Critical Review MYSID CRUSTACEANS AS POTENTIAL TEST ORGANISMS FOR THE EVALUATION OF ENVIRONMENTAL ENDOCRINE DISRUPTION: A REVIEW

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1 Coi Nb I^HIpRESSj Environm ental Toxicology and Chem istry, Vol. 23, No. 5, pp , 2004 P rinted in ihc USA /0 4 $ Critical Review MYSID CRUSTACEANS AS POTENTIAL TEST ORGANISMS FOR THE EVALUATION OF ENVIRONMENTAL ENDOCRINE DISRUPTION: A REVIEW T im A. V e r s l y c k e,*! N a n c y F o c k e d e y,j C h a r l e s L. M c K e n n e y, J r., S t e p h e n D. R o a s t,[ M a l c o l m B. Jo n e s, J a n M e e s,# and C o l in R. J a n s s e n i Laboratory o f E nvironm ental Toxicology and A quatic Ecology, G hent U niversity, J. Plateaustraat 22, B G hent, B elgium JD epartm ent o f B iology, M arine Biology Section, G hent U niversity, K rijg sk an 281-S8, B G hent, B elgium U.S. Environm ental Protection Agency, N ational H ealth and Environm ental Effects Research Laboratory, G ulf Ecology Division, 1 Sabine Island D rive. G u lf B reeze, Florida , U SA HSchool o f B iological Sciences, U niversity o f Plym outh, D rake Circus, Devon PL4 BAA, U nited K ingdom #Flanders M arine Institute, Vismijn Pakhuizen 45-52, B Ostend, Belgium (R eceived 20 June 2003; Accepted 29 Septem ber 2003) A bstract A nthropogenic chem icals that disrupt the horm onal system s (endocrine disruptors) o f w ildlife species recently have becom e a w idely investigated and politically charged issue. Invertebrates account for roughly 95% o f all anim als, y et surprisingly little effort has been m ade to understand their value in signaling potential environm ental endocrine disruption. This om ission largely can be attributed to the high diversity o f invertebrates and the shortage o f fundam ental know ledge o f th eir endocrine system s. Insects and crustaceans are exceptions and, as such, appear to be excellent candidates for evaluating the environm ental consequences o f chem ically induced endocrine disruption. M ysid shrim p (C rustacea: M ysidacea) m ay serve as a viable surrogate for many crustaceans and have been p u t forw ard as suitable test organism s for the evaluation o f endocrine disruption by several researchers and regulatory bodies (e.g., the U.S. Environm ental Protection A gency). D espite the long-standing use o f m ysids in toxicity testing, little inform ation exists on their endocrinology, and few studies have focused on the potential o f these anim als for evaluating the effects o f horm one-disrupting com pounds. T herefore, the question rem ains as to w hether the current standardized m ysid endpoints can be used or adapted to detect endocrine disruption, or if new procedures m ust be developed, specifically directed at evaluating horm one-regulated endpoints in these anim als. This review sum m arizes the ecological im portance o f m ysids in estuarinc and m arine ecosystem s, their use in toxicity testing and environm ental m onitoring, and their endocrinology and im portant horm one-regulated processes to highlight their potential use in assessing environmental endocrine disruption. K eyw ords M ysids Endocrine disruption Biom arkers Regulation Environm ental monitoring IN T R O D U C T IO N A n th ro p o g e n ic c h e m ic a ls th a t d is ru p t th e h o rm o n a l s y ste m s (e n d o c rin e d is ru p to rs ) o f w ild lif e s p e c ie s re c e n tly h a v e b e c o m e a w id e ly in v e s tig a te d a n d p o litic a lly c h a rg e d is s u e [ 1-3 ], In v e rte b ra te s a c c o u n t fo r ro u g h ly 9 5 % o f a ll a n im a ls [4], y e t s u rp ris in g ly little e ffo rt h a s b e e n in v e s te d to u n d e rs ta n d th e ir v a lu e in s ig n a lin g p o te n tia l e n v iro n m e n ta l e n d o c rin e d is ru p tio n [ ], A lth o u g h g ro w th, re p ro d u c tio n, d e v e lo p m e n t, a n d o th e r a s p e c ts o f in v e rte b ra te p h y s io lo g y a re k n o w n to b e u n d e r h o rm o n a l c o n tro l, th e e n d o c rin e s y s te m s a n d h o rm o n e s p ro d u c e d a n d u s e d in in v e rte b ra te s a re n o t d ire c tly a n a lo g o u s to th o s e o f v e rte b ra te s [1 3 ]. In in v e rte b ra te s, th e s e le c tio n o f s u ita b le te s t m e th o d s a n d s p e c ie s fo r e v a lu a tin g e n d o c rin e d is ru p tio n is c o n fo u n d e d b y d iv e rs ity. T h e u s e o f a lim ite d n u m b e r o f s p e c ie s a s re p re s e n ta tiv e o f th is d iv e rs ity is a n a iv e a p p ro a c h d e s tin e d to fa ilu r e in th e a b s e n c e o f s u ita b le s a fe g u a rd s [2,9 ]. H e n c e, th e k e y c h a lle n g e fo r e n v iro n m e n ta l a s s e s s m e n t is to fin d in v e rte b ra te s p e c ie s, s e le c te d fro m m u ltip le le v e ls o f e c o s y s te m fu n c tio n, to e ffic ie n tly m o n ito r a n d e v a lu a te th e c o m p le x ity o f p o te n tia l e n v iro n m e n ta l e ffe c ts o f e n d o c r in e -d is ru p tin g c h e m ic a ls a t a re a s o n a b le fin a n c ia l c o s t [14]. M a n y a n th ro p o g e n ic p o llu ta n ts h a v e th e w o r ld s o c e a n s a n d s e a s as a fin a l s in k, a n d a re c a r rie d th e re th ro u g h riv e rin e and e s tu a rin e c o n d u its [1 2,1 5 ], E s tu a rie s a re in tr in s ic a lly a n d c o m m e rc ia lly im p o r ta n t e c o s y s te m s a n d a re a m o n g s t th e first r e * To w hom correspondence may be addressed (tirri.verslycke(g)ugent.be). c ip ie n ts o f e n d o c rin e d is ru p to rs in th e ir s e a w a rd tra n s p o rt. O f th e e s tu a rin e o rg a n is m s th a t c o u ld b e a d v e rse ly a ffe c te d b y th e s e c o m p o u n d s, c ru s ta c e a n s a re g o o d c a n d id a te s fo r th e s tu d y o f p o te n tia l im p a c ts. C ru s ta c e a n s a re c o m m o n in fre s h w a ter, e s tu a rie s, a n d s h a llo w c o a s ta l w a te r s a n d fo rm v ita l lin k s in a q u a tic f o o d w e b s [ ]. In a d d itio n, c ru s ta c e a n s are s u s c e p tib le to th e e ffe c ts o f e n d o c r in e d is ru p to rs [1 3 ]. A n in te rn a tio n a l S o c ie ty o f E n v iro n m e n ta l T o x ic o lo g y a n d C h e m is try w o rk s h o p o n e n d o c rin e d is ru p tio n in in v e rte b ra te s h e ld in T h e N e th e r la n d s in 1998 [7] id e n tifie d in s e c ts a n d c ru s ta c e a n s as p o te n tia l o rg a n is m s fo r e v a lu a tin g c h e m ic a lly in d u c e d e n d o c rin e d is ru p tio n b y v irtu e o f th e w e a lth o f in fo rm a tio n a v a ila b le o n th e ir e n d o c r in o lo g y c o m p a re d w ith o th e r in v e r te b ra te s [ 9,1 2, ]. O f th e c ru s ta c e a n s, m y s id s h rim p h a v e b e e n p u t fo rw a rd as s u ita b le te s t o rg a n is m s fo r th e e v a lu a tio n o f e n d o c rin e d is ru p tio n [7,9,2 5 ]. T h e U.S. E n v iro n m e n ta l P ro te c tio n A g e n cy e s ta b lis h e d th e E n d o c rin e D is ru p to r S c re e n in g a n d T e stin g S ta n d a rd iz a tio n a n d V a lid a tio n T a s k F o r c e to c o o rd in a te and c o n d u c t th e s c ie n tific a n d te c h n ic a l w o rk n e c e s s a ry to v a lid a te th e s c r e e n s a n d te s ts re c o m m e n d e d b y th e E n d o c rin e D is ru p to r S c re e n in g a n d T e s tin g C o m m itte e. T h e S ta n d a rd iz a tio n and V a lid a tio n T ask F o r c e re c o m m e n d e d a tw o -tie r e d a p p ro a c h fo r d e te rm in in g w h e th e r a c h e m ic a l is a n e n d o c rin e d is ru p to r, a n d m y s id s w e re p ro p o s e d a s a s u ita b le in v e rte b ra te a ss a y in th e tie r 2 te s tin g (in v iv o te s tin g ) (h ttp ://w w w.e p a.g o v /s c ip o ly / o s c p e n d o ) fo r a tw o -g e n e ra tio n r e p ro d u c tiv e a n d d e v e lo p m e n ta l to x ic ity test. R e c e n tly, a d ra ft re v ie w p a p e r w a s c o m 1219

2 1220 Environ. Toxicol. Cheni. 23, 2004 T.A. Verslycke et al. p ile d o n m y s id lif e - c y c le to x ic ity te s tin g [1 3 ], a n d th e tw o - g e n e ra tio n m y s id lif e - c y c le a ss a y w a s p ro p o s e d to th e O rg a n iz a tio n fo r E c o n o m ic C o o p e r a tio n a n d D e v e lo p m e n t as a n e w O rg a n iz a tio n fo r E c o n o m ic C o o p e r a tio n a n d D e v e lo p m e n t te st g u id e lin e. D e s p ite th e lo n g -s ta n d in g u s e o f m y s id s in to x ic ity te s tin g, little in fo rm a tio n o n th e ir e n d o c rin o lo g y h a s b e e n p u b lis h e d a n d fe w s tu d ie s h a v e fo c u s e d o n th e p o te n tia l o f th e se a n im a ls fo r e v a lu a tin g th e e ffe c ts o f h o rm o n e -d is ru p tin g c o m p o u n d s. T h e re fo r e, th e q u e s tio n re m a in s as to w h e th e r th e c u rre n t s ta n d a rd iz e d m y s id e n d p o in ts can b e u s e d o r a d a p te d to d e te c t e n d o c rin e d is ru p tio n, o r i f n e w p ro c e d u re s m u s t be d e v e lo p e d, s p e c ific a lly d ire c te d a t e v a lu a tin g h o rm o n e -r e g u la te d e n d p o in ts in th e s e a n im a ls. T h is p re s e n t re v ie w p ro v id e s an o v e rv ie w o f th e a v a ila b le in fo rm a tio n o n m y s id s r e le v a n t to th e is s u e o f e n d o c rin e d is ru p tio n, in c lu d in g th e ir e c o lo g ic a l r o le in m a rin e a n d e s tu a rin e e c o s y s te m s, th e ir u s e in to x ic ity te s tin g a n d e n v iro n m e n ta l m o n ito rin g, a n d th e ir e n d o c rin o lo g y. A c a s e is m a d e fo r th e ir p o te n tia l u s e in a s s e s s in g th e e n v iro n m e n ta l c o n s e q u e n c e s o f e n d o c r in e -d is ru p tin g c h e m ic a ls. M YSID BIO LO G Y AND ECOLOGY M y s id s (M a la c o s tra c a : P e ra c a rid a : M y s id a c e a ) a re re la tiv e ly s m a ll (w ith th e m a jo rity o f th e s p e c ie s b e in g b e tw e e n 5 a n d 2 5 m m in le n g th ), s h rim p lik e c ru s ta c e a n s, o fte n re fe rre d to as o p o s su m s h rim p b e c a u s e th e o o s te g ite s fo rm a v e n tra l f e m a le m a rs u p iu m fo r c a r ry in g th e d e v e lo p in g e m b ry o s. T h e la tte r fe a tu re d is tin g u is h e s m y s id s fro m o th e r s h rim p lik e c ru s ta c e a n s. M y s id s a re id e n tifie d fr o m o th e r p e ra c a rid s (A m p h i p o d a, Is o p o d a, C u m a c e a, a n d T a n a id a c e a ) b y th e p re s e n c e o f a s ta to c y s t (c o n ta in in g la rg e e n d o g e n o u s s ta to lith s, th e p rim a ry e q u ilib riu m o rg a n s fo r m y s id s ) o n th e p ro x im a l p a r t o f th e u ro p o d a! e n d o p o d. M y s id s a re d is trib u te d fro m 8 0 N to 80 S a n d o c c u r in v a rio u s a q u a tic e n v iro n m e n ts, in c lu d in g fre s h w a te r, g ro u n d w a te r, b ra c k is h, e s tu a rin e, c o a s ta l, a n d o c e a n ic h a b ita ts [ ]. M a u c h lin e a n d M u ra n o [2 8 ] p u b lis h e d a w o rld lis t o f m y s id s in 1977 (7 6 5 s p e c ie s d is trib u te d b e tw e e n 120 g e n e ra ); h o w e v e r, th is n u m b e r is e v e r in c re a s in g th ro u g h im p ro v e d s a m p lin g te c h n iq u e s a n d e x p lo ra tio n o f n e w h a b ita ts. T h e p re s e n t c o u n t is m o re th a n 1,000 s p e c ie s b e lo n g in g to a p p ro x im a te ly 160 g e n e r a (h ttp ://c r u s ta c e a.n e t/). A c o m p re h e n s iv e d a ta b a s e on th e w o rld m y s id fa u n a (N e m y s, h ttp ://in tra m a r.u g e n t.b e /n e m y s ), c o n ta in in g lin k s to re le v a n t in fo rm a tio n (i.e., ta x o n o m ic a l, m o rp h o lo g ic a l, e c o lo g ic a l, b io g e o g ra p h ic, lite ra tu re, p ic to ria l, a n d m o le c u la r in fo rm a tio n ), o n th e s p e c ie s le v e l p re s e n tly is b e in g c o n s tr u c te d (T. D e p re z, G h e n t U n iv e rs ity, S e c tio n M a r in e B io lo g y, G h e n t, B e lg iu m, p e rs o n a l c o m m u n ic a tio n ). In g e n e ra l, m y s id s a re re g a rd e d as o m n iv o re s a n d fe e d on p h y to p la n k to n, Z o o p la n k to n, a n d o rg a n ic d e tritu s [ 2 6,2 7, ]. P e la g ic fo rm s filte r p a rtic le s d u rin g s w im m in g, w h e re a s b e n th ic s p e c ie s h a v e b e e n o b s e r v e d a c tiv e ly h u n tin g a n d g ra b b in g s m a ll p a rtic le s [2 7 ], M y s id s fo rm im p o r ta n t lin k s in th e fo o d w e b s o f a q u a tic e c o s y s te m s a n d o fte n fe e d s e le c tiv e ly fo r s iz e o r s p e c ie s (o r b o th ) o f p re y [2 6,3 2 ], C o n se q u e n tly, th e y h a v e th e p o te n tia l fo r s tru c tu rin g Z o o p la n k to n c o m m u n itie s [3 3,3 4 ] a n d in flu e n c in g th e s tru c tu re o f p h y to p la n k to n, ty c h o p la n k to n, a n d m e io fa u n a l c o m m u n itie s [ 3 2, ]. M o st m y s id s u tiliz e o rg a n ic d e tritu s to a c o n s id e ra b le e x te n t a n d are c a p a b le o f r e m in e ra liz in g a s u b s ta n tia l p o rtio n o f th e n o n re fra c to ry d e tritu s s u s p e n d e d in th e w a te r c o lu m n o r b u rie d in th e s u rfa c e s e d im e n ts [2 9,3 7,4 2,4 3 ]. M y sid s iz e is in te rm e d ia te b e tw e e n m e s o z o o p la n k to n ic ( p m ) a n d e n d o b e n th is o r e p i- b e n th ic (c m ) p r e y ite m s, a n d m y s id s o fte n p ro g re s s iv e ly r e p la c e c o p e p o d s in th e d ie t o f m a n y p o s tla rv a l a n d ju v e n ile c o m m e rc ia l fish s p e c ie s [1 9,2 6,4 4,4 5 ], In a d d itio n, m y s id s m a y s e r v e as p re y fo r la rg e r c ru s ta c e a n s, m a rin e m a m m a ls, o r w a d in g b ir d s [2 6,2 7,3 2, ]. E s tu a rin e m y s id s h a v e a fle x ib le p h y s io lo g y th a t re s p o n d s to a h o s t o f d y n a m ic a lly c h a n g in g e n v iro n m e n ta l v a ria b le s, c h a ra c te ris tic o f th e c o m p le x c h e m is tr y o f e stu a rie s. T e m p e r a tu re a n d s a lin ity a re th e d o m in a n t e c o lo g ic a l v a ria b le s, a n d m a y a c t e ith e r s in g ly o r in c o m b in a tio n to m o d if y th e p h y s io lo g ic a l a n d e c o lo g ic a l p ro p e rtie s o f e s tu a rin e o rg a n is m s as w e ll a s re s p o n s e s to x e n o b io tic e x p o s u re. T h e re fo r e, e m p iric a l d e te rm in a tio n o f th e o p tim a l s a lin ity a n d te m p e ra tu re c o n d i tio n s o f e s tu a rin e m y s id s is e s s e n tia l fo r th e d e v e lo p m e n t o f o p tim u m la b o ra to ry c u ltu re o f th e s e o rg a n is m s a n d th e ir u se in to x ic ity a n d h a z a r d a s s e s s m e n t. F o r e x a m p le, th e o p tim a l s a lin ity a n d te m p e ra tu re c o n d itio n s f o r g ro w th o f A m e r ic a m y s is b a h ia (fo rm e rly M y s id o p s is b a h ia ) th ro u g h its e n tire life c y c le [4 9 ] a re c o rre la te d w ith re s is ta n c e p a tte rn s to th e se d o m in a n t e n v iro n m e n ta l v a ria b le s [5 0 ] a n d d is trib u tio n o f th is s p e c ie s in e stu a rie s. M o re o v e r, te m p e ra tu re a n d s a lin ity in te r a c t to m o d ify th e re p ro d u c tiv e c a p a c ity o f th is s p e c ie s [5 1 ]. M Y SID S AND TOXICOLOGY M y s id s a re s e n s itiv e to s o m e c h e m ic a l c o n ta m in a n ts a t e n v iro n m e n ta lly r e le v a n t c o n c e n tr a tio n s a n d h a v e b e e n u s e d in re g u la to ry to x ic ity te s tin g fo r m o re th a n 2 0 y e a r s [1 5,4 3, ]. T h e U.S. E n v iro n m e n ta l P r o te c tio n A g e n c y a n d th e A m e r ic a n S o c ie ty fo r T e s tin g a n d M a te ria ls b o th h a v e a d o p te d th e s u b tro p ic a l A. b a h ia a s a k e y te s tin g s p e c ie s fo r c o a s ta l an d e s tu a rin e m o n ito r in g, a n d s ta n d a rd g u id e s fo r c o n d u c tin g life c y c le to x ic ity te sts w ith th is s p e c ie s h a v e b e e n d e v e lo p e d [1 3, ], A lth o u g h a re la tiv e ly la rg e a m o u n t o f p u b lis h e d to x ic ity d a ta is a v a ila b le fo r A m e r ic a m y s is s p e c ie s, re la tiv e ly lim ite d d a ta a re a v a ila b le on th e s e n s itiv ity o f o th e r m y s id s p e c ie s to to x ic a n ts [6 4 ]. H o w e v e r, th e a v a ila b le e v id e n c e s u g g e s ts th a t m y s id s a re g e n e r a lly m o re s e n s itiv e to to x ic s u b s ta n c e s th a n m a n y o th e r te s t s p e c ie s [4 3, ], T o x ic ity te st p ro c e d u re s h a v e b e e n p u b lis h e d fo r N e o m y s is m e r c e d is [5 2,7 6 ], M y s id o p s is in tii [5 9 ], H o lm e s im y s is c o s ta ta [6 2 ], A m e r ic a m y s is b ig e lo w i [7 7 ], N e o m y s is in te g e r [6 4,7 5,7 8 ], T e n a g o m y s is n o v a e - z e a la n d ia e [7 9 ], P r a u n u s fle x u o s u s [8 0,8 1 ], N e o m y s is a m e r ic a n a [8 2,8 3 ], a n d N e o m y s is a w a t s c h e n s is [8 4 ] (T ab le 1). In a d d itio n, m e th o d s fo r m a in ta in in g v ia b le p o p u la tio n s o f d if f e r e n t m y s id s p e c ie s u n d e r la b o ra to ry c o n d itio n s h a v e b e e n d e s c r ib e d b y s e v e r a l r e s e a r c h e r s [ 4 6,5 9,7 5,7 9, ]. R e c e n tly, a s tro n g c o rre la tio n w a s r e p o rte d b e tw e e n th e to x ic r e s p o n s e o f d a p h n id s a n d m y s id s (R 2 = , n = 2 8 ; 9 6 -h m e d ia n le th a l c o n c e n tr a tio n s fo r A. b a h ia a n d 4 8 -h m e d ia n le th a l c o n c e n tr a tio n s fo r D a p h n ia m a g n a ) fo r p e s tic id e s a n d o rg a n ic s, e m p h a s iz in g th e u s e o f m y s id s in fu tu re to x ic ity te s tin g [9 0 ]. M y s id s h a v e b e e n u s e d s u c c e s s fu lly to m e a s u re v a rio u s s u b le th a l to x ic a n t e ffe c ts, s u c h a s g ro w th, s w im m in g c a p a b ility, fe e d in g b e h a v io r, m o ltin g, e n e r g y b u d g e t, re p ro d u c tio n, s e x u a l m a tu rity, a n d v ite llo g e n e s is (d e s c r ib e d in d e ta il in th e fo llo w in g p a ra g ra p h s a n d s u m m a riz e d in T a b le 2 ). A lso, fie ld s tu d ie s a n d c a g in g e x p e r im e n ts w ith m y s id s h a v e b e e n p u b lis h e d [1 7, ]. B e c a u s e o f th e ir e c o lo g ic a l im p o r ta n c e, w id e g e o g ra p h ic d is trib u tio n, y e a r-ro u n d a v a ila b ility in th e fie ld, e a s e o f tra n s p o rta tio n, a b ility to b e c u ltu re d in th e la b o ra to ry, a n d s e n s i

3 M ysids and endocrine disruption: A review Environ. Toxicol. Cheni. 23, Table 1. Candidate mysid test species for toxicity testing w ith details on their natural habitat and culturing Species name D istribution H abitat description Com m ercial culture Culture protocol A m ericam ysis bahia ( = M ysidopsis bahia) A m ericam ys is b i g e low i ( = M ysidopsis bigelowi) A m ericam ysis alm yra (= M ysidopsis alm yra) H olm esim ysis costata (= Acanthom ysis sculpta) M ysidopsis intii M ysis mixta N eom ysis aw atschensis N eom ysis m ercedis N eom ysis integer C oastal estuaries and em baym ents ranging from the G ulf o f M exico to N arragansett (R I, U SA ) [250] E astern coast o f the U SA from M A (G eorges Bank) to FL often together w ith A. bahia [250] Eastern coast o f the USA, inshore w aters along the entire coast o f G u lf o f M exico and northw ard along Atlantic coast to Patapsco R iver (M D ) [250] Principal species o f the genus, from southern C alifornia (U SA ) to B ritish Colum bia (C anada) [122, 253] Eastern Pacific from South A m erica to the southern California coast o f the U SA [59, 250] Eastern (from W hite Sea to Iceland) and W estern (Greenland coastal w aters dow n to Cape C od) A tlantic regions [26] Pacific coast o f Japan, Korea, and USA [115] N ortheastern Pacific coast o f the U SA (southern A laska to G oviota Bay, CA) [52] N orthern E uropean estuaries and coastal waters; oligohalina and freshw ater lakes [20, 43] Praunus flexuosus Northern European coastal w a ters M arine (> 15% 0), < DC [ ] Yes [46,89,251] M arine (30-35% «), 2-30 C No [46] M arine (10-20% o), >20 C Yes [85,88,252] M arine, planktonic, lives w ithin surface canopy o f kelp M arine, epibenthic, optim al tem perature C, optimal salinity 28-35%«Brackish, low salinity, cold w a ter N o, field-collected anim als available [253] N o [59] [122,253] N o [114] M arine, estuarine No [115] Freshw ater, estuaries, and coastal lakes, planktonic/epibenthic, euryhaline (< 0.5 to >25% «), 6-22*C M arine, estuarine, freshwater, hyperbenthic, euryhaline (< 0.5 to >25% «), cold w ater (<20 C ) [116] H yperbenthic/planktonic, euryhaline, eurytherm [254, 255] No [52] No [75] No [123] tiv ity to c o n ta m in a n ts, m y s id s a re a p p ro p ria te to x ic ity te st o r g a n ism s. C A N D ID A T E M Y SID T E ST SP E C IE S FO R E N D O C R IN E - D ISR U PT IO N R E SE AR C H G e n e ra l s e le c tio n c r ite ria fo r th e m o s t a p p ro p ria te m y s id s p e c ie s fo r to x ic o lo g ic a l te s tin g a re g iv e n b y N im m o a n d H a m a k e r [1 5 ] a n d R o a s t e t al. [4 3 ]. T h e s e c r ite ria in c lu d e a v a ila b le w h e n re q u ire d ; a lre a d y a d a p te d to la b o ra to ry c o n d itio n s, e lim in a tin g a n (e x p e n s iv e ) c o n d itio n in g p h a s e ; c o lle c tio n fo r la b o ra to ry te s tin g w ill n o t d e c im a te fie ld p o p u la tio n s (o r d e s tro y h a b ita t d u rin g c o lle c tio n ); e a s ily tra n s p o rte d ; life h is to ry is s h o rt, m a k in g it p o s s ib le to s tu d y th e e ffe c ts o f a p o llu ta n t o n v a rio u s a s p e c ts o f re p ro d u c tio n ; d ie t is k n o w n a n d re a d ily c o n tro lle d ; a n d e c o lo g ic a lly im p o rta n t. In a d d itio n, th e im p o rta n t c h a r a c te ris tic s fo r th e s e le c tio n o f a s u ita b le te s t s p e c ie s fo r id e n tify in g th e e ffe c ts o f e n d o c rin e d is ru p tio n in th e e n v iro n m e n t a re g iv e n b y D e F u r e t al. [7] a n d in c lu d e p rim a ry m o d e o f re p ro d u c tio n, c u ltu re in th e la b o ra to ry, g e n e ra tio n tim e, s iz e, k n o w le d g e o f e n d o c rin o lo g y, a n d s ta n d a rd m e th o d s a v a ila b le. S o m e a ttrib u te s d e s c rib e d in th e la tte r p u b lic a tio n (e.g., m o d e o f re p ro d u c tio n o r k n o w le d g e o f e n d o c rin o lo g y ) d o n o t a llo w fo r d is c r im in a tio n a m o n g c a n d id a te m y s id s p e c ie s. A v e ry u s e fu l d o c u m e n t in th is c o n te x t is a d ra ft re v ie w p a p e r o n lif e - c y c le to x ic ity te s tin g w ith m y s id s in w h ic h s e v era l s p e c ie s (A. b a h ia, A m e r ic a m y s is a lm y r a, A. b ig e lo w i, IT. c o s ta ta, M. in tii, N. m e r c e d is, a n d N. in te g e r ) a re c o n s id e re d fo r th e ir p o te n tia l u tility in e n d o c r in e -d is ru p tio n te stin g [13]. F ro m th is re v ie w, it m a y b e c o n c lu d e d th a t, a lth o u g h A. b a h ia h a s m a n y s tre n g th s, lim ite d e c o lo g ic a l re le v a n c e fo r h ig h -la t- itu d e a n d lo w -s a lin e s y s te m s p re c lu d e its g e n e ra l u tility. H o w ev er, g iv e n th e h ig h d e g re e o f s ta n d a rd iz a tio n in A. b a h ia, p ro g r e s s in d e v e lo p m e n t o f s ta n d a rd iz e d te s t p ro to c o ls fo r e n d o c rin e -d is ru p tio n te s tin g s h o u ld b e fa s te s t in th is s p e c ie s. T a b le 1 s u m m a riz e s th e d is trib u tio n, h a b ita t d e s c rip tio n, and a v a ila b le c u ltu re p ro to c o ls fo r o th e r c a n d id a te m y s id te st s p e cies. M Y SID E N D O C R IN O L O G Y A ND H O R M O N E -R EG U L A T ED E N D P O IN T S T h e u s e o f h o rm o n e s to r e g u la te b io lo g ic a l p ro c e s s e s is a s tra te g y c o m m o n to v e rte b ra te s a n d in v e rte b ra te s. A lth o u g h th e e n d o c rin e s y s te m s o f in v e rte b ra te s re g u la te m a n y o f th e s a m e p ro c e s s e s in v e rte b ra te s (d e v e lo p m e n t, g ro w th, a n d re p ro d u c tio n ), s o m e e n d o c r in e -re g u la te d p ro c e s s e s a re u n iq u e to s p e c ific g ro u p s o f in v e rte b ra te s. F o r e x a m p le, m o ltin g, d ia p a u s e, a n d lim b re g e n e r a tio n a re e n d o c r in e -re g u la te d p ro c e s s e s a s s o c ia te d w ith s o m e in v e rte b ra te g ro u p s th a t a re ra re o r a b s e n t a m o n g v e rte b ra te s [7]. M o s t o f th e c u rre n t k n o w le d g e o f c ru s ta c e a n e n d o c rin o lo g y

4 Table 2. List o f potential endpoints for evaluating the effects o f endocrine disruptors and their use in mysids Endpoint Use in mysids References/com m ents Survival (acute) Am ericam ysis bahia (S)a [69,253] Am ericam ysis bigelowi (S) [69] Am ericam ysis alm yra (S) [69] H olm esim ysis costata (S) [62,67,253] Neom ysis m ercedis (S) [52,67,76] M ysidopsis intii [55,59] Neom ysis integer [53,64,75,78] O ther species [79,80-84] Life-cycle testing A. bahia (S) [68,70,72,253] A. bigelow i (S), A. alm yra (S) [68] H. costata (S) [56,68,71] M. intii [55,59] Ar. integer [160] Two-generation testing A. bahia, A. bigelowi, A. alm yra (S in prep) [13,219,256] M. intii M ethod should be developed H. costata, N. m ercedis, N. integer Probably im practical because o f long generation time Fecundity (brood size) A. bahia, A. bigelowi, A. alm yra (S) [68,70] IT. costa ta (S) [71,122] N. m ercedis [257] M. intii [55,59] N. integer, P raunus flexuosus [123,258] M ysis m ixta [114] N eom ysis aw atschensis [115] Em bryonic development A. bahia [205] M esopodopsis slabberi [208] Sexual m aturity A. bahia (S) [51,58,60,68,218] Tim e to first brood release H. costata [122] Egg developm ent lime N. integer, P. flexu o su s [123] M. m ixta [114] M. intii [55,59] Sex ratio and intersexuality A. bahia (S) [68,219,253] N. integer [ ] G row th, biom ass A. bahia, A. bigelowi, A. alm yra (S) [68,70] H. costata (S) [56,71,122] N. m ercedis [52] M. intii [55,59] M. m ixta [124] N. integer [107,116] N. aw atschensis [84,115,150,151] P. flexu o su s [114] T enagom ysis no vae-zealandiac [79] O ther species [117, ,132] M olt time and success A. bahia [259] N. integer [111,140] M. m ixta [140] Siriella arm ata [138] N. aw atschensis [115] Energy m etabolism A. bahia [63] 0 :N ratio, C:N ratio N. m ercedis [260] R espiration N. integer [153,155, , , ] 1222 Environ. Toxicol. Chem. 23, 2004 T.A. Verslycke et al.

5 Table 2. C ontinued Endpoint U se in mysids References/com m ents Biochemical composition M. m ixta [114,182] N. aw atschensis [ ] P. flexu o su s [81] Leptom ysis lingvura [54] M ysis relicta [176,261] G astrosaccus brevifissura [163] Ecdysteroid metabolism A. bahia S.R. Tubertyb and C.L. M ckenney,c unpublished data N. integer A. G bekiere et al.,d unpublished data S. arm ata [139] Steroid m etabolism N. integer [162,185,198,199] P450 enzym es N. integer [162,185,198] V itellogenesis A. bahia [215] S. arm ata [139] N. integer A. G hekiere et al.,j unpublished data Osm oregulation A. bahia [259,262,263] N. integer [264] P. flexuosus [265] O ther species 1248] M orphology, histology A. bahia, A. bigelow i [77] N. integer [225] Swimm ing behavior N. integer [43, ,232] Feeding behavior A. bahia [231,241] M. m ixta [239,242] Other behavioral endpoints M ating, grooming, swarm ing, burrow ing ability, predator-prcy dynamics [ , ] a S = method is standardized. b Center for Environm ental D iagnostics and Biorem ediation, U niversity o f W est Florida, Pensacola, FL, USA. CU.S. Environm ental Protection A gency, N ational H ealth and Environm ental Effects R esearch L aboratory, G u lf Breeze, FL, USA. d Laboratory o f Environm ental Toxicology and A quatic Ecology, Ghent University, Ghent, Belgium. Mysids and endocrine disruption: A review Environ. Toxicol. Cheni. 23,

6 1224 Environ. Toxicol. Cheni. 23, 2004 T.A. V erslycke et al. is b a s e d u p o n s tu d ie s w ith d e c a p o d s s u c h a s c ra b s, lo b s te rs, c ra y fis h, a n d s h rim p, a n d h a s b e e n re v ie w e d p re v io u s ly [ 7, ]. T h e m a in b io lo g ic a l p ro c e s s e s, s u c h a s g ro w th, m o ltin g, a n d re p ro d u c tio n, a re c y c lic a n d fa irly w e ll u n d e rs to o d in b e n - th ic a n d te rre s tria l m a la c o s tra c a n s, s u c h as d e c a p o d s, is o p o d s, a n d a m p h ip o d s [2 2,1 0 1,1 0 2 ], T h e s e b io lo g ic a l p ro c e s s e s a re re g u la te d b y a c o m p le x e n d o c rin e s y ste m [9 6,1 0 1 ], B a s ic a lly, in p u ts fro m th e e n v iro n m e n t a re in te g ra te d b y th e c e n tra l n e r v o u s s y ste m ; n e u ro tra n s m itte rs a n d n e u ro m o d u la to rs g o v e rn th e re le a s e o f n e u ro p e p tid e s, w h ic h c o n tro l th e p ro d u c tio n o f h o rm o n e s b y th e e n d o c r in e g la n d s [1 0 3 ]. T h e m a in c ru s ta c e a n e n d o c rin e c e n te rs in c lu d e th e Y -o rg a n, m a n d ib u la r o rg a n, a n d ro g e n ic g la n d, X -o rg a n, a n d s in u s g la n d [7,9 7 ], U n fo rtu n a te ly, e n d o c r in e g la n d s o r s ite s o f h o rm o n e p ro d u c tio n in m y s id s a re la rg e ly u n d e sc rib e d. T h e e ffe c ts o f o rg a n ic a n d in o rg a n ic c o n ta m in a n ts on c ru s ta c e a n fu n c tio n s r e g u la te d b y h o rm o n e s a re b e in g in v e s tig a te d w ith in c re a s in g fre q u e n c y a n d s e v e ra l s h o w p ro m is e a s b io - m arlcers o f e n v iro n m e n ta l c o n ta m in a tio n a n d e n d o c rin e d is ru p tio n [7,8,1 0 4,1 0 5 ]. U n fo rtu n a te ly, re la tiv e ly fe w d a ta a re a v a ila b le o n th e h o rm o n a l c o n tro l o f b io lo g ic a l p ro c e s s e s in m y s id s. H a v in g s a id th a t, c e rta in e n d p o in ts re le v a n t to th e te s tin g o f s u s p e c te d e n d o c rin e d is ru p to rs, s u ch a s s u rv iv a l, fe c u n d ity, s e x u a l m a tu ra tio n, a n d b io m a s s in c re a s e, a re a lre a d y s ta n d a rd iz e d p ro c e d u re s fo r s o m e m y s id s, s u c h as A. b a h ia, A. b ig e lo w i (p a rtly ), A. a lm y r a, I I c o s ta ta, a n d N. m e r c e d is, a n d m a n y o th e r e n d p o in ts o r s p e c ie s a re p ro m is in g [7], T h e u s e o f p o te n tia l m y s id h o rm o n e -re g u la te d e n d p o in ts as b io m a rk e rs o f e x p o s u r e o r e ffe c ts o f e n d o c r in e d is ru p to rs a re d is c u s s e d in d e ta il in th e fo llo w in g p a ra g ra p h s a n d a re s u m m a riz e d in T a b le 2. A lth o u g h m a n y, i f n o t a ll, o f th e s e e n d p o in ts m a y in d ic a te a re s p o n s e to an e n d o c rin e d is ru p to r, m o s t a ls o v a ry in re s p o n s e to e x p o s u re to o th e r s tre s s o rs a n d th is is fu rth e r c o n fo u n d e d b y th e in te rr e la te d n e s s (i.e., n o n in d e p e n d e n c e ) o f s o m e o f th e s e e n d p o in ts [1 3 ]. T h e k e y to th e in te rp re ta tio n o f th e s e e n d p o in ts as in d ic a to rs o f e n d o c rin e d is ru p tio n w ill b e to c re a te fo r e ach s p e c ie s a la rg o d a ta b a s e o f w h a t c o n s titu te s th e n o rm a l u n s tre s s e d re s p o n s e, a n d w h a t c o n s titu te s a n o rm a l re fe re n c e s ite o r p o p u la tio n w h e n w o rk in g u n d e r fie ld c o n d itio n s. G ro w th a n d m o ltin g M o s t c o m m o n ly, g ro w th is m e a s u re d e ith e r b y in c re a s e s o f d ry w e ig h t o r b o d y le n g th p e r tim e in te rv a l [ 5 9, ] a n d, fo r c ru s ta c e a n s, is o fte n e x p re s s e d in te rm s o f in te rm o lt p e rio d an d g ro w th fa c to r (p e rc e n ta g e in c re a s e in b o d y s iz e a t th e m o lt) [2 6 ]. G ro w th c u rv e s (s u c h as th e v o n B e r ta la n f fy e q u a tio n ) can b e fitte d to tire g ro w th d a ta [2 0,1 0 9,1 1 0 ], a llo w in g c o m p a ris o n s o f th e d if f e r e n t g ro w th p a ra m e te r s b e tw e e n tre a t m e n ts. A lth o u g h s e v e ra l s tu d ie s h a v e fo c u s e d o n g ro w th and m o ltin g in m y s id s u n d e r n a tu ra l c o n d itio n s [49,107, ], e x p o s u re e x p e rim e n ts a ls o h a v e c o n firm e d th e s e n s itiv ity o f th e s e e n d p o in ts in to x ic o lo g y [ 5 2,5 6,6 1,8 4,9 2,1 0 6,1 0 8, ], F o r m y s id s, re d u c e d g ro w th is th e m o s t c o m m o n su b - le th a l re s p o n s e to to x ic a n t e x p o s u re a n d th is h a s im p o rta n t im p lic a tio n s fo r re p ro d u c tiv e s u c c e s s b e c a u s e fe c u n d ity is re la te d d ire c tly to fe m a le b o d y s iz e [2 0,8 4,1 1 8,1 2 3 ]. In c ru s ta c e a n s, s ig n ific a n t g ro w th o c c u rs o n ly as a re s u lt o f m o ltin g ; th e re fo re, d is ru p tio n o f m o ltin g m a y r e s u lt in a lte ra tio n s in g ro w th [1 3,1 3 2 ], E c d y s te ro id s (th e m o ltin g h o rm o n e s in c ru s ta c e a n s ) also fu n c tio n in th e c o n tro l o f re p ro d u c tio n a n d e m b ry o g e n e s is [9 7,1 3 3 ]; th e re fo re, th e c ru s ta c e a n m o lt c y c le h a s p ro fo u n d e ffe c ts o n m a n y a s p e c ts o f o rg a n is m a l fu n c tio n, in c lu d in g p h y s io lo g y, b e h a v io r, a n d c h a n g e s in b io c h e m ic a l c o m p o s itio n [ ], M o ltin g is r e g u la te d b y a m u ltih o r m o n a l s y s te m b u t is u n d e r th e im m e d ia te c o n tro l o f m o lt- p ro m o tin g s te ro id h o rm o n e s (e c d y s te ro id s ) s e c re te d b y a n e c d y s ia l g la n d, c alled th e Y -o rg a n (th e h o m o lo g u e o f th e p ro th o r a c ic g la n d in in sec ts [ ], T h e Y -o rg a n s e c re te s e c d y s o n e, w h ic h, on re le a se in th e h e m o ly m p h, is c o n v e r te d in to a c tiv e 2 0 -h y d ro x y e c d y - s o n e (s y n o n y m s : c ru s te c d y s o n e a n d e c d y s te ro n e ). C irc u la tin g tite r s o f 2 0 -h y d r o x y e c d y s o n e v a r y im p r e s s iv e ly d u rin g th e m o lt c y c le [1 0 3,1 3 4 ]. T h e Y -o rg a n p ro d u c e s tw o o th e r e c d y s te ro id s, 3 -d e h y d ro e c d y s o n e a n d 2 5 -d e o x y e c d y s o n e, w ith he la tte r fo rm in g th e im m e d ia te p re c u rs o r to th e a c tiv e p o n a ste r- o n e A [1 3 3 ]. M o re s tu d ie s h a v e b e e n d o n e o n th e e ffe c ts o f c o n ta m in a n ts o n m o ltin g a n d lim b g e n e ra tio n th a n o n a n y o th e r h o rm o n e -m e d ia te d p ro c e s s in c ru s ta c e a n s [8,1 3 5,1 3 7 ], M o lt sta g in g, b a s e d o n c h a n g e s in th e in te g u m e n t, h a s b e en d e v e lo p e d fo r v a rio u s c ru s ta c e a n s a n d is g e n e r a lly d iv id e d in to fo u r m a jo r p e rio d s : p o s tm o lt, in te rm o lt, p re m o lt, a n d m o lt (e c - d y s is). M y s id m o lt s ta g e s h a v e b e e n d e s c rib e d fo r S ir ie lla a r m a ta [1 3 8,1 3 9 ], M y s is m ix ta [1 4 0 ], a n d N. in te g e r [1 4 0 ]. In m y s id s, e c d y s is is in s ta n ta n e o u s, w ith th e e n tire c a ra p a c e liftin g u p a n d th e m y s id s lid in g o u t o f th e o ld c u tic le w h ile s w im m in g. F o r fe m a le m y s id s, in te g u m e n ta l d e v e lo p m e n t d u r in g m o lt p re p a ra tio n, m a rs u p ia l b ro o d d e v e lo p m e n t, a n d d e v e lo p m e n t o f n e w e g g s in th e o v a ry a re s y n c h ro n iz e d, fa c ilita tin g m o lt s ta g in g [1 0 1 ]. To d a te, o n ly o n e s tu d y h a s q u a n tifie d e c d y s tc ro id tite rs d u rin g th e m y s id m o lt c y c le an d th is s tu d y w a s w ith S. a r m a ta [1 0 1 ], H o w e v e r, b o th e c d y s o n e and 2 0 -O H e c d y s o n e h a v e b e e n id e n tifie d in TV. in te g e r (A. G h e k - ie re e t a l., L a b o ra to r y o f E n v iro n m e n ta l T o x ic o lo g y a n d A q u a t ic E c o lo g y, G h e n t U n iv e rs ity, G h e n t, B e lg iu m, u n p u b lis h e d d a ta ) a n d A. b a h ia (S.R. T u b e rty, C e n te r fo r E n v iro n m e n ta l D ia g n o s tic s a n d B io re m e d ia tio n, U n iv e rs ity o f W e s t F lo rid a, P e n s a c o la, F L, U S A, a n d C.L. M c K e n n e y, Jr., U.S. E n v iro n m e n ta l P r o te c tio n A g e n c y, N a tio n a l H e a lth a n d E n v iro n m e n ta l E ffe c ts R e s e a rc h L a b o ra to r y, G u lf B re e z e, F L, u n p u b lis h e d d a ta ). A s m e n tio n e d p re v io u s ly, m o ltin g is c o n tro lle d b y e c d y s te ro id s [1 3 6 ], E c d y s te ro id s c o n tro l th e a c tiv ity o f s p ecific g e n e s a t th e tra n s c rip tio n a l le v e l b y in te ra c tin g w ith th e in tra c e llu la r e c d y s te ro id re c e p to r [1 0 3,1 3 5,1 4 1 ]. In a rth ro p o d s, th e e c d y s o n e re c e p to r is in th e s a m e g e n e fa m ily as th e v e r te b ra te th y ro id re c e p to r b u t, in te re s tin g ly, s te ro id a l e stro g e n s d o n o t a g o n iz e o r a n ta g o n iz e th e e c d y s te ro id re c e p to r [1 4 2 ]. E v id e n c e e x ists [1 4 2 ] th a t s o m e n o n s te r o id a l e n v iro n m e n ta l e s tro g e n s a re e c d y s te ro id a n ta g o n is ts (e.g.,lin d a n e, b is p h e n o l A, d ie th y lp h th a la te, a n d jö,/p -D D T ). In a d d itio n, s e v e ra l c la s s e s o f p h y to c h e m ic a ls a n ta g o n iz e e c d y s o n e a c tiv ity [1 3 7 ], T h e a p p a r e n t u b iq u ity o f th e a n tie c d y s te ro id a l a c tiv ity o f e n v iro n m e n ta l c h e m ic a ls n e c e s s ita te s in v e s tig a tio n in to th e ir p o te n tia l e ffe c ts o n c ru s ta c e a n s [1 4 3,1 4 4 ], M a n y p e s tic id e s, g e n e ra lly c la s s e d as in s e c t g ro w th re g u la to rs, fu n c tio n as e c d y s o n e a g o n is ts [7,1 1 ], A s s u g g e s te d b y Z o u a n d F in g e rm a n [1 3 6 ], fu tu re in v e s tig a tio n s o f m o ltin g in c ru s ta c e a n s s h o u ld h a v e tw o e m p h a s e s, o n e e x a m in in g in te ra c tio n s b e tw e e n p o te n tia l e n d o c rin e d is ru p to rs a n d th e e c d y s o n e re c e p to r, a n d o n e fo c u s in g o n th e p o s s ib le im p a irm e n t o f e c d y s te ro id o g e n e s is b y th e se a g e n ts. In v itro a s s a y s c a n d e te rm in e q u ic k ly w h e th e r a c h e m ic a l h a s (a n ti) e c d y s te r o id a l a c tiv ity [1 4 5,1 4 6 ]. G iv e n th e c u r re n t m e th o d s fo r q u a n tify in g e c d y s te ro id s b y u s in g im m u n o a s s a y [1 3 9,1 4 4,1 4 7 ] a n d th e a v a ila b le m e th o d s fo r m o lt s ta g in g [1 3 8,1 4 0 ], it s h o u ld b e p o s s ib le to e v a lu a te th e p o te n tia l in

7 M ysids and endocrine disruption: A review Environ. Toxicol. Cheni. 23, te ra c tio n o f th e s e c h e m ic a ls w ith th e p ro c e s s o f m o ltin g in m y s id s. B e c a u s e e c d y s te ro id s a rc u s e d as m a jo r e n d o c r in e -s ig n a lin g m o le c u le s in c ru s ta c e a n s [7 ], a n d little is k n o w n o f th e ir o th e r f u n c tio n s, it m a y b e e x p e c te d th a t a c h e m ic a l w ith (a n ti)e c d y s te ro id a l a c tiv ity a lso w ill a ffe c t o th e r h o rm o n e -re g u la te d p ro c e s s e s in c ru s ta c e a n s. S u p p o r t fo r th is h y p o th e s is is p ro v id e d b y M u a n d L e B la n c [1 4 4 ], w h o d e m o n s tra te d th a t th e fu n g ic id e fe n a rim o l a lte re d e m b ry o d e v e lo p m e n t in d a p h n - id s b y in te rfe rin g w ith e c d y s te ro id m e ta b o lis m. H o w e v e r, o n e m a jo r a d v a n ta g e o f u s in g e c d y s te ro id m e ta b o lis m as an e n d p o in t is th a t it p ro v id e s a m e a n s o f e v a lu a tin g th e im p a c t o f e n v iro n m e n ta l c h e m ic a ls o n c ru s ta c e a n s (a n d p o te n tia lly o th e r a rth ro p o d s ), w h ile n o t n e c e s s a rily a ffe c tin g v e rte b ra te s. B e c a u s e (a n ti)e c d y s te ro id a l a c tiv ity h a s b e e n p ro v e n in v itro fo r c e rta in c h e m ic a ls [1 4 5 ], a n d d is ru p tio n o f m o ltin g h a s b e e n o b s e rv e d as a r e s u lt o f c h e m ic a l e x p o s u re, th e s e c h e m ic a ls s h o u ld b e te s te d in e x p o s u re s w ith m y s id s. In th e s e e x p o su re s, e n d p o in ts s u c h as in te rm o lt p e rio d, g ro w th, m o rp h o lo g ic a l a b e rra tio n s, e c d y s o n e tite rs, p ro te in c o n c e n tra tio n s, in te g u m e n t d e v e lo p m e n t, a s w e ll a s re la te d e n d p o in ts s u ch as v ite llo g e n - e sis, s h o u ld d e te c t in v iv o e ffe c ts o f c h e m ic a ls o n e c d y s te ro id m e ta b o lis m a n d m o ltin g in m y s id s. In v itro a ssa y s s h o u ld a id th e m e c h a n is tic u n d e rs ta n d in g o f c h e m ic a l a c tio n to b e tte r a llo w d is tin c tio n b e tw e e n e n d o c rin e -s p e c ific a n d p h a rm a c o log ical effects. E n e r g y m e ta b o lis m B io m a rk e rs lin k e d w ith p h y s io lo g ic a l e n e rg e tic s p ro v id e in fo rm a tio n o n k e y p ro c e s s e s in th e o rg a n is m s e n e rg y a c q u is itio n a n d e x p e n d itu re, a n d p o s s ib ly a ls o e lu c id a te th e m o d e o f a c tio n o f th e to x ic a n t. U n d e r n o rm a l c o n d itio n s, s p e c ific a m o u n ts o f e n e rg y a re a llo c a te d to b a sa l m e ta b o lis m, g ro w th, a n d re p ro d u c tio n a n d th e re fo re, th e o re tic a lly, c h a n g e s in m e t a b o lic tu rn o v e r a n d s p e c ific a llo c a tio n s s h o u ld b e lin k e d to e ffe c ts a t h ig h e r le v e ls o f e c o lo g ic a l o rg a n iz a tio n [1 4 8 ]. A la rg e b o d y o f in fo rm a tio n is a v a ila b le o n th e n e u ro e n d o c rin e p a th w a y s o f p h y s io lo g ic a l re g u la tio n in m a c ro c ru s ta c e a n s. A l th o u g h ty p ic a l a n d w e ll-s tu d ie d c h a lle n g e s to e n d o g e n o u s e n e rg y m e ta b o lis m in c lu d e e n v iro n m e n ta l h y p o x ia, fu n c tio n a l (in te rn a l) h y p o x ia, c h a n g in g e n e r g e tic re q u ire m e n ts, d is tu r b a n c e to w a te r b a la n c e a n d io n h o m e o s ta s is a n d c h a n g e s in te m p e ra tu re (f o r re v ie w, r e fe r to M o rris a n d A irrie s s [1 4 9 ]), e x p o s u re to to x ic a n ts a ls o w ill r e s u lt in a n e n e r g e tic c h a lle n g e. B e c a u s e e n e r g e tic p ro c e s s e s a re h o rm o n e -re g u la te d, th e y are, b y d e fin itio n, s e n s itiv e to h o rm o n e d is ru p tio n a n d s e v e ra l m e a s u re m e n ts o f e n e rg y r e s e r v e s a n d c o n s u m p tio n m a y s e r v e as u s e f u l b io m a rk e rs o f e n d o c r in e -d is ru p tin g s u b s ta n c e s in c ru s ta c e a n s [1 1 ]. H o w e v e r, o n c e a g a in, th e u tility o f b io e n e rg e tic e n d p o in ts w ill d e p e n d s tro n g ly o n e s ta b lis h m e n t o f a c o n s e n su s d a ta b a s e o f w h a t th e n o rm a l b io e n e rg e tic s ta te is fo r m y s ids. A lte ra tio n s to th e e n e rg y m e ta b o lis m o f m y s id s h a v e b e en u s e d s u c c e s s fu lly as an in d ic a to r o f s tre s s to to x ic a n t e x p o s u re in A. b a h ia [6 3,1 2 5,1 2 6,1 2 9,1 3 0 ], P. fle x u o s u s [8 1 ], N. a w a t s c h e n s is [ ], a n d N. in te g e r [ ]. In A. b a h ia, P. fle x u o s u s, a n d N. a w a ts c h e n s is, w e ig h t-s p e c ific re s p ira tio n, a m m o n ia e x c r e tio n ra te s, a n d o x y g e n to n itro g e n r a tio s h a v e b e e n m e a s u re d a fte r to x ic a n t e x p o s u re. In N. in te g e r, R o a s t e t al. [1 5 3 ] u s e d s c o p e fo r g ro w th [1 5 7 ], w h e re a s V e rsly c k e a n d J a n s s e n [1 5 5 ] a n d V e rsly c k e e t al. [1 5 6 ] u s e d th e c e llu la r e n e rg y a llo c a tio n a ss a y [1 5 8 ]. B o th m e th o d s a re p ro m is in g a n d w e re re c e n tly v a lid a te d in N. in te g e r a fte r e x p o s u re to th e p e s tic id e c h lo rp y rifo s (T.A. V e rs ly c k e e t a l., u n p u b lis h e d d ata). T h e c e llu la r e n e rg y a llo c a tio n a ss a y a ls o w a s r e c e n tly v a lid a te d in th e fie ld (S c h e ld t e s tu a ry, T h e N e th e r la n d s ) [9 4 ], T h e e c o lo g ic a l re le v a n c e a n d u tility o f s h o rt-te rm b io in d ic a to rs o f m e t a b o lic p ro c e s s e s in A. b a h ia h a v e b e e n d e m o n s tra te d a fte r c h ro n ic e x p o s u re to p e s tic id e s [6 3,1 2 5,1 2 6,1 2 9,1 3 0 ]. In th e se s tu d ie s, p e s tic id e -e x p o s e d ju v e n ile m y s id s h a d a g re a te r r e lia n c e on th e m o re e n e rg y -ric h lip id s u b s tr a te s d u rin g m a tu ra tio n to s u p p o rt e le v a te d m e ta b o lic d e m a n d s, re s u ltin g in le ss lip id m a te ria l a v a ila b le fo r g a m e te p ro d u c tio n a n d re d u c e d re p ro d u c tiv e s u c c e ss. U n e x p o s e d m y s id s s h ift to w a rd m o re p ro - te in a c e o u s s u b s tra te s d u rin g m a tu ra tio n, a s d e m o n s tra te d fo r A. b a h ia [6 3 ] a n d N. in te g e r [1 5 5,1 5 9 ], T h e s e c h a n g e s in m e ta b o lic s u b s tr a te u s a g e can b e m e a s u re d b y m o n ito rin g th e o x y g e n to n itro g e n ra tio [1 2 5,1 6 0 ], th e lip id a n d p ro te in c o n te n t [1 5 5 ], o r th e c a rb o n to n itr o g e n ra tio o f th e te s t o rg a n is m [1 1 4 ], O n th e o th e r h a n d, h y p e rg ly c e m ia is a c o m m o n re s p o n se to e n v iro n m e n ta l o r fu n c tio n a l h y p o x ia a n d c o n ta m in a n t e x p o s u re in n u m e ro u s d e c a p o d s, a n d it is th o u g h t to b e trig g e re d b y th e a c tio n o f c ru s ta c e a n h y p e rg ly c e m ic h o rm o n e on v a rio u s ta r g e t tis su e s [8,1 4 9 ]. T h e a m in o a c id s e q u e n c e o f c ru s ta c e a n h y p e rg ly c e m ic h o rm o n e is h ig h ly h o m o lo g o u s w ith th a t o f th e m o lt- in h ib itin g h o rm o n e, a n o th e r p ro d u c t o f th e s in u s g la n d s in c ru s ta c e a n s, in d ic a tin g p o s s ib le in v o lv e m e n t in th e c o n tro l o f m o ltin g a n d r e p ro d u c tio n [1 4 9 ]. S e v e ra l in v e s tig a to r s h a v e e x a m in e d th e e ffe c ts o f m e ta ls a n d o rg a n ic c o n ta m in a n ts on b lo o d g lu c o s e c o n c e n tr a tio n s a n d c ru s ta c e a n h y p e rg ly c e m ic h o rm o n e tite rs in c ru s ta c e a n s [8], C h a n g e s in b lo o d g lu c o se le v e ls in m y s id s e x p o s e d to p o te n tia l e n d o c rin e d is ru p to rs m a y in d ic a te d is ru p tio n o f h o rm o n a l a c tiv ity o th e r th a n th a t a s s o c ia te d w ith m o ltin g o r re p ro d u c tio n [1 3 ], T h e m e th o d s d e s c rib e d a b o v e a re tra n s fe rre d e a s ily to o th e r m y s id s p e c ie s. E n d p o in ts re la te d to e n e r g e tic p ro c e s s e s a re re la tiv e ly e a s y to m e a s u re, b u t a b e tte r a n d h o lis tic u n d e r s ta n d in g o f tire ro le o f th e d if f e r e n t h o rm o n e s in v o lv e d in e n e rg y m e ta b o lis m, s u c h a s c ru s ta c e a n h y p e rg ly c e m ic h o r m o n e, is n e e d e d to e v a lu a te th e p o te n tia l im p a c t o f h o rm o n e - m im ic k in g s u b s ta n c e s o n m y s id s. In th is c o n te x t, n e w im m u n o a s s a y s fo r d e te rm in a tio n o f c irc u la tin g h o rm o n e s in th e h e m o ly m p h, s u c h as c ru s ta c e a n h y p e rg ly c e m ic h o rm o n e, are p ro m is in g [1 6 1 ], O th e r e n d p o in ts re la te d to m e ta b o lis m in m y s id s h a v e b e e n stu d ie d. H ig h a c e ty lc h o lin e s te r a s e a c tiv ity in S ir ie lla c la u s i, in d ic a tin g a h ig h m e ta b o lic ra te, id e n tifie d th is m y s id a s p a r tic u la rly s u ite d fo r re s e a rc h b a s e d o n b io m a rk e rs in tire m a rin e e n v iro n m e n t [1 7 ]. In a d d itio n, N. in te g e r h a s b e e n u s e d as a v ia b le a lte rn a tiv e m o d e l fo r th e p a rtia l re p la c e m e n t o f v e rte b ra te a n im a ls in m e ta b o lic s tu d ie s w ith ille g a l g ro w th p ro m o te rs a n d v e te rin a ry d ru g s [1 6 2 ], F in a lly, r e s p ira to ry r e s p o n s e s h a v e b e e n s tu d ie d in m y s id s in re la tio n to a v a ria b le e n v iro n m e n t a n d to x ic e x p o s u r e [6 3,8 1,8 4,1 1 5,1 2 5,1 6 0, ]. A lth o u g h u n d o u b te d ly h a v in g e n v iro n m e n ta l re le v a n c e an d b e in g fa irly e a s ily e x tra p o la te d to h ig h e r le v e ls o f b io lo g ic a l o rg a n iz a tio n, th e m a jo r d is a d v a n ta g e o f e n d p o in ts re la te d to e n e rg y m e ta b o lis m is th e ir d iff ic u lty in m e c h a n is tic a lly e x p la in in g h o rm o n e -re g u la te d r e s p o n s e s as c an b e e x p e c te d fro m e x p o s u re to e n d o c rin e d is ru p to rs. M a n y a b io tic a n d to x ic s tre s s o rs a ffe c t th e e n e r g y m e ta b o lic p ro c e s s e s o f o rg a n is m s [ 1 1 4,1 2 7, ,1 6 4 ], w h ile n o t n e c e s s a rily b e in g re la te d to d is ru p tio n in n o rm a l h o rm o n a l re g u la tio n. T h e re fo r e, th e s u c c e s s fu l u s e o f b io m a rk e rs fo r th e e v a lu a tio n o f e n d o c rin e d is ru p tio n w ill b e lim ite d b y th e a m o u n t o f b a c k g r o u n d d a ta on

8 1226 Environ. T o x ic o l Cheni. 23, 2004 T.A. V erslycke et al. n a tu ra l v a ria tio n a n d n o rm a l le v e ls o f th e e n d p o in ts in q u e s tio n, a n d a lso b y th e fu n d a m e n ta l u n d e rs ta n d in g o f th e to x ic a n t a c tio n a t th e (s u b )c e llu la r le v e l. In th is re g a rd, m a n y s tu d ie s c o n ta in in g b a c k g r o u n d in fo r m a tio n o n th e b io c h e m ic a l c o m p o s itio n (e.g., p ro te in s, lip id s, a n d s te r o ls ) o f m y s id s h a v e b e en p u b lis h e d [ ], b u t in fo rm a tio n o n n e u ro p e p tid e a n d h o rm o n a l le v e ls in m y s id s n e e d s d e v e lo p m e n t. Steroid metabolism and cytochrome P450 P o llu ta n ts m a y e x e r t re p ro d u c tiv e e ffe c ts th ro u g h in te rf e r e n c e w ith n o rm a l s te r o id m e ta b o lis m [ ], F o r in v e r te b ra te s, s e v e ra l s tu d ie s h a v e fo c u s e d o n p o llu ta n t-in d u c e d a l te ra tio n s in s te ro id m e ta b o lis m. T h e s e c h e m ic a ls o fte n in te r fe re w ith th e m ic ro s o m a l P m o n o o x y g e n a s e s y s te m, also c a lle d th e m ix e d -f u n c tio n o x y g e n a s e s y s te m. T h e m ix e d -fu n c - tio n o x y g e n a s e s y s te m is in v o lv e d n o t o n ly in th e m e ta b o lis m o f o rg a n ic to x ic a n ts b u t a lso in s te ro id m e ta b o lis m ; c o n s e q u e n tly, in d u c tio n o r in h ib itio n o f th e m ix e d -fu n c tio n o x y g e n a s e s y s te m a ls o m a y h a v e re p e rc u s s io n s fo r th e h o rm o n a l c o n tro l o f re p ro d u c tio n. In s e a s ta r s, a lin k a g e w a s d e m o n s tra te d b e tw e e n im p a ire d r e p ro d u c tiv e s u c c e s s, p o llu tio n - m o d u la te d e n d o c rin e fu n c tio n, a n d in d u c tio n o f th e m ix e d -fu n c tio n o x y g e n a s e s y s te m [1 8 7 ], In g a s tr o p o d s, m u c h w o rk o n s te ro id m e ta b o lis m h a s b e e n in itia te d b y th e o b s e r v a tio n o f trib u ty ltin - in d u c e d im p o s e x, a s ta te o f p s e u d o h e rm a p h ro d is m in w h ic h fe m a le s e x h ib it fu n c tio n a l s e c o n d a ry m a le c h a ra c te ris tic s. A l th o u g h th e u n d e rly in g m e c h a n is m b y w h ic h trib u ty ltin c a u s e s im p o s e x in g a s tr o p o d s h a s n o t b e e n e lu c id a te d c o n c lu siv e ly, th e w e ig h t o f e v id e n c e is in fa v o r o f th e c y to c h ro m e P d e p e n d e n t a ro m a ta s e in h ib itio n h y p o th e s is [ ], A lte ra tio n s in s te r o id m e ta b o lis m h a v e b e e n s tu d ie d in D. m a g n a [1 8 6, ] a n d in th e b lu e c ra b C a llin e c te s s a p id u s [1 9 1 ]. Tn d a p h n id s, c h a n g e s in s te r o id m e ta b o lis m c o u ld p ro v id e an e a rly in d ic a tio n o f p o te n tia l re p ro d u c tiv e to x ic ity a fte r s u b le th a l e x p o s u r e to s u s p e c te d e n d o c r in e d is r u p to r s [1 8 6,1 9 3,1 9 4,1 9 7 ]. V e rsly c k e e t al. [1 8 5 ] re p o rte d te s to s te ro n e m e ta b o lis m a n d th e p re s e n c e o f v e rte b ra te -ty p e s te ro id s in N. in te g e r a n d d e m o n s tra te d th e p re s e n c e o f a c o m p le x s te ro id h y d ro x y la s e s y s te m c o n s is tin g o f d iffe r e n t P is o z y m e s. T h e re m a rk a b le d iv e rs ity o f te s to s te ro n e h y d ro x y la tio n e x h ib ite d s h o u ld s tim u la te fu r th e r s tu d ie s o n th e in d u c tio n, s te re o s p e c - ificity, a n d re g u la tio n o f th e e n z y m e s y s te m s o f N. in te g e r a n d o th e r m y s id s. M o re re c e n tly, a lte ra tio n s in th e p h a s e I a n d II te s to s te ro n e m e ta b o lis m in N. in te g e r a fte r a c u te e x p o s u re to trib u ty ltin h a v e b e e n d e m o n s tra te d [1 9 8 ]. In a d d itio n, m e ta b o lic s tu d ie s w ith N. in te g e r h a v e b e e n u s e d re c e n tly in e x p o s u re e x p e rim e n ts w ith o th e r c h e m ic a ls, s u c h as n o n y lp h e n o l a n d m e th o p re n e, a n d a ls o in th e fie ld [1 9 9 ]. T h e p re s e n c e o f s e x h o rm o n e s h a s b e e n s u g g e s te d in m a n y, i f n o t a ll, a rth ro p o d s [1 8 3 ]. V e rte b ra te -ty p e s te ro id s (su c h as 17 ß -e s tra d io l, te s to s te ro n e, a n d p ro g e s te r o n e ) h a v e b e e n m e a s u re d in s e v e ra l m a la c o s tra c a n c ru s ta c e a n s [7,1 8 4 ]. A lth o u g h th e la c k o f a ro le fo r v e rte b ra te s e x s te ro id h o rm o n e s in arth ro p o d s h a s b e e n h ig h lig h te d [7,1 8 3 ], fra g m e n te d e v id e n c e s u g g e s ts th a t s o m e o f th e s e c o m p o u n d s m a y fu n c tio n as h o r m o n e s in c ru s ta c e a n s [7,1 4 3,1 8 5 ]. E n d o g e n o u s a n d ro g e n s m a y b e th e p re c u rs o rs fo r o th e r h o rm o n e s ; th e re fo re, e x p o s u r e to e x o g e n o u s a n d ro g e n s (o r a n d ro g e n m im ic s ) c o u ld e lic it a c tiv ity th ro u g h re c e p to rs o th e r th a n th e a n d ro g e n re c e p to r. A l th o u g h th is h a s n o t b e e n d e te rm in e d in c ru s ta c e a n s, V e rsly c k e e t al. [1 8 5 ] fo u n d e v id e n c e o f a s e x -sp e c ific p ro d u c tio n o f a n d ro g e n s, s u c h a s te s to s te ro n e a n d a n d ro s te n e d io n e, in N. in te g e r. S im ila rly, L e B la n c a n d M c L a c h la n [2 0 0 ] re p o rte d v a r io u s ra te s o f te s to s te ro n e c o n v e r s io n to a n d ro s te n e d io n e in d a p h n id s. F u tu r e s tu d ie s a re n e e d e d to r e v e a l i f th e s e c o n v e r s io n s a re a ffe c te d b y a g e, g e n d e r, r e p ro d u c tiv e s ta te, o r c h a n g e s in th e a b io tic e n v iro n m e n t. N o te th a t a n a n d ro g e n re c e p to r h as n o t b e e n fo u n d o r c lo n e d in c ru s ta c e a n s. T h e re fo r e, id e n tific a tio n a n d c h a r a c te riz a tio n o f th e a n d ro g e n re c e p to r s h o u ld b e a p rio rity fo r re s e a rc h to e x p lo re th e u s e f u ln e s s o f sex s te ro id s fo r e v a lu a tin g e n d o c rin e d is ru p tio n in c ru s ta c e a n s a n d o th e r in v e rte b ra te s. S tu d ie s o v e r th e la s t 3 0 y e a r s h a v e e s ta b lis h e d th e im p o rta n t ro le o f c y to c h ro m e P in th e b io tr a n s fo rm a tio n o f x e n o b i- o tic s a n d e n d o g e n o u s c o m p o u n d s (su c h a s e c d y s te ro id s ) in c ru s ta c e a n s (fo r a re v ie w o n c a is ta c c a n P 4 5 0, r e fe r to J a m e s a n d B o y le [1 8 4 ]). A lth o u g h n o s tru c tu ra l in fo rm a tio n o n c y to c h ro m e P in c ru s ta c e a n s is a v a ila b le, it is c le a r th a t th e y a re in v o lv e d in s e v e ra l s te p s in th e b io s y n th e s is o f e c d y s te ro id s a n d o th e r p h y s io lo g ic a lly im p o r ta n t s u b s tra te s in c ru s ta c e a n s [2 0 1 ]. M o re s tu d ie s a re n e e d e d to u n d e rs ta n d th e e ffe c ts, i f a n y, o f v a rio u s c la s s e s o f e n v iro n m e n ta l a n d o th e r c h e m ic a ls th a t a re k n o w n m o d u la to rs o f c y to c h ro m e P e x p re s s io n o r a c tiv ity. N e w m o le c u la r to o ls, s u c h a s p rim e r -b a s e d re v e rs e tra n s c r ip tio n -p o ly m e r a s e c h a in r e a c tio n p ro c e d u re s a n d e x p re s s io n o f P s in h e te ro lo g o u s s y s te m s, s h o u ld r e s u lt in b e tte r in s ig h ts in to th e fu n c tio n a n d e x p re s s io n o f P s in th e c o n te x t o f e n d o c r in e d is ru p tio n. In a d d itio n, in v iv o m e t a b o lic s tu d ie s w ith d if f e r e n t s u b s tr a te s (te s to s te r o n e a n d e c d y s o n e ) w ill p ro v id e v a lu a b le to o ls fo r e v a lu a tin g th e e ffe c ts o f to x ic a n t e x p o s u re, p a r tic u la rly w h e n lin k e d w ith e ffe c ts on h ig h e r le v e ls o f b io lo g ic a l o rg a n iz a tio n. A lth o u g h in fo rm a tio n o n th e id e n tity o f P s a n d th e ir fu n c tio n a l r o le in m y s id s is, to o u r k n o w le d g e, n o n e x is te n t, m y s id s s h o u ld b e a g o o d m o d e l to s tu d y th e s e m e c h a n is m s. F ro m th e p re lim in a ry s tu d ie s w ith N. in te g e r b y V e rs ly c k e e t al. [1 85,1 9 8,1 9 9 ], su ffic ie n t in fo rm a tio n is a v a ila b le to s u g g e s t th a t m y s id s h a v e an e n z y m a tic b io tr a n s fo rm a tio n s y s te m th a t riv a ls th a t o f o th e r in v e rte b ra te s a n d v e rte b ra te s. M e ta b o lic s tu d ie s w ith p h y s io lo g ic a lly r e le v a n t s u b s tra te s th a t a ls o m e a s u re h o rm o n e -re g u la te d e ffe c ts a t a h ig h e r le v e l o f b io lo g ic a l o rg a n iz a tio n (i.e., re p r o d u c tiv e s u c c e s s ) w o u ld b e v a lu a b le in e v a lu a tio n o f e n v i ro n m e n ta l e n d o c rin e d is ru p tio n. R e p r o d u c tio n a n d v ite llo g e n e s is A lth o u g h th e m a in n e u ro s e c re to ry c e n te rs a n d th e s in u s g la n d in m y s id s re s e m b le th e s e fro m d e c a p o d s, s e x u a l d iffe r e n tia tio n in ju v e n ile s a n d m y s id re p ro d u c tio n a re m o re lik e th o s e o f a m p h ip o d s a n d is o p o d s a n d a re s tric tly lin k e d to th e m o lt c y c le [1 0 1 ]. In m y s id s, e m b ry o n ic a n d p o s te m b ry o n ic d e v e lo p m e n t o c c u r s in th e fe m a le m a rs u p iu m a n d in c lu d e s fiv e c o n s e c u tiv e s ta g e s fro m o v ip o s itio n to th e ju v e n ile s ta g e [2 6,1 1 8, ], J u v e n ile s a re lib e ra te d im m e d ia te ly b e fo re e c d y s is o f th e m o th e r, s h o rtly a f te r w h ic h s h e la y s a n e w b a tc h o f e g g s in th e m a rs u p iu m. A s e c o n d a ry v ite llo g e n ic c y c le sta rts fo r a n e w b a tc h o f o o c y te s o n th e s e c o n d d a y o f th e m o lt cycle. S e c o n d a r y v ite llo g e n e s is is n o t o n ly c y c lic a l, a s in o th e r c ru s ta c e a n s [2 0 6 ], b u t a lso s tric tly lin k e d to th e m o lt c y c le, o ffe rin g a n e x a m p le o f th e ty p e 2 p a tte rn (e.g., A m p h ip o d a, Is o p o d a, a n d D e c a p o d a ) fo r th e re g u la tio n o f s im u lta n e o u s g o n a d a l and s o m a tic g ro w th in c ru s ta c e a n s [2 0 6,2 0 7 ], C u z in -R o u d y and S a le u d d in [1 0 1 ] p u b lis h e d a n e x c e lle n t rev iew on th e u s e o f th e m y s id S. a r m a ta as a b io lo g ic a l m o d e l fo r th e s tu d y o f h o rm o n a l c o n tro l o f m o lt a n d re p ro d u c tio n, w h ic h s h o u ld b e e x te n d e d fo r o th e r m y s id s p e c ie s. In a d d itio n, W o rth a m and P ric e [2 0 5 ] a n d G re e n w o o d e t al. [2 0 8 ] p u b lis h e d s tu d ie s on

9 M ysids and endocrine disruption: A review Environ. Toxicol. Cheni. 23, th e in v itro c u ltu re o f m y s id m a rs u p ia l d e v e lo p m e n ta l s ta g e s a t d iff e r e n t te m p e ra tu re s, T h e s e a s s a y s s h o u ld b e e v a lu a te d f u r th e r as a m e a n s o f d e te c tin g e ffe c ts o f c o n ta m in a n ts on m a rs u p ia l d e v e lo p m e n t in m y s id s. In g e n e ra l, fe w s tu d ie s h a v e b e e n c o n d u c te d o n th e e ffe c ts o f c o n ta m in a n ts o n g o n a d a l m a tu ra tio n o f c ru s ta c e a n s [8]; h o w e v er, m u c h a tte n tio n h a s b e e n g iv e n re c e n tly to v ite llo g e n in, th e p re c u rs o r to th e y o lk p ro te in v ite llin in e g g -la y in g in v e rte b ra te s a n d v e rte b ra te s, a s an in d ic a to r o f e x p o s u re to e s tro g e n ic x e n o b io tic s [5, ], C o n tro l o f v ite llo g e n e s is is b e in g s tu d ie d in te n s iv e ly b e c a u s e y o lk is a n e x c e lle n t m o d e l fo r s tu d y in g m e c h a n is m s o f h o rm o n a l c o n tro l a t th e c e llu la r an d m o le c u la r le v e ls [5,2 1 5 ], T o a s s e s s th e p o te n tia l a d v e rse e ffe c ts o f x e n o b io tic s on c ru s ta c e a n re p ro d u c tio n, it is im p o rta n t to m e a s u re a c c u ra te ly v ite llo g e n in a n d v ite llin in c ru s ta c e a n m o d e ls (an o v e rv ie w o f c ru s ta c e a n s p e c ie s fro m w h ic h v ite llin, v ite llo g e n in, o r lip o v ite llin h a s b e e n is o la te d o r p a r tia lly c h a ra c te riz e d is g iv e n b y T u b e rty e t a l. [2 1 5 ]). R e c e n tly, a q u a n tita tiv e e n z y m e - lin k e d im m u n o s o rb e n t a ss a y w a s d e v e lo p e d fo r th e m y s id A. b a h ia b y u s in g p o ly c lo n a l a n tis e ra [2 1 5 ]. In a d d itio n, s tu d ie s a re u n d e r w a y to c h a r a c te riz e a n d p u rify v ite llin o f th e m y s id N. in te g e r (A. G h e k ie re e t al., L a b o ra to r y o f E n v iro n m e n ta l T o x ic o lo g y a n d A q u a tic E c o l o g y, G h e n t U n iv e rs ity, G h e n t, B e lg iu m, u n p u b lis h e d d ata). F u tu r e la b o ra to ry a n d fie ld s tu d ie s w ith m y s id s a re n e e d e d to e v a lu a te th e u s e o f th e s e im m u n o a s s a y s fo r in v e s tig a tin g e f fe c ts o f x e n o b io tic s o n c ru s ta c e a n v ite llo g e n e s is. A g o o d e x a m p le o f th is is g iv e n b y O b c rd ö rs tc r e t al. [2 1 3,2 1 4 ], w h o re p o rte d th e e ffe c ts o f c h ro n ic p y re n e e x p o s u re o n m o ltin g a n d re p ro d u c tio n a s s e s s e d in th e g ra s s s h rim p P a la e m o n e te s p u g io b y u s in g a m o n o c lo n a l e n z y m e - lin k e d im m u n o s o rb e n t a ss a y fo r v ite llin. O th e r s tu d ie s a ls o h a v e fo u n d an im p a c t o f x e n o e s tro g e n s o n th e p ro d u c tio n o f c ru s ta c e a n p ro te in s (e.g., v ite llin a n d c y p ris m a jo r p ro te in ), w h ic h a re th o u g h t to b e u n d e r e stro g e n c o n tro l [5,2 1 7 ]. F u tu r e w o rk o n s e q u e n c e d e te rm in a tio n o f v ite llo g e n ic g e n e s a n d th e ir h o rm o n a l a c tiv ity w ill p ro v id e in te re s tin g in s ig h t in to th e v ite llo g e n ic p ro c e ss in m y s id s. S tu d y o f g e n o m ic a n d n o n g e n o m ic e ffe c ts o f e c d y s te ro id s o n o v a ria n m a tu ra tio n is a n o th e r p o te n tia l a re a o f w o rk. S y n e rg is tic a n d a n ta g o n is tic a c tio n s o f th e d iffe r e n t n e u ro p e p tid e s, a n d th e m a n d ib u la r o rg a n c o n tro l o v e r m o ltin g an d re p ro d u c tio n, a re o th e r a re a s re q u irin g fu rth e r s tu d y as a b a sis fo r u s e o f c ru s ta c e a n s fo r e n d o c rin e -d is ru p tio n te s tin g in th e fu tu re [13], L ife - c y c le te s tin g a n d p o p u la tio n a n d f i e l d s tu d ie s D e s p ite s u p e rfic ia l re s e m b la n c e to d e c a p o d s h rim p s, m y s id s a re m o re c lo s e ly re la te d to a m p h ip o d s a n d is o p o d s, a n d are g ro u p e d to g e th e r in th e s u p e r o rd e r P e ra c a rid a. A ll th re e o rd e rs a re g o o d c a n d id a te s fo r to x ic o lo g ic a l te stin g, a n d a m p h ip o d s a n d m y s id s a re u s e d ro u tin e ly. H o w e v e r, fo r e n d o c r in e -d is ru p tio n te stin g, e s p e c ia lly fo r lif c - c y c lc te s ts, m y s id s o ffe r c le a r a d v a n ta g e s o v e r a m p h ip o d s. M o s t m a rin e a m p h ip o d s u s e d in to x ic o lo g ic a l te s tin g m u s t b e c o lle c te d fro m th e ir n a t u ra l h a b ita ts b e fo re u s e in te s ts. A lth o u g h th e y can b e h e ld fo r a fe w w e e k s b e fo re te s tin g, th e y g e n e r a lly a re n o t c u ltu re d fo r te sts. C o n v e rse ly, s e v e ra l m y s id s p e c ie s h a v e b e e n c u ltu re d in th e la b o ra to ry a n d u s e d in life -c y c le te s ts [1 3 ]. S e v e ra l m e a s u re s o f re p ro d u c tiv e p e rfo r m a n c e c a n b e u s e d to a sse ss su b le th a l re s p o n s e in lif e - c y c le te s tin g, in c lu d in g sex u a l m a tu rity. th e tim e to first b ro o d re le a s e, th e tim e re q u ire d fo r eg g d e v e lo p m e n t (a n d its s e p a ra te p h a s e s ), fe c u n d ity, b ro o d s u c c ess, a n d a lte ra tio n s in r e p ro d u c tiv e c h a r a c te ris tic s in p o p u la tio n s [ 1 1,5 8,6 0,6 3,1 0 8,1 2 5,1 2 6,1 2 8,1 3 0,2 1 8,2 1 9 ] (T ab le 2). In h ib ite d re p ro d u c tio n is th e m o s t s e n s itiv e, s u b le th a l p o p u la tio n re s p o n s e o f A. b a h ia c h ro n ic a lly e x p o s e d to p e s tic id e s [6 3 ]. N u m e ro u s s tu d ie s h a v e d e s c rib e d th e u s e o f re p ro d u c tiv e e n d p o in ts in m y s id s a fte r to x ic e x p o s u r e a n d c h a n g e s in th e a b io tic e n v iro n m e n t [1 5,1 2 3,1 3 1,2 2 0 ]. A lth o u g h s ta n d a rd c h ro n ic a ssa y s, in c lu d in g r e p ro d u c tiv e e n d p o in ts, a re d e s c rib e d fo r A. b a h ia, th e s e s h o u ld b e a p p lic a b le to o th e r m y s id s, a lth o u g h th e lo n g e r life c y c le in o th e r s p e c ie s m a y re s tric t th e ir u s e in ro u tin e te stin g. T h e life h is to r y o f A. b a h ia is v e r y a m e n a b le to d e m o g ra p h ic m o d e lin g b e c a u s e o f r a p id g ro w th, e a rly sex u a l d iffe re n tia tio n (a t 14 d ) a n d re p ro d u c tio n (c o m m e n c in g a ro u n d 17 d ), a n d fre q u e n c y o f b ro o d p ro d u c tio n (a v e ra g e o f fiv e to s e v e n p e r fe m a le ) o v e r th e fu ll life s p a n o f 9 0 d [2 2 1,2 2 2 ], T h e s e e n d p o in ts p ro v id e u s e f u l in fo rm a tio n fo r p re d ic tin g p o p u la tio n -le v e l e ffe c ts o f re p ro d u c tiv e to x ic a n ts. H o w ev e r, fu r th e r v a lid a tio n is n e e d e d in m u ltig e n e ra tio n a l la b o ra to ry s tu d ie s as w e ll a s in c o rp o ra tio n o f o th e r p o p u la tio n g ro w th p a ra m e te rs s u c h as d e n s ity d e p e n d e n c e, p re d a tio n, m ig ra tio n, an d c o m p e titio n, b e fo re c o n c lu s io n s c a n b e fo r m u la te d th a t are r e le v a n t fo r n a tu ra l e n v iro n m e n ta l c o n d itio n s. P re lim in a ry tra n s g e n e ra tio n a l re s p o n s e s o f A. b a h ia to a p e s tic id e a c tin g a s a ju v e n ile h o rm o n e a g o n is t h a v e b e e n re p o rte d [2 1 9 ]. S u r v iv a l, g ro w th, d e v e lo p m e n t, a n d re p ro d u c tio n o f th is e s tu a rin e m y s id w e re m o n ito r e d th ro u g h a n e n tire life -c y c le e x p o s u re to fe n o x y c a rb a n d d u rin g th e s e c o n d g e n e ra tio n w ith o u t a d d itio n a l e x p o s u re. J u v e n ile m y s id g ro w th, a n d c a rb o n a n d n i tro g e n a c c u m u la tio n, a s w e ll as m y s id s u rv iv a l th ro u g h th e first b ro o d p ro d u c tio n, w e re s ig n ific a n tly a ffe c te d b y fe n o x y c a rb. O n th e o th e r h a n d, m a tu ra tio n tim e, s e x d e te rm in a tio n, a n d y o u n g p ro d u c tio n w e re n o t s ig n ific a n tly a lte re d d u rin g th e life -c y c le e x p o s u re. H o w e v e r, s e c o n d -g e n e ra tio n a d u lts, e x p o s e d lo fe n o x y c a rb o n ly as d e v e lo p in g e m b ry o s a n d ju v e n ile s, p ro d u c e d fe w e r y o u n g a n d c o n ta in e d s ig n ific a n tly fe w e r m a le s. T h e s e re s u lts d e m o n s tra te c le a rly th e n e e d fo r tra n s - g e n e ra tio n a l s tu d ie s w ith m y s id s to fu lly u n d e rs ta n d th e p o te n tia l c h ro n ic im p a c t o f e n d o c rin e d is ru p to rs. D e ta ile d in fo rm a tio n a n d th e s h o rt life c y c le o f A. b a h ia c le a rly fa v o r th e u s e o f th is s p e c ie s in th e in itia l d e v e lo p m e n t a n d fu rth e r v a lid a tio n o f p o p u la tio n m o d e ls b a s e d on re p r o d u c tiv e e n d p o in ts. A c o n c is e d ra ft o f a d e ta ile d re v ie w p a p e r h a s b e e n p ro d u c e d b y U.S. E n v iro n m e n ta l P ro te c tio n A g e n c y [1 3 ] o n a re c o m m e n d e d p ro to c o l a n d a d d itio n a l d a ta n e e d s fo r a tw o -g e n e ra tio n lif e - c y c le te s t w ith A. b a h ia in th e c o n te x t o f e n d o c rin e d is ru p to rs. In th is re v ie w, th e fo llo w in g e n d p o in ts an d th e ir p re fe rre d m e th o d s fo r q u a n tific a tio n a re g iv e n : s u r v iv a l, m o ltin g fr e q u e n c y, re p ro d u c tio n (s e x u a l m a tu rity, tim e to first b ro o d re le a s e, b ro o d s iz e, a n d n u m b e r o f o ffsp rin g p ro d u c e d ), m e ta b o lic d is ru p tio n, d is ru p tio n in s te ro id m e ta b o lis m, v ite llo g e n in in d u c tio n, c y to c h ro m e P le v e ls, and b lo o d g lu c o s e le v e ls. T a b le 2 s u m m a riz e s th e p o te n tia l e n d p o in ts fo r e v a lu a tin g e n v iro n m e n ta l e n d o c r in e d is ru p tio n in m y s id s. T h e u s e o f m y s id s in fie ld s tu d ie s h a s b e e n e x tre m e ly lim ite d. M c K e n n e y e t al. [9 2 ] a n d C la rk e t al. [9 1 ] p e rfo rm e d e x p e rim e n ts w ith c a g e d m y s id s to e v a lu a te th e le th a l a n d s u b le th a l re s p o n s e s o f A. b a h ia d u rin g fie ld a p p lic a tio n s o f fe n - ih io n, a n o rg a n o p h o s p h a te in s e c tic id e. T o o u r k n o w le d g e, th e s e a re th e o n ly p u b lis h e d s tu d ie s on in s itu e x p o s u re s w ith c a g e d m y s id s. In a d d itio n, s tu d ie s th a t h a v e in v e s tig a te d b io m a rk e r re s p o n s e s in fie ld -e x p o s e d m y s id s a ls o a re v e ry lim ite d

10 1228 Environ. Toxicol. Cheni. 23, 2004 T.A. V erslycke et al. [1 7,9 4 ], C le a rly, fie ld v a lid a tio n o f th e b io m a rk e rs d e s c rib e d in th is re v ie w is a s tro n g re s e a rc h n e e d fo r th e fu tu re. M o r p h o lo g y a n d h is to lo g y M o rp h o lo g ic a l c h a n g e s re s u ltin g fro m e x p o s u re to c o n ta m in a n ts h a v e b e e n d o c u m e n te d fo r m a n y ta x a, in c lu d in g a r th ro p o d s, b u t h a v e n o t b e e n c o n s id e re d w id e ly in m y s id to x ic o lo g ic a l s tu d ie s a s a m e a s u ra b le e n d p o in t [1 3 ]. G e n tile e t al. [7 7 ] re p o rte d m o rp h o lo g ic a l a b e r ra tio n s a t th e o n s e t o f sex u a l m a tu rity in A. b a h ia a n d A. b ig e lo w i e x p o s e d to c a d m iu m in th e la b o ra to ry. In a d d itio n, fie ld o b s e r v a tio n s o f in te rs e x u a lity a n d v a ria b le te ls o n m o rp h o lo g y w e re re p o rte d in N. in te g e r fro m d iffe r e n t E u r o p e a n e s tu a rie s a n d th e B a ltic S e a [ ], M o s t o f th e te ls o n d iff e r e n c e s m a y b e e x p la in e d b y r e g e n e ra tio n o f p a rts d a m a g e d b y p re d a tio n a n d c a n n o t b e re la te d d ire c tly to p h y s io lo g ic a l p e r tu rb a tio n s d u rin g m o ltin g. S till, a g e n e tic o r e p ig e n e tic b a s is c a n n o t b e r u le d o u t c o m p le te ly [2 2 5 ]. T h e d e g re e o f flu c tu a tin g a s y m m e tr y in m y s id s h a s b e e n p ro p o s e d as a q u a n tifia b le m e a s u re o f m o rp h o lo g ic a l a b e rra tio n s a n d is th o u g h t to a ris e fro m e n v iro n m e n ta l o r g e n e tic s tre s s d u rin g d e v e lo p m e n t [1 3 ], B e c a u s e th e re s u lts fro m e a r lie r s tu d ie s on m o rp h o lo g ic a l a b e rra tio n s c o u ld n o t g iv e a c le a r m e c h a n is tic e x p la n a tio n fo r th e o b s e rv e d e ffe c ts, p re lim in a ry s tu d ie s e x a m in in g d iffe r e n t p o te n tia l c h a r a c te ris tic s w o u ld first h a v e to b e p e rfo r m e d in m y s id s, b e fo re fu rth e r c o n s id e rin g this en dpoint. B e h a v io r a l a n d o th e r e n d p o in ts D is ru p tio n o f m y s id s w im m in g a n d p o s itio n m a in te n a n c e b e h a v io r h a s b e e n in v e s tig a te d in la b o ra to ry s tu d ie s w ith N. in te g e r e x p o s e d to s u b le th a l c o n c e n tr a tio n s o f c h lo rp y rifo s (an o rg a n o p h o s p o ro u s p e s tic id e ) a n d c a d m iu m [ ], A l th o u g h th e m o d e o f a c tio n o f th e to x ic a n t o n s w im m in g r e m a in s u n k n o w n, th e a u th o rs s p e c u la te d th a t th e d is ru p tio n in c h lo rp y rifo s - e x p o s e d m y s id s w a s p ro b a b ly d u e to th e in h ib i to ry a c tio n o n a c e ty lc h o lin e s te r a s e. In a d d itio n, C rip e e t al. [2 3 1 ] re p o rte d a re d u c tio n in th e m a x im u m s u s ta in e d s w im m in g s p e e d o f A. b a h ia a fte r e x p o s u re to s u b le th a l le v e ls o f tw o p e s tic id e s. O th e r a u th o rs h a v e in v e s tig a te d th e s w a rm in g b e h a v io r o f m y s id s in la b o ra to ry o r fie ld s tu d ie s [ ], F o r m y s id s, d is ru p tio n o f s w im m in g a n d s w a rm in g b e h a v io r m a y le a d to in c re a s e d p re d a tio n o r d is p la c e m e n t fro m o p tim u m s ite s in th e e s tu a ry [43], O th e r b e h a v io ra l re s p o n s e s th a t h a v e b e en m e a s u re d in m y s id s in c lu d e fe e d in g a c tiv ity [ 3 9, ], g ro o m in g b e h a v io r [2 4 3 ], b u rro w in g a b ility [2 4 4 ] a n d p r e d a to r - p r e y d y n a m ic s [2 4 5 ]. T h e u s e o f b e h a v io ra l re s p o n s e s as a m o n ito rin g to o l, h o w e v e r, h a s little u tility u n le s s b e h a v io ra l c h a n g e s are u n d e rs to o d w ith in a n e c o lo g ic a l c o n te x t; th a t is, h o w w e ll th e p a tte rn s are u n d e rs to o d w ith in th e c o n te x t o f a n a n im a l s n a t u ra l life h a b its a n d e c o lo g ic a l re q u ire m e n ts [2 4 6 ] a n d i f th e c h a n g e s c an b e re la te d c le a rly to in te rn a l re s id u e le v e ls or e n v iro n m e n ta l le v e ls o f s p e c ific c o n ta m in a n ts [2 4 7 ], S e v e ra l s tu d ie s h a v e b e e n p u b lis h e d o n o s m o tic re g u la tio n in m y s id s (W eb b e t al. [2 4 8 ] a n d re fe re n c e s th e re in ) a n d th e in te ra c tio n b e tw e e n o s m o re g u la tio n a n d c h e m ic a l e x p o s u re [6 6,2 4 9 ]. O th e r h o rm o n a l re s p o n s e s a n d d is tu rb a n c e s in c ru s ta c e a n s, su ch a s c o lo r c h a n g e s (o n e o f th e e a r lie s t s tu d ie d p h e n o m e n a th a t p ro v id e d d e fin ite p r o o f o f a h o rm o n e -m e d ia te d p ro c e s s in a c ru s ta c e a n ), re tin a l p ig m e n ts, a n d lim b re g e n e r a tio n a re d is c u s s e d in a re v ie w b y F in g e rm a n e t al. [8], H o w ever, th e u s e o f th e s e e n d p o in ts in m y s id s a w a its f u r th e r stu d y. C O N C L U SIO N T h is re v ie w c le a r ly d e m o n s tra te s th e e c o lo g ic a l re le v a n c e an d th e p o te n tia l u s e o f m y s id s h rim p a s te s t s p e c ie s fo r th e e v a lu a tio n o f e n v iro n m e n ta l e n d o c rin e d is ru p tio n a n d as a p o te n tia l s u rro g a te fo r m a n y o th e r c ru s ta c e a n s. T h e h ig h ly s ta n d a rd iz e d u s e o f m y s id s in to x ic ity te s tin g is a n im p o r ta n t a d v a n ta g e a n d re s e a rc h s h o u ld b e d ire c te d a t e v a lu a tin g th e c u r r e n t s ta n d a rd iz e d e n d p o in ts, s u c h a s s u rv iv a l, g ro w th, a n d r e p ro d u c tio n, p re fe ra b ly th ro u g h an e n tire life c y c le, w ith a n u m b e r o f e n d o c rin e d is ru p to rs. In th is c o n te x t, a n u m b e r o f re fe re n c e c h e m ic a ls, c h o s e n fo r th e ir p o s s ib le m o d e o f a c tio n (i.e., e c d y s o n e a g o n is t, e s tr o g e n a n ta g o n is t, ju v e n ile h o rm o n e a g o n is ts, a n d o th e rs ) w a s p ro p o s e d b y D e F u r e t al. [7] for e v a lu a tin g re la tiv e e n d p o in t s e n s itiv ity to p o te n tia l e n d o c rin e - d is ru p tin g c o m p o u n d s. In a d d itio n, e v id e n c e o f tra n s g e n e ra - tio n a l e ffe c ts h a s b e en p u b lis h e d a n d p re s e n tly a tw o -g e n e r atio n life -c y c le p ro to c o l is b e in g in v e s tig a te d w ith th e s ta n d a rd s p e c ie s A. b a h ia. H o w e v e r, a n e x te n s iv e lis t o f n o n s ta n d a r d iz e d e n d p o in ts h a s b e e n p u b lis h e d a n d s h o u ld b e in v e s tig a te d fu rth er. S o m e o f th e s e e n d p o in ts, su ch as d is ru p tio n o f e c d y s te ro id m e ta b o lis m a n d e m b ry o n ic d e v e lo p m e n t, m ig h t d iffe re n tia te fo r in v e rte b ra te -s p e c ific e ffe c ts o f c h e m ic a ls. T h e s e le c tio n o f w h ic h m y s id s p e c ie s to u s e w ill b e a b a la n c e o f its e c o lo g ic a l re le v a n c e a n d its e a s e o f u s e fo r m e a s u rin g th e s e le c te d e n d p o in ts. C le a rly, th e a m o u n t o f a v a ila b le in fo r m a tio n a n d th e re la tiv e ly s h o rt lif e c y c le o f A. b a h ia fa v o r th e u s e o f th is s p e c ie s, b u t its n a r r o w s a lin ity a n d te m p e ra tu re ra n g e lim it its u s e in c o ld e r w a te r o r lo w -s a lin ity te stin g. V ario u s o th e r m y s id s p e c ie s a re p ro p o s e d in th is re v ie w, to g e th e r w ith a lis t o f p o te n tia l e n d p o in ts to e v a lu a te th e e ffe c ts o f e n d o c rin e d is ru p to rs in th e s e a n im a ls. T h e s e s h o u ld s tim u la te th e s c ie n tific c o m m u n ity to e x p lo re fu r th e r th e u s e o f m y s id s h rim p as a n in v e rte b ra te m o d e l fo r th e e v a lu a tio n o f e n v i ro n m e n ta l e n d o c rin e d is ru p tio n. Acknow ledgem ent This study w as supported by a grant from the Flem ish Institute for the Prom otion o f Innovation by Science and Technology to T.A. Verslycke. R E F E R E N C E S 1. 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A bstracts, SETA C 20th Annual M eeting, Philadelphia, PA, USA, N ovem ber 14-18, 1999, p Van Sprang P, L eger P, Sorgeloos P A new test system for the evaluation o f toxic levels o f liposoluble products in the aquatic food chain using Artem ia and M ysidopsis bahia as experimental animals. A q u a t Toxicol 19: Kulin A, M unns WR Jr, Pouchcr S, Cham plin D, Lussier S Prediction o f population-level response from m ysid toxicity test data using population m odeling techniques. Environ Toxicol Chem 19: K uhn A, M unns W R Jr, C ham plin D, M ck inney R, Tagliabue M, Serbst J, G leason T E valuation o f the efficacy o f extrapolation population m odeling to predict the dynam ics o f Am ericam ysis bahia populations in the laboratory. Environ Toxico l Chem 20: C hojnacki J, C iupinski M Telson variability in N eom ysis integer (Leach, 1815) from the southern Baltic. Baltic Sea E n viron P roc 19: Hough AR, B annister N J, N ay lo r E Intersexuality in the m ysid N eom ysis integer. 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J E xp M ar B io l E col 257: Jerling HL, W ooldridge TH Feeding o f tw o m ysid species on plankton in a tem perate South A frican estuary../ E xp M ar B iol E col 188: N im m o DR, H am aker TL, M atthew s E, M oore JC An overview o f the acute and chronic effects o f first and second generation pesticides on an estuarine mysid. In V ernbergi, Thunberg F, C alabrese A. V ernberg W, eds, B iological M onitoring o f Pollutants. Academ ic, New York, NY, USA, pp V iherluoto M, V iitasalo M E ffect o f light on the feeding rates o f pelagic and littoral m ysid shrim ps: A trade-off betw een feeding success and predation a v o id a n c e../ E xp M a r B io l E col 261: Acosta CA, Poirrier M A Groom ing behavior and associated structures o f the m ysid M ysidopsis bahia. J C rustacean B iol 12: N el R, M cl achlan A, W inter D The effect o f sand particle size on the burrow ing ability o f the beach m ysid G astrosaccus p sam m odytes T attersall. E stuar C oastal S h e lf S ei 4 8 : R adem acher K, K ils U P redator/prey dynam ics offifteenspined stickleback (Spinachia spinachia) and the m ysid (N eom ysis integer). Arch Fish M ar Res 4 3 :J O lla BL, Pearson W H, Studholm e AL A pplicability o f behavioral m easures in environm ental stress assessm ent. R app P -V R e u n C ons In t E xp lo r M cr 179: O lla BL, Atem a J, Forw ard R, K ittredge J, L ivingston RJ, M cl eese DW, M iller D C, V ernberg W B, W ells PG, W ilson K T he role o f behavior in m arine pollution m onitoring. Rapp P -V R eun Cons In t E xplor M er 179: W ebb P, W ooldridge T, S chlacher T O sm oregulation and spatial distribution in four species o f m ysid shrim ps. Comp Biochem Physiol A 117: K line ER, Stekoll M S T he role o f calcium and sodium in toxicity o f an effluent to m ysid shrim p (M ysidopsis bahia). E nviron Toxicol Chem 19: Price W'W, H eard RW, Stuck L O bservations on the genus M ysidopsis Sars, 1864 w ith the designation o f a new genus, A m ericam ysis, and the description o f A m ericam ysis alleni and A. stu cki (Peracarida: M ysidacea: M ysidae), from the G u lf o f M exico. Proc B iol Soc Wash 107: W ard SH Techniques to enhance laboratory culture o f M ysidopsis bahia M olenock. Am F ish Soc Sym p 9: D om ingues PM, Turk PE, A ndrade JP, L ee PG Pilot-scalc production o f m ysid shrim p in a static w ater system. A q u a t Int 6: U.S. Environm ental Protection Agency M ethods for measuring the acute toxicity o f effluents and receiving w aters to freshw ater and m arine, 5th ed. EPA -821-R W ashington, DC M clusky D S Som e effects o f salinity and tem perature on the osm otic and ionic regulation o f Praunus flexu o su s (C rustacea, M ysidacea) from Isefjord. O phelia 18: M cl usky D S, H eard V Som e effects o f salinity on the m ysid Praunus flexuosus. 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