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Aspects of the chemical ecology of lygaeid bugs (Oncopeltus fasciatus and Lygaeus kalmii kalmii) feeding… Isman, Murray Bruce 1977

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ASPECTS OF THE CHEMICAL ECOLOGY OF LYGAEID BUGS (ONCOPELTUS FASCIATUS AND LYGAEUS KALMII KALMII) FEEDING ON MILKWEEDS (ASCLEPIAS SPECIES) IN CENTRAL CALIFORNIA by MURRAY BRUCE ISMAN B . S c , U n i v e r s i t y o f B r i t i s h Columbia, 1975 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE, i n THE FACULTY OF GRADUATE STUDIES (Department o f Zoology) We a c c e p t t h i s t h e s i s as c o n f o r m i n g to the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH'COLUMBIA A p r i l , 1977 (c) Murray Bruce Isman, 1977 In present ing th is thes is in p a r t i a l fu l f i lment of the requirements f o r an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of t h i s t h e s i s for scho la r ly purposes may be granted by the Head of my Department or by h is representat ives . It is understood that copying or p u b l i c a t i o n of th is thes is fo r f i n a n c i a l gain sha l l not be allowed without my writ ten permission. Department of ZOOLOGY The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1WS F r o n t i s p i e c e . A d u l t O n c o p e l t u s f a s c i a t u s and Lygaeus k a l m i i k a l m i i ( c e n t e r ) on a d e h i s c e n t pod o f A s c l e p i a s f a s c i c u l a r i s i n Napa County, C a l i f o r n i a . ( i i i ) ( i v ) ABSTRACT A p l a n t - i n s e c t a l l o m o n a l system was i n v e s t i g a t e d , i n v o l v i n g seed bugs ( L y g a e i d a e ) on milkweeds ( A s c l e p i a s s p p . ) . The a b i l i t y o f the i n s e c t s t o s e q u e s t e r secondary compounds from h o s t p l a n t s was s t u d i e d i n d e t a i l i n c e n t r a l C a l i f o r n i a . A c o l o r i m e t r i c a s s a y was used t o q u a n i t i f y the amount o f c a r d e n o l i d e s ( c a r d i a c g l y c o s i d e s ) i n the l y g a e i d bugs O n c o p e l t u s  f a s c i a t u s and Lygaeus k a l m i i k a l m i i and n i n e s p e c i e s o f milkweed h o s t p l a n t s . The c a r d e n o l i d e c o n t e n t o f i n d i v i d u a l a d u l t i n s e c t s , d e t e r -mined i n microgram e q u i v a l e n t s o f d i g i t o x i n , v a r i e d from z e r o to o v e r 300 pg per i n s e c t . Sources o f v a r i a t i o n o f c a r d e n o l i d e c o n t e n t i n the i n s e c t s i n c l u d e d i n t e r s p e c i f i c and i n t r a s p e c i f i c d i f f e r e n c e s i n the c a r d e n o l i d e c o n t e n t o f the h o s t p l a n t s p e c i e s , and a l s o d i f f e r e n c e s i n the c o n t e n t o f p l a n t organs on which i n s e c t s were f e e d i n g . When r e a r e d i n the l a b o r a t o r y on a d i e t o f milkweed s e e d s , the uptake o f c a r d e n o l i d e s by the bugs was p r o p o r t i o n a l to the c a r d e n o l i d e c o n t e n t o f the seeds. However, f i e l d c o l l e c t e d l y g a e i d s c o n t a i n e d fewer c a r d e n o l i d e s than were a v a i l a b l e i n the p l a n t s on which they were f e e d i n g . T h i s s u g g e s t s t h a t s e v e r a l e c o l o g i c a l p a r a m e t e r s , such as d i f f e r e n t r e p r o d u c t i v e p h e n o l o g i e s and m o r p h o l o g i e s o f the d i f f e r e n t h o s t s p e c i e s , may i n t e r f e r e w i t h the bugs' a c q u i s i t i o n o f c a r d e n o l i d e s . fj. f a s c i a t u s and L. k. k a l m i i d i f f e r i n t h e i r f e e d i n g r e q u i r e -ments, h o s t p l a n t u t i l i z a t i o n , and a b i l i t y to s e q u e s t e r c a r d e n o l i d e s from t h e i r h o s t s . However, the l a r v a l growth and development o f (v) CJ. f a s c i a t u s and L_. k_. k a l m i i i n the l a b o r a t o r y on seeds o f d i f f e r e n t milkweed s p e c i e s d i d not v a r y w i t h p l a n t s p e c i e s o r c a r d e n o l i d e c o n t e n t o f the seeds. T h e r e f o r e , seeds o f a l l p l a n t s p e c i e s were e q u a l l y s u i t a b l e as f o o d s o u r c e s f o r the bugs. The c o l o n i z a t i o n p a t t e r n o f CJ. f a s c i a t u s on s p e c i e s o f A s c l e p i a s i n n o r t h c e n t r a l C a l i f o r n i a s u g g e s t s . t h a t t h i s s p e c i e s does n o t maximize i t s o p p o r t u n i t i e s t o s e q u e s t e r l a r g e q u a n t i t i e s o f c a r d e n o l i d e s from p o t e n t i a l h o s t s . Over 50 per c e n t o f f i e l d c o l l e c t e d l y g a e i d s i n t h i s s t u d y c o n t a i n e d l e s s than 50 yg o f c a r d e n o l i d e . The e c o l o g i c a l s i g -n i f i c a n c e o f c a r d e n o l i d e s e q u e s t r a t i o n by l y g a e i d s as a d e f e n s i v e s t r a t e g y may depend on s e v e r a l modes o f p h a r m a c o l o g i c a l a c t i v i t y i n p o t e n t i a l v e r t e b r a t e p r e d a t o r s , not s i m p l y e m e t i c p o t e n t i a l . A l t e r n a t e l y , the p r e s e n c e o f v o l a t i l e s e c r e t i o n s from the s c e n t glands and h i s t a m i n e - l i k e compounds i n the d o r s o - l a t e r a l space f l u i d o f (). f a s c i a t u s and I. k_. k a l m i i c o u l d be more i m p o r t a n t than s e q u e s t e r e d c a r d e n o l i d e s as a n t i - p r e d a t o r s t r a t e g i e s . The d a t a s u g g e s t t h a t the e f f e c t i v e n e s s o f c a r d e n o l i d e seques-t r a t i o n as a c h e m i c a l d e f e n s e s t r a t e g y may v a r y o v e r the g e o g r a p h i c a l range o f t h e s e i n s e c t s p e c i e s , and may v a r y i n the c o u r s e o f a season a t a p a r t i c u l a r l o c a t i o n . T h e r e f o r e , c a r d e n o l i d e s e q u e s t r a t i o n does not appear to be a c r u c i a l a s p e c t i n the f e e d i n g and p o p u l a t i o n e c o l o g y o f t h e s e l y g a e i d s p e c i e s a t l e a s t i n c e n t r a l C a l i f o r n i a . ( v i ) TABLE OF CONTENTS Page T i t l e Page i F r o n t i s p i e c e i i i A b s t r a c t i v T a b l e o f Co n t e n t s v i L i s t o f T a b l e s v i i L i s t o f F i g u r e s i x Acknowledgements x i I INTRODUCTION 1 II MATERIALS AND METHODS A. F i e l d Study o f Milkweeds and L y g a e i d s 6 B. E x t r a c t i o n o f C a r d e n o l i d e s from P l a n t M a t e r i a l 9 C. C o l o r i m e t r i c Assay o f C a r d e n o l i d e s 11 D. E x t r a c t i o n o f C a r d e n o l i d e s from L y g a e i d s 12 E. L a b o r a t o r y R e a r i n g o f L y g a e i d s 15 I I I RESULTS A. Milkweed F l o r a and L y g a e i d Fauna 20 B. C a r d e n o l i d e Content o f P l a n t M a t e r i a l 25 C. C a r d e n o l i d e C o n t e n t o f F i e l d C o l l e c t e d L y g a e i d s 32 D. C a r d e n o l i d e C o n t e n t o f L a b o r a t o r y - R e a r e d L y g a e i d s 47 E. L a r v a l Growth and Development o f L y g a e i d s on 47 D i f f e r e n t S p e c i e s o f Milkweed i n the L a b o r a t o r y IV D i s c u s s i o n 73 R e f e r e n c e s 97 ( v i i ) LIST OF TABLES T a b l e Page I O c c u r r e n c e o f CL f a s c i a t u s and L^ . k. k a l m i i on d i f f e r e n t 12 SDecies o f A s c l e p i a s i n two a r e a s o f C a l i f o r n i a , summer 1976 II Gross c a r d e n o l i d e c o n t e n t o f seed o f A s c l e p i a s s p e c i e s 27 from C a l i f o r n i a I I I Gross c a r d e n o l i d e c o n t e n t o f p l a n t samples o f A s c l e p i a s 29 from C a l i f o r n i a IV Gross c a r d e n o l i d e c o n t e n t o f CJ_. f a s c i a t u s and L^ . k_. 36 k a l m i i c o l l e c t e d on A s c l e p i a s i n c e n t r a l C a l i f o r n i a i n 1976 V Temporal v a r i a t i o n i n g r o s s c a r d e n o l i d e c o n t e n t o f f i e l d 44 c o l l e c t e d CL f a s c i a t u s from c e n t r a l C a l i f o r n i a VI Gross c a r d e n o l i d e c o n t e n t o f (3. f a s c i a t u s and L. k. 48 k a l m i i r e a r e d i n the l a b o r a t o r y a t 25°C on milkweed seed VII Gross c a r d e n o l i d e c o n t e n t o f CJ. f a s c i a t u s r e a r e d i n the 51 the l a b o r a t o r y a t 21oC on milkweed seed V I I I D u r a t i o n o f l a r v a l development and t e n e r a l a d u l t wet 57 w e i g h t o f 0_. f a s c i a t u s r e a r e d i n the l a b o r a t o r y a t 25°C on t h e seeds o f d i f f e r e n t s p e c i e s o f A s c l e p i a s and H e l i a n t h u s IX D u r a t i o n o f l a r v a l development and t e n e r a l a d u l t wet 58 weight o f I. k. k a l m i i r e a r e d i n the l a b o r a t o r y a t 25°C on the seeds o f d i f f e r e n t s p e c i e s o f A s c l e p i a s and H e l i a n t h u s X I n f l u e n c e o f s e q u e s t e r e d c a r d e n o l i d e s on l a r v a l 59 development o f 0_. f a s c i a t u s r e a r e d i n the l a b o r a t o r y a t 21°C on the seeds o f d i f f e r e n t s p e c i e s o f A s c l e p i a s XI A d u l t body we i g h t o f CJ. f a s c i a t u s r e a r e d i n the l a b o r a t o r y 65 a t 21°C on the seeds o f d i f f e r e n t s p e c i e s o f A s c l e p i a s XII P r o n o t a l w i d t h o f a d u l t CJ. f a s c i a t u s r e a r e d i n the 69 l a b o r a t o r y a t 21°C on the seeds o f d i f f e r e n t s p e c i e s o f A s c l e p i a s Comparison o f female to male r a t i o s f o r t e n e r a l wet wei g h t and c a r d e n o l i d e c o n t e n t o f 0_. f a s c i a t u s r e a r e d on d i f f e r e n t seed d i e t s C o r r e l a t i o n s between mean c a r d e n o l i d e c o n t e n t and means o f body w e i g h t , body s i z e and l a r v a l d u r a t i o n i n fj. f a s c i a t u s r e a r e d i n the l a b o r a t o r y a t 21°C on the seeds o f d i f f e r e n t s p e c i e s o f A s c l e p i a s ( i x ) F r o n t i s p i e c e F i g u r e 1 2 4 5 6 7 8 9 10 11 LIST OF FIGURES A d u l t O n c o p e l t u s f a s c i a t u s and Lygaeus k a l m i i  k a l m i i ( c e n t e r ) on a d e h i s c e n t pod o f A s c l e p i a s f a s i c u l a r i s i n Napa County Cal i form'a S t r u c t u r e s o f some c a r d e n o l i d e s Map o f C a l i f o r n i a showing l o c a l i t i e s where milkweed p l a n t s were examined and p l a n t m a t e r i a l c o l l e c t e d F r u i t i n g season i n n a t i v e C a l i f o r n i a n A s c l e p i a s s p e c i e s , and the p o p u l a t i o n t r e n d o f fJ. f a s c i a t u s a t one milkweed s t a n d Gross c a r d e n o l i d e c o n t e n t o f seed o f A s c l e p i a s s p e c i e s from C a l i f o r n i a Gross c a r d e n o l i d e c o n t e n t o f p l a n t samples o f A s c l e p i a s from C a l i f o r n i a Gross c a r d e n o l i d e c o n t e n t o f f i e l d c o l l e c t e d CJ. f a s c i a t u s Gross c a r d e n o l i d e c o n t e n t o f f i e l d c o l l e c t e d L. k_. kalmi i Comparison o f g r o s s c a r d e n o l i d e c o n t e n t s o f f i e l d c o l l e c t e d 0. f a s c i a t u s and L. k. k a l m i i Page 111 Gross c a r d e n o l i d e c o n t e n t o f f i e l d c o l l e c t e d l y g a e i d s : d i s t r i b u t i o n o f c l a s s e s based on c a r d e n o l i d e c o n t e n t R e l a t i o n s h i p between c a r d e n o l i d e c o n c e n t r a t i o n i n t he d i e t (milkweed seeds) and the c a r d e n o l i d e c o n t e n t o f a d u l t (J. f a s c i a t u s and L_. k_. k a l m i i R e l a t i o n s h i p between c a r d e n o l i d e c o n c e n t r a t i o n i n the d i e t (milkweed seeds) and the c a r d e n o l i d e c o n t e n t o f a d u l t male and female fJ. f a s c i a t u s r e a r e d a t 21°C 3 7 24 31 34 38 40 42 46 50 53 R e l a t i o n s h i p between d u r a t i o n o f l a r v a l development and t e n e r a l wet we i g h t o f 0. f a s c i a t u s and _L. J<. k a l m i i r e a r e d i n the l a b o r a t o r y a t 25°C on seeds o f d i f f e r e n t s p e c i e s o f A s c l e p i a s and He!iant h u s Weight g a i n i n fJ. f a s c i a t u s and L k. k a l m i i d u r i n g l a r v a l development, r e a r e d i n the l a b o r a t o r y a t 25°C on the seeds o f A. f a s c i c u l a r i s R e l a t i o n s h i p between g r o s s c a r d e n o l i d e c o n t e n t and d u r a t i o n o f l a r v a l development o f fJ. f a s c i a t u s r e a r e d i n the l a b o r a t o r y a t 21°C on the seeds o f d i f f e r e n t s p e c i e s o f A s c l e p i a s R e l a t i o n s h i p between g r o s s c a r d e n o l i d e c o n t e n t and a d u l t body weight o f Cj. f a s c i a t u s r e a r e d i n the l a b o r a t o r y a t 21°C on the seeds o f d i f f e r e n t s p e c i e s o f A s c l e p i a s ( x i ) ACKNOWLEDGEMENTS I would l i k e t o e x p r e s s my s i n c e r e s t thanks to P r o f . G.G.E. Scudder, who, as my r e s e a r c h s u p e r v i s o r , p r o v i d e d i n v a l u a b l e encouragement, c r i t i c i s m , and gu i d a n c e t h r o u g h o u t the c o u r s e o f t h i s s t u d y . H i s time consuming a s s i s t a n c e i n the p r e p a r a t i o n o f m a n u s c r i p t s d e r i v e d from t h i s t h e s i s f o r p u b l i c a t i o n i s most a p p r e c i a t e d . A l t h o u g h not o f f i c i a l l y r e c o g n i z e d as a c o - s u p e r v i s o r , Dr. S.S. D u f f e y g e n e r o u s l y p r o v i d e d working space and s u p p l i e s d u r i n g a f i v e month s t a y i n h i s l a b o r a t o r y i n the Department o f Entomology, U n i v e r s i t y o f C a l i f o r n i a a t D a v i s . H i s c o m b i n a t i o n o f p a t i e n c e and p r e s s u r e h e l p e d me to overcome my i n e r t i a i n d e a l i n g w i t h t he phy t o c h e m i c a l t e c h n i q u e s r e q u i r e d to do t h i s s t u d y . I would a l s o l i k e to e x p r e s s my thanks to Dr. C. Juddson and the Department o f Entomology f o r a l l o w i n g me the use o f the s p e c t r o p h o t o m e t e r . Ken E. Evans r e c e i v e s s p e c i a l thanks f o r s e n d i n g s e v e r a l packages o f milkweed seed s , which e s t a b l i s h e d the f o u n d a t i o n f o r t h i s s tudy. I a l s o thank Ken f o r p e r m i s s i o n t o r e p r o d u c e h i s photograph which appears as the f r o n t i s p i e c e o f t h i s t h e s i s . I w i l l remember Ken not o n l y f o r p r o v i d i n g p l a n t m a t e r i a l and some i n s e c t s , but a l s o f o r our s t i m u l a t i n g d i s c u s s i o n s i n r e g a r d to our r e s p e c t i v e s t u d i e s o f l y g a e i d s . F i n a l l y , Ken's e n t h u s i a s m on our j o i n t c o l l e c t i n g t r i p s made the f i e l d work a p l e a s u r e . I would a l s o l i k e to thank Steven P. Lynch, who I c o n s i d e r t o be an e x p e r t on the A s c l e p i a s f l o r a o f western North America. Steve ( x i i ) p r o v i d e d p l a n t and i n s e c t samples, and spen t much o f h i s v a l u a b l e time t o a i d me i n l o c a t i n g milkweed s t a n d s . Drs. C.N. Roeske and J.N. S e i b e r a r e thanked f o r t h e i r h e l p w i t h t e c h n i c a l problems, and f o r g e n e r o u s l y p r o v i d i n g me w i t h a copy o f t h e i r m a n u s c r i p t d e a l i n g w i t h c a r d e n o l i d e s i n A s c l e p i a s , b e f o r e i t was p u b l i s h e d . The i n f o r m a t i o n i n t h a t m a n u s c r i p t g r e a t l y a i d e d me i n sh a p i n g my s t u d y . Dr. S e i b e r a l s o p r o v i d e d samples o f p u r i f i e d A s c l e p i a s c a r d e n o l i d e s which had been i s o l a t e d i n h i s l a b o r a t o r y . Drs. J . Myers and B.A. Bohm k i n d l y r e a d an e a r l y d r a f t o f t h i s t h e s i s , and a l s o p r o v i d e d comments and c r i t i c i s m o f a m a n u s c r i p t p r e p a r e d f o r p u b l i c a t i o n . F i n a l l y , my f e l l o w s t u d e n t s i n the entomology 'department' d e s e r v e s p e c i a l thanks f o r t h e i r c o n s t a n t encouragement and moral s u p p o r t . In p a r t i c u l a r , John Spence r a i s e d many thought p r o v o k i n g p o i n t s , and Syd Cannings p r o v i d e d c h e e r f u l encouragement both on and o f f the i c e . The r e s e a r c h was s u p p o r t e d by a N a t i o n a l Research C o u n c i l o f Canada g r a n t to P r o f . G.G.E. Scudder. 1 I INTRODUCTION Many aposematic i n s e c t s have been h y p o t h e s i z e d to s e r v e as models i n mimi c r y r i n g s o r complexes because they have the a b i l i t y to s e q u e s t e r and s t o r e b i t t e r - t a s t i n g o r t o x i c c h e m i c l a s from t h e i r h o s t p l a n t s . These c h e m i c a l s can a c t as d e t e r r e n t s t o v e r t e b r a t e p r e d a t o r s , thus p r o v i d i n g a form o f d e f e n s e which would not be a v a i l a b l e to the i n s e c t s had they f e d on a f o o d s o u r c e l a c k i n g t h e s e compounds ( R o t h s c h i l d 1972). One such c l a s s o f c h e m i c a l s i s the c a r d e n o l i d e s ( c a r d i a c g l y c o s i d e s and t h e i r genins), ( F i g u r e 1 ) , produced by s e v e r a l f a m i l i e s o f angiosperms ( S i n g h and R a s t o g i 1970, P a t t e r s o n e t aj_. 1975). These h e a r t p o i s o n s , which a r e h i g h l y t o x i c to many v e r t e b r a t e s (Hoch 1961, D e t w e i l e r 1967, Thorp and Cobbin 1967), a l s o i n d u c e emesis (Parsons and Summers 1971, B o r i s o n and Wang 1953), and a r e v e r y b i t t e r ( R e i c h s t e i n ejt aj_. 1968, Brower and G l a z i e r 1975). Temperate and s u b t r o p i c a l milkweeds ( A s c l e p i a d a c e a e ) a r e r i c h s o u r c e s o f c a r d e n o l i d e s (Roeske e t al_. 1976), and s u p p o r t a w e l l -d e f i n e d i n s e c t fauna ( P r i c e 1975, Weiss and D i c k e r s o n 1921). I t has been e s t a b l i s h e d t h a t c e r t a i n b r i g h t l y - c o l o u r e d i n s e c t s which f e e d on milkweed a t some s t a g e i n t h e i r l i f e c y c l e a r e a b l e to s e q u e s t e r and s t o r e c a r d e n o l i d e s d e r i v e d from t h i s group o f p l a n t s . In some c a s e s , the i n s e c t s a r e c a p a b l e o f s t o r i n g l a r g e q u a n t i t i e s o f t h e s e compounds i n t h e i r b o d i e s , which may have the p o t e n t i a l t o s e r v e as a chemical d e f e n s e a g a i n s t v e r t e b r a t e p r e d a t o r s (von Euw e t al_. 1967, 1971 , D u f f e y and Scudder 1972, Brower 1969, Abushama and Ahmed 1976). Well FIGURE 1: S t r u c t u r e s o f some c a r d e n o l i d e s . 1. General s t r u c t u r e (R = one o r more sugar m o i e t i e s i n the g l y c o s i d e ; R = H i n the genin ) 2. C a l o t r o p i n - c a l a c t i n ( c o n f i g u r a t i o n a l i s o m e r s ) ( a f t e r Roeske e t al_. 1976) 3. D i g i t o x i n 4 documented examples o f i n s e c t s which s t o r e c a r d e n o l i d e s o b t a i n e d from milkweeds a r e the monarch b u t t e r f l y , Danaus p l e x i p p u s L. (Brower 1969) and an A f r i c a n g r a s s h o p p e r , P o e k i l o c e r u s b u f o n i u s (Klug) (von Euw e t a l . 1967). Brower et_ al_. (1972) and Brower and M o f f i t t (1974) have shown t h a t t h e r e i s a wide v a r i a t i o n i n the c a r d e n o l i d e c o n t e n t o f f i e l d -c o l l e c t e d monarch b u t t e r f l i e s . The c a r d e n o l i d e c o n t e n t can be r e l a t e d t o the p l a n t s p e c i e s f e d upon by the l a r v a e o f the b u t t e r f l y (Brower e t al_. 1975, Roeske e t al_. 1976). F u r t h e r , Brower (1969) and Roeske ejt aj_. (1976) have demonstrated t h a t the e m e t i c p o t e n t i a l o f the monarch b u t t e r f l y t o a v e r t e b r a t e p r e d a t o r , the b l u e j a y ( C y a n o c i t t a c r i s t a t a  bromia O b e r h o l s e r ) , i s d e t e r m i n e d by the h o s t p l a n t s p e c i e s upon which the l a r v a e f e d . The e x i s t e n c e o f a r e l a t i o n s h i p between h o s t p l a n t s p e c i e s , c a r d e n o l i d e c o n t e n t and e m e t i c p o t e n t i a l o f a m i l k w e e d - f e e d i n g i n s e c t has thus been w e l l e s t a b l i s h e d . As i n the monarch b u t t e r f l y , the a b i l i t y o f the milkweed bugs O n c o p e l t u s f a s c i a t u s D a l l a s and Lygaeus k a l m i i k a l m i i St§1 to s e q u e s t e r and s t o r e c a r d e n o l i d e s from milkweed p l a n t s i n the l a b o r a t o r y has been w e l l documented ( D u f f e y and Scudder 1972, 1974). L a b o r a t o r y s t u d i e s o f IP. b u f o n i u s , D_. p l e x i p p u s and the l y g a e i d s fj. f a s c i a t u s and L. k_. k a l m i i s u g g e s t t h a t none o f t h e s e i n s e c t s a r e c a p a b l e o f s y n t h e s i z i n g c a r d e no-l i d e s , and so must o b t a i n t h e s e c h e m i c a l s from t h e i r h o s t p l a n t s (von Euw e t a l . 1967, Brower 1969, D u f f e y and Scudder 1972). Both fJ. f a s c i a t u s and IL. J < . k a l m i i have a c l o s e f e e d i n g a s s o c i a t i o n w i t h members o f A s c l e p i a s L. i n temperate North America 5 (Ralph 1976, Scudder and D u f f e y 1972, Eggermann and Bongers 1971, Simanton and Andre 1936, Andre 1934). Over t h i r t y o f the 108 d e s c r i b e d s p e c i e s o f A s c l e p i a s i n North America have been shown t o c o n t a i n c a r d e n o l i d e s ( D u f f e y and Scudder 1972, Roeske e t al_. 1976), and Roeske e t al_. (1976) have documented many examples o f i n t r a s p e c i f i c v a r i a t i o n . i n c a r d e n o l i d e c o n t e n t . I t has been s u g g e s t e d t h a t such v a r i a t i o n c o u l d g i v e r i s e t o a p a l a t a b i l i t y spectrum among l y g a e i d s f e e d i n g on t h e s e p l a n t s ( D u f f e y and Scudder 1972). In t h i s t h e s i s I have i n v e s t i g a t e d the g r o s s c a r d e n o l i d e c o n t e n t o f fj. f a s c i a t u s and j _ . k_. k a l m i i and t h e i r h o s t s ( A s c l e p i a s spp.) i n c e n t r a l C a l i f o r n i a , an a r e a w i t h a d i v e r s e milkweed f l o r a . The purpose o f t h i s s t u d y was 1.) t o e s t a b l i s h a r e l a t i o n s h i p between the c a r d e n o l i d e c o n t e n t o f the h o s t s p e c i e s and t h a t o f the l y g a e i d s f e e d i n g on i t ; 2.) t o i n v e s t i g a t e the e f f e c t s o f c a r d e n o l i d e s on the growth and d e v e l o p -ment o f l y g a e i d s ; 3.) to d e t e r m i n e i f c a r d e n o l i d e s p l a y a r o l e i n the c o l o n i z a t i o n p a t t e r n o f l y g a e i d s on a milkweed s p e c i e s complex; and 4.) t o a s s e s s the r o l e o f c a r d e n o l i d e s i n ' d e f e n s e s t r a t e g i e s o f l y g a e i d s . 6 II MATERIALS AND METHODS A. F i e l d Study o f Milkweeds and L y g a e i d s Throughout the summer o f 1976, milkweed s t a n d s were examined i n t e n c o u n t i e s i n n o r t h and c e n t r a l C a l i f o r n i a . Most o b s e r v a t i o n s and c o l l e c t i o n s were made i n two a r e a s o f the s t a t e : the n o r t h bay a r e a , r e p r e s e n t e d by Y o l o , S o l a n o , Napa and C o n t r a C o s t a c o u n t i e s , and the Fresno a r e a , r e p r e s e n t e d by Fresno and T u l a r e c o u n t i e s ( F i g u r e 2 ) . Many o f the l o c a t i o n s o f milkweed stands were p r o v i d e d by o t h e r w o r k e r s , however, new l o c a t i o n s were found d u r i n g r o u t i n e c o l l e c t i n g t r i p s . Once a milkweed s t a n d had been l o c a t e d , s e v e r a l c h a r a c t e r i s t i c s were n o t e d , i n c l u d i n g approximate number o f p l a n t s i n the p o p u l a t i o n , degree o f f l o w e r i n g o r f r u i t p r o d u c t i o n , and the p r e s e n c e o r absence o f l y g a e i d s and/or o t h e r i n s e c t h e r b i v o r e s . As the p o p u l a t i o n dynamics o f 0_. f a s c i a t u s on milkweed was c u r r e n t l y under i n v e s t i g a t i o n i n n o r t h e r n C a l i f o r n i a (K.E. Evans, u n p u b l i s h e d r e s u l t s ) , no formal attempt was made to f o l l o w the phenology and p o p u l a t i o n dynamics o f m i l k w e e d - f e e d i n g l y g a e i d s . However, s e v e r a l milkweed stands i n the v i c i n i t y o f D a vis ( Y o l o Co.) were v i s i t e d on a weekly b a s i s , and a t one l o c a t i o n (Woodland, Y o l o Co.) the t o t a l numbers o f (D. f a s c i a t u s a d u l t s and l a r v a e were r e c o r d e d by d i r e c t census. T h i s was a c c o m p l i s h e d by c o u n t i n g a l l the bugs w i t h i n a w e l l d e f i n e d s t a n d , a t a s k a i d e d by the c o n s p i c u o u s c o l o r a t i o n and b e h a v i o u r o f t h e s e i n s e c t s (Root and C h a p l i n 1976). The f r u i t i n g season o f d i f f e r e n t A s c l e p i a s s p e c i e s was e s t i m a t e d FIGURE 2: Map o f C a l i f o r n i a showing l o c a l i t i e s where milkweed p l a n t s were examined and p l a n t m a t e r i a l c o l l e c t e d . "NORTH BAY AREA" 1 Napa County 2 Y o l o County 3 S o l a n o County 4 C o n t r a C o s t a County "FRESNO AREA" 5 F r e s n o County 6 T u l a r e County SF = San F r a n c i s c o LA = Los Ang e l e s Milkweed s p e c i e s : O A s c l e p i a s f a s c i c u l a r i s Dene. A A. e r o s a T o r r e y e e r i o c a r p a Benth. ffl A. c a l i f o r n i c a g r e e n e i Woodson • A_. c o r d i f o l i a (Benth.) J e p s . • /\. v e s t i t a v e s t i t a Hook & Arn. $ A. s p e c i o s a T o r r e y s A. s o l a n o a n a Woodson c A. c u r a s s a v i c a L. I 1 1 0 0 km Map o f C a l i f o r n i a showing l o c a l i t i e s where milkweed p l a n t s were examined and p l a n t m a t e r i a l c o l l e c t e d 9 by p e r s o n a l o b s e r v a t i o n s , f i e l d notes (S.P. Lynch, p e r s . comm.) and by e x t r a p o l a t i o n from t he f l o w e r i n g p e r i o d s o f A s c l e p i a s s p e c i e s as g i v e n by v a r i o u s a u t h o r s i n the taxonomic l i t e r a t u r e (Woodson 1954, Munz 1968, Abrams 1951). B. E x t r a c t i o n o f C a r d e n o l i d e s from P l a n t M a t e r i a l P l a n t m a t e r i a l was u s u a l l y c o l l e c t e d a t l o c a t i o n s where i n s e c t s were t a k e n , to p r o v i d e d i r e c t comparisons between the c a r d e n o l i d e c o n t e n t o f the p l a n t and thos e o f the i n s e c t s where p o s s i b l e . To m i n i m i z e p o s s i b l e d i f f e r e n c e s when comparing e q u i v a l e n t organs from d i f f e r e n t milkweed s p e c i e s , i t was i m p o r t a n t to attempt to take p l a n t m a t e r i a l o f a s i m i l a r developmental s t a g e . Thus, mature o r lower l e a v e s o f s e v e r a l i n d i v i d u a l p l a n t s w i t h i n a s t a n d were c o l l e c t e d by c u t t i n g whole l e a v e s a t the p e t i o l e . Umbels were taken i n the same manner, when i n f u l l bloom o n l y . Seed samples c o n s i s t e d o f mature seeds ( t h o s e from d e h i s c e n t pods o r thos e i n which the seeds had darkened i n c o l o r i m m e d i a t e l y p r i o r to d e h i s c e n c e ) from s e v e r a l pods, u s u a l l y from d i f f e r e n t p l a n t s . Care was taken not t o s e l e c t samples from p l a n t organs on which l y g a e i d s o r o t h e r i n s e c t h e r b i v o r e s had been d i r e c t l y f e e d i n g . On r e t u r n t o the l a b o r a t o r y , p l a n t m a t e r i a l f o r a n a l y s i s was kept c o o l and d r y , but was not f r o z e n . A l t h o u g h Roeske e t al_. (1976) have sugg e s t e d t h a t c a r d e n o l i d e s may be s u b j e c t t o spontaneous breakdown, the r e s u l t s o b t a i n e d i n t h i s t h e s i s s u g g e s t t h a t any l o s s e s i n c u r r e d were no g r e a t e r than t h o s e o f Roeske e t a]_. (1976) who s t o r e d a l l f i e l d c o l -l e c t e d p l a n t m a t e r i a l on d r y i c e . P l a n t samples were d r i e d f o r 12 10 hours a t 80°C i n a f o r c e d d r a f t oven p r i o r t o e x t r a c t i o n . The e x t r a c t i o n o f c a r d e n o l i d e s from d r i e d p l a n t m a t e r i a l was based on the p r o c e d u r e s o f D u f f e y and Scudder (1972). To maximize the e x t r a c t i o n e f f i c i e n c y f o r q u a n t i t a t i v e a n a l y s i s , p l a n t m a t e r i a l , e s p e c i a l l y d r y s e e d s , r e q u i r e d m echanical breakdown t o i n c r e a s e the exposure o f the c a r d e n o l i d e - c o n t a i n i n g t i s s u e s t o the e x t r a c t i n g s o l v e n t s . Milkweed seeds were ground to a f i n e powder w i t h a commercial g r i n d e r ( C r e s c e n t Dental Mfg. C o . ) , and 0.2 grams p l a c e d i n a 12 ml screw-cap t e s t tube. The seed m a t e r i a l was e x t r a c t e d w i t h 4 ml o f p e t r o l e u m e t h e r , the t e s t tube b e i n g shaken v i g o r o u s l y a f t e r a d d i t i o n o f the s o l v e n t , then a l l o w e d t o s e t t l e f o r 6 hours. T h i s i n i t i a l e x t r a c t i o n removed l a r g e q u a n t i t i e s o f l i p i d s from the seeds which would o t h e r w i s e have i n t e r f e r e d w i t h the s p e c t r o a s s a y . T h i s hydro-carbon e x t r a c t , removed by p i p e t t e , was d i s c a r d e d because i t was found to l a c k c a r d e n o l i d e s when t e s t e d by both s p e c t r o p h o t o m e t r i c and t h i n -l a y e r c h r o m a t o g r a p h i c means. The seed r e s i d u e was then e x t r a c t e d w i t h 2 ml o f hot c h l o r o f o r m -methanol (10:1 by volume), shaken v i g o r o u s l y , and a l l o w e d t o s t a n d f o r 12 hours. The c h l o r o f o r m phase was then d e c a n t e d , and the r e s i d u e r e -e x t r a c t e d w i t h 3 ml o f 95 per c e n t aqueous e t h a n o l f o r 12 hours. The e t h a n o l e x t r a c t was added to the c h l o r o f o r m e x t r a c t , the p o o l e d e x t r a c t s e v a p o r a t e d to d r y n e s s a t room temperature and r e d i s s o l v e d i n 4 ml o f 95 per c e n t e t h a n o l . O n e - h a l f o r 1.0 ml a l i q u o t s (depending on the i n t e n s i t y o f a p r e l i m i n a r y c o l o u r r e a c t i o n ) o f p o o l e d e x t r a c t 11 were used f o r the s p e c t r o a s s a y o f c a r d e n o l i d e s . D r i e d milkweed l e a v e s and umbels c o n t a i n e d much l e s s l i p i d m a t e r i a l ; d i r e c t e x t r a c t i o n w i t h e t h a n o l p r o v i d e d s u f f i c i e n t l y pure samples. The p e t r o l e u m e t h e r e x t r a c t i o n was t h e r e f o r e u n n e c e s s a r y . D r i e d milkweed l e a v e s o r umbels were ground t o a f i n e powder i n a commercial g r i n d e r . Two-tenths o f a gram e x t r a c t e d once w i t h 4 ml o f hot 95 per c e n t e t h a n o l were a l l o w e d t o s t a n d 12 hours b e f o r e 0.5 o f 1 ml a l i q u o t s were taken d i r e c t l y f o r c a r d e n o l i d e e s t i m a t i o n . O x i d a t i v e d e g r a d a t i o n o f c a r d e n o l i d e s was kept t o a minimum by a n a l y -z i n g the f i n a l e x t r a c t s w i t h i n 24 hours. For l e a f and umbel samples, a s i n g l e e s t i m a t i o n o f each sample was made. S i n c e the l y g a e i d bugs fJ. f a s c i a t u s and I. k_. k a l m i i a r e c l o s e l y a s s o c i a t e d w i t h the seeds o f milkweed p l a n t s , i t was i m p o r t a n t to o b t a i n c o n c i s e e s t i m a t i o n s o f the c a r d e n o l i d e c o n t e n t o f t h e s e organs. T h e r e f o r e , up to f i v e e x t r a c t i o n s o f each seed sample was made. C. C o l o r i m e t r i c Assay o f C a r d e n o l i d e s The c o l o r i m e t r i c a s s a y used was t h a t o f Brower e t al_. (1972) as m o d i f i e d by Brower and M o f f i t t (1974) and Brower e t al_. (1975). T h i s a s s a y , f i r s t d e v e l o p e d by R a b i t s c h and Tambor (1969) i s based on the b l u e c o l o u r formed when 2 , 4 , 2 ' , 4 ' - t e t r a n i t r o d i p h y e n y l (TNDP) i s combined w i t h the c a r d e n o l i d e i n the p r e s e n c e o f a base (-0H i o n s i n s o l u t i o n ) . The c o l o u r i s formed by the r e a c t i o n o f the r e a g e n t s w i t h the u n s a t u r a t e d l a c t o n e ( b u t e n o l i d e ) r i n g a t the C-17 p o s i t i o n on the c a r d e n o l i d e g e n i n . C a r d e n o l i d e e s t i m a t i o n s were based on a s t a n d a r d o f d i g i t o x i n 12 ( N u t r i t i o n a l B i o c h e m i c a l Corp., C l e v e l a n d ) ( F i g u r e 1 ) ; a l l d e t e r m i n a t i o n s a r e e x p r e s s e d as e q u i v a l e n t s o f d i g i t o x i n . U s i n g t he p r o c e d u r e o u t l i n e d by Brower e t a]_. (1972), d i g i t o x i n was shown t o obey Beer's Law w i t h i n a range o f 10 t o 250 micrograms i n 4 ml. I f the absorbance o f a sample i n d i c a t e d a q u a n t i t y i n e x c e s s o f 250 micrograms, the sample s o l u t i o n was d i l u t e d to p r o v i d e a q u a n t i t y w i t h i n t h i s range. Readings were taken on a Beckman Model B s p e c t r o p h o t o m e t e r u s i n g Beckman q u a r t z sample c u v e t t e s . The maximum c o l o u r i n t e n s i t y was reached a f t e r 40 t o 45 minutes. A t t h i s p o i n t the absorbance was r e c o r d e d , and the c o n c e n t r a t i o n c a l c u l a t e d from t he l i n e a r r e l a t i o n s h i p e s t a b l i s h e d between c o n c e n t r a t i o n and absorbance u s i n g the d i g i t o x i n s t a n d a r d . The c a r d e n o l i d e e s t i m a t i o n s r e p o r t e d here a r e d i r e c t l y comparable to t h o s e o f Roeske e t aj_. (1976). The p r o c e d u r e s o f Roeske e t aj_. (1976) were c l o s e l y f o l l o w e d t o a l l o w such c o m p a r i s o n s , as t h i s l a t t e r s t u d y p r o v i d e s the o n l y q u a n t i t a t i v e d a t a on the c a r d e n o l i d e c o n t e n t o f North American milkweeds o f the genus A s c l e p i a s . The d a t a o b t a i n e d i n the p r e s e n t r e s e a r c h a r e i n c l o s e agreement w i t h t h a t o f Roeske e_t al_. (1976) f o r p l a n t organs and s p e c i e s which a r e common t o both s t u d i e s . D. E x t r a c t i o n o f C a r d e n o l i d e s from L y g a e i d s A d u l t 0_. f a s c i a t u s and I. k. k a l m i i were c o l l e c t e d on milkweed p l a n t s ( A s c l e p i a s spp.) i n c e n t r a l C a l i f o r n i a between A p r i l and September, 1976. F i e l d - c a u g h t l y g a e i d s were m a i n t a i n e d i n c o n t r o l l e d e n v i r o n m e n t a l 13 c a b i n e t s a t 27°C on a 13.5:10.5 LD l i g h t regime, and p r o v i d e d w i t h d i s t i l l e d water and husked s u n f l o w e r seeds. F i e l d - c a u g h t l y g a e i d s were as s a y e d f o r c a r d e n o l i d e s w i t h i n two weeks o f t h e i r c a p t u r e . A c o n t r o l experiment i n d i c a t e d t h a t t h e r e was no s i g n i f i c a n t l o s s o f c a r d e n o l i d e s i n l y g a e i d s f e e d i n g on s u n f l o w e r seeds (which l a c k c a r d e n o l i d e s ) f o r t h i s l e n g t h o f time a f t e r h a v i n g p r e v i o u s l y f e d on a c a r d e h o l i d e - c o n t a i n i n g d i e t . M a i n t a i n i n g f i e l d - c a u g h t i n s e c t s i n the l a b o r a t o r y on s u n f l o w e r seeds p r i o r t o the c a r d e n o l i d e a s s a y had two advantages: f i r s t , t h i s p e r i o d a l l o w e d the i n s e c t s t o c l e a r the g u t , so t h a t c a r d e n o l i d e e s t i m a t i o n s would n o t be i n f l u e n c e d by gut c o n t e n t s , and second, r e p r o d u c t i v e a d u l t s c o u l d p r o v i d e eggs f o r l a b o r a t o r y r e a r i n g o f l y g a e i d s on milkweed seeds. Only a d u l t i n s e c t s were used f o r the c a r d e n o l i d e a s s a y . D u f f e y and Scudder (1974) demonstrated t h a t c a r d e n o l i d e s a r e accumulated t h r o u g h o u t the j u v e n i l e l i f e o f the l y g a e i d fj. f a s c i a t u s , w i t h o v e r h a l f o f the a d u l t c a r d e n o l i d e complement s e q u e s t e r e d by the f i f t h and f i n a l l a r v a l i n s t a r . While some q u a n t i t i e s o f c a r d e n o l i d e s a r e s e q u e s t e r e d i n the a d u l t s t a g e , the r a t e o f uptake i s l e s s than t h a t i n the l a t e i n s t a r l a r v a e . The c o l l e c t i o n o f o n l y a d u l t i n s e c t s p r o v i d e s an e s t i m a t e o f the u l t i m a t e q u a n t i t y o f c a r d e n o l i d e s s t o r e d by t h e s e milkweed f e e d i n g i n s e c t s under n a t u r a l c o n d i t i o n s , and a l l o w e d d i r e c t comparisons between d i f f e r e n t p o p u l a t i o n s o f l y g a e i d s . To i n v e s t i g a t e the c a r d e n o l i d e c o n t e n t o f l y g a e i d s r e a r e d e x c l u s i v e l y on milkweed seed s , f i r s t i n s t a r l a r v a e o f fj. f a s c i a t u s and L. k. k a l m i i were r e a r e d t o the a d u l t s t a g e o f the seeds o f d i f f e r e n t 14 milkweed s p e c i e s . The q u a n t i t i e s o f c a r d e n o l i d e found i n t h e s e a d u l t bugs were determined and p r o v i d e an e s t i m a t e o f the u l t i m a t e amount t h a t t h e s e i n s e c t s might s e q u e s t e r i n the f i e l d , had they had an u n l i m i t e d s u p p l y o f mature seeds d u r i n g t h e i r l a r v a l l i f e . F u r t h e r , s i n c e the milkweed seeds used i n t h i s s t u d y were s u b j e c t e d t o q u a n t i t a t i v e c a r d e n o l i d e a n a l y s i s , i t was p o s s i b l e t o examine the r e l a t i o n s h i p between the c a r d e n o l i d e c o n t e n t o f the seed d i e t and the c o n t e n t o f bugs f e e d i n g on such seeds. The c o n d i t i o n s under which the l y g a e i d s were r e a r e d a r e d e s c r i b e d i n d e t a i l i n the f o l l o w i n g s e c t i o n . I d e a l l y , p h y s i o l o g i c a l e f f e c t s o f c a r d e n o l i d e s e q u e s t r a t i o n s h o u l d be s t u d i e d by i n c l u d i n g d i f f e r i n g known c o n c e n t r a t i o n s o f s i n g l e i s o l a t e d c a r d e n o l i d e s i n a s t a n d a r d i z e d , c h e m i c a l l y i n e r t d i e t which c o u l d be o f f e r e d to the i n s e c t s . T h i s s t u d y has been much s i m p l e r i n a t e c h n i c a l s e n s e , because the d i e t ( i . e . milkweed seeds) c o n t a i n s c a r d e n o l i d e s i n a pre-packaged form, and the i n s e c t s w i l l r e a d i l y f e e d on the seeds. F u r t h e r , the c a r d e n o l i d e s i n g e s t e d by the bugs from milkweed seeds a r e i n f a c t t h o s e compounds, o c c u r r i n g i n the a c t u a l c o n c e n t r a t i o n s and r e l a t i v e f r e q u e n c i e s t h a t the i n s e c t s would e n c o u n t e r i n the n a t u r a l environment. In l i g h t o f the f a c t t h a t the d i e t l e d to o v e r a t e n - f o l d d i f f e r e n c e i n c a r d e n o l i d e c o n t e n t o f the r e s u l t a n t a d u l t i n s e c t s , i t i s f e l t t h a t the r e s u l t s a r e v a l i d e v i d e n c e t h a t CJ. f a s c i a t u s a r e h i g h l y t o l e r a n t o f the compounds as they a r e e n c o u n t e r e d i n the f i e l d . The method o f Scudder & D u f f e y (1972) was used f o r e x t r a c t i n g c a r d e n o l i d e s from i n s e c t s . I n s e c t s , e i t h e r l i v e o r d r i e d , were p l a c e d 15 i n d i v i d u a l l y i n 2 ml o f c h l o r o f o r m - m e t h a n o l (2:1 by volume), a l l o w e d to s t a n d f o r 24 h r , then removed from t h i s s o l v e n t and p l a c e d i n an a d d i t i o n a l 2 ml o f c h l o r o f o r m - m e t h a n o l ( 2 : 1 ) . A f t e r a f u r t h e r 24 h r , the two e x t r a c t s were p o o l e d , e v a p o r a t e d to d r y n e s s , and d i s s o l v e d i n 1 ml o f 95 per c e n t e t h a n o l . One h a l f o f t h i s f i n a l s o l u t i o n was used f o r the s p e c t r o a s s a y o f g r o s s c a r d e n o l i d e c o n t e n t i n each i n s e c t . More than 95 per c e n t o f an i n s e c t ' s c a r d e n o l i d e complement i s e x t r a c t e d u s i n g t h i s p r o c e d u r e s i n c e r e p e a t e d e x t r a c t i o n s f a i l e d to produce more than 5 per c e n t o f the t o t a l q u a n t i t y e s t i m a t e d . The s p e c t r o a s s a y was i d e n t i c a l t o t h a t used f o r e s t i m a t i n g g r o s s c a r d e n o l i d e c o n t e n t o f p l a n t samples. U n l i k e o t h e r s t u d i e s o f the c a r d e n o l i d e c o n t e n t o f i n s e c t s , i n which the q u a n t i t i e s r e p o r t e d have been based on a f i x e d w e i g h t o f t i s s u e (Brower e t a^. 1975, R o t h s c h i l d e t al_. 1975) o r many i n s e c t s p o o l e d (von Euw e t al_. 1967), c a r d e n o l i d e d e t e r m i n a t i o n s i n t h i s s t u d y were based on whole i n d i v i d u a l i n s e c t s , i r r e s p e c t i v e o f t h e i r body weight. Two r e a s o n s f o r e s t i m a t i n g the c a r d e n o l i d e c o n t e n t o f whole i n s e c t s were because i t a l l o w e d an e x a m i n a t i o n o f the c o m p o s i t i o n o f l o c a l i z e d i n s e c t p o p u l a t i o n s which i n n a t u r e c o n s i s t o f i n d i v i d u a l s o f d i f f e r e n t w e i g h t s and body s i z e s , and o f more im p o r t a n c e , the i n d i v i d u a l i n s e c t i s the u n i t t h a t must be sampled by a p r e d a t o r . E. L a b o r a t o r y R e a r i n g o f L y g a e i d s fj. f a s c i a t u s and L_. k_. k a l m i i were r e a r e d i n the l a b o r a t o r y on the seeds o f d i f f e r e n t s p e c i e s o f A s c l e p i a s which they might e n c o u n t e r i n the f i e l d and seeds o f H e l i a n t h u s annus (which l a c k c a r d e n o l i d e s ) . 16 T h i s r e a r i n g aimed t o 1. a s s e s s , i n a g r o s s sense, the n u t r i t i o n a l v a l u e o f each milkweed s p e c i e s t o the i n s e c t s . These d a t a a r e r e l a t e d to the p a t t e r n o f c o l o n i z a t i o n o f l y g a e i d s on d i f f e r e n t milkweed s p e c i e s i n the f i e l d ; 2. d e t e r m i n e i f d i f f e r i n g c o n c e n t r a t i o n s o f c a r d e n o l i d e s i n the d i e t (seeds) a f f e c t growth and development o f l a r v a e ; 3. examine the r e l a t i o n s h i p between c a r d e n o l i d e c o n t e n t i n the d i e t ( s eeds) and the c a r d e n o l i d e c o n t e n t o f i n s e c t s r e a r e d on s p e c i f i e d d i e t s ( i . e . d i f f e r e n t milkweed s p e c i e s ) . S e p a r a t e r e a r i n g experiments were c a r r i e d out i n 1975 and 1976. In the autumn o f 1975, CJ. f a s c i a t u s and _L. k_. k a l m i i were r e a r e d i n an e n v i r o n m e n t a l c a b i n e t s e t a t 25±1°C, 60±10% RH, and c o n t i n u o u s l i g h t . Eggs o f CJ. f a s c i a t u s were o b t a i n e d from a l a b o r a t o r y c u l t u r e which had been e s t a b l i s h e d a t l e a s t t h r e e y e a r s e a r l i e r . Eggs o f j . . k_. k a l m i i were o b t a i n e d from a c u l t u r e i n i t i a t e d i n the summer o f 1975 from a d u l t s c o l l e c t e d a t Manning Park and the lower Okanagan V a l l e y , B.C. L a r v a e were r e a r e d i n c l e a r p l a s t i c p e t r i d i s h e s , a p p r o x i m a t e l y 9 cm i n d i a m e t e r , 2 cm i n h e i g h t , and e n c l o s e d by a l i d w i t h a s c r e e n e d v e n t , 3 cm i n d i a m e t e r . An excess o f milkweed seeds o f one s p e c i e s per c o n t a i n e r was added e v e r y t h r e e days d u r i n g l a r v a l development; d e c h l o r i n a t e d water was c o n s t a n t l y a v a i l a b l e from a s e c t i o n o f c o t t o n d e n t a l r o l l which was m o i s t e n e d d a i l y . C o l l e c t i o n d a t e s and l o c a l i t i e s o f the A s c l e p i a s seeds u t i l i z e d a r e as f o l l o w s . C o l l e c t e d i n September 1975: A. f a s c i c u l a r i s , Napa Co. C a l i f . , A. e r i o c a r p a , 1 > T u l a r e Co., C a l i f . , A. s p e c i o s a , 17 Cache Creek, B.C. Seeds o f A. e r o s a were c o l l e c t e d i n September 1974 i n T u l a r e Co. C a l i f o r n i a , and th o s e o f /\. c o r d i f o l i a c o l l e c t e d i n J u l y 1974 i n P l a c e r Co., C a l i f o r n i a . A l l seeds were d r i e d a t room tem-p e r a t u r e and s t o r e d i n p l a s t i c bags. S h e l l e d s u n f l o w e r seeds ( H e ! i a n t h u s ) were purchased l o c a l l y . W i t h i n 12 hr o f h a t c h i n g ten f i r s t i n s t a r l a r v a e o f one s p e c i e s were p l a c e d t o g e t h e r i n one c o n t a i n e r ; two r e p l i c a t e s were e s t a b l i s h e d f o r each d i f f e r e n t seed d i e t . The i n s e c t s were checked e v e r y 12 hr f o r i n d i v i d u a l s which had moulted to the next l a r v a l i n s t a r . Those which had moulted were weighed, and the wet wei g h t and time o f m o u l t i n g n o t e d . The i n s e c t s were then p l a c e d back i n t o t h e i r o r i g i n a l c o n t a i n e r . The d e n s i t y o f i n s e c t s was kept c o n s t a n t . In the '.autumn o f 1976, 0. f a s c i a t u s were r e a r e d i n an e n v i r o n -mental c a b i n e t s e t a t 21°C±1, 60±10% RH and a 16:8 LD l i g h t regime. T h i s temperature r e p r e s e n t s the mean ambient temperature a t D a v i s , C a l i f o r n i a between the months o f A p r i l and September. Eggs o f fJ. f a s c i a t u s were o b t a i n e d from the o f f s p r i n g o f a d u l t s which were c o l l e c t e d i n Napa County, C a l i f o r n i a i n August 1976. In t h i s e x p e r i m e n t , l a r v a e were r e a r e d i n s t y r o f o a m c o n t a i n e r s a p p r o x i m a t e l y 9 cm i n d i a m e t e r , 6 cm i n h e i g h t , and e n c l o s e d by a c l e a r p l a s t i c l i d w i t h a s c r e e n e d v e n t , 3 cm i n d i a m e t e r . An e x c e s s o f milkweed seeds o f one s p e c i e s per c o n t a i n e r was added e v e r y f i v e days d u r i n g l a r v a l development, and d i s t i l l e d water was c o n s t a n t l y a v a i l a b l e from a p l a s t i c c u l t u r e tube plugged w i t h c o t t o n , i n s e r t e d through the s i d e o f the r e a r i n g c o n t a i n e r . 18 Milkweed seeds used i n t h i s e x p e r i m e n t were c o l l e c t e d between June and September, 1976 a t the f o l l o w i n g l o c a l i t i e s i n C a l i f o r n i a : A. f a s c i c u l a r i s , Woodland, Y o l o Co.; A. e r o s a , n r . Ducor, T u l a r e Co.: A. s p e c i o s a , D a v i s , Y o l o Co.; A_. c o r d i f o l i a , n r . Moskowite C o r n e r , Napa Co.; A. e r i o c a r p a , nr. Trimmer, Fresno Co.; A. c a l i f o r n i c a g r e e n e i , Mt. D i a b l o , C o n t r a C o s t a Co.; A. y_. v e s t i t a , n r . Humphrey's S t a t i o n , Fresno Co.; A. c u r a s s a v i c a , D a v i s , Y o l o Co. ( c u l t i v a t e d ) . As i n the p r e v i o u s e x p e r i m e n t , w i t h i n 12 hours o f h a t c h i n g t e n f i r s t i n s t a r l a r v a e were p l a c e d i n one c o n t a i n e r , w i t h two r e p l i c a t e s e s t a b l i s h e d f o r each seed d i e t . The l a r v a e were checked e v e r y 12 hours f o r i n d i v i d u a l s which had moulted from f i f t h i n s t a r s i n t o a d u l t s . These t e n e r a l a d u l t s were weighed l i v e , t h e i r h e m e l y t r a marked w i t h i n k , and then r e p l a c e d i n t h e i r o r i g i n a l c o n t a i n e r s . T e n e r a l a d u l t s were p e r -m i t t e d to f e e d on t h e i r r e s p e c t i v e d i e t s f o r an a d d i t i o n a l 10-14 days. They were then f r o z e n , d r i e d f o r 24 hours a t 60°C i n a f o r c e d d r a f t oven, and kept o v e r d e s s i c a n t f o r a f u r t h e r 24 hours b e f o r e b e i n g reweighed. I t was f e l t t h a t o b t a i n i n g d r y w e i g h t s o f the a d u l t i n s e c t s might p r o v i d e more a c c u r a t e d a t a than wet w e i g h t s . F u r t h e r , the a d d i t i o n a l f e e d i n g time i n the a d u l t s t a g e a l l o w e d the i n s e c t s to s t a b i l i z e t h e i r body w e i g h t s , and perhaps t h e i r c a r d e n o l i d e c o n t e n t s as w e l l . A l l w e i g h t s were measured on a M e t t l e r M i c r o Gram-Atic e l e c t r o b a l a n c e . The p r o n o t a l w i d t h , an index o f a d u l t body s i z e , was measured u s i n g a d i s s e c t i n g m i c r o s c o p e f i t t e d w i t h an e y e p i e c e g r a t i c u l e . A d d i t i o n a l 0. f a s c i a t u s and L. k. k a l m i i , used i n c a r d e n o l i d e 19 d e t e r m i n a t i o n s , were r e a r e d from egg t o a d u l t i n an e n v i r o n m e n t a l c a b i n e t s e t a t 27°C, 70% RH and a 13.5:10.5 LD l i g h t regime ( t h i s c a b i n e t was sh a r e d w i t h o t h e r r e s e a r c h e r s who det e r m i n e d the r e a r i n g c o n d i t i o n s ) . In the f i g u r e s , e r r o r i s e x p r e s s e d as ±2 s t a n d a r d e r r o r s o f the mean ( r o u g h l y e q u i v a l e n t t o 95 per c e n t c o n f i d e n c e l i m i t s ) . Where e r r o r bars do not o v e r l a p , means a r e c o n s i d e r e d to be s i g n i f i c a n t l y d i f f e r e n t . 20 I I I RESULTS A. Milkweed F l o r a and L y g a e i d Fauna Nine s p e c i e s o f A s c l e p i a s were e n c o u n t e r e d i n c e n t r a l C a l i f o r n i a d u r i n g the summer o f 1976 ( F i g u r e 2 ) . Most o f the s p e c i e s o c c u r r e d i n a r e a s d i s t u r b e d by man, v i z . r o a d s i d e s , r a i l w a y c u t s , and i n f i e l d s l y i n g f a l l o w . Most s p e c i e s a r e members o f s e m i - a r i d c h a p a r r e l p l a n t communities (Munz 1968); e x c e p t i o n s were the montane A. c o r d i f o l i a , which o c c u r r e d i n y e l l o w Dine f o r e s t communities, and the r e l a t i v e l y r a r e A. s o l a n o a n a , which was seen on s o u t h - f a c i n g s e r p e n t i n e s l o p e s o n l y . The most common s p e c i e s , w i t h the w i d e s t range o f h a b i t a t s , was A. f a s c i c u l a r i s . A. e r o s a which i s an economic p e s t i n C a l i f o r n i a , o c c a s i o n a l l y o c c u r r e d i n l a r g e p o p u l a t i o n s on c u l t i v a t e d l a n d . D i f f e r e n t s p e c i e s o f A s c l e p i a s were o c c a s i o n a l l y found growing i n sympatry. On a ranch i n Frenso c o u n t y , A. y_. v e s t i t a , A. c o r d i f o l i a , A. e r i o c a r p a and A. f a s c i c u l a r i s were found w i t h i n a 200 meter r a d i u s . The l y g a e i d fJ. f a s c i a t u s which immigrates i n t o c e n t r a l C a l i f o r n i a e v e r y summer from o v e r w i n t e r i n g s i t e s i n the south ( p r o b a b l y M e x i c o ) , showed two c o n t r a s t i n g c o l o n i z a t i o n p a t t e r n s on s p e c i e s o f A s c l e p i a s i n the two b a s i c s t u d y a r e a s ( T a b l e I ) . In the n o r t h bay a r e a , CJ. f a s c i a t u s was o n l y found on A. f a s c i c u l a r i s - even i n l o c a t i o n s where o t h e r b r o a d ! e a f milkweeds such as A. s p e c i o s a and A. e r i o c a r p a were growing s y m p a t r i c a l l y . In c o n t r a s t , 0. f a s c i a t u s c o l o n i z e d a l l o f the milkweed s p e c i e s i n the Fresno a r e a , w i t h the n o t a b l e e x c e p t i o n o f A. c o r d i f o l i a . 21 TABLE I: O c c u r r e n c e o f 0_. f a s c i a t u s and L_. JL k a l m i i on d i f f e r e n t s p e c i e s o f A s c l e p i a s i n two a r e a s o f C a l i f o r n i a , summer 1976. Milkweed s p e c i e s North Bay Area ( Y o l o , Capa, S o l a n o , C o n t r a Costa c o u n t i e s ) Fresno Area ( F r e s n o , T u l a r e c o u n t i e s ) A. f a s c i c u l a r i s A. c o r d i f o l i a A. e r i o c a r p a A. c a l i f o r n i c a g r e e n e i A. s p e c i o s a A. e r o s a A. v e s t i t a v e s t i t a J L L L L ' L 0 0 0 0 L L L L L L 1 0 = 0 . f a s c i a t u s , L = L. k. k a l m i i 22 During 1976 t e n stands o f A. c o r d i f o l i a were examined i n f i v e c o u n t i e s , y e t CJ. f a s c i a t u s was never o b s e r v e d on t h i s p l a n t . Even where A. c o r d i f o l i a o c c u r r e d i n sympatry w i t h A. v e s t i t a and A. e r i o c a r p a and where both o f t h e s e l a t t e r s p e c i e s s u p p o r t e d CJ. f a s c i a t u s , none o f the i n s e c t s were found on the A. c o r d i f o l i a . The t i m i n g and d u r a t i o n o f f l o w e r i n g p e r i o d s v a r i e s among m i l k -weed s p e c i e s . T h i s l e a d s to temporal d i f f e r e n c e s i n the f r u i t i n g season o f the r e s p e c t i v e s p e c i e s . A l s o , f l o w e r i n g and f r u i t i n g season v a r i e s w i t h e l e v a t i o n by as much as s i x weeks. A. c o r d i f o l i a and A. c a l i f o r n i c a g r e e n e i had e a r l y , h i g h l y s y n c h r o n i z e d f l o w e r i n g and f r u i t i n g s easons, c o m p l e t i n g the r e p r o d u c t i v e c y c l e by r e l e a s i n g the comose seeds i n June and J u l y ( F i g u r e 3 ) . A. y_. v e s t i t a a l s o had a r a t h e r s h o r t f r u i t i n g season d u r i n g mid summer, c o m p l e t i n g i t s c y c l e i n August. In c o n t r a s t , A. f a s c i c u l a r i s , A. e r i o c a r p a , A. e r o s a and A_. s p e c i o s a d i d not have s y n c h r o n i z e d f l o w e r i n g and f r u i t i n g p e r i o d s which l e d t o an extended f r u i t i n g season. A. f a s c i c u l a r i s and A. e r o s a had the l o n g e s t f l o w e r i n g p e r i o d s , b e a r i n g both f l o w e r s and mature seed pods on the same p l a n t as l a t e as mid-September. A t the Woodland A_. f a s c i c u l a r i s s i t e , (3. f a s c i a t u s d i d n o t appear u n t i l mid-June, and remained i n low numbers u n t i l the second g e n e r a t i o n reached i t s peak i n e a r l y September ( F i g u r e 3 ) . Exami n a t i o n s o f p o p u l a t i o n s o f CJ. f a s c i a t u s a t o t h e r l o c a l i t i e s on A_. f a s c i c u l a r i s s t r o n g l y s u g g e s t e d t h a t t h i s was the p a t t e r n o f p o p u l a t i o n growth i n most o f n o r t h c e n t r a l C a l i f o r n i a i n 1976 (K.E. Evans, unpubl. r e s u l t s ) . In 1976 the p o p u l a t i o n s o f CJ_. f a s c i a t u s d i d not re a c h h i g h d e n s i t i e s i n FIGURE 3: F r u i t i n g season i n n a t i v e C a l i f o r n i a n A s c l e p i a s s p e c i e s and the p o p u l a t i o n t r e n d o f fj. f a s c i a t u s a t one milkweed s t a n d . Stage o f f r u i t development: immature dehi s c e n t Milkweed s p e c i e s : V = A s c l e p i a s v e s t i t a v e s t i t a CA = A_. c a l i f o r n i c a g r e e n e i SP = A_. s p e c i o s a EC = A. e r i o c a r p a CO = A. c o r d i f o l i a ES = A_. e r o s a F = A. f a s c i c u l a r i s As f r u i t i n g season can v a r y w i t h e l e v a t i o n , seasons d e p i c t e d here a r e f o r p l a n t s growing a t e l e v a t i o n s below 500 meters above sea l e v e l . Cj. f a s c i a t u s p o p u l a t i o n data from a weekly census o f a s t a n d o f A_. f a s c i c u l a r i s i n Woodland, Yolo County. P o p u l a t i o n t r e n d s o f fj. f a s c i a t u s on A. f a s c i c u l a r i s a t s e v e r a l l o c a l i t i e s i n Napa cou n t y a r e v e r y s i m i l a r . FIGURE 3 Species 25 t h i s a r e a u n t i l A. c o r d i f o l i a and A. c a l i f o r m " c a g r e e n e i had d i s p e n s e d most, i f not a l l o f t h e i r seed c r o p ( F i g u r e 3 ) . B. C a r d e n o l i d e C o n t e n t o f P l a n t M a t e r i a l The c a r d e n o l i d e c o n t e n t o f p l a n t samples o f A s c l e p i a s from C a l i f o r n i a v a r i e s o v e r a wide range. ( T a b l e s I I , I I I ) . W i t h i n s p e c i e s v a r i a t i o n i n c a r d e n o l i d e c o n t e n t o f milkweed seeds was about equal to between s p e c i e s v a r i a t i o n . Most o f the seed samples a n a l y s e d had c a r d e n o l i d e c o n t e n t s between 1.2 and 4.3 mg per gram o f d r y t i s s u e . However, t h r e e s p e c i e s o f A s c l e p i a s had c a r d e n o l i d e c o n t e n t s which o c c u r n o t a b l y o u t s i d e o f t h i s range: These a r e A_. f a s c i c u l a r i s (from 0.25 to 0.44 mg per gram) A. v_. v e s t i t a (7.1 mg per gram f o r the s i n g l e sample a n a l y s e d ) a n d A_. c u r a s s a v i c a (7.9 mg per gram, s i n g l e e s t i m a t i o n ) F i g u r e 4 ) . Both temporal and g e o g r a p h i c a l i n t r a s p e c i f i c d i f f e r e n c e s i n the c a r d e n o l i d e c o n t e n t o f the milkweed seed o c c u r ( T a b l e I I ) . For example, the c a r d e n o l i d e c o n t e n t o f seeds o f A. e r o s a , c o l l e c t e d i n the same a r e a , but i n two d i f f e r e n t y e a r s v a r i e d c o n s i d e r a b l y , as d i d the c o n t e n t o f seeds o f A. c a l i f o r m ' c a g r e e n e i , c o l l e c t e d i n the same season from two g e o g r a p h i c a l l y i s o l a t e d p o p u l a t i o n s . However, the two samples o f A. f a s c i c u l a r i s c o l l e c t e d i n 1975 from d i f f e r e n t c o u n t i e s had s i m i l a r c a r d e n o l i d e c o n t e n t s , whereas the 1976 sample from a d j a c e n t Y o l o county c o n t a i n e d a somewhat lower amount o f c a r d e n o l i d e than the f o r m e r two ( T a b l e I I ) . V a r i a t i o n a l s o e x i s t e d i n the c a r d e n o l i d e c o n t e n t o f d i f f e r e n t organs o f a s i n g l e h o s t pi a n t s p e c i e s ( T a b l e I I I ) . No s t a n d a r d TABLE I I : Gross C a r d e n o l i d e c o n t e n t o f seed o f A s c l e p i a s s p e c i e s from C a l i f o r n i a . ^ county i n C a l i f o r n i a and y e a r o f c o l l e c t i o n o f milkweed seeds 2 mg e q u i v a l e n t s o f d i g i t o x i n p er gram o f d r i e d seed 3 f o r each c o l l e c t i o n l o c a l i t y , seeds from s e v e r a l p l a n t s were p o o l e d and 1.0 g o f seed ground to a powder; sample s i z e N = number o f 0.2 g a l i q u o t s taken from the 1 g sample c u l i t v a t e d p l a n t s ; t h i s s p e c i e s i s n o t n a t i v e to C a l i f o r n i a 5 Cache Creek, B r i t i s h Columbia, Canada Milkweed S p e c i e s C o l l e c t i o n Data C a r d e n o l i d e Contents-mean S.E. N-3 A. c a l i f o r m ' c a g r e e n e i C o n t r a C o s t a 1976 4.3- + 0.40 5 Fresno 1976 3.1 + 0.08 3 A. c o r d i f o l i a Napa 1976 3.1 + 0.15 3 P l a c e r 1974 1.3 + 0.05 2 A. . 4 c u r a s s a v i c a ' Y o l o 1976 7.9 + 0.31 3 A. e r i o c a r p a Fresno 1976 2.4 + 0.07 5 Y o l o 1976 1.3 1 Fresno 1975 3.4 + 0.57 3 A. e r o s a T u l a r e 1976 2.4 + 0.22 3 T u l a r e 1974 1.9 + 0.14 5 A. f a s c i c u l a r i s Y o l o 1976 0.25 + 0.03 3 Napa 1975 0.44 + 0.04 5 Solano 1975 0.44 ± 0.04 3 A. so l a n o a n a Tehama 1973 1.2 + 0.13 3 A. s p e c i o s a S olano 1976 2.7 + 0.15 3 B.C. 5 1975 1.8 + 0.03 2 A. v. v e s t i t a F r esno 1976 7.1 + 0.29 5 TABLE I I I : Gross c a r d e n o l i d e c o n t e n t o f p l a n t samples o f A s c l e p i a s from C a l i f o r n i a . L e a f = mature o r lower l e a v e s Umbel = f l o w e r s a t time o f peak f l o w e r i n g Seed = seeds from d e h i s c e n t pods o r darkened seeds from pods c l o s e to m a t u r i t y A l l p l a n t m a t e r i a l c o l l e c t e d i n summer, 1976. mg e q u i v a l e n t s o f d i g i t o x i n per gram o f d r i e d t i s s u e ; d a t a f o r l e a f and umbel r e p r e s e n t s s i n g l e e s t i m a t i o n s !data from TABLE II 29 C a r d e n o l i d e C o n t e n t Milkweed S p e c i e s L o c a l i t y L e a f Umbel Seed A. c a l i f o r n i c a g r e e n e i C o n t r a Costa Co. 4.3 Fresno Co 0.27 A. e r i o c a r p a Fresno Co. 3.7 5.'5 3.'4 Fresno Co. 2.4 Y o l o Co. 1.7 1.3 A. e r o s a T u l a r e Co. 3.0 0.89 2.4 T u l a r e Co. 1.9 A. f a s c i c u l a r i s Y o l o Co. 0.22 0.73 0.25 Fresno Co. 1.0 Napa Co. 0.44 A. v. v e s t i t a Fresno Co. 6.8 2.6 7.1 FIGURE 4: Gross c a r d e n o l i d e c o n t e n t o f seed o f A s c l e p i a s s p e c i e s from C a l i f o r n i a . Data from TABLE I I ; e r r o r i s e x p r e s s e d as t w i c e the s t a n d a r d e r r o r o f the mean. I n t e r s p e c i f i c and i n t r a s p e c i f i c d i f f e r e n c e s a r e d e p i c t e d . 32 r e l a t i o n s h i p between the r e l a t i v e amounts o f c a r d e n o l i d e p r e s e n t and the p l a n t organs o c c u r r e d , a t l e a s t i n the few s p e c i e s examined here ( F i g u r e 4 ) . Umbels c o n t a i n e d the g r e a t e s t c o n c e n t r a t i o n s o f c a r d e n o l i d e s i n two o f the s p e c i e s examined (A. f a s c i c u l a r i s and A_. e r o s a ) , w h i l e the umbels c o n t a i n e d the l o w e s t c o n c e n t r a t i o n s o f c a r d e n o l i d e s i n two o t h e r s p e c i e s examined (A. e r i o c a r p a and A. v_. v e s t i t a ) ( F i g u r e 5 ) . C. C a r d e n o l i d e Content o f F i e l d C o l l e c t e d L y g a e i d s There was a wide v a r i a t i o n i n the c a r d e n o l i d e c o n t e n t o f the milkweed bugs 0_. f a s c i a t u s and I. k. k a l m i i c o l l e c t e d on A s c l e p i a s s p e c i e s i n the f i e l d i n c e n t r a l C a l i f o r n i a ( T a b l e I V ) . Some i n s e c t s were found t o e i t h e r l a c k c a r d e n o l i d e s o r have l e v e l s o f c a r d e n o l i d e s below the c o l o r i m e t r i c d e t e c t i o n l i m i t . Other i n s e c t s were found to c o n t a i n up to 372 yg o f c a r d e n o l i d e per i n d i v i d u a l . D i f f e r e n c e s i n c a r d e n o l i d e c o n t e n t between samples o f l y g a e i d bugs taken on a p a r -t i c u l a r h o s t s p e c i e s i n d i c a t e d t h a t i n t e r s p e c i f i c d i f f e r e n c e s i n the c a r d e n o l i d e c o n t e n t o f the h o s t s p e c i e s cannot a l o n e a c c o u n t f o r the v a r i a b i l i t y i n the i n s e c t s , even between p o p u l a t i o n s f e e d i n g on the same p l a n t organ. Both fj. f a s c i a t u s and L_. k_. k a l m i i s e q u e s t e r e d the l a r g e s t q u a n t i t i e s o f c a r d e n o l i d e s from the t h r e e b r o a d l e a f milkweed s p e c i e s A_. v_. v e s t i t a , A_. e r o s a and A. e r i o c a r p a ( F i g u r e s 6,7). Of the f i v e l o c a l i t i e s where both fJ. f a s c i a t u s and L_. k_. k a l m i i were c o l l e c t e d and a s s a y e d , I. k_. k a l m i i s e q u e s t e r e d l a r g e r q u a n t i t i e s o f c a r d e n o l i d e s than fj. f a s c i a t u s i n f o u r ( F i g u r e 8). In two l o c a l i t i e s , ( n e a r P i e d r a , Fresno Co. on A_. e r i o c a r p a ' and near Ducor, T u l a r e Co. on A. e r o s a ) , FIGURE 5: Gross c a r d e n o l i d e c o n t e n t o f p l a n t samples o f A s c l e p i a s from C a l i f o r n i a . Data from TABLE I I I . TABLE IV: Gross c a r d e n o l i d e c o n t e n t o f fj. f a s c i a t u s and L_. k_. k a l m i i c o l l e c t e d on A s c l e p i a s i n c e n t r a l C a l i f o r n i a i n 1976. ^Voucher specimens o f f l o w e r s o f the milkweed s p e c i e s a r e i n the p o s s e s s i o n o f the a u t h o r County i n C a l i f o r n i a , month o f c o l l e c t i o n , and p a r t o f p l a n t on which i n s e c t s were p r e d o m i n a n t l y f e e d i n g 3 E x p r e s s e d as e q u i v a l e n t s o f d i g i t o x i n ; NIL = no c a r d e n o l i d e s d e t e c t e d , S.E. = s t a n d a r d e r r o r o f the mean, N = number o f i n s e c t s a s s a y e d from s i t e 4 P l a n t s a t t h i s l o c a l i t y were t a x o n o m i c a l l y between /\. v_. v e s t i t a and A. v_. p a r i s h i i Woodson. 36 1 2 C a r d e n o l i d e Content Milkweed S p e c i e s C o l l e c t i o n Data (yg per i n s e c t ) mean + S.E. N ONCOPELTUS FASCIATUS A. c a l i f o r m " c a g r e e n e i Fresno May pod 5.5 + 5.5 6 A. e r i o c a r p a Fresno May umbel 20 + 8.8 9 Fresno J u l y umbel 107 + 16 9 Fresno August pod 82 + 8.3 9 Fresno August stem 15 + 7.0 6 A. e r o s a T u l a r e J u l y pod 159 + 26 9 A- f a s c i c u l a r i s Napa June umbel 37 + 7.7 9 Fresno J u l y pod 61 + 10 9 T u l a r e J u l y pod 13 + 8.3 9 Y o l o August pod 11 + 2.8 9 Solano August pod 18 + 5.4 9 A. v e s t i t a v e s t i t a Fresno May umbel 17 + 14 6 Fresno J u l y pod 195 + 32 9 LYGAEUS KALMII KALMII A. c a l i f o r n i c a g r e e n e i C o n t r a C o s t a May umbel 5.6 + 4.3 9 A. c o r d i f o l i a P l a c e r June pod 3.3 + 1.7 3 Napa June umbel MIL 2 A. e r i o c a r p a San L u i s Obispo June umbel NIL 2 Y o l o June umbel 17 + 8.4 6 Fresno J u l y umbel 152 + 13 3 Fresno J u l y umbel 162 + 22 4 Y o l o August umbel 93 + 23 9 Fresno August stem 116 + 23 7 A. e r o s a T u l a r e J u l y umbel 74 + 23 9 T u l a r e August pod 205 + 31 8 A. f a s c i c u l a r i s Y o l o August pod 5.7 + 1.4 6 A. v. v e s t i t a San L u i s O b i s p o ^ June pod 51 + 23 6 Fresno J u l y pod 256 + 35 9 FIGURE 6: Gross c a r d e n o l i d e c o n t e n t o f f i e l d c o l l e c t e d (). f a s c i a t u s . CA = A s c l e p i a s c a l i f o r n i c a g r e e n e i EC = A. e r i o c a r p a ES = A. e r o s a F = A. f a s c i c u l a r i s V = A. v. v e s t i t a Data from TABLE IV. FIGURE 6 ONCOPELTUS FASCIATUS may jn June jl July a august u umbel p pod species CA organ p month m EC u u p s m jl a a ES P jl u p p p p jn jl jl a a u m FIGURE 7: Gross c a r d e n o l i d e c o n t e n t o f f i e l d c o l l e c t e d I. k. k a l m i i . CA = A s c l e p i a s c a l i f o r n i c a g r e e n e i CO = A. c o r d i f o l i a EC = A. e r i o c a r p a ES = A. e r o s a F = A. f a s c i c u l a r i s V = A. v_. v e s t i t a Other symbols ( f o r p l a n t o r g a n s , month o f c o l l e c t i o n ) as i n FIGURE 6. FIGURE 7 LYGAEUS KALMII KALMII 300 - i species CA organ u month m 1 jn in 1 jn jn jl jl a a FIGURE 8: Comparison o f g r o s s c a r d e n o l i d e c o n t e n t s o f CJ_. f a s c i a t u s and L. k. k a l m i i c o l l e c t e d from the same l o c a l i t i e s . F = A s c l e p i a s f a s c i c u l a r i s ES = A. e r o s a EC = A. e r i o c a r p a V = A. v. v e s i t a P = d e h i s c e n t pods and seed i p = immature pods u = umbels s = stems Date from TABLE IV. O. fasciatus L. k. kalmii 300 n 200 Z a. D) 100 H o Species Organ F P ES •P EC u EC FIGURE 8 V p ro 43 J_. k_. k a l m i i s e q u e s t e r e d s i g n i f i c a n t l y more c a r d e n o l i d e than d i d CJ. f a s c i a t u s . There i s e v i d e n c e o f temporal v a r i a t i o n i n the c a r d e n o l i d e c o n t e n t o f t h e CJ_. f a s c i a t u s ( T a b l e V ) . A d u l t s o f CJ. f a s c i a t u s c o l l e c t e d i n Fresno county i n May from the t h r e e b r o a d ! e a f milkweed s p e c i e s A. c a l i f o r m ' c a g r e e n e i , A.- e r i o c a r p a and A. v_. v e s t i t a c o n t a i n e d small q u a n t i t i e s o f c a r d e n o l i d e s . However, a d u l t i n s e c t ' s c o l l e c t e d on A. e r i o c a r p a and A. y_. v e s t i t a l a t e r i n the season from the same l o c a l i t i e s had much h i g h e r l e v e l s o f c a r d e n o l i d e s i n t h e i r b o d i e s . On the o t h e r hand, t h i s t r e n d d i d not e x i s t among a d u l t s o f CJ. f a s c i a t u s c o l l e c t e d on A. f a s c i c u l a r i s from June through August. T h e r e f o r e , i f temporal v a r i a t i o n i n the c a r d e n o l i d e c o n t e n t o f l y g a e i d s i s the r e s u l t o f some a s p e c t o f the p h e n o l o g i e s o f t h e i r r e s p e c t i v e h o s t s (e.g. a v a i l a b i l i t y o f pods and s e e d ) , t h i s s i t u a t i o n i s l i m i t e d t o the c a r d e n o l i d e - r i c h b r o a d l e a f s p e c i e s o f milkweed i n C a l i f o r n i a . In g e n e r a l , both ()_. f a s c i a t u s and L^ . k_. k a l m i i c o l l e c t e d i n May and June c o n t a i n e d s u b s t a n t i a l l y lower l e v e l s o f c a r d e n o l i d e s than c o n s p e c i f i c s taken l a t e r i n the season on the same h o s t s p e c i e s ( F i g u r e s 6 and 7 ) . The f r e q u e n c y d i s t r i b u t i o n o f c a r d e n o l i d e c o n t e n t i n a l l the f i e l d - c o l l e c t e d l y g a e i d s i n t h i s s t u d y i s s t r o n g l y skewed toward the l e f t , o r low end o f the X - a x i s ( F i g u r e 9 ) . Of the 211 i n d i v i d u a l l y g a e i d s a s s a y e d , 52 p e r c e n t o f thos e had l e s s than 50 micrograms o f c a r d e n o l i d e i n t h e i r b o d i e s , and 71 per c e n t had l e s s than 100 micrograms. The o v e r a l l range i n c a r d e n o l i d e c o n t e n t o f the f i e l d - c o l l e c t e d l y g a e i d s f e l l w i t h i n the range o f c a r d e n o l i d e c o n t e n t s i n l a b o r a t o r y - r e a r e d bugs. 44 TABLE V: Temporal v a r i a t i o n i n gross c a r d e n o l i d e c o n t e n t o f f i e l d c aught fJ. f a s c i a t u s from C e n t r a l C a l i f o r n i a . C a r d e n o l i d e C ontent Milkweed S p e c i e s May-June J u l y - A u g u s t 2 Lab Reared A. c a l i f o r m ' c a g r e e n e i 5.5 - 251 A. e r i o c a r p a 20 107, 82 149 A. e r o s a - 159 151 A. f a s c i c u l a r i s 37 61, 13, 11, 18 34 A. v. v e s t i t a 17 195 281 mean i n yg e q u i v a l e n t s o f d i g i t o x i n p er i n s e c t d a t a from T a b l e IV 'reared from egg t o a d u l t e x c l u s i v e l y on seed from r e s p e c t i v e milkweed s p e c i e s ; d a t a from T a b l e VI. FIGURE 9: Gross c a r d e n o l i d e c o n t e n t o f f i e l d c o l l e c t e d l y g a e i d s : d i s t r i b u t i o n o f c l a s s e s based on c a r d e n o l i d e c o n t e n t . T o t a l sample s i z e o f 211 r e p r e s e n t s a l l i n d i v i d u a l i n s e c t s used t o f o r m u l a t e date i n T a b l e IV. While not a random sample o f l y g a e i d s on milkweed i n C a l i f o r n i a , t h e s e i n s e c t s c o n s t i t u t e a r e p r e s e n t a t i v e sample owing to the d i s p r o p o r t i o n a t e numbers o f i n s e c t s on d i f f e r e n t A s c l e p i a s s p e c i e s . Of the t o t a l sample, 52 per c e n t c o n t a i n e d l e s s than 50 yg per i n s e c t , and 71 per c e n t c o n t a i n e d l e s s t h a t 100 yg per i n s e c t . FIGURE 9 4 6 120-i 0 100 200 300 400 pg per Insect 47 D. C a r d e n o l i d e Content o f L a b o r a t o r y - R e a r e d L y g a e i d s T a b l e VI shows a n a l y s e s o f the c a r d e n o l i d e c o n t e n t o f l y g a e i d s r e a r e d from egg to a d u l t i n the l a b o r a t o r y on seed from d i f f e r e n t s p e c i e s o f A s c l e p i a s a t 25°C. CJ. f a s c i a t u s and l^. k_. k a l m i i s e q u e s t e r and s t o r e s m a l l q u a n t i t i e s o f c a r d e n o l i d e s from A. f a s c i c u l a r i s , moderate amounts from A. e r o s a , A. c o r d i f o l i a and A. e r i o c a r p a , and r e l a t i v e l y l a r g e q u a n t i t i e s from /\. c a l i f o r n i c a g r e e n e i and A. y_. v e s t i t a . There i s a p o s i t i v e c o r r e l a t i o n between c a r d e n o l i d e c o n t e n t o f l a b o r a t o r y r e a r e d i n s e c t s and the c a r d e n o l i d e c o n t e n t o f the seeds o f t h e i r r e s p e c t i v e h o s t s p e c i e s ( F i g u r e 10, T a b l e V I ) . L_. J£. k a l m i i appears t o be more e f f i c i e n t a t s e q u e s t e r i n g t h e s e compounds as i t s t o r e d l a r g e r q u a n t i t i e s o f c a r d e n o l i d e s than CJ. f a s c i a t u s when both i n s e c t s were r e a r e d on the same seed d i e t ( F i g u r e 10). S i m i l a r l y , fJ. f a s c i a t u s r e a r e d a t 21°C was more e f f i c i e n t a t s e q u e s t e r i n g c a r d e n o l i d e s than those r e a r e d a t 25°C ( F i g u r e 10). When the d a t a f o r a d u l t fj . f a s c i a t u s r e a r e d a t 21 °C a r e examined i n d e t a i l , i t i s a p p a r e n t t h a t the c a r d e n o l i d e c o n t e n t o f a d u l t bugs i s s i g n i f i c a n t l y d i f f e r e n t between the sexes i n a l l but one milkweed seed d i e t ( T a b l e V I I ) . Females were more e f f i c i e n t a t s e q u e s t e r i n g c a r d e n o l i d e s than males on a l l e i g h t s p e c i e s o f A s c l e p i a s ( F i g u r e 11). E. L a r v a l Growth and Development o f L y g a e i d s on D i f f e r e n t S p e c i e s o f Milkweed i n the L a b o r a t o r y Both fJ. f a s c i a t u s and L. k. k a l m i i were s u c c e s s f u l l y r e a r e d from eggs to a d u l t s on a d i e t o f milkweed seeds from s e v e r a l d i f f e r e n t s p e c i e s o f A s c l e p i a s as w e l l as H e l i a n t h u s ( C o m p o s i t a e ) . None o f the milkweed 48 TABLE V I : Gross c a r d e n o l i d e c o n t e n t o f 0_. f a s c i a t u s and L_. J^. k a l m i i r e a r e d i n t h e l a b o r a t o r y on milkweed seed. 3 C a r d e n o l i d e Content 1 2 0.- f a s c i a t u s l^. k. k a l m i i Milkweed S p e c i e s C o l l e c t i o n Data Seed mean + SE N mean + SE N A. c a l i f o r n i c a g r e n e e i C o n t r a C o s t a 1976 4.3 251 + 29 9 419 + 92 6 A- c o r d i f o l i a P l a c e r 1974 1.7 125 + 19 9 78 + 14 9 A- e r i o c a r p a Fresno 1975 3.4 149 + 13 9 219 + 42 9 A. e r o s a T u l a r e 1974 1.9 151 + 23 9 268 + 58 9 A. f a s c i c u l a r i s Napa 1975 0.4 34 + 5.1 9 50 + 19 9 A. v e s t i t a v e s t i t a F r e s n o 1976 7.1 281 + 20 18 765 + 72 9 C a l i f o r n i a n c o l l e c t i o n d a t a f o r milkweed seed f e d t o i n s e c t s , mg e q u i v a l e n t s o f d i g i t o x i n p er gram o f d r i e d seed; d a t a from T a b l e I I . V g e q u i v a l e n t s o f d i g i t o x i n per i n s e c t : N = no. o f i n s e c t s i n each sample, S.E. = s t a n d a r d e r r o r o f the mean. ) FIGURE 10: R e l a t i o n s h i p between c a r d e n o l i d e c o n c e n t r a t i o n i n the d i e t (milkweed s e e d s ) and the c a r d e n o l i d e c o n t e n t o f a d u l t fj. f a s c i a t u s and IL. _k. k a l m i i . I n s e c t s a s s a y e d f o r c a r d e n o l i d e s were r e a r e d from egg t o a d u l t e x c l u s i v e l y on seeds o f r e s p e c t i v e s p e c i e s o f A s c l e p i a s . V a l u e s f o r sexes combined. I n s e c t d a t a from T a b l e s VI and V I I I , seed d a t a from T a b l e I I ; r = c o r r e l a t i o n c o e f f i c i e n t , m = s l o p e . D i f f e r e n c e s between t h e sexes ( F i g u r e 11) c o n t r i b u t e to the l a r g e s t a n d a r d e r r o r s . O 0. f a s c i a t u s r e a r e d a t 21°C © 0. f a s c i a t u s r e a r e d a t 25°C L. k. k a l m i i r e a r e d a t 25°C fj. f a s c i a t u s s e q u e s t e r s more c a r d e n o l i d e s a t 21 C owing t o a l o n g e r d u r a t i o n o f l a r v a l development (approx. 40 days to the a d u l t m o l t ) compared to c o n s p e c i f i c s r e a r e d a t 25°C (approx. 22 days t o the a d u l t m o l t ) . L_. k_. k a l m i i appears t o be more e f f i c i e n t a t s e q u e s t e r i n g c a r d e n o l i d e s than fj. f a s c i a t u s a t the same tem p e r a t u r e . 51 TABLE V I I : Gross c a r d e n o l i d e c o n t e n t o f CJ. f a s c i a t u s r e a r e d i n the l a b o r a t o r y a t 21°C on milkweed seed. Seed' A d u l t 0. f a s c i a t u s ' P l a n t S p e c i e s mean. ± S.E. N cCo* mean S.E. ? ? mean ± S.E. A. f a s c i c u l a r i s 0.25 + 0.03 3 42.3 + 2.5 48.7 + 3.8 H A. e r o s a 2.4 + 0.07 3 139 + 20 204 + 14 A. s p e c i o s a 2.7 + 0.15 3 151 + 20 250 + 14 A. c o r d i f o l i a 3.1 + 0.15 3 152 + 27 363 + 30 A. e r i o c a r p a 3.4 + 0.57 3 75 + 6.7 158 + 9.3 A. c a l i f o r n i c a 4.3 + 0.40 5 215 + 5.6 387 + 32 A. v e s t i t a 7.1 + 0.29 5 291 + 81 649 + 58 A. c u r a s s a v i c a 7.9 + 0.31 4 386 + 23 707 + 51 mg per gram o f d r i e d seed; d a t a from T a b l e II yg per i n s e c t 'N = 3 f o r both sexes " d i f f e r e n c e between sexes n o t s i g n i f i c a n t FIGURE 11: R e l a t i o n s h i p between c a r d e n o l i d e c o n c e n t r a t i o n i n the d i e t (milkweed seeds) and the c a r d e n o l i d e c o n t e n t o f a d u l t male and female 0. f a s c i a t u s r e a t e d a t 21°C. Data from Tabel V I I . r = c o r r e l a t i o n c o e f f i c i e n t , m = s l o p e . FIGURE 11 0 2 4 6 8 m g per g r a m DRY SEED cn co 54 s p e c i e s t e s t e d appear t o p r e s e n t a n u t r i t i o n a l b a r r i e r t o the growth and development o f CL f a s c i a t u s and j _ . k_. k a l m i i . When r e a r e d a t 25°C, CL f a s c i a t u s completed l a r v a l development s i g n i f i c a n t l y f a s t e r on seeds o f A., f a s c i c u l a r i s than on the o t h e r milkweed seeds p r o v i d e d ( F i g u r e 12). There were no s i g n i f i c a n t d i f f e r e n c e s among t h e s e bugs when r e a r e d on seeds o f o t h e r milkweeds, v i z . A., e r o s a , /\. c o r d i f o l i a , A. e r i o c a r p a and A. s p e c i o s a ( T a b l e V I I I ) . S u r v i v a l to the a d u l t s t a g e was 75 per c e n t o r g r e a t e r on a l l d i e t s . However, developmental d u r a t i o n o f the l a r v a e was s i g n i f i c a n t l y l o n g e r f o r bugs r e a r e d on seeds o f He ! i a n t h u s annus. The small milkweed bug L_. k_. k a l m i i a l s o d e v e l o p e d f a s t e r on the seeds o f A_. f a s c i c u l a r i s than on the seeds o f o t h e r milkweeds. In c o n t r a s t t o 0. f a s c i a t u s , L_. k. k a l m i i d e v e l o p e d s i g n i f i c a n t l y f a s t e r on s u n f l o w e r seeds than on the milkweed seeds w i t h the e x c e p t i o n o f A. f a s c i c u l a r i s ( F i g u r e 12). S u r v i v a l to the a d u l t s t a g e on a l l d i e t s was lower than t h a t o f CL f a s c i a t u s ( T a b l e I X ) . At 21 UC t h e r e were few s i g n i f i c a n t d i f f e r e n c e s i n the l a r v a l developmental times o f CL f a s c i a t u s r e a r e d on e i g h t d i f f e r e n t s p e c i e s o f A s c l e p i a s ( T a b l e X). As the c a r d e n o l i d e c o n t e n t o f t h e s e i n s e c t s had been d e t e r m i n e d ( T a b l e V I I ) , i t was p o s s i b l e t o compare the c a r d e n o l i d e c o n t e n t o f the i n s e c t s w i t h the l a r v a l d u r a t i o n . In t h i s s t u d y , no s i g n i f i c a n t c o r r e l a t i o n was found between t h e s e two i n d i c e s ( T a b l e XIV, F i g u r e 14). There were no s i g n i f i c a n t d i f f e r e n c e s i n the wet weights o f t e n e r a l CJ. f a s c i a t u s o f one sex when r e a r e d on the f i v e d i f f e r e n t s p e c i e s FIGURE 12: R e l a t i o n s h i p between d u r a t i o n o f l a r v a l development and t e n e r a l wet w e i g h t o f CJ. f a s c i a t u s and L^ . k. k a l m i i r e a r e d i n the l a b o r a t o r y a t 25°C on seeds o f d i f f e r e n t s p e c i e s o f A s c l e p i a s and H e l i a n t h u s . 0. f a s c i a t u s L_. k. k a l m i i Data from T a b l e s V I I I and IX: f A. f a s c i c u l a r i s es A. e r o s a c A. c o r d i f o l i a ec A. e r i o c a r p a s A. s p e c i o s a A s c l e p i a s spp. H_. annus o • TABLE V I I I : D u r a t i o n o f l a r v a l development and t e n e r a l a d u l t wet wei g h t o f CJ. f a s c i a t u s r e a r e d i n the l a b o r a t o r y a t 25°C on the seeds o f d i f f e r e n t s p e c i e s o f A s c l e p i a s and H e l i a n t h u s . Wet Weight (mg) Developmental D u r a t i o n (days) P l a n t S p e c i e s mean ± S.E. N S u r v i v a l (%) c r+$mean ± S.E. ^ m e a n ?ftnean ?: cr A. c o r d i f o l i a 21.9 + 0.28 19 95 65.5 + 2.7 54.1 72.2 1 .33 A. e r i o c a r p a 21.7 + 0.37 18 90 68.0 + 2.4 60.9 72.6 1 .19 A. e r o s a 22.4 + 0.17 19 95 66.9 + 2.7 58.3 74.7 1 .28 A. f a s c i c u l a r i s 19.5 + 0.12 16 80 67.5 + 2.6 58.1 71.7 1 .23 A. s p e c i c o s a 22.9 + 0.63 17 85 61.6 + 2.3 54.9 69.8 1 .27 H. annus 24.4 + 0.61 15 75 52.3 + 2.4 45.9 55.6 1 .21 N = t o t a l number o f i n s e c t s c o m p l e t i n g development t o the a d u l t s t a g e ( b o t h sexes combined) See m a t e r i a l s and methods (Section D) f o r c o l l e c t i o n d a t a f o r milkweed see d s . S u r v i v a l i s the pe r c e n t a g e o f l a r v a e t h a t s u c c e s s f u l l y complete the f i n a l m o l t i n t o the a d u l t s t a g e . T e n e r a l a d u l t s were weighed w i t h i n 12 hours o f the a d u l t m o l t . on TABLE IX: D u r a t i o n o f l a r v a l development and t e n e r a l a d u l t wet w e i g h t o f L_. k_. k a l m i i r e a r e d i n the l a b o r a t o r y a t 25°C on the seeds o f d i f f e r e n t s p e c i e s o f A s c l e p i a s and H e l i a n t h u s . P l a n t S p e c i e s Developmental D u r a t i o n (days) Wet Weight (mg) mean ± S.E. N S u r v i v a l (%) o*+? mean ± S.E. A. c o r d i f o l i a 28.5 + 0.16 16 80 43.6 + 1.0 A. e r i o c a r p a 29.6 + 1.6 6 30 44.1 + 2.9 A. e r o s a 29.3 + 0.88 14 70 47.2 + 1.7 A. f a s c i c u l a r i s 25.8 + 0.12 17 85 44.1 + 0.8 A. s p e c i o s a 29.1 + 1.5 13 65 41.6 + 1.6 H. annus 26.5 + 0.43 16 80 42.9 + 1.4 M = t o t a l number o f i n s e c t s c o m p l e t i n g development to the a d u l t s t a g e (both sexes combined). See m a t e r i a l s and methods ( s e c t i o n D) f o r c o l l e c t i o n d a t a f o r milkweed seeds. S u r v i v a l i s the p e r c e n t a g e o f l a r v a e t h a t s u c c e s s f u l l y completed the f i n a l m o l t to the a d u l t s t a g e . T e n e r a l a d u l t s were weighed w i t h i n 12 hours o f the a d u l t molt. 59 TABLE X: I n f l u e n c e o f s e q u e s t e r e d c a r d e n o l i d e s on l a r v a l development o f fj. f a s c i a t u s r e a r e d i n the l a b o r a t o r y a t 21 °C on the seeds o f A s c l e p i a s . 1 2 3 P l a n t S p e c i e s C a r d e n o l i d e Content ' Developmental S u r v i v a l mean ± S.E. D u r a t i o n (days) mean ± S.E. N (%) A. f a s c i c u l a r i s 45.5 + 3.5 39.3 + 0.28 20 100 A. e r o s a 171 + 18 40.5 + 0.27 18 90 A. s p e c i o s a 200 + 25 39.2 + 0.37 19 95 A. c o r d i f o l i a 258 + 51 40.3 + 0.26 16 80 A. e r i o c a r p a 117 + 19 40.5 + 0.33 18 90 A. c a l i f o r n i c a g r e e n e i 301 + 41 40.3 + 0.20 18 90 A. v. v e s t i t a 470 + 92 40.7 + 0.25 19 95 A. c u r a s s a v i c a 547 + 76 39.8 + 0.25 18 90 yg per a d u l t i n s e c t d a t a f o r both sexes combined, N = 6 (d a t a from T a b l e V I I ) 3 s u r v i v a l i s the p e r c e n t a g e o f l a r v a e t h a t s u c c e s s f u l l y completed t he a d u l t m o l t See m a t e r i a l s and Methods ( s e c t i o n D) f o r c o l l e c t i o n d a t a f o r milkweed seeds. 60 o f A s c l e p i a s a t 25°C ( T a b l e X I I I ) . However, t h e r e were s i g n i f i c a n t d i f f e r e n c e s between the sexes when r e a r e d on the same d i e t , the females b e i n g : s i g n i f i c a n t l y h e a v i e r than t h e i r male c o u n t e r p a r t s on a l l the d i e t s t e s t e d . The r a t i o o f mean female wet wei g h t to mean male wet weig h t f o r CJ. f a s c i a t u s i n t h i s s t u d y f e l l w i t h i n t he range o f 1..2:1 t o 1.3:1. On c o p e l t u s f a s c i a t u s r e a r e d on s u n f l o w e r seeds were s i g -n i f i c a n t l y l i g h t e r than t h o s e r e a r e d on the d i f f e r e n t milkweed seeds ( F i g u r e 12). The female to male we i g h t r a t i o was s i m i l a r t o t h a t o f CJ_. f a s c i a t u s r e a r e d on the milkweed seed d i e t s . L i k e CJ. f a s c i a t u s , t h e r e were no s i g n i f i c a n t d i f f e r e n c e s i n t e n e r a l wet wei g h t o f L^ . k_. k a l m i i r e a r e d on the d i f f e r e n t s p e c i e s o f A s c l e p i a s ( T a b l e I X ) . D i f f e r e n c e s between the sexes were not i n v e s t i g a t e d . In c o n t r a s t t o 0. f a s c i a t u s , t e n e r a l a d u l t s o f l_. k_. k a l m i i r e a r e d on s u n f l o w e r seeds a r e no l i g h t e r than c o n s p e c i f i c s r e a r e d on milkweed seeds ( F i g u r e 12). T h i s f i n d i n g , t o g e t h e r w i t h t h e r e l a t i v e l y r a p i d l a r v a l development o f L. k_. k a l m i i on s u n f l o w e r s e e d s , s t r o n g l y i n d i c a t e t h a t t h i s i n s e c t i s c a p a b l e o f b e t t e r growth and development on t h i s n o n - a s c l e p a i d a c e o u s h o s t than i s CJ. f a s c i a t u s . However, CJ. f a s c i a t u s d e v e l o p s f a s t e r and a t t a i n s g r e a t e r body weigh t s a t a l l l a r v a l i n s t a r s compared to I. Jc. k a l m i i when both a r e r e a r e d on the seeds o f /\. f a s c i c u l a r i s under the same c o n d i t i o n s ( F i g u r e 13). When CJ. f a s c i a t u s was r e a r e d on e i g h t d i f f e r e n t s p e c i e s o f A s c l e p i a s a t 21°C, t h e r e were some s i g n i f i c a n t d i f f e r e n c e s between t e n e r a l wet weights w i t h i n the sexes ( T a b l e X I ) . However, a l l FIGURE 13: Weight g a i n i n CL f a s c i a t u s and L_. k_. k a l m i i d u r i n g l a r v a l development, r e a r e d i n the l a b o r a t o r y a t 25°C on the seeds o f A. f a s c i c u l a r i s . A. f a s c i c u l a r i s seed c o l l e c t e d i n Napa c o u n t y i n September, 1975. 62 FIGURE 13 • O. fasciatus o L. k. kalmii t e n e r a l a d u l t 0 10 20 30 AGE (days) FIGURE 14: R e l a t i o n s h i p between g r o s s c a r d e n o l i d e c o n t e n t and d u r a t i o n o f l a r v a l development o f fj. f a s c i a t u s r e a r e d i n the l a b o r a t o r y a t 21°C on the seeds o f d i f f e r e n t s p e c i e s o f A s c l e p i a s . Data from T a b l e X; m = s l o p e . LARVAL DURATION (days) \i9 TABLE'.XI: A d u l t body weight o f 0. f a s c i a t u s r e a r e d i n the l a b o r a t o r y a t 21 °C on the seeds o f d i f f e r e n t s p e c i e s o f A s c l e p i a s . P l a n t S p e c i e s T e n e r a l Wet Weight (mg) A d u l t Dry Weight, (mg), mean ± S.E. n mean ± S.E. n mean ± S.E. n mean ±JS.E n o* ? a* ? A. f a s c i c u l a r i s 58.6 + 1 .5 1 14 68.2 + 3.6 6 1 .16 24. 8 + 0.8 — - i 14 30. 8 + 2.42 6 A. e r o s a 59.9 + 1 .7 6 68.3 + 2.42 12 1 .14 26. 8 + 1.3 =p - 6 33. 2 + 1.5 — 1 1 2 A. s p e c i o s a 57.9 + 1 .4 11 68.6 + 5.0 8 1 .18 26. 1 + 1.1 =-i 11 31. 6 + 2.42 8 A. c o r d i f o l i a 60.7 + 1 .4 9 72.3 + 3.3 7 1 .19 25. 8 + 1.3-T 9 39. 0 + 2.1 - 7 A. e i r o c a r p a 60.2 + 1 .8 - 13 76.7 + 1.6 " 4 1 .27 21. 6 + 0.8 J-l 13 34. 6 + 1.3 -| 4 A. c a l i form'ca g r e e n e i 62.1 + 1 , 5 1 7 75.0 + 1.2 - 11 1 .21 25. 1 + 1.6 "J 7 35. 9 + 0.7 -| 11 A. v. v e s t i t a 57.3 + 1 .2 10 73.3 + 1.7, 9 1 .28 24. 1 + 1.1 1 10 35. 3 + 2.1 , 9 A. c u r a s s a v i c a 53.2 + 1 .2- • 10 64.5 + 2.0-'- 8 1 .21 19. 7 + 0.7 'J 10 26. 2 + 0.7 I •1 8 b r a c k e t ends j o i n s i g n i f i c a n t l y d i f f e r e n t means 2 no s i g n i f i c a n t d i f f e r e n c e between sexes T e n e r a l wet weight determined w i t h i n 12 hours o f the a d u l t m o l t . A d u l t d r y weig h t determined between 10 and 14 days f o l l o w i n g the a d u l t m o l t . CTl 66 d i f f e r e n c e s were owing to the l i g h t w e i g h t s o f both males and females r e a r e d on A_. c u r a s s a v i c a . As i n the 25°C r e a r i n g e x p e r i m e n t , f e m a l e s were s i g n i f i c a n t l y h e a v i e r than males on a l l d i e t s t e s t e d . A d u l t d r y w e i g h t s y i e l d e d the same r e l a t i o h s h i p as the t e n e r a l wet w e i g h t s . There were no s i g n i f i c a n t c o r r e l a t i o n s between t e n e r a l wet w e i g h t o r a d u l t d r y w e i g h t and c a r d e n o l i d e c o n t e n t f o r e i t h e r sex ( T a b l e XIV). R a t i o s o f mean female wet w e i g h t to mean male wet w e i g h t were s i m i l a r to t h o s e i n the 25°C r e a r i n g e x p e r i m e n t , f a l l i n g w i t h i n a range from 1.14 to 1.28 ( T a b l e X I ) . Measurement o f a d u l t p r o n o t a l w i d t h showed no s i g n i f i c a n t d i f f e r e n c e s f o r i n s e c t s o f the same sex r e a r e d on the d i f f e r e n t d i e t s ( T a b l e X I I ) . Thus, t h e r e were no c o r r e l a t i o n s between p r o n o t a l w i d t h and c a r d e n o l i d e c o n t e n t o f the a d u l t i n s e c t s ( T a b l e X I V ) . Females were s i g n i f i c a n t l y l a r g e r than males on a l l d i e t s . In most group r e a r i n g s , females were s i g n i f i c a n t l y h e a v i e r , l a r g e r , and c o n t a i n e d more c a r d e n o l i d e per i n s e c t than the males ( T a b l e s XI, X I I , V I I , F i g u r e s 15, 11). The d i f f e r e n c e s between the sexes i n wet w e i g h t o f t e n e r a l s does not appear to be r e l a t e d to sex d i f f e r e n c e s i n c a r d e n o l i d e c o n t e n t o f the a d u l t i n s e c t s . 0_. f a s c i a t u s r e a r e d on s u n f l o w e r seeds e x h i b i t a female t o male w e i g h t r a t i o equal t o t h a t o f m i l k w e e d - r e a r e d c o n s p e c i f i c s which v a r i e d w i t h r e s p e c t t o c a r d e n o l i d e c o n t e n t ( T a b l e X I I I ) . As t h e r e were no s i g n i f i c a n t c o r r e l a t i o n s between c a r d e n o l i d e c o n t e n t and body w e i g h t , body s i z e o r l a r v a l d e v e l o p m e n t a l i d u r a t i o n ( T a b l e XIV), i t appears t h a t t h e r e are no measurable d e t r i m e n t a l a f f e c t s FIGURE 15: R e l a t i o n s h i p between g r o s s c a r d e n o l i d e c o n t e n t and a d u l t body w e i g h t o f fj. f a s c i a t u s r e a r e d i n t h e l a b o r a t o r y a t 21°C on the seeds o f d i f f e r e n t s p e c i e s o f A s c l e p i a s . WET = t e n e r a l wet w e i g h t ( w i t h i n 12 hours o f the a d u l t m o l t ) DRY = a d u l t d r y w e i g h t (10 t o 14 days f o l l o w i n g the adul t m o l t ) . Data from T a b l e XI; m = s l o p e . 68 80 H 60 H E FIGURE 15 WET <^ • female DRY <^ o male • female a male 1 m= -.003 m=-.016 I 40 O 20 4 t m=-.003 m=-.010 0 i r 0 200 400 i r 600 pg CARDENOLIDE PER INSECT TABLE X I I : P r o n o t a l w i d t h o f a d u l t CJ. f a s c i a t u s r e a r e d i n the l a b o r a t o r y a t 21°C on the seeds o f d i f f e r e n t s p e c i e s o f A s c l e p i a s . Pronotum Width (mm) P l a n t s p e c i e s o" mean + S.E. 1 $ Mean + S.E A. f a s c i c u l a r i s 3.7 + .06 4.1 + .04 A. e r o s a 3.6 + .04 4.1 + .04 A. s p e c i o s a 3.6 + .06 3.9 + .08 A. c o r d i f o l i a 3.6 + .03 4.0 + .06 A. e r i o c a r p a 3.6 + .06 4.1 + .05 A. c a l i f o r n i c a 3.7 + .06 4.1 + .04 A. v e s t i t a 3.6 + .06 4.0 + .09 A. c u r a s s a v i c a 3.6 + .04 4.0 + .05 N = 5 f o r a l l samples e x c e p t A. e r i o c a r p a - r e a r e d females (N = 4 ) . TABLE XI I I : Comparison of female to male rat ios for teneral wet weight and cardenolide content of 0. fasciatus reared on d i f ferent seed d ie ts . Plant Species Mean Teneral Wet Weight^ y ; Mean Cardenolide Content $ : o* o* $ ? : c* a $ A. c o r d i f o l i a 60.7 72.3 1 1.19 152 363 2.39 A. curassavica 53.2 64.5 1 1.21 386 707 1.83 Helianthus annus 45.9 55.6 2 1.21 NIL 4 NIC ^data from Table XI 2 reared at 25°C; data from Table VIII 3 yg per insect ; data from Table VII 4 NIL = no cardenolides detected 71 TABLE XIV: C o r r e l a t i o n s between mean c a r d e n o l i d e c o n t e n t and means o f body we i g h t , body s i z e and l a r v a l d u r a t i o n i n 0. f a s c i a t u s . A. C a r d e n o l i d e c o n t e n t o f i n s e c t s vs. c a r d e n o l i d e c o n t e n t i n d i e t +0.789 p < .001 B. L a r v a l d u r a t i o n vs. c a r d e n o l i d e c o n t e n t ( i n s e c t s ) +0.264 p > .50 C. T e n e r a l wet w e i g h t vs. c a r d e n o l i d e c o n t e n t : males -0.647 .10 > p > .05 females -0.163 p > .50 D. A d u l t d r y wei g h t vs. c a r d e n o l i d e c o n t e n t males -0.482 .50 > p > .20 females -0.172 p > .50 E. A d u l t pronotum w i d t h v s . c a r d e n o l i d e c o n t e n t males -0.288 .50 > p > .'20 females -0.405 .50 > p > .20 r = c o r r e l a t i o n c o e f f i c i e n t o f c a r d e n o l i d e s e q u e s t r a t i o n on the l a r v a l growth and development o f 0. f a s c i a t u s . IV DISCUSSION The a b i l i t y o f the l y g a e i d bugs Q_. f a s i c a t u s and L_. k a l m i i to s e q u e s t e r and s t o r e c a r d e n o l i d e s ( c a r d i a c g l y c o s i d e s ) from t h e i r a s c l e p i a d a c e o u s h o s t s has been w e l l documented ( D u f f e y and ..Scudder 1972, 1974); t h i s a b i l i t y i s c h a r a c t e r i s t i c o f many b r i g h t l y c o l o u r e d members o f the L y g a e i n a e (Scudder and D u f f e y 1972, von Euw e t al_. 1971, Abushama and Ahmed 1976). F u r t h e r , many s p e c i e s o f A s c l e p i a s , and o t h e r n a t u r a l h o s t s o f North American L y g a e i n e s have been shown to c o n t a i n c a r d e n o l i d e s (Roeske e t al_. 1976, D u f f e y and Scudder 1972). T o g e t h e r w i t h the w e l l known p h a r m a c o l o g i c a l e f f e c t s o f c a r d i a c g l y c o s i d e s (Moe and Farah 1970, Hoch 1961, Thorp and Cobbin 1967), the h y p o t h e s i s o f a p a l a t a b i l i t y spectrum based on p l a n t - d e r i v e d c a r d e n o l i d e s i n t h e s e i n s e c t s appears t o be w e l l - f o u n d e d . T h i s t h e s i s has demonstrated t h a t a broad spectrum e x i s t s i n the c a r d e n o l i d e c o n t e n t s o f 0. f a c i a t u s and L_. k a l m i i k a l m i i f e e d i n g on A s c l e p i a s i n c e n t r a l C a l i f o r n i a . In terms o f both v a l u e s and v a r i a b i l i t y , the d a t a p r e s e n t e d here f o r C a l i f o r n i a n s p e c i e s o f A s c l e p i a s a r e l a r g e l y i n agreement w i t h t h o s e o f Roeske e t aj_. (1976). As such, the comparisons between c a r d e n o l i d e c o n t e n t o f p l a n t s and i n s e c t s i n t h i s t h e s i s , and between i n s e c t s i n t h i s t h e s i s and o t h e r r e p o r t s ( e . g . Brower e_t aX. 1975, Roeske e t a]_. 1976) a r e c o n s i d e r e d t o be v a l i d . . I t s h o u l d be r e a l i z e d t h a t i n c o l l e c t i n g a d u l t i n s e c t s i n the f i e l d t he h o s t p l a n t e x p e r i e n c e o f t h e s e i n s e c t s i n t h e i r l a r v a l s t a g e s remains u n c e r t a i n . However, i n t h i s s t u d y the p r e s e n c e o f s o f t - b o d i e d 74 a d u l t s t o g e t h e r w i t h l a t e i n s t a r l a r v a e f e e d i n g on the same seed pod has been taken t o i n d i c a t e t h a t the a d u l t s c o l l e c t e d from such a s i t e f o r a n a l y s i s r e p r e s e n t i n s e c t s which d e v e l o p e d e x c l u s i v e l y on t h a t f o o d s o u r c e . The q u a n t i t i e s o f c a r d e n o l i d e s i n f i e l d - c o l l e c t e d l y g a e i d s were o f t e n much s m a l l e r than t h o s e p r e d i c t e d by e x t r a p o l a t i n g from the r e l a t i o n -s h i p between the q u a n t i t y i n the d i e t and the q u a n t i t y s e q u e s t e r e d i n l a b o r a t o r y - r e a r e d bugs. Thus, t h e r e appear to be e c o l o g i c a l parameters i n t h e l y g a e i d - m i l k w e e d a s s o c i a t i o n which l i m i t , o r i n t e r f e r e w i t h the i n s e c t s ' o p p o r t u n i t y t o s e q u e s t e r optimum q u a n t i t i e s o f c a r d e n o l i d e s from t h e i r p o t e n t i a l h o s t s . The g r o u n d - d w e l l i n g l y g a e i d L_. k_. k a l m i i o v e r w i n t e r s i n and near milkweed s t a n d s , f e e d i n g on seed on the ground, as w e l l as the seeds o f some n o n - a s c l e p i a d a c e o u s p l a n t s , and dead i n s e c t s (R.B. Root, p e r s . comm.). Among the w i n t e r h o s t s i s the c a r d e n o l i d e - c o n t a i n i n g Merium  o l e a n d e r , a u b i q u i t o u s c u l t i v a t e d p l a n t i n c e n t r a l C a l i f o r n i a . In the e a r l y s p r i n g , b e f o r e milkweeds have begun t o s p r o u t , l ^ . k. k a l m i i r e p r o d u c e s on the seeds o f o t h e r p l a n t s , such as the common gr o u n d s e l S e n e c i o v u l g a r i s , which l a c k s c a r d e n o l i d e s (K.E. Evans, p e r s . comm.). Al t h o u g h t h i s l y g a e i d can s u c c e s s f u l l y d e v e l o p and r e p r o d u c e on p l a n t s o t h e r than milkweed, milkweed seeds enhance l a r v a l development (R.B. Root, p e r s . comm.). L_. k_. k a l m i i c o l o n i z e s most o f the s p e c i e s o f A s c l e p i a s i n C a l i f o r n i a , but remains a t low d e n s i t i e s u n t i l the seed pods d e v e l o p l a t e i n the season. Because they have the d i e t a r y f e l x i b i 1 i t y to de v e l o p i n the absence o f milkweed p l a n t s and seed, a p o r t i o n o f the 75 o v e r a l l p o p u l a t i o n o f t h e s e bugs may n o t have the o p p o r t u n i t y to s e q u e s t e r and s t o r e c a r d e n o l i d e s . The f a c t t h a t J^. k_. k a l m i i i s more e f f i c i e n t t h a t (D. f a s c i a t u s a t s e q u e s t e r i n g c a r d e n o l i d e s from p l a n t m a t e r i a l may have p e r m i t t e d t h i s s p e c i e s t o e x p l o i t a wider range o f h o s t s p e c i e s , i n c l u d i n g t h o s e which do not p r o v i d e the i n s e c t w i t h c a r d e n o l i d e s . S i n c e c a r d e n o l i d e s may be r e t a i n e d i n the d o r s o - l a t e r a l spaces o f t h e s e i n s e c t s f o r a p e r i o d o f weeks ( e x p e r i m e n t a l e v i d e n c e u n d e s c r i b e d i n t h i s t h e s i s ) , a s h o r t exposure t o a c a r d e n o l i d e - r i c h d i e t combined w i t h e f f i c i e n t s e q u e s t r a t i o n might a l l o w t h i s i n s e c t to b u i l d a c o n s i d e r a b l e c a r d e n o l i d e s t o r e which i s m a i n t a i n e d w h i l e the i n s e c t e x p l o i t s a l t e r n a t e h o s t s p e c i e s . However, the f i e l d d a t a show t h a t L_. k. k a l m i i c o l l e c t e d i n the l a t e s p r i n g on milkweed p l a n t s o f s e v e r a l s p e c i e s , e i t h e r c o n t a i n e d small q u a n t i t i e s o f c a r d e n o l i d e s , o r l a c k e d t h e s e compounds a l t o g e t h e r . T h i s f i n d i n g s u g g e s t s t h a t t h e s e i n d i v i d u a l s e i t h e r l o s t t h e i r s t o r e o f c a r d e n o l i d e s o b t a i n e d b e f o r e t he w i n t e r , o r a r e the o f f s p r i n g o f o v e r -w i n t e r i n g a d u l t s which d e p o s i t e d eggs on n o n - a s c l e p i a d a c e o u s h o s t s . In c o n t r a s t , the h o s t p l a n t range o f 0. f a s c i a t u s i n temperate North America i s r e s t r i c t e d t o members o f the genus A s c l e p i a s (Ralph 1976). In C a l i f o r n i a t h i s l y g a e i d has been seen on f i v e s p e c i e s o f milkweed (A. f a s c i c u l a r i s , A. e r o s a , A. e r i o c a r p a , A. v e s t i t a v e s t i t a and A^ . c a l i f o r n i c a g r e e n e i ) , and the l a b o r a t o r y - r e a r i n g experiments s u g g e s t t h a t a t l e a s t two o t h e r s p e c i e s (A. s p e c i o s a and A. c o r d i f o l i a ) w i l l s u p p o r t l a r v a l development, 0_. f a s c i a t u s i s known t o c o l o n i z e 76 A. s p e c i o s a i n o t h e r p a r t s o f t h i s i n s e c t ' s g e o g r a p h i c a l range (Andre 1934). CJ. f a s c i a t u s i s a l o n g d i s t a n c e m i g r a n t s p e c i e s , r e c o l o n i z i n g milkweed s t a n d s i n C a l i f o r n i a e v e r y summer from i t s s i t e s o f o v e r w i n t e r i n g i n the south (perhaps M e x i c o ) . The h o s t - p l a n t a s s o c i a t i o n s o f CJ. f a s c i a t u s a t i t s s i t e s o f o v e r w i n t e r i n g a r e unknown. I f the a d u l t s which immigrate i n t o C a l i f o r n i a had d e v e l o p e d on milkweed p l a n t s p r i o r to t h e i r l o n g d i s p e r s a l f l i g h t s , one would e x p e c t to f i n d moderate q u a n t i t i e s o f cardeno-l i d e s i n t h e s e i n s e c t s . However, the f i r s t a r r i v i n g c o l o n i z e r s a t a l l milkweed s t a n d s sampled c o n t a i n e d s m a l l q u a n t i t i e s o f c a r d e n o l i d e s . F u r -t h e r , comparison o f t h i n - l a y e r c h r o m a t o g r a p h i c p r o f i l e s o f c a r d e n o l i d e s between the e a r l y c o l o n i z e r s and l a b o r a t o r y - r e a r e d i n s e c t s s u g g e s t t h a t t h e s e s m a l l q u a n t i t i e s o f c a r d e n o l i d e s were accumulated on the p l a n t s on which the i n s e c t s were c o l l e c t e d , r a t h e r than on some o v e r w i n t e r i n g h o s t : the c a r d e n o l i d e p r o f i l e s o f i n s e c t s were i d e n t i c a l t o p r o f i l e s o f bugs r e a r e d i n the l a b o r a t o r y on the C a l i f o r n i a n s p e c i e s o f A s c l e p i a s . T h i s s u g g e s t s t h a t t h e o v e r w i n t e r i n g h o s t s e i t h e r l a c k c a r d e n o l i d e s , o r have v e r y small q u a n t i t i e s ( c f . A. f a s c i c u l a r i s ) . U n l i k e p o p u l a t i o n s o f CJ. f a s c i a t u s i n e a s t e r n North America, e a r l y c o l o n i z i n g 0_. f a s c i a t u s i n C a l i f o r n i a o f t e n a r r i v e a t milkweed st a n d s b e f o r e d e v e l o p i n g pods and seeds a r e a v a i l a b l e (Ralph 1976, K.E. Evans, unpubl. r e s u l t s , p e r s . o b s e r v . ) . Where t h i s i n s e c t a r r i v e s i n advance o f the f r u i t i n g season o f i t s h o s t , i t i s a b l e t o r e p r o d u c e and l e a v e o f f s p r i n g which must d e v e l o p on p l a n t organs o t h e r than pods. However, i t i s o n l y the seeds o f t h e milkweed p l a n t which s u p p o r t o p t i m a l growth and development o f l a r v a e (Ralph 1976), and p o p u l a t i o n s o f bugs o n l y i n c r e a s e a t a r a p i d r a t e when mature pods and seed a r e 77 p r e s e n t . D e t e r m i n a t i o n o f the c a r d e n o l i d e c o n t e n t o f l a b o r a t o r y seed-r e a r e d fj . f a s c i a t u s and bugs c o l l e c t e d on the same h o s t s i n the f i e l d shows t h a t fj. f a s c i a t u s can u s u a l l y s e q u e s t e r moderate to l a r g e q u a n t i t i e s o f c a r d e n o l i d e s from some o f t h e i r h o s t s p e c i e s d u r i n g the f r u i t i n g season. However, the l a b o r a t o r y s e e d - r e a r e d i n s e c t s o f t e n c o n t a i n more c a r d e n o l i d e than i n s e c t s c o l l e c t e d i n the f i e l d , sug-g e s t i n g t h a t bugs i n the n a t u r a l environment may n o t f e e d e x c l u s i v e l y on seed, even when i t i s a v a i l a b l e . L y g a e i d s o b t a i n water by t a p p i n g the pod w a l l , l e a v e s , and stems o f milkweed p l a n t s and i n g e s t i n g the m i l k y sap. In so d o i n g , they p r o b a b l y r e c e i v e some n u t r i e n t s i n the sap, which may not c o n t a i n the same c o n c e n t r a t i o n s o f c a r d e n o l i d e s as the seeds. A l t e r n a t e l y , the t h i c k pod w a l l o f s e v e r a l s p e c i e s o f A s c l e p i a s i n C a l i f o r n i a ( p a r t i c u l a r l y A. e r o s a , A. v_. v e s t i t a , A. c a l i f o r n i c a g r e e n e i and A_. s p e c i o s a ) may p r e v e n t l y g a e i d s from f e e d i n g on the seeds u n t i l the e a r l i e s t m a t u r i n g pods be g i n t o d e h i s c e (Ralph 1976). F u r t h e r , t he p e r i o d o f time i n which 0_. f a s c i a t u s can f e e d d i r e c t l y on seeds can be s e v e r l y l i m i t e d on milkweed s p e c i e s which have h i g h l y synchronous pod d e h i s e n c e and/or r a p i d seed d i s p e r s a l from d e h i s c i n g pods. A l l o f t h e s e f a c t o r s p r o b a b l y l i m i t t h e a b i l i t y o f both 0_. f a s c i a t u s and L. k_. k a l m i i t o s e q u e s t e r q u a n t i t i e s o f c a r d e n o l i d e s w i t h i n the p h y s i o l o g i c a l l i m i t s o f the i n s e c t s . B e f o r e pods and seed a r e a v a i l a b l e , 0_. f a s c i a t u s f e e d s a l m o s t e x c l u s i v e l y on the d e v e l o p i n g f l o w e r buds and open f l o w e r s (Brown e t a l . 1976, Evans, unpubl. r e s u l t s , p e r s . o b s e r v . ) . T h i s s t u d y has shown t h a t 78 f o r ' a t l e a s t f o u r s p e c i e s o f A s c l e p i a s i n C a l i f o r n i a , the umbels (buds and f l o w e r s ) c o n t a i n r e l a t i v e l y l a r g e q u a n t i t i e s o f c a r d e n o l i d e s , y e t i n most samples, the bugs d i d not s e q u e s t e r p r o p o r t i o n a l q u a n t i t i e s o f c a r d e n o l i d e s from t h e s e s o u r c e s . Ralph (1976) r e p o r t e d t h a t CJ. f a s c i a t u s f e e d i n g on f l o w e r s o f A. s y r i a c a d i d n o t i n g e s t m a t e r i a l from the o v a r i e s , but i n s t e a d f e d p r i m a r i l y on the n e c t a r , which may l a c k c a r d e n o l i d e s e n t i r e l y . Thus, the mode o f f e e d i n g o f CJ.' f a s c i a t u s might make the a v a i l a b i l i t y o f seed o f the h o s t p l a n t c r i t i c a l f o r the s e q u e s t r a t i o n o f c a r d e n o l i d e s . IL. k_. k a l m i i has been shown t o s e q u e s t e r g r e a t e r q u a n t i t i e s o f c a r d e n o l i d e s from t h e umbels o f milkweeds than CJ. f a s c i a t u s . Whether t h i s i s a r e f l e c t i o n o f the h i g h e r s e q u e s t r a t i o n e f f i c i e n c y o f L^ . k_. ka 1 m i i , o r a more e f f i c i e n t mode o f f e e d i n g than 0_. f a s c i a t u s r e q u i r e s more d e t a i l e d i n v e s t i g a t i o n . As p r e v i o u s l y i n d i c a t e d , l ^ . k^ . k a l m i i was seen on a l l but one o f the n i n e s p e c i e s o f A s c l e p i a s examined (A. s o l a n o a n a b e i n g the e x c e p t i o n ) , and was r e p r e s e n t e d by a t l e a s t some i n d i v i d u a l s a t a l m o s t e v e r y milkweed s t a n d v i s i t e d . The l a r g e s t numbers o f bugs were seen a t s t a n d s o f A. f a s c i c u l a r i s , A. c o r d i f o l i a and A. c a l i f o r n i c a  g r e e n e i which bore mature seed pods. On the o t h e r hand, 0. f a s c i a t u s i s a more s e l e c t i v e h e r b i v o r e , i n terms o f the numbers o f h o s t s p e c i e s u t i l i z e d . In the m i l k w e e d - r i c h Frenso a r e a , l a r g e p o p u l a t i o n s o f CJ. f a s c i a t u s o c c u r on f o u r b r o a d - l e a f s p e c i e s (A. y_. v e s i t a , A. e r o s a , A. e r i o c a r p a , and A_. c a l i f o r n i c a g r e e n e i ) as w e l l as the n a r r o w - l e a v e d 79 A. f a s c i c u l a r i s . A l l o f t h e s e h o s t s p e c i e s appeared to be c o l o n i z e d to more o r l e s s the same e x t e n t . In the more n o r t h e r l y c o l l e c t i n g a r e a (Napa v a l l e y and a d j a c e n t Y o l o , Solano and C o t n r a C o s t a c o u n t i e s ) , the o n l y milkweed c o l o n i z e d by 0. f a s c i a t u s i s A. f a s c i c u l a r i s . Large stands o f A. c o r d i f o l i a , A. s p e c i o s a , and A. e r i o c a r p a were no t c o l o n i z e d d u r i n g the summer o f 1976, even though many o f th e s e p l a n t s bore l a r g e numbers o f pods and o f t e n grew w i t h i n a few k i l o m e t e r s o f A. f a s c i c u l a r i s s t a n d s which were i n f e s t e d w i t h fj. f a s c i a t u s . F u r t h e r m o r e , f i e l d o b s e r v a t i o n s i n d i c a t e d t h a t even the s m a l l e s t , most i s o l a t e d milkweed st a n d s i n the F r e n s o a r e a were c o l o n i z e d by some bugs, s u g g e s t i n g t h a t the b r o a d - l e a f s p e c i e s i n the n o r t h bay a r e a were not s i m p l y o v e r l o o k e d by i m m i g r a t i n g Cj. f a s c i a t u s on t h e b a s i s o f t h e i r p o p u l a t i o n s i z e o r h a b i t a t . The f a i l u r e t o f i n d CJ. f a s c i a t u s on A. c o r d i f o l i a a t any time a t l o c a l i t i e s s t u d i e d i s p a r t i c u l a r l y m y s t e r i o u s . In t h e l a r v a l r e a r i n g e x p e r i m e n t s d e s c r i b e d i n t h i s t h e s i s , seeds o f A. c o r d i f o l i a s u p p o r t e d l a r v a l growth and development o f CJ. f a s c i a t u s equal to t h a t o f most o t h e r s p e c i e s o f A s c l e p i a s , s u g g e s t i n g t h a t t h e r e i s no n u t r i t i o n a l b a r r i e r p r e v e n t i n g Cj. f a s c i a t u s from e x p l o i t i n g t h i s milkweed s p e c i e s . Perhaps A. c o r d i f o l i a l a c k s some v i s u a l o r o l f a c t o r y cues which Q_. f a s c i a t u s may use i n o r i e n t a t i o n t o i t s h o s t s ( P a n t l e and F e i r 1976). One might even c o n c l u d e t h a t CJ. f a s c i a t u s i n t h i s r e g i o n does not u t i l i z e an optimum f o r a g i n g s t r a t e g y , f o r i t does no t c o l o n i z e h o s t s p e c i e s which a r e c a p a b l e o f s u p p o r t i n g p o p u l a t i o n s o f bugs. On the o t h e r hand, l a b o r a t o r y r e a r i n g o f Cj. f a s c i a t u s a t 25°C i n d i c a t e d t h a t 80 l a r v a l development was more r a p i d on A. f a s c i c u l a r i s compared t o o t h e r milkweed s p e c i e s t e s t e d , which would s u g g e s t a s h o r t e r g e n e r a t i o n time o f 0. f a s c i a t u s f e e d i n g on t h i s h o s t i n the n a t u r a l environment. A. f a s c i c u l a r i s s t ands i n the Napa V a l l e y s u p p o r t l a r g e r e p r o d u c i n g p o p u l a t i o n s o f fj_. f a s c i a t u s (K.E. Evans, unpubl. r e s u l t s ) . The a n a l y s i s o f p l a n t and i n s e c t m a t e r i a l d e s c r i b e d i n t h i s t h e s i s has shown t h a t t h e s e i n s e c t s c o n t a i n r e l a t i v e l y small q u a n t i t i e s o f c a r d e n o l i d e s i n t h e i r b o d i e s , r e f l e c t i v e o f the c a r d e n o l i d e c o n t e n t o f the h o s t p l a n t . T h e r e f o r e , i t i s suggested t h a t CJ. f a s c i a t u s i n t h i s p a r t o f i t s g e o g r a p h i c a l range does not maximize i t s o p p o r t u n i t i e s to s e q u e s t e r l a r g e q u a n t i t i e s o f c a r d e n o l i d e s , f o r i t does n o t c o l o n i z e the s p e c i e s o f A s c l e p i a s which a r e r i c h e r i n t h e s e secondary p l a n t s u b s t a n c e s . 52 per c e n t o f the f i e l d - c o l l e c t e d l y g a e i d s i n t h i s s t u d y were found to c o n t a i n l e s s than 50 yg o f c a r d e n o l i d e per i n s e c t . While t h i s sample o f l y g a e i d s i n C a l i f o r n i a i s not a random one, i t i s a r e p r e s e n -t a t i v e one, based on the o c c u r r e n c e o f 0_. f a s c i a t u s and L_k_. k a l m i i on the d i f f e r e n t milkweed s p e c i e s o b s e r v e d : a l a r g e p r o p o r t i o n o f t h i s 52 per c e n t was 0_. f a s c i a t u s from A. f a s c i c u l a r i s . T h i s p l a n t i s the most wide s p r e a d s p e c i e s o f milkweed i n C a l i f o r n i a , and a l t h o u g h i t p r o b a b l y does not c o n s t i t u t e the l a r g e s t s p e c i e s i n the s t a t e i n terms o f biomass, i t c e r t a i n l y o c c u r s i n h i g h d e n s i t i e s i n many d i f f e r e n t h a b i t a t s and p l a n t communities. I t has been su g g e s t e d t h a t the abundance and d i s t r i b u t i o n o f A_. f a s c i c u l a r i s c o u l d a c c o u n t i n p a r t f o r the h i g h p r o p o r t i o n (47 per c e n t ) o f monarch b u t t e r f l i e s c o l l e c t e d i n C a l i f o r n i a 81 w i t h o n l y small to immeasurable q u a n t i t i e s o f c a r d e n o l i d e s i n a s t u d y o f w i l d monarchs by Brower and M o f f i t t (1974). The p r e s e n t work has shown t h a t the l y g a e i d bugs 0. f a s c i a t u s and L_. k_. k a l m i i i n c e n t r a l C a l i f o r n i a c o n t a i n v a r i e d q u a n t i t i e s o f c a r d e n o l i d e s . The r o l e o f c a r d e n o l i d e s i n t h e s e m i l k w e e d - f e e d i n g l y g a e i d s remains l a r g e l y i n q u e s t i o n . I t must be emphasized t h a t o n l y when the n a t u r a l p r e d a t o r s o f t h e s e i n s e c t s a r e known w i l l we be a b l e t o t e s t the e f f e c t i v e n e s s o f c a r d e n o l i d e s as a p o t e n t i a l chemical d e f e n c e agent i n t h e s e i n s e c t s . No i n c i d e n t s o f p r e d a t i o n o f l y g a e i d s were seen d u r i n g the c o u r s e o f f i e l d work d u r i n g t h i s s t u d y . Sauer and F e i r (1972) s t u d i e d p r e d a t i o n o f 0_. f a s c i a t u s i n a s t a n d o f A. s y r i a c a i n M i s s o u r i o v e r a p e r i o d o f f i v e months and r e p o r t e d t h a t the o n l y p r e d a t o r s o f 0_. f a s c i a t u s o b s e r v e d were i n s e c t p r e d a t o r s , e.g. brown l a c e w i n g l a r v a e ( H e m e r o b i i d a e ) . In the p r e s e n t s t u d y , s e v e r a l anthropod p r e d a t o r s were ob s e r v e d f r e q u e n t l y on milkweed p l a n t s , i n c l u d i n g mantids (Stagomantis  c a l i f o r m " c a Rehn & Heb.), a s s a s i n bugs ( Z e l u s s p . ) , ambush bugs (Phymata e r o s a L . ) , c r a b s p i d e r s (Misumena sp.) and jumping s p i d e r s ( P h i d i p p u s s p . ) . I n d i v i d u a l s o f the f o r m e r t h r e e were taken i n t o the l a b o r a t o r y and o f f e r e d a d u l t , o r second and t h i r d i n s t a r 0_. f a s c i a t u s , but none o f the p r e d a t o r s c o u l d be i n d u c e d to a t t a c k t h e l y g a e i d s . M antids c o l l e c t e d on A_. e r o s a p l a n t s were a s s a y e d f o r c a r d e n o l i d e s , but were found to l a c k t h e s e compounds, s u g g e s t i n g t h a t t h e i r prey items c o n s i s t e d s o l e l y o f p o l l i n a t o r s o f A s c l e p i a s . P o l l i n a t o r s such as the honey bee, A p i s  m e l l i f e r a L. were f r e q u e n t l y caught by c r a b s p i d e r s h i d i n g i n milkweed umbels. 82 G e l p e r i n (1968) r e p o r t e d t h a t the mantids P a r a t a n o d e r a s i n e n s i s Saussure and Ma n t i s r e l i g i o s a L. would a t t a c k a d u l t CJ. f a s c i a t u s , but s u b s e q u e n t l y r e j e c t t he l y g a e i d as a prey i t e m , o o s s i b l y on the b a s i s o f o l f a c t o r y cues. M antids l e a r n e d to r e j e c t CJ. f a s c i a t u s on s i g h t a l o n e , but i n h i b i t i o n o f the s t r i k e r e s p o n s e was dependant on the n u t r i t i o n a l s t a t e o f the man t i d . No i n c i d e n c e o f p r e d a t i o n o f l y g a e i d s by v e r t e b r a t e s were ob s e r v e d i n t h i s f i e l d s t u d y , o r i n the st u d y o f Sauer and F e i r (1972). However, gut a n a l y s e s o f b i r d s and amphibians (R.T. M i t c h e l l , u n p u b l i s h e d r e s u l t s ) i n d i c a t e t h a t l a r g e numbers o f L_. k a l m i i a r e e a t e n by v e r t e b -r a t e s , i n p a r t i c u l a r t he sage gr o u s e C e n t r o c e r c u s u r o p h a s i a n u s Bona.., the s c a l e d q u a i l C a l l i p e p ! a squamata V i g o r s , and the r o a d r u n n e r Geococcyx c a l i f o r n i a n u s L e s s o n . I t was not known whether any o f th e s e i n s e c t s c o n t a i n e d c a r d e n o l i d e s o r no t . Because i t i s n o t known what l e v e l o f c a r d e n o l i d e c o n t e n t i n the C a l i f o r n i a n l y g a e i d s i s r e q u i r e d to cause an emetic o r d i s t r e s s f u l r e s p o n s e i n a v e r t e b r a t e p r e d a t o r , the f u l l i m p l i c a t i o n o f the c a r d e n o l i d e v a r i a t i o n cannot y e t be e s t a b l i s h e d . Perhaps the b e s t documented b i o a s s a y o f A s c l e p i a s c a r d e n o l i d e s and c a r d e n o l i d e s s t o r e d by an i n s e c t f e e d i n g on A s c l e p i a s i s the b l u e j a y emetic d o s e ^ t e s t o f Brower e t a l . (1968). In examining the emeti c response o f the bl u e j a y ( C y a n o c i t t a  c r i s t a t a ) t o the monarch b u t t e r f l y (D_. p l e x i p p u s ) r e a r e d on the neo-t r o p i c a l milkweed A. c u r a s s a v i c a , Brower e t aJL (1975) r e p o r t e d t h a t one emetic dose (ED^Q) r e q u i r e d between 68 and 84 yg e q u i v a l e n t s o f d i g i t o x i n f o r an 85 g b i r d . A d d i t i o n a l c a l c u l a t i o n s from the data o f Brower and 83 G l a z i e r (1975) i n d i c a t e t h a t f o r the abdomen o f such a b u t t e r f l y , a s i n g l e ED^Q c o n s i s t s o f from 44 to 54-yg o f c a r d e n o l i d e whereas a s i n g l e EDgQ from the c a r d e n o l i d e s s t o r e d i n the wings o f the same i n s e c t r e q u i r e s from 232 to 238 yg. I t has been e s t a b l i s h e d t h a t owing t o p h y s i o l o g i c a l b a r r i e r s i n v e r t e b r a t e s p o l a r c a r d e n o l i d e s tend to have lower e m e t i c p o t e n c i e s than do n o n - p o l a r ones, when a d m i n i s t e r e d v i a the o r a l r o u t e ( s e e D u f f e y 1976 f o r a complete d i s c u s s i o n ) . T h i s r e l a t i o n s h i p i s l a r g e l y because o f the more r a p i d a b s o r p t i o n o f l i p o p h i l i c c a r d e n o l i d e s a c r o s s the g u t , and t h e i r more e f f e c t i v e p r o t e i n b i n d i n g i n the b l o o d s t r e a m (White and G i s v o l d 1952, Parsons and Summers 1971). P r e l i m i n a r y s t u d i e s (Parsons 1965, R e i c h s t e i n e t al_, 1968) have shown t h a t the p r i n c i p l e c a r d e n o l i d e s s t o r e d by the monarch r e a r e d on A. c u r a s s a v i c a a r e the r e l a t i v e l y n o n - p o l a r c h e m i c a l s c a l o t r o p i n and c a l a c t i n ( F i g u r e 1 ) , which c o u l d a c c o u n t f o r the r e l a t i v e l y h i g h e m e t i c p o t e n t i a l o f the whole b u t t e r f l y (68-84 yg per ED^Q) compared to t h a t o f the wings (232-238 yg per E D 5 Q ) . I t i s c o n c e i v a b l e t h a t the lower e m e t i c i t y o f the c a r d e n o l i d e s i n the wings o f the monarch b u t t e r f l i e s may be owing to the p r e s e n c e o f a l a r g e r p r o p o r t i o n o f p o l a r c a r d e n o l i d e s i n t h e s e organs i n comparison to the c a r d e n o l i d e compliment o f the e m e t i c a l l y - p o t e n t abdomen. Thus the monarch may p a r t i t i o n i t s c a r d e n o l i d e s ' i n a f a s h i o n s i m i l a r to 0_. f a s c i a t u s which p r e f e r e n t i a l l y s e q u e s t e r s more p o l a r compounds i n the d o r s o - l a t e r a l s pace f l u i d , w h i l e more l i p o p h i l i c c a r d e n o l i d e s o c c u r i n the f a t body ( D u f f e y , unpubl. r e s u l t s ) . 84 The r e l a t i o n s h i p between emeti c a c t i v i t y and p o l a r i t y o f A s c l e p i a s c a r d e n o l i d e s may be c o n s i d e r a b l y more complex though. Roeske et^ al_. (1976) r e a r e d D_. p l e x i p p u s i n the l a b o r a t o r y on the l e a v e s o f A. c u r a s s a v i c a and the A f r i c a n a s c l e p i a d Gomphocarpus sp., and found the c a r d e n o l i d e s s t o r e d by the b u t t e r f l i e s r e a r e d on A. c u r a s s a v i c a to be 2.6 times more e m e t i c a l l y p o t e n t (per mg o f c a r d e n o l i d e ) than t h o s e s t o r e d by the monarchs r e a r e d on Gomphocarpus. However, the c a r d e n o l i d e p r o f i l e s o f t h e s e i n s e c t s show t h a t t he c a r d e n o l i d e s s t o r e d by the b u t t e r f l i e s from the d i f f e r e n t p l a n t s o c c u r o v e r the same p o l a r i t y range, w i t h r o u g h l y t he same p r o p o r t i o n o f p o l a r to n o n - p o l a r compounds. T h e r e -f o r e , t h e i r r e s u l t s i n d i c a t e t h a t the c a r d e n o l i d e s s e q u e s t e r e d by the b u t t e r f l y from the d i f f e r e n t p l a n t s d i f f e r q u a l i t a t i v e l y i n terms o f t h e i r e m e t i c p o t e n c i e s , i r r e s p e c t i v e o f t h e i r p o l a r i t y . C a l o t r o p i n and i t s s t e r e o i s o m e r c a l a c t i n a c c o u n t f o r almo s t 65 per c e n t o f the c a r d e n o -l i d e s s t o r e d by the b u t t e r f l i e s r e a r e d on A. c u r a s s a v i c a , whereas t h e s e compounds were found to be a b s e n t i n monarchs r e a r e d on Gomphocarpus (Roeske e t al_. 1976). In an e a r l i e r s t u d y , Brower and M o f f i t t (1974) r e p o r t e d a c a u s a l r e l a t i o n s h i p between c a r d e n o l i d e c o n t e n t o f w i l d monarch b u t t e r -f l i e s and t h e i r e m e t i c potency. However, b u t t e r f l i e s c o l l e c t e d i n C a l i f o r n i a were 4.6 to 6.5 times more emeti c o v e r the same range o f c a r d e n o l i d e c o n t e n t as b u t t e r f l i e s taken i n M a s s a c h u s e t t s ; As i n the r e a r i n g s t u d y , the c a r d e n o l i d e p r o f i l e s o f the C a l i f o r n i a n and M a s s a c h u s e t t s monarchs c o v e r e d the same p o l a r i t y r a n g e , w i t h a p p r o x i -m a t e l y equal p r o p o r t i o n s o f p o l a r to n o n - p o l a r compounds i n each group 85 (Roeske e_t a]_. 1976). Again the h i g h e r p o t e n c i e s per m i l l i g r a m o f c a r d e n o l i d e i n t h e C a l i f o r m " a n monarchs r e f l e c t q u a l i t a t i v e d i f f e r e n c e s i n the c a r d e n o l i d e s s t o r e d by the two groups o f b u t t e r f l i e s . C a l o t r o p i n and c a l c a t i n were a b s e n t from the b u t t e r f l i e s c o l l e c t e d i n M a s s a c h u s e t t s , w h i l e some o f the b u t t e r f l i e s i n the C a l i f o r n i a sample c o n t a i n e d a l a r g e p r o p o r t i o n o f t h e s e compounds amongst t h e i r c a r d e n o l i d e s t o r e s . S i m i l a r l y , the A f r i c a n g r a s s h o p p e r P o e k i l o c e r u s b u f o n i u s s t o r e s l a r g e q u a n t i t i e s o f c a l o t r o p i n and c a l a c t i n , when r e a r e d on the milkweeds A_. c u r a s s a v i c a and C a l o t r o p i s p r o c e r a (von Euw e t a]_. 1967); t h i s i n s e c t has been shown t o cause emesis i n w h i t e mice and the European j a y ( G a r r u l u s g l a n d a r i u s L.) ( R o t h s c h i l d 1966). While l a r g e l y c i r c u m s t a n t i a l , t h i s e v i d e n c e a l o n g w i t h the d i f f e r e n t e m e t i c p o t e n c i e s o f the s e p a r a t e body p a r t s o f JJ. p l e x i p p u s as r e p o r t e d by Brower and G l a z i e r (1975), s u g g e s t t h a t the p r e s e n c e o f c a l o t r o p i n and c a l a c t i n may l a r g e l y a c c o u n t f o r the e m e t i c r e s p o n s e seen i n the b l u e j a y . R o t h s c h i l d e t aj_. (1975 and unpubl. r e s . ) r e a r e d JJ. p l e x i p p u s on t h e i r 'Oxford s t r a i n ' o f A. c u r a s s a v i c a , which l a c k s c a l a c t i n e n t i r e l y . T h i s compound was a l s o a b s e n t from the b u t t e r f l i e s , which f a i l e d t o i n d u c e emesis i n pigeons (Columba 1 i v e a Gm.). E x a m i n a t i o n o f whole body e x t r a c t s o f f i e l d c o l l e c t e d 0_. f a s c i a t u s and J_. k_. k a l m i i ( n o t d e s c r i b e d i n t h i s t h e s i s ) shows t h a t a l l o f the c a r d e n o l i d e s s t o r e d i n t h e s e i n s e c t s a r e more p o l a r t h a t c a l o t r o p i n o r c a l a c t i n . Should t h e s e more p o l a r c a r d e n o l i d e s d e r i v e d from the C a l i f o r n i a n s p e c i e s o f A s c l e p i a s by l y g a e i d s be found t o have s i m i l a r p h a r m a c o l o g i c a l p r o p e r t i e s t o the c a r d e n o l i d e compliment 86 i n the wings o f the monarch b u t t e r f l y r e a r e d on A. c u r a s s a v i c a (Brower and G l a z i e r 1975), i t would seem p o s s i b l e t h a t few, i f any o f the i n d i v i d u a l l y g a e i d s c o l l e c t e d i n t h i s s t u d y c o n t a i n e d enough c a r d e n o l i d e o f the r e q u i r e d t y p e t o e l i c i t an e m e t i c r e s p o n s e from a p r e d a t o r w i t h a s e n s i t i v i t y s i m i l a r t o the b l u e j a y . Roughly 50 per c e n t o f the f i e l d - c a u g h t milkweed bugs examined were found to c o n t a i n l e s s than 50 yg o f c a r d e n o l i d e per i n s e c t . However, t h e s e i n s e c t s c o u l d i n c r e a s e t h e i r e m e t i c p o t e n t i a l by way o f t h e i r g r e g a r i o u s b e h a v i o u r ; a p r e d a t o r might i n g e s t s e v e r a l i n d i v i d u a l s a t one f e e d i n g , w i t h the t o t a l c a r d e n o l i d e pool o f s e v e r a l i n s e c t s e l i c i t i n g an e m e t i c r e s p o n s e (Pough e t al_. 1973). On the o t h e r hand, the number o f i n s e c t s needed to c o n d i t i o n the p r e d a t o r to a v o i d the s p e c i e s as a p r e y i t e m might outweigh the advantage o f such g r e g a r i o u s n e s s . Even i f the a d u l t s o f CJ. f a s c i a t u s and L_. k_. k a l m i i w i t h g r e a t e r than 100 yg per i n s e c t a r e found t o have some che m i c a l p r o t e c t i o n from p r e d a t o r s , the d a t a p r e s e n t e d i n t h i s t h e s i s s u g g e s t t h a t the e f f e c t i v e n e s s o f c a r d e n o l i d e s e q u e s t r a t i o n as a c h e m i c a l d e f e n s e s t r a t e g y may v a r y o v e r the g e o g r a p h i c a l range o f t h e s e s p e c i e s , and may v a r y i n the c o u r s e o f a season a t a p a r t i c u l a r l o c a t i o n . A f u r t h e r c o m p l i c a t i o n to u n d e r s t a n d i n g the e f f e c t i v e n e s s o f c a r d e n o l i d e s s t o r e d by l y g a e i d s i s the v a r i a t i o n o f s e n s i t i v i t i e s to c a r d e n o l i d e s among p o t e n t i a l v e r t e b r a t e p r e d a t o r s . The b l u e j a y (C_. c r i s t a t a ) and European j a y (G_. g l a n d a r i u s ) a r e c o n s i d e r e d to be s p e c i e s which a r e s e n s i t i v e to c a r d e n o l i d e s , the European hedgehog ( E r i n a c e u s  europaeus L.) and l a b o r a t o r y r a t a r e n o t , and the Japanese q u a i l 87 ( C o t u r n i x j a p o n i c u s Tern.) can t o l e r a t e q u a n t i t i e s o f d i g i t o x i n up t o 50 times g r e a t e r than the human l e t h a l dose ( R o t h s c h i l d and K e l l e t t 1972). D u f f e y (1970) found t h a t fj_. f a s c i a t u s r e a r e d i n the l a b o r a t o r y on seeds o f A. s y r i a c a ( t h e i n s e c t s c o n t a i n e d a mean o f 284 yg o f c a r d e n o l i d e per i n s e c t ) were c o m p l e t e l y p a l a t a b l e to both f r o g s (Rana p i p i e n s S c h r e b e r ) and toads (Bufo boreas B a i r d & G i r a r d ) . Thus i n c o n s i d e r i n g the p o s i t i o n o f m i l k w e e d - f e e d i n g l y g a e i d s i n m i m i c r y complexes one can o n l y r e p e a t the s t a t e m e n t o f Rettenmeyer (1970), who wrote: "... i n s t e a d o f r e f e r r i n g to an i n s e c t as a B a t e s i a n mimic, i t would be more n e a r l y a c c u r a t e to s t a t e t h a t under the s p e c i f i e d c o n d i t i o n s the i n s e c t , o r some p a r t o f i t s p o p u l a t i o n i n a c e r t a i n l o c a l i t y has a B a t e s i a n r e l a t i o n s h i p t o a s p e c i f i c p r e d a t o r . " I t may n o t be v a l i d to a s s e s s p l a n t - d e r i v e d c a r d e n o l i d e s as a d e f e n s i v e s t r a t e g y o f l y g a e i d s s t r i c t l y on t h e b a s i s o f t h e i r e m e t i c p o t e n t i a l to a v e r t e b r a t e . I t i s e n t i r e l y p o s s i b l e t h a t p r e d a t o r s c o u l d become c o n d i t i o n e d t o a v o i d v b r i g h t l y c o l o u r e d l y g a e i d s and o t h e r milkweed-f e e d i n g i n s e c t s on the b a s i s o f g a s t r i c d i s t r e s s ( i . e . nausea) o r d e l a y e d t o x i c o s i s , n e i t h e r o f which need be o u t w a r d l y v i s i b l e to an e x p e r i m e n t o r ( c f . e m e s i s ) . C a r d i a c g l y c o s i d e s a r e c a p a b l e o f c a u s i n g d i r e c t i r r i t a t i o n o f g a s t r i c t i s s u e ( G o l d e t aj_. 1950), and have been shown t o cause s t r o n g c o n t r a c t i o n o f v e r t e b r a t e smooth muscle i n i s o l a t e d p r e p a r a t i o n s ( H a u s t e i n ejt al_. 1966, G o d f r a i n d and G o d f r a i n d - d e Becker 1961, S h i g e i ejt a l . 1963). Rowe e t al_. (1970) s t u d i e d the t o x i c i t y o f the milkweed A. b r a c h y s t e p h a n a Englem i n sheep, and r e p o r t e d t h a t symptoms o f s u b - l e t h a l doses o f c a r d e n o l i d e - c o n t a i n i n g p l a n t m a t e r i a l (from which the animal 88 f u l l y r e c o v e r e d ) , i n c l u d e d d i a r r h e a and l a b o u r e d b r e a t h i n g . While d i f f i c u l t t o q u a n t i f y , e f f e c t s o f t h i s n a t u r e might p o s s i b l y l e a d t o the c o n d i t i o n e d a v e r s i o n o f p r e d a t o r t o c a r d e n o l i d e -c o n t a i n i n g i n s e c t s . C z a p l i c k i e_t aj_. (1975) demonstrated the t h e o r e t i c a l v i a b i l i t y o f the c o n c e p t o f o l f a c t o r y mimicry. In an a r t i f i c i a l s i t u a t i o n , g a r t e r snakes (Thamnophis sp.) were c o n d i t i o n e d to r e j e c t e d i b l e p r e y items t h a t bore an o l f a c t o r y resemblance to worms a f t e r h a v i n g e x p e r i e n c e d a d e l a y e d t o x i c o s i s f o l l o w i n g t h e i r i n g e s t i o n o f the worms. L a b o r a t o r y r a t s and q u a i l can a l s o l e a r n to a s s o c i a t e g u s t a t o r y and v i s u a l cues w i t h d e l a y e d t o x i c o s i s , l e a d i n g to t h e i r subsequent r e j e c t i o n o f f o o d and water which c o n t a i n s t h e s e cues (Wilcoxon e t a l . 1974). To d a t e , the o n l y p h a r m a c o l o g i c a l i n v e s t i g a t i o n s u s i n g c a r d e n o l i d e s from A s c l e p i a s - f e e d i n g i n s e c t s a r e t h o s e o f Brower and co-workers u s i n g the b l u e j a y e m e t i c dose^Q b i o a s s a y (Brower ejt al_. 1968). A p i l o t s t u d y o f t h e t o x i c i t y o f ' l y g a e i n i n ' ( t e n t a t i v e name f o r a compound i s o l a t e d by the a u t h o r from both 0_. f a s c i a t u s and L^ . k_. k a l m i i , s e q u e s t e r e d from s e v e r a l C a l i f o r n i a n s p e c i e s o f A s c l e p i a s ) u s i n g the embryonic c h i c k h e a r t b i o a s s a y o f Lehman and P a f f (1942), i n d i c a t e d t h a t t h i s compound was as p o t e n t as c o m m e r c i a l l y o b t a i n e d d i g i t o x i n (under-c r i b e d e x p e r i m e n t ) . However, the i n t r a v e n o u s e m e t i c p o t e n c y o f c a l a c t i n i n the c a t i s t h r e e times g r e a t e r than t h a t o f d i g i t o x i n ( P a r s ons and Summers 1971). C l e a r l y , a l l o f the p h a r m a c o l o g i c a l e f f e c t s o f A s c l e p i a s c a r d e n o l i d e s and t h o s e o b t a i n e d by i n s e c t s from milkweed p l a n t s a r e i n need o f i n v e s t i g a t i o n b e f o r e the e c o l o g i c a l r o l e o f the 89 compounds i n the i n s e c t s w i l l be u n d e r s t o o d . One p h a r m a c o l o g i c a l e f f e c t o f c a r d e n o l i d e s which may have been g r e a t l y u n d e r e s t i m a t e d i s t h e i r b i t t e r t a s t e . Most c a r d e n o l i d e s (Brower and G l a z i e r 1975 c i t e T. R e i c h s t e i n ) , l i k e o t h e r p l a n t g l y c o s i d e s ( K i n g s b u r y 1964) a r e b i t t e r t o the human t a s t e . Some b i r d s have been shown t o have w e l l d e v e l o p e d t a s t e r e c e p t o r s ( K i t c h e l l e t a]_. 1959, Duncan 1960, Yang & Kare 1968, Wenzel 1973) and have shown an a v e r s i o n to b i t t e r compounds (Wenzel 1973); such a r e s p o n s e has been used to i n v e s t i g a t e l e a r n i n g a b i l i t y i n some s p e c i e s ( e . g . A l c o c k 1971; see Brower and G l a z i e r f o r s e v e r a l o t h e r r e f e r e n c e s ) . Brower and G l a z i e r (1975) have r i g h t l y adopted t h i s argument t o s u g g e s t t h a t some b i r d s c o u l d use the b i t t e r t a s t e o f c a r d e n o l i d e s to d i s c r i m i n a t e between emetic and non-emetic monarch b u t t e r f l i e s . T h e i r f i n d i n g s t h a t the wings o f the monarch c o n t a i n the l a r g e s t c o n c e n t r a t i o n s o r c a r d e n o l i d e s , but the l e a s t e m e t i c among t h o s e i n t h e body, s u p p o r t the c o n c e p t o f an a n t i - p r e d a t o r s t r a t e g y based on the t a s t e o f c a r d e n o l i d e s . S i m i l a r l y , the h i g h e s t c o n c e n t r a t i o n s o f c a r d e n o l i d e s i n the l y g a e i d bugs fJ. f a s c i a t u s and J_. k_. k a l m i i a r e found i n the f l u i d o f the d o r s o - l a t e r a l spaces ( D u f f e y and Scudder 1974): when r o u g h l y h a n d l e d , the f l u i d from t h e s e spaces i s r e l e a s e d as d i s c r e t e d r o p l e t s onto the c u t i c l e from the d o r s o - l a t e r a l edge o f the metathorax and abdominal segments (Scudder and D u f f e y 1972, Graham and Staddon 1974). The advantage o f h a v i n g the h i g h e s t c o n c e n t r a t i o n s o f b i t t e r -t a s t i n g c a r d e n o l i d e s i n both the wings o f the monarch and the s e c r e t e d space f l u i d o f the l y g a e i d i s t h a t the p r e d a t o r can sample the i n s e c t w i t h o u t the f o r m e r s a c r i f i c i n g i t s l i f e . 90 Other v e r t e b r a t e s may be h i g h l y s e n s i t i v e to the b i t t e r t a s t e o f c a r d i a c g l y c o s i d e s . In the v e t e r i n a r y l i t e r a t u r e , most a u t h o r s s u g g e s t t h a t p l a n t m a t e r i a l o f A s c l e p i a s may l o s e i t s d i s a g r e e a b l e f l a v o r when d r y , w h i l e r e t a i n i n g i t s t o x i c i t y ( H u l b e r t and Oehme 1965, V a i l 1942). The r e a s o n f o r t h i s o b s e r v a t i o n c o u l d be t h a t the a v a i l a b i l i t y o f the b i t t e r c a r d e n o l i d e s to the t a s t e r e c e p t o r s i s l e s s e n e d when i n the d r y s t a t e . I t i s c o n c e i v a b l e t h a t c a r d e n o l i d e s i n l y g a e i d bugs, s e c r e t e d i n h i g h l y c o n c e n t r a t e d d r o p l e t s o n t o t he c u t i c l e , c o u l d g i v e r i s e t o an e x t r e m e l y r a p i d r e s p o n s e by a p r e d a t o r t a k i n g the i n s e c t i n t o i t s mouth. A l t h o u g h no q u a n t i t a t i v e s t u d i e s have been u n d e r t a k e n , s m a l l q u a n t i t i e s o f c a r d e n o l i d e s , such as t h o s e found i n many o f the C a l i f o r n i a n l y g a e i d s , may be enough t o e l i c i t an e f f e c t i v e r e s p o n s e from a v e r t e b r a t e p r e d a t o r , even though the q u a n t i t y and q u a l i t y o f t h o s e c a r d e n o l i d e s i s f a r below t h a t n e c e s s a r y to cause emesis. I f p l a n t d e r i v e d c a r d e n o l i d e s a r e not b e i n g employed by l y g a e i d bugs an an : a n t i - p r e d a t o r , s t r a t e g y , then:.what. s t r a t e g i e s might t h e s e w a r n i n g l y - c o l o u r e d i n s e c t s r e l y upon to a v o i d p r e d a t i o n ? L i k e most Hemiptera, 0_. f a s c i a t u s and _L. k_. k a l m i i p o s s e s s m e t a - t h o r a c i c s c e n t g l a n d s i n the a d u l t s t a g e (Games and Staddon 1973). These g l a n d s , which e x c r e t e v o l a t i l e a l k y l compounds (e.g. h e x a n o l s ) c o u l d p r o v i d e the b a s i s f o r u n p a l a t a b i 1 i t y i n c e r t a i n r e s p o n s i v e v e r t e b r a t e p r e d a t o r s . D u f f e y (1970) found t h a t among s e v e r a l grades o f v e r t e b r a t e p r e d a t o r s , none responded t o the c a r d e n o l i d e s o f CL f a s c i a t u s when r e a r e d on seeds o f A. s y r i a c a , whereas some p r e d a t o r s ( t u r t l e s and c h i c k e n s ) a v o i d e d t h i s 91 i n s e c t because o f i t s v o l a t i l e s c e n t g l a n d s e c r e t i o n s . S i m i l a r r e s u l t s have been shown f o r a n o l i s l i z a r d s p r e s e n t e d w i t h s u n f l o w e r - r e a r e d 0_. f a s c i a t u s (Weber 1975). I t i s c o n c e i v a b l e t h e n , t h a t some p r e d a t o r s i n the n a t u r a l environment c o u l d r e j e c t 0_. f a s c i a t u s and II. _k. k a l m i i because o f t h e s e s e c r e t i o n s , i r r e s p e c t i v e o f the c a r d e n o l i d e c o n t e n t o f the i n s e c t s . Graham and Staddon (1974) have demonstrated the p r e s e n c e o f a h i s t a m i n e analogue i n the d o r s o - l a t e r a l s p a c e , f l u i d , and o t h e r body f l u i d s o f a d u l t and l a r v a e o f fJ. f a s c i a t u s . T h i s compound was shown to produce s t r o n g c o n t r a c t i o n s i n v e r t e b r a t e smooth muscle, and as no such a c t i v i t y was found i n the s u n f l o w e r seeds on which the i n s e c t s were r e a r e d ( e x c l u d i n g the p o s s i b i l i t y o f c a r d e n o l i d e s p r o d u c i n g the e f f e c t s ) , the a u t h o r s s u g g e s t e d t h a t t h i s p r e v i o u s l y u n d e s c r i b e d compound may be s y n t h e s i z e d by the bug (Graham and Staddon 1974). H i s t a m i n e has a l s o been found i n the d e f e n s i v e s e c r e t i o n s o f the g r a s s -hopper 'P_. b u f o n i u s , which l i k e CJ. f a s c i a t u s , s e q u e s t e r s c a r d e n o l i d e s from i t s milkweed h o s t (von Euw e t al_. 1967). Perhaps t h e r e i s a s y n e r g i s t i c e f f e c t o f t h e s e compounds, i . e . c a r d e n o l i d e s p r e s e n t i n the s e c r e t i o n s enhance the c o n t r a c t i l e e f f e c t s o f h i s t a m i n e on v e r t e b r a t e smooth muscle. The p r e s e n c e o f an e f f e c t i v e compound l i k e h i s t a m i n e c o u l d o f f e r one e x p l a n a t i o n as t o why an i n s e c t such as fj. f a s c i a t u s can a f f o r d to be an i n c o n s i s t e n t s t o r e r o f c a r d e n o l i d e s , as shown i n t h i s t h e s i s , w h i l e m a i n t a i n i n g i t s c o n s p i c u o u s c o l o r a t i o n and b e h a v i o u r . Another p o s s i b i l i t y i s t h a t many o f the p o p u l a t i o n s o f 0_. f a s c i a t u s and L. k. k a l m i i , mimic t h e i r c o n s p e c i f i c s which c o n t a i n 92 p h a r m a c o l o g i c a l l y e f f e c t i v e q u a n t i t i e s o f c a r d e n o l i d e s . One problem o f t h i s h y p o t h e s i s though, i s t h a t i t r e q u i r e s s e l e c t i o n p r e s s u r e from a m i g r a t o r y p r e d a t o r , one which c o u l d sample i n s e c t s from many g e o g r a p h i -c a l l y d i s t i n c t p o p u l a t i o n s . O t h e r w i s e , a p r e d a t o r might never come i n t o c o n t a c t w i t h a p o p u l a t i o n o f l y g a e i d s which c o n t a i n s i g n i f i c a n t q u n a t i t i e s o f c a r d e n o l i d e s . W h ile t h i s t y p e o f m i m e t i c system seems a p p r o p r i a t e f o r the m i g r a t i n g monarch b u t t e r f l y (Pough e t al_. 1973, Brower and M o f f i t t 1974), i t s s t a t u s as an a n t i - p r e d a t o r s t r a t e g y i n l y g a e i d s r e q u i r e s a b e t t e r knowledge o f the p o t e n t i a l p r e d a t o r s o f t h e s e bugs. A f u r t h e r p o s s i b i l i t y i s t h a t p r e d a t o r s c a p a b l e o f v i s u a l d i s -c r i m i n a t i o n may g e n e r a l i z e t h e i r a v o i d a n c e t o a l l p r e y items w i t h c o n t r a s -t i n g c o m b i n a t i o n s o f w h i t e , y e l l o w , o r a n g e , r e d and b l a c k . P h y r r h o c o r i d bugs o f the genus Dysdercus G u e r i n M e n e v i l l e s h a r e a body s i z e , shape and c o l o r a t i o n s i m i l a r t o many l y g a e i n e s , and the g e o g r a p h i c a l ranges o f t h e s e two groups l a r g e l y o v e r l a p (van Doesburg 1968). A l t h o u g h Dysdercus s p e c i e s do not s e q u e s t e r c a r d e n o l i d e s ( D u f f e y and Scudder 1972) they do s e c r e t e v o l a t i l e ketones and a l d e h y d e s ( e . g . h e x - 2 - e n - l - a l , Calam and S c o t t , 1969) which may e f f e c t i v e l y d e t e r p r e d a t o r s (Parsons 1940), Sexton (1964) r e p o r -t e d t h a t the l i z a r d A n o l i s c a r o l i n e n s i s V o i g t r e j e c t e d a l l m u l t i - c o l o u r e d prey i t e m s . In a l a b o r a t o r y s i t u a t i o n , the common f e n c e l i z a r d S c e l o p o r u s  o c c i d e n t a l i s B a i r d & G i r a r d a v o i d s a d u l t 0. f a s c i a t u s on s i g h t a l o n e , but w i l l a c c e p t mealworm l a r v a e ( T e n e b r i o m o l i t o r L.) under the same c o n d i t i o n s ( u n d e s c r i b e d e x p e r i m e n t ) . F e i r and Suen (1971) a l s o i n d i c a t e d t h a t the f e n c e l i z a r d s w i l l not a c c e p t 0. f a s c i a t u s as a f o o d i t e m . In f a c t , most i n s e c t h e r b i v o r e s f e e d i n g on milkweed a r e b r i g h t l y c o l o u r e d , 93 s u g g e s t i n g t h a t t h e y p a r t i c i p a t e i n some form o f m i m i c r y complex ( P r i c e 1975). However, r e c e n t e v i d e n c e s t r o n g l y s u g g e s t s t h a t such a m i m i c r y complex c o u l d n o t be based on p l a n t - d e r i v e d c a r d e n o l i d e s , as few mem-bers o f t h e milkweed i n s e c t fauna s t o r e s i g n i f i c a n t q u a n t i t i e s o f t h e s e compounds ( I s m a n e t a l _ . 1977). F i n a l l y , t h r e e o b s e r v a t i o n s s u g g e s t t h a t c a r d e n o l i d e seques-t r a t i o n i s not an i m p o r t a n t a n t i - p r e d a t o r s t r a t e g y among l y g a e i d bugs i n c e n t r a l C a l i f o r n i a . F i r s t l y , i f p r e d a t o r s a r e a b l e t o e x e r t a s e l e c t i o n p r e s s u r e on l y g a e i d s t o s e q u e s t e r l a r g e q u a n t i t i e s o f c a r d e n o -l i d e s , then we would e x p e c t t h i s p r e s s u r e to be o p e r a t i n g most e f f e c -t i v e l y when the d e n s i t y o f i n s e c t s i s low. F o r l y g a e i d s , t h i s p e r i o d would be i n the l a t e s p r i n g and e a r l y summer, when i m m i g r a t i n g a d u l t s f i r s t c o l o n i z e milkweed s t a n d s . However, as the d a t a i n t h i s t h e s i s have shown, l y g a e i d s sampled i n May and June e i t h e r l a c k e d c a r d e n o l i d e s o r c o n t a i n e d s m a l l q u a n t i t i e s o f c a r d e n o l i d e s , o n l y a c q u i r i n g l a r g e r q u a n t i t i e s l a t e r i n the season when the r e p r o d u c t i v e s t r u c t u r e s o f A s c l e p i a s a r e w e l l d e v e l o p e d . S e c o n d l y , p r e d a t o r s do not appear to h i n d e r p o p u l a t i o n growth o f CJ. f a s c i a t u s on s t a n d s o f A. f a s c i c u l a r i s i n the n o r t h bay a r e a , even though t h e s e i n s e c t s have been shown by the p r e s e n t work t o c o n t a i n r e l a t i v e l y s m a l l q u a n t i t i e s o f c a r d e n o l i d e s . Once e s t a b l i s h e d , 0_. f a s c i a t u s p o p u l a t i o n s on A. f a s c i c u l a r i s i n c r e a s e i n number exponen-t i a l l y (K.E. Evans, unpubl. r e s u l t s ) . T h i s f i n d i n g i s c o n s i s t e n t w i t h p o p u l a t i o n growth o f Cj. f a s c i a t u s i n t h e midwestern U n i t e d S t a t e s , where i t most f r e q u e n t l y c o l o n i z e s A. s y r i a c a (Brown e_t aj_. 1976). 94 T h i r d l y , t h e r e does not appear to be a p h y s i o l o g i c a l o r m e t a b o l i c c o s t i n v o l v e d i n the s e q u e s t r a t i o n and s t o r a g e o f c a r d e n o l i d e s by 0_. f a s c i a t u s and L^ . Ik. k a l m i i , (which would be outweighed by the advantages o f p r o t e c t i o n from p r e d a t i o n ) . No measurable d i f f e r e n c e s i n t h e amount o r r a t e o f growth o f l a r v a l and a d u l t CJ. f a s c i a t u s were d e t e c t e d when t h i s i n s e c t was r e a r e d on the seeds o f e i g h t d i f f e r e n t s p e c i e s o f A s c l e p i a s , a l 1 o f which are p o t e n t i a l n a t u r a l h o s t p l a n t s o f t h i s i n s e c t . The f a c t t h a t c a r d e n o l i d e c o n t e n t o f the f o o d does not i n f l u e n c e the amount o r r a t e o f growth o f 0_. f a s c i a t u s i s i n agreement w i t h the d a t a o b t a i n e d by E r i c k s o n (1973). E r i c k s o n (1973) compared the l a r v a l development and growth o f t h e monarch b u t t e r f l y ID. p l e x i p p u s on f o u r d i f f e r e n t s p e c i e s o f A s c l e p i a s on which t h i s i n s e c t i s known t o f e e d , and f a i l e d t o show s i g n i f i c a n t d i f f e r e n c e s . Thus, the d a t a p r e s e n t e d i n t h i s t h e s i s p r o v i d e s t r o n g e v i d e n c e a g a i n s t t h e p o s s i b i l i t y t h a t the s e q u e s t r a t i o n and s t o r a g e o f c a r d e n o l i d e s has an a d v e r s e e f f e c t upon growth and development o f CJ_. f a s c i a t u s , even though 0_. f a s c i a t u s i n t h i s s t u d y s t o r e d up to seven times as much c a r d e n o l i d e by w e i g h t as the monarch b u t t e r f l y . F u r t h e r , the range o f c a r d e n o l i d e c o n t e n t i n the f i e l d c o l l e c t e d bugs f a l l s w e l l w i t h i n t h a t o f the l a b o r a t o r y r e a r e d i n s e c t s . The l a r g e s t q u a n t i t y o f c a r d e n o l i d e s d e t e c t e d i n a f i e l d c o l l e c t e d l y g a e i d was 372-yg, whereas l a b o r a t o r y -r e a r e d CJ. f a s c i a t u s have been known to c o n t a i n as much as 800 yg o f c a r d e n o l i d e . T h e r e f o r e , t h i s f i n d i n g i s c o n s i d e r e d t o be v a l i d f o r ly.gaeids . i - f e e d i n g on A s c l e p i a s i n the n a t u r a l environment. As s u g g e s t e d by Feeny / (1976), the t o x i c compounds o f e a r l y 95 s u c c e s s i o n a l herbs ( e . g . c a r d e n o l i d e s i n A s c l e p i a s ) may a c t as a ' q u a l i t a t i v e ' b a r r i e r to g e n e r a l i s t i n s e c t h e r b i v o r e s w h i l e h a v i n g l i t t l e o r no e f f e c t on the growth and f i t n e s s o f h i g h l y adapted h e r b i v o r e s p e c i e s . The i n a b i l i t y o f the p r e s e n t s t u d y o r the f i n d i n g s o f E r i c k s o n (1973) t o demonstrate a d e t r i m e n t a l e f f e c t o f c a r d e n o l i d e s on the growth and development o f the m i l k w e e d - f e e d i n g i n s e c t s fj. f a s c i a t u s and D_. p l e x i p p u s s u p p o r t t h i s h y p o t h e s i s . I f t h e n , c a r d e n o l i d e s a r e not s t o r e d by l y g a e i d s as an a n t i -p r e d a t o r s t r a t e g y , and t h e s e compounds do not a f f e c t the growth and development o f the i n s e c t s , then how does one e x p l a i n the p r e s e n c e o f t h e s e compounds i n the l y g a e i d s ? One p o s s i b l e e x p l a n a t i o n i s t h a t c a r d e n o l i d e s e q u e s t r a t i o n i s o r was used by t h e s e l y g a e i d s o r c l o s e l y r e l a t e d s p e c i e s i n p r e v i o u s p r e d a t o r - p r e y i n t e r a c t i o n s o v e r e v o l u t i o n a r y t i m e , o r a t the p r e s e n t day, i n o t h e r a r e a s o f the s p e c i e s ' g e o g r a p h i c a l range. Most L y g a e i n e s a r e t r o p i c a l i n s e c t s ; few s p e c i e s have s u c c e s s -f u l l y i n v a d e d temperate r e g i o n s o f the w o r l d . In the t r o p i c a l e n v i r o n -ment, where s e v e r a l s p e c i e s o f b r i g h t l y - c o l o u r e d l y g a e i d s o c c u r sympat-t i c a l l y , t h e s e i n s e c t s may depend on p l a n t - d e r i v e d c a r d e n o l i d e s as a d e f e n s i v e s t r a t e g y , as they c o u l d be s u b j e c t to p r e d a t i o n from a w i d e r v a r i e t y o f p r e d a t o r s than i n a temperate h a b i t a t ( e . g . c e n t r a l C a l i f o r n i a ) . A l t e r n a t e l y fJ. f a s c i a t u s and L. Ik. k a l m i i may have descended from a n c e s t r a l s p e c i e s which b e n e f i t e d from c a r d e n o l i d e s t o r a g e as an a n t i - p r e d a t o r d e v i c e . Such a p h y s i o l o g i c a l system, which does n o t i n t e r f e r e w i t h the normal development o f the i n s e c t , c o u l d c o n c e i v a b l y 96 e x i s t i n p r e s e n t day l y g a e i d s as an a r t i f a c t o f a p r e v i o u s p r e d a t o r - p r e y i n t e r a c t i o n , l o n g a f t e r the p r e d a t o r e v o l v e d d i f f e r e n t f e e d i n g h a b i t s which d i d not i n c l u d e l y g a e i d s . In c o n c l u s i o n , the r o l e o f c a r d e n o l i d e s i n CJ. f a s c i a t u s and I. L k a l m i i f e e d i n g on milkweeds i n c e n t r a l C a l i f o r n i a remains i n q u e s t i o n , a l t h o u g h t h e c o l o u r a t i o n and b e h a v i o u r o f t h e s e s p e c i e s s t r o n g l y s u g g e s t t h a t t h e y b e n e f i t from warning c o l o u r a t i o n and some form o f m i m i c r y which can c o n d i t i o n p r e d a t o r s t o a v o i d t h e s e i n s e c t s . 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