Insect Cuticle Structure and Metabolism - ACS Symposium Series

Chapter 12

Insect Cuticle Structure and Metabolism 1,2

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Karl J. Kramer , Theodore L. Hopkins , and Jacob Schaefer 1

U.S. Grain Marketing Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Manhattan, KS 66502 Department of Biochemistry, Kansas State University, Manhattan, KS 66506 Department of Entomology, Kansas State University, Manhattan, KS 66506 Department of Chemistry, Washington University, St Louis, MO 63130 Insects have become the most diverse and numerous animal group partly because of evolution of a multifunctional integument, which provides for growth, mobility, protection and communication. The c u t i c l e , which is secreted by the epidermis, is a composite of materials, primarily a polymeric structure of protein and chitin chains with lesser amounts of phenolics, l i p i d s and minerals. The organization and interactions of these components, which are only partially understood, confer s t a b i l i t y to the exoskeleton and provide for its varied functional properties. For several years, we have been investigating the identity and metabolism of phenolic compounds that permeate the exoskeleton and serve as precursors for agents that sclerotize and pigment the cuticle. More recently, we have used solid state nuclear magnetic resonance spectroscopy to determine cuticle composition and aromatic cross-link structure between protein amino acids and chitin. The chemical and spectroscopic data support a general scheme for assembly of cuticle with increasing amounts of protein and c h i t i n , as well as a gradual accumulation of diphenolic compounds primarily into the outer parts of the cuticle during sclerotization. Aromatic cross-links derived from quinonoid derivatives s t a b i l i z e the protein-chitin matrix against chemical and physical degradation, and confer stiffness and other essential mechanical properties. The degradation of cuticle periodically occurs as part of the molting process and is catalyzed by enzymes, which digest the less s t a b i lized portion of the exoskeleton, leaving the highly sclerotized exocuticle and outer epicuticle to be shed as the exuviae. In order to understand how the cuticle is degraded naturally, hydrolytic enzymes from insect epidermis and entomopathogens have been characterized. The primary hydrolases are proteolytic and c h i t i n o l y t i c enzymes, which facilitate digestion and recycling of

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0097-6156/88/0379-0160$07.50A) © 1988 American Chemical Society

In Biotechnology for Crop Protection; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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cuticular components and the penetration of entomopathogens through the c u t i c l e . The insect-specific metabolism that occurs in the integument is vital for growth and, therefore, is a good target for development of highly selective insecticides. Some of the physical, chemical and biological means of attacking the integument and disrupting cuticle formation and function are described in this article. A c o n t i n u i n g demand f o r more i n n o v a t i v e and s e l e c t i v e ways t o c o n t r o l i n s e c t p e s t s i s n e c e s s i t a t e d by t h e c a p a c i t y o f i n s e c t p o p u l a t i o n s to d e v e l o p r e s i s t a n c e to i n s e c t i c i d e s and by the need t o m i n i m i z e the impact of t h e s e t o x i c s u b s t a n c e s on t h e e n v i r o n m e n t . One a p p r o a c h t a k e s a d v a n t a g e o f t h e e v o l u t i o n a r y d i f f e r e n c e s i n b i o c h e m i s t r y and p h y s i o l o g y of i n s e c t s and o t h e r types of o r g a n i s m s o r between d i v e r s e groups of i n s e c t s . T h i s approach r e q u i r e s knowledge of s p e c i a l i z e d or unique l i f e p r o c e s s e s v i t a l t o the development, r e p r o d u c t i o n or s u r v i v a l o f the p e s t i n s e c t , d e f i n i t i o n o f t h e p h y s i c a l and 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 t h e systems i n v o l v e d , and d e l i n e a t i o n o f the p e r t i n e n t b i o c h e m i c a l s t e p s . With o v e r 300 m i l l i o n y e a r s s e p a r a t i n g t h e e v o l u t i o n o f i n s e c t s and h i g h e r a n i m a l s , e v o l u t i o n a r y d e s i g n has g e n e r a t e d s e v e r a l k i n d s o f i n s e c t - s p e c i f i c m e t a b o l i s m t h a t c a n be t a r g e t e d by s e l e c t i v e t o x i c a n t s or growth r e g u l a t o r s . Because the integument t h a t makes up the e x o s k e l e t o n i s so i m p o r t a n t to the maintenance and p r o t e c t i o n of the insect's i n t e r n a l environment, respiration, sensory r e c e p t i o n , l o c o m o t i o n and a m u l t i t u d e o f o t h e r f u n c t i o n s , i t i s a t i s s u e system d e s e r v i n g d e t a i l e d i n v e s t i g a t i o n , and r e s e a r c h e r s have s t u d i e d i t s d i v e r s e f e a t u r e s and f u n c t i o n s f o r s e v e r a l d e c a d e s (1-5). A l t h o u g h g r e a t p r o g r e s s has been made i n u n d e r s t a n d i n g the s e c r e t i o n and s t a b i l i z a t i o n o f t h e i n s e c t e x o s k e l e t o n and i t s m u l t i f u n c t i o n a l p r o p e r t i e s , many i m p o r t a n t q u e s t i o n s remain t o be answered t h a t c o u l d p r o v i d e new s e l e c t i v e approaches to i n s e c t p e s t control. The advantages of a c u t i c l e t h a t c o n t a i n s p r o t e i n and c h i t i n as b i o l o g i c a l s t r u c t u r a l m a t e r i a l s are i t s l i g h t n e s s of w e i g h t , s t r e n g t h and f l e x i b i l i t y . These p r o p e r t i e s have a l l o w e d i n s e c t s to d e v e l o p f l i g h t as a means o f l o c o m o t i o n and d i s p e r s i o n and t o i n h a b i t d i v e r s e e c o l o g i c a l n i c h e s . The major d i s a d v a n t a g e o f the e x o s k e l e t o n i s i t s i n a b i l i t y to expand beyond c e r t a i n p h y s i c a l l i m i t s , t h e r e b y c o n s t r a i n i n g growth and r e q u i r i n g i n s e c t s to molt p e r i o d i c a l l y i n o r d e r to grow and mature t o t h e a d u l t r e p r o d u c t i v e stage. D u r i n g e a c h m o l t i n g c y c l e , a new c u t i c l e must be s e c r e t e d and s t a b i l i z e d , and the o l d one must be p a r t i a l l y d i g e s t e d t o make p o s s i b l e escape by s p l i t t i n g the l a t t e r a l o n g predetermined l i n e s o f weakness. The c u t i c l e a l o n g these e c d y s i a l l i n e s c o n s i s t s m a i n l y o f e p i c u t i c l e a f t e r d i g e s t i o n o f the u n d e r l y i n g e n d o c u t i c l e . The new c u t i c l e t h e n b r i e f l y becomes e x t e n s i b l e t o accommodate g r o w t h , f o l l o w e d by s c l e r o t i z a t i o n and p i g m e n t a t i o n t o c o m p l e t e the f u n c t i o n a l e x o s k e l e t o n . The c u t i c l e o f some s o f t b o d i e d l a r v a l i n s e c t s h o w e v e r , c o n t i n u e s t o grow d u r i n g the i n t e r m o l t p e r i o d t o accommodate growth d u r i n g each s t a g e . The p a t t e r n s of c u t i c l e t h a t a r e s t i f f e n e d o r remain f l e x i b l e are v e r y s p e c i f i c f o r each s t a g e of



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d e v e l o p m e n t and are determined by the u n d e r l y i n g e p i d e r m a l c e l l s by mechanisms l i t t l e u n d e r s t o o d . The f o r m a t i o n and m o l t i n g of the i n s e c t e x o s k e l e t o n r e q u i r e a myriad of enzymatic and e n d o c r i n e p r o c e s s e s t h a t c e n t e r p r i n c i p a l l y i n the epidermal c e l l s . The c h i t i n o u s and p r o t e i n a c e o u s c u t i c l e i s a t e m p t i n g t a r g e t f o r c o n t r o l s t r a t e g i e s , because i t i s c h e m i c a l l y d i s t i n c t f r o m t h e i n t e g u m e n t o f v e r t e b r a t e s , which i s made up of k e r a t i n a c e o u s and c o l l a g e n o u s p r o t e i n s . A l t h o u g h many a s p e c t s o f t h e s t r u c t u r e and c h e m i s t r y of i n s e c t c u t i c l e a r e known, much s t i l l remains to be l e a r n e d about how p r o t e i n and c h i t i n a r e s e c r e t e d and a s s e m b l e d i n t o a c u t i c u l a r s t r u c t u r e and a b o u t t h e p h y s i c a l and c h e m i c a l c h a n g e s t h e s e m a c r o m o l e c u l e s u n d e r g o as t h e y become s c l e r o t i z e d and p i g m e n t e d . Some o f t h e r e s e a r c h on c u t i c l e s t r u c t u r e and metabolism w i l l be reviewed h e r e , i n c l u d i n g d i s c u s s i o n a b o u t c o m p o s i t i o n , m e t a b o l i t e s , enzymes, i n s e c t growth r e g u l a t o r s and entomopathogens t h a t a f f e c t the c u t i c l e . One a s p e c t of b i o t e c h n o l o g y t h a t may n o t be s u f f i c i e n t l y a p p r e c i a t e d by b i o l o g i s t s i s the a p p l i c a t i o n o f h i g h t e c h n o l o g i c a l a n a l y t i c a l i n s t r u m e n t a t i o n , such as n u c l e a r magnetic r e s o n a n c e , t o b i o l o g y . For s e v e r a l y e a r s , we have been u s i n g s o l i d s t a t e n u c l e a r m a g n e t i c r e s o n a n c e s p e c t r o s c o p y and c h e m i c a l a n a l y s i s t o s t u d y i n s e c t c u t i c l e s t r u c t u r e . The f o r m e r t e c h n i q u e i s e s p e c i a l l y p r o m i s i n g , because i t a l l o w s study of i n t r a c t a b l e materials i n a n o n i n v a s i v e manner. Our d a t a support the c u t i c l e m o d e l o r i g i n a l l y p r o p o s e d by P r y o r ( 6 ) i n t h e 1 9 4 0 s , which d e p i c t s p r o t e i n c h a i n s c r o s s - l i n k e d by q u i n o n o i d d e r i v a t i v e s of d i p h e n o l i c compounds. We h a v e a l s o used c h e m i c a l and k i n e t i c procedures t o s t u d y how m o l t i n g f l u i d d i g e s t s the u n s c l e r o t i z e d l a y e r s of the o l d c u t i c l e i n t o component amino a c i d s and amino s u g a r s f o r r e c y c l i n g and t h e c o n s t r u c t i o n of a new one. For the sake of t h i s d i s c u s s i o n , c u t i c l e w i l l be c l a s s i f i e d i n t o two g e n e r a l types t h a t d i f f e r i n m e c h a n i c a l properties. F i r s t , t h e r e a r e s o f t c u t i c l e s l i k e t h o s e o f most l a r v a e , which are f l e x i b l e and e x t e n s i b l e and t h e r e f o r e , h y d r o s t a t i c i n n a t u r e . Second, t h e r e a r e h a r d c u t i c l e s l i k e t h o s e o f d i p t e r a n p u p a r i a , l e p i d o p t e r a n p u p a e , and c o l e o p t e r a n a d u l t s , w h i c h a r e h i g h l y s c l e r o t i z e d or m i n e r a l i z e d , s t i f f and s e l f - s u p p o r t i n g . A l l of these s t r u c t u r e s c o n t a i n p r o t e i n , c h i t i n , d i p h e n o l s , l i p i d s , w a t e r and m i n e r a l s a l t s . D e p e n d i n g on f u n c t i o n a l demands, t h e r e l a t i v e l e v e l s of i n d i v i d u a l components can v a r y . Flexible c u t i c l e s tend to be more h y d r a t e d t h a n s t i f f e r c u t i c l e s and a l s o c o n t a i n fewer p h e n o l i c or i n o r g a n i c s t a b i l i z i n g a g e n t s . Conversely, s t i f f e r c u t i c l e s are more dehydrated and c o n t a i n h i g h e r l e v e l s o f p h e n o l i c s o r m i n e r a l s . The p h e n o l i c compounds are a s s o c i a t e d w i t h a r o m a t i c c r o s s - l i n k s between m a c r o m o l e c u l a r s u b s t i t u e n t s o f c u t i c l e or s e r v e as d e h y d r a t i n g or p r o t e i n d e n a t u r i n g agents ( 1 ) . Insect c u t i c l e i s a heterogeneous s t r u c t u r e t h a t v a r i e s i n c h e m i c a l c o m p o s i t i o n a c c o r d i n g t o s p e c i e s , s t a g e of development, appendage, l o c a t i o n and p h y s i c a l p r o p e r t y ( 1 , 7 ) . In g e n e r a l , the t h i n n e r the c u t i c l e , the weaker i t becomes, but the b l e n d of c o m p o n e n t s , d e g r e e of c o m p a c t i o n and h y d r a t i o n , as w e l l as t h e number o f c r o s s - l i n k s , c a n r e n d e r e v e n a v e r y t h i n c u t i c l e q u i t e tough. E p i c u t i c l e and the u n d e r l y i n g e x o c u t i c l e a r e t h e p r i n c i p l e b a r r i e r s t o e n v i r o n m e n t a l c h a l l e n g e s and, t h e r e f o r e , are s t a b i l i z e d by a g r e a t e r a b u n d a n c e o f a r o m a t i c c r o s s - l i n k s , p h e n o l i c s and f



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lipids. For example, the e n d o c u t i c u l a r - r i c h intersegmental membranes and u n i o n s b e t w e e n d i f f e r e n t t y p e s o f l o c u s t c u t i c l e appear to be p r e f e r e n t i a l e n t r y s i t e s f o r i n s e c t i c i d a l compounds and f u n g a l pathogens ( 8 ) . I n s e c t s u p p o r t i v e s t r u c t u r e s g e n e r a l l y c o n s i s t o f a macrom o l e c u l a r assembly of p r o t e i n , which i s s t a b i l i z e d and dehydrated t o v a r i o u s d e g r e e s e i t h e r by a r o m a t i c c r o s s - l i n k s or by d e p o s i t i o n of d i p h e n o l s or m i n e r a l s a l t s ( 1 ) . One of the s i m p l e s t n o n - c u t i c u l a r s t r u c t u r e s i n c h e m i c a l terras t h a t undergoes some form of s t a b i l i z a t i o n i s moth cocoon s i l k , such as t h a t from Bombyx m o r i , which i s p r i m a r i l y made up of two p r o t e i n s , the t h r e a d - l i k e p r o t e i n , f i b r o i n , and the g l u e - l i k e p r o t e i n , s e r i c i n ( 9 ) . Some c o c o o n s i l k s may be c r o s s - l i n k e d by d i p h e n o l i c t a n n i n g a g e n t s ( 1 0 ) o r by t r y p t o p h a n metabolites ( 1 1 ) . C h o r i o n and egg c a p s u l e s o r c a s e s f r o m c o c k r o a c h e s , m a n t i d s , g r a s s h o p p e r s and o t h e r i n s e c t s are o t h e r examples o f p r o t e i n a c e o u s s t r u c t u r e s t h a t a r e t a n n e d by c r o s s - l i n k i n g reactions i n v o l v i n g d i p h e n o l i c metabolites (6,12-15). Insect c u t i c l e , however, i s a composite of not o n l y p r o t e i n and d i p h e n o l i c compounds, but a l s o c h i t i n as a major component, which r e s u l t s i n a l a m i n a t e d framework and perhaps r e n d e r s the c u t i c l e f l e x i b l e , s t r o n g and r e c y c l a b l e . L i p i d s , w h i c h a r e r e l a t i v e l y m i n o r components, o c c u r p r i m a r i l y at o r n e a r t h e s u r f a c e o f t h e c u t i c l e and i n t h e waxy l a y e r of t h e e p i c u t i c l e . They may p r o v i d e the major b a r r i e r a g a i n s t water l o s s , but t h e y p r o b a b l y c o n t r i b u t e l i t t l e t o the s t r e n g t h and s h a p e o f the c u t i c l e . M i n e r a l c o n t e n t i s low i n most c u t i c l e s ( l e s s than a few p e r c e n t ) , except i n those d i p t e r a n s p e c i e s t h a t i m p r e g n a t e s u b s t a n t i a l amounts o f m i n e r a l s i n t o the p u p a r i a l c u t i c l e to harden i t ( 7 , 1 6 - 2 0 ) . C u t i c l e S c l e r o t i z a t i o n Versus M i n e r a l i z a t i o n . Why i s s c l e r o t i z a t i o n i n s t e a d of m i n e r a l i z a t i o n the prepondera n t m e c h a n i s m u s e d by i n s e c t s t o s t i f f e n t h e i r e x o s k e l e t o n ? S c l e r o t i z a t i o n u t i l i z e s r e l a t i v e l y h i g h l e v e l s of o r g a n i c components such as p r o t e i n s , c h i t i n and d i p h e n o l i c compounds f o r s t r u c t u r a l s u p p o r t and f o r m whereas i n o r g a n i c s a l t s such as c a l c i u m c a r b o n a t e and phosphate are i n c o r p o r a t e d i n t o an o r g a n i c m a t r i x f o r t h e same purpose d u r i n g b i o r a i n e r a l i z a t i o n . The former mechanism i s g e n e r a l l y c a p a b l e o f p r o d u c i n g a much l i g h t e r c u t i c l e w i t h r e q u i s i t e p r o p e r t i e s o f s t r e n g t h and f l e x i b i l i t y necessary for rapid t e r r e s t r i a l l o c o m o t i o n and f l i g h t . D u r i n g s c l e r o t i z a t i o n , d i p h e n o l s a r e o x i d i z e d t o r e a c t i v e m e t a b o l i t e s t h a t c r o s s - l i n k and f o r m covalent adducts w i t h m a c r o m o l e c u l a r components of the c u t i c l e . During b i o m i n e r a l i z a t i o n , inorganic s a l t s p r e c i p i t a t e i n a proteinc h i t i n m a t r i x , and i n the p r o c e s s , a h a r d r i g i d c u t i c l e i s f o r m e d . S a l t s s u c h as c o l l o i d a l c a l c i u m p h o s p h a t e h a v e b e e n r e p o r t e d to s t a b i l i z e p r o t e i n aggregates i n the presence of c h a o t r o p i c s o l v e n t s , p r o b a b l y by f o r m i n g i n t e r m o l e c u l a r c r o s s - l i n k s ( 2 1 ) . However, the s t r u c t u r e o f t h e m i n e r a l c r o s s - l i n k has n o t b e e n d e t e r m i n e d . Whether i n o r g a n i c t y p e s of c r o s s - l i n k a g e s o c c u r i n m i n e r a l i z e d i n s e c t c u t i c l e i s unknown. D u r i n g p o s t e m b r y o n i c development, the m e c h a n i c a l p r o p e r t i e s of c u t i c l e are a l t e r e d to accommodate changing f u n c t i o n a l demands. A c l a s s i c example of t h i s phenomenon i s d i p t e r a n p u p a r i a t i o n , i n which



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the s o f t l a r v a l c u t i c l e becomes s t i f f e n e d t o s u p p o r t and p r o t e c t t h e developing adult (22,23). I n t h e house f l y , Musca d o m e s t i c a , the p u p a r i a l c u t i c l e i s s t a b i l i z e d p r i m a r i l y by s c l e r o t i z a t i o n , i n w h i c h d i p h e n o l i c compounds a c c u m u l a t e i n t h e p r o t e i n and c h i t i n - r i c h l a r v a l c u t i c l e ( 1 9 ) . I n t h e f a c e f l y , Musca a u t u m n a l i s , t h e p u p a r i u m i s h a r d e n e d p r i m a r i l y by t h e d e p o s i t i o n o f c a l c i u m and magnesium p h o s p h a t e i n t o t h e l a r v a l c u t i c l e . By e x a m i n i n g t h e c h e m i c a l a n d p h y s i c a l p r o p e r t i e s o f p u p a r i a l e x u v i a e o f t h e house f l y and f a c e f l y , t h e s u p e r i o r i t y o f an o r g a n i c strengthening m a t e r i a l o v e r an i n o r g a n i c one i s demonstrated. T a b l e I l i s t s t h e major o r g a n i c and i n o r g a n i c e l e m e n t s f o u n d i n t h e s e two t y p e s o f cuticles. S c l e r o t i z e d h o u s e f l y c u t i c l e i s composed o f a p p r o x i m a t e l y f i v e t i m e s more o r g a n i c elements and t e n f o l d fewer i n o r g a n i c elements than m i n e r a l i z e d f a c e f l y c u t i c l e (19). P h y s i c a l l y , the s c l e r o t i z e d c u t i c l e i s about 80% as dense and t w i c e as f l e x i b l e a s the m i n e r a l i z e d c u t i c l e , and t h e former c u t i c l e r e q u i r e s about t h r e e t i m e s more f o r c e t o f r a c t u r e i t t h a n the m i n e r a l i z e d c u t i c l e ( T a b l e I I , 2 0 ) . A p p r o x i m a t e l y t h r e e f o l d more i n o r g a n i c mass i s r e q u i r e d t o make a c u t i c l e o f c o m p a r a b l e s t r e n g t h t o an o r g a n i c o n e . T h u s , o r g a n i c c o n s t i t u e n t s appear t o assemble and s t a b i l i z e a c u t i c l e more e f f e c t i v e l y than i n o r g a n i c e l e m e n t s . Except i n environments where a h e a v y body w e i g h t and l a c k o f m o b i l i t y and f l e x i b i l i t y a r e n o t d e t r i m e n t a l t o s u r v i v a l ( f o r example, a q u a t i c h a b i t a t s ) , s c l e r o t i z a t i o n i s t h e m e c h a n i s m o f c h o i c e f o r c u t i c l e s t i f f e n i n g . One apparent d i s a d v a n t a g e o f s c l e r o t i z a t i o n i s t h e amount o f e n e r g y required to synthesize the diverse kinds of b u i l d i n g materials. Assuming t h a t m i n e r a l s a r e r e a d i l y a v a i l a b l e , l e s s energy i s p r o b a b l y needed t o d e p o s i t m i n e r a l s i n t o a b i o l o g i c a l s t r u c t u r e t h a n t o s y n t h e s i z e and d e p o s i t b o t h l o w and h i g h m o l e c u l a r weight o r g a n i c components·

Table I .

Major Elements i n S c l e r o t i z e d and M i n e r a l i z e d Insect C u t i c l e s 3

% Dry Weight Element Carbon Nitrogen Hydrogen Oxygen Calcium Magnesium Phosphorus a

Sclerotized 45.3 9.5 6.8 29.7 0.9 0.3 0.3

Mineralized 9.7 1.5 3.3 23.7 18.5 3.0 9.9

R a t i o (S:M) 4.7 6.3 2.1 1.3 0.05 0.10 0.03

D a t a from R o s e l a n d e t a l . ( 1 9 ) . S c l e r o t i z e d and m i n e r a l i z e d c u t i c l e s a r e p u p a r i a l e x u v i a e from house f l y and f a c e f l y , r e s p e c t i v e l y .



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165


What a r e the major b i o c h e m i c a l c o n s t i t u e n t s of i n s e c t c u t i c l e s ? We h a v e u s e d s o l i d s t a t e c r o s s p o l a r i z a t i o n - m a g i c a n g l e s p i n n i n g (CPMAS) NMR to i d e n t i f y and compare t h e l e v e l s o f c o n s t i t u e n t s i n s c l e r o t i z e d and m i n e r a l i z e d c u t i c l e s and t o l o o k f o r c o v a l e n t c r o s s - l i n k s ( 2 4 ) . To e s t i m a t e the c h e m i c a l c o m p o s i t i o n , we i n t e g r a t e d resonances i n ^ C - s p e c t r a w i t h c h e m i c a l s h i f t s a t 144 ppm f o r d i p h e n o l c o n t e n t , 104 ppm f o r c h i t i n , 60 and 55 ppm f o r p r o t e i n ( a f t e r s u b t r a c t i n g t h e c o n t r i b u t i o n s from c h i t i n ) , and 33 ppm f o r l i p i d ( a f t e r s u b t r a c t i n g c o n t r i b u t i o n s f r o m d i p h e n o l and p r o t e i n c a r b o n s , see F i g . 1 and T a b l e I I I ) . Water and m i n e r a l c o n t e n t s were determined by g r a v i m e t r i c and ash a n a l y s e s , r e s p e c t i v e l y . Whereas m i n e r a l s a l t s c o m p r i s e more t h a n 60% o f t h e f a c e f l y p u p a r i a l exuvium, they make up o n l y 3% of the house f l y e x u v i u m ( T a b l e I V ) . S c l e r o t i z e d c u t i c l e c o n t a i n s s u b s t a n t i a l l y more p r o t e i n , c h i t i n and d i p h e n o l i c compounds t h a n d o e s m i n e r a l i z e d c u t i c l e . N o t e i n p a r t i c u l a r the h i g h abundance of d i p h e n o l i c carbon resonances at 116 and 144 ppm i n the spectrum of house f l y p u p a r i a but not i n t h a t o f f a c e f l y p u p a r i a ( F i g . 1 ) . A p p r o x i m a t e l y 90% o f the wet weight of h o u s e f l y p u p a r i a l c u t i c l e i s p r o t e i n , c h i t i n and diphenolic compounds, w h e r e a s t h o s e c o n s t i t u e n t s account f o r l e s s than 30% o f face f l y c u t i c l e . S i m i l a r amounts o f l i p i d and water are p r e s e n t i n b o t h types of c u t i c l e .

Table I I .

P h y s i c a l and M e c h a n i c a l P r o p e r t i e s of S c l e r o t i z e d and M i n e r a l i z e d I n s e c t C u t i c l e s a

Type of C u t i c l e Property P u p a r i a l diameter (mm) Cuticular thickness (um) D e n s i t y (g mm"~2) S t r e s s modulus of f r a c t u r e (kg mm""2) E l a s t i c modulus (kg mm~2) a

Sclerotized 2.6 26.8 2.2 23.6 711.5

Mineralized

Ratio

2.7 41.7 2.6

1.0 0.6 0.8

8.1 346.4

2.9 2.1

F r o m G r o d o w i t z et a l . ( 2 0 ) . S c l e r o t i z e d and m i n e r a l i z e d c u t i c l e s a r e p u p a r i a l e x u v i a e from house f l y and f a c e f l y , respectively.


(S:M)

166



I

ι

ιι ι

ι

·

I Ml 11 I II 10

ι

11

η—ι—ι—ι—ι—ι—ι—ι—ι—ι—ι—ι—ι—ι—ι—ι—ι—ι—ι—r~ 300

200

100

6c Figure 1

Ο

- 100

(Ppm)

13

C-CPMAS NMR s p e c t r a o f f a c e f l y and house f l y p u p a r i a l e x u v i a e (Kramer e t a l . u n p u b l i s h e d d a t a ) . See Table I I I f o r resonance a s s i g n m e n t s .



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Table I I I . Chemical Assignments o f Resonances i n t h e CPMAS- C-NMR-Spectra o f I n s e c t C u t i c l e s 13

Resonance

a

δ-Values (ppm)

1

172

2

155

3 4 5

144 129 116

6 7 8 9 10

104 82 75 74 60

11

55

12

44

13

33

14

23

15

19

3

Assignment C a r b o n y l carbon i n c h i t i n , p r o t e i n , l i p i d and d i p h e n o l a c y l groups Phenoxy carbon i n t y r o s i n e , g u a n i d i n o carbon i n a r g i n i n e Phenoxy carbon i n d i p h e n o l s A r o m a t i c carbons T y r o s i n e carbons 3 and 5, i m i d a z o l e c a r b o n 4, d i p h e n o l carbons 2 and 5 GlcNAc carbon 1 GlcNAc carbon 4 GlcNAc carbon 5 GlcNAc carbon 3 GlcNAc c a r b o n 6, amino a c i d α-carbon GlcNAc c a r b o n 2, amino a c i d α-carbon Amino a c i d , d i p h e n o l and l i p i d a l i p h a t i c carbons Amino a c i d , d i p h e n o l and l i p i d a l i p h a t i c carbons M e t h y l carbons i n c h i t i n , p r o t e i n , l i p i d and d i p h e n o l a c e t y l g r o u p s , amino a c i d methyne carbons Amino a c i d and l i p i d m e t h y l carbons

F r o m S c h a e f e r e t a l . ( 2 4 ) . θ-Values r e l a t i v e t o e x t e r n a l reference.

TMS


168


T a b l e IV.

Major Components of S c l e r o t i z e d and Insect C u t i c l e s

Mineralized

3

Types o f C u t i c l e


Component Protein Chitin Diphenol Lipid Water Mineral a

Sclerotized 31 45 13 2 6 3

Mineralized 10 19 1 2 5 63

Ratio

(S:M)

3.1 2.4 13 1.2 1.2 0.05

D a t a from Kramer e t a l . , u n p u b l i s h e d . U n i t = percentage of wet w e i g h t . S c l e r o t i z e d and m i n e r a l i z e d c u t i c l e s are p u p a r i a l e x u v i a e from house f l y and f a c e f l y , r e s p e c t i v e l y . P r o t e i n , c h i t i n , d i p h e n o l and l i p i d determined by C-NMR; water by g r a v i m e t r i c a n a l y s i s ; and m i n e r a l by ash c o n t e n t . 13

I n t e r m s o f amino a c i d and c a r b o h y d r a t e c o n t e n t l i b e r a t e d by a c i d h y d r o l y s i s , s c l e r o t i z e d c u t i c l e c o n t a i n s more than t w o f o l d more t o t a l amino a c i d s and a l s o 2-acetamido-2-deoxy-D-glucopyranoside (as c h i t i n ) than does m i n e r a l i z e d c u t i c l e ( T a b l e V ) . As p r e v i o u s l y shown, we have c o n f i r m e d t h a t the amino a c i d β-alanine i s v i r t u a l l y absent i n the m i n e r a l i z e d puparium o f t h e f a c e f l y , w h e r e a s i t i s t h e s e c o n d most a b u n d a n t amino a c i d i n t h e h o u s e f l y p u p a r i u m ( 1 9 , 2 5 ) . We h a v e a l s o d e t e r m i n e d t h a t much o f t h e β-alanine i s c o n j u g a t e d w i t h the c a t e c h o l a m i n e s N - 3 - a l a n y l d o p a m i n e (NBAD), Ν-β-alanylnorepinephrine (NBANE) or t h e i r q u i n o n o i d a d d u c t s l i n k e d to macromolecules ( 2 6 - 2 8 ) . D i p h e n o l c o n t e n t of the two t y p e s of c u t i c l e i s even more d i s s i m i l a r ( T a b l e V I ) . Whereas o n l y d o p a m i n e a t r e l a t i v e l y l o w l e v e l s i s p r e s e n t i n m i n e r a l i z e d c u t i c l e , about 1 0 0 - f o l d more d i p h e n o l s o c c u r i n s c l e r o t i z e d c u t i c l e , w i t h NBAD b e i n g t h e m a j o r compound ( 1 9 ) . O v e r a l l , s c l e r o t i z e d c u t i c l e s have h i g h e r c o n c e n t r a t i o n s of p r o t e i n s , d i p h e n o l s and c h i t i n , w h e r e a s m i n e r a l i z e d c u t i c l e s h a v e much h i g h e r l e v e l s of i n o r g a n i c s a l t s , p r i m a r i l y c a l c i u m and magnesium p h o s p h a t e s and c a r b o n a t e s . Thus, the c h e m i c a l c o m p o s i t i o n of a p a r t i c u l a r c u t i c l e r e f l e c t s the r e l a t i v e c o n t r i b u t i o n of e i t h e r s c l e r o t i z a t i o n or m i n e r a l i z a t i o n t o its physical properties. The e v o l u t i o n a r y c h o i c e o f u s i n g an o r g a n i c m a t r i x o f p r o t e i n and c h i t i n s t a b i l i z e d by i n t r a - and i n t e r m o l e c u l a r i n t e r a c t i o n s and c r o s s - l i n k s , t o g e t h e r w i t h l e s s e r amounts of d i p h e n o l s , l i p i d , m i n e r a l s , p i g m e n t s , w a t e r and other c o m p o n e n t s f o r c u t i c l e c o n s t r u c t i o n has p r o v i d e d the i n s e c t w i t h many o p t i o n s i n r e g a r d t o c u t i c l e p r o p e r t i e s and s t r u c t u r e . By v a r y i n g t h e k i n d and amount o f components as w e l l as t h e i r i n t e r a c t i o n s , the e p i d e r m i s may assemble c u t i c l e s w i t h v a r i o u s degrees o f h a r d n e s s , f l e x i b i l i t y or s t i f f n e s s and pigmentation.


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169

1

Table V· Amino A c i d C o m p o s i t i o n (Mmole g"" ) o f S c l e r o t i z e d and M i n e r a l i z e d I n s e c t C u t i c l e s


3

Amino A c i d

Sclerotized

Mineralized

ASP THR SER GLU PRO GLY a-ALA VAL ILU LEU TYR PHE 3-ALA HIS LYS ARG

208.4 + 88.6 + 78.5 + 214.5 + 115.7 + 319.5 ± 123.4 ± 134.4 + 58.3 + 76.1 + 53.6 + 47.8 ± 299.2 ± 227.4 ± 85.8 ± 41.2 ±

72.8 41.3 36.6 86.3 46.3 303.8 57.2 53.8 25.2 28.7 22.9 17.8

T o t a l amino a c i d relative ratio L

± 2.9 ± 1.5 ± 0.2 ± 3.1 ± 0.9 ±9.9 ± 0.8 ± 1.9 ± 1.2 ± 1.6 ± 0.8 ± 1.2 N 2 ] j - h i s t i d i n e or ε-[ ^ N ] - l y s i n e d e m o n s t r a t e d t h a t a s i d e c h a i n h i s t i d y l o r l y s y l n i t r o g e n becomes a t t a c h e d t o a c a r b o n atom ( N - a r y l o r N - a l k y l ) d u r i n g s c l e r o t i z a t i o n . A f t e r t h e pupal c u t i c l e was d o u b l y l a b e l e d w i t h b o t h 1 , 3 - [ ^ N 2 ] - h i s t i d i n e and ring-[ C£]dopamine and s u b j e c t e d t o NMR a n a l y s i s , the d o u b l e c r o s s p o l a r i z a t i o n s p e c t r u m r e v e a l e d t h a t one o f the a r o m a t i c catecholamine carbons i s c o v a l e n t l y bonded to a r i n g n i t r o g e n of h i s t i d i n e ( F i g . 3 ) . T h i s a r o m a t i c c a r b o n n i t r o g e n c r o s s - l i n k s t r u c t u r e i s c o n s i s t e n t w i t h an i m i d a z o y l n i t r o g e n a t t a c k i n g a p h e n y l carbon o f an o-quinone d e r i v a t i v e o f t h e d i p h e n o l i c compound. A l s o i t has b e e n p r o p o s e d t h a t bonds e x i s t between c u t i c u l a r p r o t e i n and c h i t i n ( 3 8 ) . S o l i d s t a t e N-NMR a n a l y s i s of c h i t i n p r e p a r e d by a l k a l i e x t r a c t i o n o f 1 ,3-[ N 2 l - h i s t i d i n e l a b e l e d Μ· s e x t a p u p a l e x u v i a e r e v e a l e d an ^N c h e m i c a l s h i f t expected f o r t h e substituted imidazole nitrogen c r o s s - l i n k structure depicted i n F i g . 3 ( 2 4 ) . A p p a r e n t l y , t h e c h i t i n i s not coupled d i r e c t l y t o p r o t e i n b u t , i n s t e a d , t o a d i p h e n o l i c c a r b o n , which s e r v e s as a p a r t o f t h e b r i d g e between p r o t e i n and c h i t i n macromolecules. F i g u r e 4 shows t h e p r o p o s e d m e c h a n i s m f o r the m e t a b o l i s m o f p h e n o l i c and d i p h e n o l i c compounds t o q u i n o n o i d c r o s s - l i n k i n g a g e n t s f o r m a c r o m o l e c u l e s i n t h e o u t e r p o r t i o n s o f d a r k brown c u t i c l e from A

l

A

l3

15

1 5

A


KRAMER ET A L

Insect Cuticle Structure and Metabolism (protein)


(chit La)

Figure 3

Proposed s t r u c t u r e f o r d i p h e n o l mediated c r o s s - l i n k between p r o t e i n and c h i t i n i n M. s e x t a pupal c u t i c l e ( f r o m Schaefer e t a l * 2 4 ) .

H O - ^ ^ C I ^ C H g - R - ^ H O - ^ ^ C H g ^ H g - R - - - » 0 °Ç^"

CH -CHfe- R 2

I

HO

0^^=CH-CH -R 2

H

" u H O H ^ y C H . CH-R

I

HO Figure 4

Proposed pathway o f p h e n o l m e t a b o l i s m f o r s c l e r o t i z a t i o n o f c u t i c l e i n c l u d i n g monophenol, d i p h e n o l , o-quinone ( I ) , p-quinone methide ( I I ) and α,ρ-dehydrocatechol ( I I I ) d e r i v a t i v e s . R e a c t i o n s c a t a l y z e d by p h e n o l o x i d a s e o r t y r o s i n a s e f o l l o w e d by keto-enol tautomerization.



176


s t r u c t u r e s s u c h as t o b a c c o hornworm p u p a e , house f l y p u p a r i a and f l o u r beetle e l y t r a . N-Acylcatecholamines, such as NBAD a n d NBANE, a r e c o n v e r t e d by o x i d a t i v e enzymes, such as t y r o s i n a s e s , p h e n o l o x i d a s e s , p e r o x i d a s e s o r l a c c a s e s , t o o-quinone, j>-quinone m e t h i d e , s e m i q u i n o n e o r f r e e r a d i c a l e l e c t r o p h i l i c i n t e r m e d i a t e s , which then c r o s s - l i n k c u t i c u l a r p r o t e i n s and c h i t i n ( 3 3 , 3 9 - 4 1 ) . The N-&a l a n y l c a t e c h o l a m i n e s are g e n e r a l l y more a s s o c i a t e d w i t h s t i f f brown c u t i c l e s and t h e N - a c e t y l c a t e c h o l a m i n e s with s t i f f colorless c u t i c l e , whereas e x c e s s i v e dopamine p o l y m e r i z e s i n t o b l a c k pigment, such as m e l a n i n , i n the o u t e r p a r t s o f t h e c u t i c l e ( 2 6 , 2 7 ,31 , 4 2 ) . N - A c y l a t i o n o f d o p a m i n e w i t h (3-alanine o r a c e t a t e may f a c i l i t a t e e l e c t r o n d e r e a l i z a t i o n from t h e a r o m a t i c r i n g carbons o f t h e o-quinone t o t h e a l i p h a t i c s i d e c h a i n a- o r 3-carbon and f a v o r p r o d u c t i o n o f t h e t a u t o m e r i c j ) - q u i n o n e m e t h i d e o r α,β-dehydro d i p h e n o l . These tautomers provide m u l t i p l e carbon s i t e s f o r a t t a c k by n u c l e o p h i l i c g r o u p s , w h i c h u l t i m a t e l y l e a d t o t h e f o r m a t i o n o f adducts o r c u t i c u l a r c r o s s - l i n k s (24,36,41,43). N o n c u t i c u l a r s t r u c t u r e s have been proposed as model c u t i c l e s i n w h i c h s i m i l a r h a r d e n i n g r e a c t i o n s o c c u r , b u t these m a t e r i a l s a r e g e n e r a l l y l e s s complex i n terms o f c h e m i c a l c o m p o s i t i o n , even though t h e y u n d e r g o c h e m i c a l t r a n s f o r m a t i o n s s i m i l a r t o those t h a t o c c u r during c u t i c u l a r tanning. S t r u c t u r e s such as cocoons and egg c a s e s o f i n s e c t s are composed m o s t l y o f p r o t e i n but may a l s o c o n t a i n some d i p h e n o l i c compounds, w h i c h t a n t h e p r o t e i n ( T a b l e X I I ) . They c o n s i s t o f l i t t l e o r no c h i t i n o r l i p i d . The p r o t e i n a c e o u s D r o s o p h i l a c h o r i o n i s c r o s s - l i n k e d b y an endogeneous p e r o x i d a s e t h a t c a u s e s f o r m a t i o n o f i n t e r p o l y p e p t i d e d i - and t r i - t y r o s y l adducts (44). The s i l k m o t h , Bombyx m o r i , makes a cocoon t h a t i s untanned and c o n s i s t s o f t h e f i b r o u s p r o t e i n f i b r o i n and the p r o t e i n g l u e sericin. P r o t e i n b u t no d i p h e n o l s a r e e v i d e n t i n t h e C-NMR s p e c t r u m o f B. m o r i c o c o o n s ( T a b l e X I I ) . On the o t h e r hand, t h e c o c o o n s o f Anthereae polyphemus, A. m y l i t t a and Hyalophora c e c r o p i a a r e t a n n e d a n d c o n t a i n n o t o n l y p r o t e i n b u t a l s o 2-3% d i p h e n o l s . The egg c a s e o f t h e p r a y i n g m a n t i s , Tenodera s i n e n s i s , i s composed o f 9 5 % p r o t e i n and 5% d i p h e n o l . I n t h e s e s t r u c t u r e s , which p r o v i d e p r o t e c t i v e h o u s i n g s f o r d e v e l o p i n g pupae o r e g g s , t h e d i p h e n o l i c compounds p r o b a b l y p r o v i d e s t r e n g t h by cementing ( c r o s s - l i n k i n g ) t h e p r o t e i n chains together. T h u s , d i p h e n o l i c compounds o r t h e i r o x i d i z e d m e t a b o l i t e s may s c l e r o t i z e s t r u c t u r e s t h a t c o n t a i n p r i m a r i l y b o t h p r o t e i n and c h i t i n o r o n l y p r o t e i n . 13

Table X I I .

Major O r g a n i c Components o f N o n c u t i c u l a r Insect S t r u c t u r e s 3

Cocoons Component

B. mori

Protein Diphenol

100

Insect Cuticle Structure and Metabolism - ACS Symposium Series

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