Improved techniques for rearing mud crab <i>Scylla paramamosain</i> (Estampador 1949) larvae

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t u r t i ö i e g - e p u e j s o Q

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ci J ( - '' ' S ' W W l A

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A quaculture Research, 2007, 38, 1539-1553 doi:10,llll/j.l365-2109.2007.01814.x

Improved te c h n iq u e s for rearin g m ud c ra b

Scylla

param am osain

(E stam p ad o r 1949) la rv a e

T ruong T rong N ghia1, M a th ieu W ille2, Tran C ong B in h \ H oang P h u o c T h a n h 1, N gu yen Van D a n h 1 &

P atrick S o rg elo o s2

'College of A quaculture and Fisheries, C anTho University, C an T h o City, Vietnam la b o r a t o r y of A quaculture & Artemia Reference Center, G hent University, Ghent, Belgium

C o rre s p o n d e n c e : T Trong Nghia, College of A quaculture an d Fisheries, Can Tho University, 3 rd February avenue, Cam pus II. Can Tho City,Vietnam. E-mail: ttnghia@ ctu.edu.vn

A b s tra c t

A s e rie s o f r e a r in g tr ia ls i n s m a ll 1 L c o n e s a n d la rg e t a n k s o f 3 0 - 1 0 0 L w e r e c a r r ie d o u t to d e v e lo p o p ti­ m a l r e a r in g t e c h n iq u e s fo r m u d c ra b {Scylla p a ra m a ­ m o sa in ) la rv a e . U sin g w a t e r e x c h a n g e ( d is c o n tin u o u s p a r t i a l w a te r r e n e w a l o r c o n t i n u o u s t r e a t m e n t t h r o u g h b io filtra tio n ) a n d m ic r o -a lg a e (Chlorella o r

Chaetoceros) s u p p le m e n ta tio n ( d a ily s u p p le m e n ta tio n a t 0 .1 - 0 .2 m illio n c e lls m L - 1 o r m a i n t e n a n c e a t 1 - 2 m illio n s c e lls m L - 1 ), six d iffe re n t t y p e s o f r e a r i n g s y s te m s w e r e trie d . T h e c o m b in a tio n o f a g r e e n - w a te r b a tc h s y s te m fo r e a r ly s ta g e s a n d a r e c ir ­ c u l a t i n g s y s te m w i t h m ic r o -a lg a e s u p p le m e n ta tio n fo r l a t e r s ta g e s r e s u lte d i n t h e b e s t o v e ra ll p e r f o r ­ m a n c e o f t h e c ra b la rv a e . N o c l e a r effe cts o f c ra b s to c k in g d e n s ity ( 5 0 - 2 0 0 l a r v a e L " ^ a n d r o tif e r ( 3 0 - 6 0 ro tif e r s m L ~ 1) a n d A rte m ia d e n s ity ( 1 0 - 2 0 L - 1 ) w e re o b se rv e d . A s to c k in g d e n s ity o f 1 0 0 - 1 5 0 z o e a 1 ( Z 1 ) L ~ \ c o m b in e d w i t h r o tif e r o f 3 0 - 4 5 m L ~ 1 fo r e a r ly s ta g e s a n d A rte m ia fe e d in g a t 1 0 - 1 5 n a u p l i i m L - 1 fo r Z 3 -Z 5 se e m e d to p r o d u c e t h e b e s t p e r f o r m a n c e o f S. p a ra m a m o sa in la rv a e . O p ­ ti m a l r a tio n s fo r c r a b la r v a e s h o u ld , h o w e v e r, b e a d ­ j u s t e d d e p e n d in g o n t h e sp e c ie s, la r v a l sta g e , la r v a l s ta tu s , p r e y size, r e a r in g s y s te m a n d te c h n iq u e s . A p r a c tic a l fe e d in g s c h e d u le c o u ld b e to in c r e a s e live f o o d d e n s ity fro m 3 0 to 4 5 r o tife rs m L - 1 fro m Z1 to Z 2 a n d in c r e a s e t h e n u m b e r o f A rte m ia n a u p lii m L - 1 f r o m 10 t o 15 fro m Z 3 to Z5. B a c te ria l d is e a s e r e m a in s o n e o f t h e k e y f a c to rs u n d e r ly in g t h e h ig h m o r ta lity i n t h e z o e a s ta g e s. F u r t h e r r e s e a r c h to d e v e lo p sa fe p r o p h y la c tic t r e a t m e n t s is th e r e f o r e w a r r a n te d . C o m ­ b in e d w i t h p r o p e r live fo o d e n r i c h m e n t te c h n iq u e s , a p p lic a tio n o f t h e s e fin d in g s h a s s u s t a i n e d a s u r v iv a l

r a t e fro m Z1 to c r a b 1 - 2 s ta g e s i n la r g e r e a r in g t a n k s o f 1 0 -1 5 % ( m a x im u m 30% ).

K eyw ords:

S c y lla p a ra m a m o sa in , r e a r i n g t e c h n i ­ q u e s , w a t e r e x c h a n g e , m ic r o -a lg a e , l a r v a l d e n s ity , life fo o d d e n sity , p r o p h y la x is

In tro d u ctio n

A q u a c u ltu r e o f m u d c ra b s. S cylla sp p ., c o n tr ib u te s a la rg e p r o p o rtio n to t h e w o rld p r o d u c tio n o f t h e g e n u s (FA O 1999). M o reo v er, m u d c ra b s r e p r e s e n t a v a lu a b le c o m p o n e n t o f s m a ll- s c a le d c o a s ta l f is h e rie s i n m a n y c o u n tr ie s in tr o p ic a l a n d s u b tr o p ic a l A sia , fo r w h ic h th e r e h a s b e e n a g e n e r a l t r e n d o f in c r e a s e d e x p lo ita ­ t i o n i n r e c e n t y e a r s (A n g e li 1992; K e e n a n 1 9 9 9 a). I n V ie tn a m , t h e m u d c r a b S c y lla p a ra m a m o sa in is t h e s e c o n d m o st i m p o r t a n t m a r i n e s p e c ie s n e x t to s h rim p , b e in g c u l t u r e d w id e ly i n t h e c o a s ta l a r e a . H ow ever, m u d c ra b f a r m in g c u r r e n tl y r e lie s e n tir e ly o n t h e w ild fo r se e d s to c k a n d t h e m a i n o b s ta c le fo r e x p a n s io n is t h e u n a v a ila b ility o f h a tc h e r y - r e a r e d s e e d (L iong 1992; R a tta n a c h o te & D a n g w a ta n a k u l 1992; K e e n a n 1 9 9 9 a ; S h e lle y & F ield 1999; M a n n , A s a k a w a , P iz z u to , K e e n a n & B ro ck 2 001; X u a n

2001

). R e a rin g te c h n iq u e s , d is e a s e a n d n u t r it i o n a r e th e t h r e e m a in a r e a s of r e s e a r c h t h a t h a v e s u p p o r te d c o m m e r c ia l p r o d u c tio n o f m a r i n e fish a n d c r u s t a ­ c e a n la r v a e (S o rg elo o s & L é g e r 1992). T h e s e t h r e e a s ­ p e c ts a re to a la rg e e x t e n t i n te r c o n n e c te d a n d d e v e lo p in g h a tc h e r y t e c h n iq u e s fo r a ‘n e w ’ s p e c ie s is n o t p o ssib le u n le s s a ll t h e a r e a s a r e a d d re s s e d . T h e © 2007 The Authors

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Improved re a rin g te ch n iq u es for m ud crab larvae T T N g h ia et al. A quaculture Research, 2007, 3 8 , 1539-1553

d e s ig n o f r e a r in g s y s te m s c o v e rs m o r e t h a n p u re ly t e c h n ic a l a s p e c ts . S n b - o p tim a l r e a r in g c o n d itio n s (e.g. p h y sic a l stre s s, la c k o f o x y g e n o r s u b -o p tim a l w a te r q u a lity ) a ffe c t l a r v a l h e a l t h a n d c a n c a u s e m a s s m o r ta lity d u e to d is e a s e o u tb r e a k s . Sim ilarly, s y s te m d e s ig n i n f lu e n c e s (live) fo o d q u a lity a n d its a v a ila b ility to t h e p r e d a t o r la rv a e .

T h e re h a s b e e n a g r e a t d e a l o f p ro g re s s i n m a r in e la r v a l r e a r in g t e c h n o lo g y s in c e its b e g in n in g i n th e 1 9 6 0 s ( S h e lb o u rn e 1 9 6 4 ; H o w e ll, Day, E llis & B a y n e s 1998). M a n y o f t h e s e t e c h n i c a l im p ro v e m e n ts devel­ o p e d o v e r t h e p a s t d e c a d e s c o u ld b e a p p lie d fo r m u d c r a b w i t h s o m e m o d ific a tio n s . A n o v e rv ie w o f th e r e a r in g s y s te m s c u r r e n tl y a p p lie d fo r l a r v i c u l tu r e of m u d c r a b s w a s p r e s e n t e d b y D a v is (2003). A lth o u g h k n o w le d g e h a s b e e n o b t a i n e d f r o m th e s e sy ste m s, t h e r e is a n e e d to f u r t h e r o p tim iz e r e a r in g te c h n iq u e s i n o r d e r to m a x im iz e la r v a l s u r v iv a l a n d quality. F u r th e rm o r e , te c h n i q u e s s h o u l d b e a d a p te d fo r e a c h S cylla s p e c ie s (K e e n a n , D a v ie & M a n n 1998; K e e n a n 199 9 b ) i n r e la tio n t o lo c a l c o n d itio n s (se a w a te r s o u rc e , s t a tu s o f h a t c h e r y m a n a g e m e n t, lo c a l re so u rc e s ). T h e a i m o f t h i s r e s e a r c h is to a d a p t th e e x is tin g r e a r in g s y s te m s fo r l a r v i c u l t u r e to m u d c ra b s p e c ie s (S. p a ra m a m o sa in b e i n g t h e t e s t c a se ) a n d to im p ro v e o t h e r te c h n i q u e s i n o r d e r to m a x im iz e la rv a l s u r v iv a l a n d q u a lity .

M ateria ls a n d m e th o d s

S ou rce o f larvae

G ravid c r a b s w e r e b o u g h t f ro m lo c a l m a r k e ts a n d tr a n s p o r te d to t h e h a tc h e ry . B e fo re s to c k in g i n th e h a tc h e ry , t h e c r a b s w e r e s u b je c te d to a b a th of 1 0 0 p L L - 1 o f a 4 0 % f o r m a lin s o lu tio n fo r l h . T h e c ra b s w e re s to c k e d in d iv id u a lly i n 1 0 0 L c o m p a r t­ m e n ts o f a ro o fe d 2 x 2 x 0.5 m c e m e n t ta n k , e q u ip p e d w ith a b io filter. R e a rin g w a te r of 30 ± 1 g L “ 1 s a lin ity w a s d ilu te d fro m b r in e (9 0 — 110 g L " x) w ith ta p w a t e r a n d c h lo r in a te d b e fo re use. A m b ie n t t e m p e r a tu r e f lu c tu a te d s lig h tly a r o u n d 2 8 °C. E v e ry c ra b w a s fe d a d a ily r a t i o n of 1 0 -1 5 g of fre s h m a r i n e sq u id , b iv a lv e o r s h r i m p m e a t altern ately .

A fte r 3 - 5 d a y s o f a c c lim a tio n , u n i l a t e r a l e y e s ta lk a b la tio n w a s a p p lie d to i n d u c e s p a w n in g . A fte r s p a w n in g , b e r rie d c r a b s w e r e a g a i n s u b je c te d to a 1 0 0 pL L _ 1 f o r m a lin b a t h f o r 1 h a n d t r a n s f e r r e d to a 7 0 L p la s tic t a n k c o n n e c t e d to a b io filte r fo r e g g in c u ­ b a tio n . D a ily m a n a g e m e n t c o n s i s t e d o f s i p h o n in g o u t w a s te m a te r ia ls a n d s h e d d e d e g g s fro m t h e t a n k b o t­ to m a n d c o n tr o llin g t h e t e m p e r a t u r e (3 0 °C), s a lin ity

(30 g L - 1 ) a n d a m m o n i a a n d n i t r i t e levels. E v e ry o t h e r day, t h e c r a b s w e r e p la c e d i n a 5 0 p L L - 1 fo r­ m a lin b a t h fo r 1 h to r e d u c e o r p r e v e n t i n f e s ta tio n of t h e e g g s w i t h f u n g i a n d b a c te r ia . D u r i n g e g g i n c u b a ­ tio n , t h e c r a b s w e r e n o t fed. O n e t o tw o d a y s b e fo re h a t c h i n g , t h e b e rrie d fe m a le w a s m o v e d to a 5 0 0 L fib re g la s s t a n k . W h e n th e h a t c h i n g p r o c e s s w a s c o m p le te d , l a r v a e w e re s e le c te d b a s e d o n t h e i r p h o t o t a c t i c b e h a v io u r . A e ra ­ tio n i n t h e h a t c h i n g t a n k w a s t u r n e d off f o r se v e ra l m in u te s a n d t h e a c tiv e la r v a e s w im m i n g u p to th e s u r f a c e w e r e c o lle c te d b y g e n tle sc o o p in g . T h e l a r v a e w e r e t h e n t r a n s f e r r e d to t h e r e a r in g c o n ta in e r s . A c c lim a tio n w a s p e r f o r m e d b y p la c in g t h e la r v a e i n a 5 0 L p la s tic m e s h b u c k e t a n d slo w ly r i n s i n g t h e m w i t h w a t e r f ro m t h e l a r v a l r e a r i n g c o n ­ t a i n e r s f o r 2 0 - 3 0 m i n b e fo re re le a se .

Food a n d fe e d in g

M icro -a lg a e c u ltu re

S ta r tin g c u l t u r e s o f t h e m ic r o - a lg a e C haetoceros calci­ tra n s a n d Chlorella vu lg a ris w e r e m a i n t a i n e d in d o o r s w i t h W a ln e s o lu tio n i n s e a w a t e r o f 3 0 g L “ 1 a t 25 °C. L a rg e - s c a le p r o d u c tio n w a s p e r fo r m e d i n d o o r s i n 5 0 0 L t a n k s u n d e r a t r a n s p a r e n t ro o f. A h a e m o c y t- o m e te r w a s u s e d to c o u n t m ic r o - a lg a l d e n s itie s .

R o tife r c u ltu r e and e n r ic h m e n t

T h e s a m e B ra c h io n u s p lic a tilis L -s tr a in w i t h a lo ric a l e n g th a n d w i d t h o f 1 6 4 ± 2 2 a n d 1 2 0 ± 2 2 p m , r e ­ sp ectiv ely , w a s u s e d i n a ll t h e e x p e r im e n ts . R o tifers w e r e c u l t u r e d in d o o r s in 1 0 0 L fib re g la s s t a n k s o p e r­ a te d i n a b a tc h m o d e , fo llo w in g t h e p r o c e d u r e d e ­ s c rib e d i n S o rg e lo o s a n d L a v e n s (1996). R o tife rs w e re in itia lly g r o w n o n b a k e r s y e a s t, b u t l a t e r o n fed C u ltu r e S e lc o ' (IN V E A q u a c u ltu r e , D e n d e r m o n d e B e lg iu m ) b e fo re fe e d in g to t h e la rv a e . T e m p e ra tu r e a n d s a l in i t y w e r e c o n tr o lle d a t 25 °C a n d 2 5 g L _1 re sp e c tiv e ly . T h e y w e r e h a r v e s t e d t h r o u g h a 6 0 p m s c re e n a n d r i n s e d th o ro u g h ly . R o tifers w e r e e n r ic h e d w i t h m ic r o -a lg a e o r a rtifi­ c ia l e n r i c h m e n t m e d ia b e fo re b e in g fed to t h e c ra b la rv a e . E n r i c h m e n t w i t h C hlorella w a s p e r fo r m e d a t a d e n s ity o f 5 x IO6 c e lls m L ~ 1 fo r 3 h ( D h e rt 1996). R o tife rs w e r e a ls o e n r ic h e d w i t h D ry I m m u n e S e lc o a (DIS ", IN V E A q u a c u ltu r e ) , u s i n g tw o s e p a r a te d o se s o f 0.05 g L “ 1 a t a 3 - h in te r v a l. E n r ic h m e n t w a s p e r­ fo rm e d a t a d e n s ity o f 5 0 0 r o tif e r s m L - 1 . T h e w a te r i n th e e n r i c h m e n t v e s se l w a s slo w ly h e a t e d to 1540 © 2007 The Authors Journal Compilation © 2007 Blackwell Publishing Ltd, A quaculture Research, 38, 1539-1553

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A quaculture Research, 2007, 3 8 , 1539-1553 Improved re a rin g te c h n iq u e s for m ud crab larvae T T N g h ia e t al.

T ab le 1 Overview of larval rearin g system s applied in th is study based o n th e m e th o d of w a te r exchange and m icro-algae supplem entation W ater e x c h a n g e A lg a e s u p p le m e n ta tio n D is c o n tin u o u s m a n u a l pa rtia l w a te r r e n e w a l C o n tin u o u s w a te r t r e a tm e n t t h r o u g h th e u s e o f a biofilter

No m icro-algae su p p lem en te d (indoors) C lear-B atch sy stem C lear-R ecirc sy stem

M icro-algae su p p lem en te d a t low levels to provide ex tra food for live p rey s (Indoors or outdoors)

A lgae-B atch sy stem A lgae-R ecirc sy stem

Micro-algae su p p lem en te d a t a high concentration a n d self-sustainable u n d er natural sunlight a s an ex tra food for live p rey a n d w ate r conditioning (outdoors)

G reen-B atch sy stem G reen -R ecirc sy stem

(C om bination of G reen-B atch a n d A lgae-R ecirc sy stem a t early a n d late larval s ta g e s respectively)

2 9 - 3 0 °C to a v o id e x p o s in g t h e ro tife rs to t h e r m a l s h o c k w h e n t h e y w e r e a d d e d to t h e la r v a l r e a r in g ta n k s . B efore b e in g fed to t h e la rv a e , e n r ic h e d ro tife rs w e r e r in s e d a n d r e - s u s p e n d e d i n c le a n s e a w a te r a t t h e sa m e te m p e r a t u r e a s t h e c r a b - r e a r in g ta n k s . A r te m ia c u ltu re a n d e n r ic h m e n t A rte m ia n a u p lii (V in h C h a u s tr a in ) w e r e h a tc h e d a s d e s c rib e d b y V a n S ta p p e n (1996). B o th n e w ly h a tc h e d o r e n r ic h e d A r te m ia n a u p lii w e r e u s e d i n t h e e x p e ri­ m e n ts o f t h i s stu d y . A r te m ia w e r e e n r ic h e d w i t h Chaetoceros i n t h e s a m e m ic r o - a lg a l d e n s ity a s fo r r o ­ tif e r e n r ic h m e n t. T h e n a u p lii w e r e a ls o e n r ic h e d w ith DIS “ (u s in g tw o s e p a r a te d o s e s o f 0.3 g m L “ 1 a t a 6 - h in te rv a l). W a te r t e m p e r a t u r e a n d s a lin ity w e r e m a in ­ t a i n e d a t 3 0 °C a n d 3 0 g L ~ \ resp ec tiv ely , d u r in g

A rte m ia e n r i c h m e n t . T h e d e n s ity o f A rte m ia d u r in g e n r i c h m e n t w a s 2 0 0 m L - 1 . B efore fe e d in g to th e c r a b la rv a e , t h e A r te m ia w e r e r i n s e d w i t h d isin fe c te d s e a w a te r a n d s u s p e n d e d a t a k n o w n d e n s ity in se a w a te r.

Feeding

R o tife rs w e r e fed to t h e c r a b l a r v a e fro m 0 to 6 d ay s a f te r h a t c h (DAH 0 - 6 ) [ r o u g h ly c o r re s p o n d in g to z o e a 1 (Z l)-Z 2 sta g e s]. N e w ly h a tc h e d A rte m ia o r

A rte m ia m e ta - n a u p lii w e r e o ffe re d fro m D A H 6 (Z3 sta g e ) o n w a rd s . R o tife rs a n d A rte m ia w e r e a d d e d d a ily a t 3 0 - 4 5 a n d 5 - 1 0 m L - 1 to t h e r e a r in g t a n k re sp e c tiv e ly ( e x p e r im e n ts 1, 2, 3, 4, 7 a n d 8). For e x p e r im e n ts 5 a n d 6, liv e fe e d w e re fe d a t th e re q u ire d p r e y d e n s itie s b a s e d o n t h e p la n n e d tr e a tm e n ts . W h e n e v e r t h e c r a b la r v a e w e r e fe d e n ­ r ic h e d live feed, a lg a e - o r DIS - e n r ic h e d live feed w e r e u s e d o n a l t e r n a t e d ay s.

Larval rea rin g e x p e r im e n ts: o b je c tiv e s an d

ex p e r im e n ta l d e s ig n

I n e x p e r im e n ts 1 ,2 a n d 3, t h e e ffe c t o f d iffe re n t w a te r e x c h a n g e s c h e m e s a n d t h e a d d itio n o f m ic r o -a lg a e o n la r v a l s u r v i v a l a n d d e v e lo p m e n t w e r e e v a lu a te d . I n e x p e r im e n ts 4 - 8 , o t h e r c u l t u r e a s p e c ts s u c h a s Z1 s to c k in g d e n s ity , live fo o d d e n s ity a n d t h e effe ct of d iffe re n t p r o p h y la c tic t r e a t m e n t s w e r e in v e s tig a te d . T h e w a t e r q u a lity m a n a g e m e n t s c h e m e s t e s te d i n ex­ p e r im e n ts 1 - 3 a r e s u m m a r i z e d i n T ab le 1. A n o v e r­ v ie w o f t h e e x p e r i m e n t a l d e s ig n a n d c u l t u r e c o n d itio n s o f a ll t h e e x p e r im e n ts is p r e s e n te d in T ab le 2. T h e s m a ll- s c a le e x p e r im e n ts ( 1 - 3 0 L) w e re c a r r ie d o u t i n a t e m p e r a tu r e - c o n tr o lle d ro o m ( 2 8 - 3 0 °C). T h e e x p e r i m e n t s i n 1 0 0 L t a n k s w e re p e r fo r m e d o u t d o o r s a t a m b i e n t te m p e r a t u r e ( 2 7 - 31 °C). T h e s o u r c e a n d t h e d is in f e c tio n p r o c e d u r e of t h e s e a w a te r f o r l a r v a l r e a r in g w e r e s im ila r t o th o s e u s e d fo r b r o o d s to c k r e a r in g . F o rm a lin a t a c o n c e n t r a ­ ti o n o f 2 0 pL L ~ 1 w a s a p p lie d e v e ry o t h e r d a y a s a p ro p h y la c tic t r e a t m e n t i n e x p e r im e n ts 1 - 6 . E x p e rim e n t 1 L a rv a l s u r v iv a l a n d g r o w t h i n a c le a r w a te r s y s te m w i t h d a ily p a r t i a l w a t e r e x c h a n g e (C lear-B atch ) w a s c o m p a r e d w i t h t h o s e i n a c l e a r w a t e r r e c irc u la tin g s y s te m (C lear-R ecirc). I n t h e firs t r e a r in g sy ste m , 3 0 - 5 0 % o f t h e w a te r w a s r e p la c e d daily. I n t h e r e c ir ­ c u la tin g s y s te m , a ll r e a r in g t a n k s w e r e c o n n e c te d to a c e n t r a l b io filter. W a te r w a s r e c ir c u la te d a t a r a te o f a p p r o x im a te ly 1 0 0 % o f t h e t a n k v o lu m e e v e ry 3 - 4 h. Live fo o d a n d c r a b la rv a e w e r e r e t a i n e d i n t h e r e a r in g t a n k s w i t h b y a m e s h s c r e e n o f 7 0 a n d 3 0 0 p m d u r ­ in g t h e r o tif e r a n d A rte m ia fe e d in g s ta g e resp ec tiv ely . L a rg e r m e s h s c r e e n s (250 a n d 5 0 0 - 1 0 0 0 p m fo r ro ti­ fer a n d A rte m ia s ta g e re sp e c tiv e ly ) a n d h i g h e r flow

© 2007 The Authors

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Table 2 O v er v ie w of th e e x p e ri m e n ta l c o n d it io n s an d w ater q ua li ty p a ra m e te rs (m ea n ± st a n d a rd d ev iat io n ) in e x p e ri m e n ts 1 -8

Improved rearin g techniques for mud crab larvae T T N g h ia e t al. A quaculture R esearch. 2007, 38, 1539-1553 T - o > ï - c o m m o o ) i n o 5 c o c or q q q c o r - r n c o w c ' j q Ö Ó Ö Ó Ö Ó Ö Ö Ó Ó Ó Ó - H - H - H - H - H - H - H - H - H - f l - H - H - j r T f O N O i f j c o c o n ^ t o T - T - O T - i n T r i - L n T r ^ c o o d d d d ó ö d ó d d d ó ^ r r ^ T f o D O o r ^ c D C M c o c o i n co in o o o o t-1 o -H -H -H -H +1 -H tí) (O (\J N CM ’T r-co o o o in o o o o ^ (O (Û ^ ▼ " d o d o ■tí -H -H -H +1 o s Œ co to in o oí r^- q o o o o O O UJ O O ». 0) * .£ a) JS E <3 V, _ o o P s : s z ÿ . sz -C d d ,!r o o ‘73 O ■S S m 0 ® »

I 1 1 I I I I i

0 0 U < 0 0 ( 3 0

ra

- E S’ ¡5

-I

"c E

I f

o 13 ' " § O o -S o >• 0J io E CvJ a. S o 's 0 -3 q c 4 1 ca <u -d _ e 0 v 3 a c .S ~ d .2 E c 2 d, h V *[“« {-< QJ ■S S u ¡2 © 2007 The Authors

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A quaculture Research, 2007, 3 8 , 1539-1553 Im proved rearing te c h n iq u e s for m ud crab larvae T T N g h ia e t al. r a te s w e r e u s e d u p o n d a ily flu s h in g o u t o f u n e a t e n live fo o d a n d w a s te . E x p e r im e n t 2 A C le a r-R e c irc s y s te m w a s c o m p a r e d w i t h tw o s y s te m s w h e r e m ic r o -a lg a e w e r e a d d e d . R e a rin g c o n ­ d itio n s fo r t h e C le a r-R e c irc s y s te m w e r e s im ila r to t h o s e d e s c rib e d i n e x p e r im e n t 1. I n t h e A lg a e -R e c irc s y s te m , m ic r o - a lg a e w e r e a d d e d d a ily to t h e r e c ir c u ­ la tin g s y s te m a t a lo w c o n c e n tr a tio n r a n g i n g f ro m 0.1 t o 0 .2 m illio n c e lls m L ~ \ T h e o p e r a tio n o f t h e r e a r ­ i n g t a n k s w a s s im ila r to th e C le a r-R e c irc tr e a tm e n t. I n t h e G re e n -B a tc h tr e a tm e n t, a c la s s ic a l ‘g r e e n - w a t e r ’sy s te m , m ic r o -a lg a e c o n c e n tr a tio n s i n t h e c u l ­ t u r e t a n k s w e r e k e p t a t a te n fo ld h i g h e r level o f 1 - 2 m illio n c e lls m L - 1 . I n t h i s sy ste m , t h e c u l t u r e t a n k s w e r e in itia lly o n ly filled to 50% o f t h e i r c a p a ­ c ity a n d g r a d u a lly in c r e a s e d to 100% by t h e e n d o f t h e Z 2 s ta g e b y a d d i n g w a t e r a n d a lg a e daily. L a te r o n , 1 0 - 3 0 % o f t h e r e a r in g w a te r w a s r e p la c e d d a ily b y c l e a n s e a w a te r a n d / o r a lg a e , d e p e n d in g o n th e d e n s i ty o f m ic r o - a lg a e r e m a in in g i n t h e r e a r in g t a n k s . U p o n w a te r e x c h a n g e , u n e a t e n live fo o d w a s a ls o f lu s h e d o u t t h r o u g h a m e s h s c re e n ( m e s h siz e s a s d e s c rib e d i n e x p e r im e n t 1). T h e s a m e a m o u n t of liv e fo o d ( 3 0 - 4 5 r o tif e r s m L “ 1 a n d 5 - 1 0 A rte m ia

n a u p lii m L ~ L) w a s fe d i n a ll t h e tr e a tm e n ts . I n t h e s y s te m s u s i n g a lg a e , Chlorella w a s u s e d f o r Z 1 -Z 3 s ta g e s ( w h ic h is u n s u i t a b l e a s a fo o d s o u r c e fo r

A rtem ia ); fro m Z 4 o n w a r d s . Chlorella w a s g r a d u a lly r e p la c e d w i t h Chaetoceros. E x p e r im e n t 3 I n t h is e x p e r im e n t, a G re e n -B a tc h a n d a G re e n -R e c irc s y s te m w e r e s e t u p i n o r d e r to f u r t h e r e v a lu a te th e a p p lic a tio n o f m ic r o -a lg a e o n t h e p e r fo r m a n c e o f c r a b la rv a e . T h e f irs t r e a r i n g s y s te m w a s a b a t c h s y s ­ te m w i t h a d d itio n o f h i g h c o n c e n tr a tio n s o f a lg a e a s d e s c r ib e d i n e x p e r i m e n t 2. T h e s e c o n d s y s te m c o n ­ s is te d o f a c o m b in a tio n o f t h e G r e e n -B a tc h s y s te m fo r e a r ly c r a b s ta g e s (Z 1 -Z 2 ) a n d a A lg a e -R e c irc sy s ­ t e m fo r l a t e r s ta g e s (Z3 o n w a rd s). E x p e r im e n ts 4 - 6 I n t h e s e e x p e r im e n ts , t h e e ffe c t o f Z1 s to c k in g d e n s ity (50, 1 0 0 , 1 5 0 a n d 2 0 0 L ~ \ e x p e r im e n t 4), ro tife r fe e d in g d e n s itie s a t 30, 4 5 a n d 6 0 m L - 1 fo r Z 1 -Z 2 ( e x p e r im e n t 5) a n d A r te m ia d e n s itie s a t 10, 15 a n d 2 0 m L “ 1 fo r Z 3 o n w a r d s (e x p e r im e n t 6) w a s e v a lu ­ a te d . T h e s e e x p e r i m e n t s w e r e r u n in a G r e e n -B a tc h ( e x p e r im e n t 4) o r a C le a r-R e c irc s y s te m ( e x p e r im e n ts 5 a n d 6) a s d e s c r i b e d above. E x p e rim e n t 7 I n e x p e r im e n t 7, t h e effect o f p r o p h y la c tic c h e m ic a ls o n t h e s u r v iv a l o f t h e la rv a e w a s in v e s tig a te d . T h r e e tr e a tm e n ts , c o n s i s t i n g o f a c o n tr o l (n o c h e m ic a ls u se d ), d a ily a d d i t i o n o f f o rm a lin a t 2 0 pL L ~ 1 a n d d a i­ ly a d d itio n o f O x y te tr a c y c lin e a t 10 m g L “ \ w e r e r u n i n 1 L p la s tic c o n e s . A ll c o n e s w e r e p la c e d i n a w a t e r b a t h i n o r d e r to m a i n t a i n t h e r e a r in g t e m p e r a t u r e a t 3 0 °C. W a te r w a s r e p l a c e d a lm o s t c o m p le te ly daily. U p o n w a te r e x c h a n g e , th e s u r v iv a l w a s d e te r m in e d . E x p e rim e n t 8 To a v o id t h e u s e o f d r u g s a s a p ro p h y la x is, d ir e c t o z o ­ n a t i o n o f th e c u l t u r e t a n k s w a s te s te d i n a e r a te d 1 L p la s tic c o n e s. O z o n e ( 0 3) w a s in je c te d d ir e c tly v ia a n a i r s t o n e in to e v e r y l a r v a l r e a r in g e o n e u p o n c h a n ­ g in g w a te r a n d fe e d daily. S ix tr e a t m e n t s w i t h t h r e e r e p lic a te s w e r e a r r a n g e d c o n s is tin g o f a c o n tr o l ( w ith o u t 0 } a p p lic a tio n ) a n d 0 3 in je c tio n f o r 2, 4, 6, 8 a n d 10 m i n ( e q u iv a le n t to a r e s id u a l 0 3 lev el i n t h e w a t e r o f 0 ,0 6 ,0 ,1 2 ,0 .1 5 ,0 .1 7 a n d 0.19 m g L " 1 a s m e a ­ s u r e d b y a t e s t k it u p o n f in is h in g t h e in je c tio n ). O th e r r e a r in g c o n d itio n s a n d d a ily m a n a g e m e n t w e r e s im i­ l a r to th o s e d e s c rib e d f o r e x p e r im e n t 7.

E valu ation c r ite r ia

T h e s u r v iv a l r a te s i n t h e e x p e r im e n ts u s in g la rg e ( 3 0 - 1 0 0 L) c o n ta in e r s (e x p e r im e n ts 1 - 6 ) w e r e e s ti­ m a te d b y v o lu m e tric s a m p lin g . D e p e n d in g o n t h e t a n k v o lu m e a n d t h e d e n s ity o f t h e s u r v i v i n g la rv a e , tr ip lic a te 3 0 0 - 1 0 0 0 m L s a m p le s w e r e t a k e n fro m e a c h t a n k . M e g a lo p a e (M) (DAH 1 5 - 1 8 ) a n d first c r a b s (C l) (DAH 22) w e r e c o u n te d in d iv id u a lly . I n e x ­ p e r im e n ts 7 a n d 8 ( u s in g s m a ll co n e s), t h e a v e ra g e s u r v iv a l ra te w a s c a lc u la te d b y i n d iv id u a lly c o u n t i n g a ll s u r v iv in g la r v a e i n e a c h re p lic a te . L a rv a l d e v e lo p m e n t w a s m o n ito re d e v e ry 3 d a y s b u t d a ily i n e x p e r im e n ts 7 a n d 8 b y id e n tify in g t h e a v e r a g e z o e a l i n s t a r s ta g e o f a s a m p le o f l a r v a e a n d a s s i g n i n g i t a v a lu e : Z1 = 1 , Z 2 = 2, etc . M e g a lo p a s ta g e w a s a s s ig n e d a v a l u é o f 6. To c o m p a r e t h e la r v a l d e v e lo p m e n t i n e a c h t r e a t m e n t , a n a v e ra g e la rv a l s ta g e in d e x (LSI) w a s c a lc u la te d fro m t h e a v e ra g e LSI v a lu e o f a ll r e p lic a te c o n t a i n e r s i n t h e s a m e tr e a t­ m e n t. F o r la rg e c o n ta in e r s ( e x p e rim e n ts 1 -6 ), five o r 10 l a r v a e (in 3 0 a n d 1 0 0 L t a n k s re sp e c tiv e ly ) w e re © 2007 The Authors

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Improved re a rin g techniques for m ud crab larvae T T N g h ia e t al. A quaculture Research. 2007, 38, 1539-1553

T a b le 3 E xperim ent 1: survival rates a nd larval stage index (LSI) values o f Scylla paramamosain la rv a e c u ltu red in tw o differ­ en t re a rin g system s D ays a fte r h a tc h T re atm e n t 3 6 9 12 15 18 Survival rate (%)* C lear-B atch 85 ± 6a 7 9 ± 9a 7 0 ± 6 a 6 4 ± 7a 4 2 ± 6b 32 ± 5b C lear-R ecirc 84 ± 4 a 7 8 ± 8a 72 ± 5 a 70 ± 5a 63 ± 9 a 4 7 ± 6 a LSI’

C lear-B atch 1.5 ± 0.2a 2 .7 ± 0 .1 a 3.5 ± 0.4a 4 .0 ± 0.0a 4 .6 ± 0.2a ND

C lear-R ecirc 1.5 ± 0 ,2 a 2 .7 ± 0 .1 a 3.6 ± 0.3a 4 .2 ± 0.3a 4 .8 ± 0.1a ND

’ Survival rates or LSI values in the sam e colum n followed by the sam e superscript letter are not statistically different (P > 0.05). For treatm ent descriptions, refer to Table 1.

ND, not determ ined.

s a m p le d f ro m e a c h t a n k to c a lc u la te t h e a v e ra g e LSI. T h e s a m p le d l a r v a e w e r e s ta g e d u n d e r a d is s e c tin g m ic ro s c o p e . I n e x p e r im e n t 8, u s i n g s m a ll c o n ta in e r s , a ll la r v a e w e r e s ta g e d v is u a lly u p o n c o u n t i n g d a ily s u rv iv a l. I n th i s r e s e a rc h , six la r v a l r e a r in g s y s te m s w e re a p p lie d fo r t h e e x p e r im e n ts . E a c h r e a r in g s y s te m h a d its o w n f e a tu re s , i.e. w a t e r q u a lity a n d ‘e a s e of o p e r a tio n ’. T h e re fo re , th e s e f e a tu r e s i n c o m b in a tio n m a d e u p a t r e a t m e n t a s a ty p e o f r e a r in g s y s te m . T h e y w e r e n o t c o n s id e r e d a s v a ria b le s.

S ta tistic a l a n a ly sis

O n e -w a y a n a ly s is o f v a r i a n c e (a n o v a ) w a s u s e d to c o m p a r e d a ta . H o m o g e n e ity o f v a r i a n c e w a s te s te d w i t h t h e L e v e n e s ta tis tic (P o r a v a lu e w a s s e t a t 0.05). If n o s ig n if ic a n t d iffe re n c e s w e r e d e te c te d b e ­ tw e e n t h e v a r ia n c e s , t h e d a t a w e r e s u b m itte d t o a o n e - w a y a n o v a . T u k e y 's h o n e s t ly s ig n if ic a n t d iffe r­ e n c e p o s t h o c a n a ly s is w a s u s e d t o d e te c t d iffe re n c e s b e tw e e n m e a n s a n d to in d ic a te a r e a s o f s ig n if ic a n t d iffe re n c e . I f s ig n if ic a n t d iffe re n c e s w e r e d e te c te d b e tw e e n v a r ia n c e s , d a t a w e r e t r a n s f o r m e d u s i n g t h e a r c s in e - s q u a r e r o o t (for p e r c e n ta g e d a ta , i.e. s u r v iv a l r a te ) o r l o g a r i th m i c t r a n s f o r m a tio n s (for LSI v a lu e ) (S o k al & R o h lf 1995). A ll a n a ly s e s w e r e p e r fo r m e d u s i n g t h e s ta tis tic a l p r o g r a m s t a t i s t i c a 6.0.

R e su lts

E xp erim en t 1

S u rv iv a l i n t h e C le a r-R e c irc s y s te m a t Z 4 - Z 5 s ta g e s o n DAH 15 a n d i n t h e m e g a lo p a s ta g e o n D A H 18 w a s s ig n if ic a n tly h i g h e r t h a n t h o s e i n t h e C le a r-B a tc h s y s te m ( b o th a t P < 0.01) (T ab le 3). A l th o u g h s lig h tly h i g h e r i n t h e r e c i r c u la ti n g sy ste m , LSI w a s n o t s ig n if ic a n tly d iffe re n t b e t w e e n tr e a tm e n ts . T h e b e tte r l a r v a l p e r f o r m a n c e i n t h e C le a r-R e c irc s y s te m w a s a c c o m p a n ie d b y s ig n if ic a n tly lo w e r a v e ra g e a m m o n ia lev els (P c O .O l) a n d s lig h tly lo w e r n i t r i t e lev els (see T ab le 2).

E x p erim en t 2

O n DAH 9, la r v a l s u r v iv a l i n t h e C le a r-R e c irc s y s te m w a s s ig n if ic a n tly lo w e r (P < 0 .0 5 ) t h a n i n b o t h tr e a t­ m e n ts w i t h m ic r o -a lg a e s u p p le m e n ta tio n (A lg ae-R e­ c irc a n d G re e n -B a tc h s y s te m s ) (Table 4). O n D A H 12, s u r v iv a l i n t h e C le a r-R e c irc t r e a t m e n t w a s lo w e r (P < 0.05) t h a n i n t h e A lg a e -R e c irc sy ste m , w h e r e a s t h e G re e n -B a tc h s y s te m h a d in te r m e d ia te re su lts . T h e LSI v a lu e s o n DAH 15 s h o w a s im ila r tre n d , a lt h o u g h n o t s ig n ific a n tly d iffe re n t.

T h e a v e ra g e lev els o f a m m o n ia a n d n i t r i t e i n t h e C le a r-R e c irc a n d A lg a e -R e c irc s y s te m s w e r e sig n ifi­ c a n tly lo w e r (P < 0.01) t h a n t h o s e in t h e G r e e n -B a tc h s y s te m (see T ab le 2). I n t h e G re e n -B a tc h sy ste m , p e a k s o f a m m o n ia a n d n i t r i t e c o n c e n tr a tio n s o f 3 a n d 1 m g L " \ resp ec tiv ely , w e r e r e c o rd e d a t t h e e n d o f t h e e x p e r im e n t.

E xp erim en t 3

T ab le 5 p r e s e n ts t h e la r v a l p e r fo r m a n c e o f t h e c ra b la r v a e c u l t u r e d i n tw o r e a r in g sy ste m s. T h e s u rv iv a l r a te s a n d LSI v a lu e s o f b o th r e a r in g sy s te m s w e r e n o t sig n ific a n tly d iffe re n t. H o w ev er, t h e s u rv iv a l r a te s o n l a t e r d a y s (fro m DAH 1 2 - 2 2 ) i n t h e G re e n -R e c irc sy s­ te m te n d e d to b e h i g h e r t h a n th o s e i n t h e G re e n - B a tc h sy s te m . T h e b io filte r h a d a p o sitiv e im p a c t o n

1544

© 2007 The Authors Journal Compilation © 2007 Blackwell Publishing Ltd. Aquaculture Research, 38, 1539-1553

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A quaculture Research, 2007, 3 8 , 1539-1553 Improved rearing te c h n iq u e s for m ud crab larvae T T N g h ia e t al.

T a b le 4 E xperim ent 2: survival rates and larval stage index (LSI) values of Scylla param am osain larvae c u ltu red in three different rearin g systems

T re atm e n t

D ays a f te r hatch

3 6 9 12 15

Survival rate (%)*

C lear-Recirc 7 4 ± 12a 6 3 ± 7a 44 ± 6 b 26 ± 11b 8 ± 7 a

Algae-R ecirc 7 4 ± 12a 6 3 ± 9a 61 ± 7 a 43 ± 7 a 15 ± 8a

G reen-B atch 7 4 ± 11a 6 7 ± 9 a 5 8 ± 9 a 35 ± 9 ab 13 ± 6 a

LSI*

C lear-Recirc 1.9 ± 0 .1 a 2 ,7 ± 0 .2 a 3.9 ± 0.1a 5.0 ± 0.1a 5.1 ± 0.1b

Algae-R ecirc 2 .0 ± 0 .1 a 2 .8 ± 0.3a 4 .0 ± 0.1a 5.0 ± 0.1a 5 .6 ± 0.2a

G reen-B atch 2 .0 ± 0 .1 a 2 .8 ± 0 .2 a 4 .0 ± 0 .1 a 5 .0 ± 0.1a 5.1 ± 0.1ab

‘ Survival rates or LSI values in the same column followed by the same superscript letter are n o t statistically different (P > 0.05). For treatment descriptions, refer to Table 1.

T a b le 5 E xperim ent 3: su rv iv al rates a nd larval stage index (LSI) values o f Scylla param am osain larvae cultured in two dif­ ferent rearin g system s

D ays a fte r h a tc h

TVeatment 3 6 9 12 15 22

Survival rate (%)*

G reen-B atch 94 ± 6 a 88 x 9 a 80 ± 3 a 6 6 ± 15a 4 4 ± 2 0 a 9 ± 1a

G reen-R ecirc 9 4 ± 6 a 89 ± 8 a 8 0 ± 5a 6 8 ± 11a 5 6 ± 1 1 a 12 ± 3 a

LSI*

G reen-B atch 1.4 ± 0.3a 2 .7 ± 0 .1 a 3 .8 ± 0.4a 5 .0 ± 0 .0 a 5 .2 ± 0 .2 a ND

G reen-R ecirc 1.4 ± 0 .2 a 2 .6 ± 0 .2 a 3 .9 ± 0 .3 a 5 .0 ± 0 .0 a 5 .3 ± 0 .1 a ND ‘ Survival rates or LSI values in the same column followed by the same superscript letter are not statistically different (P > 0.05). For treatment descriptions, refer to Table 1.

ND, not determined. t h a t LSI w a s a lw a y s t h e lo w e s t i n tr e a t m e n t 3 0 r o tife rs m L ” 1 a n d t h e h ig h e s t i n tr e a t m e n t 6 0 r o tife rs m L " \

E xp erim en t 6

T a b le 8 p r e s e n ts t h e s u r v i v a l a n d d e v e lo p m e n t r a t e o f c r a b la r v a e fe d A r te m ia n a u p lii a t th r e e d e n s itie s (10, 15 a n d 2 0 m L “ :) fro m t h e Z 3 s ta g e o n w a rd s . N o sig­ n if ic a n t d iffe re n c e s w e r e o b s e rv e d b e tw e e n t h e t r e a t ­ m e n ts . T h e r e se e m e d , h o w e v e r, to b e a w e a k tr e n d to w a r d s h i g h e r s u r v i v a l a n d LSI w i t h in c r e a s in g

A rte m ia d e n s ity to w a r d s t h e e n d o f t h e tria l.

E x p erim en t 7

T ab le 9 s h o w s t h e s u r v iv a l a n d d e v e lo p m e n t r a t e to t h e m e g a lo p a s ta g e (DAH 2 2 ) o f la r v a e re c e iv in g dif­ f e r e n t p ro p h y la c tic tr e a tm e n ts . F ro m D A H 6 o n ­ w a rd s, t h e s u r v iv a l r a te o f la r v a e i n t h e t r e a tm e n t w a te r q u a lity i n t h e s e c o n d p a r t o f t h e e x p e r im e n t, w i t h s ig n if ic a n tly r e d u c e d a m m o n ia ( P < 0 .0 5 ) a n d n i t r i t e (P c O .O l) c o n c e n t r a t i o n s a s a c o n s e q u e n c e (se e T a b le 2).

E x p erim en t 4

T ab le 6 s h o w s t h e s u r v iv a l a n d d e v e lo p m e n t r a t e of c ra b l a r v a e s to c k e d a t f o u r d iffe re n t Z1 d e n s itie s (50, 100, 150 a n d 2 0 0 L - *). T h e s u r v iv a l r a te s w e r e n o t s ig n if ic a n tly d if fe r e n t a m o n g tr e a tm e n ts . O n ly o n DAH 6 w a s a n e g a tiv e c o r r e la tio n b e tw e e n LSI a n d la r v a l d e n s ity o b se rv e d .

E x p erim en t 5

T ab le 7 s h o w s t h e s u r v i v a l r a te s a n d t h e LSI v a lu e s o f c ra b l a r v a e fe d t h r e e d if fe r e n t r o tif e r d e n s itie s i n t h e Z 1 -Z 2 s ta g e s. N o s ig n if ic a n t d iffe re n c e s w e r e fo u n d fo r a n y o f t h e p a r a m e te r s . I t c a n , h o w e v e r, b e n o tic e d © 2007 The Authors

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Improved re a rin g techniques for m ud crab larvae T T N g h ia e t al. A quaculture R esearch, 2007, 38, 1539-1553

T a b le 6 E xperim ent 4: survival rates a nd larval stage index (LSI) values of Scylla paramamosain la rv a e stocked a t four differ­ e n t 21 densities (21L ~ *)

T re atm e n t

D ay s a fte r h atch

3 6 9 12 15 22

Survival ra te (%)*

50 7 9 ± 9a 5 6 ± 19a 4 2 ± 16a 31 ± 17a 2 8 ± 12a 4 ± 6 a

100 80 ± T 74 ± 6a 71 ± 10a 5 6 ± 11a 4 5 ± 8 a 5 ± 4a

150 79 ± 2 a 57 ± 12a 4 5 ± 9a 31 ± 12a 2 8 ± 10a 5 ± 1a

200 85 ± 5 a 5 3 ± 17a 42 ± 8 a 3 4 ± 3 a 3 0 ± 5 a 5 ± 1a

LSI’

50 1.7 ± 0 .2 3 3 .0 ± 0.1a 3 .9 ± 0.1a 5 .0 ± 0.1a ND ND

100 1.8 ± 0.1a 3 .0 ± 0.1ab 4 .0 ± 0.0a 5 .0 ± 0.1a ND ND

150 1.8 i 0.2a 3 .0 ± 0.1ab 3 .9 ± 0.2a 5 .0 ± 0.1a ND ND

200 1.8 ± 0.2a 2 .7 ± 0.1b 3 .7 ± 0.2a 4 .8 ± 0.1a ND ND

’ Survival rates or LSI values in the sam e colum n followed by the sam e superscript letter are not statistically different (P > 0.05). ND, n o t determ ined.

T a b le 7 E xperim ent 5: survival rates and larval stage index (LSI) values o f Scylla paramamosain larvae-fed th re e different rotifer densities (rotifers mL ~ ) from day 0 to day 6 after h atch

T re atm e n t D ays a fte r h a tc h 3 6 9 12 15 Survival rate (%)* 30 89 ± I a 5 3 ± 10a 3 0 ± 8 a 13 ± 7 a 10 ± 7 a 45 8 7 ± 6 a 5 8 ± 9 a 3 5 ± 7 a 18 ± 9 a 14 ± 8 a 60 8 7 ± 5 a 5 5 ± 7 a 32 ± 6 a 16 ± 11a 11 ± 10a LSI*

30 1.8 ± 0 .2 a 2 .7 ± 0.1a 3 .6 ± 0 .2 a 3.8 ± 0.2a 4 .0 ± 0.4a

45 1.8 ± 0 .2 a 2.8 ± 0.2a 3 .8 ± 0 .1 a 3 .9 ± 0.1a 4 .3 ± 0.5a

60 1.8 ± 0 .2 a 2 .8 ± 0.2a 3 .8 ± 0 .2 a 4 .0 ± 0.1a 4 .4 ± 0 ,5 a

’ Survival rates or LSI values in th e sam e colum n followed by the sam e superscript letter are not statistically different (P > 0.05).

u s in g a n tib io tic s w a s s ig n if ic a n tly h i g h e r t h a n th o s e i n th e r e m a i n i n g tr e a tm e n ts . T h e s u r v iv a l r a te s o f th e c o n tr o l a n d f o r m a lin t r e a t m e n t s w e r e s im ila r on m o s t d a y s. F ro m D A H 6 o n w a r d s , t h e LSI v a lu e s of t h e f o r m a lin t r e a t m e n t w e r e g e n e r a lly h i g h e r t h a n fo r t h e o t h e r t r e a t m e n t s (n o t a lw a y s sig n ific a n t). O n DAH 15 a n d 18, t h e a n tib io tic t r e a t m e n t r e s u lte d in lo w e r LSI v a lu e s (P < 0.01).

E x p erim en t 8

T able 10 p r e s e n t s t h e s u r v iv a l a t t h e Z2, Z3 a n d Z 4 s ta g e s o f l a r v a e t h a t w e r e d a ily e x p o s e d to d iffe re n t lev els o f 0 3. O n D A H 3 (Z2 sta g e ), s u r v iv a l i n th e c o n ­ tr o l a n d t h e t r e a t m e n t w i t h 0 3 in je c tio n fo r 2 m in (O zo n 2 ) w a s h i g h e r ( 6 7 -7 8 % ) c o m p a r e d w ith t h e o t h e r t r e a t m e n t s (2 4 -4 5 % ). O n D A H 6 (Z3 stage),

t h e s u r v iv a l o f t r e a t m e n t O z o n 2 b e c a m e th e h ig h e s t (52% ); h o w e v e r, th is w a s n o t s ta tis tic a lly d iffe re n t f ro m t h e c o n t r o l (25% ). O n DAH9, t h e r e w e re n o sig ­ n if ic a n t d if fe re n c e s i n s u r v iv a l i n a ll t r e a tm e n ts .

D isc u ssio n

R earin g sy s te m

R ecircu la tio n W a te r r e c i r c u la ti o n t h r o u g h a b io filte r i n th e C lear- R e c irc s y s te m p o sitiv e ly a ffe c te d la r v a l p e r fo r m a n c e c o m p a r e d w i t h m a n u a l p a r t i a l w a te r r e p la c e m e n t in t h e C le a r-B a tc h s y s te m (e x p e r im e n t 1). T h e a d v a n ­ ta g e s o f r e c i r c u la ti n g sy s te m s i n c o m m e rc ia l fish a n d c r u s t a c e a n la r v a l p r o d u c tio n h a v e b e e n p ro v e n b e fo re fo r o t h e r sp e c ie s. R e s e a r c h in to r e c irc u la tin g 1546 © 2007 The Authors Journal Compilation © 2007 Blackwell Publishing Ltd, Aquaculture Research. 38, 1539-1553

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A quaculture Research, 2007, 3 8 , 1539-1553 Improved rearin g te c h n iq u e s for m ud crab larvae T T N g h ia et al.

T a b le 8 E xperim ent 6: survival rates and larval stage index (LSI) values of Scylla paramamosain larvae-fed th re e different instar-1 Artemia densities (Artemia mL ^ 1) from 6 days a fter h atch

T re atm e n t

D ays afte r hatch

9 12 15 Survival rate (%)* 10 2 6 ± 10a 12 ± 5 a 8 ± 3“ 15 3 0 ± 6a 13 ± 7 a 10 ± 6 a 20 3 2 ± 8 a 19 ± 9a 18 ± 9 a LSI* 10 3.1 ± 0.2° 3 .7 ± 0.4a 4 .3 ± 0 .5 a 15 3.1 ± 0.1a 3.7 ± 0.2a 4 .3 ± 0 .5 a

20 3 .2 ± 0.1a 3 .8 ± 0.3a 4 .6 ± 0.3a

* Survival rates or LSI values in the same column followed by the same superscript letter are not statistically different (P > 0.05).

T a b le 9 E xperim ent 7: survival rates and larval stage index (LSI) values of Scylla paramamosain larvae treated daily w ith prophylactic chem icals

D ays a f te r h a tc h T re atm e n t 3 6 9 12 15 18 22 Survival ra te s (%)* Control 85 ± 3a 6 4 ± 4 8 ± 8 b 3 4 + 13b 28 ± 11b 1 7 ± 7 b 9 ± 5 b Formalin 8 4 ± 7 a 6 6 ± 8 b 4 7 ± 7 b 3 4 ± 12b 26 ± 8 b 13 ± 10b 11 ± 8 ab Antibiotics 91 ± 4 a 8 0 ± 2 a 7 4 ± 4 a 66 ± 8 a 52 ± 6 a 3 4 ± 3 a 21 ± 5a LSI*

Control 1.8 ± 0.1° 2 .6 ± 0 .3 a 3 .4 ± 0 .2 ab 4 .5 ± 0.3a 5.1 ± 0.1a 5 .5 ± 0 .1 b 5 .8 ± 0 .2 a Formalin 1.8 ± 0 .1 a 2 .8 ± 0 .1 a 3 ,7 ± 0 .1 a 4 .3 ± 0.3a 5.1 ± 0.1a 5 .8 ± 0 .1 a 6 .0 ± 0.1a Antibiotics 1.9 ± 0 .0 a 2 .7 ± 0 .2 a 3 .3 ± 0 .2 b 4 .3 ± 0.1a 4 .9 ± 0 .1 b 5 .3 ± 0 .2 b 5 .6 ± 0.2a "■Survival rates or LSI values in the same column followed by the same superscript letter arc not statistically different (P > 0.05).

T a b le 10 E xperim ent 8: survival rates of Scylla paramamosain larvae treated daily by ozone for different durations of tim e (min)

T re a tm e n t DAH 3 (Z2) DAH 6 (Z3) DAH 9 (Z4)

Control 7 8 ± 6a 2 5 ± 9ab 9 ± 6a

O zon2 6 7 ± 6ab 5 2 ± 14a 11 ± 10a

O zon4 4 0 ± 17abc 3 3 ± 11ab 5 ± 2 a

O zon6 4 5 ± 25abc 2 7 ± 15ab 5 ± 5 a

O zon8 31 ± 2bc 19 ± 3 b 0 ± 0 a

O zonlO 2 4 ± 15c 12 ± 9 b 0 ± 1a

Survival rates in the same column followed by the same superscript letter are not statistically different (P > 0.05).

DAH, days after hatch; Z, zoea; Control, without ozonation; Ozon2,4, 6,8 and 10, duration of ozone injection from 2 to 10 min, which is equivalent to 0.06,0.12, 0.15, 0.17 and 0.19 mg L " 1 of the residual ozone respectively.

s y s te m s h a s a ls o b e e n id e n tifie d a s a p r io rity fo r s h r im p c u l t u r e ( L a w re n c e & L ee 1997). I n th e s e sy s ­ te m s , w a t e r e x c h a n g e is m in im iz e d th r o u g h t h e u s e o f b io lo g ic a l, c h e m i c a l a n d / o r m e c h a n ic a l f iltra tio n to m a i n t a i n g o o d w a t e r q u a lity c o n tin u o u s ly . A s th e y p r o v id e le s s s t r e s s a n d c o n f e r c o n s t a n t g o o d w a te r q u a lity to t h e la r v a e , th e s e s y s te m s a r e a b le to m a in ­ t a i n a h i g h b io lo g ic a l c a r r y in g c a p a c ity i n re la tiv e ly little s p a c e (Q u ille re , M a rie, R o u x , G o sse & M o ro t- g a u d r y 1993; T w a ro w s k a , W e s te rm a n & L o so rd o 1997). F o r c r a b la r v ic u ltu r e , r e c ir c u la tin g sy s te m s a ls o a p p e a r to w a r r a n t f u r t h e r in v e s tig a tio n i n o r d e r to d e c r e a s e la b o u r r e q u ire m e n ts a n d s e a w a te r c o n ­ s u m p tio n , p r o v id in g a m o r e s ta b le c u l t u r e m e d iu m © 2007 The Authors

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Im proved rearin g techniques for m ud crab larvae T T N g h ia e t al. A quaculture R esearch. 2007, 3 8 , 1539-1553

a n d t h u s r e d u c in g la r v a l stre s s. If t h e s y s te m d e s ig n is k e p t sim p le, r e c ir c u la tin g s y s te m s c o u ld a ls o b e s u i ­ ta b le f o r la r g e -s c a le p ro d u c tio n . R o le o f su p p le m e n te d m icro-algae T h e a d d itio n o f m ic r o -a lg a e to t h e r e c ir c u la tio n s y s te m s r e s u lte d i n b o th h i g h e r s u r v i v a l a n d fa ste r d e v e lo p m e n t i n th is stu d y . M ic ro - a lg a e h a v e b e e n p r o v e n to b e b e n e fic ia l by v a r io u s m o d e s o f a c tio n . T h e y c o u ld h e lp m a in ta in t h e q u a lity o f live fo o d . As i n t h e c u l t u r e o f m a r in e fis h la rv a e , u n c o n s u m e d r o tif e r s m a y r e sid e i n t h e t a n k s fo r s e v e ra l d a y s a n d t h e i r n u t r i t i o n a l v a lu e m a y b e c o m e s e v e re ly re d u c e d (M a k rid is & O lse n 1999). F u r th e r m o r e , a c c o r d in g to t h e s e a u th o r s , p o o rly fed r o tife rs w e r e m o r e s e n sitiv e to s t a r v a t i o n t h a n w ell-fed ro tife rs, a s t h e i r n itr o g e n c o n t e n t d e c r e a s e d a t a h i g h e r ra te .

M ic ro - a lg a e a ls o p lay a n im p o r t a n t ro le i n s ta b iliz ­ i n g w a t e r q u a lity v ia e ith e r a m m o n ia u p t a k e o r oxy­ g e n p r o d u c t i o n (Tseng, H u a n g & L iao 1991). B e c a u s e t h e C le a r-R e c irc s y s te m a lr e a d y p r o v id e d o p tim a l w a t e r q u a lity , i t is u n lik e ly t h a t t h e s ta b iliz in g effect o n w a t e r q u a lity is re sp o n s ib le f o r t h e im p ro v e d p e r­ f o r m a n c e i n t h e a lg a e - s u p p le m e n te d s y s te m . In b a t c h c u l t u r e sy ste m s, t h i s e ffe c t w o u ld p r o b a b ly be m u c h m o r e p r o n o u n c e d . A d ir e c t c o m p a r is o n b e t w e e n a g r e e n a n d c le a r w a t e r b a t c h s y s te m w as, h o w e v e r, n o t m a d e in th is stu d y . I n a s t u d y o n t h e effect o f Chlorella o n t h e p o p u la ­ t i o n o f lu m i n o u s b a c te r ia Vibrio h a rv e y i, n o l u m i n o u s b a c t e r i a w e r e re c o v e re d o n d a y s 2 a n d 3 i n flask s w i t h Chlorella, w h ile th o s e w i t h o u t t h e m ic r o -a lg a e still h a r b o u r e d lu m in o u s b a c te r ia a t d a y 3 (T e n d e n c ia & d e la P e n a 2 003). Also, th e d ia to m Chaetoceros h a s b e e n s h o w n to p r o d u c e n a t u r a l a n tib io tic s a n d h ig h c o n c e n tr a tio n s o f th is m a r in e d ia to m w ill e lim in a te

Vibrio v u ln ific u s a n d o th e r p a th o g e n ic b a c te r ia , w h ic h c o n tr ib u te to t h e p ro p a g a tio n o f v ir u s e s i n t h e s h r im p p r o d u c tio n e n v ir o n m e n t (W a n g 2003). I n c o n c lu s io n , m ic r o -a lg a e i n m u d c r a b l a r v a l r e a r ­ i n g m a y p la y a ro le i n im p ro v in g a n d m a i n t a i n i n g live fo o d q u a l i t y a n d c o n tr o llin g b a c t e r i a levels. Choice o f s y s te m I n e x p e r i m e n t 3. t h e G re e n -R e c irc s y s te m ( w h ic h is a c o m b i n a t i o n o f a G re e n -B a tc h s y s te m d u r i n g t h e r o ­ tif e r f e e d in g s ta g e a n d a A lg a e -R e c irc s y s te m t h e r e ­ a fte r) s e e m e d to b e b e tte r t h a n t h e G re e n - B a tc h s y s te m . T h e G re e n -B a tc h s y s te m s e e m s t o b e m o re a p p r o p r i a t e fo r e a r ly s ta g e s o f c r a b l a r v a e (Z 1 -Z 2 ) a s it is le s s s tr e s s f u l fo r t h e e a r ly z o e a e a n d e a s i e r t o g r a ­ d u a lly fill u p t h e t a n k s w i t h f r e s h s e a w a te r, a lg a e a n d ro tif e r s t h a n f lu s h in g o u t old r o t i f e r s i n t h e r e c i r c u la ­ tio n sy ste m . I n th e r e c i r c u la ti n g sy s te m , t h e y o u n g la r v a e m a y b e p ro n e to p h y s i c a l d a m a g e a n d m a y s p e n d c o n s id e ra b le e n e r g y t r y i n g t o s w im u p a g a i n s t t h e c u r r e n t. E a rly c ra b l a r v a e a r e d e lic a te d u e to t h e i r s m a ll siz e a n d th e t h r e e lo n g s p i n e s o n t h e c a r a p a c e t h a t a r e e a s ily d a m a g e d iv h e n t h e y a r e e n tr a p p e d o n t h e m e s h s c re e n d u r in g f l u s h in g o u t o f u n e a t e n fe e d i n t h e r e c irc u la tio n s y s te m (D a v is 2 003). T h e n u t r i ­ tio n a l effe ct of m ic r o -a lg a e is p r o b a b l y a ls o m o r e p r o ­ n o u n c e d d u r in g t h e r o tif e r f e e d in g s ta g e t h a n d u r i n g t h e A rte m ia fe e d in g sta g e . F u r t h e r m o r e , it is n o t n e ­ c e s s a r y to r e c irc u la te w a t e r d u r i n g th e s e firs t d ay s, a s t h e c o n c e n tr a tio n s o f a m m o n i a a n d n i t r i t e a r e s till low. U sin g t h e A lg a e -R e c irc s y s t e m i n l a t e r s ta g e s is m o re fa v o u ra b le fo r r e d u c in g t h e i n c r e a s i n g a m m o ­ n ia a n d n i t r i t e c o n c e n tr a tio n s a s m o r e w a s te m a t e r i ­ a l is p ro d u c e d by t h e c ra b la r v a e . M o re o v e r, a s t h e la r v a e d ev elo p in to m o re e ffic ie n t p r e d a to r s , fe e d is c o n s u m e d fa ste r, a n d m a i n t e n a n c e o f o p tim a l fe e d q u a lity is less o f a n issu e . M a n y s t u d i e s s u c c e s s fu lly a p p lie d a s im ila r c o m b in e d r e a r i n g te c h n i q u e d u e to its b e n e fit fo r t h e la rv a e a n d c o n v e n ie n c e fo r m a n ­ a g e m e n t. p a r tic u la r ly fo r la r g e r e a r i n g c o n ta in e r s . U n d e r g r e e n - w a te r c u l t u r e c o n d itio n s , w a t e r is n o t e x c h a n g e d fo r th e first 3 d ay s. T h e re a fte r , w a t e r e x ­ c h a n g e is slo w ly in c r e a s e d fro m 1 0 - 2 0 % d a y “ 1 fo r Z 2 -Z 3 to b e tw e e n 4 0 % a n d 50 % d a y ~ 1 a t t h e e n d o f t h e r e a r in g c y cle (Z 4 -M ) (M a n n , A s a k a w a & P iz z u to 1999; Q u in itio . P a ra d o -E ste p a , M illa m e n a . R o d rig u e z & B o r lo n g a n 2001). I n J a p a n , a m e s o c o s m s y s te m is u s e d fo r c u l t u r in g la r v a e i n l a r g e r t a n k s ( > 10 m 3). T h e t a n k s a r e p a r tia lly filled w i t h g r e e n w a t e r a t Z1 ( 2 0 -2 5 % v olum e), t a n k s a r e t h e n fille d u p w i t h c le a n s e a w a te r d u r in g t h e c o u r s e o f t h e Z 2 - Z 3 s ta g e s a n d d u r in g t h e Z 4 a n d M s ta g e s w a te r is e x c h a n g e d o n a flo w -to -w a ste b a s is (H a m a sa k i, S u p ra y u d i & T a k e u c h i 2002).

O ther rea rin g te c h n iq u e s

ZI sto c k in g d e n sity N o s ig n if ic a n t effect o f la r v a l d e n s ity w a s o b s e rv e d fro m 50 to 2 0 0 Z 1 L - 1 . T h is w o u ld s u g g e s t t h a t th e l a r v a e c a n b e g r o w n a t 2 0 0 Z l L ~ \ V a ria tio n i n t h e fin a l s u r v iv a l b e tw e e n re p lic a te t a n k s a ls o s e e m e d to d e c r e a s e a t h ig h e r d e n s itie s . F o r S. p a ra m a m o sa in , D ju n a id a h , M a rd jo n o , W ille. K o n ta r a a n d S o rg e lo o s (2001) f o u n d a te n d e n c y o f i n c r e a s e d s u r v iv a l to Z5 a s a f u n c tio n o f t h e Z l s to c k in g d e n s ity (i.e. s u r v iv a l

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A quaculture Research. 2007, 3 8 , 1539-1553 Improved rearin g te c h n iq u e s for m ud crab larvae T T N g h ia e t al.

r a te s o f 2 7 % , 3 9% a n d 6 3% b e in g o b ta in e d a t d e n s i­ tie s o f 5 0 ,7 5 a n d 1 0 0 Z l L “ 1 resp ec tiv ely ). B a y lo n a n d F a ila m a n (1999) a ls o r e p o r te d h i g h e r s u r v iv a l a n d m e t a m o r p h o s is o f S cylla se rra ta a t 5 0 Z 1 L - 1 c o m ­ p a r e d w i t h lo w e r d e n s itie s o f 10 a n d 25 Z l L ~ x. I n ­ c r e a s e d s u r v iv a l a t h i g h e r la r v a l d e n s itie s s o m e h o w s e e m s c o n tra d ic to ry . H o w ev er, in d ire c tly , fo o d r a t i o n m ig h t b e re sp o n s ib le . E x c e ss fo o d in t r e a t m e n t s w i t h lo w la r v a l d e n s itie s m a y p o llu te t h e w a te r a n d m a y t h u s c a u s e m o rta lity . I n o u r stu d y , w e n o te d h ig h e r c o n c e n tr a tio n s o f a m m o n ia a n d n i tr ite i n t h e t r e a t ­ m e n t h a v in g 5 0 Z 1 L - 1 (se e T a b le 2). F o r t h e h ig h e s t s to c k in g d e n s itie s te s te d i n o u r s tu d y (2 0 0 Z l L ~ x), t h e la r v a l d e v e lo p m e n t r a te s e e m e d slig h tly im p a ire d . T h is h i g h s to c k in g d e n s ity m a y h a v e c a u s e d c o m p e ti­ t i o n fo r feed, r e s u l t i n g in s lo w e r d e v e lo p m e n t. T h e r e ­ fo re, Z l s to c k in g d e n s itie s i n th e r a n g e o f 1 0 0 - 1 5 0 Z l L “ 1 m ig h t b e o p tim a l.

R o tife r d e n s ity f o r fee d in g early la rv a l stages (Z 1 -Z 2 stages)

A l t h o u g h t h e r e w a s a t r e n d t o w a r d s in c r e a s e d s u r v i­ v a l a n d g r o w t h w i t h in c r e a s in g r o tif e r d e n sity , n o sig ­ n if ic a n t d iffe re n c e s i n l a r v a l s u r v iv a l o r g r o w t h w e re f o u n d b e tw e e n th e d iffe re n t r o tif e r d e n s itie s te s te d . A l t h o u g h n o t sig n ific a n t, t h e h ig h e s t s u r v iv a l w a s g e n e r a lly o b s e rv e d a t 4 5 r o tif e r s m L - \ w h ile a d e n ­ s ity o f 6 0 r o tife rs m L ~ 1 r e s u lte d i n th e f a s te s t la rv a l d e v e lo p m e n t. T h e d iffe re n c e s w e re , h o w ev er, n o t v e ry m a rk e d , a n d m o r e o v e r s u c h h i g h fe e d in g r a te s m ig h t b e e c o n o m ic a lly u n r e a lis tic . W e c a n th e r e fo r e c o n ­ c lu d e t h a t f e e d in g 3 0 r o tife rs m L - 1 is e n o u g h fo r o p ­ t i m a l la r v a l p e r fo r m a n c e . I n p ra c tic e , h o w e v e r, th e i n te r m e d ia te d e n s ity o f 4 5 r o tif e r s m L _ 1 w a s fre ­ q u e n t l y u s e d f o r fe e d in g e a r ly la r v a l sta g e s. O th e r s t u d ie s in d ic a te d t h a t h i g h ro tife r d e n s itie s ( 3 0 - 8 0 m L - x ) a r e r e q u ir e d fo r o p tim a l g r o w t h a n d s u r v iv a l o f S. p a ra m a m o sa in (D ju n a id a h , M a rd jo n o , L a v e n s & W ille 1998; Z e n g & Li 1999) a n d S. serrata

(S u p ra y u d i, T a k e u c h i, H a m a s a k i & H iro k a w a 2 0 0 2 ). F o r S. p a ra m a m o sa in la rv a e , fe e d in g 3 0 a n d 6 0 r o tife rs m L ~ 1 r e s u lte d i n a sig n ific a n tly h ig h e r s u r v i v a l c o m p a r e d w i t h fe e d in g o n ly 15 r o tif e r s m L “ 1 ( D ju n a id a h e t al. 2001). T h e s e a u t h o r s f o u n d t h a t t h e in d iv id u a l d r y w e ig h t o f Z 5- fe d 15 r o tif e r s m L ~ 1 w a s s ig n ific a n tly lo w e r t h a n t h o s e o f Z 5 fe d w i t h h i g h e r r o tif e r d e n s itie s . P r a c t i ­ cally , fe e d in g 3 0 r o tife rs m L " 1 a t Z l a n d in c r e a s in g g r a d u a l l y t o 4 5 m L " 1 a t Z 2 p ro v e d to b e su ffic ie n t f o r a s to c k in g d e n s ity o f 1 0 0 Z l L ~ 1 in o u r tr ia ls i n la r ­ g e r r e a r in g t a n k s ( 5 0 0 - 1 0 0 0 L). I n c r e a s in g t h e ra ­ tio n b y la r v a l s t a g e s i n t h is w a y c o m p e n s a te s fo r t h e i n c r e a s e d i n g e s t i o n o f c ra b l a r v a e a s t h e y g ro w (B ay lo n , B ravo & M a n ig o 2 0 0 4 ). F o r e a r ly la rv a e , h o w e v e r, fo o d a m o u n t c a n n o t b e r e d u c e d to th e ir m a x im u m i n g e s t i o n p o te n tia l a s t h e y a r e q u ite in effi­ c ie n t p r e d a to r s a n d th e r e fo r e m i g h t r e q u ir e a m in i­ m a l d e n s ity t o m a x i m i z e e n c o u n te r .

S im ila r to o u r s t u d y m o s t s tu d ie s i n v e s tig a tin g t h e effe ct o f r o tif e r d e n s i t y a d d e d t h e live fo o d i n o n e s in ­ g le r a tio n . U n d e r t h e s e c ir c u m s ta n c e s , th e o r e t i c d e n ­ sitie s a r e o n ly a t t a i n e d u p o n fe e d in g a n d g r a d u a lly d e c r e a s e a s l a r v a e c o n s u m e t h e prey. O p tim a l live fo o d q u a n titie s c a n n o t , h o w e v e r, b e s e p a r a te d fro m fe e d in g fre q u e n c y . B e c a u s e z o e a la r v a e c a n c o n s u m e t h e i r o p tim a l r a t i o n w i t h i n l h , G e n o d e p a , S o u th g a te a n d Z en g (2 0 0 4 ) s u g g e s te d t h a t th e y c a n b e fe d o n c e a day. B e c a u s e o f t h e se v e re r e d u c tio n i n t h e n u t r i ­ tio n a l v a lu e o f r o tif e r s w i t h lo n g e r r e t e n t i o n tim e s i n r e a r in g c o n t a i n e r s (M a k rid is & O lse n 1999) a n d th e f a c t th e r e is a m i n i m u m p re y d e n s ity n e e d e d fo r th e p a ssiv e f e e d in g b e h a v i o u r o f z o e a la r v a e ( H e a s m a n & F ie ld e r 1983; Z e n g & L i 1999), t h e i n t e r a c t i o n b e tw e e n t h e o p tim a l r a tio n a n d fe e d in g f re q u e n c y s h o u ld be f u r t h e r in v e s tig a te d . A r te m ia f o r fe e d in g la te r la rv a l sta g e s (from Z 3 onw ards) W e f o u n d n o d iffe re n c e b e tw e e n fe e d in g Z 3 a d a ily fe e d r a t i o n o f 10, 15 o r 2 0 A rte m ia n a u p lii m L - 1 . E sp e c ia lly in la t e r la r v a l s ta g e s (Z 4 -Z 5 ), th e r e w a s, h o w e v e r, a te n d e n c y to w a r d s h i g h e r s u r v iv a l w i t h in c r e a s in g ra tio n . I n t h i s re s p e c t, it m i g h t b e b e n e fi­ c ia l to in c r e a s e t h e A r te m ia d e n s ity b y c r a b s ta g e f ro m 10 to 15 m L- 1 . H ig h liv e fe e d d e n s itie s w o u ld i n ­ c r e a s e t h e c h a n c e fo r e a r ly la r v a e to e n c o u n t e r a n d c a p t u r e fe e d o r g a n is m s (Z e n g & Li 1 9 9 9 ) a n d t h e r e ­ fo re w o u ld im p ro v e t h e la r v a l p e r fo r m a n c e (B rick 1974; H e a s m a n & F ie ld e r 1983; Q u in itio e t al. 2001). O n t h e o t h e r h a n d , o ld e r la r v a l h a v e a h i g h e r i n g e s ­ tio n c a p a c ity . O p tim a l r a tio n s s h o u ld th e r e fo r e b e d e ­ te r m in e d fo r e a c h la r v a l s ta g e se p a ra te ly . I n t h is re s p e c t, s tu d ie s o n in d iv id u a l la r v a e a r e v e r y u s e fu l to d e te r m in e p r e y c o n s u m p tio n . A c c o rd in g to o u r p re v io u s e x p e r im e n ts (N g h ia 2 0 0 4 ), e a c h Z3, Z4, Z 5 a n d m e g a lo p a la r v a w a s c a p a b le o f c o n s u m in g o n a v e ra g e 15. 25, 37 a n d 114 n e w ly h a t c h e d

A rte m ia d a y - 1 resp ec tiv ely . T h e re fo re , a t a s to c k in g o f 1 0 0 l a r v a e L - \ t h e d a ily A rte m ia fe e d in g d e n s itie s th e o r e tic a lly s h o u ld b e a t le a s t 1.5, 2.5, 3.7 a n d 11.4 m L ~ 1 fo r Z3, Z4, Z5 a n d m e g a lo p a s ta g e s r e s p e c ­ tively. F o r Z l, Z 2 a n d Z 3 s ta g e s o f S. serrata, t h e

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