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Some aspects of the sequestration of cardenolides in the large milkweed bug, Oncopeltus fasciatus (Dallas)… Moore, Lynn Marie Vasington 1985

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SOME ASPECTS OF THE SEQUESTRATION OF CARDENOLIDES IN THE LARGE MILKWEED BUG, ONCOPELTUS FASCIATUS (DALLAS) (HEMIPTERA: LYGAEIDAE) by LYNN MARIE VASINGTON MOORE B.Sc. (Magna Cum Laude) UNIVERSITY OF CONNECTICUT, 1975 M.Sc. UNIVERSITY OF MASSACHUSETTS, 1978 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF ZOOLOGY We accept t h i s t h e s i s as conforming to the-f5e.auired standard A p r i l , 1985 (c) Lynn Marie Vasington Moore, 1985 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 O E - 6 ( 3 / 8 1 ) i i ABSTRACT S p e c i f i c a s p e c t s o f the s e l e c t i v e s e q u e s t r a t i o n , e x c r e t i o n and t o l e r a n c e o f c a r d e n o l i d e s i n the l a r g e mi lkweed b u g , O n c o p e l t u s f a s c i a t u s have been s t u d i e d u s i n g s p e c t r o p h o t o m e t r y a s s a y s , t h i n - l a y e r c h r o m a t o g r a p h y , t r a c e r s t u d i e s , i n v i v o t o l e r a n c e a s s a y s , and enzyme i n h i b i t i o n t e c h n i q u e s . The c a r d e n o l i d e c o n t e n t o f t h e d o r s o l a t e r a l s p a c e , g u t , wings and f a t body o f O n c o p e l t u s f a s c i a t u s was e x a m i n e d . The r e s u l t s i n d i c a t e t h a t t h e m a j o r i t y o f c a r d e n o l i d e s s e q u e s t e r e d i n the i n s e c t a r e c o n c e n t r a t e d i n t h e d o r s o l a t e r a l s p a c e , w h i c h c o n f i r m s t h e b a s i c p a t t e r n o f q u a n t i t a t i v e 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 s i n fJ. f a s c i a t u s d e t e r m i n e d i n e a r l i e r w o r k . L a r g e amounts o f c a r d e n o l i d e s were n o t f o u n d i n t h e g u t , wings and f a t b o d y . The f emale f a t body c o n t a i n e d 4-5% o f the t o t a l 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 . The c a r d e n o l i d e c o n t e n t o f male f a t b o d y , and gut and wings o f bo th sexes was below t h e d e t e c t i o n l i m i t o f t h e c a r d e n o l i d e a s s a y . T h i n - l a y e r c h r o m a t o g r a p h y was used t o d e t e r m i n e t h e c a r d e n o l i d e a r r a y o f v a r i o u s t i s s u e s and s e c r e t i o n s o f fJ. f a s c i a t u s r e a r e d on seeds o f a s i n g l e s p e c i e s o f mi lkweed ( A . s p e c i o s a ) and a d u l t e x t r a c t s and d o r s o l a t e r a l space f l u i d o f 0. f a s c i a t u s r e a r e d on seeds o f two s p e c i e s o f mi lkweed w i t h d i f f e r e n t c a r d e n o l i d e a r r a y s ( A . s p e c i o s a and A . s y r i a c a ) • The r e s u l t s i n d i c a t e t h a t c a r d e n o l i d e s a r e n o t s e q u e s t e r e d i n the i n s e c t s i m p l y on t h e b a s i s o f p o l a r i t y and t h a t m e t a b o l i s m and d i f f e r e n t i a l e x c r e t i o n o f c a r d e n o l i d e s a r e i n v o l v e d i n t h e 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 i n fJ. f a s c i a t u s . . The s i m i l a r i t i e s i n t h e c a r d e n o l i d e p r o f i l e s 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 f o o d s o u r c e s , and t i s s u e s o f fj. f a s c i a t u s r e a r e d on a s i n g l e f o o d s o u r c e i n d i c a t e s t h a t t h e r e i s r e g u l a t i o n o f the c a r d e n o l i d e a r r a y i n 0. f a s c i a t u s . An i n v i t r o p r e p a r a t i o n o f M a l p i g h i a n i 1* i tubules was used to investigate the excretion of the polar cardenolide, ouabain, in 0. fasciatus. Both segments of the tubules were found to metabolize ouabain. The distal Segment (Segment II) secreted primary urine and ouabain. Secretion of ouabain by Segment II was not observed to occur against a concentration gradient and increased with increasing f lu id secretion. The proximal segment (Segment I) reabsorbed f lu id and ouabain but not metabolites of ouabain. Ouabain was reabsorbed against a strong concentration gradient (23-fold), was independent of f lu id reabsorption, and increased with increasing f lu id secretion by Segment II . In rapidly secreting Malpighian tubules (a situation of high cardenolide secretion by Segment II ) , the presence of Segment I reduced the excretion of ouabain by 84 - 93%, mainly by reducing ouabain concentration. It appears excretory loss of cardenolides can be reduced in 0. fasciatus and thus may be a factor in the sequestration of cardenolides in this insect. 0. fasciatus tolerated 1954x and 7288x, respectively, the LD50 ouabain dose of Schistocerca gregaria and Periplaneta americana when ouabain was injected into the hemocoel of these insects. The maximum ouabain dose that could be injected into 0_. fasciatus (200 nmoles) resulted in no mortality; this dose is higher than the lethal ouabain doses recorded for vertebrates and invertebrates. The ouabain concentration resulting in 50% inhibition (I50) of Na,K-ATPase act ivi ty was determined in lyophilates of nervous tissue of 0. fasciatus and brain and recta of j>. gregaria and were 2.0 x l O " 4 , 2.0 x 10" 6, and 1.0 x 10~6 M, respectively. The I 5 0 value for ouabain inhibition of Na,K-ATPase act ivity in the nervous tissue of 0_. fasciatus is higher than the I50 values for nervous tissue in most other insects as well as many other invertebrate and vertebrate i v t i s s u e s . T h u s , the p r e s e n c e o f o u a b a i n r e s i s t a n t N a , K - A T P a s e s a p p e a r s to be a f a c t o r i n t h e t o l e r a n c e and s e q u e s t r a t i o n o f p l a n t c a r d e n o l i d e s i n 0. f a s c i a t u s . V Table of Contents Page Ab s t r a c t . i i Table of Contents v L i s t of Tables v i i i L i s t of Figures x Acknowledgements x i i CHAPTER 1: GENERAL INTRODUCTION 1 Toxic p l a n t compounds in plants and i n s e c t s 1 Cardenolide chemistry and t o x i c i t y 2 Cardenolides and the Asclepiadaceae (milkweeds) 5 Ouabain and d i g i t o x i n 6 Cardenolide sequestration i n i n s e c t s 10 Oncopeltus f a s c i a t u s 11 Cardenolide sequestration i n Oncopeltus f a s c i a t u s 13 CHAPTER 2: SELECTIVE SEQUESTRATION OF MILKWEED (ASCLEPIAS sp.) CARDENOLIDES IN ONCOPELTUS FASCIATUS 17 Summary 17 Introduct i o n 18 Materi a l and Methods 20 1) Insects 20 2) C o l l e c t i o n of samples 20 3) E x t r a c t i o n of cardenolides 21 4) Cardenolide determinations 22 a) To t a l cardenolide concentrations 22 b) Determinations of cardenolide p r o f i l e s with t h i n - l a y e r chromatography (TLC) 23 v i Page R e s u l t s 25 1) T o t a l c a r d e n o l i d e c o n t e n t o f the f a t body, wings, gut and d o r s o l a t e r a l space f l u i d o f fj. f a s c i a t u s r e a r e d on A. s p e c i o s a 25 2) C a r d e n o l i d e p r o f i l e s o f 0 f a s c i a t u s r e a r e d on A s p e c i o s a and A. s y r i a c a seeds 28 3) C a r d e n o l i d e p r o f i l e s o f a d u l t e x t r a c t s , t i s s u e s and d o r s o l a t e r a l space f l u i d o f 0. f a s c i a t u s r e a r e d on A. s p e c i o s a seeds 38 4) Ge o g r a p h 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 p r o f i l e o f A. s y r i a c a seeds 41 D i s c u s s i o n 43 CHAPTER 3: EXCRETION OF OUABAIN BY MALPIGHIAN TUBULES OF ONCOPELTUS FASCIATUS 50 Summary 50 I n t r o d u c t i o n 51 M a t e r i a l and Methods 52 1) I n s e c t s 52 2) S a l i n e s 53 3) In v i t r o M a l p i g h i a n t u b u l e p r e p a r a t i o n 53 4) Chromatography 55 R e s u l t s 59 1) C h a r a c t e r i s t i c s o f ou a b a i n t r a n s p o r t by Segment II 59 a) S e c r e t i o n o f ouabain by Segment II 59 b) M e t a b o l i s m o f ouabain by Segment II 65 2) Ouabain R e a b s o r p t i o n i n Segment 1 65 vii Page a) Direct analysis 65 b) Set droplets 72 3) Modification of Segment II secretion by Segment 1 76 Discussion 79 CHAPTER 4: OUABAIN RESISTANT NA.K ATPASES AND CARDENOLIDE  TOLERANCE IH THE LARGE MILKWEED BUG, ONCOPELTUS FASCIATUS 84 Summary 84 Introduction 85 Material and Methods 86 1) Insects 86 2) Ouabain injections 87 3) Na++K+ -dependent ATPase activity and ouabain sensitivity 88 Results 90 1) Sensitivity of insects to injections of ouabain into the hemocoel 90 2) Activity and ouabain sensitivity of Na++K+ -dependent ATPases in tissue lyophilates of 0. fasciatus and S^ . gregaria 92 Discussion 99 CHAPTER 5: GENERAL DISCUSSION 108 Literature cited 113 v i i i List of Tables Page Table 2.1 Total cardenolide content of adult an 5th instar Oncopeltus fasciatus samples 26 Table 2.2 Rn, values of cardenolides and non-cardenolides detected in extracts of 0. fasciatus and its food sources, A. speciosa or A. syriaca 32 Table 2.3 The variability in cardenolide profiles of 0_. fasciatus and A..speciosa..'. 40 Table 2.4 Distribution of cardenolides in Oncopeltus fasciatus expressed as % of the adult total 44 • Table 3.1 Chromatographic systems used for the separation of cardenolides and their metabolites 57 Table 3.2 Ionic composition (mM) of whole-tubule secretions, reabsorbed fluid and bathing saline 73 Table 4.1 Survival of _0. fasciatus injected with 10 - 200 nmoles ouabain 91 Table 4.2 Post-injection recovery times of 0_. fasciatus 93 Table 4.3 Ouabain tolerance of CJ. fasciatus, _S. gregaria and P_. americana 96 Table 4.4 Total, Mg2+ and Na,K-ATPase activity of tissue lyophilates of 0_. fasciatus and S^ . gregaria 98 Table 4.5 Inhibition of Na,K-ATPase activity by ouabain in nervous tissue of 0. fasciatus and brain and rectum of S. gregaria 102 ix Page Table 4.6 Effects of ouabain on tissue processes and survival in insect and millipede species 103 Table 4.7 Toxicity values for ouabain in invertebrates and vertebrates 105 X L i s t o f F i g u r e s Page F i g u r e 1.1 G e n e r a l s t r u c t u r e o f c a r d e n o l i d e s 4 F i g u r e 1.2 G e n e r a l s t r u c t u r e s o f t h e t h r e e g r o u p s o f A s c l e p i a s c a r d e n o l i d e s 7 F i g u r e 1.3 The c h e m i c a l s t r u c t u r e s o f o u a b a i n and d i g i t o x i n . . . . 9 F i g u r e 2.1 C a r d e n o l i d e p r o f i l e s o f A . s p e c i o s a and A . s y r i a c a seeds and a d u l t e x t r a c t s and d o r s o l a t e r a l space f l u i d o f fJ. f a s c i a t u s r e a r e d on each seed 30 F i g u r e 2.2 C a r d e n o l i d e p r o f i l e s o f 0. f a s c i a t u s a d u l t s , t i s s u e s and s e c r e t i o n s and A . s p e c i o s a seeds 35 F i g u r e 2.3 C a r d e n o l i d e p r o f i l e s o f A . s y r i a c a seeds from f o u r g e o g r a p h i c l o c a t i o n s 42 F i g u r e 3.1 In v i t r o p r e p a r a t i o n o f rj. f a s c i a t u s M a l p i g h i a n t u b u l e s 54 F i g u r e 3.2 The e f f e c t o f v a r y i n g e x t e r n a l o u a b a i n c o n c e n t r a t i o n on u r i n e - t o - p l a s m a ( U / P ) r a t i o s 60 F i g u r e 3.3 E f f e c t o f v a r y i n g e x t e r n a l o u a b a i n c o n c e n t r a t i o n on f l u i d s e c r e t i o n r a t e 61 F i g u r e 3.4 E f f e c t o f f l u i d s e c r e t i o n r a t e on t h e r a t e o f o u a b a i n s e c r e t i o n by Segment II a l o n e 63 F i g u r e 3.5 E f f e c t o f f l u i d s e c r e t i o n r a t e on o u a b a i n c o n c e n t r a t i o n i n f l u i d s e c r e t e d by Segment II a l o n e 64 F i g u r e 3.6 D i s t r i b u t i o n o f r a d i o a c t i v i t y i n t y p i c a l chromatograms o f e x p e r i m e n t a l and c o n t r o l f l u i d s 66 xi Page Figure 3.7 Amount of f l u i d , ouabain, metabolised ouabain, and ouabain concentration in Segment II secretion, reabsorption droplets and whole-tubule secret ion. . . 68 Figure 3.8 Amount of f lu id secreted by whole tubule in 2 h in relation to ouabain reabsorption by Segment I during same time period 71 Figure 3.9 Change in f lu id volume, radioactive label , and radioactive label concentration in four set droplets and whole-tubule secretion over 2 h 74 Figure 3.10 Rate of ouabain secretion with time 77 Figure 3.11 Ouabain concentration in secreted f lu id with time.. 78 Figure 4.1 Mortality (24 h) of S^ . gregaria and P_. americana injected with varying amounts of ouabain 94 Figure 4.2 Effect of varying ouabain concentrations on Na,K-ATPase act ivity in lyophilate preparations of fj. fasciatus nervous tissue and ^. gregaria brain and rectum 100 xi i Acknowledgements I would like to thank my research supervisor, Dr. 6.G.E. Scudder, for his advice and support throughout my Ph.D. program. I would also like to thank the members of my research committee, Drs. J . Gosline, W. Milsom, J . Phillips and G.H.N. Towers, for their constructive criticism and interest throughout my research program, and for reviewing this thesis. A special thanks to Joan Martin for sharing her scientific expertise, providing-lots of encouragement and masterfully slogging through the sludge that became my manuscripts and thesis! The advice of Dr. J .N. Seiber on chromatography systems and the advice of Dr. J .H. Anstee on techniques for determining Na,K-ATPase activity in insect tissues is gratefully acknowledged. Carl Edman cheerfully synthesized the 2,4,2',4'-tetranitrodiphenyl and Dr. M.B. Isman provided advice and encouragement. To the 4th floor critters: J i l l Lancaster, Jennifer Robinson, Edie Bijdemast, Dick Cannings, Syd Cannings, John Ford, Deb Cavanaugh, Garry Stenson and Rich Lechleitner, and Liz and Bob Hancock; thanks for loads of fun, friendship, advice and encouragement. A special thanks to my husband, Richard, for his love, patience and fun. This work was supported by operating Grant A0865 from the Natural Sciences and Engineering Research Council of Canada to Dr. G.G.E. Scudder and by a University Graduate Summer Scholarship from the University of British Columbia to L.V. Moore. 1 J CHAPTER 1: GENERAL INTRODUCTION The l a r g e milkweed bug, Oncopeltus f a s c i a t u s ( D a l l a s ) feeds e x c l u s i v e l y on p l a n t s that contain cardenolides (Duffey and Scudder, 1972). Cardenolides are s p e c i f i c i n h i b i t o r s of Na,K-ATPase a c t i v i t y , and are t o x i c to many verte b r a t e s and i n v e r t e b r a t e s (Detweiter, 1967; B i e k i r c h , 1977; R a f a e l i - B e r n s t e i n and Mordue, 1978; Benson et al_., 1979; Stekhoven and Bonting, 1981; Anstee and Bowler, 1984) at concentrations as low as 10" 8 M (Glynn, 1964). 0. f a s c i a t u s , however, stores l a r g e q u a n t i t i e s of these compounds i n i t s body (up to 707 j j g / i n s e c t ; Isman, 1977), and seems to s u f f e r no ill e f f e c t s from them (Isman, 1977; Chaplin and C h a p l i n , 1981; Jones e t j a l _ . 1983). I t has been suggested that the majority of cardenolides in the i n s e c t are sequestered in the d o r s o l a t e r a l space, a h i g h l y vacuolated e p i t h e l i a l l a y e r i n the integument, and that these cardenolides f u n c t i o n i n chemical defense a g a i n s t p o t e n t i a l predators of fj. f a s c i a t u s (Duffey and Scudder, 1974; Scudder and Meredith, 1982a). The work described in t h i s t h e s i s was undertaken to i n v e s t i g a t e some of the p o s s i b l e processes involved in the sequestration and t o l e r a n c e of p o t e n t i a l l y t o x i c cardenolides i n £ . f a s c i a t u s . Therefore, I have provided below a b r i e f summary of various aspects of the cardenolide system p e r t i n e n t to the chapters that f o l l o w . Toxic p l a n t compounds in plants and i n s e c t s Toxic p l a n t compounds defend plants from general attack by microorganisms, f u n g i , i n s e c t s and other herbivores ( f o r review see Whittaker and Feeny, 1971). However, despite t h e i r t o x i c i t y , p l a n t s r a r e l y 2 escape insect herbivory. Many insects are resistant to the toxic effects of a specific group or array of plant chemicals (for review see Dowd et a l . , 1983), and like 0_. fasciatus feed specifically on plants containing these chemicals. In many of these insects, toxins from the host plants are sequestered in various tissues and secretory glands where they function in the insect's defense against its own predators (for review see Blum, 1981; Blum, 1983). Insects that sequester plant toxins are often brightly colored and part of large mimicry complexes, presumably to warn potential predators of their distastefulness or toxicity (for review see Rothschild, 1972; Huheey, 1984). Cardenolides are one of the better studied groups of plant chemicals that are involved in defense systems of plants and insects, as well as in insect mimicry complexes. Cardenolide chemistry and toxicity Cardenolides are a class of cardiac glycosides, a group of compounds well known for their bitter, emetic, and toxic qualities (Hoch, 1961). Cardenolides are toxic to many vertebrates and invertebrates (Hoch, 1961; Treherne, 1966; Detweiler, 1967; Anstee and Bell , 1975; Biekirch, 1977; Rafaeli-Bernstein and Mordue, 1979; Stekhoven and Bonting, 1981; Anstee and Bowler, 1984), at concentrations as low as 10 - 8 M (Glynn, 1964). In vertebrates, the different toxicities of individual cardenolides have been related to differences in their polarities and/or partition coefficients (Detweiler, 1967; Okita, 1967; Lullmann and Peters, 1973; Smith and Haber, 1974). Cardenolides are classified as cardiac glycosides owing to their toxic effects upon vertebrate heart tissue and the presence of one to four sugars attached to the genin. Cardenolides are 23 carbon steroids and characterized by a 5 carbon ,8-unsaturated ^-lactone (butenolide) ring 3 a t t a c h e d a t C-17, a c i s j u n c t u r e o f r i n g s C and D and a 14j£-uH group ( F i g . 1.1, S e i b e r et _al_., 1983); a l l t h r e e c h a r a c t e r i s t i c s a r e e s s e n t i a l f o r the t o x i c a c t i v i t y o f t h e s e compounds ( W r i g h t , 1960; Nover, 1972). Sugars a r e g e n e r a l l y a t t a c h e d t o the g e n i n a t C-3 through an hydroxy group, and a r e o f t e n unusual d e r i v a t i v e s o f deoxymethylpentose, normal hexoses such as g l u c o s e a r e r a r e l y e n c o u n t e r e d ( P a r i s , 1963; S e i b e r ^ t , 1983). C a r d e n o l i d e s e x e r t t h e i r t o x i c e f f e c t s by s p e c i f i c a l l y i n h i b i t i n g the enzyme Na ++K +-ATPase (Na,K-ATPase; E.C. 3.6.1.3; A k e r a , 1977; Bodeman, 1981). T h i s enzyme, a l s o known as the sodium pump, i s u b i q u i t o u s among animal s p e c i e s and a l l animal c e l l s , and i s e s s e n t i a l i n m a i n t a i n i n g the e l e c t r o c h e m i c a l g r a d i e n t s f o r N a + a c r o s s the c e l l plasma membrane (Jtfrgensen, 1980; Stekhoven and B o n t i n g , 1981). The enzyme i s r e s t r i c t e d t o the b a s a l a t e r a l plasma membrane o f e p i t h e l i a , w i t h h i g h e s t a c t i v i t i e s o c c u r r i n g i n e x c i t a t o r y and s e c r e t o r y t i s s u e s (Stekhoven and B o n t i n g , 1981) . Na,K-ATPases a r e i n v o l v e d i n numerous v e r t e b r a t e t i s s u e f u n c t i o n s , i . e . s a l t r e g u l a t i o n i n a v i a n s a l t g l a n d s ( E a s t i n e t , 1982), b i c a r b o n a t e and a c i d s e c r e t i o n i n g a s t r i c mucosa ( H e l a n d e r and D u r b i n , 1982) , s o l u t e r e a b s o r p t i o n ( p a r t i c u l a r l y Na +) and r e g u l a t i o n o f i n t r a c e l l u l a r pH and C a 2 + l e v e l s i n k i d n e y t u b u l e s (Jtfrgensen, 1980) and p r o p e r f u n c t i o n i n g o f nervous t i s s u e (Stekhoven and B o n t i n g , 1981). In i n v e r t e b r a t e s , Na,K-ATPases have been demonstrated t o f u n c t i o n i n M a l p i g h i a n t u b u l e s e c r e t i o n ( P i l c h e r , 1970; A t z b a c h e r et j a K , 1974; Anstee and B e l l , 1975), p r o p e r f u n c t i o n i n g o f nervous t i s s u e ( T r e h e r n e , 1966; F a r q u h a r s o n , 1974), midgut i o n f l u x e s and t r a n s e p i t h e l i a l p o t e n t i a l s ( O ' R i o rdan, 1969; P r u s c h , 1978) and l a b i a l g l a n d s e c r e t i o n ( K a f a t o s , 1968). C a r d e n o l i d e i n d u ced m o r t a l i t y i n v e r t e b r a t e s o c c u r s when i n h i b i t i o n o f 4 F i g . 1.1. G e n e r a l s t r u c t u r e o f c a r d e n o l i d e s . 5 Na,K-ATPase act iv i ty in cardiac ce l l s results in such large net losses of K + from the cytoplasm, the heart becomes arrhythmic and no longer functions danger, 1981). The actual cause of cardenolide induced death in invertebrates has not been determined, but may also be the result of cardiac arrest. Cardenolides and the Asclepiadaceae (milkweeds) Cardenolides have been isolated from twelve families of Angiosperms (Singh and Rastogi, 1970) and are particularly abundant in the milkweed (Asclepiadaceae) and dogbane (Apocynaceae) families (Paris, 1963). Most of the information available on the chemistry and distribution of plant cardenolides is from the Asclepiadaceae, the primary food plants of 0. fasciatus. Nearly a l l the Asclepiadaceae examined to date contain cardenolides (Seiber et ji l_., 1983). The individual cardenolides in the plant (as many as 23; Brower et al_., 1984a) and their relative concentrations determine the plant's cardenolide array or prof i l e . Comparative studies of the cardenolide content and profi le of Asclepias species have revealed large species differences in terms of the quantity and array of cardenolides present; some differences within populations of the same species have also been noted (Seiber et ^1_., 1978; Brower et a l . , 1982; Brower et a l . , 1984a; Moore and Scudder, 1985). Cardenolides are present in a l l parts of the plant (Roeske et _al_., 1976). There is a great deal of intraplant variation in terms of cardenolide content and array (Nelson et al_., 1981; Nishio et a l . , 1983), as well as seasonal variation in the gross cardenolide content of the various plant parts (Seiber et al_., 1983). Plants synthesizing cardenolides appear to prevent autotoxicity by 6 p h y s i c a l l y i s o l a t i n g t h e s e t o x i n s i n l e a f v a c u o l e s ( L o f f e l h a r d t e t a l . , 1979) o r i n t h e l a t i c i f e r sys tem where c a r d e n o l i d e s a r e s t o r e d and t r a n s p o r t e d i n t h e l a t e x i n l a t i c e s i n d e p e n d e n t o f t h e p l a n t ' s v a s c u l a r system ( N e l s o n et a\_., 1981; N i s h i o e t j H . , 1983). S t o r a g e i n t h e l a t i c i f e r system i s p a r t i c u l a r l y c h a r a c t e r i s t i c o f A s c l e p i a s s p e c i e s w i t h h i g h c a r d e n o l i d e c o n t e n t ( N e l s o n e t ^1_., 1981). T h r e e groups o f c a r d e n o l i d e s have been i s o l a t e d i n t h e A s c l e p i a d a c e a e , s i m p l e c a r d e n o l i d e g e n i n s ( i . e . u z a r i g e n i n and s y r i o g e n i n ) and t h e i r s u g a r d e r i v a t i v e s , and two groups o f 2 , 3 - d i h y d r o x y c a r d e n o l i d e d e r i v a t i v e s w i t h c y c l i c b r i d g e d s u g a r s ( F i g . 1.2, S e i b e r e t _al_. , 1983). The two l a t t e r groups a r e (1) t h e g l y c o s i d e s o f c a l o t r o p a g e n i n ( g o m p h o s i d e , a f r o s i d e , c a l a c t i n , c a l o t r o p i n , a s c l e p i n , u s c h a r i d i n , u s c h a r i n , v o r u s c h a r i n , and c a l o t o x i n ) f ound i n C a l o t r o p i s s p . and A . c u r a s s a v i c a , A . f r u t i c o s a , A . v e s t i t a , A . c o r d i f o l i a and A . c a l i f o r n i c a , and (2) d e s g l u c o s y r i o s i d e and i t s d e r i v a t i v e s , w i t h a 7,8 epoxy f u n c t i o n ( l a b r i f o r m i d i n , l a b r i f o r m i n , s y r i o s i d e , s y r i o b i o s i d e ) f ound i n A . l a b r i f o r m i s , A . e r i o c a r p a , A . , s y r i a c a , A . e r o s a and A . s p e c i o s a . Many o f t h e a s c l e p i a d c a r d e n o l i d e s a r e u n i d e n t i f i e d ; w i t h f u t u r e i n v e s t i g a t i o n a d d i t i o n a l c a r d e n o l i d e groups may be d i s c o v e r e d ( S e i b e r e t a l _ . , 1983). O u a b a i n and D i g i t o x i n Commerc ia l p r e p a r a t i o n s o f h o s t p l a n t c a r d e n o l i d e s o f 0_. f a s c i a t u s a r e n o t a v a i l a b l e . T h e r e f o r e , e x p e r i m e n t s i n v e s t i g a t i n g t h e 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 i n t h i s i n s e c t have g e n e r a l l y used two n o n - A s c l e p i a d c a r d e n o l i d e s , o u a b a i n and d i g i t o x i n , owing to the c o n t r a s t i n g p o l a r i t i e s o f t h e s e two compounds and t o t h e i r a v a i l a b i l i t y i n p u r e and r a d i o l a b e l e d p r e p a r a t i o n s o f known s t r u c t u r e ( F i g . 1.3). In a d d i t i o n , a number o f the 7 F i g . 1.2. G e n e r a l s t r u c t u r e s o f t h e t h r e e groups o f A s c l e p i a s c a r d e n o l i d e s : A) s i m p l e c a r d e n o l i d e g e n i n s , B) g l y c o s i d e s o f c a l o t r o p a g e n i n , and C) d e s g l u c o s y r i o s i d e and i t s d e r i v a t i v e s . 8 9 10 metabolites of these compounds have been i d e n t i f i e d and are a l s o a v a i l a b l e i n pure pre p a r a t i o n s . Ouabain and d i g i t o x i n have a 5 ^ c i s A/B s t e r o i d r i n g j u ncture rather than the 5 ot trans A/B s t e r o i d r i n g juncture c h a r a c t e r i s t i c of A s c l e p i a d c a r d e n o l i d e s . Cardenolide sequestration in i n s e c t s F i f t y i n s e c t species in seven orders are known to sequester cardenolides from t h e i r host plants ( R o t h s c h i l d and R e i c h s t e i n , 1976). L i k e fJ. f a s c i a t u s , these i n s e c t s are warningly c o l o r e d and many are members of various mimicry complexes (Duffey and Scudder, 1972; R o t h s c h i l d , 1972; R o t h s c h i l d , 1976; Scudder and Duffey, 1972; Isman et al_., 1977). Various s t u d i e s suggest that sequestered cardenolides are an e f f e c t i v e part of the defense system of these i n s e c t s ( R o t h s c h i l d , 1966; von Euw e t al_., 1967; R o t h s c h i l d and K e l l e t , 1972; R o t h s c h i l d et ^1_., 1973; R o t h s c h i l d et a K , 1978; Fink and Brower, 1981; Cohen and Brower, 1983; Malcolm and R o t h s c h i l d , 1983). In most cases, i n s e c t s appear to s u f f e r no ill e f f e c t s from sequestering l a r g e amounts of cardenolides ( E r i k s o n , 1973; Isman, 1977; Smith, 1978; Dixon e t ^ l _ . , 1978; Chaplin and C h a p l i n , 1981; Jones ^ t a K , 1983; Brower et al_., 1984a; but see Seiber et al_., 1980). A number of s i m i l a r i t i e s i n the o v e r a l l process of cardenolide sequestration have been noted in the i n s e c t s s t u d i e d so f a r . In general, cardenolides are sequestered in i n s e c t s in proportion to the cardenolide content of the food source (Roeske e t al_., 1976; Isman, 1977; Cohen and Brower, 1983), although in the monarch cardenolide l e v e l s are r e l a t i v e l y independent of the cardenolide content of the food source (Brower et a l . , 1982, 1984a,b). Comparisons of host p l a n t and i n s e c t cardenolide p r o f i l e s i n d i c a t e t h a t in general, not a l l of the cardenolides a v a i l a b l e in the host p l a n t are found in the i n s e c t ; t h i s i s a r e s u l t , in p a r t , of s e l e c t i v e feeding on p a r t i c u l a r p l a n t parts by the i n s e c t , as well as metabolism of 11 ingested cardenolides i n the i n s e c t ( R o t h s c h i l d e t a l . , 1970; von Euw e t al_., 1971; Roeske ^ t ail_., 1976; Duffey et al_., 1978; Seiber ^ t al_., 1980; Brower e t a l . , 1982; Scudder and Meredith, 1982b). Sequestration of cardenolides i n i n s e c t s a l s o seems to be c h a r a c t e r i z e d by d i f f e r e n t i a l d i s t r i b u t i o n of these compounds among the i n s e c t s ' t i s s u e s i n terms of quanti t y , and in the monarch, a l s o i n terms of emetic potency (Duffey and Scudder, 1974; Brower and G l a z i e r , 1975; Blum, 1983). However, despite the s i m i l a r i t i e s i n the o v e r a l l process of cardenolide sequestration i n i n s e c t s stated above, the d e t a i l s of the d i f f e r e n t mechanisms involved appear to vary g r e a t l y between i n d i v i d u a l s p e c i e s , l e a d i n g Blum (1983) to suggest that each species of i n s e c t has a unique process of se q u e s t r a t i o n , t o l e r a n c e and defensive use of the toxi n s ingested with i t s food p l a n t . Oncopeltus f a s c i a t u s 0_. f a s c i a t u s i s a member of the Lygaeidae (seed bugs), one of the l a r g e s t f a m i l i e s i n the i n s e c t order, Hemiptera. The majority of Lygaeids feed upon mature seeds of p l a n t s . In common with other Hemipterans, the Lygaeids have p i e r c i n g and sucking mouthparts and feed by l i q u e f y i n g t h e i r food with i n j e c t i o n s of s a l i v a p r i o r to i n g e s t i o n . A l l Hemipterans undergo incomplete metamorphosis; there i s l i t t l e change during development except in s i z e and reproductive maturity, and immatures and adults u s u a l l y occupy s i m i l a r h a b i t a t s and u t i l i z e s i m i l a r food resources. Hemipteran adults are a c t i v e feeders and l o n g - l i v e d . In a d d i t i o n , both a d u l t s and immatures possess scent glands which may deter predators. 0. f a s c i a t u s i s a r e l a t i v e l y l a r g e Lygaeid (17-18 mm in l e n g t h ) . I t i s widely d i s t r i b u t e d i n the New World from Maine and Ontario, west to S. Dakota and C a l i f o r n i a , and south throughout the Neotropics. The i n s e c t i s l e s s common in the northern 12 parts of i t s range, does not overwinter there, but migrates north each year from the south (Dingle, 1978). Six to seven a d d i t i o n a l Oncopeltus sp. occur i n the southern and southeastern s t a t e s ( f o r f u r t h e r d e t a i l s of the Lygaeidae and Hemiptera see S l a t e r , 1964 and S l a t e r and Baranowski, 1978). The conspicuously c o l o r e d orange and black 0. f a s c i a t u s belongs to the Lygaeinae, a c l a s s of Lygaeids c h a r a c t e r i z e d by t h e i r b r i g h t c o l o r a t i o n . In general, Lygaeids are c r y p t i c a l l y c o l o r e d ; i t has been suggested that the b r i g h t l y c o l o r e d Lygaeinae are warningly c o l o r e d to a d v e r t i s e t h e i r d i s t a s t e f u l n e s s or t o x i c i t y to predators (Scudder and Duffey, 1972). B r i g h t l y c o l o r e d Lygaeinae are involved i n mimicry complexes i n A f r i c a , North America and the Caribbean (Scudder and Duffey, 1972; Duffey and Scudder, 1972). The presence of cardenolides i n the major food plants of the warningly c o l o r e d Lygaeinae, as well as the presence of these p l a n t - d e r i v e d cardenolides i n the Lygaeinae themselves, e s t a b l i s h e d a chemical b asis f o r these mimicry complexes (Scudder and Duffey, 1972; Duffey and Scudder, 1972). 0. f a s c i a t u s feeds e x c l u s i v e l y on plants c o n t a i n i n g c a r d e n o l i d e s . The i n s e c t i s found p r i m a r i l y on members of the Asclepiadaceae but a l s o on members of the Apocynaceae. Mature seeds are an e s s e n t i a l p a r t of the d i e t of fj. f a s c i a t u s . Although nymphs feed on vegetative parts of the p l a n t u n t i l mature seeds are a v a i l a b l e , fJ. f a s c i a t u s must feed on mature seeds to complete i t s development (Ralph, 1976; Blakley and Dingle, 1978). Seeds are a patchy food source, however, the high m o b i l i t y and migratory behavior of fJ. f a s c i a t u s a d u l t s , as well as the o v i p o s i t i o n behavior of the female, enable the i n s e c t to e x p l o i t t h i s type of food resource (Ralph, 1977; B l a k l e y , 1980; Dingle e t a l . , 1980; Klausner e t a l . , 1980; Chaplin and 13 C h a p l i n , 1981). Cardenolide Sequestration in Oncopeltus f a s c i a t u s 0_. f a s c i a t u s sequesters cardenolides from the seeds and vegetative parts of i t s host p l a n t throughout i t s l i f e c y c l e (Duffey and Scudder, 1974; F e i r and Suen, 1971; Moore and Scudder, 1985). Cardenolides are a l s o found i n the i n s e c t ' s warningly c o l o r e d eggs (Duffey and Scudder, 1974). The wide range of cardenolide contents detected in f i e l d caught, as well as l a b o r a t o r y reared, i n s e c t s (42 to 707 jiq cardenolide per i n s e c t ) r e f l e c t s both i n t r a - and i n t e r s p e c i e s d i f f e r e n c e s in the cardenolide content of the i n s e c t s ' host p l a n t s , and provides evidence that 0. f a s c i a t u s sequesters y cardenolides in proportion to the cardenolide content of i t s food source (Duffey and Scudder, 1974; Isman, 1977; Isman et j i l _ . , 1977; Duffey e t al_., 1978). Q u a n t i t a t i v e r e g u l a t i o n of cardenolide content in 0_. f a s c i a t u s has been i n d i c a t e d by the demonstration that a greater percentage of the seed cardenolide content i s sequestered in i n s e c t s reared on seeds of low cardenolide content than in i n s e c t s reared on seeds of high cardenolide content (Vaughan, 1979). Evidence to date suggests that uptake of ingested cardenolides across the midgut i s passive (Scudder and Meredith, 1982b). The presence of a d o r s o l a t e r a l space, a h i g h l y vacuolated e p i t h e l i a l l a y e r i n the integument, appears to be a s p e c i a l i z a t i o n of the integument of 0_. f a s c i a t u s , and other members of the Lygaeinae, that i s i n t i m a t e l y involved with the sequestration of cardenolides in these i n s e c t s (Scudder and Duffey, 1972; Scudder and Meredith, 1982a). The majority of cardenolides sequestered in 0_. f a s c i a t u s adults appear to be concentrated in the d o r s o l a t e r a l space where they are thought to f u n c t i o n in the i n s e c t ' s defense a g a i n s t predators (Duffey and Scudder, 1974; Scudder and 14 Meredith, 1982a). When 0. f a s c i a t u s a d u l t s are roughly handled, c a r d e n o l i d e - r i c h d o r s o l a t e r a l space f l u i d i s r e l e a s e d in d i s c r e t e d r o p l e t s from c u t i c u l a r openings along the thorax and abdomen where the f l u i d presumably r e p e l s predators by i t s emetic, b i t t e r and/or t o x i c p r o p e r t i e s (Duffey and Scudder, 1974; Scudder and Meredith, 1982a). The d o r s o l a t e r a l space i s present in the immatures, however, there are no c u t i c u l a r openings, thus r e l e a s e of d o r s o l a t e r a l space f l u i d does not appear to be part of the defensive strategy of immatures (Duffey and Scudder, 1974). I n v e s t i g a t i o n s of the r o l e of sequestered cardenolides i n the defense of fJ. f a s c i a t u s a g a i n s t i t s p o t e n t i a l predators i s complicated by the presence of the i n s e c t ' s other defense mechanisms. A d d i t i o n a l defenses of fJ. f a s c i a t u s include the r e l e a s e of v o l a t i l e aldehydes and cardenolides from scent glands i n the a d u l t and immatures, the r e l e a s e of copious amounts of r e c t a l f l u i d i n immatures (Games and Staddon, 1973; Duffey and Scudder, 1974; Staddon and Daroogheh, 1981), and the presence of histamine or a histamine analogue in the r e c t a l f l u i d and hemolymph of immatures and the hemolymph and d o r s o l a t e r a l space f l u i d of a d u l t s (Graham and Staddon, 1974). A f u r t h e r complication in assessing the r o l e of sequestered cardenolides i n the defense of fj. f a s c i a t u s i s the v a r i a t i o n i n the cardenolide s u s c e p t i b i l i t y of the i n s e c t ' s p o t e n t i a l predators (Duffey, 1977). Therefore, the r o l e of cardenolides i n the i n s e c t ' s defense i s a t t h i s date, ambiguous. The actual s e q u e s t r a t i o n system i n the d o r s o l a t e r a l space i s not understood, although i n t e r a c t i o n with an emulsion phase has been suggested (Duffey e t al_., 1978). U l t r a s t r u c t u r a l and k i n e t i c a n a l y s i s i n d i c a t e t h a t cardenolide uptake i n t o the d o r s o l a t e r a l space i s passive, in proportion to the c a r d e n o l i d e content in the food source and the hemolymph, and 15 nonsaturable (Duffey e t al_., 1978; Scudder and Meredith, 1982a, b). I t has been suggested that the r a p i d uptake and accumulation of cardenolides i n the d o r s o l a t e r a l space, which can occur when cardenolide l e v e l s in the hemolymph are as low as 3.5 x 1 0 - 7 M, may f u n c t i o n in the i n s e c t ' s t o l e r a n c e of these toxins by maintaining low l e v e l s of these compounds i n the hemolymph ( l e s s than 6.5 x 10~ 6 M, Moore and Scudder, 1985). In a d d i t i o n , the very low, i f any turnover of sequestered cardenolides in the d o r s o l a t e r a l space in the a d u l t (Isman et^ aJL, 1977) may a l s o be a f a c t o r in the i n s e c t ' s t o l e r a n c e to these t o x i n s . In v i v o and in v i t r o studies with d i g i t o x i n and ouabain, and i n vivo studies with milkweed c a r d e n o l i d e s , provide evidence of s e l e c t i v e uptake of cardenolides by the gut and i n t o the d o r s o l a t e r a l space, as well as evidence of d i f f e r e n t i a l metabolism of cardenolides a t the l e v e l of the gut and elsewhere i n the i n s e c t (Duffey and Scudder, 1974; Yoder e t al_., 1976; Duffey e t al_., 1978; Scudder and Meredith, 1982b). The chemical or s t r u c t u r a l b a s i s f o r t h i s s e l e c t i v i t y i s not known, however the r o l e of p o l a r i t y in the sequestration of cardenolides in 0. f a s c i a t u s has been suggested (Duffey, 1977; Duffey e t a l . , 1978). Blum (1981, 1983) has s t r e s s e d the need f o r d e t a i l e d s t u d i e s of the s p e c i f i c s of sequestration of p l a n t t o x i n s by i n s e c t s . This t h e s i s examines s p e c i f i c aspects of the sequestration and t o l e r a n c e of cardenolides in 0_. f a s c i a t u s . Chapter 2 c l a r i f i e s c e r t a i n aspects of the d i f f e r e n t i a l d i s t r i b u t i o n of natural cardenolides in 0. f a s c i a t u s and examines the cardenolide content of three organs, the gut, wings and f a t body, t h a t have not been i n v e s t i g a t e d p r e v i o u s l y as p o s s i b l e s i t e s of cardenolide accumulation. Chapter 2 a l s o documents some of the c a p a b i l i t i e s of the sequestration process of cardenolides in 0. f a s c i a t u s 16 by determining and comparing the cardenolide array of various t i s s u e s and s e c r e t i o n s of rj. f a s c i a t u s reared on seeds of a s i n g l e species of milkweed, and of a d u l t e x t r a c t s and d o r s o l a t e r a l space f l u i d of i n s e c t s reared on seeds of two species of milkweed with very d i f f e r e n t cardenolide p r o f i l e s . Chapter 3 i n v e s t i g a t e s the r o l e of e x c r e t i o n in the sequestration of cardenolides in fJ. f a s c i a t u s by examining the f u n c t i o n of the Malpighian tubules in the sequestration of the p o l a r c a r d e n o l i d e , ouabain. F i n a l l y , s i nce the r e l a t i v e s e n s i t i v i t y of 0. f a s c i a t u s to the t o x i c e f f e c t of c a r d e n o l i d e s , as well as the p o s s i b i l i t y t h at the Na,K-ATPases in the i n s e c t are r e s i s t a n t to the i n h i b i t o r y e f f e c t s of cardenolides have never been determined, Chapter 4 compares the r e l a t i v e in v i v o s e n s i t i v i t y of fJ. f a s c i a t u s to ouabain and the ouabain s e n s i t i v i t y of i t s Na,K-ATPases to that of two i n s e c t s which do not normally encounter cardenolides in t h e i r d i e t . 17 CHAPTER 2: SELECTIVE SEQUESTRATION OF MILKWEED (ASCLEPIAS sp.)  CARDENOLIDES IN ONCOPELTUS FASCIATUS Summary The cardenolide content of the gut, wings and f a t body of Oncopeltus  f a s c i a t u s was examined. The female f a t body contained 4-5% of the t o t a l c ardenolide content of the i n s e c t . The cardenolide content of male f a t body, and gut and wings of both sexes was below the d e t e c t i o n l i m i t of the cardenolide assay. T h i n - l a y e r chromatography was used to determine the cardenolide array of various t i s s u e s and s e c r e t i o n s of 0. f a s c i a t u s reared on seeds of a s i n g l e species of milkweed (A. speciosa) and a d u l t e x t r a c t s and d o r s o l a t e r a l space f l u i d of 0_. f a s c i a t u s reared on seeds of two species of milkweed with d i f f e r e n t cardenolide arrays (A. speciosa and A,, s y r i a c a ) . The r e s u l t s i n d i c a t e that cardenolides are not sequestered in the i n s e c t simply on the b a s i s of p o l a r i t y and that metabolism and d i f f e r e n t i a l e x c r e t i o n of cardenolides are involved i n the sequestration of cardenolides i n 0_. f a s c i a t u s . The s i m i l a r i t i e s i n the cardenolide p r o f i l e s of _0. f a s c i a t u s reared on d i f f e r e n t food sources, and t i s s u e s of 0_. f a s c i a t u s reared on a s i n g l e food source i n d i c a t e s that there i s r e g u l a t i o n of the cardenolide array i n 0_. f a s c i a t u s . 18 INTRODUCTION In t h i s chapter, the cardenolide content of the gut, wings and f a t body of fJ. f a s c i a t u s i s determined to provide a more complete understanding of the q u a n t i t a t i v e d i s t r i b u t i o n of cardenolides i n t h i s i n s e c t . In a d d i t i o n , the p o s s i b i l i t y of s e l e c t i v e sequestration of host plant cardenolides in fJ. f a s c i a t u s in terms of the i n d i v i d u a l cardenolides sequestered and t h e i r r e l a t i v e concentrations i s i n v e s t i g a t e d . F i n a l l y , to document some of the c a p a b i l i t i e s of the sequestration of cardenolides i n fJ. f a s c i a t u s , the cardenolide p r o f i l e of a d u l t e x t r a c t s and d o r s o l a t e r a l space f l u i d of fj. f a s c i a t u s reared on seeds of two species of milkweed with very d i f f e r e n t cardenolide p r o f i l e s , i s determined. The gut, wings and f a t body have not been i n v e s t i g a t e d p r e v i o u s l y as p o s s i b l e s i t e s of cardenolide accumulation in fJ. f a s c i a t u s . I t has been suggested that r e t e n t i o n of t o x i c compounds in the gut lumen or t i s s u e and in the f a t body may a i d in the t o l e r a n c e of t o x i n s i n i n s e c t s ( K i l b y , 1963; Brooks, 1976). Furthermore, the monarch b u t t e r f l y (Danaus plexippus) which a l s o sequesters cardenolides from i t s food p l a n t s , i s known to sequester s u b s t a n t i a l amounts of p l a n t cardenolides in both the gut lumen and the f a t body (see Blum, 1981; 1983). Thus, i t i s of i n t e r e s t to see i f cardenolides accumulate in the f a t body and gut of 0. f a s c i a t u s . I t i s p o s s i b l e that the wings of fJ. f a s c i a t u s contain cardenolides since l a r g e concentrations of these compounds, thought to f u n c t i o n in defense a g a i n s t predators, are sequestered in the wings of two l e p i d o p t e r a n s , Danaus plexippus (Brower and G l a z i e r , 1975; see Blum, 1981) and Cycnia inopinatus (Hy. Edwards) (see Blum, 1983) and in the e l y t r a of the cerambycid b e e t l e Tetraopes melanurus Schon. (N i s h i o e t al_., 1983). 19 Therefore, i n t h i s chapter I a l s o determine the cardenolide content of the wings of 0_. f a s c i a t u s . S e l e c t i v e sequestration i n i n s e c t s of host p l a n t c a r d e n o l i d e s , both i n terms of the i n d i v i d u a l cardenolides sequestered and t h e i r r e l a t i v e c o n c e n t r a t i o n s , has been demonstrated i n the aphid, Aphis n e r i i , the monarch b u t t e r f l y , and two l y g a e i d bugs, Caenocoris n e r i i and S p i l o s t e t h u s pandurus ( R o t h s c h i l d e t a l . , 1970; von Euw e t al_., 1971; Roeske e t a l . , 1976; Seiber e t j H . , 1980; Brower e t al_., 1982). In a d d i t i o n , there i s evidence that cardenolides of d i f f e r i n g emetic potencies are s e l e c t i v e l y sequestered i n various t i s s u e s of the monarch b u t t e r f l y (Brower and G l a z i e r , 1975). Therefore, i n t h i s chapter I i n v e s t i g a t e the p o s s i b i l i t y of s e l e c t i v e sequestration of host p l a n t cardenolides i n 0_. f a s c i a t u s , as well as s e l e c t i v e sequestration of cardenolides among the various t i s s u e s of the i n s e c t , by using t h i n - l a y e r chromatography to determine the cardenolide array of various t i s s u e s and s e c r e t i o n s of _0. f a s c i a t u s , as well as the cardenolide array of the i n s e c t ' s food source. In t h i s chapter, I a l s o determine the cardenolide array of adult e x t r a c t s and d o r s o l a t e r a l space f l u i d of fJ. f a s c i a t u s reared on seeds of two species of milkweed with very d i f f e r e n t cardenolide p r o f i l e s to document some of the c a p a b i l i t i e s of the sequestration process of cardenolides i n 0. f a s c i a t u s . The cardenolide p r o f i l e s are als o used to determine the p o l a r i t y d i s t r i b u t i o n of cardenolides i n 0. f a s c i a t u s and i t s t i s s u e s since the t o x i c i t y of cardenolides has been c o r r e l a t e d with t h e i r p o l a r i t y (Duffey, 1977; Smith and Haber, 1974; Detweiler, 1967) and i t has been suggested that the sequestration of cardenolides i n 0. f a s c i a t u s may be a fu n c t i o n of t h e i r ' p o l a r i t y (Duffey, 1980). T h i s chapter provides evidence that cardenolides are d i f f e r e n t i a l l y 20 d i s t r i b u t e d i n fj. f a s c i a t u s i n terms of q u a n t i t y , but not p o l a r i t y , and that cardenolides are not sequestered in the i n s e c t simply on the basis of p o l a r i t y . T h i s chapter a l s o provides evidence that metabolism and d i f f e r e n t i a l e x c r e t i o n of cardenolides are part of the s e l e c t i v e sequestration process of cardenolides in fJ. f a s c i a t u s . In a d d i t i o n , the s i m i l a r i t i e s i n the cardenolide p r o f i l e s of fJ. f a s c i a t u s reared on d i f f e r e n t food sources, and between t i s s u e s of fj. f a s c i a t u s reared on a s i n g l e food source i n d i c a t e s t h a t there i s r e g u l a t i o n of the cardenolide array i n fj. f a s c i a t u s . MATERIALS AND METHODS 1) Insects Adult male and female and f i f t h i n s t a r l a r v a e of Oncopeltus  f a s c i a t u s taken from a l a b o r a t o r y c u l t u r e maintained at 26° C under a 16:8 l i g h t : d a r k c y c l e were used in a l l experiments. The i n s e c t s were reared e i t h e r on commercial sunflower seeds (Heliothus annuus L . ) , or on milkweed seeds ( A s c l e p i a s speciosa T o r r . or A. s y r i a c a L . ) . Insects reared on sunflower seeds provided c o n t r o l s since t h i s food source does not contain c a r d e n o l i d e s . 2) C o l l e c t i o n of samples Gut (with contents) and f a t body samples were d i s s e c t e d from i n s e c t s in Berridge's d i s s e c t i n g s a l i n e i n i t i a l l y (Berridge, 1966), and in l a t e r experiments i n a s a l i n e based on the c o n s t i t u e n t s of fj. f a s c i a t u s hemolymph (Meredith, jet j i K , 1984). Tissues were r i n s e d in three 0.5 ml a l i q u o t s of s a l i n e ^ n d placed d i r e c t l y i n e x t r a c t i n g solvent or b l o t t e d 1 second on each s i d e and wet weight determined: no cardenolides were detected in the 21 s a l i n e r i n s e s of f a t body or gut t i s s u e . No attempt was made to remove tracheae or remnants of the membrane that encloses the f a t body. Insects used to assay gut (with contents) were anesthetized f o r 30 seconds with a low volume of CO2 or cooled at 4°C. The gut was l i g a t u r e d with s u r g i c a l thread ( E t h i c o n , Inc.) at the Malpighian tubule-pylorus j u n c t i o n s , the a n t e r i o r end of the 1st v e n t r i c u l u s and the p o s t e r i o r end of the hind gut, and removed by c u t t i n g d i s t a l to the l i g a t u r e s : the l i g a t u r e s ensured removal of the gut with i t s contents i n t a c t . D i s s e c t i o n s were completed w i t h i n 20 minutes. D o r s o l a t e r a l space f l u i d and hemolymph were c o l l e c t e d from the i n s e c t as described by Duffey and Scudder (1974). Urine/feces samples were c o l l e c t e d by p l a c i n g i n s e c t s f o r 48-72 hours i n small r e a r i n g dishes l i n e d with Whatman Chromatography paper. An excess of seeds was provided and 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 . A f t e r the c o l l e c t i o n p e r i o d the f i l t e r paper, which had absorbed the u r i n e / f e c e s , was removed and e x t r a c t e d f o r c a r d e n o l i d e s . F i l t e r paper from a c o n t r o l r e a r i n g dish (N=l) set up i d e n t i c a l l y to the r e a r i n g dishes f o r u r i n e / f e c e s samples but without i n s e c t s was c o l l e c t e d a f t e r 72 hours, e x t r a c t e d as f o r u r i n e / f e c e s samples and assayed f o r c a r d e n o l i d e s : none was detected. Wings were removed from i n s e c t s a f t e r immobilization at 4°C. 3) E x t r a c t i o n of Cardenolides With the exception of d o r s o l a t e r a l space f l u i d and hemolymph, cardenolides were extracted from a l l samples by e i t h e r the i n s e c t or the seed method (Isman et al_., 1977). The seed method was modified by r e t a i n i n g the CHCI3 phase of the CHCl3:MeOH 10:1 e x t r a c t i o n f o r cardenolide determination. L i p i d s in some samples i n t e r f e r e d with both TLC 22 a n a l y s i s and c o l o r i m e t r i c determination of t o t a l cardenolide content. The seed method removed the i n t e r f e r i n g l i p i d s and t h e r e f o r e , was used ( i ) to e x t r a c t a l l samples f o r TLC a n a l y s i s (except u r i n e / f e c e s ) , and ( i i ) with seed and f a t body samples, used f o r t o t a l cardenolide determination. The i n s e c t method was used to e x t r a c t whole i n s e c t , wing and gut samples f o r t o t a l c a r d enolide determinations and u r i n e / f e c e s samples f o r TLC a n a l y s i s . A l l samples were ext r a c t e d i n a shaking incubator to f a c i l i t a t e d i f f u s i o n of cardenolides and e x t r a c t i o n l i q u o r s were concentrated to 10-20 jul by evaporation under N2 before a p p l i c a t i o n to TLC p l a t e s . Tests showed no d i f f e r e n c e i n the cardenolide array i n whole i n s e c t samples extracted by e i t h e r method. For a l l samples, the petroleum ether d i s c a r d of the seed method was assayed by TLC f o r car d e n o l i d e s : none was detected. Even a f t e r seed method e x t r a c t i o n , f a t body e x t r a c t s were d i f f i c u l t to apply to the TLC p l a t e . Therefore, the e x t r a c t was c e n t r i f u g e d (12000 g, 2 min) and the r e s u l t i n g two phases a p p l i e d s e p a r a t e l y . Only one f a i n t blue spot at the o r i g i n was detected i n the lower l i q u i d phase. D o r s o l a t e r a l space f l u i d and hemolymph were c o l l e c t e d from the i n s e c t , p r e c i p i t a t e d with acetone (5:1 acetone:hemolymph) and 95% EtOH (1 ^ il d o r s o l a t e r a l space f l u i d / m l 95% EtOH), c e n t r i f u g e d (12000 g, 2 min) to remove p r o t e i n s and the supernatant used f o r t o t a l c a r d enolide determination and TLC a n a l y s i s of hemolymph. Resuspension of the p e l l e t e d hemolymph p r o t e i n a f t e r 1 min of s o n i c a t i o n i n 95% EtOH y i e l d e d n e g l i g i b l e c a r d e n o l i d e . D o r s o l a t e r a l space f l u i d was a p p l i e d d i r e c t l y a f t e r c o l l e c t i o n to TLC p l a t e s . 4) Cardenolide Determinations a) Tot a l Cardenolide Concentrations Total cardenolide concentrations were determined by a spectrophoto-23 metric assay using a Lambda 3 UV/VIS spectrophotometer (Perkin-Elmer) and the i n d i c a t o r 2,4,2',4'-tetranitrodiphenyl (TNDP) in the presence of base (NaOH) (Brower and M o f f i t t , 1974; Brower e t al_., 1975). The TNDP r e a c t i o n was run a t room temperature. Sample absorbance at 626 nm was recorded 40 minutes a f t e r the c o l o r i m e t r i c r e a c t i o n was s t a r t e d , with 95% EtOH as the reference. Two c o n t r o l s were used to monitor any absorbance at 626 nm owing to substances i n the i n d i v i d u a l sample e x t r a c t s t e s t e d , as well as the r e a c t i o n reagents TNDP and NaOH: Control 1 = the absorbance of the p a r t i c u l a r i n s e c t sample being t e s t e d , i n the absence of TNDP; Control 2 = the absorbance of TNDP and NaOH, in the absence of i n s e c t sample. The absorbance of the two c o n t r o l s was subtracted from the absorbance of the experimental cuvette ( i n s e c t sample, i n the presence of TNDP and NaOH). Cardenolide concentrations were determined by comparison to a d i g i t o x i n standard, and expressed as molar or jjg equivalent amounts of d i g i t o x i n (jugn) to f a c i l i t a t e comparisons with other s t u d i e s (Roeske e t al_., 1976). Brower and G l a z i e r (1975), using the TNDP r e a c t i o n , found only minor d i f f e r e n c e s i n the e x t i n c t i o n c o e f f i c i e n t s of nine cardenolides and d i g i t o x i n . Therefore, i t was assumed that d i g i t o x i n and the i n d i v i d u a l cardenolides ingested by the i n s e c t s have s i m i l a r e x t i n c t i o n c o e f f i c i e n t s . b) Determination of Cardenolide P r o f i l e s with T h i n - l a y e r  Chromatography (TLC) T h i n - l a y e r chromatography p l a t e s prepared with S i l i c a Gel G (Red i / P l a t e , F i s h e r S c i . Co., gel 250 jm t h i c k ) were used. P l a t e s were a c t i v a t e d over concentrated H2SO4 24 h p r i o r to and 12 h a f t e r applying samples (Duffey and Scudder, 1972). The r e l a t i v e l y nonpolar cardenolide d i g i t o x i n was spotted as a standard on both s i d e s of each sample. P l a t e s 24 were developed i n f i l t e r paper- l i n e d , saturated chambers c o n t a i n i n g methylene chloride:methanol:formamide (105:15:1) (Isman, pers. communication) to a distance of 15-16 cm. TNDP followed by NaOH (Brower et a l . , 1982) were sprayed on the p l a t e s to detect c a r d e n o l i d e s . To standardize r e s u l t s w i t h i n and between TLC p l a t e s and obtain a measure of p o l a r i t y of the cardenolides detected, TLC r e s u l t s were recorded as R e l a t i v e - t o - d i g i t o x i n (Rp,) values: Rp spot R D = — X Rp of d i g i t o x i n on each side of sample distance spot moved from o r i g i n where Rp = distance solvent f r o n t moved from o r i g i n . The Rp of d i g i t o x i n was 0.51 + 0.003 (n=18). The cardenolides of A. speciosa and A. s y r i a c a seeds have not been i d e n t i f i e d . In a d d i t i o n , since Rp, values vary between d i f f e r e n t samples of a given t i s s u e , and between TLC p l a t e s , we used the f o l l o w i n g c h a r a c t e r i s t i c s to i d e n t i f y i n d i v i d u a l c a r d e n o l i d e s : (1) c o l o r (red or blue, red i n d i c a t i n g a non-cardenolide); (2) RQ value; (3) pattern (based on c o l o r , r e l a t i v e p o s i t i o n to other cardenolides in sample and i n t e n s i t y ) . I n t e n s i t y i s an i n d i c a t i o n of concentration (Brower et al_., 1982). The d e t e c t i o n l i m i t of the TLC assay was 0.3-0.5yjg of d i g i t o x i n . For a f u r t h e r i n d i c a t i o n of p o l a r i t y , the RQ values of the very p o l a r cardenolide ouabain and the nonpolar cardenolide d i g i t o x i g e n i n were determined and found to be 0.081, (n=3) and 1.22 + 0.010 (n=9), r e s p e c t i v e l y . Although samples from males and females were assayed s e p a r a t e l y , no sexual d i f f e r e n c e s were found (except where noted in F i g . 25 2.1) and r e s u l t s from both sexes were pooled. The cardenolide p r o f i l e s were compiled from TLC a n a l y s i s of from 4 to 16 d i f f e r e n t e x t r a c t s of each t i s s u e or s e c r e t i o n . To ensure d e t e c t i o n of cardenolides present in low c o n c e n t r a t i o n s , the amount of i n s e c t material used f o r the d i f f e r e n t e x t r a c t s of the i n d i v i d u a l t i s s u e s was v a r i e d . The f o l l o w i n g c o n t r o l samples from i n s e c t s reared on H. annuus were assayed f o r cardenolide p r o f i l e s : a d u l t whole i n s e c t s (1 male (M), 1 female ( F ) ) , u r i n e / f e c e s (10F, 10M e x c r e t i n g f o r 72 hours), eggs (100 and 200), hemolymph (2 r e p l i c a t e s each of 1/2 e x t r a c t of 33M and 25F), and d o r s o l a t e r a l space f l u i d (3F and 3M). No cardenolides were detected i n any of the samples. RESULTS 1) Total Cardenolide Content of the Fat Body, Wings, Gut and D o r s o l a t e r a l  Space F l u i d of 0; f a s c i a t u s reared on A. speciosa The r e s u l t s i n d i c a t e that l a r g e amounts of cardenolides do not accumulate i n the f a t body, wings or gut of a d u l t 0_. f a s c i a t u s reared on A. speciosa (Table 2.1). The concentration of cardenolides i n gut and wing e x t r a c t s of both sexes and f a t body samples of a d u l t males was below the d e t e c t i o n l i m i t of the assay (6.5 x 1 0 - 6 M or 13.0 nmoles d i g i t o x i n e q u i v a l e n t s ) . Fat bodies of a d u l t females contained only 4-5% (mean 8 ^ugn) of the t o t a l cardenolide content detected in whole females. S i m i l a r l y , the f a t body of male and female f i f t h i n s t a r l a r v a e does not appear to be a major s i t e of cardenolide accumulation. Using 1/2 of the t o t a l c a r d e n o l i d e content of adults as a conservative estimate f o r the concentration of cardenolides in f i f t h i n s t a r l a r v a e (Duffey and Scudder, 1974), the c a r d e n o l i d e s sequestered i n the f a t body of such l a r v a e account f o r l e s s than 12% of the t o t a l cardenolides stored at t h i s stage. 26 T«b1e 2.1. Total cardenolide content of adult and 5th instar Oncopeltus fasciatus samples. Cardenolide content Is measured 1n digitoxin equivalents tyjgpland 1s reported as mean *_ S.E. (range). BDL • below detec-tion limit of assay. All Insects were reared on Asdepias speciosa seeds collected 1n Pentlcton, B.C. Sample t In Total each * of Sample Samples ^9D P e r Insect or organ per mg wet weight Insect or organ Whole Insect (adult) Female" 1 10 Male' 1 10 Female^  1 8 Hale0 1 8 Fat Bodyc Female adult 6-7 3 Female 5th Instar 6-7 2 Male adult 6 3 Male 5th Instar 7 2 Gut With Contents (adult)c Female 4 2 Male 4 2 Wings (set « fore • h1nd)b Female 1 set 4 Male 1 set 4 Dorsolateral Space Fluid (adult)1* Female 1 ^ 1 9 Male 1 ^ Jl 11 208.3 • 23.6 (100.8 - 336.1) 208.8 • 21.1 (99.6 - 293.8) 152.5 • 9.6 (103.4 - 196.1) 148.0 • 4.3 (66.8 - 191.6) 8.0 (4.0 - 10.6)0" 8.4 (8.0 - 9.0)<f BDL1' 5.3 (4.9 - 5.6)° BDL BDL BDL BDL 69.8 - 104.7« 88.0 - 132.0« 7.5) 7.9) 4.9 + 0.4 (2.9 5.8 • 0.6 (2.2 3.4 • 0.3 (1.6 - 5.9) 4.5 + 0.2 (1.3 - 7.8) 4.0 • 1.0 (2.0 - 5.3) 2.5 • 0.1 (2.4 - 2.7) BDL 1.4 • 0.1 (1.3 BDL BDL BDL BDL 1.5) * Reared on seeds collected September 1981 b Reared on seeds collected September 1982. c Reared on seeds collected September 1980. d Calculated by multiplying digitoxin equivalent per mg wet weight by mean fat body mass/insect. e Calculated by multiplying u^g digitoxin equivalent per^jl dorsolateral space fluid by estimated volume of dorsolateral space fluid/insect (2-3^jl; Duffey and Scudder, 1974). Mean determined concentration of cardenolides in dorsolateral space fluid for adult females and males is 34.9 * 4.4 (range 24.2 - 67.0) and 44.0 + 3.9 (range 17.0 - 58.3)yigo/ul fluid, respectively. 27 In both sexes, cardenolide accumulation in the f a t body appears to change during development from f i f t h i n s t a r l a r v a e to a d u l t (Table 2.1). The f a t body of female f i f t h i n s t a r l a r v a e and a d u l t s contained s i m i l a r t o t a l amounts of cardenolides (mean 8.4, range 8.0 - 9.0 and mean 8.0, range 4.0 - 10.6, j i g p / f a t body, r e s p e c t i v e l y ) . However, when expressed as mg per wet weight, on average 1/3 more cardenolides were detected i n the a d u l t (mean 4.0, range 2.0 - 5.3, vs mean 2.5, range 2.4 - 2.7,jugn/mg f a t body). Measureable amounts of cardenolides were detected only in the f a t body of male f i f t h i n s t a r l a r v a e : none was detected in the a d u l t . In both the f i f t h i n s t a r and a d u l t , the male f a t body contained lower concentrations of cardenolides than the female. The cardenolide content of the f a t body of a d u l t females may be a f f e c t e d by reproductive s t a t e . The cardenolide content of three samples of 6-7 f a t bodies was determined; the average cardenolide content of the f a t bodies in two of the samples was almost twice that found i n the t h i r d sample (9.1 and 8.2 vs 4.8 jugn/fat body). In the t h i r d sample, one haM of the i n s e c t s used had well developed o v a r i o l e s and eggs, whereas none of the i n d i v i d u a l s in the other two samples contained eggs in the o v a r i o l e s and were probably young females p r i o r to egg development. The g r e a t e s t concentration of cardenolides was found in the d o r s o l a t e r a l space f l u i d , with a mean of 34.9 +_ 4.4 (range 24.2 - 67.0) ^ign/^ul f l u i d i n females and a mean of 44.0 + 3.9^jgp//Jl (range 17.0 -58.3) f l u i d i n males. The d i f f e r e n c e between the cardenolide content of d o r s o l a t e r a l space f l u i d i n males and females i s not s i g n i f i c a n t (Scheffe's t e s t f o r m u l t i p l e comparisons). Using 2-3 JJI as an estimated t o t a l volume of d o r s o l a t e r a l space f l u i d i n a d u l t _0. f a s c i a t u s (Duffey and Scudder, 28 1974), the cardenolides in the d o r s o l a t e r a l space f l u i d account f o r 46-89% of the cardenolides in the i n s e c t . The sequestration s i t e s of the cardenolides unaccounted f o r in t h i s study of cardenolide d i s t r i b u t i o n in t i s s u e s of fJ. f a s c i a t u s are unknown. I t i s p o s s i b l e t h a t the cardenolides in the d o r s o l a t e r a l space f l u i d account f o r a greater percentage of the cardenolides in the i n s e c t than we estimated, since i t i s d i f f i c u l t to determine the t o t a l vacuolar volume of the inner e p i t h e l i a l l a y e r of the epidermis. The t o t a l cardenolide content of a d u l t 0. f a s c i a t u s was determined f o r i n s e c t s reared on A. speciosa c o l l e c t e d from P e n t i c t o n , B.C. in September 1981 and September 1982. Less cardenolides were sequestered by i n s e c t s reared on seeds c o l l e c t e d in 1982 (mean 150 (range 67 - 196) jig^ vs. mean 208 (range 100 - 336) j u g ^ / i n s e c t ) . The cardenolide content of males and females d i d not d i f f e r s i g n i f i c a n t l y on a per i n s e c t basis when reared on seeds c o l l e c t e d e i t h e r year or on a wet weight b a s i s f o r i n s e c t s reared on Sept. 1981 seeds (Student's t - t e s t ) . However, the cardenolide content/mg wet weight of males was s i g n i f i c a n t l y greater than females f o r i n s e c t s reared on September 1982 seeds (4.5 vs 3.4 ^ign,/mg wet weight, p<0.01, Student's t - t e s t ) . 2) Cardenolide P r o f i l e s of 0. f a s c i a t u s Reared on A. speciosa and  A. s y r i a c a Seeds To i n v e s t i g a t e the p o t e n t i a l c a p a b i l i t i e s and l i m i t a t i o n s of cardenolide sequestration in fj. f a s c i a t u s , the d i f f e r e n c e s and s i m i l a r i t i e s i n the cardenolide array of d o r s o l a t e r a l space f l u i d and a d u l t e x t r a c t s from i n s e c t s reared on the seeds of two d i f f e r e n t species of milkweed (A. speciosa c o l l e c t e d in P e n t i c t o n , B.C. and A. s y r i a c a c o l l e c t e d in W i l l i m a n t i c , Connecticut) were determined by t h i n - l a y e r chromatography 29 (TLC) a n a l y s i s . Since only minor d i f f e r e n c e s were detected between males and females (6,(j), F i g . 2.1), data from both sexes were pooled. Table 2.2 l i s t s the RQ values and i d e n t i t i e s of the cardenolides i n each p r o f i l e . The cardenolides of A. speciosa and A. s y r i a c a seeds have not been i d e n t i f i e d , t h e r e f o r e , cardenolides were i d e n t i f i e d i n t h i s study by c o l o r , Rn. value and pattern (based on c o l o r , r e l a t i v e p o s i t i o n to other cardenolides i n sample and i n t e n s i t y ) . Cardenolides of a wide p o l a r i t y range were a v a i l a b l e from A. speciosa seeds (RQ 0.14 - 1.31; A, F i g . 2.1). Insects reared on t h i s food source, however, p r e f e r e n t i a l l y sequestered cardenolides of a more l i m i t e d p o l a r i t y range: very nonpolar (RQ> 1.20) and p o l a r cardenolides (RQ<0.50) were absent or in low c o n c e n t r a t i o n s . ^ , C, F i g . 2.1, Table 2.2). A nonpolar cardenolide (RQ 1.32) was detected i n male fJ. f a s c i a t u s and p o l a r cardenolides were present i n the f a t body, but these were i n very low concentrations (see Section 3 ) . A more l i m i t e d p o l a r i t y range of cardenolides was present in A. s y r i a c a seeds (RQ 0.38 - 1.24; D, F i g . 2.1). Very nonpolar cardenolides were not detected in i n s e c t s reared on t h i s food source e i t h e r . However, polar cardenolides (RQ<0.50) were sequestered i n greater concentrations in i n s e c t s reared on A. s y r i a c a than in those reared on A. speciosa (E, F, F i g . 2.1). r The cardenolide of highest concentration in i n s e c t s reared on e i t h e r seed was of intermediate p o l a r i t y (RQ approx. 0.62,0, F i g . 2.1, Spot 19, Table 2.2). Another cardenolide of intermediate p o l a r i t y (RQ approx. 0.57, Spot 16, Table 2 . 2 ; © , F i g . 2.1) and l e s s p o l a r cardenolides i n the same p o l a r i t y range as d i g i t o x i n (RQ 1.0,@, F i g . 2.1) were concentrated i n i n s e c t s reared on e i t h e r species of milkweed seed. In a d d i t i o n , f a i r l y nonpolar cardenolides (RQ 1.12 & 1.17, Spot 32 & 33, Table 2.2) were 30 FIGURE 2.1. Cardenolide p r o f i l e s of A. speciosa and A. s y r i a c a seeds and a d u l t e x t r a c t s and d o r s o l a t e r a l space (DLS) f l u i d of 0_. f a s c i a t u s reared on each seed. The r e l a t i v e concentrations of the cardenolides of the d i f f e r e n t samples are not comparable. Symbols: red (non-cardenolide) ; C j , f a i n t blue ;Q » blue;^§), dark b l u e ; ^ , very dark b l u e , ^ , dark blue or very dark blue depending on the e x t r a c t ; /\t seen in only one e x t r a c t ; " j - * seen in >50% of e x t r a c t s ; ^ , seen in a l l samples; (5^ , seen in male only; ^ , seen in female only; J , l i g h t blue t a i l i n g . Sample s i z e s : A. speciosa seeds and samples of i n s e c t s reared on A. speciosa as described i n Figure 2.2. A. s y r i a c a seeds, N=4; A d u l t s , N=8 (4C)!,4c)); D o r s o l a t e r a l space f l u i d , N=8 (40^4^), f l u i d from 1-3 a d u l t s per sample. v 31 1.50 A. speciosa reared 1.25 1.00-Q § 0.75 CD 0.50 0.25 0 A. o+ o o+ o+ o o+ ®* 0+ o+ 0+ o+ o+ o+ o o •* •+ o O 8: A. syriaca reared / \ O* o* o* o* o* o o* o* o o* digitoxigenin O digitoxin >* O ouabain O A B C D E F speciosa Adult DLS A. syriaca Adult DLS Seeds Fluid Seeds Fluid Standards TABLE 2.2. RQ values of cardenolides and non-cardenoUdes ( N C ) A detected In extracts of 0. fasciatus and Its food sources, A. speciosa or A. syriaca. (N) » number of samples. Rp values are reported as mean *_ S.E. TLC spot number A. speciosa seeds (9) Extracts from Insects reared on A. speciosa seeds A. syriaca seeds (4) Extracts reared on A. from Insects syriaca seeds 0. fasciatus adults (10) Dorsolateral space fluid (15) Hemolymph (4) Fat body Eggs (females) (7) (7) Urine/feces (16) 0. fasciatus adults (7) Dorsolateral space fluid (8) 1 0.00 • .000 — _ 2 - - - - 0.05 4 .003 - - - -3 0.14 • .005 - - - - - - - -4 NC - - - - 0.14 4 .004 - - - -5 0.19 • .002 - - - 0.19 4 .005 0.18 4 .003 - - -6 0.22 .003 - - - - - - - -7 MC - - - - 0.23 4 .009 - - - -8 0.26 4 .004 - - - - - - - -9 - - - - 0.31 4 .005 - - 0.27 4 .006 -10 NC 0.32 • .008 - - - - - - - -11 0.35 + .002 - - - 0.36 4 .005 - - - -12 0.40 4 .009 - - - 0.41 0.42 4_ .003 0.38 4 .007 0.34 4 .004 0.34 4 .005 13 NC 0.41 • .007 - - - - - - - -14 0.46 • .006 - - - 0.48 0.46 4 .005 0.46 -15 0.52 4 .012 - - - - 0.52 4 .007 - - -16 - 0.56 • .008 0.58 • .007 0.56 • .009 0.55 4 .008 0.56 4 .005 0.57 4 .007 0.50 4 .004 0.52 4 .004 17 0.59 .016 - - - - - - - -IB NC - - - - - 0.60 4 .010 - - -19 - 0.64 4_ .007 0.66 • .008 0.66 + .009 0.63 4 .008 0.64 4 .003 0.61 4 .006 0.65 4_ .007 0.59 4 .005 0.62 4 .012 20 NC 0.63 • .011 - - - - - -21 . . . . . . . . o.66 22 NX - - - - - - 0.67 23 0.71 • .006 . . . 0.71 . . . 0 .70 • .008 24 - 0.73 + .009 - - 0.76 + .009 - 0.75 • .015 - - 0.73 + .008 25 - 0.80 • .009 0.80 • .008 0.79 .007 0.81 0.78 - 0.78 • .003 0.79 • .004 0.79 +_ .007 26 . . . 0.86 • .010 0.86 • .002 - 0.86 • .008 . . . 27 - 0.89 + .009 0.90 • .006 - 0.88 - - 0.87 • .003 0.90 • .003 0.87 • .009 28 . . . 0.94 • .010 0.94 . . . . . 29 - 0.96 • .005 0.97 • .003 - 0.96 + .006 0.96 0.95 + .009 - 0.97 • .009 0.97 •• .007 30 - 1.04 • .012 1.02^ .005 1.00 006 1.02 • .010 1.02 1.01 +.018 - - 1.02 • .007 31 - - - 1.08 + .003 . . . . . 32 1.10 +.009 1.14 + .010 1.13 +.009 1.11 + .004 - - 1.12 +.004 - 1.10 • .007 1.12 • .008 33 1.19 - 1.20 +.009 - - - - 1.16 + .006 - 1.17 • .007 34 - - - - - - 1.24 • .007 35 1.31 • .014 1.32 - -Total number of cardenolides 13 9 8 7 11 13 12 8 11 10 OO OJ a NoncardenoHdes (NC) are compounds that turned red when sprayed with colorimetric reagents, cardenolides color blue or purple. 34 c o n c e n t r a t e d i n i n s e c t s r e a r e d on A . s y r i a c a . C a r d e n o l i d e s o f t h e s e Rn. v a l u e s were p r e s e n t i n A . s p e c i o s a r e a r e d i n s e c t s , but were i n lower c o n c e n t r a t i o n s . In A . s y r i a c a r e a r e d i n s e c t s , f o u r c a r d e n o l i d e s i n the Rn range o f 1 . 0 - 1 . 2 were c o n c e n t r a t e d i n the d o r s o l a t e r a l space f l u i d , b ut, o n l y two d i f f u s e s p o t s were d e t e c t e d i n t h i s range i n whole a d u l t e x t r a c t s . I t i s p o s s i b l e t h a t a l l f o u r c a r d e n o l i d e s were p r e s e n t i n the a d u l t e x t r a c t s , b ut c o u l d n o t be s e p a r a t e d owing t o i n t e r f e r e n c e from waxes, l i p i d s o r o t h e r compounds i n the a d u l t e x t r a c t s . When r e a r e d on e i t h e r A . s y r i a c a o r A . s p e c i o s a seeds, (D. f a s c i a t u s s e q u e s t e r e d most o f the i n d i v i d u a l c a r d e n o l i d e s p r e s e n t i n the seeds. Nine o f t he t h i r t e e n c a r d e n o l i d e s d e t e c t e d i n e x t r a c t s o f A . s p e c i o s a seeds and s i x o f the e i g h t c a r d e n o l i d e s d e t e c t e d i n e x t r a c t s o f A . s y r i a c a seeds c o r r e s p o n d e d t o c a r d e n o l i d e s i n i n s e c t s r e a r e d on t h e s e seeds ( F i g . 2 . 1 & 2 . 2 , T a b l e 2 . 2 ) . However, twelve c a r d e n o l i d e s d e t e c t e d i n i n s e c t s r e a r e d on A . s p e c i o s a d i d n o t c o r r e s p o n d t o A . s p e c i o s a c a r d e n o l i d e s , t e n o f t h e s e o c c u r r i n g i n Rn, ranges where no c a r d e n o l i d e s were d e t e c t e d i n the seeds ( R Q 0 . 0 0 - 0 . 0 5 and 0 . 7 5 - 1 . 0 5 , F i g . 2 . 1 and 2 . 2 , T a b l e 2 . 2 ) . S i m i l a r l y , e i g h t o f the c a r d e n o l i d e s found i n i n s e c t s r e a r e d on A . s y r i a c a d i d not c o r r e s p o n d t o A . s y r i a c a seed c a r d e n o l i d e s ( F i g . 2 . 1 , T a b l e 2 . 2 ) . Four o f th e s e i n s e c t c a r d e n o l i d e s o c c u r r e d i n Rn. ranges where no c a r d e n o l i d e s were d e t e c t e d i n A . s y r i a c a seeds (Rn. 0 . 2 7 and 0 . 9 7 - 1 . 1 2 ) . The p r e s e n c e o f c a r d e n o l i d e s i n 0. f a s c i a t u s t h a t were not d e t e c t e d i n i t s f o o d source i n d i c a t e s m e t a b o l i s m o f seed c a r d e n o l i d e s i n the seeds by s a l i v a r y enzymes o f 0. f a s c i a t u s , metabolism w i t h i n the i n s e c t , o r c o n c e n t r a t i o n i n the i n s e c t o f seed c a r d e n o l i d e s t h a t were i n such low l e v e l s i n the seeds they were n o t d e t e c t e d i n TLC a n a l y s i s . 35 FIGURE 2.2. Cardenolide p r o f i l e s of 0. f a s c i a t u s a d u l t s , t i s s u e s and s e c r e t i o n s and A. speciosa seeds. The r e l a t i v e concentrations of the cardenolides of the d i f f e r e n t samples are not comparable. Symbols as described in Figure 2.1; ^ , red t a i l i n g . Sample s i z e s : A. speciosa seeds, N=9, 0-5 r e p l i c a t e s per sample; A d u l t s , N=10 3o), 1-5 adults and 0-1 r e p l i c a t e per sample; D o r s o l a t e r a l space (DLS) f l u i d , N=15 (76,8(j)), f l u i d from 1-3 i n d i v i d u a l s and no r e p l i c a t e s per sample. i n d i v i d u a l s and 3 r e p l i c a t e s per sample; Eggs, N=7, 50-200 eggs and no r e p l i c a t e s per sample; Fat body, N=7 ( a l l females), 10-15 f a t bodies and 0-1 r e p l i c a t e per sample; Ur i n e / f e c e s , N=16 (T3,9(J}), 180-1920 excrement hours (EH) = # of i n s e c t s e x c r e t i n g x # hours i n s e c t s excreted, 0-4 r e p l i c a t e s per sample. hemolymph from 24-30 1.50 r 1.25-1.00-Q DC <u 5 0.50 0.25 O O O 8: OcV o+ o+ o ©• o+ o+ o+ o+ o+ o+ o+ o+ o+ o °? •* •* •* o o* 0 A. speciosa Adult DLS Hemo Seeds Fluid lymph o+ O* 8: O o* OA o* o* o o+ o+ o o o o ©A o e digitoxigenin O digitoxin O •+ o+ ouabain O Eggs Fat Urine/ Standards Body Feces 37 The cardenolide p r o f i l e s of a d u l t e x t r a c t s and d o r s o l a t e r a l space f l u i d of i n s e c t s reared on the two d i f f e r e n t milkweed seeds were qu i t e s i m i l a r . Nine of the ten cardenolides detected i n adults and d o r s o l a t e r a l space f l u i d of i n s e c t s reared on A. speciosa seeds corresponded c l o s e l y to cardenolides found i n the same e x t r a c t s of A. s y r i a c a reared i n s e c t s (Table 2.2). Only four of the t h i r t e e n cardenolides present i n a d u l t e x t r a c t s and d o r s o l a t e r a l space f l u i d of fj. f a s c i a t u s reared on A. s y r i a c a were not found i n the same e x t r a c t s of A. speciosa reared i n s e c t s . However, they were detected i n low concentrations i n e x t r a c t s of other t i s s u e s of A. speciosa reared i n s e c t s (Spots 9, 12, 14, 24; Table 2.2). In a d d i t i o n , the highly concentrated cardenolides in i n s e c t s reared on A. speciosa (fe *®> F i g . 2.1; Spots 16, 19, 30, Table 2.2) corresponded to three of the most concentrated cardenolides i n i n s e c t s reared on A. s y r i a c a . The s i m i l a r i t i e s i n the cardenolide p r o f i l e s of fj. f a s c i a t u s reared on the two d i f f e r e n t milkweed seeds do not r e f l e c t s i m i l a r i t i e s in the cardenolide array of the two seeds. Only two of the nine cardenolides found i n common i n ad u l t e x t r a c t s and d o r s o l a t e r a l space f l u i d of fj. f a s c i a t u s reared on e i t h e r food source were found i n both species of milkweed seeds. Furthermore, two of the highly concentrated cardenolides i n A. s y r i a c a reared i n s e c t s were a l s o the most concentrated cardenolides in t h e i r food source (Spots 16 & 19, Table 2.2), whereas these same ca r d e n o l i d e s , although highly concentrated i n A. speciosa reared i n s e c t s , were not hig h l y concentrated i n A. speciosa seeds. 38 3) Cardenolide P r o f i l e s of Adult E x t r a c t s , Tissues and D o r s o l a t e r a l Space  F l u i d of 0. f a s c i a t u s Reared on A. speciosa seeds To determine i f d i f f e r e n t cardenolides were p r e f e r e n t i a l l y sequestered i n d i f f e r e n t t i s s u e s of 0_. f a s c i a t u s , the cardenolide array of a d u l t e x t r a c t s , d o r s o l a t e r a l space f l u i d , u r i n e / f e c e s , f a t body, hemolymph and egg e x t r a c t s of i n s e c t s reared on A. speciosa seeds was i n v e s t i g a t e d by TLC a n a l y s i s . The cardenolide p r o f i l e s of a l l the e x t r a c t s t e s t e d were s i m i l a r in terms of the i n d i v i d u a l cardenolides sequestered and t h e i r r e l a t i v e concentrations ( F i g . 2.2, Table 2.2). The cardenolide array of both sexes was analyzed f o r each e x t r a c t , but, since only minor d i f f e r e n c e s were detected between male and female samples ($, (j), F i g . 2.2) the data from both sexes were pooled. The cardenolide arrays of the a d u l t , d o r s o l a t e r a l space f l u i d , hemolymph and eggs showed the g r e a t e s t s i m i l a r i t y : 7/8, 5/7 and 7/11 (87, 71 and 64%) of the cardenolides detected i n the d o r s o l a t e r a l space f l u i d , hemolymph, and eggs, r e s p e c t i v e l y , corresponded to the a d u l t c a r d e n o l i d e s . The most concentrated cardenolide in the i n s e c t (Rn 0.64) was a l s o the most concentrated cardenolide i n the d o r s o l a t e r a l space f l u i d , hemolymph and eggs. T h i s cardenolide was a l s o concentrated i n the f a t body and u r i n e / f e c e s , but both e x t r a c t s contained two more po l a r cardenolides of s i m i l a r c o n c e n t r a t i o n . The c a r d e n o l i d e arrays of the f a t body and u r i n e / f e c e s were s i m i l a r to the other i n s e c t t i s s u e s in the intermediate p o l a r i t y range Rn 0.55-1.10, but d i f f e r e d owing to the presence of low concentrations of cardenolides in the p o l a r range Rn<0.50 and the concentration of both intermediate and more p o l a r cardenolides (A ® , F i g . 2.2). Large amounts of u r i n e / f e c e s , f a t body and eggs were needed to obtain e x t r a c t s with cardenolide 39 concentrations high enough to v i s u a l i z e in TLC a n a l y s i s . As a consequence, the d e t e c t i o n of cardenolides in low concentrations was enhanced i n these t i s s u e s and may e x p l a i n , in p a r t , the greater number of cardenolides detected in these samples and the d e t e c t i o n of cardenolides i n the p o l a r range RQ<0.50 i n f a t body and u r i n e / f e c e s samples. Large sample s i z e s could not be used f o r the other i n s e c t samples because of severe t a i l i n g of h i g h l y concentrated c a r d e n o l i d e s . Cardenolides in the p o l a r range RQ<0.50 may be present in low concentrations in the other samples s i n c e f a i n t blue t a i l i n g was detected between the o r i g i n and RQ 0.55 in most samples (j , F i g . 2.2). A l t e r n a t i v e l y , t h i s t a i l i n g may represent n o n - s p e c i f i c binding of cardenolides to various compounds such as p r o t e i n s , waxes, pigments or l i p i d s . Very nonpolar cardenolides were absent or i n low concentrations in a l l e x t r a c t s of the i n s e c t . Adult and d o r s o l a t e r a l space f l u i d e x t r a c t s contained cardenolides of the lowest p o l a r i t y (RQ 1.32 and 1.20, r e s p e c t i v e l y ) , which correspond to the most nonpolar cardenolides detected in the seeds. However, the very nonpolar cardenolide (RQ 1.32) i n the a d u l t male was apparently in very low c o n c e n t r a t i o n s : i t was only detected in a pooled sample of 5 males, never i n e x t r a c t s of s i n g l e males. Low concentrations of very nonpolar cardenolides may a l s o occur i n the f a t body since e x t r a c t s produced f a i n t blue t a i l i n g from RQ 1.04 to approximately 1.32 (j , F i g . 2.2). With the exception of u r i n e / f e c e s , the array of cardenolides detected i n d i f f e r e n t e x t r a c t s of the same i n s e c t t i s s u e was q u i t e constant (Table 2.3, + and * in F i g . 2.1 and 2.2). Much of the v a r i a b i l i t y between e x t r a c t s of the same t i s s u e was the r e s u l t of the d i f f i c u l t y i n d e t e c t i n g cardenolides of low concentrations; owing to t h e i r weak i n t e n s i t y and/or 40 Table 2.3. The v a r i a b i l i t y i n cardenolide p r o f i l e s of fj. f a s c i a t u s and A. speciosa. V a r i a b i l i t y i s expressed as the percentage of i n d i v i d u a l c ardenolides seen i n 50% or more of the e x t r a c t s of each sample. Sample % A. speciosa seeds 56 Urine/feces 25 Adults 70 Eggs 64 Hemolymph 100 D o r s o l a t e r a l space f l u i d 100 Fat body 62 V 41 masking by t a i l i n g of cardenolides of higher concentrations they were d i f f i c u l t to detect. However, most of the cardenolides detected i n u r i n e / f e c e s occurred in l e s s than h a l f of the e x t r a c t s t e s t e d , i n d i c a t i n g a real d i f f e r e n c e in cardenolide p r o f i l e s between samples. In a d d i t i o n , the cardenolides of g r e a t e s t concentration v a r i e d between samples of u r i n e / f e c e s F i g . 2.2), whereas, i n d i f f e r e n t e x t r a c t s of a l l other t i s s u e s the cardenolide of g r e a t e s t concentration was constant (fe F i g . 2.1 and 2.2). 4) Geographic D i f f e r e n c e s in the Cardenolide P r o f i l e of A. s y r i a c a seeds Previous studies of sequestration of A s c l e p i a d cardenolides i n CJ. f a s c i a t u s used A. s y r i a c a seeds from Missouri and Ontario as the i n s e c t s ' food source ( F e i r and Suen, 1971; Duffey and Scudder, 1974). I t i s p o s s i b l e t h a t d i f f e r e n c e s in the r e s u l t s between these s t u d i e s and the present study are a r e s u l t of geographic v a r i a t i o n in the cardenolide p r o f i l e of seeds of A. s y r i a c a . Geographical v a r i a t i o n has not been e s t a b l i s h e d f o r the seeds of A. s y r i a c a , t h e r e f o r e , I i n v e s t i g a t e d t h i s by determining the cardenolide array of A. s y r i a c a seeds from the f o l l o w i n g four l o c a t i o n s : Montebello, Quebec, ( c o l l e c t e d October, 1976); Ottawa, Ontario ( c o l l e c t e d September, 1976); Cleveland, Ohio ( c o l l e c t e d September, 1975); and W i l l i m a n t i c , Connecticut ( c o l l e c t e d September 1981). The cardenolide array of the seeds c o l l e c t e d in Ontario, Ohio and Quebec showed only minor d i f f e r e n c e s i n the i n d i v i d u a l cardenolides present and t h e i r r e l a t i v e c o n c e n t r a t i o n s , but were d i s s i m i l a r to the cardenolide p r o f i l e of the Connecticut seeds ( F i g . 2.3). 42 1.50 r 1.25 1.00 O DC 0.50 0.25 -O* O* o* o* o* o* O A •* O A ©• ©* 0* O A o* ©« o* ®* ©* o* )* digitoxigenin O digitoxin O ouabain O Connecticut Ontario Ohio Quebec Standards FIGURE 2.3. Cardenolide profiles of A. syriaca seeds from four geographic locations. Symbols as described in Figure 2.1. Sample sizes: N=2 for Ontario, Ohio and Quebec seeds; N=4 for Connecticut seeds. 43 DISCUSSION This study was undertaken to c l a r i f y c e r t a i n aspects of the d i f f e r e n t i a l d i s t r i b u t i o n of natural cardenolides i n 0. f a s c i a t u s , both i n terms of quantity and p o l a r i t y , and to document some of the c a p a b i l i t i e s of the sequestration process of cardenolides in 0_. f a s c i a t u s . My r e s u l t s show t h a t l a r g e amounts of cardenolides do not accumulate i n the f a t body, gut and wings of 0. f a s c i a t u s ; the g r e a t e s t amount of cardenolides in the i n s e c t i s i n the d o r s o l a t e r a l space. The d i s t r i b u t i o n of cardenolides in 0_. f a s c i a t u s i s summarized in Table 2.4. Blum (1983) has suggested that each species of i n s e c t has a unique process of s e q u e s t r a t i o n , t o l e r a n c e and defensive use of the t o x i n s ingested with i t s food p l a n t . The d i f f e r e n c e s in the d i s t r i b u t i o n of cardenolides in 0_. f a s c i a t u s and two other i n s e c t s , Danaus plexippus and Cycnia inopinatus, support t h i s argument. Low amounts of cardenolides are sequestered i n the wings, f a t body, gut and hemolymph of 0_. f a s c i a t u s , whereas, s u b s t a n t i a l amounts of cardenolides are found in these t i s s u e s i n the monarch (see Blum, 1981, 1983). C_. inopinatus d i f f e r s from both the monarch and 0. f a s c i a t u s in the presence of l a r g e amounts of cardenolides in the hemolymph and wings, but n e g l i g i b l e amounts of these compounds in the gut (see Blum, 1983). I t appears that 0. f a s c i a t u s , u n l i k e £ . plexippus and C_. inopinatus, may not t o l e r a t e l a r g e q u a n t i t i e s of cardenolide in the hemolymph. Thus, the r a p i d uptake and accumulation of cardenolides i n the d o r s o l a t e r a l space may f u n c t i o n i n the i n s e c t ' s t o l e r a n c e of sequestered cardenolides by maintaining low l e v e l s of cardenolides i n the hemolymph (Duffey e t aj_., 1978) and other t i s s u e s , i . e . f a t body and wings ( t h i s study). Furthermore, the low cardenolide content of the wings i n 0. f a s c i a t u s i n d i c a t e s that the a n t i - p r e d a t o r s t r a t e g i e s 44 Table 2.4. D i s t r i b u t i o n of cardenolides in Oncopeltus f a s c i a t u s expressed as % of the a d u l t t o t a l . BDL = below de t e c t i o n l i m i t of assay (13 nmoles or 6.5 x I O - 6 M d i g i t o x i n e q u i v a l e n t s ) . Sample % of Adult Total Reference 9 D o r s o l a t e r a l space 60-95 1 D o r s o l a t e r a l space f l u i d 46-89 2 b Hemolymph BDL 1,2 C Urine/feces BDL 1 Metathoracic gland BDL 1 Fat body (male) BDL 2 Fat body (female) 4-5 2 Gut with contents BDL 2 Wings BDL 2 a 1 = Duffey and Scudder, 1974 using A s c l e p i a s s y r i a c a f o r the food source. 2 = T h i s study, using A. speciosa f o r the food source. b C a l c u l a t e d using estimate of 2-3 jul f o r volume of d o r s o l a t e r a l space f l u i d i n a d u l t (Duffey and Scudder, 1974). c Unpublished r e s u l t s . 45 of fJ. f a s c i a t u s may d i f f e r from D_. plexippus and C. inopinatus. In the monarch, the g r e a t e s t concentration of cardenolides i s i n the wings and t h i s i s thought to f u n c t i o n as an a n t i - p r e d a t o r s t r a t e g y , causing avian predators to r e j e c t the b u t t e r f l y r e l a t i v e l y unharmed and preventing attack of the more c r i t i c a l areas, the thorax and abdomen (Brower and G l a z i e r , 1975). I t i s u n l i k e l y that the wings of fJ. f a s c i a t u s would f u n c t i o n e f f e c t i v e l y i n t h i s manner. Owing to the small body s i z e of fJ. f a s c i a t u s 17-18 mm) and the p o s i t i o n i n g of the wings at r e s t f l a t along the back, l a r g e predators probably snatch the e n t i r e i n s e c t upon attac k . Thus, f o r fj. f a s c i a t u s , concentration of cardenolides i n the d o r s o l a t e r a l space and r e l e a s e of d o r s o l a t e r a l space f l u i d along the thorax and abdomen i s probably a more e f f e c t i v e a n t i - p r e d a t o r strategy (Duffey and Scudder, 1974; Scudder and Meredith, 1982a). The r e s u l t s from the TLC a n a l y s i s i n d i c a t e that there are only minor d i f f e r e n c e s i n the cardenolides sequestered and concentrated i n various t i s s u e s and s e c r e t i o n s of CJ. f a s c i a t u s reared on seeds of a s i n g l e species of milkweed. The presence i n the f a t body of low concentrations of p o l a r cardenolides not detected in other t i s s u e s may i n d i c a t e a d i f f e r e n c e in the cardenolide array sequestered i n t h i s t i s s u e . A l t e r n a t i v e l y , t h i s d i f f e r e n c e may be the r e s u l t of enhanced de t e c t i o n of cardenolides in low concentrations i n the f a t body (see R e s u l t s ) . Only minor d i f f e r e n c e s were detected i n the cardenolide p r o f i l e s of a d u l t e x t r a c t s and d o r s o l a t e r a l space f l u i d of CJ. f a s c i a t u s reared on two food sources which appear to have d i f f e r e n t cardenolide a r r a y s . T h i s i n d i c a t e s t h a t the cardenolide array of the i n s e c t can remain f a i r l y constant despite d i f f e r e n c e s i n the cardenolide p r o f i l e s of i t s food p l a n t s . These r e s u l t s d i f f e r from st u d i e s in which v a r i a t i o n i n 46 cardenolide p r o f i l e s of monarchs has been r e l a t e d to i n t e r s p e c i f i c d i f f e r e n c e s in the cardenolide array of t h e i r food p l a n t s (Roeske e t a l . , 1976; Brower et a l . , 1984a). However, two other s t u d i e s of sequestration of cardenolides in 0. f a s c i a t u s reared on A. s y r i a c a seeds ( F e i r and Suen, 1971; Duffey and Scudder, 1974) suggest that the cardenolide p r o f i l e of 0_. f a s c i a t u s may e x h i b i t d i f f e r e n c e s r e l a t e d to i n t r a s p e c i f i c v a r i a t i o n in the cardenolide p r o f i l e s of i t s food p l a n t . In my study seven c a r d e n o l i d e s were detected i n i n s e c t s reared on A. s y r i a c a seeds c o l l e c t e d i n Connecticut and most of the cardenolides i n the seeds were sequestered in the i n s e c t . In c o n t r a s t , F e i r and Suen (1971) found that only four cardenolides were sequestered in i n s e c t s reared on A. s y r i a c a seeds c o l l e c t e d in M i s s o u r i and only one of these corresponded to a seed c a r d e n o l i d e . I found only minor d i f f e r e n c e s in the cardenolide p r o f i l e s of a d u l t e x t r a c t s and d o r s o l a t e r a l space f l u i d of i n s e c t s reared on A. s y r i a c a seeds from Connecticut, and that the h i g h l y concentrated cardenolides in the i n s e c t covered a wide range of p o l a r i t i e s . However, i n s e c t s reared on A. s y r i a c a seeds from Ontario e x h i b i t e d a predominance of p o l a r cardenolides and the cardenolides of the d o r s o l a t e r a l space f l u i d e x h i b i t e d a much smaller p o l a r i t y range than seen in the whole i n s e c t (Duffey and Scudder, 1974). The d i f f e r e n c e s i n the cardenolide p r o f i l e s of 0_. f a s c i a t u s i n these s t u d i e s may be explained, i n p a r t , by the d i f f e r e n t e x t r a c t i o n methods, solvent "systems and d e t e c t i o n reagents used in the TLC analyses. In a d d i t i o n , the d i f f e r e n c e s in the cardenolide arrays of the i n s e c t may r e f l e c t v a r i a t i o n in the cardenolide p r o f i l e s of the A. s y r i a c a seeds the i n s e c t s were reared on. Geographic v a r i a t i o n in the cardenolide array of v e g e t a t i v e parts of A. s y r i a c a has been well e s t a b l i s h e d (see Roeske e t al_., 1976 f o r review). My r e s u l t s e s t a b l i s h i n g a d i f f e r e n t c ardenolide array in A. s y r i a c a seeds c o l l e c t e d i n Connecticut than in 47 seeds of A. s y r i a c a c o l l e c t e d in Quebec, Ontario, and Ohio, and the d i f f e r e n c e s i n the numbers of cardenolides detected i n the seeds of the d i f f e r e n t populations of A. s y r i a c a used in the s t u d i e s mentioned above, i n d i c a t e t hat geographic v a r i a t i o n a l s o occurs in the seeds of A. s y r i a c a . In v i v o and in v i t r o evidence with two non-Asclepiad c a r d e n o l i d e s , ouabain and d i g i t o x i n , i n d i c a t e s t h a t d i f f e r e n t i a l e x c r e t i o n and metabolism of cardenolides i n CJ. f a s c i a t u s as well as p r e f e r e n t i a l uptake of i n d i v i d u a l cardenolides across the gut and i n t o the d o r s o l a t e r a l space are involved in the s e l e c t i v e sequestration of cardenolides in £ . f a s c i a t u s (Scudder and Meredith, 1982b; Duffey e t ^1_., 1978; Meredith et al_., 1984). However, ouabain and d i g i t o x i n do not occur in the host plants of fj. f a s c i a t u s . The present study provides evidence f o r d i f f e r e n t i a l e x c r e t i o n and metabolism in CJ. f a s c i a t u s of cardenolides present in i t s natural food p l a n t s . The presence of many cardenolides in CJ. f a s c i a t u s that were not detected in i t s food sources suggests metabolism of seed cardenolides or concentration in the i n s e c t of seed cardenolides present in such low concentrations they were not detected by TLC a n a l y s i s . Metabolism of seed cardenolides may occur i n the seed while i t i s being digested by the s a l i v a of the i n s e c t , i n the i n s e c t gut by b a c t e r i a or the gut m i l i e u , and/or i n other t i s s u e s w i t h i n the i n s e c t . Two other i n s e c t species are known to metabolize cardenolides ingested with t h e i r food p l a n t (Seiber e t a l . , 1980; Brower e t a l . , 1982; Levey, 1983): i n one of these, the monarch, homogenates of both the gut and f a t body metabolized the A s c l e p i a d c a r d e n o l i d e , u s c h a r i d i n (Marty and K r i e g e r , 1984). Very p o l a r and very nonpolar cardenolides were absent or in very low concentrations in fJ. f a s c i a t u s reared on A. speciosa or A. s y r i a c a . The d i f f e r e n t i a l e x c r e t i o n of l a r g e amounts of intermediate and higher p o l a r i t y 48 cardenolides r e l a t i v e to cardenolides of lower p o l a r i t y in the u r i n e / f e c e s may e x p l a i n , i n p a r t , the low l e v e l s of p o l a r cardenolides in 0_. f a s c i a t u s when feeding upon seeds of A. speciosa. Rapid metabolism of nonpolar cardenolides i n the i n s e c t may a l s o e x p l a i n the absence of these cardenolides in 0. f a s c i a t u s . Seiber e t al_. (1980) have shown i n the monarch t h a t r a p i d metabolism of several l e s s polar cardenolides to more pol a r metabolites r e s u l t s i n the absence of or very low concentrations of these cardenolides in the l a r v a l t i s s u e . The sequestration and concentration of cardenolides of a wide p o l a r i t y range in _0. f a s c i a t u s reared on A. s y r i a c a or A. speciosa suggests t h a t cardenolides are not sequestered in the i n s e c t simply on the basis of p o l a r i t y . The importance of physical-chemical c h a r a c t e r i s t i c s other than p o l a r i t y i n cardenolide sequestration in Q_. f a s c i a t u s i s a l s o i n d i c a t e d by the s e q u e s t r a t i o n and concentration of intermediate and more po l a r cardenolides in the f a t body of 0_. f a s c i a t u s reared on A. s p e c i o s a . T h i s was unexpected since the f a t body often accumulates nonpolar compounds and toxins owing to t h e i r l i p o p h i l i c nature ( K i l b y , 1963), and e a r l i e r observations i n d i c a t e d p r e f e r e n t i a l sequestration of the nonpolar cardenolide d i g i t o x i n in the f a t body of a d u l t 0. f a s c i a t u s (Duffey e t a l . , 1978). The importance of physical-chemical c h a r a c t e r i s t i c s other than p o l a r i t y in cardenolide sequestration has a l s o been i n d i c a t e d in the monarch b u t t e r f l y (Seiber ^ t ^ . , 1980; Brower et a l . , 1982) and Seiber e t a l . , (1980) have suggested that physical-chemical c h a r a c t e r i s t i c s that i n f l u e n c e the chemical s t a b i l i t y , s o l u b i l i t y and ease of t r a n s p o r t of i n d i v i d u a l cardenolides and t h e i r binding a f f i n i t y to blood p r o t e i n s and sequestration systems in the i n s e c t are involved. In summary, t h i s study provides evidence that cardenolides are 49 d i f f e r e n t i a l l y d i s t r i b u t e d i n fJ. f a s c i a t u s i n terms of t o t a l q u a n t i t y . In c o n t r a s t , only minor d i f f e r e n c e s were detected i n the cardenolide array of adults and f i v e t i s s u e s and s e c r e t i o n s of fJ. f a s c i a t u s . T h i s study a l s o provides evidence t h a t cardenolides of a wide p o l a r i t y range can be sequestered i n fj. f a s c i a t u s , however, very nonpolar and very p o l a r cardenolides are not sequestered or are present i n extremely low conc e n t r a t i o n s . In a d d i t i o n , the r e s u l t s suggest that metabolism and d i f f e r e n t i a l e x c r e t i o n of cardenolides may be part of the s e l e c t i v e sequestration process of cardenolides i n fj. f a s c i a t u s . F i n a l l y , the constancy of the cardenolide p r o f i l e s of fj. f a s c i a t u s reared on seeds of two species of milkweed with very d i f f e r e n t c a r denolide a r r a y s , and between t i s s u e s of fJ. f a s c i a t u s reared on a s i n g l e species o f milkweed seeds i n d i c a t e s t h a t there i s q u a l i t a t i v e r e g u l a t i o n of the cardenolide array i n CJ. f a s c i a t u s , as i n the monarch (Brower e t a l . , 1 9 8 2 ) . (Material i n t h i s chapter i s in press: Moore, L.V. and G.G.E. Scudder. 1 9 8 5 . S e l e c t i v e sequestration of milkweed ( A s c l e p i a s sp.) cardenolides i n Oncopeltus f a s c i a t u s (Dallas)(Hemiptera: Lygaeidae). J . Chem. Ecol.) 50 CHAPTER 3: EXCRETION OF OUABAIN BY MALPIGHIAN TUBULES  OF ONCOPELTUS FASCIATUS Summary An i n v i t r o preparation of Malpighian tubules was used to i n v e s t i g a t e the e x c r e t i o n of the p o l a r c a r d e n o l i d e , ouabain, i n 0. f a s c i a t u s . Both segments of the tubules were found to metabolize ouabain. The d i s t a l segment (Segment II) secreted primary urine and ouabain. S e c r e t i o n of ouabain by Segment II was not observed to occur a g a i n s t a concentration gradient and increased with i n c r e a s i n g f l u i d s e c r e t i o n . The proximal segment (Segment I) reabsorbed f l u i d and ouabain but not metabolites. Ouabain was reabsorbed a g a i n s t a strong concentration gradient ( 2 3 - f o l d ) , was independent of f l u i d r e a b s o r p t i o n , and increased with i n c r e a s i n g f l u i d s e c r e t i o n by Segment I I . In r a p i d l y s e c r e t i n g Malpighian tubules (a s i t u a t i o n of high cardenolide s e c r e t i o n by Segment I I ) , the presence of Segment I reduced the e x c r e t i o n of ouabain by 84 - 93%, mainly by reducing ouabain c o n c e n t r a t i o n . I t appears excretory l o s s of cardenolides can be reduced i n 0. f a s c i a t u s and thus may be a f a c t o r i n the sequestration of cardenolides i n t h i s i n s e c t . 51 INTRODUCTION In the previous chapter, s e l e c t i v e sequestration of host p l a n t cardenolides in CJ. f a s c i a t u s was demonstrated. In t h i s chapter, the s e c r e t i o n , metabolism and reabsorption of the p o l a r c a r d e n o l i d e , ouabain, i s i n v e s t i g a t e d under a v a r i e t y of c o n d i t i o n s to determine the p o s s i b l e r o l e of the Malpighian tubules i n the s e l e c t i v e sequestration of cardenolides in 0. f a s c i a t u s . Since the Malpighian tubules are an important p a r t of the excretory system in i n s e c t s , they are a p o t e n t i a l s i t e of metabolism and l o s s of cardenolides that might otherwise be sequestered in CJ. f a s c i a t u s . The r o l e of Malpighian tubules i n i n s e c t excretory systems has been e x t e n s i v e l y reviewed by P h i l l i p s (1981) and the gross morphology and" physiology of the Malpighian tubules of fj. f a s c i a t u s have been well described by Meredith et j»l_. (1984). However, general aspects of i n s e c t Malpighian tubules, as well as s p e c i f i c aspects of the Malpighian tubules of CJ. f a s c i a t u s p e r t i n e n t to the work described in the present chapter w i l l be reviewed here. In i n s e c t excretory systems, the formation of primary urine from the hemolymph occurs i n the Malpighian tubules. The primary urine contains many of the s o l u t e s present i n the blood and i s secreted i n t o the hindgut where, in most i n s e c t , s o l u t e s and water can be conserved by r e a b s o r p t i o n . In general, reabsorption of s o l u t e s and f l u i d does not occur in the tubules. The Malpighian tubules a l s o f u n c t i o n i n the r a p i d clearance of toxins from the hemolymph by a c t i v e l y s e c r e t i n g these compounds. fj. f a s c i a t u s adults commonly have four s i m i l a r Malpighian tubules, approximately 15 mm i n length, that empty d i r e c t l y i n t o the f o u r t h 52 v e n t r i c u l u s (pylorus) of the gut at t h e i r proximal ends. Each tubule c o n s i s t s of at l e a s t two morphologically and p h y s i o l o g i c a l l y d i s t i n c t segments. The long (11 mm) d i s t a l Segment II fu n c t i o n s i n the s e c r e t i o n of primary u r i n e , which appears to be secreted by the process common to many i n s e c t s , a c t i v e K + s e c r e t i o n with C l " as the accompanying ion ( P h i l l i p s , 1982). The proximal Segment I fun c t i o n s i n f l u i d reabsorption from the Malpighian tubule lumen to the hemolymph side of the tubule. In the f o l l o w i n g study, an i n v i t r o preparation of Malpighian tubules i s used to i n v e s t i g a t e the e x c r e t i o n of the po l a r c a r d e n o l i d e , ouabain, i n 0. f a s c i a t u s . P r e l i m i n a r y experiments (Meredith et , 1984) i n d i c a t e d that ouabain i s secreted by Segment II and reabsorbed by Segment I of the tubules. In t h i s chapter, the s e c r e t i o n and metabolism of ouabain by Segment II and the reabsorption of ouabain and i t s metabolites i n Segment I i s examined under, a v a r i e t y of c o n d i t i o n s . In a d d i t i o n , the m o d i f i c a t i o n by Segment I of the primary s e c r e t i o n from Segment II i s determined. Thin l a y e r chromatography i s used to detect the presence of ouabain and i t s metabolites i n the secreted and reabsorbed f l u i d s of the Malpighian tubules. Ouabain with [ 3H]ouabain i s used owing to i t s r e l a t i v e l y high s o l u b i l i t y i n s a l i n e which f a c i l i t a t e s the de t e c t i o n of the cardenolide and i t s metabolites i n the extremely small volumes ( n l ) of f l u i d secreted and reabsorbed by the Malpighian tubules. MATERIALS AND METHODS 1) Insects Adult male and female Oncopeltus f a s c i a t u s taken from a la b o r a t o r y c u l t u r e maintained at 26 °C under a 16:8 l i g h t : d a r k c y c l e were used i n a l l 53 experiments. The i n s e c t s were reared on milkweed seeds ( A s c l e p i a s  s p e c i o s a ) . 2) S a l i n e s The s a l i n e s used i n t h i s study were based on the composition of fj. f a s c i a t u s hemolymph (unpublished observations; F l o r k i n and Jeuniaux, 1974; Staddon and Everton, 1980) and contained the f o l l o w i n g : NaCl, 20 mM; KC1, 24 mM; MgCl 2, 2 mM; C a C l 2 , 2 mM; glucose, 6.7 mM; NaH 2P0 4, 2.5 mM; Naj^HPO^, 3.5 mM. In l a t e r experiments glutamine, 2.7 mM; a l a n i n e , 5 mM; and p r o l i n e , 5 mM were included i n s a l i n e s . The pH equaled 6.9 i n a i r and osmotic pressure was v a r i e d by adding sucrose. Tubules were d i s s e c t e d i n s a l i n e with an osmotic pressure equal to t h a t of CJ. f a s c i a t u s hemolymph (326 mOsm). Tubules were stimulated to secrete by i n c l u d i n g c y c l i c AMP in the bathing s a l i n e s . C y c l i c AMP (2.5 x 10"^ M) served to both increase (approximately 40 f o l d ) and prolong f l u i d s e c r e t i o n . The ex c r e t i o n of ouabain was followed using [ ^ o u a b a i n (general l a b e l , s p e c i f i c a c t i v i t y 14.0 or 18.0 Ci/mM, 98.5% radiochemical p u r i t y ) obtained from New England Nuclear. 3) In v i t r o Malpighian tubule preparation Malpighian tubules were prepared using an in v i t r o technique modified from Szibbo and Scudder (1979). Tubules were d i s s e c t e d using f i n e g l a s s needles with ends that were sealed and s l i g h t l y curved i n a gas flame. The requi r e d tubule segments were severed, t r a n s f e r r e d to a drop of s a l i n e held in a depression i n a watch glas s l i n e d with Sylgard (Dow Corning), and covered with l i q u i d p a r a f f i n o i l ( F i g . 3.1). Tubule segments were tethered at t h e i r proximal end by a f i n e s i l k l i g a t u r e , then drawn out into the o i l 54 bathing saline paraffi oil V reabsorbed fluid secreted f luid igature weights Segment I Sylgard resin Segment H Figure 3.1. In v i t r o preparation of 0. f a s c i a t u s Malpighian tubules. 55 and anchored i n t o p o s i t i o n by small weights. S e c r e t i o n was c o l l e c t e d by n i c k i n g the tubules with a sharp m i c r o p i p e t t e immediately d i s t a l to the 1igature. In a l l cases d i s s e c t i o n r e q u i r e d 1.5 - 2 h and time 0 was considered to be the s t a r t of c o l l e c t i o n of s e c r e t i o n . Experiments were conducted at 21-23 °C and the bathing s a l i n e changed hourly. Under these c o n d i t i o n s Malpighian tubules continued to secrete f o r at l e a s t 18 h, although most experiments were concluded by 8 h. Volumes and r a t e s of f l u i d s e c r e t i o n and reabsorption were c a l c u l a t e d according to the method of Maddrell (Maddrell, 1969), from measurements of the diameters of secreted drops. Radioactive samples were e i t h e r counted d i r e c t l y i n 10 ml of commercial s c i n t i l l a t i o n f l u i d using a Beckman LS 9000 s c i n t i l l a t i o n counter or chromatographed. C o r r e c t i o n f o r varying counting e f f i c i e n c e s was performed using the "H number method" (Anonymous, 1979). Ionic composition of whole tubule s e c r e t i o n , reabsorbed f l u i d and bathing s a l i n e was determined by e l e c t r o n microprobe a n a l y s i s as described in Strange e t al_. (1982). 4) Chromatography Thin l a y e r chromatography p l a t e s prepared with S i l i c a Gel G (Redi/ P l a t e , F i s h e r S c i . Co., gel 250 ^im t h i c k ) or c e l l u l o s e (Eastman Kodak Chromatogram Sheet, adsorbent 160 ^im t h i c k ) were used. S i l i c a Gel G p l a t e s were a c t i v a t e d f o r 24 hours over concentrated H2SO4 i n a c l o s e d chromatography tank p r i o r to and 12 h a f t e r sample a p p l i c a t i o n . Samples 11 were a p p l i e d 1 from the bottom of the p l a t e . F i f t y to seventy-fivejugm each of three c o l d standards, ouabain, ouabagenin and rhamnose (Sigma Chemical Co.), were spotted on top of the samples. Both ouabagenin and rhamnose are known metabolites of ouabain. Chromatography tanks were l i n e d 56 with Whatman Chromatography Paper #1 (W&R Ba l s t o n . Ltd.) and e q u i l i b r a t e d with 300 mis of the solvent system. P l a t e s were developed to a distance of 15 - 16 cm from the o r i g i n . To detect the ca r d e n o l i d e s , ouabain and ouabagenin, p l a t e s were sprayed with 2.5 ml of 2,4,2',4'-tetranitrodiphenyl (TNDP, 0.5 gm/100 ml toluene) followed by NaOH (10% in MeOH) (Neher, 1969). Under these c o n d i t i o n s , TNDP re a c t s with a lactone r i n g to form a blue c o l o r and i s r e l a t i v e l y s p e c i f i c f o r cardenolides (Nover, 1972). To detect rhamnose, p l a t e s were sprayed with a p-anisaldehyde-H2S04 reagent (Stahl and Kaltenbach, 1965). P l a t e s could be sprayed f o r rhamnose then a i r d r i e d and sprayed f o r cardenolide d e t e c t i o n , but not v i c e versa. Samples were analyzed i n three chromatographic systems (Table 3.1). Three a d d i t i o n a l ethyl acetate:MeOH systems were t r i e d (EA:M 95:5, 90:10, and 50:50), however, they d i d not c l e a r l y separate the three standards. A l l c a l c u l a t i o n s and s t a t i s t i c a l a n a l y s i s were done with r e s u l t s from System I because i t produced d i s t i n c t spots with very l i t t l e t a i l i n g . T h i s system d i d not separate rhamnose and ouabain, but no peak of r a d i o a c t i v i t y i n d i c a t i n g rhamnose was detected i n any of the samples i n System II and I I I . The p l a t e s were analyzed f o r r a d i o a c t i v i t y as f o l l o w s . Chromatograms were d i v i d e d i n t o columns 1 cm wide extending from 1 cm below the o r i g i n to the s o l v e n t f r o n t and i n c l u d i n g the standard spots. These were d i v i d e d i n t o 0.5 cm s e c t i o n s , each of which was scraped i n t o a s c i n t i l l a t i o n v i a l c o n t a i n i n g 0.5 ml H2O. Samples were leached overnight and counted as described p r e v i o u s l y . Neither incubation i n o i l nor the mixing with c o l d Malpighian tubule s e c r e t i o n followed by overnight incubation i n o i l a l t e r e d the chromatographic behavior of s a l i n e s c o n t a i n i n g pure ouabain l a b e l l e d with [ 3H]ouabain. R a d i o a c t i v i t y i n these c o n t r o l s and a nonincubated TABLE 3.1. Chromatographic systems used for the separation of cardiac glycosides and their metabolites. Solvent Retardation factor (Rf) System Stationary Phase Mobile Phase Front ouabain ouabagenin rhamnose Reference SIHca Gel G activated CHCl3:MeOH:H20 over H 2S0 4 24 h prior (65:30:5) to and 12 h after applying samples (10) 15-16 cm 0.33 0.42 0.33 Dutta, 1963 II Cellulose Impregnated with water saturated with n-butyl alcohol n-Butyl alcohol saturated with water 14 cm 0.45 0.64 0.34 modified from von Schenker, 1954 III Si l ica Gel G Ethyl acetate:MeOH 15 cm (75:25) developed 2 times 0.39 0.13 0.70 modified from Brower, et a l . , 1982 58 s a l i n e c o n t r o l always chromatographed as a s i n g l e peak c o i n c i d e n t with c o l d ouabain and could be recovered with 89.9% e f f i c i e n c y i n System I. The remaining l a b e l (10.1%) was evenly d i s t r i b u t e d throughout the chromatogram and judged to be a r e s u l t of n o n - s p e c i f i c adsorption to the s i l i c a gel rather than to i m p u r i t i e s . The amount of ouabain (nmoles) i n experimental s o l u t i o n s ( 0 e ) was c a l c u l a t e d as f o l l o w s : 0 e = [(Do* D t _ 1 ) + k] • D e • D s " l where D 0 = d i s i n t e g r a t i o n s per minute (dpm) recovered from the ouabain standard spot in the chromatogram of the experimental s o l u t i o n D+; = dpm recovered between the o r i g i n and s o l v e n t f r o n t i n the experimental s o l u t i o n chromatogram k = a c o r r e c t i o n f o r the f r a c t i o n of ouabain which i s n o n - s p e c i f i c a l l y adsorbed to the TLC support (= 0.101 and i s derived from (D+; - D 0) • D-t~l f o r chromatograms of pure ouabain s o l u t i o n s D e = dpm measured i n the experimental s o l u t i o n D s = dpm per nmole ouabain measured i n standard ouabain s o l u t i o n s . From t h i s value and the measured volume of experimental s o l u t i o n s , ouabain concentrations can be c a l c u l a t e d . Metabolism (0M e), the amount of ouabain that must have been changed to give r i s e to the observed metabolism, was c a l c u l a t e d as f o l l o w s : 0M e = ( D e - D s " l ) - 0 e where 0 e i s the amount of unchanged ouabain i n the experimental 59 s o l u t i o n . A l l r e s u l t s are reported as mean +_ standard e r r o r of the mean. The v a r i a b i l i t y among animals was found to be no greater than the v a r i a b i l i t y among tubules from one animal, hence the number of tubules i s reported. RESULTS 1) C h a r a c t e r i s t i c s of Ouabain Transport in Segment II a) S e c r e t i o n of ouabain by Segment II The e f f e c t of external ouabain concentrations on the s e c r e t i o n of ouabain by Segment II was i n v e s t i g a t e d . Urine/plasma (U/P) r a t i o s of r a d i o a c t i v e l a b e l and the s e c r e t i o n r a t e s of f l u i d and r a d i o a c t i v e l a b e l were determined in i s o l a t e d Segment II's bathed i n s a l i n e (321 mosmol) con t a i n i n g .003 - 5.0 mM ouabain (plus [ 3H]ouabain) + 2.5 x 10~ 4 M cAMP without amino a c i d s . F l u i d and r a d i o a c t i v e l a b e l s e c r e t i o n r a t e s were followed over 5 h and found to be r e l a t i v e l y constant during the time period of the experiment, 2 - 5 h. Figures 3.2 & 3.3 summarize the r e s u l t s of these experiments. U/P r a t i o s of l e s s than 1 were always observed, even assuming no metabolism of ouabain, see Section II below ( F i g 3.2,#). U/P r a t i o s c o r r e c t e d f o r measured % metabolism are i n d i c a t e d (A). Increasing external ouabain concentrations d i d not c o n s i s t e n t l y a f f e c t rates of f l u i d s e c r e t i o n although segments bathed i n 1 mM ouabain secreted at reduced rates ( F i g . 3.3). In p r e l i m i n a r y experiments with unstimulated Segment I I ' s , 1 mM ouabain d i d not a f f e c t rates of f l u i d s e c r e t i o n (data not shown). The e f f e c t of f l u i d s e c r e t i o n r a t e s on the rate of ouabain s e c r e t i o n 60 i i i 1 1 + 1.0 0 -1 .0 "2.0 "3 .0 log ouabain concentration (mM) in bathing saline Figure 3.2. The effect of varying external ouabain concentration on urine-to-plasma (U/P) ratios, (%, i f we assume no ouabain metabolism; A, corrected for measured ouabain metabolism) in Segment II. Each point represents mean + S.E. (where larger than symbol); number in parentheses indicates number of determinations from 4 - 8 tubules, h 2 and 3 are pooled. Figure 3.3. E f f e c t of varying external ouabain concentration on f l u i d s e c r e t i o n r a t e , d e t a i l s as i n Figure 3.2. 62 and i t s concentration i n f l u i d secreted by i s o l a t e d Segment U ' s was a l s o determined. F l u i d s e c r e t i o n r a t e s were a l t e r e d by varying the osmotic pressure of the s a l i n e (134, 223, 312, 401 mOsm). Bathing s a l i n e contained ImM ouabain (plus [ 3H]ouabain). The r e s u l t s of these experiments are shown in F i g s . 3.4 & 3.5. The r a t e of ouabain s e c r e t i o n by Segment II was dependent on the rate of f l u i d s e c r e t i o n ( F i g . 3.4). C o r r e c t i o n s f o r metabolism (A) reduced these r a t e s only s l i g h t l y . The concentration of ouabain i n secreted f l u i d ( F i g . 3.5) approached 1 mM at the lowest rate of f l u i d s e c r e t i o n but decreased with higher f l u i d s e c r e t i o n r a t e s . A permeability c o e f f i c i e n t (b, cm-sec - 1) was c a l c u l a t e d using the data i n F i g s . 3.2 and 3.4 according to the equation b = a ( U 7 P ) ( l - U / P ) - 1 (Ramsay, 1958) where U/P i s the urine/plasma r a t i o of ouabain concentration and a i s the rate at which water i s a c t i v e l y pumped through u n i t area of wall i n t o the lumen (^l«mrn - 2.min - 1, c a l c u l a t e d by R = 2Tfrl_a where R i s the rate at which urine issues from the tubule and 2irrL i s the area of the tubule taken from gross morphological measurement, r = radius [1 /2 the outer diameter of the whole tubule] and L = l e n g t h ) . The permeability c o e f f i c i e n t ranges from 0.25 - 2.7 x 10 -6 cm-sec - 1. Assuming constant metabolism, we found the permeability c o e f f i c i e n t to be l i t t l e changed ( i . e . , w i t h i n experimental v a r i a b i l i t y ) by e i t h e r changes in f l u i d s e c r e t i o n rate or bathing s a l i n e concentration of ouabain, suggesting ouabain t r a n s p o r t i s mainly by d i f f u s i o n . / 63 cn o E c C o a> u to re ro 3 O ro or .30 n .25 . 2 0 -.15 ' . 1 0 -"E .05' ( 1 8 ) (39) 1 1 1 1 .2 .4 .6 .8 Fluid s e c r e t i o n rate (g l - h" 1 ) F i g u r e 3.4. E f f e c t o f f l u i d s e c r e t i o n r a t e on the r a t e o f ouabain s e c r e t i o n by Segment II a l o n e . # , l a b e l assumed t o be 100% o u a b a i n ; A , c o r r e c t e d f o r measured metabolism o f o u a b a i n . Each p o i n t r e p r e s e n t s the mean + S.E. (where l a r g e r than symbol); number i n p a r e n t h e s e s i n d i c a t e s number o f d e t e r m i n a t i o n s from 4 - 8 t u b u l e s , h 3 - 5 are p o o l e d . 64 Figure 3.5. E f f e c t of f l u i d s e c r e t i o n rate on ouabain concentration i n f l u i d secreted by Segment II alone. Horizontal arrow, ouabain concentration of bathing s a l i n e . D e t a i l s as i n Figure 3.4. 65 b) Metabolism of ouabain by Segment II When s e c r e t i o n c o l l e c t e d from Segment II bathed i n 326 mosmol s a l i n e c o n t a i n i n g 1 mM ouabain plus [ 3H]ouabain was chromatographed, 69.7 + 0.19 % of the r a d i o a c t i v e l a b e l chromatographed as unchanged ouabain (74% i n System I I , 54% i n System I I I , F i g . 3.6). Increasing the ouabain concentration of the bathing s a l i n e to 5 mM or i n c r e a s i n g the rate of f l u i d s e c r e t i o n r e s u l t e d i n Segment II s e c r e t i n g a s l i g h t l y greater percentage of unchanged ouabain (75% and 82%, r e s p e c t i v e l y , i n System I ) , although t h i s d i f f e r e n c e was not s i g n i f i c a n t (Student's t - t e s t a t the .05 l e v e l ) 2) Ouabain Reabsorption i n Segment I a ) D i r e c t a n a l y s i s Whole tubules were placed i n 326 mosmol bathing s a l i n e and the e n t i r e length of Segment I drawn out i n t o p a r a f f i n o i l . A f t e r the tubules e q u i l i b r a t e d f o r 45 minutes the s a l i n e was replaced by 326 mosmol bathing s a l i n e with 1 mM ouabain plus [ 3H]ouabain. Tubules secreted f l u i d a t the proximal end f o r at l e a s t 4 h and a reabsorption d r o p l e t often formed on the outside surface of Segment I over the f i r s t 3 h ( F i g . 3.7). Tubules not s e c r e t i n g continuously throughout the experimental period were discarded. Reabsorption d r o p l e t s were c o l l e c t e d two hours a f t e r changing the bathing s a l i n e ; whole tubule s e c r e t i o n s were c o l l e c t e d and analyzed every h a l f hour during t h i s p e r i o d . E i g h t - s i x and one-half percent of the r a d i o a c t i v e l a b e l i n the reabsorbed f l u i d chromatographed as unchanged ouabain i n System I (92% and 67% i n System,II and I I I , r e s p e c t i v e l y , F i g . 3.6). T h i s percentage i s not s i g n i f i c a n t l y d i f f e r e n t from the 1 mM ouabain plus [ 3H]ouabain s a l i n e c o n t r o l s (Student's t - t e s t , p > 0.05), i n d i c a t i n g t h a t only ouabain was 66 Figure 3.6. D i s t r i b u t i o n of r a d i o a c t i v i t y i n t y p i c a l chromatograms of experimental and c o n t r o l f l u i d s . Bathing s a l i n e i n a l l cases was 326 mosmol co n t a i n i n g 1 mM ouabain plus [ 3H]ouabain. P o s i t i o n s of o r i g i n (0 on x - a x i s ) , ouabain ( c l e a r o v a l ) , ouabagenin (cross-hatched o v a l ) , rhamnose ( s t i p p l e d oval) and solvent f r o n t (16, 14, 15) are i n d i c a t e d . Percentages i n d i c a t e r a d i o a c t i v i t y co-chromatographing with ouabain standard. S, Segment II s e c r e t i o n ; Rn, natural reabsorption d r o p l e t s ; R, set reabsorption d r o p l e t s ; W, whole-tubule s e c r e t i o n ; C, bathing s a l i n e c o n t r o l . 67 S Y S T E M I S Y S T E M TI S Y S T E M TJI CM F R O M O R I G I N 68 Figure 3.7. Amount of f l u i d , ouabain, metabolized ouabain, and ouabain concentration i n Segment II s e c r e t i o n , reabsorption d r o p l e t s and whole-tubule s e c r e t i o n . Malpighian tubules were bathed i n 326 mosmol bathing s a l i n e with 1 mM ouabain plus [ 3H] ouabain. Values are f o r a 2 h period (n = 8 ) . Segment II s e c r e t i o n values are c a l c u l a t e d from measured determinations of reabsorption d r o p l e t s and whole- tubule s e c r e t i o n . bathing saline segment II-secretion segment II reabsorption J'\ droplet segment I — whole tubule ff\ secretion — ligature • 5 0 0 1 4 ± 3 4 8 6 ± 3 6 Ouabain pmo les Metabol ized ouabain pmoles (%) Ouabain concentration m M 0 (0 ) 1 . 0 0 1 5 1 . 3 3 8 . 3 ( 2 0 . 2 ) 0 . 3 0 9 8 . 8 + 2 3 0 ( 0 ) 6 .91 2 9 . 2 ± 6 5 8 . 1 ( 6 6 . 6 ) 0 . 0 6 70 reabsorbed by Segment I. In c o n t r a s t , only 23.3 + 0.54% of the l a b e l i n whole tubule s e c r e t i o n s chromatographed as ouabain i n System I (35% and 33% in System II and I I I , r e s p e c t i v e l y , F i g . 3.6). The amount of ouabain i n whole tubule s e c r e t i o n s increased to 67% when the tubules were bathed e n t i r e l y i n s a l i n e . The concentrations of unchanged and metabolized ouabain i n whole tubule s e c r e t i o n and reabsorbed f l u i d s were c a l c u l a t e d using the equations given in M a t e r i a l s and Methods. To estimate Segment II s e c r e t i o n the f o l l o w i n g assumptions were made: a) the volume of Segment II s e c r e t i o n equals the volume of whole tubule s e c r e t i o n and reabsorption d r o p l e t b) the t o t a l r a d i o a c t i v e l a b e l present i n Segment II s e c r e t i o n equals the sum of t h a t present i n whole tubule and reabsorption d r o p l e t s c) the r a t i o of unchanged and metabolized ouabain present i n Segment II s e c r e t i o n equals that observed in chromatograms of s e c r e t i o n s c o l l e c t e d from Segment II alone. F i g . 3.7 summarizes these r e s u l t s . Concentrations of ouabain i n the reabsorption d r o p l e t s (6.9 mM) were s u b s t a n t i a l l y higher than those i n whole tubule (.06 mM) or Segment II (.30 mM) s e c r e t i o n or in the bathing s a l i n e (1.0 mM). S i x t y - f i v e percent of the ouabain and 3% of the f l u i d secreted by Segment II was reabsorbed by Segment I. A s i n g l e Malpighian tubule can remove 189.6 (151.3 + 38.3) pmoles of ouabain from the bathing s a l i n e i n 2 h. Approximately 31% of t h i s was metabolized: 20% by Segment II (38.3/189.6) and 10% by Segment I (58.1 -38.3/189.6), 52% (98.8/189.6) was reabsorbed by Segment I, while 15% (29.2/189.6) was f i n a l l y excreted into the py l o r u s . The amount of ouabain reabsorbed may be p o s i t i v e l y c o r r e l a t e d with whole tubule s e c r e t i o n rate (r= .95 excluding p o s s i b l e a t y p i c a l tubules at f l u i d s e c r e t i o n r a t e s of 600 n l , F i g . 3.8) but i s 71 . 3 - , .21 100 200 300 400 500 600 700 Fluid secreted in two hours (nl) Figure 3.8. Amount of f l u i d secreted by whole tubule i n 2 h i n r e l a t i o n to ouabain reabsorption by Segment I during same time p e r i o d . Values are f o r i n d i v i d u a l tubules. 72 independent of f l u i d reabsorption rate (data not shown). Ionic composition of whole tubule s e c r e t i o n , reabsorbed f l u i d and bathing s a l i n e was determined i n these experiments by e l e c t r o n microprobe a n a l y s i s . The r e s u l t s are shown in Table 3.2. The i o n i c composition of the three f l u i d s d i f f e r e d s u b s t a n t i a l l y , i n d i c a t i n g s e l e c t i v e s e c r e t i o n and reabsorption of ions and/or d i f f e r i n g p e r m e a b i l i t i e s of the two segments. b) Set d r o p l e t s To f u r t h e r i n v e s t i g a t e reabsorption along the length of Segment I and to provide c o n t r o l s f o r the d i r e c t a n a l y s i s of reabsorption p r e v i o u s l y described, a second experiment was conducted. Tubules were s e t up as before and small (6 - 18 n l ) d r o p l e t s of s a l i n e without cAMP or ouabain were placed along the length of Segments I and II as diagrammed i n F i g . 3.9. Droplets could be r e t r i e v e d from tubules a f t e r 3 min with l i t t l e change i n volume. Although placed r e l a t i v e l y c l o s e together they d i d not coalesce with each other or with the bathing s a l i n e or secreted f l u i d . S i m i l a r s i z e d d r o p l e t s incubated i n p a r a f f i n o i l d i d not change volume or ouabain concentration over the experimental p e r i o d . These c o n t r o l s i n d i c a t e t h a t measured f l u i d volumes of set and reabsorbed d r o p l e t s were not a f f e c t e d by our c o l l e c t i o n techniques, evaporation or c o a l e s c i n g of d r o p l e t s . Droplets placed on the outside of Segment I immediately wet the surface of the tubule while those on Segment II d i d not. A f t e r 2 h, s e t dro p l e t s were removed from tubules and analysed. The r e s u l t s are presented in F i g s . 3.6 and 3.9. The volume of d r o p l e t s placed on the outside of Segment II decreased ( i n d i c a t e d by a negative sign) over 2 h. L i t t l e r a d i o a c t i v i t y was detected i n these d r o p l e t s despite a chemical gradient favouring ouabain entry from the lumen. A l l d r o p l e t s placed on the outside 73 Table 3.2. Ionic composition (mM) of whole-tubule secretions, reabsorbed fluid and bathing saline. Ion i*l Reabsorbed f luid (n=3) Whole tubule secretion (n - 3) Bathing saline (n - 1) Ca2+ 1.53 • 0.55 0.76 + 0.10 1.58 Na+ 52.71 6.65 8.50 • 0.55 27.66 65.35 + 7.88 170.37 4 3.86 23.06 c i - 119.83 + 11.68 126.53 4 0.47 43.33 2.84 + 0.77 3.28 4 0.17 1.93 Total S 2.03 • 0.58 1.34 4 0.09 0.16 Total P 10.91 • 1.43 37.28 4 2.91 5.96 Values are mean 4 SE. S, sulfur, P, phoshorus. 74 Figure 3.9. Change in f l u i d volume, r a d i o a c t i v e l a b e l , and r a d i o a c t i v e l a b e l c o n centration in four s e t d r o p l e t s and whole-tubule s e c r e t i o n over 2 h. Negative values i n d i c a t e a decrease in f l u i d volume. Malpighian tubules were bathed i n 326 mosmol bathing s a l i n e c o n t a i n i n g 1 mM ouabain plus [ 3H]ouabain. Values are mean + S.E. (n= 8 ) . bathing saline segment II-segment I whole tubule secretion I igature • Radioactive Radioactive label Fluid label concentration nl pmoles mM - 4 . 9 ± . 6 0 0 " 5 . 4 ± 1.1 0 . 3 + 0 . 2 0 +2.4+ 1.2 37.1 + 6 . 8 1 5 . 4 +4 .4+1.4 4 . 7 ± 0 . 9 1.1 110.8 ± 2 7 . 4 1 3 . 4 1 2 . 5 0 .1 76 of Segment I increased i n volume a f t e r 2 h. The concentration of r a d i o a c t i v i t y i n t h i s reabsorbed f l u i d was always higher than that in the bathing s a l i n e and chromatographic a n a l y s i s ( F i g . 3.6, System I and II) showed t h a t a l l of the r a d i o a c t i v e l a b e l was ouabain. In a l l cases i t appeared most of the ouabain (approximately 88%) was reabsorbed from the lumen by the d i s t a l d r o p l e t thus reducing the amount of ouabain "seen" by the more proximally set d r o p l e t on Segment I. 3) M o d i f i c a t i o n of Segment II s e c r e t i o n by Segment I In the previous experiments only the luminal side of Segment I was bathed in s a l i n e . Moreover reabsorption d r o p l e t s covered only about 10% of the tubule s u r f a c e . Both of these f a c t o r s could r e s u l t i n low estimations of r e a b s o r p t i o n . To overcome these problems we were able to analyze tubules s e q u e n t i a l l y since p r e l i m i n a r y experiments had i n d i c a t e d that at f a s t s e c r e t i o n r a t e s both whole tubules and Segment I I ' s secreted f l u i d and ouabain at r e l a t i v e l y constant r a t e s between 2 - 5 h. Accordingly s e c r e t i o n was c o l l e c t e d from whole tubules ( l e s s the 1.5 mm length of Segment I re q u i r e d to separate s e c r e t i o n ) bathed e n t i r e l y i n 132 mosmol s a l i n e over the f i r s t 3 h. Then Segment I was withdrawn as described p r e v i o u s l y and s e c r e t i o n c o l l e c t e d from Segment II alone. F i g . 3.10 & 3.11 present the r e s u l t s of these experiments. The presence of Segment I sharply reduces the rate of r a d i o a c t i v e l a b e l e x c r e t i o n in whole Malpighian tubules by 85 - 94% (p < .001, a n a l y s i s of variance, Scheffe's t e s t , F i g . 3.10). F i g . 3.11 i n d i c a t e s t h i s reduction i s accompanied by a reduction of the ouabain concentration in the excreted f l u i d by about 71% (p < .001, a n a l y s i s of variance, Scheffe's t e s t ) . Chromatography of whole tubule and Segment II s e c r e t i o n produced under 77 ^ S e g m e n t I , U ^ S e g m e n t CD 1 2 3 4 5 Time Ch) Figure 3.10. Rate of ouabain s e c r e t i o n with time. At 3 h (stand dotted l i n e ) Segment I i s removed. Traces are f o r i n d i v i d u a l tubules bathed i n 132 mosmol s a l i n e c o n t a i n i n g 1 mM ouabain plus [ 3H]ouabain. 78 .Segment I , I L > <-Segment H . o CC c O C O o 03 . Q CD Z J O 0.2 H 0.1 H 2 3 T 4 1 5 Time Ch) Figure 3.11. Ouabain concentration i n secreted f l u i d with time. At 3 h ( and dotted l i n e ) Segment I i s removed. D e t a i l s as i n Figure 3.10. 79 these c o n d i t i o n s of f a s t s e c r e t i o n rates i n d i c a t e d 58 and 82% r e s p e c t i v e l y of the r a d i o a c t i v e l a b e l was unchanged ouabain. Although the percentage of metabolized ouabain i s greater in whole tubule s e c r e t i o n than i n i s o l a t e d Segment U ' s , t h i s can be more than accounted f o r by reabsorption of unchanged ouabain, suggesting under these c o n d i t i o n s l i t t l e metabolism i s o c c u r r i n g in Segment I. DISCUSSION This study demonstrates t h a t in in v i t r o preparations the d i s t a l Segment II of the Malpighian tubules of 0. f a s c i a t u s secreted primary urine and ouabain i n t o the Malpighian tubule lumen. The high K +/Na + r a t i o and high C I " concentration of the f l u i d secreted by CJ. f a s c i a t u s Malpighian tubules suggest that the s e c r e t i o n of primary urine i s by the process common to many i n s e c t s - a c t i v e K + s e c r e t i o n with C I " as the accompanying anion ( P h i l l i p s , 1982). The proximal Segment I f u n c t i o n s i n reabsorption as evidenced by the a b i l i t y of t h i s segment to reabsorb f l u i d and ouabain in v i t r o . The discovery in fJ. f a s c i a t u s of a Malpighian tubule segment t h a t reabsorbs ouabain suggests that excretory l o s s of cardenolide from the i n s e c t could be minimized, p o s s i b l y a i d i n g in the i n s e c t ' s sequestration of large q u a n t i t i e s of c a r d e n o l i d e s . S e c r e t i o n of ouabain by Segment II against a concentration gradient was never observed. Under a l l c o n d i t i o n s t e s t e d , external ouabain concentrations of .03 - 5 mM and f l u i d s e c r e t i o n rates of .043 - .812 j j l / h , U/P r a t i o s f o r ouabain were l e s s than one. The s e c r e t i o n rate of ouabain increased with i n c r e a s i n g f l u i d s e c r e t i o n . The permeability c o e f f i c i e n t remained r e l a t i v e l y constant throughout these experimental manipulations 80 (0.25 - 2.7 x 10" 6 cm-sec - 1) suggesting passive ouabain t r a n s p o r t i n t o the tubule lumen. Malpighian tubule segment II appears f a r more permeable than the midgut [Pouabain = 8 , 5 x 1 0 _ 1 1 c m ' s e c " l , (Scudder and Meredith, 1982b)] but caution must be used i n such comparisons since gross measurement of t i s s u e surface areas are used to estimate pe r m e a b i l i t y c o e f f i c i e n t s and do not take i n t o account true membrane area. Passive e x c r e t i o n of ouabain has been reported in Locusta m i g r a t o r i a and Zonocerus v a r i e g a t a fed on a cardenolide f r e e d i e t ( R a f a e l i - B e r n s t e i n and Mordue, 1978). Indeed, the Malpighian tubules of a number of other i n s e c t s are permeable to organic compounds, a process Maddrell and Gardiner (1974) suggest i s an automatic way f o r i n s e c t s to c l e a r the hemolymph of t o x i n s . Ouabain i s a well known i n h i b i t o r of (Na + + K +)-dependent ATPases (Stekhoven and Bonting, 1981). High external concentrations of ouabain (up to 5 mM) do not appear to i n h i b i t f l u i d s e c r e t i o n r a t e s i n 0. f a s c i a t u s Malpighian tubules as one would expect i f (Na + + K +)-dependent ATPases are e s s e n t i a l in the formation of primary urine in i n s e c t s [reviewed by P h i l l i p s (1981)]. T h i s may r e f l e c t a) suboptimal temperature or ion concentrations f o r ouabain i n h i b i t i o n of (Na + + K +)-dependent ATPases [ f o r d i s c u s s i o n concerning c o n f l i c t i n g r e p o r ts of i n s e c t t i s s u e s e n s i t i v i t y to cardenolides and experimental methodology see Anstee and Bowler (1979)]. b) the absence of (Na + + K +)-dependent ATPases or the presence of (Na + +K +)-dependent ATPases i n s e n s i t i v e to ouabain (Jungreis and Vaughan, 1977; Vaughan and J u n g r e i s , 1977; but see Anstee and Bowler, 1979). c) s e c r e t i o n of primary urine by ion pumps other than Na-K exchange [reviewed by P h i l l i p s ( P h i l l i p s , 1981)]. 81 One might expect to f i n d ouabain i n s e n s i t i v e Malpighian tubules i n CJ. f a s c i a t u s , however, since the i n s e c t ingests and a s s i m i l a t e s cardenolides from i t s natural food sources (Duffey and Scudder, 1972). The a b i l i t y of both Malpighian tubule segments to metabolize ouabain i s i n t e r e s t i n g since such metabolism was not found i n mammals (Dutta, e t a l , 1963; Kolenda, et &]_, 1971) nor in fJ. f a s c i a t u s gut, hemolymph or d o r s o l a t e r a l space f l u i d , although a nonpolar c a r d e n o l i d e , d i g i t o x i n , was metabolized (Scudder and Meredith, 1982b). Further study i s necessary to e l u c i d a t e the f u n c t i o n of cardenolide metabolism i n CJ. f a s c i a t u s . When Segment I was d i r e c t l y analyzed i n slowly s e c r e t i n g tubules, i t reabsorbed 52% of the ouabain excreted by Segment I I . This reabsorption occurred a g a i n s t a strong concentration gradient (23 f o l d ) and was s p e c i f i c to ouabain, i . e . no l a b e l l e d m e tabolites, although present i n the lumen, were reabsorbed. Reabsorption of ouabain was independent of f l u i d reabsorption^but increased with i n c r e a s i n g f l u i d s e c r e t i o n by Segment I I . In r a p i d l y s e c r e t i n g whole Malpighian tubules, the presence of Segment I reduced the e x c r e t i o n of ouabain by 84 - 93%, mainly by reducing ouabain c o n c e n t r a t i o n . These r e s u l t s suggest that ouabain reabsorption i s an a c t i v e process and remains unsaturated under the l i m i t e d c o n d i t i o n s used i n t h i s study. Moreover, the r e s u l t s suggest that i n in viv o s i t u a t i o n s r e q u i r i n g f a s t e x c r e t i o n r a t e s (a s i t u a t i o n of high cardenolide s e c r e t i o n by Segment II) CJ. f a s c i a t u s i s able to recover by reabsorption i n Segment I most of the cardenolide that otherwise would be l o s t . The high concentration of Na + i n the reabsorption f l u i d i n d i c a t e s that ouabain may be l i n k e d to Na + t r a n s p o r t as seen i n the reabsorption of glucose and tre h a l o s e i n the Malpighian tubules of L_. m i g r a t o r i a (Ramsay, 1958). R a f a e l i - B e r n s t e i n and Mordue (1978) report that e x c r e t i o n of ouabain 82 ( i . e . , r a d i o a c t i v e l a b e l ) by Malpighian tubules of 2. v a r i e g a t a i s increased, with U/P r a t i o s about 3, when the i n s e c t ' s d i e t contains c a r d e n o l i d e s . The authors suggest they have induced an a c t i v e ouabain pump. No evidence was found in the present study f o r a c t i v e e x c r e t i o n in (). f a s c i a t u s Malpighian tubules. The a c t i v e pump we have described i n Segment I d i f f e r s from that proposed f o r 2. variegatus in both i t s o r i e n t a t i o n , r e s u l t i n g in a c t i v e r e a b s o r p t i o n , and a l s o i n the higher concentration gradient achieved. I t would be i n t e r e s t i n g to determine whether t h i s pump i s i n d u c i b l e as in 1. v a r i e g a t u s . Reabsorption of an organic molecule such as ouabain appears to be rare in Malpighian tubules of i n s e c t s studied so f a r . Only a few other organics, glucose and t r e h a l o s e have been shown to be reabsorbed by Malpighian tubules (Knowles, 1975; R a f a e l i - B e r n s t e i n and Mordue, 1979); Maddrell and P h i l l i p s (1976) s t a t e that amino acids are a c t i v e l y reabsorbed in the lower tubule of Rhodnius. Other than these examples, reabsorption by Malpighian tubules appears to be l i m i t e d to ion r e c y c l i n g [ K + (Irvine v, 1969; Maddrell and P h i l l i p s , 1976) or Na + i n the case of some blood feeders (Gee, 1976)] f l u i d reabsorption ( I r v i n e , 1969) and p o s s i b l y bicarbonate reabsorption (Wigglesworth, 1931). Reabsorption by Segment I may a i d i n f l u i d reabsorption in 0. f a s c i a t u s . Although under c o n d i t i o n s of d i r e c t a n a l y s i s f l u i d reabsorption in 0. f a s c i a t u s i s only 3% of e x c r e t i o n , i t may be s i g n i f i c a n t l y higher i n v i v o . Sequential a n a l y s i s suggests the presence of Segment I reduces s e c r e t i o n r a t e s 1.1 - 5.1 times, although t h i s could be p a r t l y a r e s u l t of r e s t r i c t e d f l u i d flow. Segment I could a l s o play a r o l e in i o n i c r e g u l a t i o n since the Na +/K + r a t i o i n the reabsorbate (0.61) more c l o s e l y resembles that in the hemolymph (0.96) than that in whole tubule s e c r e t i o n (0.05). 83 Th i s study demonstrates that i n in v i t r o preparations the d i s t a l segment of the Malpighian tubules of fJ. f a s c i a t u s secretes the polar cardenolide ouabain whereas the proximal Segment I reabsorbs ouabain. Both segments were found to metabolize t h i s c a r d e n o l i d e . Each of these processes occurs simultaneously and provides p o t e n t i a l p o i n t s of c o n t r o l f o r c a r denolide accumulation i n v i v o . fJ. f a s c i a t u s may be forc e d to los e cardenolides by e x c r e t i o n since Malpighian tubules are n e c e s s a r i l y permeable s t r u c t u r e s . The discovery of a s p e c i a l i z e d Malpighian tubule segment that can a c t i v e l y reabsorb a ca r d e n o l i d e , however, suggests t h i s excretory l o s s could be minimized. Further i n v e s t i g a t i o n i s necessary to e s t a b l i s h the r o l e in vivo of the Malpighian tubules i n the o v e r a l l process of sequestration of cardenolides i n fJ. f a s c i a t u s . (Material i n t h i s chapter was done i n c o l l a b o r a t i o n with J . Meredith and i s in Meredith, J . , Moore, L. and G.G.E. Scudder. 1984. Ex c r e t i o n of ouabain by Malpighian tubules of Oncopeltus f a s c i a t u s . Am. J . P h y s i o l . 246 (Regulatory I n t e g r a t i v e Comp. P h y s i o l . 15): R705-715). 84 CHAPTER 4: OUABAIN RESISTANT NA.K-ATPASES AND CARDENOLIDE TOLERANCE IN  THE LARGE MILKWEED BUG, ONCOPELTUS FASCIATUS Summary Oncopeltus f a s c i a t u s t o l e r a t e d 1954x and 7288x, r e s p e c t i v e l y , the LD50 ouabain dose of S c h i s t o c e r c a g r e g a r i a and P e r i p l a n e t a americana when ouabain was i n j e c t e d i n t o the hemocoel of these i n s e c t s . The maximum ouabain dose t h a t could be i n j e c t e d i n t o Ch f a s c i a t u s (200 nmoles) r e s u l t e d i n no m o r t a l i t y ; t h i s dose i s higher than the l e t h a l ouabain doses recorded f o r vertebrates and i n v e r t e b r a t e s . The ouabain concentration r e s u l t i n g i n 50% i n h i b i t i o n (I50) of Na,K-ATPase a c t i v i t y was determined i n l y o p h i l a t e s of nervous t i s s u e of fJ. f a s c i a t u s and b r a i n and r e c t a of ^ . g r e g a r i a and were 2.0 x 10~4, 2.0 x 10~6, and 1.0 x 10"6 M, r e s p e c t i v e l y . The I5Q value f o r ouabain i n h i b i t i o n of Na,K-ATPase a c t i v i t y i n the nervous t i s s u e of CJ. f a s c i a t u s i s higher than the I50 values f o r nervous t i s s u e i n most other i n s e c t s as well as many other i n v e r t e b r a t e and ve r t e b r a t e t i s s u e s . Thus, the presence of ouabain r e s i s t a n t Na,K-ATPases appears to be a f a c t o r i n the tol e r a n c e and sequestration of pl a n t cardenolides in 0. f a s c i a t u s . 85 INTRODUCTION Cardenolides are t o x i c to both vertebrates and i n v e r t e b r a t e s (Hoch, 1961; Treherne, 1966; Detweiler, 1967; Anstee and B e l l , 1975; B i e k i r c h , 1977; R a f a e l i - B e r n s t e i n and Mordue, 1978; Benson et a K , 1979; Stekhoven and Bonting, 1981; Anstee and Bowler, 1984), at concentrations as low as 1 0 - 8 M (Glynn, 1964). Cardenolides e x e r t t h e i r t o x i c e f f e c t s by s p e c i f i c a l l y i n h i b i t i n g the enzyme Na ++K +-ATPase (Na,K-ATPase; E.C. 3.6.1.3; Akera, 1977; Bodeman, 1981), which i s e s s e n t i a l in maintaining the electrochemical gradients f o r Na + across the c e l l (Jtfrgensen, 1980; Stekhoven and Bonting, 1981). In i n v e r t e b r a t e s , the c a r d e n o l i d e , ouabain, has been demonstrated to i n t e r f e r e with Malpighian tubule s e c r e t i o n ( P i l c h e r , 1970; Atzbacher e t a K , 1974; Anstee and B e l l , 1975), proper f u n c t i o n i n g of nervous t i s s u e (Treherne, 1966; Farquharson, 1974) midgut ion f l u x e s and t r a n s e p i t h e l i a l p o t e n t i a l s (O'Riordan, 1969; Prusch, 1978) and l a b i a l gland s e c r e t i o n (Kafatos, 1968). However, de s p i t e the p o t e n t i a l t o x i c i t y of c a r d e n o l i d e s , l a r g e amounts of these compounds are ingested by, and sequestered i n , (). f a s c i a t u s from i t s host p l a n t s with no apparent ill e f f e c t s (Isman, 1977; Chaplin and C h a p l i n , 1981; Jones e t _ a K , 1983). Among v e r t e b r a t e s , species d i f f e r e n c e s in cardenolide s e n s i t i v i t y are known to p a r a l l e l d i f f e r e n c e s i n the cardenolide s e n s i t i v i t y of t h e i r Na,K-ATPases (Akera, 1977; Schwalb et al_., 1982). However, n e i t h e r the r e l a t i v e s e n s i t i v i t y of 0. f a s c i a t u s to the t o x i c e f f e c t s of cardenolides or the p o s s i b i l i t y that the presence of cardenolide r e s i s t a n t Na,K-ATPases in 0_. f a s c i a t u s i s a f a c t o r in the apparent i n s e n s i t i v i t y of t h i s i n s e c t to cardenolide i n t o x i c a t i o n has been determined. Therefore, in t h i s chapter, the in v i v o r e s i s t a n c e of 0. f a s c i a t u s to ouabain i s compared to that of two i n s e c t s , namely S c h i s t o c e r c a g r e g a r i a and P e r i p l a n e t a americana, which 86 do not normally encounter cardenolides i n t h e i r d i e t . In a d d i t i o n , the ouabain s e n s i t i v i t y of Na,K-ATPases i s determined i n t i s s u e l y o p h i l a t e s of CJ. f a s c i a t u s and j>. g r e g a r i a . The r e s u l t s of t h i s chapter provide evidence t h a t CJ. f a s c i a t u s i s more r e s i s t a n t to cardenolides than S. gre g a r i a and IP. americana, as well as many other i n v e r t e b r a t e s and v e r t e b r a t e s . Furthermore, the r e l a t i v e i n s e n s i t i v i t y of Na,K-ATPases in the nervous t i s s u e of CJ. f a s c i a t u s i n d i c a t e s that the presence of cardenolide r e s i s t a n t Na,K-ATPases may be another f a c t o r i n the a b i l i t y of t h i s i n s e c t to ing e s t and sequester c a r d e n o l i d e s . MATERIALS AND METHODS 1) Insects Adult Oncopeltus f a s c i a t u s ( D a l l a s ) were obtained from l a b o r a t o r y c o l o n i e s maintained a t 26°C with 65 % r e l a t i v e humidity (RH) and a 16 h l i g h t : 8 h dark (L:D) l i g h t regime. Insects were reared on one of two d i e t s : 1) milkweed seeds ( A s c l e p i a s speciosa T o r r . c o l l e c t e d Sept. 1982 i n Pe n t i c t o n , B.C.), which contain cardenolides and 2) sunflower seeds (Heliothus annuus L . ) , which are c a r d e n o l i d e - f r e e . Sunflower-reared bugs were obtained from a colony r a i s e d on sunflower seeds f o r two years; no cardenolides were detected i n these i n s e c t s (Moore and Scudder, 1985). S c h i s t o c e r c a g r e g a r i a L. adults were obtained from a l a b o r a t o r y colony reared on a c a r d e n o l i d e - f r e e d i e t (bran, milk powder, a l f a l f a and l e t t u c e ) and were maintained a t 29°C, 55 % RH, 12:12 L:D. P e r i p l a n e t a americana L. adults were su p p l i e d by C a r o l i n a B i o l o g i c a l Supply Company ( B u r l i n g t o n , N. C.) and were maintained a t room temperature on a c a r d e n o l i d e - f r e e d i e t 87 (potato, l e t t u c e , bran, milk powder, a l f a l f a and r a t chow). 2) Ouabain I n j e c t i o n s Female 0_. f a s c i a t u s t o l e r a t e d l a r g e r i n j e c t i o n volumes than males, th e r e f o r e a d u l t females o f " a l l three i n s e c t s p e c i e s , (). f a s c i a t u s and P_. americana of random ages, and j>. g r e g a r i a 16-18 days a f t e r e c d y s i s , were used. Ouabain s o l u t i o n s (ouabain octahydrate, Sigma Chem. Co.) i n 0.9% NaCl were i n j e c t e d i n t o the hemocoel by f i n e g l a s s p i p e t t e s ( F i s h e r a l k a l i - f r e e c o a g u l a t i on c a p i l l a r y tubes). 0_. f a s c i a t u s were i n j e c t e d between abdominal sterna 5 and 6, ^. g r e g a r i a between abdominal sterna 1 and 2, and £ . americana between abdominal terga 9 and 10. These i n j e c t i o n s i t e s minimized backwelling of hemolymph and i n j e c t e d s o l u t i o n s , and hemorrhaging i n P_. americana. Oneyul volumes were i n j e c t e d i n t o J>. g r e g a r i a and P_. americana; one to f i v e jul volumes were i n j e c t e d i n t o (). f a s c i a t u s . To f a c i l i t a t e i n j e c t i o n s i n t o 0. f a s c i a t u s , hemolymph volume was reduced by maintaining i n s e c t s with food, but no water, f o r 3 h f o r i n j e c t i o n s of 1-2 ^ul and 13-20 h f o r i n j e c t i o n s of 3-5 j u l . Insects were immobilized by c o o l i n g at -20°C (5, 8, and 9 min f o r 0. f a s c i a t u s , P_. americana and Si. g r e g a r i a , r e s p e c t i v e l y ) , i n j e c t e d , and then placed in cages under 100 watt bulbs, with food and water. Recovery time was defined as walking to the top of the cage. A f t e r recovery, i n s e c t s were returned to t h e i r usual l a b o r a t o r y maintenance regime ( s t a t e d above) and s u r v i v a l was monitored f o r 8 days; only those i n s e c t s that were upr i g h t and moved when di s t u r b e d were considered s u r v i v o r s . Control i n s e c t s were i n j e c t e d with 1-5 ^1 volumes of s a l i n e (0.9% NaCl) and t r e a t e d in the same manner as ouabain-injected i n s e c t s . 88 3) Na + + K +-dependent ATPase A c t i v i t y and Ouabain S e n s i t i v i t y T i s s u e l y o p h i l a t e s of a d u l t male CJ. f a s c i a t u s (random ages) and S_. gr e g a r i a (1-4 weeks a f t e r ecdysis) reared on c a r d e n o l i d e - f r e e d i e t s were prepared according to the methods of Peacock (1979, 1981a, 1981b) and Tolman and S t e e l e (1976); a l l s o l u t i o n s and procedures were at 4°C. Tissues were d i s s e c t e d and r i n s e d i n dH 20 (gut t i s s u e s were s l i t l o n g i t u d i n a l l y and contents removed p r i o r to r i n s i n g ) , pooled by t i s s u e type in 0.5 ml dH 20 u n t i l a l l t i s s u e s were d i s s e c t e d , homogenized with a t e f l o n p e s t l e f o r 3 min at 3000 rpm, frozen immediately i n a MeOH/dry i c e bath, l y o p h i l i z e d overnight and used immediately or stored at -80°C f o r a maximum of 18 days. L y o p h i l a t e s were r e c o n s t i t u t e d by homogenization with a t e f l o n p e s t l e a t 0°C f o r 1 min at 1500 rpm in b u f f e r (30 mM imidazole, 250 mM mannitol, 5 mM EDTA, pH 7.2; modified from Anstee and Bowler 1984). Incubation and assay c o n d i t i o n s f o r Na,K-ATPase a c t i v i t y were modified from the methods of Anstee and B e l l (1975), Jtfrgensen (1974), and Peacock (1979). F i v e r e a c t i o n media were used: 1) 4 mM MgCl 2, 100 mM NaCl, 20 mM KC1 2) 4 mM MgCl 2 3) 4 mM MgCl 2, 100 mM NaCl 4) 4 mM MgCl 2, 20 mM KCL 5) 4 mM MgCl 2, 100 mM NaCl, 20 mM KC1 with varying concentrations of ouabain (10~ 8 to 1.2 x 10-3 M ) # A l l media were buffered with 50 mM imidazole, pH 7.4, and contained 3 mM ATP ( T r i s salt,' Sigma Chem. Co.) and 0.1 mM EDTA. Anion concentration was kept constant by the a d d i t i o n of c h o l i n e CI" (Hanrahan and P h i l l i p s , 1983). Total ATPase a c t i v i t y was determined i n medium 1 and ouabain 89 i n h i b i t i o n of Na,K-ATPase a c t i v i t y was determined i n medium 5. Four d i f f e r e n t determinations were averaged to estimate Mg2 +-ATPase a c t i v i t y (media 2-5; Anstee and Bowler, 1984); the common estimate of Mg 2 +-ATPase a c t i v i t y (medium 5 with 10" 3 M ouabain, Anstee and Bowler, 1984) was only used with t i s s u e s of g r e g a r i a s i n c e the presence of o u a b a i n - i n s e n s i t i v e Na,K-ATPases was suspected in 0. f a s c i a t u s . Na,K-ATPase a c t i v i t y was determined by t o t a l ATPase a c t i v i t y (medium 1) minus Mg2 +-ATPase a c t i v i t y (average a c t i v i t y i n media 2-4 or 2-5, Anstee and Bowler, 1984). Reaction media (1 ml) were preincubated at r e a c t i o n temperature (30°C) f o r 10 min i n 1.5 ml Eppendorf tubes, the r e a c t i o n s t a r t e d by the a d d i t i o n of 10-15 ^il of l y o p h i l a t e suspension (20 to 75 ^ug protein) and terminated a f t e r 20 min by the a d d i t i o n of 0.1 ml of 50% t r i c h l o r o a c e t i c a c i d . Under these assay c o n d i t i o n s , the r e a c t i o n was l i n e a r with respect to both time and enzyme c o n c e n t r a t i o n . Tubes were placed on i c e , c e n t r i f u g e d at 4°C at 12,000 g f o r 5 min to remove any p r e c i p i t a t e d p r o t e i n and kept on i c e u n t i l use. Enzyme a c t i v i t y was measured by a s e n s i t i v e microdetermination of inorganic phosphate ( P i ) r e l e a s e (lower l i m i t 0.15 nmoles P i ; Chen et a l . , 1956), using a Lambda 3 UV/VIS spectrophotometer (Perkin-Elmer). Contaminating Pi present in the l y o p h i l a t e suspension or r e l e a s e d by ATP degradation was determined by two c o n t r o l tubes t r e a t e d in the same manner as experimental tubes: 1) r e a c t i o n medium 1 with l y o p h i l a t e suspension added, i n the absence of ATP, and 2) r e a c t i o n medium 1 i n the absence of l y o p h i l a t e suspension. P i r e l e a s e d in experimental tubes was c o r r e c t e d by s u b t r a c t i n g the P i present in both c o n t r o l tubes. P r o t e i n was determined by a modified Lowry assay ( S c h a c t e r l e and P o l l a c k , 1973) with bovine plasma 90 gamma g l o b u l i n standard (BioRad). RESULTS 1) S e n s i t i v i t y of Insects to I n j e c t i o n s of Ouabain i n t o the Hemocoel 0. f a s c i a t u s (N=16) s u f f e r e d no m o r t a l i t y 48 h a f t e r i n j e c t i o n with the maximum dose of ouabain (200 nmoles, Table 4.1): 200 nmoles was the maximum dose of ouabain that could be i n j e c t e d i n t o the hemocoel of 0. f a s c i a t u s owing to the l i m i t e d s o l u b i l i t y of ouabain i n the s a l i n e c a r r i e r (1 gm/75 ml H2O) and to the l i m i t e d volume that could be i n j e c t e d i n t o t h i s small i n s e c t (5 jul = 45% of the t o t a l hemolymph volume). Previous exposure to cardenolides or the presence of l a r g e amounts of sequestered p l a n t cardenolides (150 jxq per i n s e c t ; Moore and Scudder, 1985) did not a f f e c t the t o l e r a n c e of £. f a s c i a t u s to ouabain since a l l i n s e c t s i n j e c t e d with 200 nmoles of ouabain survived (48 h) whether they were reared on a d i e t c o n t a i n i n g cardenolides or were obtained from a colony that had not been exposed to cardenolides f o r 2 years (Table 4.1). S i m i l a r l y , longer term s u r v i v a l (8 days) was unaffected by the presence or absence of cardenolides i n the d i e t or by the l a r g e amounts of ouabain i n j e c t e d i n t h i s study. Although s l i g h t m o r t a l i t y a t 8 days occurred in one group of i n s e c t s i n j e c t e d with 200 nmoles of ouabain (11%, N=9), s i m i l a r m o r t a l i t y was seen in a s a l i n e - i n j e c t e d c o n t r o l group of i n s e c t s reared on the same food source (10%, N=10). The p o s s i b i l i t y of sublethal t o x i c e f f e c t s of ouabain was i n v e s t i g a t e d i n 0. f a s c i a t u s by measuring the time taken f o r recovery from i n j e c t i o n s of i n c r e a s i n g doses of ouabain. Although recovery times from i n j e c t i o n s were v a r i a b l e , no s i g n i f i c a n t d i f f e r e n c e s were found among recovery times from i n j e c t i o n s of 10 - 200 nmoles of ouabain, nor d i d recovery times of ouabain 91 Table 4.1. S u r v i v a l of fJ. f a s c i a t u s i n j e c t e d with 10 - 200 nmoles ouabain. 3 Ouabain I n j e c t i o n (nmoles) Presence of cardenol ides i n d i e t Number of i n s e c t s i n j e c t e d % S u r v i v a l 48 h 8 days 10 20 40 200 200 S a l i n e control' 5 S a l i n e c o n t r o l b 4 4 5 9 7 10 8 100 100 100 100 100 100 100 100 100 100 89 100 90 100 a Adult females of random ages. b Maximum i n j e c t i o n volume (5 jx\) 92 i n j e c t e d i n s e c t s d i f f e r s i g n i f i c a n t l y from recovery times of control i n d i v i d u a l s reared on the same food source and i n j e c t e d with the same volume of s a l i n e (Student's t - t e s t , p> 0.05; Table 2 ) . In c o n t r a s t to the i n s e n s i t i v i t y of 0. f a s c i a t u s to i n j e c t i o n s of as much as 200 nmoles of ouabain, m o r t a l i t y occurred i n groups of S_. g r e g a r i a and P_. americana i n j e c t e d with as l i t t l e as 3.0 and 0.5 nmoles of ouabain, r e s p e c t i v e l y ( F i g . 4.1). The ouabain dose causing 50% m o r t a l i t y (LDgg) i n S_. g r e g a r i a and P_. americana was c a l c u l a t e d by p r o b i t a n a l y s i s ( F i g . 4.1) and i s reported in Table 4.3. 0. f a s c i a t u s i s c l e a r l y i n s e n s i t i v e to ouabain, and t o l e r a t e d 33x and 222x the LD^Q ouabain dose of ^. g r e g a r i a and P_. americana (6.1 and 0.9 nmoles ouabain per i n s e c t , r e s p e c t i v e l y ) . C o r r e c t i n g f o r weight and hemolymph volume d i f f e r e n c e s between the three i n s e c t s p e c i e s , 0. f a s c i a t u s t o l e r a t e d 1954x and 7288x the L D 5 0 ouabain dose of J5. g r e g a r i a and P_. americana, r e s p e c t i v e l y . 2) A c t i v i t y and Ouabain S e n s i t i v i t y of Na"1" + K +-dependent ATPases in  Tissue L y o p h i l a t e s of 0. f a s c i a t u s and S. gregaria Na,K-ATPase a c t i v i t y was determined i n l y o p h i l a t e s of the f o l l o w i n g four t i s s u e s in £ . f a s c i a t u s , the number in parentheses i n d i c a t i n g the number of t i s s u e s pooled per l y o p h i l a t e : 1) 1st v e n t r i c u l u s of the gut (30), 2) 2nd and 3rd v e n t r i c u l i of the gut (30), 3) nervous t i s s u e (head capsule contents plus p r o t h o r a c i c and c e n t r a l ganglion) (15), and 4) dorsal blood vessel (15). Na,K-ATPase a c t i v i t y was a l s o determined i n l y o p h i l a t e s of two t i s s u e s of the o u a b a i n - s e n s i t i v e j>. g r e g a r i a : 1) b r a i n (4) and 2) rectum ( 2 ) . 93 Table 4.2. P o s t - i n j e c t i o n recovery times of fj. f a s c i a t u s . Ouabain I n j e c t i o n Presence of Number of Recovery I n j e c t i o n volume cardenolides i n s e c t s time (nmoles) (jul) in d i e t i n j e c t e d X+SE (min) 10 1 - 4 1.5+0.29 20 1 - 4 1.7+0.86 40 2 5 5.4 + 2.96 200 5 - 9 3.2 + 1.33 200 5 + 7 9.8+5.68 S a l i n e i n j e c t i o n 1 - 5 4.6 +_ 4.04 2 - 5 1.8+0.13 5 - 7 1.9+0.47 " 5 + 7 1.5 + 0.24 94 Figure 4.1. M o r t a l i t y (24 h) of S_. g r e g a r i a and P_. americana i n j e c t e d with varying amounts of ouabain. Each data p o i n t represents the % m o r t a l i t y , expressed i n p r o b i t s , seen in a group of 5-6 i n s e c t s . 9 000) i 00 / O A O P americana A s gregaria 7 (977) 5(50.0) H O O O o/o A A/ A 3(2.3) H 1 0 4--1D -05 6 ' 05 ' 10 log l 0 nmoles ouabain injected oi 96 Table 4.3. Ouabain toler a n c e of fJ. f a s c i a t u s , j>. g r e g a r i a and P . americana 3 I n i t i a l ouabain nmoles (^ig) nmoles (jig) ouabain concentration in ouabain injected/mg wet hemolymph a f t e r Insect i n j e c t e d / i n s e c t weight i n s e c t i n j e c t i o n (M) b 0. f a s c i a t u s 200.0 (145.7) 4.3 (3.2) 12.5 x 10" 3 100% s u r v i v a l S. g r e g a r i a 6.1 (4.4) 2.2 x I O - 3 (1.6 x 10' ' 3) 3.0 x IO" 5 L D 5 0 P . americana 0.9 (0.6) 5.9 x I O - 4 (4.3 x 10" • 4) 5.4 x I O - 6 L D 5 0 3 Adult females - CJ. f a s c i a t u s and P_. americana random ages, S_. gr e g a r i a 16-18 days a f t e r e c d y s i s . S u r v i v a l a f t e r 24 h (48 h s u r v i v a l i s the same). b C a l c u l a t e d using the f o l l o w i n g hemolymph volume estimates: fj. f a s c i a t u s and !>. gr e g a r i a 11 and 200 j j l , r e s p e c t i v e l y (Meredith, unpublished r e s u l t s ) , P_. americana 165 jul (Guthrie and T i n d a l l , 1968), and assuming complete mixing of i n j e c t e d ouabain with hemolymph. 97 The r e s u l t s are shown in Table 4.4. No ATPase a c t i v i t y of any kind was detected i n a l y o p h i l a t e of 15 dorsal blood v e s s e l s (data not shown) and Na,K-ATPase a c t i v i t y i n the 1st v e n t r i c u l u s and 2nd plus 3rd v e n t r i c u l i l y o p h i l a t e s was low, only 19.3 and 15.8 nm Pi/mg protein/min, r e s p e c t i v e l y . Much higher Na,K-ATPase a c t i v i t y was detected i n l y o p h i l a t e s of the nervous t i s s u e of CJ. f a s c i a t u s (148.6 nm Pi/mg protein/min) and the brai n and rectum of ^ . gr e g a r i a (258.5 and 225.2 nm Pi/mg protein/min, r e s p e c t i v e l y ) . Preincubation with the s o l u b i l i z i n g agent sodium deoxycholate (NaDOC) or NaDOC (0.1%) and 2mM sodium iodide ( N a l ) , increases Na,K-ATPase a c t i v i t y in many microsomal Na,K-ATPase preparations (Nakao e t al_., 1965; Peacock e t a l . , 1972; J0rgensen, 1974; Anstee and Bowler, 1984). Therefore, preincubation with these s o l u b i l i z i n g agents was used i n an attempt to increase Na,K-ATPase a c t i v i t y i n the two gut l y o p h i l a t e s of CJ. f a s c i a t u s . Gut l y o p h i l a t e s were preincubated i n r e c o n s t i t u t i o n b u f f e r (see M a t e r i a l and Methods) c o n t a i n i n g various concentrations of NaDOC or NaDOC (0.1%) plus 2mM Nal f o r 30 min at 4°C. Preincubation with NaDOC (0.1%) plus 2 mM Nal d i d not e f f e c t Na,K- or Mg 2 +-ATPase a c t i v i t y i n e i t h e r gut l y o p h i l a t e of fJ. f a s c i a t u s (N=l, data not shown). S i m i l a r l y , no e f f e c t was seen i n the Na,K- and Mg 2 +-ATPase a c t i v i t y of 1st v e n t r i c u l u s l y o p h i l a t e s preincubated with concentrations of NaDOC ranging from .04 to .35% (5 co n c e n t r a t i o n s , N=l-2, data not shown) or in a 2nd plus 3rd v e n t r i c u l i l y o p h i l a t e preincubated with concentrations of NaDOC ranging from .18 to .52% (4 con c e n t r a t i o n s , N=l, data not shown). To i n v e s t i g a t e the p o s s i b l e r o l e of ouabain r e s i s t a n t Na,K-ATPases i n the ouabain r e s i s t a n c e of 0. f a s c i a t u s , ouabain i n h i b i t i o n curves Table 4.4. Total, rig* and Na,K-ATPase activity of tissue lyophilates of 0. fasciatus and ^ . gregaria. X ATPase activity (nmPj/mg protein/minute) + S.E. (N» number of lyophilates) 0. fasciatus 0. fasciatus 0. fasciatus j>. gregaria S. gregaria 1st ventrlculus 2nd A 3rd ventrlculus nervous tissue brain rectum Ionic Media3 (N • 3) (M - 3) (N - 6) (N • 5) (N - 5) 1. Total (Mg.Na.K) 111.2 + 5.1 83.7 + 3.6 203.0 + 18.4 362.0 + 21.2 336.3 • 32.0 2. Mg 90.1 • 2.3 65.5 • 2.9 56.9 + 5.3 106.6 + 7.5 122.7 + 11.2 3. Mg.Na 95.6 • 7.0 72.5 + 8.1 53.4 + 6.4 109.8 • 8.7 115.7 • 8.5 4. Mg.K 89.8 • 3.1 65.6 + 2.3 52.8 + 2.4 95.2 • 7.3 99.2 • 6.9 5. Mg.Na,K plus 1.2 x 10"3 ouabain b b b 102.1 + 10.8 106.4 • 11.8 X Mg2+-ATPase act iv i ty 0 91.9 + 2.2 67.9 + 2.5 54.4 • 2.6 103.4 + 3.8 111.0 + 4.6 Na.K-ATPase act1v1tyd 19.3 15.8 148.6 258.5 225.2 for details of Ionic composition see Materials and Methods Medium 5 was not used to determine Mg2+-ATPase activity 1n 0. fasciatus because the presence of ouabain Insensitive Na,K-ATPases was suspected. Average of the Mg2+-ATPase activity determined In media 2-4 for 0. fasciatus tissues and media 2-5 for S. gregaria tissues. Na.K-ATPase activity was determined by total ATPase activity (medium 1) minus x Mg2+-ATPase act ivity. In al l cases, total ATPase activity was significantly greater than Mg2+-ATPase activity (p < 0.05, Student's t-test). 99 (10-8 to 1.2 x 1 0 - 3 M) of Na,K-ATPase a c t i v i t y were determined f o r the f o l l o w i n g l y o p h i l a t e s : 1) nervous t i s s u e of rj. f a s c i a t u s , 2) br a i n of S_. g r e g a r i a , and 3) rectum of j>. g r e g a r i a ( F i g . 4.2). As a r e s u l t of t h e i r low Na,K-ATPase a c t i v i t y , ouabain i n h i b i t i o n curves could not be constructed f o r dorsal blood vessel or gut l y o p h i l a t e s of 0. f a s c i a t u s . PI50 and I50 values (the - l o g i n molar concentration and the molar c o n c e n t r a t i o n , r e s p e c t i v e l y , of ouabain t h a t i n h i b i t s 50% of the Na,K-ATPase a c t i v i t y ) were c a l c u l a t e d by l i n e a r r e g r e s s i o n of ouabain concentration vs. % i n h i b i t i o n of Na,K-ATPase a c t i v i t y expressed as p r o b i t s . The r e s u l t s are reported i n Table 4.5. The ouabain concentration r e s u l t i n g i n 50% i n h i b i t i o n of the Na,K-ATPase a c t i v i t y in. nervous t i s s u e l y o p h i l a t e s of 0. f a s c i a t u s i s 2.0 x 1 0 - 4 M. T h i s concentration i s lOOx greater than the ouabain concentration r e s u l t i n g i n 50% i n h i b i t i o n of the Na,K-ATPase a c t i v i t y of l y o p h i l a t e s of _S. g r e g a r i a b r a i n (2.0 x 1 0 - 6 M), and 200x greater than the ouabain concentration r e s u l t i n g i n 50% i n h i b i t i o n of Na,K-ATPase a c t i v i t y i n S. gre g a r i a rectum (1.0 x 1 0 - 6 M). DISCUSSION I t i s c l e a r that £ . f a s c i a t u s i s very r e s i s t a n t to the t o x i c e f f e c t s of ouabain. High concentrations of ouabain (_> 1 0 - 3 M) do not a f f e c t s u r v i v a l or Malpighian tubule f l u i d s e c r e t i o n i n t h i s i n s e c t ( t h i s chapter, Chapter 3 ) , whereas lower ouabain concentrations (10~ 4 to 10 -6 M) e f f e c t various t i s s u e processes and s u r v i v a l i n many other i n s e c t s and a m i l l i p e d e (Table 4.6). Tissue processes i n a number of i n s e c t species have been reported to be unaffected by high concentrations 100 Figure 4.2. E f f e c t of varying ouabain concentrations on Na,K-ATPase a c t i v i t y i n l y o p h i l a t e preparations of fj. f a s c i a t u s nervous t i s s u e and _S. g r e g a r i a b r a i n and rectum (means +_ S.E. where l a r g e r than symbol; n = number of l y o p h i l a t e s ) . 101 XX) >» < O. fasciatus nervous tissue p i s o " 3 7 n«6 ~'°8io ouabain concentration C M ) 102 Table 4.5. I n h i b i t i o n of Na,K-ATPase a c t i v i t y by ouabain i n nervous t i s s u e of 0. f a s c i a t u s and br a i n and rectum of S. g r e g a r i a . a b Species Tissue p i ™ I c n (M) CL f a s c i a t u s nervous t i s s u e 3.7 2.0 x 10" _S. gr e g a r i a b r a i n 5.7 2.0 x 10" S. gr e g a r i a rectum 6.0 1.0 x 10" 9 P^O = ( _ ^ ° 9 l 0 °^ t n e m ° l a r concentration, r e s u l t i n g i n 50% i n h i b i t i o n . b I 5 0 = the molar concentration r e s u l t i n g i n 50% i n h i b i t i o n . 103 Table 4.6. Effects of ouabain on tissue processes and survival 1n Insect and millipede species. Ouabain concentration Species Tissue process (M); effect Reference Carausius morosus Malpighian tubule fluid secretion 2.5 x IO* 4 ; inhibition Pilcher (1970) Na efflux from nerve cord I O - 5 ; Inhibition Treherne (1966) Glomeris marginata Na efflux from nerve cord 5 x IO"6 - I O - 3 ; inhibition Farquharson (1974) Drosophila hydei Malpighian tubule dye excretion 1 ^jl 3 x I O - 4 Injection; Inhibition Atzbacher e1 al_. (1974) Locusta migratoria Malpighian tubule fluid secretion IO"6 - I O ' 3 ; Inhibition Anstee and Bell (1975) Peripi aneta americana Midgut ion fluxes and transepi-thelial potential IO"5 - 10"3; reduces potential; I O - 4 reduces 1on fluxes O'Riordan (1969) 5.4 x I O - 6 this study Na efflux from nerve cord IO" 5; Inhibition Treherne (1966) Schistocerca gregaria L D50 3.0 x I O- 5 this study Sarcophaga bullata Midgut transepi-thelial potential I O - 5 ; potential to zero Prusch (1978) Antherea pernyi Labial gland secretion 5 x IO" 4; 50% inhibition Kafatos (1968) 104 of ouabain (10~ 3 M), however, these r e s u l t s are c o n t r o v e r s i a l ( f o r review, see Anstee and Bowler, 1979). 0. f a s c i a t u s i s a l s o i n s e n s i t i v e to much l a r g e r ouabain doses than those r e s u l t i n g i n death i n at l e a s t nine v e r t e b r a t e s p e c i e s , i n c l u d i n g r a t s which are considered r e s i s t a n t to cardenolide i n t o x i c a t i o n (Table 4.7; Stekhoven and Bonting, 1981). Although l e t h a l i n j e c t i o n doses of ouabain i n i n s e c t s and vertebrates may not be d i r e c t l y comparable owing to the d i f f e r e n c e s between open and c l o s e d c i r c u l a t o r y systems, the LD50 doses f o r j>. g r e g a r i a and £ . americana are w i t h i n the range of l e t h a l doses recorded f o r v e r t e b r a t e s , i n d i c a t i n g that l e t h a l doses i n these taxa may be s i m i l a r . I f t h i s i s true, the very l a r g e d i f f e r e n c e between the ouabain s e n s i t i v i t y of 0. f a s c i a t u s and that recorded f o r J>. g r e g a r i a , _P. americana, and the ver t e b r a t e s species l i s t e d in Table 4.7 st r o n g l y suggests that 0. f a s c i a t u s i s more r e s i s t a n t to ouabain than most v e r t e b r a t e s , as well as many i n v e r t e b r a t e s . Among ve r t e b r a t e s , species d i f f e r e n c e s i n cardenolide s e n s i t i v i t y are known to p a r a l l e l d i f f e r e n c e s i n the cardenolide s e n s i t i v i t y of t h e i r Na,K-ATPases (Akera, 1977; Schwalb e t al_., 1982). The PI50 value (3.7) f o r ouabain i n h i b i t i o n o f Na.K-ATPase a c t i v i t y i n 0. f a s c i a t u s nervous t i s s u e i s much lower than the PI50 values f o r nervous t i s s u e in most other i n s e c t s (PI50 5 - 6 ; Anstee and Bowler, 1984) as well as many other i n v e r t e b r a t e and vertebrate t i s s u e s (pi5 0 5 - 8, t h i s study; Anstee and Bowler, 1984; P h i l ippot j i t , 1972; Bonting, 1966; Bakkeeren and Bonting, 1968; Ridderstrap and Bonting, 1969; P f e i l e r and Ki r s c h n e r , 1972; S c h w a l b ^ t ^ K , 1982). Thus, as i n many ve r t e b r a t e s , the cardenolide r e s i s t a n c e seen i n /J. f a s c i a t u s may be r e l a t e d to Na,K-ATPase cardenolide r e s i s t a n c e . Ouabain r e s i s t a n t Na,K-ATPases have been i s o l a t e d from the nervous 105 Table 4-7. Toxicity values for ouabain In invertebrates and vertebrates. Animal Type of Injection Dose (mg/kg)a References 0. fasciatus hemocoel 100% survival 3200.0 this study S. gregaria hemocoel L D50 1.63 H hemocoel L D L 0 0.80 M P. americana hemocoel L D50 0.43 M L D L 0 0.24 •t Cat Intravenous MLD 0.11-0.15 Duffey (1977) Dog Intravenous L D L 0 0.054 Lewis « Tatken (1979) Dog subcutaneous L D L0 150. H Rat Intravenous L D50 14. H Rat intraperitoneal L D50 47. t l Monkey Intravenous L D L 0 0.106 H Rabbit subcutaneous L D L 0 0.120 M Rabbit intravenous LD 0.1-0.2 Hoch (1961) Guinea pig Intravenous L D L 0 0.288 Lewis & Tatken (1979) Pigeon Intravenous L D L 0 0.285 M Mouse intraperitoneal L D50 12. H Frog subcutaneous L D50 0.417 Hoch (1961) A LDJQ: dose resulting in 50% mortality. LDJ_Q: lowest dose given over any period of time in one or more portions known to cause death. MLD: mean lethal dose. LD: lethal dose. 106 t i s s u e of one other i n s e c t , the monarch b u t t e r f l y (PI50 <3; Vaughan and J u n g r e i s , 1977) and in a mutant c e l l l i n e of the mosquito Aedes  al bop i c t u s (Mento e t al_., 1979). I t i s i n t e r e s t i n g that the monarch s p e c i a l i z e s on food sources c o n t a i n i n g c a r d e n o l i d e s , and l i k e fJ. f a s c i a t u s , sequesters l a r g e amounts of these compounds (Brower and G l a z i e r , 1975). In v e r t e b r a t e s , ouabain r e s i s t a n t Na ,K-ATPases have been i s o l a t e d from r a t s and ouabain r e s i s t a n t mutant c e l l l i n e s of several other species (Bakkeren and Bonting, 1968; Schwalb e t al_., 1982; Robbins and Baker, 1977; Baker e t a l . , 1974; Landolph et al_., 1980; Mankovitz et al_., 1974). The ouabain r e s i s t a n c e of these vertebrate Na ,K-ATPases and the mutant mosquito c e l l l i n e appear to be the r e s u l t of s t r u c t u r a l changes i n the enzymes r e s u l t i n g in unstable enzyme-ouabain complexes, a decrease i n the number of ouabain binding s i t e s on o u a b a i n - r e s i s t a n t c e l l s , and/or a changed response to bound ouabain. I t i s u n l i k e l y that the low Na ,K-ATPase a c t i v i t y of fJ. f a s c i a t u s midgut t i s s u e i s the r e s u l t of the predominance of K +-ATPases in the f u n c t i o n i n g of t h i s t i s s u e as has been suggested f o r the midgut e p i t h e l i a l t i s s u e s of several lepidopteran species in which Na ,K-ATPase a c t i v i t y could not be demonstrated (Jungreis and Vaughan, 1977; Harvey ^ t ^ . , 1983). Harvey jst al_. (1983) suggest that K +-ATPases f u n c t i o n i n ion t r a n s p o r t i n the midgut of these i n s e c t s since the gut lumen and hemolymph have high K + / N a + r a t i o s and, i n one sp e c i e s , midgut Mg 2 +-ATPase a c t i v i t y was stimulated by K + . However, in fJ. f a s c i a t u s the Na /K r a t i o i n the hemolymph i s >_ one (Mullen, 1957; Staddon and Everton, 1980; Meredith, unpublished r e s u l t s ) , and midgut Mg 2 +-ATPase a c t i v i t y i s not stimulated by K + ( t h i s study). I t i s p o s s i b l e that i n l y o p h i l a t e preparations of CJ. f a s c i a t u s midgut t i s s u e Na ,K-ATPase a c t i v i t y i s l a t e n t ; 107 indeed, f o r some i n s e c t gut t i s s u e s an elaborate p u r i f y i n g procedure, r e q u i r i n g l a r g e r t i s s u e volumes than a v a i l a b l e f o r the present study, i s necessary to obtain preparations with high Na.K-ATPase a c t i v i t y (Anstee, pers. comm.). In summary, t h i s chapter provides evidence that 0. f a s c i a t u s i s r e s i s t a n t to the t o x i c e f f e c t s of c a r d e n o l i d e s . T h i s t o l e r a n c e does not seem to depend upon previous exposure to c a r d e n o l i d e s , nor does i t appear to be a f f e c t e d by the usual presence of l a r g e amounts of sequestered plant cardenolides in the a d u l t i n s e c t . The ouabain r e s i s t a n c e of Na,K-ATPases in the nervous t i s s u e of 0. f a s c i a t u s i n d i c a t e s that t h i s t i s s u e i s well protected from cardenolide t o x i c i t y . I f the ouabain r e s i s t a n c e of nervous t i s s u e Na,K-ATPases i s i n d i c a t i v e of ouabain r e s i s t a n c e of Na,K-ATPases in the r e s t of the i n s e c t then other t i s s u e s in fJ. f a s c i a t u s w i l l a l s o be protected. Thus, the presence of Na,K-ATPases r e s i s t a n t to cardenolide i n t o x i c a t i o n appears to be one more f a c t o r in the a b i l i t y of t h i s i n s e c t to t o l e r a t e and sequester cardenolides from i t s food p l a n t s . 108 CHAPTER 5: GENERAL DISCUSSION The work described i n the previous three chapters examined s p e c i f i c aspects of the sequestration and tolera n c e of cardenolides i n £ . f a s c i a t u s . Chapter 2, S e l e c t i v e sequestration of milkweed cardenolides i n 0. f a s c i a t u s , i s concerned with several aspects of the q u a n t i t a t i v e d i s t r i b u t i o n of cardenolides i n 0. f a s c i a t u s and i s the f i r s t i n v e s t i g a t i o n of the s e l e c t i v e sequestration of i n d i v i d u a l milkweed cardenolides i n the i n s e c t . Chapter 3, E x c r e t i o n of ouabain by the Malpighian tubules of 0. f a s c i a t u s , i s the f i r s t i n v e s t i g a t i o n of the r o l e of the excretory system in cardenolide sequestration i n i n s e c t s . In Chapter 4, Ouabain  r e s i s t a n t Na,K-ATPases and cardenolide t o l e r a n c e i n (). f a s c i a t u s , the r e l a t i v e i n s e n s i t i v i t y of the i n s e c t and i t s Na,K-ATPases to cardenolides was e s t a b l i s h e d f o r the f i r s t time. My i n v e s t i g a t i o n of the s e l e c t i v e sequestration of milkweed cardenolides i n £ . f a s c i a t u s (Chapter 2) confirms the b a s i c pattern of q u a n t i t a t i v e d i s t r i b u t i o n of cardenolides i n _0. f a s c i a t u s determined by Duffey and Scudder (1974). Duffey and Scudder found t h a t the majority of cardenolides sequestered i n 0. f a s c i a t u s are concentrated i n the d o r s o l a t e r a l space while low l e v e l s are maintained i n the hemolymph. The demonstration i n Chapter 2 of the same pattern of cardenolide d i s t r i b u t i o n in £ . f a s c i a t u s reared on a d i f f e r e n t species of milkweed than that used i n the Duffey and Scudder study, suggests that the sequ e s t r a t i o n of cardenolides p r i m a r i l y i n the d o r s o l a t e r a l space and the maintenance of low cardenolide l e v e l s i n the hemolymph i s c h a r a c t e r i s t i c of t h i s i n s e c t . The work i n Chapter 2 a l s o i n d i c a t e s that minimal amounts of cardenolides are sequstered i n the gut, wings and f a t body of £ . f a s c i a t u s . This not only 109 provides a more complete understanding of the q u a n t i t a t i v e d i s t r i b u t i o n of cardenolides i n (h f a s c i a t u s , i t a l s o provides evidence that the sequestration of cardenolides i n £ . f a s c i a t u s d i f f e r s markedly from that of the two other i n s e c t s whose cardenolide d i s t r i b u t i o n i s known (C_. inopinatus and D. plexippus; Blum, 1981, 1983). T h i s lends f u r t h e r credence to Blum's hypothesis t h a t each i n s e c t has a unique way of handling the toxins i t encounters in i t s host p l a n t (Blum, 1983). Brower et a l . (1982) suggest t h a t e a r l i e r arguments advocating the prominent r o l e of p o l a r i t y in the sequestration of cardenolides in 0. f a s c i a t u s are premature. My r e s u l t s in Chapter 2 support t h i s suggestion. The cardenolide p r o f i l e s of 0. f a s c i a t u s , i t s t i s s u e s and s e c r e t i o n s c l e a r l y i n d i c a t e t h a t cardenolides of a wide p o l a r i t y range are sequestered and concentrated i n the i n s e c t . This provides f u r t h e r evidence that a number of physical-chemical c h a r a c t e r i s t i c s of cardenolides must be involved in t h e i r s e q u e s t r a t i o n in i n s e c t s as suggested by Seiber e t a l . (1980), and i s s i m i l a r to the s i t u a t i o n described in the monarch b u t t e r f l y (Brower e t al_., 1982). Brower et al_. (1982, 1984a, 1984b) have a l s o suggested t h a t i t might be p o s s i b l e to i d e n t i f y the species of milkweed an i n s e c t has been reared on by the i n s e c t ' s cardenolide p r o f i l e , and give evidence f o r t h i s i n monarch b u t t e r f l i e s feeding on three species of A s c l e p i a s . The s i m i l a r i t y of the cardenolide p r o f i l e s of 0. f a s c i a t u s reared on seeds of two species of A s c l e p i a s (Chapter 2 ) , however, i n d i c a t e that i d e n t i f i c a t i o n of an i n s e c t ' s food p l a n t by the i n s e c t ' s cardenolide p r o f i l e i s e i t h e r not p o s s i b l e with a l l species of A s c l e p i a s , or may not be p o s s i b l e with CL f a s c i a t u s . The presence of cardenolides in rj. f a s c i a t u s t h a t were not detected i n i t s food source, and the high concentration of intermediate and higher p o l a r i t y c a r d e n o l i d e s i n the u r i n e / f e c e s of fj. f a s c i a t u s (Chapter 2) i s the 110 f i r s t evidence suggesting metabolism and d i f f e r e n t i a l e x c r e t i o n of host p l a n t cardenolides i n fj. f a s c i a t u s . Metabolism of host p l a n t cardenolides has been well documented i n the monarch b u t t e r f l y (Seiber e t al_., 1 9 8 0 ; Brower et a l . , 1 9 8 2 ; Marty and K r e i g e r , 1 9 8 4 ) . Chapter 2 a l s o provided the f i r s t evidence of the r o l e of e x c r e t i o n in the sequestration of cardenolides in i n s e c t s , and i n i t i a t e d the i n v e s t i g a t i o n of the e x c r e t i o n of ouabain by the Malpighian tubules of CJ. f a s c i a t u s i n Chapter 3 . In Chapter 3 the s e c r e t i o n , metabolism and reabsorption of ouabain was i n v e s t i g a t e d under a v a r i e t y of c o n d i t i o n s to determine the p o s s i b l e r o l e of the Malpighian tubules in the sequestration of cardenolides i n CJ. f a s c i a t u s . The r e s u l t s suggest t h a t s e c r e t i o n , metabolism and reabsorption of cardenolides by the Malpighian tubules are a l l p o t e n t i a l p o i n t s of c o n t r o l f o r cardenolide accumulation i n CJ. f a s c i a t u s . The passive s e c r e t i o n and metabolism of ouabain by the Malpighian tubules of CJ. f a s c i a t u s i n d i c a t e s t h a t the tubules are a p o t e n t i a l s i t e of c ardenolide l o s s i n the i n s e c t ; £ . f a s c i a t u s may be f o r c e d to l o s e cardenolides by Malpighian tubule s e c r e t i o n since tubule permeability to organic compounds appears to be an automatic way f o r i n s e c t s to c l e a r the hemolymph of t o x i n s (Maddrell and Gardiner, 1 9 7 4 ) . However, the a c t i v e reabsorption of ouabain i n the proximal segment of the tubules suggests that t h i s excretory l o s s could be minimized. Indeed, i n r a p i d l y s e c r e t i n g Malpighian tubules, a s i t u a t i o n of high cardenolide s e c r e t i o n by the tubules, reabsorption recovered 84-93% of the ouabain t h a t otherwise would be excreted. Reabsorption of an organic molecule such as ouabain appears to be rare in Malpighian tubules of i n s e c t s s t u d i e d so f a r ; only two other o r g a n i c s , glucose and t r e h a l o s e , have been shown to be reabsorbed by Malpighian tubules (Knowles, 1 9 7 5 ; R a f a e l i - B e r n s t e i n and Mordue, 1 9 7 9 ) . I t may be I l l t h a t the a b i l i t y of the Malpighian tubules of 0. f a s c i a t u s to reabsorb cardenolides that would otherwise be excreted i s a unique s p e c i a l i z a t i o n , and of major importance i n the sequestration of cardenolides in (). f a s c i a t u s . Chapter 4 documents two a d d i t i o n a l s p e c i a l i z a t i o n s in fJ. f a s c i a t u s that may f u n c t i o n in the a b i l i t y of t h i s i n s e c t to t o l e r a t e and sequester cardenolides from i t s food p l a n t s ; in vivo t o l e r a n c e of the i n s e c t to l a r g e amounts of cardenolides i n j e c t e d i n t o the hemolymph and the presence of cardenolide r e s i s t a n t Na,K-ATPases i n the i n s e c t ' s nervous t i s s u e . The r e s u l t s in Chapter 4 provide evidence that 0. f a s c i a t u s i s r e s i s t a n t to doses of i n j e c t e d ouabain much l a r g e r than the l e t h a l doses recorded f o r both v e r t e b r a t e s and i n v e r t e b r a t e s . Furthermore, i n s e c t s reared on c a r d e n o l i d e - f r e e d i e t s as well as those reared on t h e i r normal c a r d e n o l i d e - r i c h d i e t were both extremely r e s i s t a n t to the t o x i c e f f e c t s of i n j e c t e d ouabain. Thus, the in vivo cardenolide t o l e r a n c e of 0. f a s c i a t u s does not seem to depend upon previous exposure to c a r d e n o l i d e s , nor does i t appear to be a f f e c t e d by the usual presence of l a r g e amounts of sequestered cardenolides in the i n s e c t . This apparent independence of the cardenolide t o l e r a n c e of the i n s e c t from the cardenolide content of i t s food p l a n t s , as well as the cardenolides present in i t s t i s s u e s , may be a f a c t o r in the i n s e c t ' s a b i l i t y to feed on food plants that vary g r e a t l y in t h e i r cardenolide content (Isman, 1977; Vaughan, 1979; Chaplin and Chaplin, 1981; Jones e t al_., 1983). Among v e r t e b r a t e s , species d i f f e r e n c e s in cardenolide s e n s i t i v i t y are known to p a r a l l e l d i f f e r e n c e s i n the cardenolide s e n s i t i v i t y of t h e i r Na,K-ATPases (Akera, 1977; Schwal b e t al_., 1982). The r e s u l t s in Chapter 4 suggest t h a t s i m i l a r l y , the i n v i v o s e n s i t i v i t y of 5>. g r e g a r i a and 112 fJ. f a s c i a t u s to cardenolides a l s o p a r a l l e l s the s e n s i t i v i t i e s of t h e i r Na,K-ATPases to cardenolide i n h i b i t i o n . T h is provides the f i r s t evidence that cardenolide s e n s i t i v i t y i n i n v e r t e b r a t e s may be r e l a t e d to the cardenolide s e n s i t i v i t y of t h e i r Na.K-ATPases. Furthermore, the i s o l a t i o n of ouabain r e s i s t a n t Na,K-ATPases from the nervous t i s s u e of 0. f a s c i a t u s i n d i c a t e s t h a t ouabain r e s i s t a n t Na,K-ATPases are another f a c t o r i n the toleran c e and sequestration of p l a n t cardenolides i n t h i s i n s e c t . Chapter 4 i s the second documentation of ouabain r e s i s t a n t Na,K-ATPases in an i n s e c t that ingests and sequesters cardenolides from i t s host p l a n t ; ouabain r e s i s t a n t Na,K-ATPases have a l s o been i s o l a t e d from the nervous t i s s u e of the monarch b u t t e r f l y (Vaughan and J u n g r e i s , 1977). I t the r e f o r e seems p o s s i b l e t h a t cardenolide r e s i s t a n t Na,K-ATPases may be c h a r a c t e r i s t i c of i n s e c t s that ingest and sequester l a r g e amounts of cardenolides from t h e i r host p l a n t s . In summary, the work described i n t h i s t h e s i s has c l a r i f i e d several aspects of the q u a n t i t a t i v e d i s t r i b u t i o n of cardenolides i n fj. f a s c i a t u s , as well as provided evidence that cardenolides are not sequestered i n the i n s e c t simply on the b a s i s of p o l a r i t y . In a d d i t i o n , t h i s work suggests that metabolism and d i f f e r e n t i a l e x c r e t i o n of host p l a n t cardenolides may be involved i n the s e l e c t i v e sequestration of these compounds i n fj. f a s c i a t u s . F i n a l l y , t h i s t h e s i s documents three s p e c i a l i z a t i o n s that may f a c t o r i n the to l e r a n c e and sequestration of host p l a n t cardenolides i n 0. f a s c i a t u s - a c t i v e reabsorption of cardenolides by the Malpighian tubules, high i n vivo t o l e r a n c e to cardenolides and the presence of cardenolide r e s i s t a n t Na,K-ATPases. 113 LITERATURE CITED Akera, T. 1977. Membrane adenosinetriphosphatase: a d i g i t a l i s receptor? Science 198: 569-574. Anonymous. 1979. LS 8000 S e r i e s L i q u i d S c i n t i l l a t i o n System Manual, C a l i f o r n i a : Beckman Instruments, Inc., 169pp. Anstee, J.H. and B e l l , D.M. 1975. 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