Chapter 11
Variability in Steroid Metabolism Among Phytophagous Insects James A. Svoboda and Malcolm J. Thompson Insect Physiology Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705
Members of the class Insecta require an exogenous source of sterol to support normal development and reproduction. Cholesterol will satisfy this need in nearly all species studied, but many phytophagous and omnivorous insects thrive on diets containing little or no cholesterol. Most of these species that have been critically examined are able to dealkylate and convert dietary 24-alkyl (C and C ) phytosterols to cholesterol. However, significant variations in the utilization and metabolism of dietary sterols between phytophagous species have been discovered in recent years. Thus, it is becoming increasingly difficult to generalize about sterol metabolism even among members of the same Order. These differences in the utilization of neutral sterols can often be correlated with ecdysteroid (molting hormone) production. Certain of the most significant variations in insect steroid utilization and metabolism in phytophagous insects will be discussed with respect to phylogenetic relationships. 28
29
S i n c e i n s e c t s l a c k the c a p a c i t y to b i o s y n t h e s i z e the s t e r o i d n u c l e u s , they g e n e r a l l y r e q u i r e a d i e t a r y source o f s t e r o l f o r normal development and r e p r o d u c t i o n 11). This i s an important area o f b i o c h e m i c a l d i f f e r e n c e , between i n s e c t s and many o t h e r o r g a n i s m s , t h a t might be e x p l o i t e d t o develop new p e s t c o n t r o l s t r a t e g i e s . C h o l e s t e r o l w i l l s a t i s f y t h i s d i e t a r y requirement i n a l l but two known cases i n which d i e t a r y Δ ' - s t e r o l s a r e e s s e n t i a l (,2,2)· a d d i t i o n , some i n s e c t s may o b t a i n an adequate supply o f s t e r o l from symbionts o r i n t e s t i n a l m i c r o o r g a n i s m s . For many y e a r s , i t was b e l i e v e d t h a t phytophagous i n s e c t s i n general were c a p a b l e o f d e a l k y l a t i n g and c o n v e r t i n g d i e t a r y C28 and C29 p h y t o s t e r o l s t o c h o l e s t e r o l t o s a t i s f y t h e i r need f o r c h o l e s t e r o l ( 4 ) . A l s o , a number o f omnivorous s p e c i e s of i n s e c t s a r e known t o be c a p a b l e o f t h i s c o n v e r s i o n (5). Thus, c h o l e s t e r o l I
This chapter not subject to U.S. copyright. Published 1987, American Chemical Society
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11. SVOBODA AND THOMPSON
Steroid Metabolism
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can be made a v a i l a b l e f o r s t r u c t u r a l needs i n membranes (I) and f o r e s s e n t i a l p h y s i o l o g i c a l purposes such as s e r v i n g as a p r e c u r s o r f o r the C27 m o l t i n g hormones ( e c d y s t e r o i d s , F i g u r e 1 ) , e . g . ecdysone (j>). I t has become i n c r e a s i n g l y e v i d e n t t h a t c o n s i d e r a b l e v a r i a b i l i t y i n s t e r o i d u t i l i z a t i o n and metabolism e x i s t s among phytophagous s p e c i e s of i n s e c t s . In r e c e n t y e a r s , we have d i s c o v e r e d s e v e r a l phytophagous s p e c i e s t h a t are unable to c o n v e r t C28 o r C29 p h y t o s t e r o l s to c h o l e s t e r o l . T h i s i n c l u d e s one s p e c i e s t h a t d e a l k y l a t e s the C - 2 4 s u b s t i t u e n t of the s i d e c h a i n but produces mostly s a t u r a t e d s t e r o l s and s e v e r a l s p e c i e s t h a t t o t a l l y l a c k the a b i l i t y to d e a l k y l a t e the s t e r o l s i d e c h a i n . Certain members o f t h i s l a t t e r group are of p a r t i c u l a r i n t e r e s t because they have adapted t o u t i l i z i n g a COQ s t e r o l as an e c d y s t e r o i d p r e c u r s o r and makisterone A (C28) has been i d e n t i f i e d as the major e c d y s t e r o i d of c e r t a i n developmental stages of these s p e c i e s . We w i l l d i s c u s s some of our comparative s t e r o l metabolism s t u d i e s and p r o v i d e s p e c i f i c examples to i l l u s t r a t e some of these unusual v a r i a t i o n s i n s t e r o i d u t i l i z a t i o n and metabolism i n i n s e c t s , and to show how t h i s i n f o r m a t i o n i s u s e f u l i n p r e d i c t i n g d i f f e r e n c e s i n e c d y s t e r o i d b i o s y n t h e s i s i n c e r t a i n s p e c i e s . We w i l l a l s o p o i n t out i n s t a n c e s i n which these v a r i a t i o n s i n n e u t r a l s t e r o l metabolism can be r e l a t e d to p h y l o g e n e t i c r e l a t i o n s h i p s between s p e c i e s . Phytophagous I n s e c t s That Convert C to C h o l e s t e r o l
0 Q
and C
o 0
Phytosterols
'
Lepidoptera. The most e x t e n s i v e s t u d i e s of the u t i l i z a t i o n and metabolism of d i e t a r y s t e r o l s i n phytophagous i n s e c t s have been c a r r i e d out w i t h two L e p i d o p t e r a , the tobacco hornworm, Manduca s e x t a , i n our l a b o r a t o r y (7) and the s i l k w o r m , Bombyx m o r i , by Ikekawa and coworkers i n Japan ( 8 ) . These were p i o n e e r i n g s t u d i e s u t i l i z i n g a r t i f i c i a l d i e t s , r a d i o l a b e l e d s t e r o l s , and s t a t e - o f - t h e - a r t a n a l y t i c a l t o o l s . Manduca l a r v a e r e a d i l y c o n v e r t C28 and C29 p h y t o s t e r o l s ( e . g . c a m p e s t e r o l , s i t o s t e r o l , and s t i g m a s t e r o l ) to c h o l e s t e r o l ( F i g u r e 2) and desmosterol i s the t e r m i n a l i n t e r m e d i a t e i n the c o n v e r s i o n of each of these s t e r o l s to c h o l e s t e r o l ( 9 ) . This was the f i r s t i n t e r m e d i a t e to be i d e n t i f i e d i n the metaboTic c o n v e r s i o n of p h y t o s t e r o l s to c h o l e s t e r o l i n i n s e c t s . F u c o s t e r o l was l a t e r determined to be an i n t e r m e d i a t e between s i t o s t e r o l and desmosterol a n d , a n a l o g o u s l y , 2 4 - m e t h y l e n e c h o l e s t e r o l was found to be the f i r s t i n t e r m e d i a t e between campesterol and c h o l e s t e r o l ( 4 ) . Stigmasterol i s d e a l k y l a t e d and c o n v e r t e d t o 5 , 2 2 , 2 4 - c h o l e s t a t r i e n - 3 3 - o l which i s r e d u c e d , f i r s t to d e s m o s t e r o l , and then to c h o l e s t e r o l (4·). The A 4 - b o n d i s necessary f o r enzyme s p e c i f i c i t y i n o r d e r to reduce the A22-bond. R e s u l t s from research i n our l a b o r a t o r y a l s o i n d i c a t e t h a t o t h e r L e p i d o p t e r a such as the corn earworm, H e l i o t h i s z e a , the f a l l arrçyworm, Spodoptera f r u g i p e r d a ( 1 0 ) , and the I n d i a n meal moth, P l o d i a i n t e r p u n c t e l l a , (11) m e t a b o l i z e C23 and C29 p h y t o s t e r o l s i n a manner s i m i l a r to Manduca. These pathways have been shown to be s i m i l a r i n B. mori and, i n a d d i t i o n , f u c o s t e r o l 2 4 , 2 8 - e p o x i d e has been i d e n t i f i e d as an i n t e r m e d i a t e between f u c o s t e r o l and desmosterol i n mori ( 8 ) . 2
178
ECOLOGY AND METABOLISM OF PLANT LIPIDS
Desmosterol
î
t
5,22,24-Cholestatrienol
24-Methylenecholesterol
Stigmasterol
{*
Fucosterol
Cholesterol
F i g u r e 2 . Pathways o f c o n v e r s i o n o f C - 2 4 a l k y l s t e r o l s t o c h o l e s t e r o l i n the tobacco hornworm and o t h e r phytophagous insects. * F u c o s t e r o l 2 4 , 2 8 - e p o x i d e has been shown t o be an i n t e r m e d i a t e between f u c o s t e r o l and desmosterol i n Bombyx mori and Tenebrio m o l i t o r .
Campesterol
Sitosterol
ξ-
s
8
Ο ξ
Η
< ο 00 Ο α > > ζ α
180
ECOLOGY AND METABOLISM OF PLANT LIPIDS
C o l e o p t e r a . The confused f l o u r b e e t l e , T r i b o l i u m confusum, was the f i r s t phytophagous i n s e c t we found t h a t produces an a p p r e c i a b l e amount of a s t e r o l o t h e r than c h o l e s t e r o l from r a d i o l a b e l e d d i e t a r y C28 and C29 p h y t o s t e r o l s . We found t h i s i n s e c t produced l a r g e q u a n t i t i e s o f 7 - d e h y d r o c h o l e s t e r o l , e q u i v a l e n t to as much as 70% o f the t o t a l t i s s u e s t e r o l s i s o l a t e d ( 1 2 ) . I t was f u r t h e r determined t h a t c h o l e s t e r o l and 7-dehydrocTiôlesterol were i n e q u i l i b r i u m i n t h i s f l o u r b e e t l e . Another new i n t e r m e d i a t e , 5 , 7 , 2 4 - c h o l e s t a t r i e n - 3 B - o l was i d e n t i f i e d as an i n t e r m e d i a t e between desmosterol and 7 - d e h y d r o c h o l e s t e r o l ( F i g u r e 3 ) . We found very s i m i l a r pathways of s t e r o l metabolism to e x i s t i n the c l o s e l y r e l a t e d f l o u r b e e t l e , T r i b o l i u m castaneum ( Γ 3 ) . However, another f l o u r b e e t l e , T e n e b r i o ' m o l i t o r , had only about o n e - t h i r d or l e s s of the l e v e l s o f 7 - d e h y d r o c h o l e s t e r o l as the two T r i b o l i u m s p e c i e s , but s t i l l much h i g h e r l e v e l s of t h i s s t e r o l than has been found i n most species. F u c o s t e r o l 2 4 , 2 8 - e p o x i d e was a l s o i m p l i c a t e d as an i n t e r m e d i a t e i n the s y n t h e s i s of c h o l e s t e r o l from s i t o s t e r o l i n T. molitor (14). J T v e r y unique m i x t u r e of s t e r o l s was found i n the Mexican bean b e e t l e , E p i l a c h n a v a r i y e s t i s , when s t e r o l s from i n s e c t s fed soybean l e a v e s were a n a l y z e d (15)"! The s t e r o l s from bean b e e t l e pupae c o n s i s t e d o f >70% s a t u r a t e d s t e r o l s and c h o l e s t a n o l was the major s t e r o l i s o l a t e d from the i n s e c t . M e t a b o l i c s t u d i e s w i t h r a d i o l a b e l e d s t e r o l s demonstrated t h a t the Mexican bean b e e t l e does d e a l k y l a t e C28 and C29 p h y t o s t e r o l s , but reduces the Δ -bond f i r s t ( F i g u r e 4 ) ( 1 6 ) . In a d d i t i o n , the A - b o n d can be i n c o r p o r a t e d i n t o c h o l e s t a n o l , and thus a p p r e c i a b l e amounts (>10% of the t o t a l s t e r o l s ) o f l a t h o s t e r o l ( Δ ' - c h o l e s t e n o l ) o c c u r i n the s t e r o l s of t h i s s p e c i e s . Recent s t u d i e s (13) have c o n f i r m e d t h a t i n the metabolism of s i t o s t e r o l and s t i g m a s t e r o l , the s i d e c h a i n d e a l k y l a t i o n and c o n v e r s i o n to a 2 4 - d e s a l k y l s i d e c h a i n i n the Mexican bean b e e t l e p a r a l l e l s the mechanism i n Manduca. A A 2 4 - s t e r o l i s i n v o l v e d as a t e r m i n a l i n t e r m e d i a t e i n the metabolism of both s t e r o l s , and the s i d e c h a i n of s t i g m a s t e r o l i s f i r s t deal k y l ated to form a A 2 2 , 2 4 . - t r m e d i a t e , and then the A22-bond i s reduced. 7
1
Phytophagous Phytosterols
n
e
I n s e c t s Unable to Convert (^Q and C Q Q to Cholesterol
Hemiptera. The l a r g e milkweed bug, Qncopeltus f a s c i a t u s , was the f i r s t phytophagous i n s e c t d i s c o v e r e d to be i n c a p a b l e of c o n v e r t i n g the major p h y t o s t e r o l s (C28 and C29) t o c h o l e s t e r o l ( Γ 7 ) . D i e t a r y s t e r o l s of s u n f l o w e r seeds were i n c o r p o r a t e d e s s e n t i a l l y unchanged i n t o the t i s s u e s and, when i n j e c t e d , n e i t h e r r a d i o l a b e l e d campesterol nor s i t o s t e r o l was m e t a b o l i z e d to c h o l e s t e r o l . A p p a r e n t l y , t h e r e was some s e l e c t i v e uptake of d i e t a r y c h o l e s t e r o l , i n d i c a t e d by an enrichment of c h o l e s t e r o l i n the i n s e c t s t e r o l s compared to the c h o l e s t e r o l c o n c e n t r a t i o n i n the seed s t e r o l s . A C28 e c d y s t e r o i d , m a k i s t e r o n e A ( F i g u r e 1 ) , was i d e n t i f i e d as the major e c d y s t e r o i d of milkweed bug eggs (18) and, s u b s e q u e n t l y , m a k i s t e r o n e A was i d e n t i f i e d as the major e c 3 y s t e r o i d i n hemolymph of l a s t stage milkweed bug nymphs and two o t h e r phytophagous s p e c i e s o f the Pentatomomorpha group of Hemiptera ( 1 9 ) . M a k i s t e r o n e A was
Sitosterol
Fucosterol HO
7-Dehydrocholesterol
HO"
5,7,24-Cholestatrienol
F i g u r e 3. D e a l k y l a t i o n and c o n v e r s i o n o f s i t o s t e r o l t o 7 - d e h y d r o c h o l e s t e r o l and c h o l e s t e r o l i n T r i b o l i u m confusum.
Desmosterol
HO'
Cholesterol
Ζ
>
Ο CO Ο D
to
00
11.
SVOBODA AND THOMPSON
Steroid Metabolism
183
a l s o c a . 10 times more a c t i v e than 20-hydroxyecdysone i n s t i m u l a t i n g c u t i c l e s y n t h e s i s and i n h i b i t i n g v i t e l l o g e n e s i s i n a d u l t milkweed bugs ( 2 0 ) . I t was l a t e r determined t h a t t h i s a d a p t a t i o n t o u t i l i z i n g a C20, p r e c u r s o r f o r e c d y s t e r o i d b i o s y n t h e s i s c o r r e l a t e d w e l l w i t h n e u t r a l s t e r o l metabolism i n each of t h e s e three species (21). S i m i l a r r e s u l t s on s t e r o l u t i l i z a t i o n and o c c u r r e n c e o f màFisterone A have been r e p o r t e d f o r the c o t t o n s t a i n e r bug, Dysdercus f a s c i a t u s ( 2 2 ) . These s p e c i e s a l l have i n common the i n a b i l i t y t o d e a l k y l a t e the C - 2 4 a l k y l s u b s t i t u e n t s o f C28 and C29 p h y t o s t e r o l s . Coleoptera. S t e r o l metabolism s t u d i e s w i t h another i m p o r t a n t s t o r e d p r o d u c t s p e s t , the khapra b e e t l e , Trogoderma g r a n a r i u m , r e v e a l e d a n o t h e r phytophagous i n s e c t t h a t i s unable t o d e a l k y l a t e and c o n v e r t C28 and C29 p h y t o s t e r o l s t o c h o l e s t e r o l ( 2 3 ) . Similar r e s u l t s were o b t a i n e d whether a d i e t c o n s i s t i n g of c r a c k e d wheat and b r e w e r ' s y e a s t o r an a r t i f i c i a l d i e t c o a t e d w i t h r a d i o l a b e l e d s t e r o l s was used ( 2 4 ) . There was some s e l e c t i v e uptake o f c h o l e s t e r o l from tfie d i e t a r y s t e r o l s , as i n d i c a t e d by an enrichment of c h o l e s t e r o l i n the pupal s t e r o l s (1.2% of t o t a l ) , compared to the d i e t a r y s t e r o l s (0.5% of t o t a l ) . U n l i k e the p r e v i o u s l y d i s c u s s e d s t o r e d product c o l e o p t e r a n p e s t s , T. confusum and T. castaneum, both o f which had h i g h l e v e l s o f 7 - d e h y ï ï r o c h o i e s t e r o l , no 7 - d e h y d r o c h o l e s t e r o l c o u l d be i d e n t i f i e d i n the s t e r o l s from the khapra b e e t l e . Hymenoptera. While examining the e f f e c t s of v a r i o u s d i e t a r y s t e r o l s on brood p r o d u c t i o n i n honey b e e s , A p i s m e l l i f e r a , we d i s c o v e r e d t h a t the honey bee u t i l i z e d d i e t a r y C28 and C29 p h y t o s t e r o l s unchanged ( 2 5 , 2 6 ) . R e g a r d l e s s of the d i e t a r y s t e r o l added to a chemical 1 y-deTTnëcT d i e t , o r even w i t h no s t e r o l added, 2 4 - m e t h y l e n e c h o l e s t e r o l was always the major s t e r o l of the next g e n e r a t i o n of b e e s , and s i t o s t e r o l and i s o f u c o s t e r o l were a l s o p r e s e n t i n a p p r e c i a b l e amounts. Detailed studies with e i t h e r r a d i o l a b e l e d c a m p e s t e r o l , s i t o s t e r o l , or 2 4 - m e t h y l e n e c h o l e s t e r o l added t o the a r t i f i c i a l d i e t p r o v i d e d no e v i d e n c e f o r the metabolism o f any of these p h y t o s t e r o l s to c h o l e s t e r o l o r o t h e r s t e r o l s ( 2 7 ) . In f a c t , H - 2 4 - m e t h y l e n e c h o l e s t e r o l has been t r a c e d unchanged through two g e n e r a t i o n s of bees ( 2 8 ) . Thus, t h e r e i s a very unusual mechanism t h a t e n a b l e s the w o r k e r T e e to s e l e c t i v e l y t r a n s f e r c e r t a i n d i e t a r y s t e r o l s or s t e r o l s c y c l e d from t h e i r endogenous p o o l s t o the brood food t o m a i n t a i n a c o n s t a n t supply o f c e r t a i n s t e r o l s f o r the brood f o o d . The u t i l i z a t i o n of n e u t r a l s t e r o l s by the honey bee and the i n a b i l i t y t o produce c h o l e s t e r o l from the d e a l k y l a t i o n o f 2 4 - a l k y l C g and C29 p h y t o s t e r o l s i s r e f l e c t e d i n the r e c e n t i s o l a t i o n of m a k i s t e r o n e A as the major e c d y s t e r o i d a t peak t i t e r i n the honey bee pupa ( 2 9 ) . We have a l s o found another phytophagous hymenopteran, the a l f a l f a l e a f c u t t e r bee, M e g a c h i l e rotunda t a , t h a t u t i l i z e s d i e t a r y p h y t o s t e r o l s s i m i l a r l y to the honey bee [SB). As i n the honey bee, 2 4 - m e t h y l e n e c h o l e s t e r o l was a major component ( 3 4 . 1 % o f the t o t a l s t e r o l s ) of the s t e r o l s of newly-emerged a d u l t s . In a d d i t i o n , t h e r e was l i t t l e c h o l e s t e r o l (
