Molecular Mechanisms of Insecticide Resistance - American Chemical

Chapter 14 Plant-Allelochemical-Adapted Glutathione Transferases in Lepidoptera Downloaded by CALIFORNIA INST OF TECHNOLOGY on September 14, 2017 | http://pubs.acs.org Publication Date: September 22, 1992 | doi: 10.1021/bk-1992-0505.ch014

S. J. Yu Department of Entomology and Nematology, University of Florida, Gainesville, F L 32611

Glutathione transferases metabolized toxic allelochemicals, including α,β-unsaturated carbonyl compounds, isothiocyanates and organothiocyanates in lepidopterous insects. These transferase activities in the specialist velvetbean caterpillar are lower than i n the generalist fall armyworm; the activity toward the isothiocyanates in the crucifer-adapted cabbage looper was 2- to 6-fold higher than that i n the fall armyworm. Host plants such as crucifers and umbellifers, and allelochemicals such as coumarins, indoles, flavonoids, isothiocyanates and monoterpenes induced glutathione transferases in these insects. The highly polyphagous Lepidoptera, fall armyworm and corn earworm, possessed multiple glutathione transferases containing six and four isozymes, respectively, whereas the more specialized Lepidoptera, tobacco budworm, cabbage looper and velvetbean caterpillar, had a single form of the enzyme. The results suggest that glutathione transferases play an important role i n allelochemical resistance i n phytophagous Lepidoptera. G l u t a t h i o n e t r a n s f e r a s e s a r e a group o f d e t o x i c a t i o n enzymes c a t a l y z i n g t h e c o n j u g a t i o n o f g l u t a t h i o n e (GSH) with various xenobiotics possessing a reactive e l e c t r o p h i l i c center (1). Since the conjugates are subsequently transformed i n animals t o give excretable m e r c a p t u r i c a c i d s (2.) , g l u t a t h i o n e - d e p e n d e n t c o n j u g a t i o n has been r e g a r d e d as an i m p o r t a n t d e t o x i c a t i o n mechanism i n i n s e c t s a s w e l l a s i n mammals. These enzymes p e r f o r m a v a r i e t y o f r e a c t i o n s i n c l u d i n g (a) t h e S - a l k y l a t i o n o f

0097-6156y92y0505-O174$06.00/0 © 1992 American Chemical Society

Mullin and Scott; Molecular Mechanisms of Insecticide Resistance ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

Downloaded by CALIFORNIA INST OF TECHNOLOGY on September 14, 2017 | http://pubs.acs.org Publication Date: September 22, 1992 | doi: 10.1021/bk-1992-0505.ch014

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GSH by a l k y l h a 1 i d e s and r e l a t e d compounds; (b) t h e r e p l a c e m e n t o f l a b i l e a r y l h a l o g e n o r n i t r o g r o u p s by GSH; (c) t h e r e p l a c e m e n t o f l a b i l e a r a l k y l h a l o g e n by GSH; (d) t h e a d d i t i o n o f GSH t o v a r i o u s e p o x i d e s ; (e) the a d d i t i o n o f GSH t o a,6-unsaturated compounds i n c l u d i n g a l d e h y d e s , k e t o n e s , l a c t o n e s , n i t r i l e s and n i t r o compounds; a n d (f) t h e O - a l k y l and O - a r y l c o n j u g a t i o n o f p h o s p h o r o t h i o a t e s a n d p h o s p h a t e s w i t h GSH (2). Because of t h e i r broad substrate s p e c i f i c i t i e s , glutathione t r a n s f e r a s e s are r e s p o n s i b l e f o r the d e t o x i c a t i o n of numerous t o x i c a n t s . G l u t a t h i o n e t r a n s f e r a s e s are important i n the phase I I m e t a b o l i s m o f r e a c t i v e m e t a b o l i t e s f o r m e d by m i c r o s o m a l o x i d a t i o n s . They a r e a l s o i m p o r t a n t i n t h e p h a s e I m e t a b o l i s m o f o r g a n o p h o s p h o r u s i n s e c t i c i d e s (OP) and a r e b e l i e v e d t o p l a y a n i m p o r t a n t r o l e i n OP r e s i s t a n c e i n i n s e c t s ( 4 , 5 ) . O u r r e c e n t work h a s a l s o shown t h a t g l u t a t h i o n e t r a n s f e r a s e s a r e i n v o l v e d i n t h e metabolism of p o t e n t i a l l y t o x i c a l l e l o c h e m i c a l s i n c l u d i n g a , B - u n s a t u r a t e d c a r b o n y l compounds, i s o t h i o c y a n a t e s a n d organothiocyanates i n lepidopterous insects (6-8). E v i d e n c e a c c u m u l a t e d so f a r i n d i c a t e s t h a t g l u t a t h i o n e t r a n s f e r a s e s p l a y important r o l e s i n the d e t o x i c a t i o n of a l l e l o c h e m i c a l s and may be r e l a t e d t o h o s t p l a n t r a n g e i n phytophagous i n s e c t s . T h i s p a p e r r e v i e w s t h e c u r r e n t knowledge o f t h e b i o c h e m i c a l i n t e r a c t i o n s between g l u t a t h i o n e t r a n s f e r a s e s and a l l e l o c h e m i c a l s i n l e p i d o p t e r o u s s p e c i e s a n d p r e s e n t s experimental evidence suggesting that glutathione transferases play a significant role in allelochemical resistance i n these insects.

D e t o x i c a t i o n o f T o x i c A l l e l o c h e m i c a l s by G l u t a t h i o n e T r a n s f e r a s e s i n Lepidoptera Many a l l e l o c h e m i c a l s p o s s e s s s t r u c t u r a l r e q u i r e m e n t s t o s e r v e as s u b s t r a t e s f o r g l u t a t h i o n e t r a n s f e r a s e s . For example, the a , B - u n s a t u r a t e d c a r b o n y l moiety i s commonly f o u n d i n p l a n t c o n s t i t u e n t s s u c h a s c o u m a r i n s , q u i n o n e s , t e r p e n o i d s , c a r d e n o l i d e s , a l k a l o i d s , e t c . We found t h a t the a l l e l o c h e m i c a l s trans-cinnamaldehyde, trans-2-hexenal t r a n s , t r a n s - 2 , 4 - d e c a d i e n a l and b e n z a l d e h y d e were m e t a b o l i z e d by g l u t a t h i o n e t r a n s f e r a s e s f r o m t h e s o l u b l e f r a c t i o n o f t h e m i d g u t homogenates o f f a l l armyworm (FAW, S p o d o p t e r a f r u q i p e r d a ) l a r v a e with trans-cinnamaldehyde being a preferred substrate ( T a b l e I ) . U s i n g t r a n s - 4 - p h e n y 1 - 3 - b u t e n - 2 - o n e (TPBO) a s a m o d e l s u b s t r a t e , i t was f o u n d t h a t t h e TPBO t r a n s f e r a s e a c t i v i t y was d i f f e r e n t f r o m t h a t t o w a r d t h e m o d e l s u b s t r a t e 1 , 2 - d i c h l o r o - 4 - n i t r o b e n z e n e (DCNB) b a s e d on i t s d i s t r i b u t i o n p a t t e r n i n t h e p r o t e i n f r a c t i o n s i s o l a t e d b y ammonium s u l f a t e f r a c t i o n a t i o n o f t h e c y t o s o l (6). I n a d d i t i o n , p l a n t i s o t h i o c y a n a t e s were f o u n d t o be m e t a b o l i z e d by g l u t a t h i o n e t r a n s f e r a s e f r o m l e p i d o p t e r o u s f



a b

Adapted from Mean + S E .

r e f s . 6-8.

1 3 . 6 + 1.20

Benzyl

thiocyanate

95.0 ± 45.0 80.0 ± 30.0

0.70 0.46 0.67 0.48

A l l y l isothiocyanate Benzyl isothiocyanate

± ± ± +

4.67 10.05 3.59 4.08

Fall armyworm

16.6 + 4.00

57.0 + 20.0 170.0 ± 65.0

Cabbage looper

9.20 + 4.00

0 0

Velvetbean caterpillar

b

Glutathione transferase (nmol/min/mg p r o t e i n )

Glutathione transferase a c t i v i t y i n lepidopterous i n s e c t s toward p l a n t a l l e l o c h e m i c a l s *

Trans.trans-2,4-decadienal Trans.trans-cinnamaldehyde Benzaldehyde Trans-2-hexena1

Substrate

Table I .



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111

l a r v a e u s i n g t h e m i d g u t s o l u b l e f r a c t i o n a s t h e enzyme s o u r c e ( T a b l e I ) . A l l y l and b e n z y l i s o t h i o c y a n a t e s w e r e m e t a b o l i z e d by g l u t a t h i o n e t r a n s f e r a s e from t h e t w o g e n e r a l i s t s , t h e f a l l armyworm and cabbage l o o p e r ( C L , T r i c h o p l u s i a n i ) , b u t no a c t i v i t y was d e t e c t e d from t h e s p e c i a l i s t v e l v e t b e a n c a t e r p i l l a r (VBC, A n t i c a r s i a a e m m a t a l i s ) . The g e n e r a l i s t s , b u t n o t t h e s p e c i a l i s t , a r e a d a p t e d t o f e e d i n g on i s o t h i o c y a n a t e - c o n t a i n i n g c r u c i f e r s . The t r a n s f e r a s e a c t i v i t y t o w a r d t h e s e a l l e l o c h e m i c a l s i n t h e c r u c i f e r - a d a p t e d cabbage l o o p e r was 2 - t o 6 - f o l d h i g h e r t h a n t h a t i n t h e f a l l armyworm. The t r a n s f e r a s e s y s t e m o f f a l l armyworm a l s o metabolized 2 - p h e n y l e t h y l i s o t h i o c y a n a t e , but a c t i v i t y c a n o n l y be o b s e r v e d a f t e r i n d u c t i o n ( 7 ) . Data i n Table I a l s o demonstrated t h a t the o r g a n o t h i o c y a n a t e a l l e l o c h e m i c a l b e n z y l t h i o c y a n a t e was m e t a b o l i z e d by g l u t a t h i o n e t r a n s f e r a s e from l a r v a e o f t h r e e l e p i d o p t e r o u s s p e c i e s , t h e f a l l armyworm, c a b b a g e l o o p e r and v e l v e t b e a n c a t e r p i l l a r , u s i n g t h e m i d g u t s o l u b l e f r a c t i o n a s t h e enzyme s o u r c e . From T a b l e I I , i t c a n be s e e n t h a t t h e s e a , B u n s a t u r a t e d c a r b o n y l compounds, i s o t h i o c y a n a t e s and a n o r g a n o t h i o c y a n a t e were a l l t o x i c t o t h e f a l l armyworm, c a u s i n g a c u t e t o x i c i t y i n n e o n a t e s . Among t h o s e t e s t e d , b e n z y l and 2 - p h e n y l e t h y l i s o t h i o c y a n a t e s w e r e t h e most a c u t e l y t o x i c t o t h e armyworm. The i s o t h i o c y a n a t e s a n d t h e o r g a n o t h i o c y a n a t e were a l s o t o x i c t o o t h e r L e p i d o p t e r a s u c h as t h e v e l v e t b e a n c a t e r p i l l a r and cabbage l o o p e r ( 6 - 8 ) . P u r i f i c a t i o n and C h a r a c t e r i z a t i o n of G l u t a t h i o n e T r a n s f e r a s e s from Lepidoptera G l u t a t h i o n e t r a n s f e r a s e s have been p u r i f i e d and c h a r a c t e r i z e d i n numerous s p e c i e s o f i n s e c t s w i t h p a r t i c u l a r e m p h a s i s on t h e house f l y . M o l e c u l a r w e i g h t s of the g l u t a t h i o n e transferases studied are w i t h i n the r a n g e 3 5 , 0 0 0 - 6 3 , 0 0 0 . They c o n s i s t o f two s u b u n i t s (homodimers and h e t e r o d i m e r s ) o f m o l e c u l a r w e i g h t b e t w e e n 1 9 , 0 0 0 and 3 5 , 0 0 0 ( 9 ) . I n L e p i d o p t e r a , C l a r k g t a l . (10) were t h e f i r s t t o p u r i f y g l u t a t h i o n e t r a n s f e r a s e from a l e p i d o p t e r o u s i n s e c t , t h e g r e a t e r wax moth ( G a l l e r i a m e l l o n e l l a ) , u s i n g a f f i n i t y c h r o m a t o g r a p h y on g l u t a t h i o n e - s u l f o b r o m o p h t h a l e i n - a g a r o s e . Because o f i t s s e l e c t i v e a b i l i t y to bind with glutathione transferase, t h i s enzyme was i s o l a t e d from t h i s s p e c i e s i n s u b s t a n t i a l l y p u r e f o r m . The t r a n s f e r a s e h a d a m o l e c u l a r w e i g h t o f 4 1 , 0 0 0 w i t h two s u b u n i t s o f M 2 5 , 0 0 0 . I t s s u b s t r a t e s p e c i f i c i t y was f o u n d t o r e s e m b l e t h a t o f t h e g l u t a t h i o n e t r a n s f e r a s e B from r a t l i v e r ( 1 1 ) . S e v e r a l g l u t a t h i o n e t r a n s f e r a s e s from t h e p o r i n a moth ( W i s e a n a c e r v i n a t a ) were p u r i f i e d by a f f i n i t y c h r o m a t o g r a p h y , c a t i o n - e x c h a n g e c h r o m a t o g r a p h y and p r e p a r a t i v e i s o e l e c t r o f o c u s i n g (12) . The m a j o r r



178

MOLECULAR MECHANISMS OF INSECTICIDE RESISTANCE

Table I I .

T o x i c i t y of a l l e l o c h e m i c a l s t o neonate armyworm l a r v a e * *

fall

b

Allelochemical

a b

c

L C

50

C

Trans,trans-2,4-decadienal Trans-cinnamaldehyde Benzaldehyde Trans-2-hexenal

0.033 0.077 0.089 0.214

A l l y l isothiocyanate Benzyl isothiocyanate 2-Phenylethyl isothiocyanate

0.017 0.006 0.006

Benzyl thiocyanate

0.041

A d a p t e d from r e f s . 6 - 8 . G r o u p s o f 20 n e o n a t e l a r v a e were f e d a r t i f i c i a l d i e t c o n t a i n i n g a l l e l o c h e m i c a l s f o r 24 h r b e f o r e m o r t a l i t y c o u n t s were made. % i n d i e t (w/w).


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t r a n s f e r a s e was p u r i f i e d t o h o m o g e n e i t y by a f a c t o r o f 5 3 0 - f o l d . The m o l e c u l a r w e i g h t s o f t h e s e t r a n s f e r a s e s w e r e e s t i m a t e d t o be 4 5 , 0 0 0 - 5 0 , 0 0 0 . They a p p e a r e d t o be homodimers o f e i t h e r o f two t y p e s o f s u b u n i t s o f M 22,800 and 2 4 , 6 0 0 . G l u t a t h i o n e t r a n s f e r a s e from t h e d i a m o n d b a c k moth ( P l u t e l l a x y l o s t e l l a ) was p u r i f i e d by e i t h e r t h e t r a d i t i o n a l method i n v o l v i n g ammonium s u l f a t e f r a c t i o n a t i o n , g e l f i l t r a t i o n and h y d r o x y a p a t i t e c h r o m a t o g r a p h y o r by a f f i n i t y c h r o m a t o g r a p h y ( G S H s u l f o b r o m o p h t h a l e i n - a g a r o s e ) . The n a t i v e g l u t a t h i o n e t r a n s f e r a s e h a d m o l e c u l a r w e i g h t s o f 4 5 , 0 0 0 - 4 6 , 5 0 0 . The p u r i f i e d t r a n s f e r a s e a c t i v i t y t o w a r d DCNB i n t h e i n s e c t i c i d e r e s i s t a n t s t r a i n was h i g h e r t h a n i n t h e s u s c e p t i b l e s t r a i n (13.) . T h a t t h i s s p e c i e s p o s s e s s e d a s i n g l e f o r m o f t h e enzyme was c o n f i r m e d by B a l a b a s k a r a n e t a l . (14) who u s e d i o n - e x c h a n g e c h r o m a t o g r a p h y (DEAESephadex A - 5 0 and CM-Sephadex C-50) and a d s o r p t i o n c h r o m a t o g r a p h y ( B i o - G e l HTP h y d r o x y a p a t i t e ) . A c c o r d i n g t o t h e s e a u t h o r s , the p u r i f i e d g l u t a t h i o n e t r a n s f e r a s e had an i s o e l e c t r i c p o i n t o f 9 . 2 6 and a m o l e c u l a r w e i g h t o f 36,400. G l u t a t h i o n e t r a n s f e r a s e s from l a r v a e o f t h e s u g a r b o r e r ( D i a t r a e a s a c c h a r a l i s ) and t h e M e x i c a n r i c e b o r e r (Eoreuma l o f t n i ) w e r e p u r i f i e d by G S H - a f f i n i t y c h r o m a t o g r a p h y and i s o e l e c t r i c f o c u s i n g ( 1 5 ) . Two g l u t a t h i o n e t r a n s f e r a s e s were p u r i f i e d f r o m s u g a r b o r e r l a r v a e . The i s o z y m e w i t h a p i v a l u e o f 9 . 3 was a homodimer o f two s u b u n i t s o f M 2 5 , 0 0 0 , w h e r e a s t h e i s o z y m e w i t h a p i v a l u e o f 8 . 0 was a h e t e r o d i m e r o f s u b u n i t s h a v i n g M 2 5 , 0 0 0 and 2 7 , 0 0 0 . On t h e o t h e r h a n d , t h r e e g l u t a t h i o n e t r a n s f e r a s e s were p u r i f i e d f r o m M e x i c a n r i c e b o r e r l a r v a e . The i s o z y m e s w i t h p i v a l u e s o f 9 . 7 a n d 7 . 7 w e r e homodimers o f s u b u n i t s w i t h M 2 5 , 0 0 0 a n d 2 6 , 0 0 0 , r e s p e c t i v e l y . However, t h e isozyme w i t h a p i v a l u e o f 5 . 3 was a h e t e r o d i m e r o f s u b u n i t s h a v i n g M 26,000 and 2 7 , 0 0 0 . P e p t i d e f i n g e r p r i n t a n a l y s i s r e v e a l e d primary s t r u c t u r a l differences i n these isozymes. We h a v e r e c e n t l y p u r i f i e d and c h a r a c t e r i z e d g l u t a t h i o n e t r a n s f e r a s e s from f i v e s p e c i e s o f L e p i d o p t e r a w i t h d i f f e r i n g d e g r e e s o f p o l y p h a g y . The f a l l armyworm and c o r n earworm (CEW, H e l i c o v e r p a zea) a r e h i g h l y p o l y p h a g o u s i n s e c t s . The t o b a c c o budworm (TBW, H e l i o t h i s v i r e s c e n s ) and c a b b a g e l o o p e r a r e a l s o p o l y p h a g o u s i n s e c t s b u t a r e more s p e c i a l i z e d . The f o r m e r f e e d s m a i n l y on t o b a c c o and c o t t o n , w h i l e t h e l a t t e r p r e f e r e n t i a l l y f e e d s on p l a n t s i n t h e cabbage f a m i l y . The v e l v e t b e a n c a t e r p i l l a r i s a s p e c i a l i s t i n s e c t f e e d i n g m a i n l y on c e r t a i n s p e c i e s o f legumes. We h a v e f o u n d t h a t g l u t a t h i o n e t r a n s f e r a s e s c a n be p u r i f i e d from t h e s e l e p i d o p t e r o u s i n s e c t s t o apparent h o m o g e n e i t y u s i n g a t w o - s t e p p r o c e d u r e i n v o l v i n g ammonium s u l f a t e f r a c t i o n a t i o n and a f f i n i t y c h r o m a t o g r a p h y on a g l u t a t h i o n e - a g a r o s e column ( 1 6 ) . P u r i f i c a t i o n s were 22, 4 5 , 3 7 , 9 and 3 3 - f o l d i n t h e FAW, CEW, TBW, CL and V B C ,


r

r

r

r

r



180


r e s p e c t i v e l y , a s compared w i t h t h e s p e c i f i c a c t i v i t y i n t h e r e s p e c t i v e s o l u b l e f r a c t i o n ( T a b l e I I I ) . The s p e c i f i c a c t i v i t y of the p u r i f i e d g l u t a t h i o n e transferases ranged f r o m 2 . 2 9 /xmol/min/mg p r o t e i n i n t h e VBC t o 5 8 . 3 5 /xmol/min/mg p r o t e i n i n t h e CEW, a d i f f e r e n c e o f 2 5 - f o l d i n enzyme a c t i v i t y . The b i o c h e m i c a l c h a r a c t e r i s t i c s o f p u r i f i e d g l u t a t h i o n e t r a n s f e r a s e s from t h e f i v e l e p i d o p t e r o u s s p e c i e s a r e shown i n T a b l e I V . A n a l y s i s o f t h e a f f i n i t y p u r i f i e d p r e p a r a t i o n s by p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (PAGE) u n d e r n o n d e n a t u r i n g c o n d i t i o n s r e v e a l e d t h e p r e s e n c e o f 6, 4 , 1, 1 and 1 p r o t e i n s p e c i e s i n t h e FAW, CEW, TBW, CL and VBC, r e s p e c t i v e l y . T h e s e p r o t e i n s p e c i e s were r e g a r d e d as g l u t a t h i o n e t r a n s f e r a s e i s o z y m e s s i n c e e a c h band c a t a l y z e d g l u t a t h i o n e c o n j u g a t i o n w i t h CDNB. When t h e p r e p a r a t i o n s were a n a l y z e d by i s o e l e c t r i c f o c u s i n g i n p o l y a c r y l a m i d e g e l s , t h e same number o f p r o t e i n bands a s f o u n d i n PAGE a r e a l s o o b s e r v e d i n e a c h s p e c i e s . The p i v a l u e s r a n g e d f r o m 4 . 4 5 t o 6 . 0 0 among t h e f i v e s p e c i e s , i n d i c a t i n g t h a t t h e s e t r a n s f e r a s e s were a l l a c i d i c i s o z y m e s . SDS-PAGE o f t h e p u r i f i e d enzymes showed 4 , 3 , 3 , 2 and 2 p r o t e i n b a n d s i n t h e FAW, CEW, TBW, CL and VBC, r e s p e c t i v e l y . The s u b u n i t m o l e c u l a r w e i g h t s r a n g e d from 2 7 , 0 0 0 t o 3 2 , 0 0 0 w i t h t h e s u b u n i t ( M 29,000) b e i n g found i n each s p e c i e s . Since i n s e c t g l u t a t h i o n e transferases e x i s t i n dimers (homodimers a n d h e t e r o d i m e r s ) ( 9 ) , t h e m o l e c u l a r w e i g h t s of the n a t i v e g l u t a t h i o n e t r a n s f e r a s e s would l i k e l y range f r o m 5 5 , 0 0 0 t o 6 4 , 0 0 0 b a s e d on t h e i r s u b u n i t m o l e c u l a r w e i g h t s . I t i s n o t known why t h e TBW, w h i c h p o s s e s s e d a s i n g l e f o r m o f g l u t a t h i o n e t r a n s f e r a s e , showed t h r e e s u b u n i t s i n S D S - P A G E . The p o s s i b i l i t y e x i t s t h a t one o f t h e s u b u n i t s may h a v e been an a r t i f a c t r e s u l t i n g f r o m d e g r a d a t i o n o f t h e enzyme. K i n e t i c s t u d i e s showed t h a t t h e p u r i f i e d g l u t a t h i o n e t r a n s f e r a s e s among t h e s e i n s e c t s were q u a l i t a t i v e l y L d i f f e r e n t b a s e d on t h e i r K v a l u e s e x c e p t f o r t h e e q u i v a l e n t K s o f CEW and TBW. T h a t g l u t a t h i o n e transferases are q u a l i t a t i v e l y d i f f e r e n t i n the f i v e s p e c i e s was a l s o s u p p o r t e d by t h e r e s u l t s o b t a i n e d f r o m t h e enzyme i n h i b i t i o n s t u d y . A s shown i n T a b l e I V , t h e TBW g l u t a t h i o n e t r a n s f e r a s e was most s e n s i t i v e t o i n h i b i t i o n b y q u e r c e t i n , f o l l o w e d by CEW, C L , FAW a n d VBC. I t h a s r e c e n t l y been d e m o n s t r a t e d f o r h o u s e f l i e s t h a t some g l u t a t h i o n e t r a n s f e r a s e i s o z y m e s p o s s e s s i n g DCNB a c t i v i t y do n o t n e c e s s a r i l y b i n d t o G S H - a g a r o s e ( C l a r k e t a l . , P r o t e i n E x p r e s s i o n and P u r i f i c a t i o n , i n p r e s s ) . Therefore, the p o s s i b i l i t y of the presence of o t h e r i s o z y m e s i n t h e s e l e p i d o p t e r o u s s p e c i e s c a n n o t be ruled out. r

m

m



VBC

CL

TBW

CEW

FAW

b

a

Species

Adapted from r e f . Mean ± S E .

16.

Cytosolic fraction Ammonium s u l f a t e (45-75%) GSH-agarose Cytosolic fraction Ammonium s u l f a t e GSH-agarose Cytosolic fraction Ammonium s u l f a t e GSH-agarose Cytosolic fraction Ammonium s u l f a t e GSH-agarose Cytosolic fraction Ammonium s u l f a t e GSH-agarose

0.47 1.05 10.37 1.30 1.93 58.35 0.47 1.41 17.50 1.35 2.73 12.16 0.07 0.11 2.29

69 12 100 80 8 100 62 10 100 69 3 100 54 26

+ + + + + + + + + + + + + +

0.13 1.49 0.04 0.08 2.29 0.10 0.09 3.72 0.27 0.47 1.86 0.01 0.07 0.33

+ 0.06

b

Specific activity (/imol CDNB/min/mg protein)

100

Yield (%)

2.2 22.1 1.0 1.5 44.9 1.0 3.0 37.2 1.0 2.0 9.0 1.0 1.6 32.9

1.0

Purification factor

P u r i f i c a t i o n of g l u t a t h i o n e t r a n s f e r a s e s from f i v e lepidopterous i n s e c t s *

Purification step

Table I I I .



K

ra

0.40

0.40

0.56 9.09

CEW

TBW

CL VBC

12.5 18.2

50.0

50.0

17.2

Vmax (/xmol/min/ mg p r o t e i n )

22 100

0

0

23

D

46 100

0

12

71

10~ M

32,000 29,000 27,500 31,000 31,000 29,000

32,000 29,000 27,500

30,000 29,500 29,000 28,200

r

Subunit M

SOURCE: Reprinted with permissionfromref. 16. Copyright 1989 Academic.

5.10 4.85

6.0 5.10 5.00 4.75 4.55 4.45 5.10 5.05 4.90 4.75 5.45

PI

enzyme

0.53 0.44

0.34 0.42 0.46 0.51 0.55 0.59 0.38 0.42 0.46 0.54 0.47

Isozyme (Rf)

f r o m e a c h s p e c i e s was u s e d a s

10~* M

I n h i b i t i o n by quercetin (% o f c o n t r o l )

Biochemical c h a r a c t e r i s t i c s o f p u r i f i e d g l u t a t h i o n e t r a n s f e r a s e s from f i v e lepidopterous i n s e c t s *

A f f i n i t y purified preparation s o u r c e t o w a r d CDNB.

1.33

(mM)

FAW

Species

T a b l e IV.


14. YU Plant-Allelochemical-Adapted Glutathione Transferases

183


I n d u c t i o n o f G l u t a t h i o n e Transferases by A l l e l o c h e m i c a l s i n Lepidoptera G l u t a t h i o n e t r a n s f e r a s e s a r e i n d u c e d by x e n o b i o t i c s s u c h a s b a r b i t u r a t e s and p e s t i c i d e s i n mammals (17) and i n i n s e c t s . O t t e a and P l a p p (18) f o u n d t h a t g l u t a t h i o n e t r a n s f e r a s e was i n d u c e d n e a r l y 3 - f o l d i n h o u s e f l i e s by d i e t a r y p h e n o b a r b i t a l . H a y a o k a and Dauterman (19) a l s o o b t a i n e d i n d u c t i o n i n house f l i e s by v a r i o u s d i e t a r y i n s e c t i c i d e s w i t h c h l o r i n a t e d h y d r o c a r b o n s b e i n g most a c t i v e . More r e c e n t l y , Capua e t a l . (20) h a v e demonstrated i n d u c t i o n of g l u t a t h i o n e transferase i n the b u l b m i t e ( R h i z o q l y p h u s r o b i n i ) by numerous x e n o b i o t i c s , i n c l u d i n g p e n t o b a r b i t a l and t h e i n s e c t i c i d e s f e n p r o p a t h r i n and p r o p o x u r . Glutathione transferases i n lepidopterous insects a r e i n d u c e d by numerous h o s t p l a n t s . U m b e l l i f e r s and c r u c i f e r s w e r e b e t t e r i n d u c e r s o f t h e s e enzymes t h a n o t h e r p l a n t s s c r e e n e d ( T a b l e V ) . P a r s n i p was t h e b e s t i n d u c e r among t h o s e t e s t e d , c a u s i n g a 3 9 - f o l d i n c r e a s e compared w i t h a c t i v i t y i n l a r v a e f e d a r t i f i c i a l d i e t . However, p l a n t s such as soybeans, sorghum, m i l l e t , B e r m u d a g r a s s , c o r n , p o t a t o , c u c u m b e r , c a r r o t and b r o c c o l i h a d no e f f e c t on t h i s enzyme (21,22.) . Time c o u r s e s t u d i e s showed t h a t t h e maximum i n d u c t i o n o f t h e t r a n s f e r a s e by cowpeas o c c u r r e d two d a y s a f t e r f e e d i n g began ( 2 1 ) . The m e t h a n o l l e a f e x t r a c t o f a r e s i s t a n t s o y b e a n v a r i e t y (PI227687) i n d u c e d g l u t a t h i o n e t r a n s f e r a s e i n the soybean l o o p e r ( P s e u d o p l u s i a includens) (23). Induction of glutathione transferases also occurs i n d e c i d u o u s t r e e - f e e d i n g i n s e c t s . L i n d r o t h (24.) f o u n d t h a t g l u t a t h i o n e t r a n s f e r a s e a c t i v i t i e s i n t h e l u n a moth ( A c t i a s l u n a ) l a r v a e f e d b l a c k w a l n u t , b u t t e r n u t and s h a g b a r k h i c k o r y were 2 t o 3 - f o l d h i g h e r t h a n i n those fed paper b i r c h . Furthermore, microsomal g l u t a t h i o n e t r a n s f e r a s e a c t i v i t y v a r i e d up t o 5 - f o l d among e a s t e r n t i g e r s w a l l o w t a i l ( P a p i l i o g l a u c u s glaucus) l a r v a e fed b l a c k c h e r r y , t u l i p t r e e , paper b i r c h , w h i t e a s h and basswood (2j>) . The i n d u c t i o n i s b e l i e v e d t o be due t o a l l e l o c h e m i c a l s i n t h e p l a n t s . The i d e n t i t y o f t h e g l u t a t h i o n e t r a n s f e r a s e i n d u c e r i n p a r s n i p l e a v e s was d e t e r m i n e d by t h i n - l a y e r c h r o m a t o g r a p h y , h i g h - p r e s s u r e l i q u i d c h r o m a t o g r a p h y , g a s c h r o m a t o g r a p h y , and mass s p e c t r o m e t r y as x a n t h o t o x i n , a l i n e a r furanocoumarin (26). O t h e r a l l e l o c h e m i c a l s such as i n d o l e 3 a c e t o n i t r i l e , i n d o l e 3 - c a r b i n o l , i n d o l e 3-B-D g l u c o s i d e , flavone, a l l y l isothiocyanate, benzyl isothiocyanate, 2p h e n y l e t h y l i s o t h i o c y a n a t e , b e n z y l t h i o c y a n a t e and s i n i g r i n a l s o i n d u c e d t h e t r a n s f e r a s e i n f a l l armyworms (Table V ) . Xanthotoxin a l s o induced g l u t a t h i o n e transferase i n the black s w a l l o w t a i l ( P a p i l i o polyxenes) and c a b b a g e l o o p e r and h a r m i n e , an i n d o l e a n a l o g , i n d u c e d t h e t r a n s f e r a s e i n t h e cabbage l o o p e r ( 2 7 ) .



184


I n a d d i t i o n , d i e t a r y c o u m a r i n and m o n o t e r p e n e s (cepinene, B-pinene, limonene, terpinene) induced g l u t a t h i o n e t r a n s f e r a s e i n s o u t h e r n armyworm ( S p o d o p t e r a e r i d a n i a ) l a r v a e (2IL) . H o w e v e r , m o n o t e r p e n e s were n o t i n d u c e r s o f t h e t r a n s f e r a s e i n f a l l armyworm l a r v a e (21) • C o u m e s t r o l , a coumarin analog found i n a r e s i s t a n t soybean c u l t i v a r , induced g l u t a t h i o n e t r a n s f e r a s e i n s o y b e a n l o o p e r s (22.) • 2 - T r i d e c a n o n e f o u n d i n w i l d t o m a t o l e a v e s i n d u c e d g l u t a t h i o n e t r a n s f e r a s e i n t o b a c c o budworm larvae (30). K i n e t i c s t u d y r e v e a l e d a q u a n t i t a t i v e , b u t no q u a l i t a t i v e d i f f e r e n c e between t h e g l u t a t h i o n e t r a n s f e r a s e o f s o y b e a n - and c o w p e a - f e d f a l l armyworms ( 2 1 ) . These r e s u l t s support the n o t i o n t h a t t h e s e i n d u c e d t r a n s f e r a s e s a r e n o t s e p a r a t e i s o z y m e s . Our r e c e n t w o r k also i n d i c a t e d that induction of glutathione transferase i n f a l l armyworm l a r v a e by x a n t h o t o x i n i n c r e a s e d l e v e l s of the e x i s t i n g isozymes but d i d not r e s u l t i n p r o d u c t i o n o f a new i s o z y m e ( 1 6 ) . G l u t a t h i o n e t r a n s f e r a s e s toward the t o x i c a l l e l o c h e m i c a l s trans-cinnamaldehyde. benzaldehyde, a l l y l i s o t h i o c y a n a t e , b e n z y l i s o t h i o c y a n a t e and b e n z y l t h i o c y a n a t e c a n be i n d u c e d by v a r i o u s a l l e l o c h e m i c a l s i n c l u d i n g r e s p e c t i v e s u b s t r a t e s ( T a b l e V I ) . Hence a l l e l o c h e m i c a l i n d u c t i o n o f i t s own m e t a b o l i s m c a n o c c u r i n p h y t o p h a g o u s i n s e c t s . However, x a n t h o t o x i n , a p o t e n t i n d u c e r o f t h e enzyme, was n o t m e t a b o l i z e d by g l u t a t h i o n e t r a n s f e r a s e f r o m l a r v a e o f t h e f a l l armyworm (6) a n d b l a c k s w a l l o w t a i l (27) . U s i n g t w o a l l e l o c h e m i c a l s , x a n t h o t o x i n and i n d o l e 3 a c e t o n i t r i l e , as i n d u c e r s o f g l u t a t h i o n e t r a n s f e r a s e ( t o w a r d DCNB), m a r g i n a l i n d u c t i o n (16-39%) was f o u n d i n t h e s p e c i a l i s t VBC compared t o 1580-2544% i n t h e g e n e r a l i s t FAW ( F i g u r e 1 ) . Enzyme a s s a y s i n i n d i v i d u a l l a r v a e showed t h a t i n d u c i b i l i t y o f g l u t a t h i o n e t r a n s f e r a s e v a r i e d considerably i n the populations of the s p e c i a l i s t and g e n e r a l i s t s p e c i e s ( F i g u r e s 2 and 3 ) . A l l i n d i v i d u a l s w e r e i n d u c e d i n t h e FAW p o p u l a t i o n c o m p a r e d t o o n l y one t h i r d o f t h e VBC p o p u l a t i o n b e i n g i n d u c e d . M o r e o v e r , t h e n e t i n c r e a s e i n enzyme a c t i v i t y due t o i n d u c t i o n was much h i g h e r i n t h e FAW t h a n i n t h e V B C . The o b s e r v e d d i f f e r e n c e s i n enzyme i n d u c i b i l i t y c o u l d be a t t r i b u t e d t o t h e q u a l i t a t i v e a s w e l l as q u a n t i t a t i v e d i f f e r e n c e s i n g l u t a t h i o n e t r a n s f e r a s e isozymes i n these species. Role o f G l u t a t h i o n e T r a n s f e r a s e s i n Insect Herbivory As mentioned above, g l u t a t h i o n e t r a n s f e r a s e s a r e i n v o l v e d i n the i n v i t r o metabolism of t o x i c a l l e l o c h e m i c a l s i n p h y t o p h a g o u s L e p i d o p t e r a . The a l l e l o c h e m i c a l s t r a n s cinnamaldehyde, trans-hexena1, trans,trans-2,4d e c a d i e n a l and b e n z a l d e h y d e , a l l o f w h i c h c o n t a i n t h e a,B-unsaturated c a r b o n y l m o i e t y , were m e t a b o l i z e d by g l u t a t h i o n e t r a n s f e r a s e from m i d g u t homogenates o f FAW.


14. YU Plant-Allelochemical-Adapted Glutathione Transferases Table V.

Glutathione t r a n s f e r a s e a c t i v i t y o f f a l l armyworm l a r v a e f e d host p l a n t s and a l l e l o c h e m i c a l s *


Inducer

a

Glutathione transferase (% o f a r t i f i c i a l d i e t - f e d

Cotton Peanuts C h i n e s e cabbage Cabbage Collards Cowpeas Radish Turnip Mustard Parsley Parsnip

142 179 221 404 472 493 636 731 782 1944 3909

I n d o l e 3 - a c e t o n i t r i l e (0.2%) I n d o l e 3 - c a r b i n o l (0.2%) I n d o l e 3 - B - D - g l u c o s i d e (0.2%) F l a v o n e (0.2%) S i n i g r i n (0.2%) 2 - P h e n y l e t h y l i s o t h i o c y a n a t e (0.025%) X a n t h o t o x i n (0.01%)

1801 388 202 687 342 217 2644

Adapted from r e f s .

—

185

(DCNB) insects)

2 1 , 22 a n d 2 6 .

3.000

c

VBC

FAW

Xanthotoxin (0.01%)

VBC

FAW

Indole 3-acetonitrile (0.1-0.2%)

F i g . 1. Induction of glutathione transferase a c t i v i t y by a l l e l o c h e m i c a l s i n v e l v e t b e a n c a t e r p i l l a r a n d f a l l armyworm l a r v a e . Mullin and Scott; Molecular Mechanisms of Insecticide Resistance ACS Symposium Series; American Chemical Society: Washington, DC, 1992.


13

237

249

Benzaldehyde

542

1092 550 625 558

Allyl isothiocyanate

1006

550

Benzyl isothiocyanate

162

515 412 426 456

Adapted from r e f s . 6 - 8 . N e w l y m o l t e d s i x t h - i n s t a r l a r v a e were f e d a r t i f i c i a l d i e t s c o n t a i n i n g t h e a l l e l o c h e m i c a l s (0.2%, u n l e s s o t h e r w i s e s t a t e d ) f o r t w o d a y s b e f o r e enzyme a s s a y s were c o n d u c t e d .

93

217

218

Transcinnamaldehyde

Benzyl thiocyanate

transferase larvae*

Glutathione transferase with substrate (% o f c o n t r o l a c t i v i t y )

E f f e c t o f d i e t a r y a l l e l o c h e m i c a l s on g l u t a t h i o n e a c t i v i t y toward a l l e l o c h e m i c a l s i n f a l l armyworm

Indole 3 - a c e t o n i t r i l e Indole 3-carbinol Flavone X a n t h o t o x i n (0.01%) T r a n s - c innamaldehyde Benzaldehyde A l l y l isothiocyanate (0.025%) Benzyl isothiocyanate (0.025%) Benzyl thiocyanate

Treatment

Table V I .



14. YU

Plant-AUelochemical-Adapted Glutathione Transferases

40

Control 16

12

8

-

8

LL

Xanthotoxin (0.01%)

0

0.4

0.8

1.2

1.6

2.0

2.4

Glutathione transferase (nmol/min/larva)

F i g . 2. Histograms f o r g l u t a t h i o n e t r a n s f e r a s e a c t i v i t y (DCNB) i n i n d i v i d u a l larvae of the f a l l armyworm f e d xanthotoxin (0.01%) (Yu, S. J . J . Econ. Entomol.. i n p r e s s ) .


187



16

-

Control 12

8

-

o c

D WMMM.mM Indole 3-acetonitrile (0.1%)

0

20

40

60

80

100

Glutathione transferase (nmol/min/larva) F i g . 3. H i s t o g r a m s f o r g l u t a t h i o n e t r a n s f e r a s e a c t i v i t y (DCNB) i n i n d i v i d u a l l a r v a e o f t h e velvetbean c a t e r p i l l a r fed i n d o l e 3 - a c e t o n i t r i l e (0.1%).



14. YU

Plant-AUelochemical-Adapted Glutathione Transferases

189

I s o t h i o c y a n a t e s s u c h a s a l l y l , b e n z y l and 2 - p h e n y l e t h y l i s o t h i o c y a n a t e s and o r g a n o t h i o c y a n a t e s s u c h a s b e n z y l t h i o c y a n a t e w e r e m e t a b o l i z e d by g l u t a t h i o n e t r a n s f e r a s e f r o m l a r v a e o f t h e FAW, CL and V B C . F u r t h e r m o r e , numerous a l l e l o c h e m i c a l s induced glutathione transferase a c t i v i t y w h i c h i s p r e s u m e d t o enhance t h e d e t o x i c a t i o n o f a l l e l o c h e m i c a l s . In a d d i t i o n , glutathione transferase a c t i v i t y t o w a r d v a r i o u s a l l e l o c h e m i c a l s was l o w e r i n t h e s p e c i a l i s t VBC t h a n t h a t i n t h e g e n e r a l i s t s FAW and C L , and a c t i v i t y t o w a r d t h e i s o t h i o c y a n a t e s i n t h e c r u c i f e r a d a p t e d c a b b a g e l o o p e r was 2 - t o 6 - f o l d h i g h e r t h a n t h a t i n t h e f a l l armyworm. The r e s u l t s s u p p o r t t h e n o t i o n t h a t g l u t a t h i o n e t r a n s f e r a s e s p l a y an i m p o r t a n t r o l e i n t h e f e e d i n g s t r a t e g i e s o f l e p i d o p t e r o u s i n s e c t s . The h i g h l y p o l y p h a g o u s i n s e c t s may h a v e e v o l v e d m u l t i p l e g l u t a t h i o n e transferases to d e t o x i f y the d i v e r s e t o x i c a l l e l o c h e m i c a l s e n c o u n t e r e d i n t h e i r h o s t p l a n t s . On t h e o t h e r h a n d , s p e c i a l i z e d i n s e c t s w h i c h f e e d on a n a r r o w r a n g e o f h o s t p l a n t s and e n c o u n t e r more s p e c i f i c a l l e l o c h e m i c a l s h a v e a s few as one form o f g l u t a t h i o n e t r a n s f e r a s e . T h e r e f o r e , the isozyme c o m p o s i t i o n o f glutathione transferase i n lepidopterous species i s r e l a t e d t o h o s t p l a n t f e e d i n g . More work i s n e e d e d t o l e a r n the s u b s t r a t e s p e c i f i c i t i e s of these isozymes b e f o r e one c a n f u l l y u n d e r s t a n d t h e m o l e c u l a r m e c h a n i s m s of glutathione-dependent d e t o x i c a t i o n i n these L e p i d o p t e r a . These r e s u l t s suggest t h a t g l u t a t h i o n e t r a n s f e r a s e s p l a y an i m p o r t a n t r o l e i n a l l e l o c h e m i c a l r e s i s t a n c e i n phytophagous L e p i d o p t e r a . Acknowledgments The o r i g i n a l r e s e a r c h r e p o r t e d h e r e was s u p p o r t e d i n p a r t by USDA ( C o m p e t i t i v e R e s e a r c h G r a n t s O f f i c e ) G r a n t s N o . 8 2 - C R C R - 1 - 1 0 9 1 and 8 5 - C R C R - 1 6 8 5 . I w o u l d l i k e t o t h a n k D r s . F . S l a n s k y and G . S . W h e e l e r f o r c r i t i c a l r e a d i n g o f t h i s manuscript. F l o r i d a A g r i c u l t u r a l Experiment S t a t i o n J o u r n a l S e r i e s No.R-01745. Literature Cited 1. 2. 3. 4. 5. 6. 7.

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Molecular Mechanisms of Insecticide Resistance - American Chemical

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