Insect Neuropeptides - American Chemical Society

relations of the insect neuropeptides that can trigger color changes ... of melanin in the cuticle and ommochromes in the epidermis of armyworm specie...
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Chapter 11 Insect Neuropeptides Influence on Color Change in Insects and Chromatophoral Pigment Movements in Crustaceans K. Ranga Rao, Carl J. Mohrherr, Sherman L. Bonomelli, John P. Riehm, and Timothy G. Kingan Downloaded by UNIV OF ROCHESTER on August 31, 2017 | http://pubs.acs.org Publication Date: January 25, 1991 | doi: 10.1021/bk-1991-0453.ch011

1

Department of Biology, University of West Florida, Pensacola, FL 32514

This report focuses on the identification and sequence relations of the insect neuropeptides that can trigger color changes in crustaceans, with emphasis on the newly identified family of pigment-dispersing peptides. The latter show no relation to the insect melanization and reddish coloration hormone (MRCH) and are distinct from the adipokinetic hormone (AKH) family peptides, related to the crustacean red pigment concentrating hormone (RPCH). The pigment-dispersing factors (PDFs), identified from the insects Romalea microptera and Acheta domesticus, as well as the pigment-dispersing hormones (PDHs) of Crustacea, are octadecapeptides sharing several features. The function of PDFs in insects is unknown, although immunocytochemical localization points to a neuromodulatory role. In orthopterans PDH-immunoreactive neurons are located in the optic lobes, with fiber projections into the brain as well as arborizations in the optic lobes. Enzyme-linked immunosorbant assays (ELISA) confirm the optic lobes as a rich source of immunoreactive PDF in Romalea. Among a r t h r o p o d s , c r u s t a c e a n s a r e w e l l known f o r t h e i r a b i l i t y t o display reversible integumental color changes a n d eye pigment movements. The former a r e b r o u g h t about by d i s p e r s i o n o r c o n c e n t r a t i o n ( a g g r e g a t i o n ) o f pigment g r a n u l e s w i t h i n e p i t h e l i a l chromatophores. Eye pigment movements, o c c u r r i n g d u r i n g t h e c o u r s e o f l i g h t o r dark a d a p t a t i o n , i n v o l v e the m i g r a t i o n o f s c r e e n i n g pigment g r a n u l e s w i t h i n p h o t o r e c e p t o r c e l l s ( r e t i n u l a r c e l l s ) and i n c e r t a i n e x t r a r e t i n u l a r c e l l s a s s o c i a t e d w i t h t h e ommatidia. whereas t h e pigment movements w i t h i n t h e r e t i n u l a r c e l l s appear t o be d i r e c t l y i n f l u e n c e d by c o n d i t i o n s o f i l l u m i n a t i o n , the pigmentary r e s p o n s e s o f e x t r a r e t i n u l a r 1

Current address: Insect Chemical Ecology Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705 0097-6156/91/0453-0110$06.00/0 © 1991 American Chemical Society

Menn et al.; Insect Neuropeptides ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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11. RAO ET AL.

Color Change and Chromatophoral Pigment Movements

eye pigment c e l l s and e p i t h e l i a l chromatophores i n C r u s t a c e a a r e c o n t r o l l e d by n e u r o s e c r e t o r y hormones (1-3). These hormones a r e r e a d i l y s e p a r a b l e i n t o two sets: the hormones r e s p o n s i b l e f o r c h r o m a t o p h o r a l pigment c o n c e n t r a t i o n and o m m a t i d i a l d a r k a d a p t a t i o n belong t o one s e t , and they are d i s t i n c t from those causing chromatophoral pigment d i s p e r s i o n and o m m a t i d i a l l i g h t adaptation (2, 4). Many i n s e c t s show l i g h t / d a r k - a d a p t i v e eye pigment movements ( 5 ) , b u t o n l y a few s p e c i e s can undergo r a p i d i n t e g u m e n t a l c o l o r changes (6) . S c r e e n i n g pigment movements i n i n s e c t compound eyes were once thought t o be under nervous c o n t r o l , b u t r e c e n t e v i d e n c e p o i n t s t o a d i r e c t l i g h t - i n d u c e d pigment m i g r a t i o n i n r e t i n u l a r c e l l s (7) and i n e x t r a r e t i n u l a r eye pigment c e l l s (8). In c o n t r a s t to Crustacea, there i s no e v i d e n c e f o r hormonal c o n t r o l o f eye pigment movements i n insects. Neurohormones a r e , however, i m p l i c a t e d i n the c o n t r o l o f c o l o r changes i n i n s e c t s . The somewhat slow, m o r p h o l o g i c a l c o l o r changes r e s u l t i n g from q u a l i t a t i v e o r q u a n t i t a t i v e a l t e r a t i o n s i n p i g m e n t a t i o n a r e r e g u l a t e d by m u l t i p l e h o r m o n e s - - j u v e n i l e hormone, ecdysone, and neurohormones--the i n t e r p l a y o f which seems t o v a r y w i t h the i n s e c t s p e c i e s (6). T h i s paper summarizes c u r r e n t knowledge o f the c h e m i s t r y o f neurohormones i n v o l v e d i n the r e g u l a t i o n o f c o l o r changes i n i n s e c t s , as well as the nature of other insect neuropeptides with c h r o m a t o p h o r o t r o p i c a c t i o n s i n C r u s t a c e a - - w i t h emphasis on pigmentdispersing peptides. M e l a n i z a t i o n and R e d d i s h C o l o r a t i o n Hormone (MRCH) Among the neurohormones r e g u l a t i n g insect pigmentation, primary s t r u c t u r e i s known f o r o n l y MRCH. T h i s hormone promotes the f o r m a t i o n o f m e l a n i n i n the c u t i c l e and ommochromes i n the e p i d e r m i s o f armyworm s p e c i e s (9, 10). M u l t i p l e forms o f MRCH have been i s o l a t e d from the heads o f a d u l t Bombyx mori (11-13) and one o f t h e s e , MRCH-1, was found t o be structurally identical t o PBAN-I (pheromone b i o s y n t h e s i s a c t i v a t i n g neurohormone) i s o l a t e d from the same s o u r c e (14) . A l t h o u g h t h i s hormone a c t s o n l y as PBAN i n Bombyx, the same m o l e c u l e s e r v e s d u a l f u n c t i o n s i n the cutworm Spodoptera l i t u r a by p r o m o t i n g cuticular m e l a n i z a t i o n i n the l a r v a l s t a g e and sex pheromone p r o d u c t i o n i n the a d u l t s t a g e (Matsumoto, S.; Kitamura, A.; Nagasawa, H.; Kataoka, H.; O r i k a s a , C ; M i t s u i , T.; S u z u k i , A. Insect Biochem., i n press). The PBAN-I (MRCH-I) i s o l a t e d from Bombyx i s s t r u c t u r a l l y r e l a t e d t o the PBAN c h a r a c t e r i z e d from H e l i o t h i s zea (15; see a l s o a r t i c l e by R a i n a et al., t h i s volume). As i n d i c a t e d below, t h e s e p e p t i d e s have common t e r m i n i ( N - t e r m i n a l Leu, C - t e r m i n a l Leu-amide), c o n s e r v e d c h a i n l e n g t h (33 r e s i d u e s ) , and show r e s i d u e d i f f e r e n c e s a t o n l y 7 p o s i t i o n s . Heliothis: Bombyx:

LSDDMPATPADQEMYRQDPEQIDSRTKYFS PRLamide LSiH)MPATPADQEMYQPDPE£/f£SRT!?YFSPRLamide

The N - t e r m i n a l sequence o f MRCH/PBAN shows some s i m i l a r i t y t o a p o r t i o n o f i n s u l i n - l i k e growth f a c t o r - I I , whereas the C - t e r m i n a l sequence of MRCH/PBAN i s nearly identical to the C-terminal p e n t a p e p t i d e sequence o f l e u k o p y r o k i n i n ( p G l u - T h r - S e r - P h e - T h r - P r o - A r g -

Menn et al.; Insect Neuropeptides ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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Leu-amide) i s o l a t e d from c o c k r o a c h heads ( 1 6 ) . MRCH/PBAN p e p t i d e s do n o t resemble any o f the c r u s t a c e a n c h r o m a t o p h o r o t r o p i n s , and the former p e p t i d e s may n o t be a c t i v e on c r u s t a c e a n chromatophores and eye pigment cells. I t remains unknown whether MRCH i n f l u e n c e s r a p i d c o l o r changes b r o u g h t about by i n t r a c e l l u l a r pigment m i g r a t i o n i n i n s e c t s .

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Control

of Rapid Color

Changes i n

Insects

R a p i d c o l o r changes, c a u s e d by pigment m i g r a t i o n s w i t h i n chromatophores o r w i t h i n e p i d e r m a l c e l l s , a r e d i s p l a y e d by o n l y a few s p e c i e s of i n s e c t s , e.g., Corethra and the s t i c k i n s e c t Carausius. Ameboid chromatophores a s s o c i a t e d w i t h a n t e r i o r and p o s t e r i o r t r a c h e a l b l a d d e r s contribute t o c o l o r change i n Corethra. Migration o f the dark ommochromes from the base o f the e p i d e r m a l c e l l t o i t s s u r f a c e c a u s e s a r a p i d c o l o r change, body d a r k e n i n g , i n Carausius. A darkening neurohormone p r o d u c e d i n the b r a i n i s i m p l i c a t e d i n the c o n t r o l o f t h e s e r a p i d c o l o r changes (17-19). T h i s c h r o m a c t i v a t i n g hormone i s cross-reactive: the e x t r a c t s o f Corethra heads cause d a r k e n i n g i n Carausius, and Carausius o r g a n e x t r a c t s are a c t i v e on Corethra as w e l l as on Carausius. These r e a c t i o n s can a l s o be t r i g g e r e d by brain e x t r a c t s from a v a r i e t y o f i n s e c t s l a c k i n g c o l o r change, p o i n t i n g t o a wide d i s t r i b u t i o n o f the d a r k e n i n g hormone among i n s e c t s (18). A t t e m p t s have been made t o c h a r a c t e r i z e t h i s hormone (20, 21), b u t i t s s t r u c t u r e remains unknown. D i f f e r e n t i a t i o n o f Chromactive F a c t o r s

i n Insect

Nervous

Tissues

B e s i d e s the i n s e c t d a r k e n i n g hormone d i s c u s s e d above, the insect n e r v o u s t i s s u e s c o n t a i n f a c t o r s which can t r i g g e r c o l o r changes i n crustaceans. I n i t i a l s t u d i e s examined the a b i l i t y o f i n s e c t t i s s u e extracts t o cause body b l a n c h i n g (due t o c h r o m a t o p h o r a l pigment c o n c e n t r a t i o n ) i n prawns and shrimp (22) . T h i s b l a n c h i n g s u b s t a n c e was f o u n d i n the c o r p o r a c a r d i a c a o f many i n s e c t s p e c i e s (23-25), whereas the s u b s t a n c e c a u s i n g d a r k e n i n g i n i n s e c t s (Corethra, Carausius) was p r e s e n t i n the c e n t r a l nervous system. I n i t i a l s e p a r a t i o n by paper e l e c t r o p h o r e s i s (26, 27) i n d i c a t e d t h a t the o b s e r v e d a c t i v i t i e s a r e due t o two d i s t i n c t substances: F a c t o r A, causing concentration of c h r o m a t o p h o r a l pigments i n c r u s t a c e a n s and showing o n l y a s l i g h t e f f e c t on color change i n Carausius; Factor C, which lacks pigmentc o n c e n t r a t i n g e f f e c t i n c r u s t a c e a n s b u t causes d a r k e n i n g i n Carausius. F a c t o r A i s p r e s e n t i n c r u s t a c e a n s i n u s g l a n d s and i n s e c t c o r p o r a c a r d i a c a , whereas F a c t o r C i s found i n the b r a i n o f Carausius and a b s e n t i n the c o r p o r a c a r d i a c a (26, 27). P e p t i d e s o f the RPCH/AKH F a m i l y The s i m i l a r i t y o f the c r u s t a c e a n b l a n c h i n g s u b s t a n c e ( F a c t o r A) found i n c r u s t a c e a n s i n u s g l a n d s and i n s e c t c o r p o r a c a r d i a c a has been c o n f i r m e d by the s t r u c t u r a l i d e n t i f i c a t i o n o f a f a m i l y o f n e u r o p e p t i d e s common t o a r t h r o p o d s . The f i r s t i d e n t i f i e d member o f t h i s f a m i l y i s RPCH ( r e d pigment c o n c e n t r a t i n g hormone) , an o c t a p e p t i d e (pGlu-Leu-AsnP h e - S e r - P r o - G l y - T r p - a m i d e ) i s o l a t e d from e y e s t a l k s o f the shrimp Pandalus borealis (28). Shortly thereafter, a r e l a t e d peptide-d e s i g n a t e d as a d i p o k i n e t i c hormone (AKH)--was p u r i f i e d from l o c u s t

Menn et al.; Insect Neuropeptides ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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11. RAO ET AL.

Color Change and Chromatophoral Pigment Movements

c o r p o r a c a r d i a c a and shown t o be a d e c a p e p t i d e : pGlu-Leu-Asn-Phe-ThrP r o - A s n - T r p - G l y - T h r - a m i d e (29). B e s i d e s t h e i r sequence s i m i l a r i t y , the two p e p t i d e s were found t o be a c t i v e i n c r o s s - t e s t s . I n i t i a l tests i n d i c a t e d t h a t RPCH and AKH a r e a b l e to mimic each o t h e r and e l i c i t h y p e r l i p e m i a i n l o c u s t s and b u t t e r f l i e s , h y p e r g l y c e m i a i n c o c k r o a c h e s , and c o n c e n t r a t i o n o f chromatophore pigments i n C r u s t a c e a (29-32). The r e l a t i v e p o t e n c i e s o f the p e p t i d e s v a r i e d w i t h the t e s t system, each hormone b e i n g g e n e r a l l y more r e a c t i v e i n i t s own system. The sequence o f RPCH i s i d e n t i c a l among the c r u s t a c e a n s p e c i e s examined, the shrimp Pandalus borealis (28) and the c r a b s Cancer magister and Carcinus maenas (33). I n c o n t r a s t , as r e v i e w e d by K e e l e y et al. i n t h i s volume, the A K H - r e l a t e d p e p t i d e s c o n t a i n 8 t o 10 r e s i d u e s and show c o n s i d e r a b l e sequence h e t e r o g e n e i t y , w i t h as many as 13 d i f f e r e n t p e p t i d e s i d e n t i f i e d so f a r from v a r i o u s i n s e c t s p e c i e s (34, 35). D e s p i t e t h i s h e t e r o g e n e i t y , the RPCH/AKH p e p t i d e s a r e members o f a f a m i l y w i t h the f o l l o w i n g common f e a t u r e s : p G l u as the b l o c k e d amino-terminus, Phe a t p o s i t i o n 4, Trp a t p o s i t i o n 8, and an amidated c a r b o x y l terminus. The A K H - r e l a t e d p e p t i d e s show v a r i e d functions i n insects--hyperglycemia, hypertrehalosemia, hyperlipemia, c a r d i o a c c e l e r a t i o n , myotropic a c t i o n , or a combination o f these r o l e s - depending on the s p e c i e s . Thus, w h i l e m a i n t a i n i n g some c r i t i c a l degree o f sequence s i m i l a r i t y , t h e s e p e p t i d e s e v o l v e d a p p r o p r i a t e s t r u c t u r a l m o d i f i c a t i o n s to s e r v e unique f u n c t i o n s i n d i f f e r e n t s p e c i e s . It remains u n c l e a r whether the A K H - r e l a t e d p e p t i d e s a i d i n the c o n t r o l o f pigmentation or pigmentary migrations i n i n s e c t s . Pigment-Dispersing Actions C r u s t a c e a n Chromatophores

of

Insect

Nervous

Tissue

Extracts

on

Early workers n o t e d that i n s e c t head e x t r a c t s cause n o t only c h r o m a t o p h o r a l pigment c o n c e n t r a t i o n i n shrimp and prawns, b u t a l s o c h r o m a t o p h o r a l pigment d i s p e r s i o n i n c r a b s (22). The i n s e c t f a c t o r s r e s p o n s i b l e f o r the l a t t e r a c t i o n d i d n o t r e c e i v e much a t t e n t i o n u n t i l r e c e n t l y . The d i s c o v e r y o f s t r u c t u r a l s i m i l a r i t y o f RPCH and AKH (28, 29) has t r i g g e r e d renewed i n t e r e s t i n d e t e r m i n i n g the r e l a t i o n s h i p o f i n s e c t p i g m e n t - d i s p e r s i n g f a c t o r s and c r u s t a c e a n p i g m e n t - d i s p e r s i n g hormones. I n t e s t s f o r melanophore pigment d i s p e r s i o n i n e y e s t a l k l e s s i n d i v i d u a l s o f the f i d d l e r c r a b Uca pugllator, head e x t r a c t s from the c o c k r o a c h Periplaneta americana and the honey bee Apis m e l l i f e r a showed s t r o n g a c t i v i t y , as d i d the c r u s t a c e a n e y e s t a l k e x t r a c t s (36). Head e x t r a c t s o f the b u t t e r f l y Danaus plexippus and the mealworm Tenebrio molitor were, however, i n a c t i v e i n the c h r o m a t o p h o r a l d i s p e r s i o n a s s a y . I n g e l f i l t r a t i o n chromatography, the pigment d i s p e r s i n g hormone (PDH) a c t i v i t y o f Uca e y e s t a l k e x t r a c t s emerged i n a s i n g l e peak, whereas the a c t i v i t y o f Periplaneta head e x t r a c t s e l u t e d i n two peaks w i t h a p p a r e n t m o l e c u l a r masses o f 3500 and 1000 d a l t o n s . The Uca PDH and the l a r g e r f a c t o r from Periplaneta showed n e a r l y i d e n t i c a l e l u t i o n profiles. D e s p i t e t h i s apparent s i m i l a r i t y , the c o r r e s p o n d i n g f a c t o r from Periplaneta d i d n o t seem to be i d e n t i c a l t o Uca PDH; the former was o x i d a t i o n - r e s i s t a n t (36), whereas the l a t t e r was oxidations e n s i t i v e (37). The f a c t o r from Apis appeared t o be an o x i d a t i o n s e n s i t i v e l a r g e r p o l y p e p t i d e , about 20,000 d a l t o n s (36). These i n i t i a l

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e v a l u a t i o n s s u g g e s t e d t h a t the p i g m e n t - d i s p e r s i n g assemblage o f h e t e r o g e n e o u s p o l y p e p t i d e s .

factors

are

an

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Characterization of Pigment-Dispersing Factors i n Insects L a r g e - s c a l e i s o l a t i o n and p u r i f i c a t i o n o f p i g m e n t - d i s p e r s i n g f a c t o r s (PDFs) was u n d e r t a k e n i n our l a b o r a t o r y , u t i l i z i n g l y o p h i l i z e d heads o f the c r i c k e t Acheta domesticus (38, 39) and the l u b b e r g r a s s h o p p e r Romalea microptera (40) as s t a r t i n g m a t e r i a l , and i d e n t i f y i n g the a c t i v e compound by a s s a y s f o r melanophore pigment d i s p e r s i o n i n e y e s t a l k l e s s Uca. The p r o t o c o l s employed were a c o m b i n a t i o n o f methods p r o v e n s u c c e s s f u l f o r the i s o l a t i o n o f PDHs from c r u s t a c e a n e y e s t a l k s (41, 42). The i n s e c t PDFs were i s o l a t e d as f o l l o w s . L y o p h i l i z e d heads (100-150 g l o t s ) were powdered and e x t r a c t e d i n b o i l i n g water. The e x t r a c t was c o o l e d , a c i d i f i e d (by a d d i t i o n o f a c e t i c a c i d up t o 5 % ) , c e n t r i f u g e d , and the s u p e r n a t a n t s u b j e c t e d t o b a t c h chromatography w i t h Bio-Rex 70, a weakly a c i d i c c a t i o n - e x c h a n g e r e s i n (Bio-Rad) . The r e s i n was added t o the e x t r a c t , the m i x t u r e s t i r r e d f o r 4 t o 6 h o u r s , and t h e n p o u r e d i n t o a column. The PDF a d s o r b e d t o the r e s i n was r e c o v e r e d by e l u t i o n w i t h 50% a c e t i c a c i d . About 90-95% o f the PDF a c t i v i t y i n the o r i g i n a l e x t r a c t c o u l d be r e c o v e r e d by t h i s b a t c h chromatography. T h i s m a t e r i a l was t a k e n up i n water and e x t r a c t e d w i t h the o r g a n i c phase o f 1 - b u t a n o l / 0.1 M NH (1:1, v/v) . R e p e t i t i v e e x t r a c t i o n w i t h the o r g a n i c phase (up t o f i v e t i m e s ) b r o u g h t most o f the PDF i n t o t h i s phase, which was t h e n d r i e d and s u b j e c t e d t o an a d d i t i o n a l l i q u i d p a r t i t i o n i n g step. The d r i e d m a t e r i a l was t a k e n up i n 0.2 M HC1 and p a r t i t i o n e d w i t h c y c l o h e x a n e / b u t a n o l (4:1, v / v ) . Most o f t h e PDF a c t i v i t y was r e t a i n e d i n the aqueous phase, and t h i s was l y o p h i l i z e d and f u r t h e r p u r i f i e d by a s e r i e s o f c h r o m a t o g r a p h i c s t e p s : a) g e l f i l t r a t i o n , Sephadex G-25-50, e l u a n t 1 M a c e t i c a c i d ; b) ion-exchange chromatography, CM-Sephadex C-25-120, e l u a n t 0.075 M ammonium a c e t a t e pH 4.9; c ) p a r t i t i o n chromatography, Sephadex G-25-50, g e l bed i n an aqueous phase o f b u t a n o l / a c e t i c a c i d / w a t e r (4:1:5, v / v ) , e l u a n t o r g a n i c phase o f the s o l v e n t system; d) r e v e r s e - p h a s e HPLC, Whatman ODS-2, 3045% a c e t o n i t r i l e w i t h 0.1% TFA o v e r a 30-minute g r a d i e n t . W i t h head e x t r a c t s from Acheta (37) , as w e l l as Romalea (39) , the g e l f i l t r a t i o n s t e p y i e l d e d two peaks o f p i g m e n t - d i s p e r s i n g a c t i v i t y , emerging a t about 0.5 and 0.8 column volumes, r e s p e c t i v e l y . The f i r s t peak c o i n c i d e d w i t h the e l u t i o n p r o f i l e o f PDH from Uca; the PDF i n t h i s zone, l i k e the Uca PDH, c a u s e d d i s p e r s i o n o f c h r o m a t o p h o r a l pigments i n Uca and d i d n o t cause pigment c o n c e n t r a t i o n . The s e c o n d a c t i v e peak, emerging a t about 0.8 column volume i n g e l f i l t r a t i o n , t r i g g e r e d n o t o n l y melanophore pigment d i s p e r s i o n b u t a l s o l e u c o p h o r e ( w h i t e ) pigment c o n c e n t r a t i o n i n Uca. The two b i o l o g i c a l a c t i v i t i e s were inseparable from each other, even after ion-exchange chromatography, p a r t i t i o n chromatography, and r e v e r s e - p h a s e HPLC. T h i s f a c t o r c a n be c a l l e d p i g m e n t - d i s p e r s i n g / c o n c e n t r a t i n g f a c t o r (PDCF). Initial s e q u e n c i n g attempts w i t h H P L C - p u r i f i e d Acheta PDCF i n d i c a t e d a b l o c k e d amino-terminus. I t was d e b l o c k e d by p y r o g l u t a m y l amino p e p t i d a s e and s u b j e c t e d t o gas-phase s e q u e n c i n g . The r e s u l t i n g d a t a , a l o n g w i t h s p e c t r a l a n a l y s i s , i n d i c a t e d t h a t the p u r i f i e d p e p t i d e has the sequence: p G l u - V a l - A s n - P h e - S e r - T h r - G l y - T r p - a m i d e . T h i s was a l s o t h e deduced sequence f o r Gryllus AKH (43). The s y n t h e t i c p e p t i d e , 3

Menn et al.; Insect Neuropeptides ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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11. RAO ET AL.

Color Change and Chromatophoral Pigment Movements

however, f a i l e d t o induce melanophore pigment d i s p e r s i o n o r l e u c o p h o r e pigment concentration. This indicates that t h e melanophored i s p e r s i n g / l e u c o p h o r e - c o n c e n t r a t i n g c h r o m a t o p h o r o t r o p i n i s o n l y a minor component o f t h e p u r i f i e d m a t e r i a l , and a l t e r n a t i v e s t r a t e g i e s have t o be d e v e l o p e d f o r s e p a r a t i n g i t from the Gryllus/Acheta AKH o c t a p e p t i d e . Our e v a l u a t i o n s i n d i c a t e t h a t the c o r p o r a c a r d i a c a a r e a r i c h e r s o u r c e o f PDCF i n Acheta, and we hope t o r e i n v e s t i g a t e t h i s p e p t i d e . F o r t u n a t e l y , the PDFs from Roma lea (40) and Acheta (38, 39) were amenable t o 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 . The f i n a l y i e l d o f PDF was h i g h e r from Romalea (11 nmol from 3,000 heads) t h a n Acheta (