib i t e d [E Fpatterns which were, generally, do m bta ted by fe w er prote ins; b isexualpopu la t ions exh ib i ted more even ly d is t r ibu ted , complex pro te in pa t te rns .There wer e similarities refle cte d in II: 'F pro files o f geographically isolatedArtemia desc end ed fro m a co m m on ancestry . Yearly col lec t ions o]"Artemia exh ibi te d no apparen t d i fferences in IE F prote in patterns. Therewere d i fJOrences Hoted in three deve lop m enta l stages o f Artemia. A hhzarymLx'ntre o]" geographic po pu latio ns yi eM ed It: 'F pa tterns bttermediare to#rose o f t t te co m po ne n t pure samples.

I N T R O D U C T I O NI n t h e p a s t d e c a d e , i s o e l e c t r i c f OC L'S ing ( I E F ) h a s re c e i v e d w i d e a p p l i c a -t i o n in t il e i d e n t i f i c a t i o n a n d / o r c l a r i f ic a t i o n o f m o r p h o l o g i c a l l y s i m i la rm a r i n e s p e c i e s , s u c h a s w h i t e f i s h ( D j u s p s u n d , 1 9 7 6 ) , t r o u t ( C h u a e t a l . ,1 9 7 8 ) , s e a b r e a m ( T a n i g u c h i e t a l . , 1 9 8 2 ) a n d h a k e ( b l a c k i e a n d J o n e s .1 9 7 8 ) . A n u m b e r o f c o m p a r a t i v e s t u d ie s h av e e m p l o y e d [ E F t o s uc c es s-f u ll y d i f f e r e n t i a t e b e t w e e n a s m a n y a s 2 2 fis h s p e c ie s ( H a m i l t o n , 1 9 8 2 :

T o o m e t a l . , 1 9 8 2 ; Y a m a d a a n d S u z u k i , 1 9 8 2 ).I n v ie w o f t h e d e p e n d e n c e o f t il e a q u a c u l t u r e i n d u s t r y a n d sc i e n ti f i c

c o m m u n i t y o n A r t e m i a spp . a s a l i ve d i e t f o r l a r v a l f i s h a n d i n v e r t e -3o3

Aquacultural Engineering 014 4-86 09/8 4/S0 3.00 Elsev ier Applied Sc ienceP u b l i s h e r s L t d , E n g l a n d , 1 9 8 4 . P r i n t e d i n G r e a t B r it a in


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304 C . R . S i e d e l , K . L . S i m p s o n

bra t e s , t he ne e d a r i s e s t o i de n t i fy a r e l i a b l e A r t e m i a p r o d u c t o f h i g hf e ed q u a li ty . H o w e v e r , t h e o u t c o m e o f a r e se a r ch e f fo r t t o b i o c h e m i c a l lyc h a r ac t er iz e a n u m b e r o f A r t e m i a p o p u l a t i o n s s t r o n g l y s u g g e s t s t h a ti n t e r - p o p u l a t i o n v a r i a b il it y e x i st s w i t h i n t h e g e n u s , e s p e ci al ly w i t hr e g ar d t o f a t t y a c id s , a m i n o a c id s a n d c h l o r i n a t e d h y d r o c a r b o n s ( O i n e ye t a l . , 1 9 8 0 ; S c h a u e r e t a l . , 1 9 8 0 ; S e i d e l e t a l . , 1982) . B io log i c a l f e e d ings t u d i e s w e r e p e r f o r m e d w h o s e r e s u lt s r e v e al e d si g n i fi c a n t d i f f e r e n c e s int h e g r o w t h a n d s u r v i v a l o f l a r v a e f e d c e r t a i n A r t e m i a p o p u l a t i o n s a sa t o t a l d i e t ( J o h n s e t a l . , 1 9 8 0 ; K l e in - M a c P h e e e t a l . , 1 9 8 2 ) . H e n c e , t h en o n - s t a n d a r d i z e d b i o c h e m i c a l m a k e - u p p r e s e n t in A r r e m i a p o s e sa d e f i n i t e r i s k t o c o n s u m e r l a r v a e i n c u l t u r e f a c i l i t i e s , w h i l e c r e a t i n gi n c o n v e n i e n c e a n d / o r s e r i o u s s to c k lo s se s f o r t h e i n d i v id u a l s i n v ol v ed .

T h i s p a p e r e v a lu a t e s I E F i n t h i n l ay e rs o f p o l y a c r y l a m i d e g el a sa r a p i d m e t h o d f o r i d e n t i f y i n g A r t e m i a s p p . p o p u l a t i o n s a n d t h ep o t e n t i a l f o r q u a n t i f y i n g t h e s e I E F r e s u lt s us in g so f t l as er d e n s i t o m e t r y .T h e r e i s a n e e d t o i d e n t i f y A r t e m i a a t t h e g e o g r a p h i c p o p u l a t i o n l e v e li n o r d e r t o h e l p i n s u r e a sa l e a n d n u t r i t i o n a l l y s o u n d la rv a l d i e t . T h e r ei s a l s o s c i e n t i f i c b e n e f i t g a i n e d b y i d e n t i f y i n g n e w l y - a c q u i r e d A r t e m i ap o p u l a t i o n s o r a n c e s t o r s t h e r e o f , a n d , in t u r n , t e s ti n g t h e ir f e ed v a lu ef o r a q u a c u l t u r a l l y i m p o r t a n t s p e c i e s .

M A T E R I A L S A N D M E T H O D SA r t e m i a c u l t u r e

D ry A r r e m i a c y s t s , o b t a i n e d f r o m t h e E P A L a b o r a t o r y , N a r r a g a n s e t t ,R h o d e I s l a n d , w e r e h y d r a t e d i n 30 ~'0 0 , U V - t r e a t e d , f i l te r e d s e a w a t e rf r o m N a r r a g a n s e t t B a y , R h o d e I s l a n d , in 2 0 0 m l s e p a r a t o r y f u n n e l ss u p p l i e d w i t h v i g o r o u s a e r a t i o n . A r t e m i a we re ha rve s t e d a t t i l e i n s t a rI s ta g e o f d e v e l o p m e n t ( H e a t h , 1 9 2 4 ) , a p p r o x i m a t e l y 3 0 h l at er . N a u p l i in o t u s ed i m m e d i a t e l y w e re f r o z e n u n d e r a n i tr o g e n a t m o s p h e r e .

T w o p o p u l a ti o n s o f A r t e m i a w e r e l a b o r a t o r y c u l t u r e d t o 7 d a y s p o s t-h a t c h a n d 14 d a y s p o s t - h a t c h . D e - f a t t e d ri ce b r a n w a s f e d d u r i n g t h ec u l t u r e p e r i o d . A d e t a i l e d d e s c r i p t i o n o f t h e m a s s c u l t u r e o f A r t e m i ao n r i ce b r a n m a y b e f o u n d in R o n s iv a U i ( 1 9 8 3 ) .

O n e g r a m ( w e t w e i g h t ) A r t e m i a n a u p l i i w a s h o m o g e n i z e d w i t h 3 m lo f a 1% g l y c in e s o l u t i o n a d j u s t e d t o p H 7 -0 0 ( + 0 - 0 5 ) u s in g a P o l y t r o nU l t ra s o n ic H o m o g e n i z e r ( B r i n k m a n n I n s t r u m e n t s , W e s tb u r y , N e w


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A rernia analys is b y th in layer isoelectr ic foc us in g 305York). The homogenate was filtered through a medium porositysintered glass funnel, the surface of which was thinly coated with HyfloSuper Cel. Samples were analyzed for protein concentration and appro-priate dilutions were made with 1% glycine (pi-I = 7.00) to yieldsamples of 4.00 (+0-10) mg protein m1-1. This concentration was foundto be very satisfactory, for IEF analysis. Diluted samples were sealedunder nitrogen and stored at 4C for no longer than five days.G e l p r e p a r a t i o n

Thin layer polyacrylamide gels used as tile IEF medium were preparedby adding the following reagents sequentially: distilled water (20.5ml), 50% v/v glycerol (7-5 rot), 20% acrylamide in distilled water: 0.8%methylene-A,N'-bisacrylamide in distilled water (12.5mi ), carrierampholyte (Pharmacia Fkqe Chemicals, Piscataway, New Jersey) pH4.0-6.5 (3.0 ml). This mixture was deaerated under a vacuum for 10rain, 10% ammonium persulfate in distilled water (0.13 ml) was added,and deaerat ion was continued I min longer. The gel solution wasrapidly transferred, by careful pouring, into a preformed 260 X 125 cmglass mold, with a 1.0 mm top edge opening. Polymerization wascomplete after 45 min at room temperature and, at that time, the gelwas carefully demolded.

Isoelectric focusing

IEF was accomplished using a Multiphor IEF basic unit in combinationwith a 2000-V constant power supply (model 2103), both supplied byLKB Instruments Inc., Gaithersburg, Maryland. The gel was cooled to4C during electrofocusing. Prepared samples were applied in 10/~1volume to 1.0 X0.5 mm sample application tabs (LKB InstrumentsInc.), placed on tile gel surface approximately 1.0 cm from the cathodeedge. Electroly te filter paper wicks, 21.5 cm X 1-0 cm 1.0 mm (MRACorp., Clearwater, Florida) were evenly wetted with 1 M H3PO4 and I MNaOH for anode and cathode edges, respectively.Initially, 20 mA constant current was applied to the gel. After 10rain, 25 W constant power was applied, and the mA limit was raised to200. 30 min later, the sample applicat ion tabs were carefully removedand, approximately 90 min after tab removal, the protein system


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306 C. R. S iedel , K. L . S impsonattained equilibrium. The voltage limit was set at 1000 V throughoutthe experiment.

The gel gradient was determined with a surface pH electrode (IngoldElectrodes, Andover, Massachusetts) at 1.0 cm intervals. Immediate lyafterwards, the gel was immersed in a Coomassie Brilliant Blue G-250staining solution. The staining procedure was modified from Righettiand Drysdale (1974). Following destaining, gels were immersed ina preserving solution, air-dried, wrapped in cellophane and stored ina humidity chamber at 4C until analyzed by densitometry. The proteinzones of gels stored in this manner have been found to be stable for sixmonths (Toom e t a l . , 1982).

Qu antitative de nsitome tryStained protein bands were scanned using an Ultro Scan laser densito-meter (LKB Instruments Inc.). A model 3390A reporting integrator(Hewtett-Packard, Avondale, Pennsylvania) indirectly monitored lightabsorbance, as the 633 nm, fixed wavelength laser passed over each 9.0cm sample lane, from anode to cathode edge at a speed of 6 mm min-LEach sample lane of two replicate gels were scanned, in the mannerdescribed, three times within the inner 2/3 of the lane and the mean ofthese integrated values was calculated for each protein band. This way,true bands could be distinguished from artifacts such as bubbles,scratches, dust or stain particles. Area percent values were assigned toproteins, identified on the basis of isoelectric point.

Visual examinat ion was employed to interpret stained gels along withdensi tometric analysis; the former was most valuable in detecting 'falsebands' (i.e. artifacts) f rom true scan-integration data and the latter wereessential in quantifying sample protein bands.

Experimental studies performed

Four IEF studies were performed; for each study, two replicate gelswere run in the pH 4-0-6.5 range (see Table 1). The first ('geograptlic')study compared instar I nauplii from China (CHN), France (FRN),Australia (AUS), Reference A r t e m i a cysts (RAC) and San FranciscoBay (SFB), San Pablo Bay, California (SPB) and Great Salt Lake, Utah(GSL) populations. The second ('temporal') study group included instar


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TABLE1

D

pooAemiaCeoU

Geacogn

Ln

Y

Cmmeza

A

ao

Su

h

e

sue

goa

AaaSkB

l1

'~

WodO

LdKwVebn

A

G

A~aa

CnTesn

1

GeWaba-C

ETesn

CN

G

PesR

coCn

Fa

Ld

1

Ncmmecayawae

FR

G

ReeAemiacsz

AemiaeeeceG

Bgum

R

GDB

UASFasB

2

3

1

MeaameSFasBBaIn

S1:B-2018

T

Nwk

SB3

T

SFmcsB

2

1

MeaameSFasBBaIn

SB2

T

Nwk

SIqnsB

3

3

1

MeaameS

asBBaIn

SB3

GT

Nwk

SB3

T

SmPaoky

1

1

LvnWodSFasBBaIn

SB

GT

Nwk

GeSL

1

S

BnSnmpCO

/G7

T

GeSLk

1

.G7

G"DB

GeSL

IOJ

[G7

T

aE

measugop(5gacsuTempasu

=doe

ttldy;

1{=baymixue

sudtTRhb

o

aecbaomaeansueunAemiaaafosoc(SmpoaB

198o)

E" n o,q O ~


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A rremia analys is by th in laver isoelecrr ic foc us in g 309T A B L E 2

Isoelectric Focusing Profiles o f A r t e m i a Nauplii in the pH 4-30-6.70 Range ShowingVariation Between Duplicate Gels (Results Expressed As Relative Area Percent)

P I A U S C H N F R z V G S L - 7 7 S P B S F B - 3 1 5 0 R A C4-304-35 14.404.60 I 54.75 4 4 3 5 2 4 14 11 i 14.805.00 10 9 6 i1 8 85.10 16 155-15 9 6 26 17 13 11 7 85-20 14 95.25 22 20 l l I0 23 36 34 315.275.30 23 20 18 26 14 15 13 8 12 185.33 8 65.35 13 175-40 6 55.45 8 7 8 7 7 85.48 7 4 10 10 7 65.50 8 8 4 55.55 11 11 9 6 1 15.58 24 19 13 11 5 25.60 4 15.63 5 5 4 35.70 4 7 5 5 ~ 3 v 15.83 2 5 2 15 -906.07 2 4 1 16-10 4 6 5 6 3 4 1 3 1 16.17 2 56.556-63 i 2 1 1

33 35 3

10 1118 2010 137 116 43 43 1

11 93 I

12 10

2 21 1

1 1

"~ 5 45 43 2

3 7 4

23 168 126 11

6 106 7

10 1114 12

6 4

4 4

similarities were observed h~ IE F profiles for geogr aphic ally isolatedA r t e m i a with a common ancestry (SFB, SPB, RAC); however , each ofthe three IEF patterns was electrophoret ical ly dis t inct .


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310 C. R. Siedel, K. L. SimpsonResults for IEF of temporal collections of SFB Arternia (lots 2018,

3348, 2149, 3150, 3490) and GSL Ar temia (lots from 1970, 1977,1978) in the pH 4.0-6.5 range revealed no intrapopulation differences(see Fig. 2). Both qualitative and quanti tat ive data supported thesefindings.

Quantitative results for ttle developmental stage study are presentedin Table 3. For the GSL-77 and RAC populations. IEF profiles demon-strated a general increase in ttle low isoelectric point (P~) proteins in theinstar VII and XIV Ar temia compared to the newly-hatched animals.One protein (Pt = 4.37) was found to occur in tile instar XIV Ar temiafrom GSL-77 whictl was not detected in other GSL-77 groups; tworelatively major proteins (P[ = 4.90; PI = 4.95) were present in instarXIV animals from RAC, but absent in other groups from that poptt-lation.The binary mixture study revealed IEF protein patterns that wereintermediate to those of the pure GSL-77 and RAC samples. Tile effectof mixing was most dramatic in proteins which focused at Pt values of5.25 and 5.27 (see Fig. 3). In the RAC sample, tile P[ = 5.27 protein(s)contribut ed 91% of the two-band total, and tile Pt = 5.25 protein(s),9%. As the mixture contained a greater proportion ot" GSL-77 and lesser

0-

1 -

2

Eu4-t.-

~ 6 -07 -

8-

F i g . 2 .

9 -

III

!3 3 4 8 2 1 4 9 3 1 5 0 3 4 9 0

I

1.6.5fI

- 6 - 0

5 - 5

5 " 0

i

2 0 1 8 7 0 7 7 7 8S F B G S L

-4.5

Cathode

PI

Anode

Visual IEF patterns of yearly collections of Artemia nauplii in the pH4.00-6.70 range (sketched t'rom experimental gel).


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.4rrernia analysis by thin laye r isoelecrric fbcusg ng 311T A B L E 3

I s o e l e c t r i c F o c u s i n g P r o f i l e s o f A r r e m i a a t T h r e e D e v e l o p m e n t a l S t ag e s ( p H R a n g e4 . 0 0 - 6 - 6 0 ; R e l a t i v e A r e a P e r c e n t S h o w n )

G S L - 7 7 R A CP! /a up l i i 7 -day 14 -day P~ /a u p l i i 7 -day 14 -day

2 , " ~ " ) ,2 ,4 . , 0 - _ 1 3 4.35 . 6 __ 64 . 2 5 - 1 3 4 - 4 0 6 1 9 l 04 - 3 0 - 1 2 4 . 6 0 4 1 1 34 . 3 2 - 1 2 4 - 7 5 5 5 14 . 3 5 - 5 4 4 - 9 0 - - 1 34 . 3 7 - - 2 4 - 9 5 - - 1 64 . 4 0 - 1 5 t 3 5 . 2 5 2 3 1 2 1 84 . 7 5 9 ~ I 5 - 2 7 9 1 3 1 95 . 1 5 9 5 I 5 - 3 0 8 1 0 65 - 2 5 3 1 2 3 2 4 5 . 4 5 6 t r a c e a 2,5 . 3 0 1 4 1 7 7 5 .48 5 t r a c e a t r a c e a5 .33 9 12 7 5.55 10 3 75 - 4 5 7 3 2 5 . 6 0 f 2 2 t r a c e a5 - 4 8 6 1 2 5 . 9 0 3 t r a c e a t r a c e a5 .5 5 7 6 2 6- 55 4 "~ 15 . 6 3 4 3 I5 . 7 0 2 4 76 . 1 0 2 I t r a c e aa D e n o t e s < 0 .5 ?2 - or" t o t a l p r o t e i n s r e p o r t e d .

p r o p o r t i o n o f R A C n a u p l i i , t h e c o n t r i b u t i o n o f P~ = 5 . 2 7 p r o t e i n ( s )w a s g r a d u a l l y r e d u c e d , a n d P l = 5 . 2 5 p r o t e i n ( s ) w a s g r a d u a l l y i n c r e a s e du n t i l , i n t i l e G S L - 7 7 s a m p l e p a t t e r n , t i l e P I = 5 . 2 5 p r o t e i n ( s ) c o n t r i b u t e d1 0 0 % o f t h e t w o - b a n d t o t a l .

D I S C U S S I O NA s a r a p i d i d e n t i f i c a t i o n m e t h o d f o r A r t e m i a p o p u l a t i o n s , I E F r e p r e -s e n t s a n a d v a n c e m e n t o v e r t h e r e s u l t s o f A b r e u - G r o b o i s a n d Beardmore( 1 9 8 0 ) w h o s e e l e c t r o p h o r e t i c c h a r a c t e r i z a t i o n o f A r t e m i a i s o z y m e s w a s


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312 C. R. S iedel , K. L . S im ps on

~ 91"fo

NAC

~ 8 3 %

751oRAC 2 5 % GSL-7751% ,9 - , .

50%RAC 50"/.GSL-776 6 % ~ 34%

25%RAC 75%GSL-77100%_ ~

GSL-77F ig . 3 . D e n s i t o me t r ic s c a n a n aly sis o f a b i n a r y m i x t u r e o f R A C a n d G S L - T 7A r t e m i a nauplii showing the effect of mixing on two proteins. Peak at left repre-

sents protein(s)with Pt = 5,25; peak at r ight represents protein(s)with PI = 5.27.

u n a b le t o d i f f e r e n t i a t e b e t w e e n S F B , S P B . R A C a n d A U S p o p u l a ti o n s .T h e u s e o f I E F e n a b l e d a p o p u l a t i o n - s p e c i f i c i d e n t i f ic a t i o n o f t h e s ev e ng e o g r a p h i c A r t e m i a p o p u l a t i o n s a n a l y z e d .T e m p o r a l s a m p l e a n a ly s i s b y I E F l e n d s f u r t h e r c re d e n c e t o t h eg e o g r a p h i c p o p u l a t i o n r e su l ts , s i nc e n o y e a r l y i n t r a p o p u l a t i o n v a r ia t io nw a s r e f l e c t e d f o r e i t h e r S F B o r G S L - 7 7 A r t e m i a c o l le c t i o n s. T e m p o r a ls a m p l e r e s u l t s f o r A r t e m i a a gr ee w i t h t h o s e o f T o o m e t al. ( 1 9 8 2 ) w h o


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A rtemia analysis by th in layer i soelectr ic focu sing 313reported no seasonal variability to exist in Atlantic croaker, blue crab,Southern flounder, brown shrimp or red snapper when they weresampled monthly over a twelve-month period, and subjected to IEFanalysis.Heath (1924) described the morphological changes that A r r e r n i aundergo in their development from newly-hatched nauplius through tomature adult. For each of their 13-15 instar stages, a host of bio-chemical changes has also been known to occur as a result of geneticand/or environmental factors (Bagshaw, 1980; Clegg and Conte, 1980).The IEF results for instar I, VII and XIV animals from RAC andGSL-77 A r t e m i a populations may vary due to the increased digestiveenzyme activity found to be present at certain A r t e m i a life stages.There is strong evidence that the digestive enzymes o f A r t e m i a includean acid protease (Warner and Shridhar, 1980, T. Nagatani, personalcommunication), thus, possibly explaining the increase in low PIproteins for the instar VII and XIV groups of both populations. Theappearance o f one or more of the three electrophoretically distincthemoglobin molecules synthesized by A r t e m i a (Bowen e t a l . , 1969,1978) after hatching into a nauplius, may also be partially responsiblefor the observed increase in low PI proteins.

The fact that binary mixtures o f A r t e r n i a were detected by IEF, andthese results were verified using quantitative dens itometry , representsa valuable contribution to the description of the genus. Only twoaccounts of quantitative dens itometry of IEF results for marine speciesidentification were found in the literature (Hamilton, 1982: Toom e t a l . ,1982). IEF is largely being used as a qualitative taxonomic index at thepresent time. With the use of soft laser densitometry, quantitativeapplications of IEF will become more important in the future. The useof a rapid identification technique, as reported here, along with A r r e m i aquality indices such as polyunsaturated fatty acid levels and chlorinatedhydrocarbon levels provide aquaculture and aquatic research scientistswith valuable tools to select the best larval diet.

ACKNOWLEDGEMENTSWe gratefully acknowledge the A r t e m i a Reference Center, Ghent,Belgium lbr providing the A r t e m i a cysts used in this research andmembers of the Culture Team at the EPA Environmental Research


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314 C. R. Siedel. K. L. SimpsonL a b o r a t o ry " i n N a r r a g a n s e t t , R h o d e I s la n d f o r h e l p i n g w i t h Artemiah a t c h i n g a n d c u l t u r e .

T h i s p u b l i c a t i o n w a s p r e s e n t e d a t th e 4 3 r d A n n u a l M e e t in g o f t h eI n s t i t u t e o f F o o d T e c h n o l o g i s t s , N e w O r l ea n s , L o u i s i a n a , 19-22 J u n e1 9 8 3 . It is t h e r e s u lt o f re s e a r c h s p o n s o r e d b y N O A A O f f i c e o f S e aG r a n t , U S D e p a r t m e n t o f C o m m e r c e , u n d e r g r an t n o . N A 8 1 A A - D -0 0 0 7 3 , a n d U S E P A g ra n t n o . C R 8 0 6 7 3 5 - 0 3 - 0 . R h o d e I sla n d A g ri-c u l t u ra l E x p e r i m e n t S t a t i o n c o n t r i b u t i o n n o . 2 1 6 3 .

I n t e r n a t i o n a l i n t er d i s ci p l in a r y s t u d y o n Artemia s t r a i n s c o o r d i n a t e db y t h e Arremia R e f e r e n c e C e n t e r , S t a t e U n i v e r s i ty o f G h e n t , B e l g iu m .

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Siedel 1984 Aquacultural-Engineering

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