Spotte 1984 Aquacultural-Engineering 1

7/28/2019 Spotte 1984 Aquacultural-Engineering 1

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A q u a c u l t u r a l E n g i n e e r in g 3 (1984) 1-13

P R O B I T A n a l y s is P r e d i ct s H a t c h i n g R a t es o f B r i n eS h r i m p Ar temi a sp. Cy st s*Stephen Spotte, Gary Adam s

Mystic Marinelife Aquarium, Sea Research Foundation Inc., Mystic,Connecticut 06355, USAand

Paul E. StakeDepartment of Nutritional Sciences, University of Connecticut, Storrs,Connecticut 06268, USA

A B S T R A C TThi r t y rep l i ca tes o f San Fran c i sco Bay br ine shr imp Artemia: sp. c y s t sf r o m o n e l o t w e r e w e i g h e d t o 0 . 0 1 m g . T h e m e a n m a s s o f a s in g l e c y s t w a s2. 59 7 rig (+- O. 04 0 t lg, 95% c on f ide nc e level) . Ba tches o f I g f r o m the sa mel o t w e r e h a t c h e d i n t h e l ab o r a to r y , a n d f r a c t i o n h a t c h e d v e rs u s t i m e d a taw e r e f i t t e d t o a n o n l in e a r c u r v e u s in g P R O B I T a na ly sis . M e d i a n t i m e o fha t ch was 24 .8 h w i th a s tandard error o f + 3 .92 h. Cys t s and naupl i i in1-ml a l i quo t s were p ipe t t ed a t regu lar i n t erva l s and counted under a d i s -s e c ti n g m i c ro s c o p e. T h e n u m b e r o f c y s t s p i p e t t e d a t t h e b e g in n in g o f th ee x p e r i m e n t w a s 1 9% le ss th a n e s t im a t e s b a s e d o n t h e k n o w n n u m b e r p e rv o l u m e o f h a t c h i n g m e d i u m , i n d ic a t in g t h a t h a t c h i n g d at a d e r i v e d f r o mpip e t t i ng proce dures m ay con ta in l arge sampl ing er rors . P R O B IT analysi sa l l o w s f r a c t i o n h a t c h e d t o b e p r e d i c t e d w h e n a p a r ti c u la r l o t o f c y s t s ish a t c h e d u n d e r c o n t r o l l e d c o n d i ti o n s .

INTRODUCTIONNauplii of the brine shrimp A r t e m i a s p p . are ideal food organisms forlarvae of plankton-feeding fishes and invertebrates. Nutrient assays have* Contribution no. 33 of Sea Research Foundation, Inc. and no. 977 of the StorrsAgricultural Experiment Station.

1A q u a c u l t u r a l E n g i n e e r i n g 0144-8609/84/$03.00-Elsevier Applied SciencePublishers Ltd, England, 1984. Printed in Great Britain


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2 S . Spo t t e , G . A d a m s , P . E . S t a k es h o w n t h a t n e w l y h a t c h e d n a u p l ii c o n t a in f u ll c o m p l e m e n t s o f a m i n oa c i d s ( S e i d e l e t a l . , 1 9 8 0 ) a n d m o s t o f t h e c o3 -s er ie s p o l y u n s a t u r a t e df a t t y a c i d s c o n s i d e r e d e s s e n ti a l t o s e a w a t e r f is h e s ( S c h a u e r e t a l . , 1 9 8 0 ) .Ben i j t s e t a l . ( 1 9 7 6 ) s h o w e d t h a t t h e n u t r i t i o n a l v a lu e o f a n a u p l i u sd e c l i n e s m a r k e d l y a f t e r t h e f i rs t m o l t a t ~ 2 4 h , s u g g e s ti n g t h a t n a u p l i is h o u l d b e h a r v e s t e d s o o n a f t e r h a t c h i n g . T h i s w a s c o n f i r m e d b y A b l e t ta n d R i c h a r d s ( 1 9 8 0 ) . B r in e s h r i m p c y s ts h a t c h a t n o n l i n e a r r at e s, a n de f fi c ie n t h a t c h i n g m e t h o d s m u s t a c c o u n t f o r th e f r a c ti o n o f c y st sh a t c h e d v e rs u s t i m e ( t h e n u m b e r o f c y s ts t h a t b e c o m e n a u p l ii ), ina d d i t i o n t o t h e n u m b e r o f n a u p l i i u l t i m a t e l y o b t a i n e d . B e c a u se t h en u m b e r o f c y s t s i s a n e s t i m a t e b a s e d o n c o l l e c ti v e m a s s , t h e f r a c t i o nh a t c h e d i s n o t p r e d i c t a b l e u n l e s s r e p r e s e n t a t i v e c y s t s a r e w e i g h e da c c u r a t e l y b e f o r e h a n d .

M e t h o d s o f h a t c h i n g la rg e q u a n t i t i e s o f n a u p l i i fo r u s e i n a q u a c u l t u r ew e r e g i v e n b y B o s s u y t a n d S o r g e l o o s (1 9 8 0 ) , K i n n e ( 1 9 7 7 ) , N a sh ( 1 9 7 3 ) ,P e r s o o n e a n d S o r g e l o o s ( 1 9 7 2 ) a n d S o r g e lo o s a n d P e r s o o n e (1 9 7 5 ) .N o n e o f t h e se r e p o r t s , h o w e v e r , p r o v i d e d d a t a o n c y s t m a s s o r a q u a n t i -t a ti v e p r o c e d u r e f o r p r e d i c t i n g t h e f r a c t i o n h a t c h e d .

M A T E R I A L S A N D M E T H O D SE x p e r i m e n t a l d e s i gnC o m m e r c i a l l y p a c k e d , d e s i c c a t e d c y s t s w e r e u s e d ( S a n F r a n c i s c o B a yB r a n d I n c . , N e w a r k , C a l i f o r n i a 9 4 5 6 0 , U S A , l o t n u m b e r 2 8 5 1 ) . T h i r t yr e p l i c a t e s w e r e w e i g h e d t o 0 - 0 1 m g o n a n a n a l y t i c a l b a l a n c e ( S a r t o r i u sm o d e l 2 6 0 4 , d i s t r i b u t e d b y B r i n k m a n n I n s t r u m e n t s I n c . , W e s t b u r y ,N e w Y o r k 1 1 5 9 0 , U S A ) . T h e c y s ts i n e a c h r e p l ic a t e w e r e t h e n c o u n t e du n d e r a d i s se c t in g m i c r o s c o p e ( T a b le 1). T o k e e p t h e c y s ts f r o m a b so r b -i ng m o i s t u r e t h e y w e r e l e f t in t h e v a c u u m - s e a le d c o n t a i n e r u n t i l j u s tb e f o r e w e i g h in g .

H a t c h i n g w a s c a r r i e d o u t i n f o u r l - l i t e r b o i l i n g f l a s k s , e a c h c o n t a i n -i n g 5 0 0 m l n e w a r ti fi c ia l s e a w a t e r ( S p o t t e , 1 9 7 9 ) d i l u t e d t o a s a l in i t yo f 2 0.1 ~6 o w i t h d i s ti l le d w a t e r . T h e f l as k s w e r e p l u g g e d w i t h t h r e e - h o l er u b b e r s t o p p e r s a n d s e t u p a s i l lu s t r a te d i n F ig . I . A t h e r m o m e t e r w a si n s e rt e d t h r o u g h o n e h o l e , a n d a n o t h e r ( 8 m m I .D . ) w a s l e ft o p e n t ov e n t a ir. A i r w a s i n j e c t e d t h r o u g h 2 7 . 9 c m o f s t a n d a r d g la ss l a b o r a t o r yt u b i n g i n s e r te d in t h e t h i rd h o l e . F l e x ib l e p o l y e t h y l e n e a ir li ne tu b i n g


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PR OBIT analysis predicts hatching rares o: brine shrimp cysts 3T A B L E 1

Cyst Masses from 30 ReplicatesMass ( r a g ) Number of cysts

0-60 1980.73 2490.60 2150-57 2660.66 2760.54 2350.57 2390.55 2980.57 1820.53 2260.75 2730-54 2030.59 2500.59 2040-60 2360.56 208O.54 2O20.58 2020-61 2550.57 1890.70 2610.57 2320.56 2000.58 2010.58 220O-58 2OO0.58 2280.61 2060-54 2640.59 213

(4 mm I.D.) was con nec ted to the ex pose d end of the glass tubing;a rigid pol ypr opy len e ' T' was attached to the submerged end tokeep it above the bottom of the flask. Air was supplied from the maincompressors at Mystic Marinelife Aquarium through a splinter line to


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4 S . S p o t t e , G . A d a m s , P . E . S t a k e

F i g . 1 .

" I I I

~Vent ~e /FlaskC

Airline ubing

Air o u r c e

Experimental apparatus used to hatch brine shrimp cysts

t h e l a b o r a t o r y a n d d i s t r i b u t e d f r o m a s in g le s o u r c e t o t h e f la s k s t h r o u g ht h r e e ri gi d p o l y p r o p y l e n e ' Y ' c o n n e c t i o n s w i t h a t t a c h e d a i rl in e t u b i n g .A e r a t i o n r a t e w a s s e t a t 1 .9 l it e rs m i n -~ ( 0 . 4 7 l i te r m i n - I t o e a c h f l a s k )a n d m o n i t o r e d b y a n a ir f l o w m e t e r ( L a b o r a t o r y S u p p l i e s , H i c k sv i ll e,N e w Y o r k 1 1 8 0 1 , U S A ) . C o n s t a n t t e m p e r a t u r e ( 3 0 C ) w a s m a i n t a i n e db y i m m e r s i n g t h e f la s k s in an u n c o v e r e d , t h e r m o s t a t i c , c i r cu l a ti n g w a t e rb a t h . T h e a e r a t e d fl as k s c o n t a i n i n g h a t c h i n g m e d i u m b u t n o t c y s t s w e r ep l a c e d i n t h e w a t e r b a t h s e v e ra l h o u r s b e f o r e s t ar t in g t h e e x p e r i m e n t t oa l l o w t h e t e m p e r a t u r e t o e q u i l i b r a t e .

T h e e x p e r i m e n t d e s c r ib e d b e l o w w a s p e r f o r m e d f o u r ti m e s. T h e f ir stt ri al w a s te r m i n a t e d a f t e r 2 4 h ; t h e n e x t t h r e e w e r e c o n t i n u e d t h r o u g h


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P RO B IT analys is pred i c t s ha t ch ing ra t es o f br ine shr imp cys t s 53 0 h . F l a s k s w e r e r e m o v e d f r o m t h e w a t e r b a t h a n d 1 g o f c y s ts w e r ea d d e d t o e a c h . T h e s e c y s t s w e r e n o t t h e o n e s w e i g h e d i n t h e 3 0 r e p li -c a te s , b u t w e r e t a k e n f r o m t h e s a m e lo t . L ig h t i n t e n s i t y a n d d u r a t i o na re k n o w n t o a f f e c t h a tc h i n g r a t e ( S o r g e l o o s , 1 9 7 3 ; V a n h a e c k e e t a l . ,1 9 8 1) . T h e o v e r h e a d l ig h ts w e r e t u r n e d o f f a n d c y s t d e v e l o p m e n t w a si n i t i a t e d b y i l l u m i n a t i n g t h e f l a sk s f o r 1 0 r a in w i t h 2 0 0 0 l u x p r o v i d e db y o n e 4 0 -W , c o o l - w h i t e f l u o r e s c e n t l a m p p l a c e d 2 1 - 2 5 c m a w a y .S i m u l t a n e o u s a e r a t i o n p r e v e n t e d s t ra t i f i c a t io n , w h i c h w o u l d h a v es h i e l d e d s o m e c y s t s f r o m t h e l i gh t. T h e l ev e l o f i l l u m i n a t i o n w a sm e a s u r e d w i t h a l ig h t m e t e r ( P h o t o - M e t e r 1, Q u a n t u m I n s t r u m e n t s I n c. ,G a r d e n C i ty , N e w Y o r k 1 1 5 3 0 , U S A ) . A f t e r th i s i n it ia l il l u m i n a t i o n t h ec ei li ng l i gh t s w e r e t u r n e d o n , a n d t h e f la s k s w e r e r e t u r n e d t o t h e w a t e rb a t h . T h e l ig h t l ev e l a t t h e s u r f a c e o f t h e w a t e r r e m a i n e d c o n s t a n t a t1100 l u x .

R e s u l t s o f p r e l i m i n a r y t r ia ls s h o w e d t h a t n o c y s t s h a t c h e d b e f o r e8 h , a n d t h a t m e d i a n t i m e o f h a t c h o c c u r r e d a t "~ 2 4 h . S a m p l e s o f 1 m lt h e r e f o r e w e r e t a k e n a t 2 - h i n t e r v a ls s t a r t in g a t 8 h u n t i l t e r m i n a t i o n o fa t r ia l a t 2 4 o r 3 0 h . B e f o r e s a m p l i n g , f l a s k s w e r e s w i r l e d t o d i s t r i b u t ec y s t s a n d n a u p l i i e v e n l y i n th e h a t c h i n g m e d i u m , a n d to f r ee c y s t st r a p p e d a g a in s t t h e g l as s a b o v e t h e w a t e r l in e. S a m p l e s w e r e c o l l e c te di n 1 -m l s e r o l o g i c a l p i p e t t e s i n s e r t e d t h r o u g h t h e v e n t h o l e s i n t h es t o p p e r s , t r a n s f e r r e d t o l a b e l e d v ia ls , fi x e d w i t h 0 . 5 m l f o r m a l d e h y d es o l u t i o n a n d r e f r i g e r a te d . P i p e t t e s w e r e f l u sh e d a f t e r e a c h u s e w i t hd i s t il l e d w a t e r t o c o l l e c t c y s t s a n d n a u p l i i t r a p p e d i n s id e . A v ia l w a ss h a k e n g e n t ly , a n d 0-1 m l w a s p i p e t t e d i n t o e a c h o f 10 w e ll s o f a B o e r n e rs l i d e . N a u p l i i w e r e t h e n c o u n t e d u n d e r a d i s s e c t i n g m i c r o s c o p e . T h e l a s ts t e p w a s r e p e a t e d u n t i l a ll n a u p l i i in t h e v ia l w e r e c o u n t e d . A f t e r w a r dt h e w a ll s o f t h e v ia l a n d p i p e t t e w e r e e x a m i n e d b y m i c r o s c o p e f o r a n yr e m a i n i n g n a u p l i i .Com putational proceduresA i m p o r t a n t o p e r a t i o n a l p r o b l e m i n b r i n e s h r im p c u l t u r e is p r e d i c t in gw h e n a p a r t i c u l a r f r a c t i o n w i l l h a t c h . F i n n e y ( 1 9 7 1 , p . 2 1) w r o t e : ' A ni n t e re s t i n g p o s s i b i l i ty is t h e u s e o f q u a n t a l r e s p o n s e t e c h n i q u e s i n t h es t u d y o f s o m e p h e n o m e n o n in a m a t u r i n g a n im a l th a t c a n n o t b e d a t e de x a c t l y b u t c a n r e a d il y b e r e c o r d e d a s h a v in g o c c u r r e d o r n o to c c u r r e d . . . . ' T o e s t i m a t e t h e f r a c t i o n o f c y s ts h a t c h e d a t a g i ve n t i m e ,t h e e x p e r i m e n t a l d a t a m u s t b e f i t t e d t o a n a p p r o p r i a t e n o n l i n e a r c u r v e.


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6 S . S p o t t e , G . A d a m s , P . E . S t a k e

i 0.5 . . . . . . . . . . . . .0 T i m e t ~

(a )

2 .

- - " I n ( t i m e )


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PR O BIT analys is pred i c t s ha t ch ing rares o f br ine shr imp cys t s 7w h e r e t = t i m e , Co = 2 . 5 1 5 5 1 7 , c 1 = 0 - 8 0 2 8 5 3 , c z = 0 - 0 1 0 3 2 8 , a l l =1 . 4 3 2 7 8 8 , d 2 = 0 . 1 8 9 2 6 9 , a n d d 3 = 0 - 0 0 1 3 0 8 . T h i s a p p r o x i m a t i o n isl i m i t e d t o p ~< 0 . 5 . I f t h e v a l u e is > 0 - 5 r e p l a c e p b y 1 - - p , c a l c u l a t ey , a n d r e v e r se t h e s ig n . T h e a b s o l u t e v a lu e o f t h e m a x i m u m e r r o r int h is a p p r o x i m a t i o n is 4 -5 1 0 -4 . D i s c u ss i o n s o f P R O B I T a n a ly s i s a r ea v a i la b l e in F i n n e y ( 1 9 71 ) a n d S o k a l a n d R o h l f ( 1 9 6 9 ) .

R E S U L T SD a t a f r o m w e i g h i n g 3 0 r e p l i c a t e s o f c y s t s a r e s h o w n in T a b l e 1. V a l u e sr a n g e d f r o m 0 . 5 3 t o 0 . 7 5 r a g , a n d t h e n u m b e r o f c y s t s i n e a c h r e p l ic a t ev a r i e d f r o m 1 8 2 t o 2 9 8 . T h e m e a n m a s s o f a s in g l e c y s t w a s 2 . 5 9 7 tag(-+ 0 - 0 0 8 ta g, 9 5 % c o n f i d e n c e l e v e l) .

N u m b e r s o f c y s t s h a t c h e d v e r su s t i m e a re s h o w n in T a b l e 2 . F r a c t i o nh a t c h e d w a s t r a n s f o r m e d t o P R O B I T s , a n d t i m e w a s t r a n s f o r m e d t o t h e

T A B L E 2

N a u p l ii p e r m l V e r su s I n c u b a t i o n T i m eEla pse d Trial I Trial 2 Trial 3 Trial 4 M ea n ~o ha tch edt i m e ( h )

0 0 a 0 0 0 0 08 0 0 - - 0 09 _a _ 0 0 0 0

10 18 0 1 - 8.5 1.3512 56 18 27 33 33-5 5.3514 186 84 87 109 116-5 18-616 190 132 132 177 157-7 25-218 224 193 158 194 192.3 30.720 301 238 198 247 246 .0 39 .322 248 296 250 267 274 .3 43-82 4 3 4 6 3 1 8 3 0 7 2 7 6 3 1 1 - 8 4 9 . 826 - 366 312 293 323 .7 51-728 - 345 354 348 349-0 55 .83 0 - 3 6 3 3 1 6 3 3 6 3 3 8 . 3 5 4 -0

a Dashes and ze ros i nd i ca t e no da t a and no naup l i i , r e spec t i ve ty .


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8 S . S p o t r e , G . A d a m s , P . E . S t a k e

F i g . 3 .

0.70.60 .5

u 0 .4

"~ 0.30.2

?0.1~-

.S ~ e / * (/

5 1 0 1 5 2 0 2 5 3 0 3 5T ime ( h )

Fraction hatched versus time. The curve is a least squares fit using PROBITanalysis. Horizontal bar is the standard error of estimate at 50% hatch.

logarithm of time. Transformed data were fitted to a least squarescurve, and the standard error was calculated. The curve and original dataare shown in Fig. 3. Data are shown numerically in Table 2. The bestestimate of the median time of hatch, as indicated by the PROBIT tech-nique, is 24.8 h with a standard error of + 3.92 h.

The original fraction hatched versus time data are tabulated in Table3, columns 1 and ". Column 3 is the natural logarithm of time, t.Column 4 is the PROBIT of fraction hatched. Column 5 is the squareof column 3. Column 6 is the prod uct of columns 3 and 4. The sums ofcolumns 3 through 6 can then be calculated.

The slope, m, o f the linear curve fit is given bym = (n Zx y- - Zy Zx )/ (n Zx 2- (Y.x) 2) (4)

where n is the number of data points. The intercept, b, is given byb = ( Z y - - m Z x ) / n (5)

Median time o f hatch, t l / 2 , is calculated fromt l / 2 = e- ~/" (6)


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TABLE 3CaLcuLation o f Su m s for Least-Squares Curve Fit

Time, F r a c t i o nh a t c h e d ,

P

In (t), P R O B I T , x 2 x y ( x - X ~ s t) 2x y

10 0432875 2-30258 -1 -8 9985 5-30189 -4 -374 57 0-0009310 0-0016 2.30258 -2- 94 866 5.30189 -6 .78 95 4 0.3008212 0-05271 2-48490 -1 -6 19 42 6-17476 -4432411 0.0055312 0434313 2.48490 -1 -7 15 83 6-17476 -4 .2 63 68 0-0148812 0-02875 2-48490 - 1 "89985 6-17476 -4 .7 20 95 0.0453112 0438945 2"48490 - 1-34432 6.17476 - 3-34051 0.0037814 0.17412 2"63905 -0 .9 37 92 6-96462 -2 .4 75 22 043116714 0.13897 2-63905 -14384 96 6.96462 -2 .8 63 27 043012514 0-13418 2"63905 -1 -1 06 87 6"96462 -2 .9 21 10 0-0006014 0-29712 2'63905 -0" 5322 9 6-96462 -1-4 047 5 0-0950716 0.28274 2-77258 -0 .5 74 32 7-68724 - 1-59237 043237316 0-21086 2.77258 -0 .8 03 23 7-68724 -2- 227 04 0-0016816 0"21086 2"77258 -0"803 23 7.68724 -2 .2 27 04 0-0016816 0.30351 2-77258 -0.5 1391 7.68724 - 1 4 2 4 8 7 0-0338118 0.30990 2.89037 -0 -4 95 70 8-35424 - 1 43278 0-0056418 0.25239 2-89037 -0 -6 66 66 8.35424 - 1-92690 0-0000918 0-30830 2-89037 -0 .5 00 24 8.35424 - 1-44589 0-0053118 0.35782 2"89037 -0 .3 63 82 8-35424 -1- 051 59 0-0196620 0-39456 2.99573 -0 -2 67 00 8-97441 -0 -79988 0-0068320 0-31629 2"99573 -0"4 77 66 8.97441 - 1-43096 0-0004620 0-38019 2.99573 -0 -3 04 54 8-97441 -0"91 232 0-0041120 0-48083 2.99573 -0 -0 47 94 8.97441 -0-14 361 0-0364222 0-45367 3439104 -0 .116 11 9-55454 -0 -35892 0-0038322 0-47284 3-09104 -0 .6 79 5 9.55454 -0- 210 03 0-0073422 0-39936 3-09104 -0- 254 58 9-55454 -0. 78 693 0-0000422 0-42651 3.09104 -0 .1 84 88 9-55454 -0. 57 149 0.0007824 0.44089 3.17805 -0 .1 48 38 10-10002 -0 -47 158 0-0016924 0.49041 3-17805 -0 -2 395 10.10002 -0 -0 76 14 043004224 0-50798 3.17805 0-01996 10.10002 0-06345 0-0017924 0.55271 3.17805 0.13223 10.10002 0-42024 0-0095126 0.58466 3.25809 0.21345 10.61519 0.69545 043033126 0-49840 3.25809 -0-0 160 0 10.61519 -0 .01 30 0 0.0024826 0-46805 3.25809 -0- 079 97 10.61519 -0. 26 05 5 0-0076328 0.55591 3.33220 0.14030 11.10358 0-46752 043027728 0-56549 3-33220 0.16458 11.10358 0.54841 0-0016528 0-55111 3-33220 0-12820 11.10358 04 2719 0.0034430 0-42102 3-40119 -0 .2 01 19 11.56814 -0 -6 84 29 043842730 0.50479 3-40119 0431197 11.56814 0-04073 0-0342430 0.53674 3-40119 0.09200 11.56814 0.31292 0.02117

~ x = Y . y = Y ..x 2 = Y . x y = Y ~ ( x - x ~ a ) 2 =114-79 -2 1-09 341-7 54-25 0-8056


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1 0 S . S p o t t e , G . A d a m s , P . E . S t a k eT h e e l e m e n t s o f c o l u m n 7 a r e c a lc u l a t e d f r o m

( X - - X e s t ) 2 = [ X - y / m + b / m ]2 (7)T h e s u m o f c o l u m n 7 ca n n o w b e c al c u la t e d . T h e s t a n d a r d e r r o r o fe s t i m a t e , S x , y , i s c a l c u l a t e d f r o m

=,,/z x - - 2 ) ( 8 )T o c o n v e r t s t a n d a r d e r r o r t o t h e o r ig i n a l ti m e s c a le f i n d

S t ,y = e l t n ( r " ) + S x , y 1 - - t l / 2 (9 )T h e l i n e a r c u r v e f i t r e s u l t s i n t h e e q u a t i o n

y = m I n ( t ) + b ( 1 0 )A t t i m e , t , t h e e x p e c t e d h a t c h , p , c a n b e p r e d i c t e d b y f ir s t o b t a i n i n gy f r o m e q n ( 1 0 ) a n d t h e n u s in g e q n ( 1 l )

p = I - - 0 - 5 ( 1 + y ( e l + y ( e 2 + Y 2 ( e 3 + y e 4 ) ) ) ) - 4 ( 1 1 )w h e r e e t = 0 . 1 9 6 8 5 4 , e 2 = 0 . 1 1 5 1 9 4 , e 3 = 0 - 0 0 0 3 4 4 a n d e4 = 0 .0 1 9 5 2 7 .T h is a p p r o x i m a t i o n ( A b r a m o w i t z a n d S t e g u n , 1 9 64 , s e ct . 2 6 . 2 . 1 8 ) h a sa m a x i m u m e r r o r o f -+ 2 -5 l 0 -4 .

D I S C U S S I O NA s o f 1 9 79 b r i n e s h r i m p h a d b e e n d e s c r i b e d f r o m 1 64 l o c a t i o n s a r o u n dt h e w o r l d ( S i m p s o n , 1 9 7 9 ). C y s t m a s s va r ie s e v e n w i t h i n t h e s a m e l o tc o l l e c t e d f r o m a s in g le s tr a in . O b t a i n i n g t h e a c c u r a t e m a s s o f a k n o w nn u m b e r o f c y st s is th e r e f o r e a p r e r e q u is i te t o p r e d ic t i n g h o w m a n yn a u p li i c a n b e p r o d u c e d in a g iv e n t i m e . H a t c h i n g c h a r a c t e r i s t ic s a r es t r a in - d e p e n d e n t , in a d d i t i o n t o b e in g a f f e c t e d b y e n v i r o n m e n t a lf a c t o r s ( e .g . l ig h t , t e m p e r a t u r e , s a l in i ty , d i s s o lv e d o x y g e n c o n c e n t r a -t io n , e n v i r o n m e n t a l p H ) . A c h a n g e i n o n e f a c t o r m u s t b e c o m p e n s a t e db y a s h i ft in o t h e r s t o m a i n t a i n o p t i m u m h a t c h i n g r a t e. I d e a l t e m p e r a -t u r e , f o r e x a m p l e , m u s t b e re - e s ta b l i sh e d w i t h e a c h c h a n g e i n s a l in i ty( I v a n o v s k i i e t a l . , 1 9 80 ). T h e s h if t fr o m d o r m a n c y t o r e s u m e d d e v e lo p -m e n t o f th e g a s tr u la i s a f f e c t e d p r o f o u n d l y b y i n te r n a l p H . T h e e x t r e m ea l k a li n i z a ti o n t h a t o c c u r s d u r i n g t r a n s i t io n ( > 1 p H u n i t ) e x c e e d s t h a to f a n y o r g a n i s m y e t s t u d i e d ( B u s a e t a l . , 1 9 8 2 ) . T h e i m p o r t a n c e o f


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P R O B I T a n a l ys is p r e d i c t s h a t c h i n g r ar es o f b r i n e s h r i m p c y s t s 1 1i n te r n al a l k a li n i za t io n o n a r o u s a l f r o m c r y p t o b i o s i s w a s d e m o n s t r a t e dc o n v i n c i n g l y b y B u s a an d C r o w e ( 1 9 8 3 ) . W h e n t h e y a d d e d 4 0 m MN H 4C 1 t o t h e h a t c h i n g m e d i u m ( 1 6 0 m M N aC 1 a n d 5 0 m M t r i ci n e a tp H 8 -5 a n d 2 3 C ), t h e p e r c e n t m e a n h a t c h a t 68 h i n c re a s e d m o r e t h a ns i x f o l d . A c c o r d i n g t o S p o t t e a n d A d a m s ( 1 9 8 3 ) , u n d e r t h e s e c o n d i t i o n s

1 3% o f t h e t o t a l a m m o n i a w o u l d e x i s t in t h e f r e e s t a t e (N H 3 ) . F r e ea m m o n i a is a w e a k b a s e c a p a b l e o f p e n e t r a t i n g t h e c y s t m e m b r a n e s .A c c o r d i n g t o S o r g e t o o s ( 1 9 8 0 ) h a t c h in g r a te c a n d e p e n d o n p r o c es s in gm e t h o d s u s e d o n t h e c y s t s p r i o r t o p a c k a g i n g t h e m ( e. g. l e ve l a n d d u r a -t i o n o f i l lu m i n a t i o n , r e m o v a l o f t h e c y s t c h o r i o n w i t h a s t r o n g o x i d iz i n gs o l u t i o n ) .

A s s h o w n i n F i g. 3 s o m e c y s t s h a d h a t c h e d b y 1 0 h , w h e r e a s 4 0 %s til l h a d n o t h a t c h e d a f t e r 3 0 h . O u r e x p e r i m e n t s e s t a b l is h e d t h a tP R O B I T a n a ly s is is a u s e f u l t o o l f o r p r e d i c ti n g f r a c t i o n h a t c h e d o f ak n o w n n u m b e r o f c y s ts in a p a r t i c u la r l o t u n d e r g i ve n c o n d i t i o n s .N u m b e r s o f c y s t s a re t r a d i t io n a l l y e s t i m a t e d b y m a s s. W e t h e r e f o r es t a r te d b y d e t e r m i n i n g t h e m e a n c y s t m a s s o f t h e l o t s e l e c te d f o r t h ee x p e r i m e n t . T h e t o t a l n u m b e r o f c y s t s a d d e d t o e a ch f la sk w a s 3 -8 5 x l 0 s( m e a n c y s t m a s s e x t r a p o l a t e d t o 1 g ). A f t e r w a r d s c y s t s a n d n a u p l ii w e r er e m o v e d r e g u la r ly in t - m l a li q u o t s a nd c o u n t e d . W e s o m e t i m e s f o u n d i td i f fi c u l t t o d i st in g u i s h a n e m p t y c y s t sh e ll f r o m a n u n h a t c h e d c y s t , s oc y s t c o u n t d a t a w e r e n o t u s e d , e x c e p t in a l i q u o t s t a k e n a t 8 h . T h e s eg a v e t r u e c o u n t s b e c a u s e n o n a u p l i i h a d h a t c h e d . A 1 -m l a l i q u o t t h e r e -f o r e s h o u l d h a v e c o n t a i n e d 7 7 0 c y s t s, b u t t h e a v er ag e c o u n t o f t h e 1 6a l i q u o t s w a s 6 2 6 ( - 6 1 , 95 % c o n f i d e n c e l ev e l) , o r 8 1% o f t h e e s t im a t e dn u m b e r p r e s e n t. I n t h e s e e x p e r i m e n t s t h e f r a c ti o n h a t c h e d d a t a p l o t t e di n F ig . 2 a r e b a s e d o n t h e c o u n t s o f p i p e t t e d s a m p l e s , a n d t h e d is -c r e p a n c y d i d n o t a f f e c t t h e r e su l ts .

T h e r e m o v a l o f cy s t s b y p i p e t t i n g a p p a r e n t l y u n d e r e s t i m a t e d t h en u m b e r a c t u a ll y p r e s e n t, t h u s a c c o u n t i n g f o r t h e 19% d i f f e r e n c eb e t w e e n a l i q u o t c o u n t s a n d t h e n u m b e r k n o w n t o h a ve b e e n a d d e d t ot h e f la sk s . T o o u r k n o w l e d g e t h e re a re n o p u b l i s h e d r e p o r t s o f t h ea c c u r a c y o f p i p e t t i n g t e c h n i q u e s u s e d t o c o u n t e i t h e r c y s t s o r na u p li i.A m i x t u r e o f c y s t s o r n a u p l i i i n w a t e r h a s tr a d i t i o n a l l y b e e n tr e a t e da s a tr u e s o l u t i o n i n s t e a d o f a s u s p e n s i o n o f l a rg e p a r ti c l e s . It s e e m sl og ic al t h a t t h e d i s t r i b u t i o n o f s u c h p a r t ic l e s w o u l d b e u n e v e n b e c a u s eo f s t ra n d i n g o n t h e g la ss a n d v o r t e x e f f e c t s , a n d p e r h a p s n o t r e p r e s e n t e da c c u r a t e l y b y p r o c e d u r e s t h a t r e l y o n p i p e t ti n g .


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12 S. Spotte, G. Adams, P. E. StakeA C K N O W L E D G E M E N T S

P a t r i c i a M . B u b u c i s a n d P a u l G a j p r o v i d e d l a b o r a t o r y a n d a r t is t ic a ss is t-a n c e , r e s p e c t iv e l y , L o u i s M e r c u r i h e l p e d w i t h t h e m a t h e m a t i c s , a n d C a rlJ . B e rg J r. , C a r o l E . B o w e r a n d J o h n D . B u c k r e v i e w e d t h e m a n u s c r i p t .T h e i r c o n t r i b u t i o n s a r e g r a t e f u l l y a c k n o w l e d g e d .

R E F E R E N C E SAb let t , R. F . & Richards, R. H . (1980 ) . Suitabi l i ty of twenty-four-hour and for ty-eight-hour unfed Artemia as an ear ly foo dst uff for 'O ' group Dover sole Solea

solea L. production. Aquaculture, 19, 371-7 .Abramowitz , M. & Stegun, I . (1964) . Handbook of Mathematical Functions with

Formulas, Graphs and Mathematical Tables. US Gov ernmen t Printing Office,Washington DC.

Benijts, F., van Vo ord en, E. & Sorgeloos, P. (19 76). C hanges in the biochemicalcom posit ion o f the early larval s tages of the br ine shrimp Artemia salina L. In:lOth European Symposium on Marine Biology, Vol. 1, eds G. Persoone and E.Jaspers, U niversity Press, W etteren, p p. 1 -9.Bo ssuyt, E. & Sorgeloos, P. (1980 ). Technological aspects o f the batch culturingo f Artemia in h ig h densities. In: The Brine Shrimp Artemia, Vol. 3, eds G.Persoone, P. Sorgeloos, O . Roels and E. Jaspers, University Press, W etteren, pp.133 - 52 .

Busa, W. B. & Crowe, J. H. (1983). lntraceUular pH regulates transitions betweendormancy and development of br ine shr imp Artemia salina embryos . Science,2 2 1 , 3 6 6 - 8 .

Busa, W. B., Crowe, J. H. & Matson, G. B. (1982). lntraceUular pH and the meta-bolic status of dormant and developing Artemia embryos . Arch. Biochem.Biophys., 2 1 6 , 7 1 1 - ! 8 .Finney, D. J . (1971) . PROBITAnalysis, 3rd ed n, Cambridge University Press, NewYork.

lvanovskii, Y u. A., M itrofanov, Y u. A. & Chaga, 1. L. (19 80 ). H atching of nauplii o fArtemia salina at d ifferent salinity and tem pera ture regimes. Soy. J. Mar. Biol.,6 , 2 0 2 - 7 .

Kinne, O. (1977). Marine Ecology: A Comprehensive, Integrated Treatise on Lifein Oceans and Coastal Waters,Vol. 3, Part 2, Wiley, Chichester.Nash, C. E. (1973). Automated mass-production of Artemia salina nauptii forhatcheries. Aquaculture, 2 , 2 8 9 - 9 8 .


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PR OBIT analysis predicts hatching rates of brine shrimp cysts 13Persoone, G. & Sorgeloos , P . (1972) . An im pr ov ed separator bo x fo r Artemia

naupl i i and o the r photo tac t i c inver t ebra tes . Helgoliind. wiss. Meeresunters., 2 3 ,2 4 3 - 7 .Schauer , P . S . , Johns , D. M. , Olney, C. E. & Simpson, K. L. (1980) . Internat ional

s t u d y o n Artemia. IX. L ip id l eve l, ene rgy con ten t an d f a t ty ac id comp os i t ion ofthe cys ts and new ly ha tched naupl ii f rom f ive geographica l s t ra ins of Artemia.In: The Brine Shrimp Artemia , Vol . 3 , eds G. Persoone, P . Sorgeloos , O. Roelsand E. Jaspers, Univers i ty Press, W et teren, pp. 3 65-7 1.

Seidel, C. R. , Kry zno we k, J . & Simpso n, K. L. (1980 ) . Intern at iona l s tu dy onArtemia. XI. Amino ac id compos i t ion and e l ec t rophore t i c p ro te in pa t t e rns ofArtemia from five geographic locations. In: The Brine Shrimp Artemia , Vol . 3 ,eds G . Perso one , P. Sorgeloos, O. R oels and E. Jaspers, Un iversi ty Press, W etteren ,pp . 375-82 .S impson , K . L . (1979) . Focus ing on the m odes t an d m inute brine shr imp. Marl-times, 23 (4) , 9 -11 .

Soka l , R . R. & Ro hl f , F . J . (1969) . Biometry, Freem an, San F ranc isco .Sorgeloos, P . (19 73) . F i rs t report o f the tr iggering effe ct of l ight on th e hatch ing

mechani sm ofArtemia salina dry cys t s. Mar. Biol., 22 , 75 - 6 .Sorgeloos , P . (1980) . The use of the br ine shr imp Artemia in aquacul ture . In: The

Brine Shrimp Artemia, Vol. 3, eds G. Persoone, P. Sorgeloos, O. Roels and E.Jaspers, Un iversi ty Press, W etteren , pp. 25 -4 6.

Sorgeloos , P . & Persoone, G. (1975) . Techn ological imp rovem ents for the cul tiva-t ion o f inver tebra tes as f oo d for f ishes and crus taceans . I I. Hatching and cul tur-ing of the br ine shr imp Artemia saline L. Aquaculture, 6 , 3 0 3 - 1 7 .

Spot te , S . (1979) . Fish and Invertebrate Culture: Water Management in ClosedSystems, 2nd edn , Wi ley , New Y ork .Spo t t e , S . & Ad ams , G . (1983) . Es t ima t ion of the a l lowable upper l imi t o f am mo niain saline waters. Mar. Ecol. Prog. Ser., 1 0 , 2 0 7 - 1 0 .Vanh aecke , P ., C oorem an, A . & Sorge loos , P . (1981) . In t e rna t iona l s tud y on

Artemia. XV. Ef fec t s o f l i gh t in t ens i ty on ha tch ing ra t e o f Artemia cys t s f romdi f fe ren t geographica l origin.Mar. Ecol. Prog. Ser., 5, 111-14 .

Spotte 1984 Aquacultural-Engineering 1

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