Life Sciences, Vol. 51, pp. 1479-1484 Printed in the USA
Pergamon Press
CONTINUOUS LIGHT INCREASES N-ACETYLTRANSFERASE ACTIVITY IN T H E O P T I C L O B E O F T H E G I A N T F R E S H W A T E R P R A W N M a c r o b r a c h i u m r o s e n b e r g i i de M a n ( C R U S T A C E A : D E C A P O D A ) Boonsirm
Withyachumnarnkul, Anchalee Pongsa-Asawapaiboon S u p a p o r n Ajpru, P a n u S i a m w a l l a Wantanee Trakulrungsi and Chuanpis Samritthong* D e p a r t m e n t of A n a t o m y a n d D e p a r t m e n t of B i o l o g y * F a c u l t y of Science, M a h i d o l U n i v e r s i t y R a m a 6 Rd., B a n g k o k 10400, T h a i l a n d (Received in final form September 3, 1992)
Summary Giant freshwater prawns, Macrobrachium rosenbergii de Man, were reared under three different lighting c o n d i t i o n s : c o n t i n u o u s d a r k n e s s (DD), 12 hr of l i g h t a n d 12 h r of d a r k n e s s (LD 12:12) and c o n t i n u o u s l i g h t (LL). A f t e r o n e month, the prawns were sacrificed and optic l o b e s i s o l a t e d f r o m t h e e y e s t a l k s w e r e d e t e r m i n e d for Nacetyltransferase (NAT) activities and melatonin concentrations. Gonads were weighed and examined under light microscopy. The optic lobes from LL prawns contained significantly higher activities of N A T t h a n t h o s e f r o m LD 1 2 : 1 2 prawns. T h e m e l a t o n i n c o n c e n t r a t i o n s and size and histological f e a t u r e s of t h e g o n a d s f r o m t h e t h r e e g r o u p s of p r a w n s d i d n o t d i f f e r . T h e r e s u l t s indicate that continuous light increases NAT activities in t h e o p t i c l o b e of M. r o s e n b e r g i i b u t h a s n o d r a s t i c e f f e c t o n g o n a d a l growth. The enzyme arylalkylamine N-acetyltransferase (NAT, E.C. 2 . 3 . 1 . 5 ) h a s b e e n i d e n t i f i e d in t h e o p t i c l o b e of g i a n t f r e s h w a t e r prawns, M a c r o b r a c h i u m r o s e n b e r g i i de M a n (i). T h e a c t i v i t i e s w e r e f o u n d to b e m u c h h i g h e r t h a n t h o s e of rat p i n e a l g l a n d s u n d e r s t i m u l a t i o n b y i s o p r o t e r e n o l , a b e t a - a d r e n e r g i c a g o n i s t k n o w n t o s t i m u l a t e NAT activity via the stimulation of the enzyme adenylate cyclase (2,3,4). In c o n t r a s t to t h e d i u r n a l r h y t h m of N A T a c t i v i t y w h i c h is h i g h at n i g h t t i m e in m o s t v e r t e b r a t e s (5,6,7), t h e o p t i c l o b e N A T w a s f o u n d to be d i u r n a l l y n o n - r h y t h m i c (i). T h e d a y t i m e N A T l e v e l s t e n d e d to be h i g h e r t h a n t h e n i g h t t i m e levels, t h o u g h t h e y did not significantly differ. This prompted us to e x p l o r e if increasing light exposure would significantly increase the enzyme activity. And since light-dark cycle has been shown to play a s i g n i f i c a n t r o l e in r e p r o d u c t i v e f u n c t i o n s of s e v e r a l v e r t e b r a t e s b y m o d u l a t i o n o f t h e p i n e a l f u n c t i o n (7), w e t h e r e f o r e e x a m i n e d g o n a d s of t h e p r a w n s r e a r e d u n d e r d i f f e r e n t l i g h t i n g c o n d i t i o n s as well. 0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights reserved.
1480
N-acetyltransferase and Light
Materials
Vol. 51, No. 19, 1992
and Methods
Juvenile M. rosenbergii, aged three months, were reared in laboratory aquarium under aerated flowing water and provided fresh m u s s e l s as f e e d s ad l i b i t u m . T h e a q u a r i u m w a s k e p t in c a b i n e t s with controlled light-dark cycles. The prawns were reared in groups of 20-25 under different lighting conditions; continuous darkness (DD), a l t e r n a t i n g 12 h r of l i g h t a n d 12 h r of d a r k n e s s (LD 12:12, light was on between 06.00-18.00 hr) a n d c o n t i n u o u s l i g h t (LL). T h e a q u a r i u m l i g h t w a s a p p r o x i m a t e l y 600 l u x o n the water surface. A f t e r o n e month, t h e a n i m a l s w e r e w e i g h e d , e y e s t a l k s c u t a n d o p t i c lobes isolated under dissecting microscope. The optic lobe included lamina ganglionalis, medulla externa, medulla interna and m e d u l l a t e r m i n a l i s . T h e t i s s u e s w e r e k e p t f r o z e n at -74 °C p e n d i n g a s s a y s for N A T a c t i v i t i e s a n d m e l a t o n i n levels. G o n a d s ( o v a r i e s or testes) were isolated, weighed, fixed in B o u i n ' s fixative and processed for light microscopic study. All the prawns were sacrificed between 21.00-22.00 hr to m i n i m i z e l i g h t e x p o s u r e to t h e D D group. C u t t i n g of t h e e y e s t a l k s of t h e D D a n d LD 1 2 : 1 2 p r a w n s w a s p e r f o r m e d u n d e r d i m r e d light. D e t e r m i n a t i o n s of N A T a c t i v i t i e s a n d m e l a t o n i n l e v e l s in t h e o p t i c lobes were carried out according to the methods described previously (i). Tissue samples were sonicated with i00 ~i p h o s p h a t e b u f f e r e d s a l i n e (PBS), 0.i M, p H 6.8. T w e n t y m i c r o l i t e r of t h e s o n i c a t e w a s i n c u b a t e d w i t h 30 ~ i of a m i x t u r e c o n t a i n i n g tryptamine (0.6 ~ m o l ) , a c e t y l C o A (0.2 n m o l ) and 3H-acetyl CoA (0.5 ~Ci, specific activity 3.84 Ci/mmol, Amersham, UK). The r e a c t i o n m i x t u r e w a s i n c u b a t e d for 20 m i n at 37 °C. At t h e e n d of the period the acetylated (3H)-tryptamine was extracted into one ml of w a t e r - s a t u r a t e d chloroform a n d w a s h e d w i t h 200 ~ i of the buffer. A 500 ~i aliquot of chloroform was placed into a scintillation v i a l a n d a l l o w e d to e v a p o r a t e d . Ten milliliter of scintillant was added and the sample was counted in a l i q u i d scintillation counter (Beckman LS60OOTA, USA). Blanks, not containing tisssue, were simultaneously carried through the same procedure. Melatonin antibody was purchased from Stockgrand Ltd., U K and melatonin radioimmunoassay w a s p e r f o r m e d a c c o r d i n g to a t e c h n i q u e slightly modified from that recommended by the company. Thirty m i c r o l i t e r of t h e s o n i c a t e w a s a l i q u o t e d a n d d i l u t e d w i t h t r i c i n e b u f f e r , 0.i M, pH 5.5 to m a k e 500 ~i. T w o h u n d r e d m i c r o l i t e r of diluted melatonin antibody was added into the sample and the mixture incubated at r o o m t e m p e r a t u r e for 30 min. One hundred microliter of 3H-melatonin (specific activity 86 Ci/mmol, A m e r s h a m , UK) w a s t h e n a d d e d a n d t h e m i x t u r e i n c u b a t e d at 4 °C for 72 hr. B o u n d a n d f r e e l i g a n d s w e r e s e p a r a t e d b y i n c u b a t i o n w i t h 500 ~i of dextran-coated charcoal for 15 min at 4 °C and centrifuged at 1,500xg, 4°C for 30 min. S e v e n h u n d r e d m i c r o l i t e r of the supernate was aliquoted into a scintillation vial containing i0 ml of t h e s c i n t i l l a n t , t h e m i x t u r e w a s s h a k e n for one hour before count. The standard curve was simultaneously c a r r i e d t h r o u g h t h e s a m e p r o c e d u r e b u t u s i n g 500 ~ i o f m e l a t o n i n standard, containing 0 to 250 p g of m e l a t o n i n in m e l a t o n i n - f r e e optic lobe sonicate, i n s t e a d of t h e u n k n o w n . The melatonin-free optic lobe sonicate was prepared by using charcoal-stripped
Vol. 51, No. 19, 1992
technique
N-acetyltransferase and Light
as d e s c r i b e d
1481
in the p r o t o c o l .
Protein contents were determined in individual samples using Lowry's method (8). N - A c e t y l t r a n s f e r a s e activities and melatonin contents were expressed as p m o l product/hr/~g p r o t e i n a n d p g / ~ g protein, respectively. All data were expressed as mean +/standard e r r o r and s t a t i s t i c a l l y analyzed b y A N O V A and N e w m a n K u e l s test.
Results The LL p r a w n s , b u t not t h e DD prawns, h a d h i g h e r N A T a c t i v i t i e s than those of t h e LD 12:12 p r a w n s (Fig. i). M e l a t o n i n levels varied considerably a m o n g i n d i v i d u a l p r a w n s but t h e g r o u p v a l u e s d i d n o t differ. 301
~ ] NAT
~ ] Melatonln
- 1.6
~ P ( O.OI, compored to L I 2 : DI2 NAT >.
o
K
+
c
m Ar-
I - 0,8
/
z
E
I
z ~Z v~
m z
-0
DD
LDI2:I2 FIG.
LL
1
N-Acetyltransferase and m e l a t o n i n l e v e l s in o p t i c l o b e s of prawns reared under continuous darkness (DD), alternating 12 hr of light and d a r k n e s s (LD 12:12) and c o n t i n u o u s l i g h t (LL). F i g u r e 2 d e p i c t s the r e l a t i v e w e i g h t s of t h e g o n a d s of the t h r e e groups which did not differ statistically. G o n a d s of t h e p r a w n s reared under different lighting conditions had similar microscopic appearance. T h e t e s t e s and o v a r i e s revealed normal features of c e l l types, s p e r m a t o g e n e s i s and o o g e n e s i s ( p i c t u r e n o t shown).
Discussion Findings in t h i s and the p r e v i o u s s t u d y (i) s u g g e s t t h a t light i n c r e a s e s N A T a c t i v i t y in t h e o p t i c l o b e of M. r o s e n b e r g i i . Light has b e e n s h o w n to i n d u c e an i n c r e a s e of d i s t a l p i g m e n t lightadapting hormone (DPLH) and p r o b a b l y erythrophore-concentrating hormone (ECH), crustacean hyperglycemic hormone (CHH) and the neurodepressive hormone (NDH) (9,10,11). Darkness also increased the accumulation of light-dispersing hormone (LDH) in some
1482
N-acetyltransferase and Light
4.0
[ ~ TESTIS
rlJ
Pergamon Press
CONTINUOUS LIGHT INCREASES N-ACETYLTRANSFERASE ACTIVITY IN T H E O P T I C L O B E O F T H E G I A N T F R E S H W A T E R P R A W N M a c r o b r a c h i u m r o s e n b e r g i i de M a n ( C R U S T A C E A : D E C A P O D A ) Boonsirm
Withyachumnarnkul, Anchalee Pongsa-Asawapaiboon S u p a p o r n Ajpru, P a n u S i a m w a l l a Wantanee Trakulrungsi and Chuanpis Samritthong* D e p a r t m e n t of A n a t o m y a n d D e p a r t m e n t of B i o l o g y * F a c u l t y of Science, M a h i d o l U n i v e r s i t y R a m a 6 Rd., B a n g k o k 10400, T h a i l a n d (Received in final form September 3, 1992)
Summary Giant freshwater prawns, Macrobrachium rosenbergii de Man, were reared under three different lighting c o n d i t i o n s : c o n t i n u o u s d a r k n e s s (DD), 12 hr of l i g h t a n d 12 h r of d a r k n e s s (LD 12:12) and c o n t i n u o u s l i g h t (LL). A f t e r o n e month, the prawns were sacrificed and optic l o b e s i s o l a t e d f r o m t h e e y e s t a l k s w e r e d e t e r m i n e d for Nacetyltransferase (NAT) activities and melatonin concentrations. Gonads were weighed and examined under light microscopy. The optic lobes from LL prawns contained significantly higher activities of N A T t h a n t h o s e f r o m LD 1 2 : 1 2 prawns. T h e m e l a t o n i n c o n c e n t r a t i o n s and size and histological f e a t u r e s of t h e g o n a d s f r o m t h e t h r e e g r o u p s of p r a w n s d i d n o t d i f f e r . T h e r e s u l t s indicate that continuous light increases NAT activities in t h e o p t i c l o b e of M. r o s e n b e r g i i b u t h a s n o d r a s t i c e f f e c t o n g o n a d a l growth. The enzyme arylalkylamine N-acetyltransferase (NAT, E.C. 2 . 3 . 1 . 5 ) h a s b e e n i d e n t i f i e d in t h e o p t i c l o b e of g i a n t f r e s h w a t e r prawns, M a c r o b r a c h i u m r o s e n b e r g i i de M a n (i). T h e a c t i v i t i e s w e r e f o u n d to b e m u c h h i g h e r t h a n t h o s e of rat p i n e a l g l a n d s u n d e r s t i m u l a t i o n b y i s o p r o t e r e n o l , a b e t a - a d r e n e r g i c a g o n i s t k n o w n t o s t i m u l a t e NAT activity via the stimulation of the enzyme adenylate cyclase (2,3,4). In c o n t r a s t to t h e d i u r n a l r h y t h m of N A T a c t i v i t y w h i c h is h i g h at n i g h t t i m e in m o s t v e r t e b r a t e s (5,6,7), t h e o p t i c l o b e N A T w a s f o u n d to be d i u r n a l l y n o n - r h y t h m i c (i). T h e d a y t i m e N A T l e v e l s t e n d e d to be h i g h e r t h a n t h e n i g h t t i m e levels, t h o u g h t h e y did not significantly differ. This prompted us to e x p l o r e if increasing light exposure would significantly increase the enzyme activity. And since light-dark cycle has been shown to play a s i g n i f i c a n t r o l e in r e p r o d u c t i v e f u n c t i o n s of s e v e r a l v e r t e b r a t e s b y m o d u l a t i o n o f t h e p i n e a l f u n c t i o n (7), w e t h e r e f o r e e x a m i n e d g o n a d s of t h e p r a w n s r e a r e d u n d e r d i f f e r e n t l i g h t i n g c o n d i t i o n s as well. 0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights reserved.
1480
N-acetyltransferase and Light
Materials
Vol. 51, No. 19, 1992
and Methods
Juvenile M. rosenbergii, aged three months, were reared in laboratory aquarium under aerated flowing water and provided fresh m u s s e l s as f e e d s ad l i b i t u m . T h e a q u a r i u m w a s k e p t in c a b i n e t s with controlled light-dark cycles. The prawns were reared in groups of 20-25 under different lighting conditions; continuous darkness (DD), a l t e r n a t i n g 12 h r of l i g h t a n d 12 h r of d a r k n e s s (LD 12:12, light was on between 06.00-18.00 hr) a n d c o n t i n u o u s l i g h t (LL). T h e a q u a r i u m l i g h t w a s a p p r o x i m a t e l y 600 l u x o n the water surface. A f t e r o n e month, t h e a n i m a l s w e r e w e i g h e d , e y e s t a l k s c u t a n d o p t i c lobes isolated under dissecting microscope. The optic lobe included lamina ganglionalis, medulla externa, medulla interna and m e d u l l a t e r m i n a l i s . T h e t i s s u e s w e r e k e p t f r o z e n at -74 °C p e n d i n g a s s a y s for N A T a c t i v i t i e s a n d m e l a t o n i n levels. G o n a d s ( o v a r i e s or testes) were isolated, weighed, fixed in B o u i n ' s fixative and processed for light microscopic study. All the prawns were sacrificed between 21.00-22.00 hr to m i n i m i z e l i g h t e x p o s u r e to t h e D D group. C u t t i n g of t h e e y e s t a l k s of t h e D D a n d LD 1 2 : 1 2 p r a w n s w a s p e r f o r m e d u n d e r d i m r e d light. D e t e r m i n a t i o n s of N A T a c t i v i t i e s a n d m e l a t o n i n l e v e l s in t h e o p t i c lobes were carried out according to the methods described previously (i). Tissue samples were sonicated with i00 ~i p h o s p h a t e b u f f e r e d s a l i n e (PBS), 0.i M, p H 6.8. T w e n t y m i c r o l i t e r of t h e s o n i c a t e w a s i n c u b a t e d w i t h 30 ~ i of a m i x t u r e c o n t a i n i n g tryptamine (0.6 ~ m o l ) , a c e t y l C o A (0.2 n m o l ) and 3H-acetyl CoA (0.5 ~Ci, specific activity 3.84 Ci/mmol, Amersham, UK). The r e a c t i o n m i x t u r e w a s i n c u b a t e d for 20 m i n at 37 °C. At t h e e n d of the period the acetylated (3H)-tryptamine was extracted into one ml of w a t e r - s a t u r a t e d chloroform a n d w a s h e d w i t h 200 ~ i of the buffer. A 500 ~i aliquot of chloroform was placed into a scintillation v i a l a n d a l l o w e d to e v a p o r a t e d . Ten milliliter of scintillant was added and the sample was counted in a l i q u i d scintillation counter (Beckman LS60OOTA, USA). Blanks, not containing tisssue, were simultaneously carried through the same procedure. Melatonin antibody was purchased from Stockgrand Ltd., U K and melatonin radioimmunoassay w a s p e r f o r m e d a c c o r d i n g to a t e c h n i q u e slightly modified from that recommended by the company. Thirty m i c r o l i t e r of t h e s o n i c a t e w a s a l i q u o t e d a n d d i l u t e d w i t h t r i c i n e b u f f e r , 0.i M, pH 5.5 to m a k e 500 ~i. T w o h u n d r e d m i c r o l i t e r of diluted melatonin antibody was added into the sample and the mixture incubated at r o o m t e m p e r a t u r e for 30 min. One hundred microliter of 3H-melatonin (specific activity 86 Ci/mmol, A m e r s h a m , UK) w a s t h e n a d d e d a n d t h e m i x t u r e i n c u b a t e d at 4 °C for 72 hr. B o u n d a n d f r e e l i g a n d s w e r e s e p a r a t e d b y i n c u b a t i o n w i t h 500 ~i of dextran-coated charcoal for 15 min at 4 °C and centrifuged at 1,500xg, 4°C for 30 min. S e v e n h u n d r e d m i c r o l i t e r of the supernate was aliquoted into a scintillation vial containing i0 ml of t h e s c i n t i l l a n t , t h e m i x t u r e w a s s h a k e n for one hour before count. The standard curve was simultaneously c a r r i e d t h r o u g h t h e s a m e p r o c e d u r e b u t u s i n g 500 ~ i o f m e l a t o n i n standard, containing 0 to 250 p g of m e l a t o n i n in m e l a t o n i n - f r e e optic lobe sonicate, i n s t e a d of t h e u n k n o w n . The melatonin-free optic lobe sonicate was prepared by using charcoal-stripped
Vol. 51, No. 19, 1992
technique
N-acetyltransferase and Light
as d e s c r i b e d
1481
in the p r o t o c o l .
Protein contents were determined in individual samples using Lowry's method (8). N - A c e t y l t r a n s f e r a s e activities and melatonin contents were expressed as p m o l product/hr/~g p r o t e i n a n d p g / ~ g protein, respectively. All data were expressed as mean +/standard e r r o r and s t a t i s t i c a l l y analyzed b y A N O V A and N e w m a n K u e l s test.
Results The LL p r a w n s , b u t not t h e DD prawns, h a d h i g h e r N A T a c t i v i t i e s than those of t h e LD 12:12 p r a w n s (Fig. i). M e l a t o n i n levels varied considerably a m o n g i n d i v i d u a l p r a w n s but t h e g r o u p v a l u e s d i d n o t differ. 301
~ ] NAT
~ ] Melatonln
- 1.6
~ P ( O.OI, compored to L I 2 : DI2 NAT >.
o
K
+
c
m Ar-
I - 0,8
/
z
E
I
z ~Z v~
m z
-0
DD
LDI2:I2 FIG.
LL
1
N-Acetyltransferase and m e l a t o n i n l e v e l s in o p t i c l o b e s of prawns reared under continuous darkness (DD), alternating 12 hr of light and d a r k n e s s (LD 12:12) and c o n t i n u o u s l i g h t (LL). F i g u r e 2 d e p i c t s the r e l a t i v e w e i g h t s of t h e g o n a d s of the t h r e e groups which did not differ statistically. G o n a d s of t h e p r a w n s reared under different lighting conditions had similar microscopic appearance. T h e t e s t e s and o v a r i e s revealed normal features of c e l l types, s p e r m a t o g e n e s i s and o o g e n e s i s ( p i c t u r e n o t shown).
Discussion Findings in t h i s and the p r e v i o u s s t u d y (i) s u g g e s t t h a t light i n c r e a s e s N A T a c t i v i t y in t h e o p t i c l o b e of M. r o s e n b e r g i i . Light has b e e n s h o w n to i n d u c e an i n c r e a s e of d i s t a l p i g m e n t lightadapting hormone (DPLH) and p r o b a b l y erythrophore-concentrating hormone (ECH), crustacean hyperglycemic hormone (CHH) and the neurodepressive hormone (NDH) (9,10,11). Darkness also increased the accumulation of light-dispersing hormone (LDH) in some
1482
N-acetyltransferase and Light
4.0
[ ~ TESTIS
rlJ
