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Plant (Asclepias) - insect (Oncopeltus) chemical relationship Duffey, Sean Stephen 1970

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A PLANT (ASCLEPIAS) - INSECT (ONCOPELTUS) CHEMICAL RELATIONSHIP by SEAN STEPHEN DUFFEY B.Sc, University of British. Columbia, 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Zoology We accept this thesis as conforming to the required standards THE UNIVERSITY OF BRITISH COLUMBIA October 1970 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l m a k e i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e H e a d o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, C a n a d a D e p a r t m e n t D a t e i ABSTRACT The association of the Large Milkweed Bug, Oncopeltus fasciatus Dallas, with, a potentially poisonous Asclepiad, Asclepias, has "been investigated to determine the fate of sequestered cardiac glycosides i n this insect and to investigate the p o s s i b i l i t y that these compounds and/or odorous and v o l a t i l e alkyl secretions of this insect may be serving as (an) anti-predator device(s). Nineteen species of Asclepias from diverse parts of North America have been shown to contain cardiac glycosides. Evidence i s also given that Oncopeltus plus several other species of brightly coloured Coleopterans and Hemipterans, which are associated with Asclepias as a food-host, contain cardenolides which could function as the chemical basis for a Mullerian mimicry complex. The large quantities of polar cardiac glycosides sequestered by Oncopeltus fasciatus (approximately 111 micron grams) from the seeds of Asclepias syriaca were found to be concentrated i n a complex of dorso-lateral abdominal and thoracic secretory glands. Various parameters of the uptake and entry of the natural cardiac glycosides of Asclepias  syriaca and unnatural isotopic cardiac glycosides into the dorso-lateral glands were examined. The high levels of polar glycosides i n Oncopeltus i s also related to other aspects of the insect's physiology and the cardenolide composition of i i the food-host. The l i t e r a t u r e c i t e s that l i p i d , cardenolid.es are more emetic to bir d s than are the polar glycosides: therefore, the high l e v e l s of p o l a r glycosides i n t h i s Hemipteran feeding on the above plant could make i t non-emetic. Oncopeltus f a s c i a t u s was shown to be aposematic to chickens, t u r t l e s , l i z a r d s and s t a r l i n g s because of the v o l a t i l e secretions of the v e n t r a l metathoracic glands. Frogs and toads d i d not consider t h i s insect to be aposematic. The cardiac glycosides that had been sequestered from the seeds of t h i s northern; Asciepiad by Oncopeltus were not shown to be e f f e c t i v e i n causing r e j e c t i o n by the above predators i n laboratory conditions. The. p r e d a t i o n , studies on Oncopeltus suggest that the responses of various predators to a complex of glycoside containing mimics are not equivalent. This study also shows that along with predator responses being a c r i t i c a l feature i n a p a l a t a b i l i t y spectrum, the i n s e c t ' s physiology and i t s behavioural a s s o c i a t i o n with the plant are poignant aspects of the ins e c t ' s p o t e n t i a l to be unpalatable. i i i TABLE OF CONTENTS Page INTRODUCTION 1 MATERIAL AND METHODS 5 THE INSECT STUDIES 5 REARING 6 EXTRACTION AND PURIFICATION 7 CHROMATOGRAPHY 10 SPECTROPHOTOMETRY AND COLORIMETRY 13 BIOASSAY 18 ISOTOPE EXPERIMENTS 19 (a) The uptake of isotopes of ouabain and d i g i t o x i n throughout the l i f e c ycle 20 (b) The r e t e n t i o n of ouabain-3H and digitoxin - 3 H 23 (c) Gland transport experiments 25 (d) Uptake of cardenolide l a b e l from the 29 gut PREDATION EXPERIMENTS 29 VALIDITY OF GLYCOSIDE EXTRACTION, PURIFICATION AND IDENTIFICATION METHODS 37 RESULTS 4-5 CARDIAC GLYCOSIDES IN PLANT TISSUE 4-5 CARDIAC GLYCOSIDES IN ONCOPELTUS AND VARIOUS INSECTS FEEDING UPON ASCLEPIADS 50 THE DISTRIBUTION OF CARDIAC GLYCOSIDES IN ONCOPELTUS FASCIATUS 54-CARDIAC GLYCOSIDES IN THE SECRETORY GLANDS OF ONCOPELTUS FASCIATUS 55 THE POLARITY EFFECT IN THE DORSO-LATERAL GLAND COMPLEX IN ONCOPELTUS FASCIATUS (a) The possible sequestering of the more polar cardenolides (b) The selective sequestering a b i l i t y of the dorso-lateral meso- and meta-thoracic glands (c) The fate of non-polar glycosides i n the body PARAMETERS OF GLYCOSIDE ACCUMULATION IN ONCOPELTUS FASCIATUS (a) The uptake and carry-over of natural cardenolides i n the l i f e cycle. (b) The retention of specific cardiac glycosides (c) Glycoside dependant retention (d) The uptake capability of adults PREDATION EXPERIMENTS DISCUSSION SUMMARY REFERENCES LIST OF TABLES Table I II III IV V VI VII VIII IX X Results of chemical assay and bioassay of plant extracts of some Asclepiad species and other plants for cardiac glycosides Numbers of cardiac glycosides detected i n various Asclepiads from North America Results of chemical assay and bioassay of insect whole animal extracts for cardiac glycosides Plants from which Oncopeltus fasciatus was able to sequester cardiac glycosides Distribution of cardiac glycosides and alkyl aldehydes and ketones i n the various defensive glands and body fluid s of Oncopeltus fasciatus Changes in,the levels of ouabain- H and digitoxin-^H i n the dorso-lateral meso-and methathoracic gland fluids and the haemolymph of Oncopeltus fasciatus at various developmental stages when reared on labelled seeds of Asclepias syriaca Distribution of cardenolide label i n Oncopeltus fasciatus i l l u s t r a t i n g the concentration of cardenolides by the various secretory glands Cardiac glycoside concentrations i n various stages of development of Oncopeltus fasciatus grown on seeds of Asclepias syriaca, as determined by colorimetry with reference to ouabain, digitoxin, and strophanthin K as standard cardiac glycosides The polarity effect on the uptake of cardiac glycosides from labelled seeds of Asclepias  syriaca into the dorso-lateral meso- and metathroacic glands of Oncopeltus fasciatus Predation results with frogs (Rana pipiens), toads (Bufo borealis), and chamelions (Anolis carolinensis) as predators to determine the palatability of Oncopeltus fasciatus Page 46 49 51 52 57 66 72 75 ,86 91 Predation results with turtles (Pseudemys  scripta) as predators to determine the palatahility of Oncopeltus fasciatus Predation results with chicks (Gallus) as predators to determine the palatability of Oncopeltus fasciatus VX1 LISO? OP FIGURES Figure Page 1 Map of North America showing the distribution of the various species of brightly coloured insects and species of plants assayed for cardiac glycosides " 4-6 2 General distribution of the major southern and northern Apocynad and Asclepiad species 4 0 considered i n this thesis 3 The distribution of cardiac glycosides of Asclepias syriaca i n the various tissues of 5th instar nymphs and adults of Oncopeltus 56 fasciatus Diagram of the mid-dorsal abdominal glands of a f i f t h instar nymph of Oncopeltus fasciatus 58 5 Diagram of the thoracic and abdominal cardenolide concentrating glands of Oncopeltus fasciatus 59 6 The uptake of the natural glycosides of Asclepias syriaca compared to the uptake of the cardenolide isotopes, oubain-^H and digitoxin-*!!, from a crushed seed preparation of seeds of Asclepais syriaca 64 x 7. 7 The uptake of ouabain- H and digitoxin- H from the haemolymph into the dorso-lateral meso- and metathoracic glands of the adult Oncopeltus fasciatus 70 8 The uptake of the natural cardiac glycosides of the seeds of Asclepias syriaca by Oncopeltus fasciatus, as determined by colorimetry with reference to ouabain as a standard cardenolide 78 9a The retention of ouabain- H by Oncopeltus  fasciatus when feeding on foods with and without cardiac glycosides 81 9b The retention of digitoxin- H by Oncopeltus  fasciatus when feeding on foods with and without cardiac glycosides 81 The uptake of ouabain-H from seeds of Asclepias syriaca into the dorso-lateral "thoracic glands of 1 week and older adults of Oncopeltus fasciatus The movement of cardiac glycosides (ouabain-^H) from the gut to the dorso-lateral thoracic glands of Oncopeltus fasciatrus after drinking labelled water X X LIST OF PLATES Plate Page 1 Adults of Oncopeltus f a s c i a t u s , the Large Milkweed Bug, r e s t i n g on leaves of Nerium oleander. x i i X ACKNOWLEDGEMENTS A thesis i s the product of more than one mind; therefore, the author extends special thanks to Dr. G.G.E. Scudder for the inception of this project and for his valuable criticisms and encouragement throughout the course of study. The author also acknowledges Dr. G.G.E. Scudder as the supervisor. The assistance of Drs. A.M. Perks, J.E. P h i l l i p s and G.H.N. Towers for c r i t i c a l l y reading the manuscript i s much appreciated. The author i s also appreciative of the many people through whose generosity f i e l d samples of insects from various regions of the continent were made available: Dr. R.M. Baranowski, Mr. W.D. Barkley, Mr. R. Gannings, Dr. D. Feir, Mr. D. Lane and Mr. D. Tanner. The author i s indebted to Dr. D. Feir, Dr. W.G. Friend and Dr. E.H. Salkeld for provid-ing new cultures of insects and Asclepiad seeds i n moments of need. Dr. K.I. Beamish i s thanked for many plant specimens. Thanks are extended to Mr. K. Clark for his suggestions and assistance i n carrying out the predation experiments, and to Mrs. M.D. Jensen for assistance i n the turtle experiments. Despite misfortune the author i s grate-f u l for the time and energy of Mr. S. Johnston to provide starlings. To Mr. J.W. Kamp, a fellow student i s passed an eternal smile. The valuable discussions with Mr. R. Neil are also appreciated. The research, was supported "by a National Research Council of Canada grant to Dr. G.G.E. Scudder. x i i PLATE 1: Adults of Oncopeltus f a s c i a t u s , the Large Milkweed Bug, r e s t i n g on leaves of Nerium oleander. 1 INTRODUCTION The chemical association between plants and phytophagous insects can be a more subtle relationship than that whereby the plant merely provides sustenance for the insect (Hewitt et a l . , 1969; Thorsteinson,I960). The non-nutritional role of primary and secondary plant compounds i s complex, with such effects as attracting insects and controll-ing their feeding behaviour (Beck, 1965). Many plants have a propensity for synthesizing repellant and/or toxic chemicals (Ehrlich et a l . , 1967). Insects have i n some cases benefitted from.these attempts of plants to be disagreeable. This benefit can either be derived by various casual associations with obnoxious plants or by selection of the plant as a food-host and perhaps from i t sequestering noxious principles or precursors of these. The noxious substances i n the insect then may serve as a defence against predators (v. Euw et a l . , 1967). Roth and Eisner (1961) discuss the occurrence of defensive mechanisms i n insects though,-they place considerable doubt i n most cases of the defensive secretions actually being dir e c t l y absorbed from the plant. Instead simple precursors are absorbed from the plant and further metabolized and stored i n well defined organ systems (Filshie et a l . , 1969; Fishelson, I960; Happ, 1968; Remold, 1963). This appears to be the case for the phenolic secretions of the 2 beetles Phyllodecta v i t e l l i n a e and Melasoma populi (see Roth et a l . , 1961 for details); though i t i s established that Eleodes l o n g i c o l l i s (Coleoptera: Tenebrionidae) produces i t s own phenolic secretions (Meinwald et a l . , 1966). In the case of the i r i d o i d a l monoterpenes of Dolichoderine ants (McGurk et a l . , 1968), the alkyl ketone and aldehyde secretions of various Hemiptera. (Gordon et a l . , 1963; Waterhouse et a l . , 1961, 1964-; Youdeowei et a l . , 1969) and cockroaches (Roth et a l . , 1961), and the cyanogenic glycosides of millipedes (Eisner et a l . , 1963) i t has not been thoroughly established i f these compounds are sequestered directly, derived from basic precursors i n the food, or synthesized by the biochemical ingenuity of the insects with or without the aid of alimentary or intracellular microbes. Recently attention has been brought to a powerful group of poisonous drugs, the cardiac glycosides or cardenolides (Wilbrandt, 1963), particularly i n Apocynaceous and Asclepiadaceous plants, as secondary plant compounds that are sequestered by Danaid butterflies, particularly the Monarch butterfly, Danaus plexippus (Brower, 1969; v. Euw et a l . , 1967; Parsons, 1965) and by the Orthopteran Poekilocerus bufonis (v. Eiiw et a l . , 1967; Fishelson, I960) for defensive purposes. It i s doubtful that the Monarch butterfly can synthesize the cardenolide molecule since the butterfly, i f raised on plants lacking cardiac glycosides w i l l contain no obnoxious chemicals and be palatable (Brower et a l . , 1967; Brower, 1969); 3 s i m i l a r i l y , P. bufonis appears to sequester rather than synthesize cardenolides (v. Euw et a l . , 1967; Rothschild, 1966). These l a t t e r two cases represent the best documented cases of a subtle r e l a t i o n s h i p between a plant and an animal i n which the animal besides gaining n u t r i t i o n and residence, acquires an array of h i g h l y repugnant chemicals to avoid being preyed upon. However, no d e t a i l e d biochemical or p h y s i o l o g i c a l information i s a v a i l a b l e on how these insects harbour these undesirable and very t o x i c compounds i n t h e i r bodies; t h e i r e f f e c t s can be profound (Lee et a l . , 1970; Vuilliemoz, et a l . , 1968). Around the Asclepiad dependant b u t t e r f l i e s (Danaids) at l e a s t has evolved a Batesian and M u l l e r i a n mimcry complex of considerable magnitude (Brower et a l . , 1964; Wickler, 1968). However the Lepidoptera do not form the only mimicry complex associated with Apocynaceae i n North and South America. There i s a large fauna of b r i g h t l y coloured Coleoptera and Hemiptera i n South arid North America associated with these plants (Weiss et a l . , 1921) and these have been implicated i n Mullerian and Batesian mimicry complexes. However l i t t l e d e f i n i t i v e work has been done to substantiate any of these claims (van Doesburg, 1968; L i n i l e y , I960; L i n s l e y ;et a l . , 1961; Jones, 1937, 1934, 1932), although chemical evidence has been provided that the so c a l l e d defensive secretions of many Hemipterans may be a basis f o r aposematism (van Doesburg, 1968; Calam et a l . , 1968; G i l c h r i s t et a l . , 1966 Remold, 1963). 4 Oncopeltus fasciatus i s widely distributed i n the nearctic (see Slater, 1964) and occurs with other brightly coloured Lygaeidae (Lygaeus kalmii; Slater et a l . , 1969), and beetles (Tetraopes; Weiss et a l . , 1921), a l l of which are associated with Asclepias. This thesis therefore examines the relationship between Oncopeltus and i t s Asclepiad host. It attempts to determine i f the cardiac glycosides known to be present i n the host plant (Bauer et a l . , 1964; Duffey, 1970) are u t i l i z e d by the insect i n any manner for protection, which could qualify this insect as a model for a mimicry complex. Brower's work (1969) implies glycosides are synonymous with unpalatability; this aspect i s also examined i n reference to Oncopeltus and various grades of predators. How Oncopeltus copes with undesirable compounds i s also investigated. 5 MATERIALS AND METHODS THE INSECT STUDIED The Lygaeids (Lygaeidae: Hemiptera) are t y p i c a l l y d u l l brown i n s e c t s ; t h i s ,colouration i s a c r y p t i c adaptation which matches the s o i l surface where they feed upon seeds ( M i l l e r , 1956). However, of the Lygaeids, the sub-family Lygaeinae i s exceptional being composed mostly of large and b r i g h t l y coloured species (contrasting oranges, reds, yellows and blacks) ( M i l l e r , 1956). Many such species of the Lygaeinae exis t i n North and South America, l i v i n g openly upon the a e r i a l regions of p r i m a r i l y Asclepiadaceous plants (Blatchley, 1924; S l a t e r , 1964, 1969; Torre-bueno, 1946; Weiss et a l . , 1921). Oncopeltus f a s c i a t u s i s a common b r i g h t l y coloured Lygaeinae somehow prospering by i t s obvious exposure; t h i s species i s unusually large making i t s e l f even more apparent to a predator's eye. Nymphs and adults occur together generally at the t i p s of the plants i n c l u s t e r s forming an e a s i l y v i s i b l e mass of i n s e c t s . Oncopeltus f a s c i a t u s appears throughout i t s l i f e to be mostly associated with the genus Asclepias s y r i a c a (Blatchley, 1924; Hussey, 1952; Robertson, 1887; Weiss, et a l . , 1921). This bug also occurs on Nerium oleander (Hussey, 1952; Packanian, 1955) and on Apocynum (Froeshner, 1944) both plants containing cardiac glycosides (Hoch, 1961). However Oncopeltus  f a s c i a t u s i s not obliged to feed upon Asclepias. In the spring or l a t e f a l l i t i s o c c a s i o n a l l y found upon willows and other plants (Blatchley, 1924) when lus h milkweed t i s s u e and/or seeds are not a v a i l a b l e : Lygaeus ka l m i i p a r a l l e l s t h i s s i t u a t i o n (Simanton, et a l . , 1936). Dingle (1968) reports that 0. fa s c i a t u s migrates. This may be to acquire new food-plants or just moisture from other plants a f t e r which they return to dried up milkweeds. This a b i l i t y to acquire moisture or food from other plants may be an advantage, but the period during which they feed upon these a l i e n plants (no glycosides) may be a c r i t i c a l period i n the insect's l i f e because they may become palatable. In f a c t a f u l l - l i f e a s s o c i a t i o n with Asclepias, from the f i r s t , i n s t a r nymph to the adult stage (5 nymphal i n s t a r s ) may be c r i t i c a l i n e s t a b l i s h i n g p a l a t a b i l i t y . The ease of re a r i n g Oncopeltus i n the laboratory permits some of these aspects to be investigated. REARING Oncopeltus f a s c i a t u s was reared i n an environmental control chamber at 27°G., R.H. 30% and a light/dark regime of 16:8, and was fed e i t h e r Asclepias s y r i a c a seeds or husked sunflower seeds. L i q u i d was normally presented from cotton wicks i n Erlenmeyer f l a s k s , and consisted of a d i l u t e s a l t s o l u t i o n (KCl - 3 gm./lit., NaCl - 1 gm./lit., MgCl 2 - 0.63 gm./lit., and C a C ^ - 0.50 g m . / l i t . ) . Some insects were reared under s i m i l a r conditions but were contained i n glass preserving j a r s or cardboard, food containers with screened l i d s , watered from wicked v i a l s , and fed on d r i e d leaves, seeds, or pods of various plant species. 7 EXTRACTION AND PURIFICATION The e x t r a c t i o n and p u r i f i c a t i o n procedures used were designed f o r approximately 1-30 grams of fr e s h t i s s u e weight, and were f o r r a p i d separation of cardiac glycosides and f o r estimations of the numbers of cardenolides i n t i s s u e of plants (seeds) or i n s e c t s . Insects c o l l e c t e d from the wil d were k i l l e d by pl a c i n g them i n 95% ethanol and stored at -20°C. i n t h i s f l u i d u n t i l used. Laboratory reared insects were k i l l e d by pl a c i n g them d i r e c t l y i n t o chloroform-methanol (2:1). The extraction of cardiac glycosides was a modification of the method f o r l i p i d e x traction of Folch et a l . , (1957), as described below. Both the insects and t h e i r ethanolic preserving f l u i d were used i n the extraction of the glycosides as follows. The f i l t e r e d preserving f l u i d was dr i e d i n vacuo at 45°C. and dissolved i n a minimal volume of chloroform-methanol (2:1).> The i n s e c t s ( f r e s h or preserved) were homogenized i n chloroform-methanol (2:1) (10 - 15 mls/gm) twice. The residue each time was f i l t e r e d o f f and f i n a l l y r i n s e d with 25 mis. of chloroform, a l l e x t r a c t i o n volumes being pooled, i n c l u d i n g the resolved preservative f r a c t i o n . The pooled extracts were p a r t i t i o n e d overnight against 1/5 volume s a l i n e (0.9% NaCl and 0.1% NaHCO^). The chloroform l a y e r was recovered and the sa l i n e l a y e r washed at l e a s t twice with 25-30 mis. of chloroform, since the most polar cardiac glycosides remained i n the s a l i n e phase i n s i g n i f i c a n t q u a n t i t i e s . A l l chloroform extracts were pooled and dr i e d i n vacuo at 4-5°C, and then redissolved 8 i n a minimal volume of ethanol free chloroform. The latte r was made "by partitioning chloroform against an equal volume of d i s t i l l e d water overnight after thorough mixing, and drying the chloroform with anhydrous Na2S0^. Seeds (1-2 gms.) were extracted by crushing the seeds i n a mortar and pestle with 25 mis. of n-hexane. The n-hexane and the seed pulp were transferred to a soxhlet thimble and refluxed for 3# hours with ethanol-methanol (1:1) (100-150 mis), the temperature being kept s u f f i c i e n t l y hot to keep the extract boiling. The extract was then dried i n vacuo and thereafter treated the same as an insect tissue extract. Leaf and pod extracts were made from dried plant tissues; therefore, special precautions could not be taken to prevent glycoside hydrolysis. As l i t t l e as 50 mgs. of tissue could be extracted by the method below, but i f larger quantities of material were available, 5-10 gms. of dry tissue were hand crushed to a coarse powder and soaked overnight i n 50-100 mis., of 50% aqueous ethanol at 45°C. The ethanolic f l u i d was then decanted and the tissue rinsed with 50 mis. of aqueous ethanol which was added to the former volume. An excess of lead acetate was added to this f l u i d , i t was shaken well, and allowed to stand for 1 hour. An excess of Na2HP0^ was added, the shaken, and allowed to settle. The supernatant was collected i n a separatory funnel, the residue rinsed with a further 25 mis*of aqueous ethanol, allowed to settle, and then this f l u i d added to the .separatory funnel. The extract was then partitioned three times with 50-75 mis. of chloroform, these extracts being pooled, dried by an air jet, and dissolved in. a minimal volume of ethanol-free chloroform i f i t was to be purified by column chromatography. Otherwise, i t was dissolved i n a minimal volume of chloroform-methanol (2:1) and used directly for thin layer chromatography. This method i s not suitable for quantitative extraction of cardiac glycosides from plant tissue. For purification of insect and leaf extracts by column chromatography, the extracts were applied to a column of F l o r i s i l (Floridin Company, Tallahassee, Florida), 60-100 mesh, 15 cm. x 1.5 cm. c h i l l e d by a water jacket. The F l o r i s i l was treated as outlined by Carrol (1961) and the elution technique was a modification of the method outlined by the above author. The elution sequence was as follows: (flow rate was approximately 4 mis./min., 10°C.) Fraction 1/ Hexane-diethylether (1:1) 50 mis. Fraction 2/ Diethyl ether - methanol (50:1) 50 mis. ( f i r s t 18 mis. discarded after addition to column when the above eluent reaches bed level) Fraction 5/ Diethyl ether - methanol (1:1) 50 mls .<r Fraction 4/ Methanol 100 mis. The last 52 mis. of fraction 2 and fraction 5 and 4 contained the cardiac glycosides; individual glycosides could not be separated by this technique, even at slower flow rates, nor with a more gradual increase i n eluent polarity such as benzene - methanol 100:1, 100:1.5, 20:1, 50:1, though there 10 was a demarcation between the l i p i d and polar cardenolides. This column partitioning does not completely r i d the glycosides of contiminating steroids and fats. However, further resolution could be obtained by thin layer chromatography (TLC). This elution technique easily separated extracts of 60 insects. After elution the fractions containing the glycosides were pooled and dried i n vacuo at 45°C. and resolved i n 1-2 mis. of chloroform-methanol (2:1) and stored at 0°C. u n t i l required for TLC or bioassay. CHROMATOGRAPHY Por thin layer chromatography of cardiac glycosides, S i l i c a gel G plates were prepared 250 microns thick, dried openly overnight, and then stored i n a desiccator containing sulphuric acid u n t i l used. S i l i c a gel G plates were highly dependant upon proper activation for separation; thus, once a plate was removed from the desiccator i t was spotted as rapidly as possible so as not to exceed the 15 minute requisite exposure time to the laboratory atmosphere and to obtain somewhat constant Rf values ( £ 0 . 0 5 : Rf front = 1.0). The solvent systems employed at 16°C. were ethylene dichloride-methanol-formamide (80:25:1) or methylene dichloride-methanol-formamide (80:10:1). Ouabain and digitoxin (Nutritional Biochemical Company) were used to define the approximate limits of cardenolide TLC behaviour i n the various solvent systems over a 10 cm. solvent migration, though several cardenolides from seeds had Rf values higher than digitoxin. Cardiac glycosides were detected by one or more of the following reagents, l i s t e d i n order of excellence of reactivity and sp e c i f i c i t y . Reagent 1/ Eluted plates were sprayed with 3,5-dinitrobenzoic acid (2% i n 70% ethanol), heated for 1}& minutes at 110°C, and then sprayed with 10% NaOH. Glycosides appeared as purplish spots which faded rapidly. It was found that i f the plates were sprayed with 95% ethanol saturated with 2,4-dintirophenylsulphone immediately after they were sprayed with NaOH, the colours were intensified and s l i g h t l y prolonged. Reagent 2/ As above but employing 3,5-dinitrobenzene. This reagent i s very evanescent and not as sensitive as the former. Reagent 3/ As above but with a 1,2-naphthaquinone-4-sulphonic acid saturated solution of 70% ethanol. Glycosides appear as purple spots. Reagents 1, 2 and 3 give v i s i b l e spots when approximately 5 micrograms are present i n an Rf zone. Reagent 4/ Eluted plates were sprayed with a 1% solution of aqueous p i c r i c acid followed by 10% NaOH. Orange spots (due to cardenolides) could be accentuated by spraying the plates with concentrated sulphuric acid u n t i l the back-ground becomes white. It i s not as sensitive as 1 or 3, (10-20 migrograms). Reagent 5/ Eluted plates were heated for 5-10 minutes at 110°C. and then sprayed with a 1:1 mixture of 0.125 % xanthydro1 i n gl a c i a l acetic acid and concentrated HG1. This reagent i s very sensitive but i s not as specific for cardenolides as are the above reagents, since many compounds well outside the cardenolide Rf zone and tissue extracts, which contain no cardiac glycosides (even i n the cardenolide zone) react to give red spots. It detects approximately 1 microgram. If an extract was limited these reagents were applied to one plate i n sequence, that i s , 1 ,3,5: 2 ,3,5: 3,4,5: but time was allowed for reagents 1, 2 and 3 "to fade before the application of any other reagent. For purposes of chromatography the column purified extracts of plant and insect tissue (column elution of extracts of small amounts of leaf and pod tissue were not warranted) after being dried down i n vacuo were resolved i n chloroform-methanol (2:1) i n three 1 ml. aliquots to suf f i c i e n t l y wash the evaporation flask, and then l e t to dry i n open a i r to approximately 0.5 mis. If 20 or more insects, or 2 or more gms. of seed were extracted 50 microliters of the 0.5 mis. was usually sufficient to give a good reagent response on a TLC 13 plate. The extraction of cardenolides from seeds posed no problems with interfering pigments, and extracts could be used di r e c t l y for chromatography. However, extracts of leaves and pods, though they were cleared of the majority of interfer-ing pigments by lead acetate precipitation, s t i l l contained enough pigment to interfere with the chromatographing. Therefore, i n spotting the plates precautions had to be taken to ensure that the plate was not overloaded with pigment which would result i n severe streaking and obscuring of the cardiac glycosides* reaction with the reagents. The leaf and pod extracts contained very l i t t l e l i p i d material and the TLC solvent systems could be used dire c t l y regardless of whether the extracts were purified on the column or not. With seed and insect extracts that had not been purified on the column but merely resolved i n chloroform-methanol (2:1) after partitioning against saline (to scan tissues for cardenolides), i t was found advisable to f i r s t elute the plates i n a chloroform-methanol (10:1) system, l e t them a i r dry for 5 minutes, and then elute with the cardenolide solvent systems. The application of column purified extracts of insect or plant tissue was done carefully to avoid overloading of the plate which would result i n streaking of the cardenolides. This was complicated by the fact that the cardiac glycosides present i n the seeds of Asclepias examined had closely approximating Rf values. SPECTROPHOTOMETRY AND COLORIMETRY To be more conclusive about the identification of 14 m-dinitrobenzoic a c i d p o s i t i v e compounds i n the extracts, spectrophotometry (Spectronic 620) was undertaken using reagents 5, 4 and 1. Reagent 4 was employed as described by Rowson (1952), but the r e a c t i o n volume was reduced to 2 mis. and approximately 50 m i c r o l i t e r s of column p u r i f i e d extract (from the 0.5 ml. volume) was added d i r e c t l y . The maximum peak of absorption occurred at 490 to 500 nm. Reagent 5 was used i n the following manner: 1.9 mis. of 0.125% xanthydrol i n g l a c i a l a c i d and 0.1 mis. of concentrated HC1 were mixed, 50 m i c r o l i t e r s of the extract added, heated i n b o i l i n g water f o r 1 minute and then placed i n an i c e bath. The maximum absorption peak occurred at approximately 625 nm. Both t h i s and the above reagent were stable f o r % to 1 hour, but the 3 , 5-dintrobenzoic a c i d NaOH reagent colour described below l a s t e d only 1-3 minutes. Reagent 1 was employed as a modification of the method outli n e d by Rowson (1952) so that i t could be applied to the col o r i m e t r i c estimation of cardenolide content i n whole s i n g l e insect extracts. The method was as follows: 1 ml. of 2% 3,5-d i n i t r i b e n z o i c a c i d i n 95% ethanol was mixed with 1 ml. of the insect extract (described below) resolved i n chloroform-methanol (2:1), 0.5 ml. of 1 M NaOH added, shaken and read at 565 nm. on a Spectronic 620 with maximum colour occurring at 1 minute and 40 seconds. If only the presence of a peak at 565 nm.was required and extract was l i m i t i n g , the same o u t l i n e was followed but the extract was added i n the form of 50 m i c r o l i t e r s to the 1 ml. of m-dinitrobenzoic a c i d s o l u t i o n and 1 ml. of chloroform-methanol. The peak was read at 565 nm. on the Spectronic 620. If the sample "being scanned on the Spectronic 620 turned milky on the addition of NaOH the solution was cleared by the addition of 95% ethanol to the cuvette; this shifted the peak to shorter wave lengths, closer to the values stated for the original procedure of Rowson (1952). Standard curves were obtained with ouabain, digitoxin and strophanthin K. Each standard curve obeyed Beer's Law up to 50 mgm. %. The modifications of the colorimetry procedures for the reaction of cardiac glycosides with alkaline 3,5-dinitrobenzoic acid from those directions prescribed by Rowson (195?), were necessitated since the f a c i l i t o u s method of extracting 1-2 bugs i n chloroform-methanol (2:1) was not applicable i n resolving the dried extract i n 50% ethanol, because a constant time could not be obtained for the maximum color intensity. The u t i l i z a t i o n of chloroform-methanol (2:1) as a portion of the reaction volume stabilized the time for maximum colour and permitted the dissolution of l i p i d soluble material which would not otherwise be soluble i n Rowson's reaction mixture. This method also alleviates the problem of dissolving l i p i d soluble fats,cardiac glycosides or agly-cones. It was also found i n preparatory experiments for modifying the colorimetry method that one could carry out the procedure i n an almost anhydrous condition. Solvents such as chloroform, ethylene dichloride or hexane could be employed 16 i f the alkaline reagent, 1 N NaOH, was made with d i s t i l l e d methanol; 2% 3,5-dinitrobenzoic acid i n 95% ethanol was s t i l l employed, or absolute alcohol as an alternative. Extractions of insects for colorimetry were carried out as follows: 2 adult Oncopeltus fasciatus (or larger numbers of the other nymphal instars up to 150 mgms.) were anaesthetized with methylene dichloride, weighed, transferred to a glass tube containing 3 mis. of chloroform-methanol (2:1), crushed with a glass rod, heated b r i e f l y i n a water bath u n t i l l i g h t l y boiling (tube agitated while heating to avoid explosion of contents), and f i l t e r e d over a tig h t l y packed wad of cottonwool. The tube was rinsed three times with 1.5 mis.of the extraction f l u i d , and dried down overnight at 65°C-with metal pins used as boiling chips. The extract was then resolved i n 1.5 mis. of chloroform-methanol (2:1) and used for colorimetry, from which the 1 ml. aliquot was taken. The extraction f l u i d was efficient i n removing a l l ranges of cardiac glycoside polarity. Two adults represented one sample, 2-5 f i f t h instar nymphs, 4-8 third and fourth instar nymphs, 7-16 second and f i r s t instar nymphs, 13-17 f i r s t instar nymphs and 11-30 eggs likewise represented one sample. Prom egg to second instar 5-5 samples were taken and from third instar to adults 7-12 samples were taken for each stage of develop-ment. The author expresses his awareness of the theoretical d i f f i c u l t y of comparing these colorimetric results to the standard curves. 17 To estimate the cardiac glycoside content of the adult l a t e r a l dorsal metathoraic glands, the secretions of these glands were collected i n disposable 10 lambda pipettes (the meso- and metathoracic secretions were pooled since the two glands mix their contents naturally at the posterior dorso-lateral margins of the metathorax). For the dorso-lat e r a l abdominal glands of the adult (segments III - VII) the secretions were also pooled. Secretions from the mid-dorsal abdominal glands of nymphs (segments IV ant'V were mixed. It was found that 2-5 microliters of secretion was adequate for colorimetric determinations. However, each individual gland was checked by TLC methods to verify the presence of cardiac glycosides. Fluid from the anus and haemolymph (of insects with transected legs) were likewise collected, but 5-10 micro-l i t e r quantities were employed. The micropipettes containing the various flui d s were treated as i f they were an insect for extraction and colorimetry. To determine the presence of cardenolides i n the various individual glands, 0.1 - 0.2 u l were collected i n 1 lambda pipettes and applied to TLC plates and sprayed with the above mentioned reagents. In order to show the presence of alkyl aldehydes and ketones i n the gland fluids of Oncopeltus 0.1 - 0.2 microliters of gland f l u i d was applied to a sheet of f i l t e r paper dampened with 2N ethanolic HC1 saturated with 2,4-dinitrophenylhydrazine. The reagent gave orange spots i f the alkyl compounds were present. The 18 cardenolides showed no immediate reaction. To further establish chemical evidence for cardiac glycosides, extracts of Oncopeltus fasciatus plus a standard (a mixture of omabain, digitoxin, and strophanthin K) were acid and base hydrolyzed alternatively i n 50% aqueous ethanol at pH 1 and pH 14 with HC1 and W&OH at 60°C. for 2 hours each. For the bioassay the extracts were similarly hydrolyzed, but this was carried out i n their saline solution and neutralized with HC1 before use. The dilution resulting during the hydrolysis of bioassay samples was kept to a minimum. Also the extracts and the standardswere methylated (Morrison et a l . , 1964), and heated i n the 2,4-dmitrophenylhydrazine reagent to give the corresponding derivatives. A l l these compounds on elution i n the solvent system (chloroform-methanol 9:1) followed by spraying with the described reagents maintained their identities as cardiac glycosides. BIOASSAY For the bioassay of cardiac glycosides the inotropic response of a rat heart was recorded on a Gilson polygraph. Column purified extracts were dried and resolved i n 0.9% NaCl saline (5% ethanol); the appropriate volume of ethanol was f i r s t added to the dried extract to f a c i l i t a t e dissolution. A l l seed and leaf extracts were diluted to 5 mls/2-5 gms. tissue; insect extracts of 2-10 animals were diluted to 5 mls»;> and 11-60 animals to 10 mis. Upon total dilution some 19 precipitation occurred; this was removed by centrifugation. The diluted samples were stored at 5°C. u n t i l required. For the determination of an inotropic effect i n plant and insect extracts male and female rats, 200-350 gms., were anaesthesized with Nembutal (2.6 mgms^ /100 gms.), and the blood pressure recorded v i a a polyethylene cannula i n the carotid. Samples were injected through a polyethylene cannula i n the jugular. Injection volumes ranged from 0.1 -0.4 mis. Each rat was used for only 4-6 unknown injections, and no attempt at quantification was made. Dibenzyline was not injected with the anaesthetic' so as to minimize sensitization and therefore make the inotropic response less exaggerated. Ouabain (3 x .10"^) was used as a standard; also 1 mgrt\. each of ouabain and digitoxin were subjected to the column elution procedure and separated from equal quantities of cholesterol and palmitic acid. The inotropic a c t i v i t y occurred only i n the glycoside fraction off the column. For the antagonism of the inotropic effect induced by cardiac glycosides (Thorp et a l . , 1967) aldosterone (3 x 10~^Mi) was mixed 1:1 with the assay solution and injected, rather than pretreatment as suggested by Lefer et a l . (1964). Inhibition of the inotropic response was almost complete. ISOTOPE EXPERIMENTS Radio-isotopes of two cardiac glycosides were used to study the uptake and retention of cardenolides (ouabain-G-^H, 11.7 curies/mK. and digitoxin-G-%, 4.5 curies/mM.) i n 20 Oncopeltus fasciatus to confirm estimates of the uptake and retention of natural glycosides of seeds of Asclepias syriaca as determined "by colorimetry. The isotopes (New England Nuclear) were counted by a Nuclear Chicago Mark 1 li q u i d s c i n t i l l a t i o n counter. The s c i n t i l l a t i o n solvent, toluene-ethanol (99:1) with 0.5% PH) (2 ,5-diphenyloxazole) and 0.03% dimethyl POPOP (l,4-bis-[2-(4-methyl-5-phenoxazolyl)] -benzene), was dehydrated with Na2S0^ before dissolving the fluors. A l l extracts of animals, seeds, and micropipette samples of haemolymph and gland f l u i d were ground i n chloroform-methanol (2:1) and treated as described for the preparation of insect extracts for colorimetry, except that the extracts were dried i n the s c i n t i l l a t i o n v i a l s by means of a fan. The dried extract of one insect (one sample) was resolved i n 10 mis. of s c i n t i l l a t i o n f l u i d . (a) The uptake of isotopes of ouabain and digitoxin throughout the l i f e cycle To study the kinetics of uptake of two cardenolides, one polar, one l i p i d , throughout the l i f e cycle of Oncopeltus  fasciatus (egg to egg) ouabain and digitoxin isotopes were independently applied to Asclepias syriaca seeds. Ten grains of seeds were crushed i n a grinder to a course powder (particles 0 .5 - 1.0 mm.in diameter) and added to 25 mis. of chloroform-methanol (2:1) containing the appropriate amount of label to impart approximately 440 d.p.m./mgm. seed of 3 z ouabain- H and 2800 d.p.m./mgm. seed of digitoxin - H. The 21 seed-solvent mixture was stirred well for fiv e minutes, l e f t to soak for 4 hours at 45°C., and then dried at that temperature with frequent s t i r r i n g to prevent unequal distribution of the cardiac glycoside label and the natural Asclepias cardenolides. This method resulted i n a seed preparation with a standard error heterogeneity of 4 _ 88 d.p.m./mgm. and +_ 550 d.p.m./mgm. for ouabain and digitoxin respectively. The amount of cold glycoside included with the isotope on adding the l a t t e r to the seed-solvent mixture would maximally not exceed 0.1 micrograms/gram of seed, which was considered negligible compared to the natural glycoside concentration of the seeds which was 415 micrograms/gram of whole seed. The material used i n this experiment were newly l a i d eggs that had been stored at 10°C. for two weeks prior to use. Eggs stored for a month or more would not hatch on being placed i n a benign environment. These diapausing eggs were placed i n plastic lined pint cardboard food containers with a 2 inch square nylon mesh window i n the l i d of the container. Liquid was presented i n a 25 ml. v i a l with a cotton wick. The radioactive crushed seeds were presented i n 3/4 inch diameter shallow lipped p l a s t i c v i a l s (approx. 500 mgm./vial), two of these v i a l s being put i n each container to feed up to 15 bugs; the water and the food were replaced every third day. The insects otherwise were reared under the conditions previously described. As a control to ensure that the amount of feeding 22 was not abnormally disturbed by the treatment i n labelling the crushed seeds, 4- samples were taken of both f i f t h instars and one day-old adults and the level of natural cardiac glycosides measured by colorimetry to compare with the levels of cardiac glycosides present i n insects fed on whole unlabelled milkweed seeds. To estimate the uptake of the labelled cardenolides from the crushed seed preparations, 5-8 insects were collected at each of the 5 nymphal instars and at 1 day-old and 1, week-old adult stages. Each nymphal instar was collected at approximately the middle of i t s stadium, while the adult samples were collected within 12 hours of moulting or at the age stated. One insect represented one sample. Also, gland f l u i d (nymphal mid-dorsal abdominals and adult dorso-lateral meso- and metathoracic), haemolymph and anal f l u i d were collected from at least three f i f t h instar nymphs, 1 day-old, and 1 week-old adults from both types of glycoside label feeding regimes. In both nymphs and adults the fluids collected from the glands were pooled (nymph's segments IV and ..V of abdomen, and adult's meso- and metathorax). The a c t i v i t i e s of the glandular, haemocoelic and anal fluids were obtained and compared only as ratios when a l l f l u i d s were derived from one insect. Eggs l a i d by the radioactive bugs and also f i r s t instar nymphs emerging from these eggs were measured for label within hours of emergence; no food was available to the insects. 23 (b) The retention of ouabain- H and digitoxin- H 7. A study of the retention of ouabian- H and digitoxin-H by Oncopeltus fasciatus was carried out by applying 2 micro-l i t e r s of either compound i n ethanol-benzene (95:5) directly to one whole uncrushed seed, attempting to place most of the volume on the endosperm area of the seeds on both sides. This method was chosen i n preference to the labelled crushed seed preparation since large amounts of label could be applied easily to a few seeds and then one type of labelled seed given to newly hatched f i r s t instar. This guaranteed that they would be highly labelled by their third instar; the specific a c t i v i t y of the seeds was not calculated. Not a l l the label would soak into the inner regions of the seed; thus, the uptake of the label was not complete or l i k e l y even throughout three instars (feeding time), but the purpose of the radioactive pulse (approximately 1 million d.p.m./seed of digitoxin and 150,000 d.p.m./seed of ouabain) was merely to produce larvae with a high specific activity. The amount of cold ouabain and digitoxin added to the seed with the isotope would not maximally exceed 0.1 micrograms/seed, which was considered insignificant i n diluting the natural glycosides of the Asclepias seeds. Eggs from cold storage were placed i n food containers (previously described) and permitted to develop u n t i l the middle of their third instar feeding on the labelled whole seeds (10 seeds/10 larvae). These pulsed third instar larvae were then transferred to whole unlabelled milkweed seeds. A 24 large excess of seeds was added and these and the l i q u i d changed every third day. At least 4-6 samples were taken at the third, fourth and f i f t h nymphal instars, and at the 1 day-old and 1 week-old adult stages. The a c t i v i t y was counted i n the extracts of each insect. Also, pulsed third instar larvae were transferred to an excess of sunflower seeds, the l i q u i d and seeds being changed every third day. The insects were grown to the f i f t h instar nymph, 3-5 samples being taken to measure the retention of isotopes. However, since the insects i n their transformation from the late nymphal instars to a 1 day-old adult were found to lose a large percentage of their sequestered label, f i f t h instar nymphs (arising from the pulsed third instar that had been fed on whole unlabelled milkweed seeds to their f i f t h nymphal instar) were transferred to an excess of sunflower seeds and managed as previously stated, i n order to determine i f the rate of loss of label would be greater than the rate of loss from the third instar to the f i f t h instar (on sunflower seed) and greater than the loss from f i f t h instar to a 1 day-old adult,(on whole unlabelled milkweed seeds). In this experi-ment 4-6 samples were taken at the 1 day-old and 1 week-old adult stages and the amount of label present determined. A© a control experiment the loss of the natural cardiac glycosides sequestered from the Asclepias seeds was determined by placing third instar nymphs and 1 day-old adults (both grown 25 on unlabelled whole Asclepias seed) on an excess of sunflower seeds. The third instar nymphs were allowed to develop on the sunflower seeds to their f i f t h instar and 1 day-old adult stages at which 3-4 samples were measured colorimetrically for cardiac glycoside content. Also, the 1 day-old adults that had been placed on an excess of sunflower seeds were fed for a week on that regime and their cardiac glycoside content measured by colorimetry. A blank for the extracts of Oncopeltus fasciatus which had been fed on sunflower seeds was employed i n these colorimetric estimations, though this was not found to be necessary for insect extracts that had originated from the insect fed only on Asclepias seeds. It was found that often the extracts of Oncopeltus fed on sun-flower turned cloudy on the addition of the 0.5 mis. of NaOH to the colorimetric reaction mixture. This problem was often alleviated by immediately doubling the reaction volume. (c) Gland transport experiments In the study of cardiac glycosides i n the dorso-lateral and ventral metathoracic glands of the adult, i n the adult dorso-lateral abdominal glands (segments III - VII), and i n the larval mid-dorsal abdominal glands several approaches were taken. Oncopeltus adults were fed for 10 days on whole Milkweed seeds containing unspecified amount of ouabain-%. Samples of dorso-lateral metathoracic gland f l u i d and haemolymph (maximally 1 microliter) were collected from three 26 insects, comparing the gland and haemolymph content from one insect, at 1, 3, 5 and 10 days. The concentration of label i n the adult dorsal l a t e r a l abdominal glands ( f l u i d from segments III-VII pooled) was determined at 5 days only. To estimate the approximate concentration of cardiac glycoside label i n the larval mid-dorsal abdominal glands, and to show that i t i s more concentrated than the haemolymph, 3 three f i f t h mstar nymphs that had been reared on the ouabain- H labelled crushed seeds (the same insects from which the haemolymph, and anal f l u i d were collected) were chosen. These gland secretions were collected i n f i n e l y drawn glass micro-pipettes that were gently pressed against the o r i f i c e of the two abdominal glands (segments IV and V). Only 0.005 - 0.01 microliters was collected, but this volume was sufficient to determine the radioactive content. Further, to show that the dorso-lateral meso- and metathoracic glands of the adult could concentrate cardenolides, besides the evidence from the colorimetric determinations, 1 microliter of insect saline (Strong et a l . , 1968) containing label was injected into the haemolymph through the abdomen vi a 3 5 f i n e l y drawn glass micro-pipettes. Ouabain- H and digitoxin- H were injected into separate test animals to also determine i f a polarity effect (higher levels of polar glycosides maintained i n the gland fl u i d ) was e l i c i t e d at the gland l e v e l . The injection f l u i d contained 10 micrograms of cold ouabain dissolved i n 0.1 mls.of the insect saline. 10 migrograms of 27 digitoxin would not dissolve i n this volume of saline therefore, both isotopes were dissolved i n the ouabain-saline mixture so that 1 microliter of injection of either label gave approximately 2780 d.p.m.,; Three samples (from 3 insects) of gland f l u i d and haemolymph were taken at }& nr., 1 hr., 2 hrs., 3 hrs., 4 hrs., 5 hrs., and 8 hrs. after injection into the haemolymph unless otherwise indicated. This experiment was not undertaken for a quantitative estimate of uptake, but served to i l l u s t r a t e a polarity effect at the gland level and verify a concentration mechanism. To prove that the ventral metathoracic gland of the adult Oncopeltus fasciatus could also concentrate cardiac glycosides, 4 samples of gland f l u i d were collected at the ' 5 hour interval of the above ouabain- H injection experiment. These four animals were also used to take the haemolymph and meso- and metathoracic gland f l u i d samples. Also from 3 animals i n both the ouabain- H and digitoxin-^H injection experiment above (from which the 5 hour interval samples were taken) were sampled for the presence of label i n the urine or anal secretion. This was collected by gently squeezing the abdomen of the animal after the gland fluids had been collected, but before the haemolymph samples were taken. To show that Oncopletus fasciatus were capable of sequestering higher levels of cardiac glycoside label than the level obtained from the uptake experiment (a) adults 1 week-28 o l d or older were presented with h i g h l y l a b e l l e d whole seeds of Asclepias s y r i a c a . These adults were allowed to feed on seeds i n d i v i d u a l l y l a b e l l e d with approximately 1 m i l l i o n d.p.m. of ouabain- H applied evenly on both sides of the seed endosperm (3 seeds/adult). The l a b e l was applied to the seed i n m i c r o l i t e r quantities of ethanol.benzene (95«5)» Haemolymph, and d o r s o - l a t e r a l meso- and metathoracic gland f l u i d (pooled) samples were c o l l e c t e d a f t e r 1 , 3 , 5, and 10 days of feeding. Also a f t e r 5 days of feeding gland f l u i d from the d o r s o - l a t e r a l abdominal" glands were.collected. The p o s s i b i l i t y remained that i f a p o l a r i t y e f f e c t 3 3 was observed i n the uptake of ouabain- H and d i g i t o x i n - H from the crushed seed source (experiment a) a f t e r feeding from the f i r s t nymphal i n s t a r to the 1 day-old or 1 week-old adult that t h i s p o l a r i t y e f f e c t would be more d i r e c t l y r e l a t e d to feeding behaviour and morphogenesis throughout that time rather than to d i r e c t e f f e c t s of the d o r s o - l a t e r a l meso- and meta-thoracic glands. Therefore, to be assured that older adults were e l i c i t i n g a p o l a r i t y preference of a magnitude s u f f i c i e n t to account f o r any detected differences from the uptake experiment, adults, 1 week ol d and older, were fed highly l a b e l l e d seeds of Asclepias; one group of adults was fed only ouabain or d i g i t o x i n l a b e l . To each seed (4.3 mgm./seed) eit h e r 3 x 10^ d.p.m. of digitoxin-^H or 0.5 x 10^ d.p.m. of ouabain- H was applied to the endosperm area of the seed, on both sides, and soaked a f t e r the ethanol-benzene (95:5) solvent 29 had dried with several microliters of chloroform-methanol (2:1) several times. By this treatment the seed was assumed to be homogeneous i n content of label. Five such seeds were given to every three insects and the insects allowed to feed upon these seeds for three days; whence, the dorso-lateral meso-and metathoracic gland f l u i d (pooled) and haemolymph samples were taken from 3 insects for determination of gland/haemolymph activity ratios. (d) Uptake of cardenolide label from the gut To determine i f the gut might be a limiting factor i n the rate of uptake of cardiac glycosides, and that they could be taken up from the gut without the presence of food i n the gut a crude experiment was undertaken involving the drinking of glycoside labelled water by 1 day water and food fasted adults. Ten micrograms of ouabain (unlabelled) was added to 0.5 mis. of tap water i n which had been dissolved enough cardenolide to impart 1000 d.p.m./microliter of water of ouabain-^H. The fasted insects were allowed to drink for 4-5 minutes after which the cotton wads i n which the l i q u i d was presented was removed. Samples of gland f l u i d and haemolymph were taken from 3 insects at 1, 2, 3 and 4 hours after commencement of drinking. PREDATION EXPERIMENTS Predation experiments were carried out using the following animals: 6 toads, Bufo boreas; 4 frogs, Rana pipiens; 30 4- t u r t l e s , Pseudemys s c r i p t a sub-species; 6 green anoles, Anolis c a r o l i n e n s i s ; 2 northern a l l i g a t o r l i z a r d s , Gerrhontius  coeruleus p r i n c i p i s ; and 3 f o u r - f i v e week old chickens, Gallus. Toads, frogs, green anoles (supposedly from F l o r i d a area) and chickens were purchased through l o c a l dealers, and thus the f i r s t three could not be considered naive. The a l l i g a t o r l i z a r d s were captured l o c a l l y (Vancouver, B r i t i s h Columbia); t h e i r h i s t o r y was not known. The 4- t u r t l e s (four years o l d , both males and female) were domestic pets and were considered to be naive predators. The toads and frogs were kept i n d i v i d u a l l y i n 5 g a l l o n aquaria with sand covered bottoms, perforated cardboard sheets as l i d s , and a pan of water i n a corner of the tank. They were fed one 1 - \% inch dewinged grasshopper or several Tenebrio molitor larvae and/or adults once a week f o r two weeks before use i n the predation experiments. The green anoles were kept i n a large screen topped aquaria p r i o r to use and fed on f r u i t f l i e s . For the experiment they were i n d i v i d u a l l y placed i n the same 5 g a l l o n aquaria used f o r the f r o g experiments and kept there during the t o t a l duration of the experiment. Each a l l i g a t o r l i z a r d was kept separately i n a 5 g a l l o n aquarium f o r the whole experiment. The t u r t l e s were kept together i n a large 2* x 3' glass tank. They had been previously fed on scraps of meat, vegetables, and various stages of Tenebrio. For these predation experiments they were fed simultaneously i n the same 31 container, but each animal was presented with i t s own tes t food i n a d i f f e r e n t region of the tank. They were fed on no other food than the t e s t foods during the en t i r e course of the experiments. The chickens were contained i n 2#' x 2#>l x 3' card-board boxes with a 1 foot square )&" mesh wide door i n the front of the cage. A l l other sides of the boxes were closed. Light was provided from a 60 V/att desk lamp positioned i n the top of the back wall of the cage. They were given 12 hours of a r t i f i c i a l l i g h t (9:00 a.m. - 9:00 p.m.), but no attempt was made to exclude natural l i g h t from the laboratory (the fluorescent l i g h t s of the laboratory provided s u f f i c i e n t l i g h t f o r the other predators). The chickens had been r a i s e d on Chick-starter, and were maintained on t h i s d i e t from 1:00 p.m. t i l l 8 p.m. a f t e r which only water was a v a i l a b l e to them from a glass bowl. The feeding experiments were c a r r i e d out i n the morning a f t e r a night of food d e p r i v a l . I n i t i a l experiments ind i c a t e d that the l e v e l of s a t i e t y d i d not a f f e c t the eager-ness of the chickens to accept Tenebrio larvae and adults presented to them. The frogs and toads were both fed i n an i d e n t i c a l manner. On each succeeding day of the experiment the t e s t -food animals were presented skewered on a p i n and suspended i n the f i e l d of v i s i o n of the predator by a black thread. On each day randomized p a i r s of two test-food animals were presented to each predator, u n t i l s i g n i f i c a n c e i n choice was a c h i e v e d t o 95% c o n f i d e n c e l e v e l a s o u t l i n e d "by C o l e (1962). T h e f i r s t p a i r o f p r e y p r e s e n t e d t o t h e t o a d s a n d f r o g s c o n s i s t e d o f a n a d u l t T e n e b r i o m o l i t o r a n d a n a d u l t O n c o p e l t u s f a s c i a t u s r e a r e d o n s u n f l o w e r s e e d s ( n o c a r d i a c g l y c o s i d e s ) . T h e s e t w o p r e d a t o r s m a d e n o d i n t i n c t i o n b e t w e e n O n c o p e l t u s r e a r e d o n s u n f l o w e r s e e d s t h a t w e r e a l i v e o r w h i c h h a d b e e n s t o r e d a t -20°C. T h e s e c o n d r a n d o m i z e d p a i r e x p e r i m e n t c o n s i s t e d o f a d u l t T e n e b r i o a n d a n a d u l t O n c o p e l t u s f e d o n A s c l e p i a s s y r i a c a s e e d s . I n a d d i t i o n , s i n c e b o t h t h e f r o g s a n d t o a d s a c c e p t e d a l l o f t h e s e s p e c i m e n s e q u a l l y , a n d w o u l d a c c e p t a n y n u m b e r o f O n c o p e l t u s r e a r e d o n s u n f l o w e r o r m i l k w e e d s e e d s p r e s e n t e d t o i t o n s u c c e s s i v e d a y s , i t w a s d e c i d e d t o t e s t i f h i g h e r l e v e l s o f c a r d i a c g l y c o s i d e i n t h e p r e y w o u l d c a u s e r e j e c t i o n b y t h e p r e d a t o r s . F o r t h i s T e n e b r i o a d u l t s w e r e p r e s e n t e d t o f r o g s w i t h a t h i n l a y e r o f p e t r o l e u m j e l l y o n t h e i r e l y t r a t o w h i c h h a d b e e n a d d e d a p p r o x i m a t e l y 2 - 5 m g m s . o f a c a r d i a c g l y c o s i d e m i x t u r e c o n s i s t i n g o f s t r o p h a n t h i n K - o u a b a i n - d i g i t o x i n (2:1:1). U p t o f o u r T e n e b r i o o f t h i s t y p e w e r e p r e s e n t e d t o o n e p r e d a t o r a t a t i m e . 33 The anoles were used i n an experiment of a s i m i l a r design to that of the frogs and toads except that i n a l l presentations l i v e animals were used. The prey was dropped i n the cage i n the v i s u a l range of the predator. Tenebrio adults randomly paired with both Oncopeltus reared on sunflower seeds and milkweed seeds were presented u n t i l a 9 5 % l e v e l of s i g n i f i c a n c e of r e j e c t i o n or acceptance was obtained as ou t l i n e d by Cole (1962). Tenebrio adults coated with petroleum j e l l y and s p r i n k l e d with the glycoside mixture were not presented to these animals. Preliminary studies i n d i c a t e d that the s a t i e t y l e v e l of the animal would not become a s i g n i f i c a n t f a c t o r i n causing r e j e c t i o n of a palatable prey unless the animals were fed three or more Tenebrio adults per day. In a second experiment with t u r t l e s , 60 Oncopeltus  f a s c i a t u s adults reared on sunflower seeds were extracted of t h e i r l i p i d content; the l i p i d s were p a r t i t i o n e d against s a l i n e as described i n the extraction methods but they were not column p u r i f i e d . This extract was resolved i n chloroform-methanol (2:1) so 10 m i c r o l i t e r s of t h i s represented the l i p i d content of one i n s e c t . Also, the cardiac glycoside content of 600 Oncopeltus reared on milkweed seeds was column p u r i f i e d and t h i s extract resolved i n chloroform-methanol (2:1) such that a 10 m i c r o l i t e r a l i q u o t represented the cardenolide content of one i n s e c t . These 10 m i c r o l i t e r aliquots were i n j e c t e d into Tenebrio adults; t h i s preparation was allowed to dry over-night. 34 The controls consisted of injecting 10 microliters of solvent containing 100 micrograms of a strophanthin K-ouabain-digitoxin (2:1:1) mixture into Tenebrio adults. After drying over-night the Tenebrio preparations were kept frozen prior to presentation. Unfortunately this technique produced many prey insects that were pulpy, but the turtles s t i l l ate the pulpy solvent controls. The prey were presented i n the following order: 1 - Tenebrio adult, solvent control injection 2 - Tenebrio adult, sunflower injection 3 - Tenebrio adult, milkweed injection ^ ~ Tenebrio adult, glycoside mixture injection The seven food specimens of each test group were presented every second to third day u n t i l a l l were consumed by the predator. Several days (3-5) were allowed to intervene before starting a new group and during this perbd the turtles were not fed. The two all i g a t o r lizards were not employed i n a planned experiment as for the frogs and toads. To each l i z a r d which had been fed intermittently Tenebrio adults for several weeks was presented an Oncopeltus fasciatus reared on sunflower seeds; the insect was dropped into the aquarium. This was done several times but not carried out to significance as directed by Cole (1962). After having been deprived of food a l l night, the chickens were fed at 10:30 a.m. and 12:30 p.m. with the test-food animals. The chickens were trained to accept palatable 3 5 Tenebrio adults and larvae from forceps placed through the wire mesh, and were not timorous owing to the presence of the experimenters. Afte r having eaten to s a t i e t y on chicken-s t a r t e r the chickens would s t i l l r e a d i l y accept i\any palatable insect passed through the wire mesh. Five experiments described below were undertaken with the chickens, a l l t e s t s except the f i f t h being rim. u n t i l s i g n i f i c a n c e i n choice was obtained as ou t l i n e d by Cole (1962). F i r s t l y , Tenebrio adults and Oncopeltus reared on sunflower seeds were presented as randomized p a i r s , followed by a Tenebrio l a r v a f w i t h each p a i r . Secondly, an extract of 170 Oncopeltus reared on Milkweed seeds was column p u r i f i e d and t h i s extract then resolved i n the appropriate volume of a thoroughly mixed chicken egg-white-egg-yolk paste thickened by the addition of corn starch so that an i n j e c t i o n of 65 m i c r o l i t e r s into a d r i e d abdominal husk of a Tenebrio adult contained the amount of cardiac glycoside equivalent to one Oncopeltus. This preparation was randomly paired with a Tenebrio l a r v a with the p a i r followed by another Tenebrio l a r v a . T h i r d l y , adult Oncopeltus reared on sunflower seeds were anaesthesized with CO2 and t h e i r v e n t r a l metathoracic scent glands were seared out with a hot probe, taking care not to squeeze the insect so that the d o r s o - l a t e r a l thoracic glands exuded t h e i r f l u i d . These insects were frozen and l e f t to dry i n the open a i r over-night; the following morning 36 they were painted t o t a l l y black with a c r y l i c paint. A f t e r the paint had d r i e d the insects were replaced i n the freezer u n t i l used. This preparation was randomly paired with a Tenebrio l a r v a , the p a i r followed by a Tenebrio l a r v a . Fourthly, Oncopeltus reared on milkweed seeds were treated i n i d e n t i c a l manner to the above Oncopeltus reared on sunflower seeds, and presented i n an i d e n t i c a l manner. F i f t h l y , three chickens were deprived of any food, but not water, f o r Vk days a f t e r which they were only presented with Oncopeltus reared on sunflower seeds. The experimental period l a s t e d f o r 12 hours during which they were presented f i v e such i n s e c t s . 37 VALIDITY OF GLYCOSIDE EXTRACTION, PURIFICATION AND IDENTIFICATION METHODS In order to prove that cardiac glycosides are present i n extracts of animal or plant t i s s u e not only does chemical evidence have to be supplied, but i t i s customary to complement t h i s v e r i f i c a t i o n with p h y s i c a l , and most importantly, b i o l o g i c a l assays (Thorp et a l . , 1967). Since d i f f i c u l t y was encountered i n employing the methods av a i l a b l e i n the l i t e r a t u r e f o r the various analyses of cardiac glycosides of Asclepias  s y r i a c a i t i s f e l t necessary to j u s t i f y the v a l i d i t y of the methods employed i n t h i s thesis as tools f o r i n d i c a t i n g the presence of cardiac glycosides. A s s u r i t y i n the r e s u l t s obtained from these methods i s the basis of t h i s t h e s i s . The procedure f o r the extraction of cardiac glycosides o u t l i n e d by Parsons (1965) was not e f f i c i e n t i n removing a l l ranges of p o l a r i t y of the cardiac glycosides; the method used by v. Euw et a l . , (1967) was not considered r a p i d enough f o r large numbers of samples of t i s s u e . The procedures, out-l i n e d i n the Methods, f o r the extraction of cardiac glycosides from insect t i s s u e are considered more thorough and rapid i n i s o l a t i n g a l l ranges of p o l a r i t y of these molecules. The r e f l u x i n g of the seeds i n the described solvent system was t y p i c a l of most methods a v a i l a b l e (Adams et a l . , 1961), and since no precautions had been taken i n obtaining the dr i e d seeds to prevent hydrolysis of the sugar residues, any possible 38 e f f e c t s of the r e f l u x i n g temperature were not accounted f o r . However, p u r i f i e d extracts of "both plant and ins e c t t i s s u e showed no observable differences i n the detectable number of glycosides present i f the extracts were a i r d r i e d at 100°C. The extraction of the cardiac glycosides from l e a f and pod t i s s u e i n v o l v i n g the p r e c i p i t a t i o n of the majority of contaminating pigments by lead acetate i s a crude procedure i n approximating the number of cardiac glycosides or aglycones present. Since cardiac glycosides could be i s o l a t e d from as l i t t l e as 50 mgms. of le a f t i s s u e (Table II) using t h i s method i t i s considered that the approximations of numbers of major cardenolides a v a i l a b l e i n the l e a f to an ins e c t feeding upon them i s v a l i d . Even acid hydrolysis of the glycosides of Asclepias to the aglycones does not permit t h e i r separation from the contaminating pigments e i t h e r by column elutions with attenuated rates of p o l a r i t y change, or by TLC. C e l l u l o s e , DEAE and S i l i c a gel G columns were employed i n attempts to r i d the cardiac glycoside containing f r a c t i o n of these pigments without success. The author i s unaware of any method used i n extracting cardiac glycosides which would a l l e v i a t e the probelm of i n t e r f e r i n g components. The i s o l a t i o n and complete separation of the 15 or more glycosides from seed t i s s u e and the 5 or more glycosides from Asclepias s y r i a c a and other Asclepiads poses a problem beyond the scope of t h i s thesis.. An extract of 600 Oncopeltus adult insects reared on A. s y r i a c a seed (approximately '60 mgms. 39 of cardenolide), p u r i f i e d on a F l o r i s i l column, was not amenable to the p r e c i p i t a t i o n of c r y s t a l l i n e cardiac glycosides from hexane or benzene. Also, even though the F l o r i s i l column e l u t i o n sequence o u t l i n e d i n the Methods, was e f f i c i e n t i n i s o l a t i n g cardiac glycosides i n a small t o t a l e l u t i o n volume (182 mls^ very r a p i d l y , no i n d i v i d u a l r e s o l u t i o n of a glycoside or groups of glycosides could be obtained; and fur t h e r , the presence of contaminating steroids and l i k e l y saponins (Xahthydrol p o s i t i v e due to sugar residues) i n the cardenolide f r a c t i o n could not be eliminated. This method was more applicable (and more rapid) to the separation of Asclepias glycosides than the methods employed by Sauer et a l . (1969) and v. Euw et a l . (1967). The use of neither F l o r i s i l nor S i l i c a Gel G columns with benzene, chloroform or ethylene d i c h l o r i d e as the main solvent could separate s a t i s f a c t o r i l y the s t e r o i d f r a c t i o n from the cardiac glycoside f r a c t i o n . The F l o r i s i l column method employed i n t h i s research was therefore most b e n e f i c i a l f o r i t s r a p i d i t y , ( S i l i c a Gel or C e l l u l o s e and i t s d e r i v a t i v e s columns being painstakingly slow) since large numbers of extracts had to be p u r i f i e d f o r bioassay purposes. D i g i t o n i n p r e c i p i t a t i o n was avoided since i t would also p r e c i p i t a t e -OH-aglycones; whether aglycones i n Asclepias s y r i a c a t i s s u e were present was not investigated, though they have been reported jfco occur (Bauer et a l . , 1964). The TLC eluent systems employed (ethylene d i c h l o r i d e -40 methanol-formamide, 80:25-1 and methylene dichloride-methanol-formamide, 80:10:10 permitted the observation of at l e a s t 15 cardenolides i n the seeds of three species of Asclepias and fewer numbers i n other tissues of various other plants (Table I I ) . Other proportions of the above solvent components were t r i e d both as s i n g l e and multiple developments procedures, but none were successful i n broadening the Rf range of the glycosides. More polar solvent systems extended the Rf range but d i s t o r t e d the separation or causedexcessive d i f f u s i o n of the compounds. This d i f f u s i o n lowered the accuracy of marking the evanescent intermediates on the TLC pla t e a f t e r being sprayed with a l k a l i n e 5,5-dinitrobenzoic a c i d . Both increases and decreases i n the p o l a r i t y of the TLC solvent system also f a i l e d to separate d i s t i n c t l y the accompanying steroids from the cardenolides since the most l i p i d cardenolides had Rf values s l i g h t l y lower than that of c h o l e s t e r o l , and greater than that of many of the more polar s t e r o i d s . Many other TLC solvent systems described i n ' <Stahl (1965) were t r i e d but were unsuccessful i n r e s o l v i n g the many cardenolides of Asclepias. I t appears that formamide as a stationary phase was e s s e n t i a l . Also, i t was found that the degree of hydration ( a c t i v a t i o n ) of the TLC plates had to be r i g i d l y c o n t r o l l e d , i n order to obtain reproducible Rf values. However, with the proper precautions the glycosides could be resolved into f a i r l y d i s t i n c t bands that were easy to mark (immediately a f t e r spraying with reagents) with a p i n point on the TLC p l a t e , before the colour faded. Some of the cardenolide bands separated on TLC were wide such that i t was not discernable whether more than one component was present at that Rf value. Many bands had close Rf values and marking these bands accurately was d i f f i c u l t . Base or ac i d hydrolysis of the cardiac glyco-sides d i d not permit any advantage i n separating the eight evident h y d r o l y s i s products. Thus, a conclusive separation of i . . . the t o t a l cardiac glycoside composition of Asclepias s y r i a c a and the other species was not po s s i b l e . The four reagents employed (reagents 1-4) are f a i r l y s p e c i f i c reagents f o r the butenolide r i n g or cardiac glycosides (Kaul et a l . , 1967; Wright, I960). Reagents which react with the s t e r o i d and sugar portions of the cardenolide molecule w i l l also be subsequently discussed. Wright ( i 9 6 0 ) discusses these a l k a l i n e reagents (1-4) as being the most s p e c i f i c methods of l o c a l i z i n g cardenolides. The hydrolysis of cardiac glycosides by base or acid, or T methylation of such (ouabain, strophanthin K, and d i g i t o x i n ) d i d not a l t e r the r e a c t i o n of these spray reagents. These d r a s t i c chemical procedures were u t i l i z e d i n the chemical assay of cardenolides i n the t i s s u e extracts. Steroids generally give coloured products when sprayed with concentrated sulphuric a c i d or phosphoric a c i d and heated at approximately 110°C. (Matsumoto, 1963; Stahl, 1965; Takeuchi, 1 9 6 3 ) . Extracts of plants known to contain cardiac glycosides or pure glycosides gave p o s i t i v e reactions. 42 The colours produced "by the spraying of the extracts of cardiac glycoside containing plants and the standards were reds, greys, purples and l i g h t greens. This t e s t served to confirm the s t e r o i d a l nature of the components i d e n t i f i e d as cardiac glyco-sides. A comparison of the spectra ( u l t r a - v i o l e t ) of strophanthin K dissolved i n concentrated sulphuric acid to that of the i s o l a t e d cardenolides from a known plant source showed a s i m i l a r i t y ; t h i s method has been employed f o r the assaying of cardiac glycosides (Brown, et a l . , I960). This i s considered to be valuable secondary evidence to the general r e a c t i o n of steroids with sulphuric a c i d or phosphoric acid as described above. Further spectrophotometric evidence f o r the presence of cardiac glycosides i n plant and animal t i s s u e extracts was sought by taking the spectra of the coloured product obtained by the r e a c t i o n with the following reagents: 3 , 5 - d i n i t r o b e n z o i c acid plus NaOH, 1,2-dinitronaphthoquinone-4-sulphonic a c i d plus NaOH, p i c r i c a c i d plus NaOH, and Xanthydrol plus HC1 as outli n e d by Wright (I960). Pure glycosides and glycosides derived from known plant sources reacted as indicated i n the Methods. These r e a c t i o n colours v i s u a l l y appear to be s i m i l a r to those colours obtained by the re a c t i o n of cardenolides with the reagents on the TLC plates, but t h i s extra evidence without doubt establishes that the colour peaks are s i g n i f y i n g the presence of cardiac glycosides. 43 X a n t h y d r o l was a l s o used as a supplementary reagent to d e t e c t c a r d i a c g l y c o s i d e s because of i t s r e a c t i v i t y w i t h sugars and i t s h i g h s e n s i t i v i t y f o r t h e s e compounds (Wright, I960). The i n t e n s e c o l o u r produced i n the c a r d e n o l i d e Rf range of the TLC p l a t e by a p p l i c a t i o n o f t h i s reagent adds f u r t h e r evidence t h a t l a r g e q u a n t i t i e s o f sugars are p r e s e n t , which i s i n d i c a t i v e o f the presence of a s t e r o i d a l g l y c o s i d e . The c o l o r i m e t r i c procedure used to determine the amount of c a r d i a c g l y c o s i d e p r e s e n t i n i n s e c t t i s s u e c o u l d be used o n l y as a crude approximation because of a l a c k of knowledge of t h e e x t i n c t i o n c o e f f i c i e n t s o f the s e compounds i n A s c l e p i a s s y r i a c a . B i o l o g i c a l assay was a l s o used t o demonstrate the presence of c a r d i a c g l y c o s i d e s i n t i s s u e e x t r a c t s i n accordance w i t h normal p h a r m a c o l o g i c a l procedure (Thorp et a l . , 1967). A p o s i t i v e i n c r e a s e i n the b l o o d p r e s s u r e of a c a r o t i d - c a n n u l a t e d r a t was jrhe c r i t e r i o n employed ( L e f e r et a l . , 1964; Wright, I960); the r a t was chosen because of i t s f a c i l i t y f o r f a s t and s h o r t -termed o p e r a t i o n s and a l s o because i t i s r e a s o n a b l y i n s e n s i t i v e t o c a r d i a c g l y c o s i d e i n d u c e d c a r d i a c i n o t r o p i s m (Hoch, 1961; Parsons, 1965). Thus, when an i n o t r o p i c response was indu c e d by i n j e c t i o n of a t i s s u e e x t r a c t i t c o u l d be c o n s i d e r e d t o be due to the presence of l a r g e amounts of c a r d i a c g l y c o s i d e s and not some o t h e r h e a r t modulator. The almost complete i n h i b i t i o n o f the i n o t r o p i c response i n d u c e d i n mammalian h e a r t s by c a r d i a c g l y c o s i d e s through the use of a l s o s t e r o n e 44 (Lefer et a l . , 1964) was considered by the author to be a more s p e c i f i c demonstration that the i n o t r o p i c response i s due to cardenolides, and not some other compound(s). The coupling of aldosterone i n the assay was used i n only a few extracts, but the r e s u l t s of such assays were used as an inference that other i n o t r o p i c responses would show s i m i l a r i n h i b i t i o n i f treated with aldosterone as ou t l i n e d i n the Methods. Attempts were made to follow changes i n heart rate v i a an ECG ( e l e c t r o -cardiogram) but with glycoside standards no differences were observed, p o s s i b l y because the amount i n j e c t e d into each r a t was well below the d i g i t a l i z a t i o n l e v e l (Thorp et a l , , 1967). By means of extraction methods, column p u r i f i c a t i o n procedures, t h i n layer chromatography techniques, the employ-ment of s p e c i f i c reagents f o r cardiac glycosides and general reagents f o r steroids coupled with spectrophotometry, and concisive pharmacological evidence, with comparative reference to glycoside standards the author considers that i t i s poss i b l e unambiguously to recognize cardiac glycosides i n the t e s t materials. I t i s on t h i s assumption that the r e s t of the the s i s i s based. 4-5 RESULTS CARDIAC GLYCOSIDES IN PLANT TISSUE Nineteen species of Asclepias and one species of Acerates (Asclepiadaceae) from diverse points about North America (Figure 1) were examined and a l l were shown to contain cardiac glycosides i n t h e i r l e a f t i s s u e s (Table I ) . A study of the a e r i a l portions of Asclepias s y r i a c a , A. incarnata and A. speciosa, showed that l e a f , pod, and seed t i s s u e s a l l contained these compounds (Tables I and II) and that these three portions of the plant contain a d i f f e r e n t array of cardenolides. TLC examination of the leaf,pods, and seed t i s s u e indicated that the seeds of the above three species had at l e a f 1 5 cardiac glycosides with s i m i l a r Rf values; the 5 glycosides present i n the l e a f and pod t i s s u e s of these three species also had s i m i l a r Rf. values, and were v e r y ' l i k e l y represented i n the seeds. A d e t a i l e d r e s o l u t i o n of the s i m i l a r i t i e s and differences between cardiac glycoside components i n Table II was not undertaken. Even though based on a study of small quantities of l e a f t i s s u e , Table I I f u r t h e r c l e a r l y suggests that species d i f f e r i n t h e i r cardenolide composition. Further, when spraying the TLC separated extracts with a l k a l i n e 3 , 5-dinitrobenzoic a c i d , obvious differences i n i n t e n s i t y of colour r e a c t i o n were apparent. Some extracts of amounts of t i s s u e of approximately 50 mgms. showed more 46 FIGURE 1: Map of North America showing the d i s t r i b u t i o n of the various species of b r i g h t l y coloured insec t s and species of plants assayed f o r cardiac glycosides. For the d i s t r i b u t i o n of Oncopeltus f a s c i a t u s see S l a t e r (1964). For the d i s t r i b u t i o n of the Lygaeus ka l m i i complex see S l a t e r (1964),and S l a t e r et a l . (1969). Oncopeltus f a s c i a t u s has a range which extends as f a r north as southern Ontario and reaches down the eastern seaboard and middle states to and into the Caribbean. I t also occurs i n South America. The Lygaeus ka l m i i complex has i t s northern l i m i t s i n most southerly portions of the provinces of Canada, and occurs i n almost a l l the states of the United States. I t extends into Mexico and part of South America. o S p e c i e s a s s a y e d f o r c a r d i a c . g l y c o s i d e s ( T a b l e s I'•& . I I . ) Oncopeltus f a s c i a t u s A.Oncopelt.us .sandarachatus a S u b s p e c i e s o f L y g a e u s • k a l m i i . • S p e c i e s o f T e t r a opes -••'Danaus. p l e x i p p u s J ^ S p e c i e s o f Dysdercus A S p e c i e s o f A s c l e p i a s d * o FIGURE 1. 4-7 FIGURE 2: General d i s t r i b u t i o n of the major southern and northern Apocynad and Asclepiad species considered i n t h i s t h e s i s . The northern species (see Brower, 1969) are: A. incarnata, tuberosa, s y r i a c a , speciosa (See Table I) The major southern species examined and relevant to Oncopeltus are: Asclepias curassayica, Nerium oleander, Apocynum For a d e t a i l e d synopsis of As c l e p i a s see Woodson (1954-). N o r t h e r n s p e c i e s T r o p i c a l s p e c i e s FIGURE 2 TABLE I: Results of chemical assay and "bioassay of plant extracts of some Asclepiad species and other plants f o r cardiac glycosides. + = p o s i t i v e response Blank =* not determined p = Spectrophotometric peak determined (See Table II) * Ouabain and d i g i t o x i n define the approximate Rf ranges of cardenolides. See Figure 3 INO = Inotropic response i n r a t heart DNBoic = 3,5-dinitrobenzoic a c i d + NaOH XANTH = Xahthydrol + HCL ALDO = Antagonism of INO response DNBzene = 3,5-dinitrobenzene + NaOH PICR = P i c r i c a c i d + NaOH NAPH = l,2-naphthoquinone-4-sulphonic acid + NaOH H +- 0H~ = Alternate base and a c i d hydrolysis with HC1 and NaOH. A p o s i t i v e response means the cardenolides maintain t h e i r i n o t r o p i c as well as TLC behaviour. TABLE I RESULTS OF CHEMICAL ASSAY AND BIOASSAY OF PLANT EXTRACTS OF SOME ASCLEPIAD SPECIES AND OTHER PLANTS FOR CARDIAC GLYCOSIDES D e s c r i p t i o n B i o a s s a y Reagent Response i n C a r d e n o l i d e Rf Zone* (TLC) INO ALDO DNBoi'c DNBzene NAPH PICR XANTH H -OH" Chemical C o n t r o l s Ouabain 3 x 10"?M. . A l d o s t e r o n e 3 x 10~?M. P l a n t s ( A s c l e p i a d a c e a e ) A s c l e p i a s s y r i a c a Peterborough, O n t a r i o 1 Midland, O n t a r i o A s c l e p i a s i n c a r n a t a M i d l a n d , O n t a r i o A s c l e p i a s s p e c i o s a Summerland, B r i t i s h Columbia (B.C.) P e n t i c t o n , B.C. Cache Creek, B.C. A s c l e p i a s a m p l e x i c a u l i s N o r t h C a r o l i n a Seeds Seeds Leaves Pods Seeds Leaves Pods Seeds Leaves Leaves Seeds Pods Leaves + + + P + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + P + P + P + + + + + + + + + + + + + + A s c l e p i a s a r e n a r i a Nebraska, A s c l e p i a s c a l i f o r n i c a C a l i f o r n i a A s c l e p i a s c a p r i c O r n u Utah A s c l e p i a s c u r a s s a v i c a Jamaica A s c l e p i a s c o r d i f o l i a C a l i f o n r x a A s c l e p i a s e x a l t a t a. North C a r o l i n a A s c l e p i a s f a s i c u l a r i s C a l i f o r n i a A s c l e p i a s l a b r i f o r m e s Utah A s c l e p i a s mexicana C a l i f o r n i a A s c l e p i a s o v a l i f o l i a Manitoba A s c l e p i a s t u b e r o s a O n t a r i o A s c l e p i a s v a r i e g a t a North C a r o l m a A s c l e p i a s v e r i d i f l o r a M i s s o u r i A s c l e p i a s v e r t i c i l l a t a Manitoba A c e r a t e s a n g u s t i f o l i a Nebraska P l a n t s (Non-Asclepiadaceous D i g i t a l i s p u r p urea Nerium o l e a n d e r Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves ) Leaves Leaves + + + + + + + + p + + p + + TABLE I I : Number of cardiac glycosides detected i n various Asclepiad species from North America. > The numbers of cardenolides stated are only the number observed from the extraction of these minimal amounts of ti s s u e and they do represent the actual number of cardenolides present i n the plant t i s s u e . *** The 15 glycosides i n these seeds appeared to have s i m i l a r Rf values. The pods and leaves of these three species had 5 glycosides present which had s i m i l a r Rf values; these components were probably present i n the seeds. * These weights represent a comparative estimate of the amount of l e a f t i s s u e extracted crudely approximated to the nearest 50 milligrams. ** This s i g n i f i e s the Rf range i n which the 3 , 5-^-nitrobenzoic acid + NaOH p o s i t i v e spots were observed. The Rf of ouabain was 0.08 and d i g i t o x i n was 0.4-0 i n a solvent system of ethylene dichloride-methanolr-f ormamide (80 :25:1). Species Number Weight of Rf Range** Tissue* A. s y r i a c a *** Seeds 15 1 - 2 gms. 0.08 - 0.62 Leaves 5 2 - 5 gms. 0.20 - 0.43 Pods 5 2 - 5 gms. 0.20 - 0.43 A. incarnata *** Seeds !5 1 - 2 gms. 0.08 - 0.62 Leaves 5 2 - 5 gms. 0.20 - 0.43 Pods 5 2 - 5 gms. 0.20 - 0.43 A. speciosa *** Seeds 15 1 - 2 gms. 0.08 - 0.62 Leaves 5 2 - 5 gms. 0.20 - 0.43 Pods 5 2 - 5 gms. 0.20 - 0.43 A. amplexicaulis Leaves 8 100 mgms. 0.25 - 0.75 A. arenaria Leaves 3 100 mgms. approx. . 0.6 A. c a l i f o r n i c a Leaves 6 100 mgms. 0.25 - .0.50 A. capricornu Leaves 3 100 mgms. 0.10 - 0.45 A. c o r d i f o l i a Leaves 6 100 mgms. 0.10 - 0.50 A. exaltata Leaves 4 50 mgms. 0.35 - 0.45 A. f a s i c u l a r i s Leaves 3 50 mgms. 0.35 - 0.45 A. labriformes Leaves 1 100 mgms. 0.35 - 0.45 A' mexicana Leaves 4 100 mgms. 0.35 - 0.45 A. o v a l i f o l i a Leaves 4 100 mgms. 0.35 - 0.45 A. tuberosa Leaves 3 300 mgms. 0.10 - 0.40 A. variegata Leaves 4 50 mgms. 0.20 - 0.65 A. v e r i d i f l o r a Leaves 8 300 mgms. 0.20 - 0.65 A. v e r t i c i l l a t a Leaves 8 50 mgms. 0.20 - 0.65 Acerates a n g u s t i f o l i a Leaves 8 50 mgms. 0.20 - 0.65 Asclepias curassaviceau Leaves 11 1 - , 2 gms. 0.10 - 0.65 50 intense r e a c t i v i t y with the above reagent than extracts of l a r g e r q u antities of t i s s u e . Such observations may i n d i c a t e varying concentrations of i d e n t i c a l or d i s s i m i l a r cardenolides, but t h i s was not d e f i n i t e l y established. The r e s u l t s i n Table II i n d i c a t e that the Asclepiads wherever they occur on the North American continent are an i n s e c t food source that contains cardiac glycosides. CARDIAC GLYCOSIDES IN ONCOPELTUS AND VARIOUS INSECTS FEEDING UPON ASCLEPIADS Having confirmed that many Asclepiad species possess cardiac glycosides, i t i s reasonable to assume that these could provide a source of cardiac glycosides f o r i n s e c t s feeding upon them. While chewing i n s e c t s , feeding on whole l e a f , stem, pod or other parts, might be expected to ingest the glycosides, t h i s does not n e c e s s a r i l y happen i n the p i e r c i n g and sucking Hempitera, since these l a t t e r i n s e c t s can s e l e c t i v e l y feed. I t i s necessary to a s c e r t a i n by analysis whether the various insects feeding on Asclepias do a c t u a l l y take up cardenolides i n t h e i r bodies. Table I I I presents the r e s u l t s of such an analysis; both sucking and chewing insects were analyzed. A l l insects examined which feed on Asclepias i n the w i l d were found to contain cardiac glycosides. The chewing insect s Tetraopes  oregonensis, T... tetraophthalmus, and Danaus piexippus (which has chewing larvae) a l l contained cardiac glycosides, while the chewing i n s e c t controls (Schistocerca gergaria, Tenebrio TABLE I I I : Results of chemical assay and bioassay of insect whole animal extracts f o r cardiac glycosides. + = p o s i t i v e response 0 = no response Blank = not determined o = no spectrophotometric peak, p = Spectrophotometric peak determined DNBoic = 565 m i l l i -microns PICR = 550 millimicrons XANTH = 625 m i l l i -microns # = peak present but r e s u l t s from non-cardenolide components i f very high concentrations of extract i n r e a c t i o n volume used compared to concentrations required to give s i g n i f i c a n t peak with cardenolide containing extracts * Ouabain and d i g i t o x i n were used to define the approximate range of cardenolide Rf behaviour i n a solvent system of ethylene dichloride-methanol-formamide (80:25:1) Rf range of cardiac glycosides found i n t i s s u e s was 0.08 - 0.60. For key to reagents see Table I. H +- OH i n d i c a t e that the glycoside maintained t h e i r i n o t r o p i c as well as chromatographic behaviour under ac i d and base h y d r o l y s i s . A l l extracts are from adult i n s e c t s . TABLE I I I RESULTS OP CHEMICAL ASSAY AND BIOASSAY OP INSECT WHOLE ANIMAL EXTRACTS FOR CARDIAC GLYCOSIDES Description ~~HbZ ~ of Bioassay Insects Reagent Response i n Cardenolide Rf Zone (TLC)* Chemical Controls Ouabain 3 x 10"^M. Aldosterone 3 x 10"'M. Animals Feeding on Asclepias spp. et a l . Oncopeltus f a s c i a t u s (Lygaeidae) L i t c h f i e l d , I l l i n o i s Point Pelee, Ontario Oncopeltus sandarachatus Runaway Bay, Jamaica 2 7 8 Lygaeus k a l m i i angustomarginatus (Lygaeidae) L i t c h f i e l d , I l l i n o i s Midland, Ontario Peterborough, Ontario Lygaeus ka l m i i k a l m i i Summerland, B r i t i s h Columbia, (B.C.) Tetraopes oregonensis (C erambycidaej Summerland, B.C. Tetraopes tetraophthalmus (CerambycidaeJ L i t c h f i e l d , I l l i n o i s Midland, Ontario 10 5 40 16 30 7 60 INO ALDO DNBoic DNBzene NAPH PICR XANTH H+-0H" + + + + + + + + P + P + P + P + + P + P + P + P +. P + + + + + + + + + + + + P + + + + + P + + -/Tit*:* Danaus plexippus (Danainae) Midland, Ontario (indigene: Aug. 20 & 31 ) 2 Picton, Ontario (indigene: Aug. 1 3 ) 1 Picton, Ontario (migrant: J u l y 4 & 23) 2 Manitoba (data unknown) 1 Animal Controls Oncopeltus f a s c i a t u s (Lygaeidae) Reared on sunflower seeds 60 Leptocoris rubrolineatus (Rhopalidael Kamloops, B.C. 12 Schistocerca gregaria (Acrididae) Lab animal 2 Tenebrio molitor (Tenebrionidae) Lab animal 20 Chauliognathus pennsylvanicus (Cantharidae) Midland, Ontario 2 Limenitis archippus (Nymphalidae) Data unknown 2 Dysdercus andreae (Pyrrhocoridae) Runaway Bay, Jamaica 6 Dysdercus Jamaicensis (Pyrrhocoridae) Runaway Bay, Jamaica 10 Dysdercus mimuloides (Pyrrhocoridae) — - Runaway Bay, Jamaica " 6 0 0 + + 0 o 0 o 0 o 0 o 0 0 0 0 0 0 0 + + 0 0 0 0 + + + p# o " + p# + p# 52 TABLE IV: Plants from which Oncopeltus f a s c i a t u s was able to sequester cardiac glycosides. F i r s t i n s t a r nymphs of Oncopeltus were placed i n preserving jars and permitted to feed upon the s a i d t i s s u e u n t i l 5th i n s t a r nymphs or u n t i l death occurred. Not a l l these food sources were, advantageous f o r growth. Whole animal extracts were used; the presence of cardiac glycosides was confirmed by TLC analysis i n a solvent system of ethylene d i c h l o r i d e -methanol-formamide (80:25:1). Three to f i v e i n s e c t s were used f o r each determination: See Table I f o r reagent legend + = p o s i t i v e response 0 = no response blank = not determined P = spectrophotometric peak at appropriate wave length: see Table III,; Food P l a n t Reagent Response i n C a r d e n o l i d e Rf Zone (TLC)* ' DNBb'ic NAPH DNBzene XANTH INO ALDO H + - OH A s c l e p i a s s y r i a c a Seeds Pods Leaves A s c l e p i a s i n c a r n a t a Seeds Pods Leaves A s c l e p i a s s p e c i o s a Seeds Pods Leaves C a l a t r o p i s p r o c e r a Seeds + P + + + + + + + + + + + + + + + + 4P + + + + + + + H e l i a n t h u s annuus Sunflower Seeds 0 0 0 + S. seeds + g l y c o s i d e s + + + + Nerium o l e a n d e r Leaves + + + + 53 molitor, Chauliognathus pennsylvanicus and Limemtis arcliippus) did not. Likewise, in. the sucking Hemipterans, Oncopeltus  f a s c i a t u s , 0. sandarachatus, Lygaeus ka l m i i k a l m i i and L.k. angustomarginatus contained cardenolides while control forms such as Leptocoris rubrolineatus and the three species of Dysdercus d i d not. Further, i t was shown that 0. f a s c i a t u s fed on Asclepias, C a l o t r o p i s or Nerium i n the laboratory took up cardiac glycosides from these plants, while the same species fed on seeds of sunflower d i d not contain any cardenolides (Table IV). When 0. f a s c i a t u s was fed on sunflower seeds which had had glycosides added, the insects contained such glycosides. I t was also shown that 0. f a s c i a t u s was capable of taking up cardenolides from the various a e r i a l protions (seeds, leaves, and pods) of three Asclepiad spe'cies, A. incarnata, A. speciosa, and A. incarnata. In reference to the bioassay of in s e c t t i s s u e (and plant m a t e r i a l ) , extracts that contained no cardiac glycosides caused no i n o t r o p i c response, though o c c a s i o n a l l y an i n i t i a l drop i n blood pressure d i d occur i n a l l extracts; the return to normality was r a p i d . The i n o t r o p i c response induced by the extracts and the standard glycoside u s u a l l y l a s t e d 2-5 minutes. Besides the glycoside causing an i n o t r o p i c response i n ve r t e -brate hearts, the aglycone (derived from acid or base hy d r o l y s i s ) w i l l also cause an i n o t r o p i c response. This was found to be the case where hydrolyzed extracts of seeds of Asclepias s y r i a c a 54 and Oncopeltus reared on these seeds caused the c r i t i c a l response to i n d i c a t e that these substances d i d b i o l o g i c a l l y contain cardiac glycosides (Table I and I I I ) . The i n h i b i t i o n of the i n o t r o p i c response by aldosterone i s more s p e c i f i c evidence that the heart modulators assayed are cardiac glyco-sides. The response induced by a 0.2 - 0.3 ml. i n j e c t i o n volume of an assay f l u i d r e s u l t i n g from an extract of 12 adult Oncopeltus (reared on Milkweed seeds) was roughly equivalent to _7 a s i m i l a r volume i n j e c t i o n of 6 x 10 rM. ouabain, i n d i c a t i n g that there would be approximately 43 micrograms (based on ouabain molecular weight) i n each i n s e c t . This value i s representative;of the microgram l e v e l s determined by colorimetry (Table VIII) i n Oncopeltus. THE DISTRIBUTION OF CARDIAC GLYCOSIDES IN ONCOPELTUS FASCIATUS Insects, both sucking and chewing, have been shown to be able to take up cardiac glycosides from a food source; but i t does not n e c e s s a r i l y follow from t h i s that these substances w i l l get in t o the t i s s u e s of the i n s e c t ; the gut might act as a b a r r i e r . '.: Examination of extracts of the whole l a r v a l or adult Oncopeltus. showed that they contained apparently a l l the types of cardenolides present i n the seeds though the most l i p i d soluble glycosides appeared i n l i m i t e d concentrations i n these extracts. The extracts of faeces also contained the same irange of glycosides present i n the seeds but a higher 55 concentration of the l i p i d glycosides compared to the whole insect extract was evident. 0. f a s c i a t u s was thus studied i n more d e t a i l to determine the occurrence of glycosides i n s i d e the body. The r e s u l t s (Table V) showed cardenolides to be present i n the haemolymph, anal f l u i d , and also i n considerable amounts i n the various defensive glands. The extracts of haemolymph (50 - 100 m i c r o l i t e r s ) indicated that the more polar glycosides are well represented i n t h i s f l u i d compared to the numbers and concentration of the more l i p i d soluble glycosides represented i n the seeds. Figure 3 i l l u s t r a t e s the differences i n the presence of reagent (1) detectable cardenolides. The haemolymph appeared to have a higher l e v e l of the more l i p i d soluble glycosides than d i d the defensive gland f l u i d s . As mentioned previously, the anal f l u i d (urine and gut contents) contained a l l the glycosides that were v i s u a l i z e d from the seed extracts; the more l i p i d soluble glycosides were on the contrary to the haemolymph and gland f l u i d s well represented. CARDIAC GLYCOSIDES IN THE SECRETORY GLANDS OF ONCOPELTUS FASCIATUS Figures 4- and 5 i l l u s t r a t e the po s i t i o n s of the various secretory glands i n the adult and l a r v a l Oncopeltus. The o r i f i c e s of these glands, opening to the e x t e r i o r , are positioned i n the d o r s o - l a t e r a l margins of the meso- and meta-thorax i n male and female adults, and on the d o r s o - l a t e r a l FIGURE 3: The d i s t r i b u t i o n of cardiac glycosides of Asclepias s y r i a c a i n various t i s s u e s of f i f t h i n s t a r nymphs or adults of Oncopeltus f a s c i a t u s . The l e a f l i k e shapes i n d i c a t e the r e l a t i v e concentration (x-axis) of the various cardenolides i n each extract as detected by 3 , 5-dinitrobenzoic acid; each -leaf i s not q u a n t i t a t i v e l y comparable to any other. The f i g u r e i l l u s t r a t e s mainly the d i s t r i b u t i o n of cardiac glycosides unequally throughout the body of Oncopeltus. The numbers of cardenolides present i n the extracts are not i n d i c a t e d . The d i v i s i o n between polar and non-polar glycosides i s an a r b i t r a r y designation, with d i g i t o x i n being considered very l i p i d soluble and ouabain very polar. The t a b l e below indicates the approximate amounts of the various t i s s u e s required to show the presence of cardiac glycosides as i l l u s t r a t e d by the leaves. I t gives some approximation of the quantity of glycoside i n the extracts. At l e a s t 15 cardenolides were detected i n the extracts of seeds of Asclepias s y r i a c a and i n the anal f l u i d (faeces). Because of the d i f f i c u l t i e s of separation and the nature of t h i s study i t i s not considered relevant to c i t e the observed Rf values f o r the various cardenolides. FIGURE 3 TLC - PLATE <D •H H O CD o <H O CQ =H 0.8 0i'.6 0.4 0.2 L I P I D SOLUBLE Di g . A POLAR Oua. Seed Whole Eggs Haemolymph Glands Faeces a n i m a l o f nymphs a n d . a d u l t s . Di g = D i g i t o x i n Oua. ~ Ouabain T i s s u e Amount o f t i s s u e E x t r a c t volume M i c r o l i t e r s a p p l i e d t o TLC p l a t e Seed Eggs Body Haemolymph Glands Faeces 1 . - 2 gms. kO eggs 25 - 60 i n s e c t s 100 m i c r o l i t e r s • 1 - 2 m i c r o l i t e r s approx. 0.5 mis m i n i m a l approx. 0.5 mis, minimal. 25 - 50 . t o t a l 2 5 - 5 0 t o t a l a p p l y d i r e c t l y e x t r a c t t a k e n from f i l t e r paper l e f t . i n cage 57 TABLE V: D i s t r i b u t i o n of cardiac glycosides and a l k y l aldehydes and ketones i n the various defensive glands and body f l u i d s of Oncopeltus f a s c i a t u s . The error i s expressed as Standard E r r o r of the Mean determined from 5-8 samples. For the determination of the cardenolide content i n the d o r s o - l a t e r a l glands 1-2 m i c r o l i t e r s of f l u i d was used per sample. Each in s e c t contained about 2-3 m i c r o l i t e r s of f l u i d . Each animal provided 3-5 m i c r o l i t e r s of haemolymph. For the chemical reagent t e s t s only 0.05-0.2 micro-l i t e r s of gland f l u i d from any gland was required to obtain a colour response. The r e a c t i o n with 2,4—dinitrophenylhydrazine (2,4—DPH) was obtained by applying 0.05-0.1 m i c r o l i t e r s of the gland f l u i d d i r e c t l y to a piece of f i l t e r paper moistened with the reagent. - = no response + = p o s i t i v e response blank = not determined Micrograms of Cardenolide/ microliter  Ouabain Strophanthin "K Glands or Fluid Chemical Test Reagents DNBoic NAPH XANTH 2,4-DPH Nymph (5th Instar) Adult Mid-dorsal abdominals Segment IV + Segment V + Haemolymph + Urine-anal Fluid + Ventral Metathoracic + Dorso-lateral Mesothoracic + Dorso-lateral Metathoracic + Dorso-lateral Abdominals Segment III + Segment IV + Segment V + Segment VI + Segment VII + Haemolymph + Urine-anal Fluid + + + + + + + + + + + + + + + + + + + + + + + + + + + Digitoxin + + greater than 10; see Note 1 II i t i t . i t II II less than 1; see Note 2 II II i t . i t i t i t greater than 10; see Note 1 55.2 + 4.50 49.6 ± 4.82 ~ 90.1 i 7.20 see Note 3 A l l f i v e segments' f l u i d pooled 30.5 + 7.20 43.0 + 7.70 " 78.1 + 11.5" less than 1; see Note 2 i t i t i t . i t II 2 Note 1. Not determined colorimetrically, but only .01 microliters required to give reagent colour 2. An extraction of 5-10 microliters gave an insignificant reagent response 3. Same as for meso-thoracic values. The fluids of these two glands were pooled FIGURE 4-: Diagram of the mid-dorsal abdominal glands of a f i f t h i n s t a r nymph of Oncopeltus f a s c i a t u s . A,. = Abdominal segment V Ag = Abdominal segment VI G = P o s i t i o n s of the o r i f i c e s of the mid-dorsal abdominal glands. F I G U R E 4 59 FIGURE 5: Diagram of thoracic and abdominal cardenolide concentrating glands of Oncopeltus f a s c i a t u s . A = L a t e r a l view of adult Oncopeltus f a s c i a t u s showing general positions of the metathoracic scent gland, the thoracic d o r s o - l a t e r a l glands and the d o r s o - l a t e r a l abdominal glands. B = An enlargement of the meso- and metathorac showing the po s i t i o n s of the various secretory glands. IG An enlargement of a abdominal d o r s o - l a t e r a l gland. S = Metathoracic s t i n k gland G = Dorso-lateral complex of glands A c = Abdominal segment V = Pro their ax = Mesothorax T, = Metathorax 60 margins of segments I I I - VII i n both adults. Adults (both) also have l a t e r a l v e n t r a l metathoracic gland openings, but they are not present i n the larvae. Larvae have a se r i e s of mid-dorsal abdominal glands at the p o s t e r i o r of terga IV and V,: and such glands are also present i n the adult, but thought to be non-functional. The study of the various gland secretions showed that a l l glands, i n c l u d i n g the v e n t r a l metathoracic scent glands, contain cardiac glycosides i n much higher concentrations than the haemolymph. The contents of the v e n t r a l glands of the metathorax are estimated to be about 1000 times as concentrated as the haemolymph since on a TLC pl a t e 0.005 m i c r o l i t e r s of t h i s gland f l u i d gives a reagent (1) colour response s i m i l a r to that of an extract of about 30-50 m i c r o l i t e r s of haemolymph. On a u n i t volume basis the adult d o r s o - l a t e r a l thoracic (meso- andmeta-pooled) and abdominal (pooled) gland f l u i d s are also at l e a s t 300-500 times as concentrated as the le v e l s of glycoside i n the haemolymph as determined by colorimetry based on ouabain as a standard reference. R e f e r r a l to other standards i s also possible by f a c t o r s of 1.41 and 2.56 f o r strophanthin K and d i g i t o x i n r e s p e c t i v e l y . The d o r s o - l a t e r a l thoracic and abdominal gland f l u i d s of the adult were found to contain 35-2 - 4.50 and 30.5 +. 7«70 micrograms of cardiac glycoside r e f e r r e d to ouabain per m i c r o l i t e r of gland f l u i d , when t h i s i n s e c t was fed on seeds of Asclepias syriaca* Observation in d i c a t e s that the average Oncopeltus 61 adult contains approximately 2-3 m i c r o l i t e r s of f l u i d i n a l l the above glands, which i s i n close agreement with a t o t a l value of 73-111 micrograms of cardenolide adult i n s e c t (Table V I I I ) . Thus i t appears that v i r t u a l l y a l l the cardiac glycosides sequestered from the food source are stored i n the described glands, causing a higher l e v e l , 120 micrograms, per i n s e c t . Preliminary observations (not discussed i n t h i s t h e s i s ) show that the gland f l u i d appears to contain cardiac glycosides, several ninhydrin p o s i t i v e substances, and at l e a s t nine proteins (acrylamide gel electrophoresis) some resembling those of the haemolymph with a cardenolide isotope (ouabain-^H) associated with major protein bands. No steroids were detectable on TLC plates from 1-2 m i c r o l i t e r s of gland f l u i d ; whereas, the above mentioned compounds were detectable. In the t h i n l a y e r separation of the cardiac glycosides from the f l u i d of the d o r s o - l a t e r a l thoracic and abdominal glands of Oncopeltus i t was observed that the g l y c o s i d i c content of these glands was l i m i t e d to the more polar components that were present i n the extracts of whole Oncopeltus, faeces, or seeds of Asclepias s y r i a c a . Polar glycosides were also dominant i n the haemolymph, but the gland f l u i d appeared to have a more l i m i t e d representation of the more l i p i d soluble glycosides than did the haemolymph (Figure 3)« THE POLARITY EFFECT IN THE DORSO-LATERAL GLAND COMPLEX IN ONCOPELTUS FASCIATUS . Since Oncopeltus f a s c i a t u s accumulates large 62 quantities of cardiac glycosides i n i t s adult defensive glands, as determined by colorimetry, and also concentrates these compounds to unknown l e v e l s i n the nymphal glands (Tablet'iV) i t was considered of i n t e r e s t to study various parameters of t h i s process. The f o l l o w i n g aspects were examined i n d e t a i l : (a) The possible sequestering of the more polar cardenolides. (b,) The s e l e c t i v e sequestering a b i l i t y of the d o r s o - l a t e r a l meso- and metathoracic glands. (c) The f a t e of non-polar glycosides i n the body. (a) The possib l e s e l e c t i v e sequesting of the more polar cardenolides. To f u r t h e r i n v e s t i g a t e the p o s s i b i l i t y that Oncopeltus s e l e c t i v e l y sequesters the more polar glycosides from a food source, t r i t i a t e d - " ouabain and d i g i t o x i n were applied to crushed seeds of Asclepias s y r i a c a and the i n s e c t s , from f i r s t i n s t a r nymphs allowed to feed on them. These (Figure 3) i n i t i a l observations from TLC analysis suggesting s e l e c t i v e uptake of the more polar cardenolides i n the d o r s o - l a t e r a l thoracic glands were confirmed by t h i s experiment. The r e s u l t s (Figure 6'); show that, though digitoxin-^H was 2.5 times more concentrated i n the seed preparation than the equivalent preparation of ouabain- H, the l a t t e r glycoside was accumulated and stored i n the body at much higher l e v e l s than d i g i t o x i n - H. A f t e r feeding on t h i s t r i t i a t e d g l y c o s i d i c food source u n t i l 1 week of 63 adulthood Oncopeltus (male or female) sequestered 237 +. 30 d.p.m./ x mgm. wet weight of digitoxin- H and 234 +_ 22 d.p.m. per mgm. wet weight of ouabain -^H (without the concentration correction factor of 2.5; see Figure 6). Figure 6 also i l l u s t r a t e s the kinetics of the uptake of both ouabain and digitoxin label. It should be stressed that the curves for ouabain-^H and d i g i t o x i n - % do not represent the uptake of these glycosides s p e c i f i c a l l y but the uptake of ouabain or digitoxin i n the presence of the cardiac glycosides of Asclepias syriaca seeds. Therefore, since ouabain and digitoxin have not been reported to be cardenolides present i n species of Asclepias, and examination of this genus does not give an indication that they are present, i t can only be suggested that these curves (Figure 6) for the triated compounds represent trends of the uptake of glycosides. It might be questioned as to whether the treatment of the crushed Asclepias syriaca seeds (to incorporate as homogeneously as possible the label into the endosperm) resulted i n a detrimental effect on the uptake of natural cardiac glycosides from the seeds thus affecting the coinciding uptake of the la b e l . Figure 6 shows that the colorimetric determinations of insects sampled from the radio-isotope experiment had levels of cardenolide present which were accepted to be representative of the levels expected, though they were abnormally high. A more detailed examination of the concentration of cardiac glycosides i n the f i f t h instar nymph, 1 day-old adult 64-FIGURE 6: The uptake of the natural glycosides of Asclepias syriaca compared to the uptake of the cardenolide isotopes, x x ouabain- H and digitoxin- H, from a crushed seed preparation of seeds of Asclepias syriaca. • uptake of ouabain-^H from a seed food with 4-4-0 d.p.m./mgm. of seed of Asclepias syriaca: d.p.m./mgm. of wet weight. A uptake of digitoxin-^H from a seed food with 2800 d.p.m./ mgm. of wet weight of seed of Asclepias syriaca: d.p.m./ mgm. of wet weight. • uptake of the natural cardiac glycosides of seeds of Asclepias syriaca without label present (whole uncrushed seeds): micrograms/mgm. wet weight. D uptake of the natural cardiac glycosides of seeds of Asclepias syriaca (crushed seed preparation) with label present; migrograms/mgm. wet weight. Note the ouabain isotope i s less concentrated than :the digitoxin isotope (by a factor of 2.5). This includes a specific a c t i v i t y factor of 2.6. These values (• A ) were derived from extracts of one whole insect which had been fed from the f i r s t instar on a crushed seed preparation containing either of the two labels; see experiment (a) i n Methods. One sample represents one insect; each point was derived from at least 5-8 insects. These values ( • ) were derived from Figure 5 merely for comparison to the curves for the uptake of the two labels. These values ( a ) were derived from at least four colorimetric determinations of labelled insects that had been fed on the crushed seed preparation from which these values (•* ) were derived. The error i s expressed as the Standard Error of the Mean. The weights of the insects enclosed by this line are expressed i n Table VI, since they are dissimilar to Z-axis values. FIGURE 6 65 and 1 week-old adult stages f o r both ouabain and d i g i t o x i n isotopes was undertaken and the r e s u l t s are presented i n Table VI. The a c t i v i t y values f o r the gland and haemolymph f l u i d s were derived from i n s e c t s which had been r a i s e d i n the uptake k i n e t i c s experiment previously described (Figure 6). The values presented i n Table VI have not been corrected f o r the 2.5 times concentration d i f f e r e n c e of d i g i t o x i n above the l e v e l of ouabain i n the food. These r e s u l t s of glycoside d i s t r i b u t i o n are i n agreement with the d i s t r i b u t i o n of cardenolides i n the body of Oncopeltus as detected by chromatography (Figure 3 and Table V). Accompanying the drop i n r a d i o a c t i v i t y of l i v e insects, of both o u a b a i n - a n d d i g i t o x i n - H from the 5th i n s t a r to the 1 day o l d adult (Table VI), are changes i n the l e v e l s of r a d i o a c t i v i t y i n the d o r s o - l a t e r a l meso- and metathoracic gland secretions, i n the haemolymph, and the r a t i o s of the r a d i o -a c t i v i t y present i n the gland f l u i d compared to the l e v e l s i n the haemolymph. In comparing the decrease of d.p.m./mgm. (Table VI and Figure 6) f o r d i g i t o x i n - ^ H from the 5th i n s t a r to the 1 day-ol d adult and subsequently to a 1 week-old adult i n s i g n i f i c a n t changes i n the d.p.m./microliter of gland f l u i d and also the gland f l u i d to haemolymph r a t i o s occur. However, from the data f o r ouabain- H i t can be seen that i n the i n t e r v a l between the 5th i n s t a r and 1 week-old adult i n s e c t the increase i n the concentration of cardiac glycoside l a b e l i n the d o r s o - l a t e r a l thoracic glands i s s i g n i f i c a n t , and also concurrent with t h i s 66 TABLE VI: Changes i n the l e v e l s of ouabain- H and digito x i n - ^ H i n the d o r s o - l a t e r a l meso- and metathoracic gland f l u i d s and the haemolymph of Oncopeltus f a s c i a t u s at various developmental stages when reared on l a b e l l e d seeds of Asclepias syriaca* These values were derived from the uptake k i n e t i c s experi-ment (a) i n which d i g i t o x i n - % was .2,5 'times more concentrated (see Figure 6) i n the seed preparation than ouabain-^H, These values are not corrected f o r t h i s concentration d i f f e r e n c e . Each value was derived from at l e a s t three samples, one sample being the extract, a sample of f l u i d , or the r a t i o of gland f l u i d activity/haemolymph a c t i v i t y from one i n s e c t . Every set of determinations had at l e a s t 1 male or 1 female represented (except f o r the 5th i n s t a r larvae; not sexable). The error i s expressed as the Standard E r r o r of the Mean. * These values were determined from gland f l u i d samples c o l l e c t e d i n f i n e micro-pipettes. The volume c o l l e c t e d was estimated to be approximately 0.005 - 0.01 m i c r o l i t e r s ; the c a l c u l a t i o n i n terms of d.p.m./microliter was based on a sample considered to be 0.005 m i c r o l i t e r s . This serves only to prove that the nymphal glands concentrate the cardenolides ( l a b e l ) above the l e v e l s present i n the haemolymph. Thus, they are not comparable to the other l i s t e d values. Insect D.p.m./mgm. of Stage L i v e Insect D.p.m./Microliter of Gland F l u i d Ouabain _ D i g i t o x i n Ouabain D i g i t o x i n 5th Instar 92.2 + 11.3 262 + 28.8 662 + 4-22* 9900 + 5000* 1 Day-ol d Adult . 124- + 2.60 209 + 28.3 34-25 + 2605 2003 ± 24-5 1 Week-ol d Adult 234 + 21.9 237 ± 30.3 2006 + 548 708 + 14-5 Insect Stage D.p.m./Microliter of Haemolymph Gland Fluid/Haemolymph Ratio of D.p.m./ M i c r o l i t e r 5th Instar Ouabain D i g i t o x i n Ouabain D i g i t o x i n 352 + 61 662 + 305 1.93 + 1-22* 1 19.5 + 10.1 1 Day-old Adult 24-44- + 1584- 104-2 + 258 2.62 + 1.05 2.27 + 1.30 1 Week-ol d Adult 110 + 23 460 + 121 20.7 + 8.30 1.80 + 0.19 T o t a l Wet Weight (mgms.) of Live Animal from Label Uptake Experiments  5th Instar 28.2 + 4.74 1 Day-old Adult 29.4- + 2.60 1 Week-old Adult 26.1 + 1.50 35.1 ± 2.08 32.5 + 2.70 34.2 + 1.80 Ouabain- H Digitoxin-<H 6?,, there i s a s u b s t a n t i a l increase i n the r a t i o of the gland f l u i d to the haemolynph a c t i v i t y . There i s also a d e f i n i t e increase i n the haemolymph l e v e l s of both these l a b e l s i n the t r a n s i t i o n from a 1 day-old adult to a 1 week-old adult. Further, the actual sequestering of the more polar glycosides i s l i k e l y •z 7. mediated at the glandular l e v e l , since ouabain--'!! and d i g i t o x i n - H occur i n the haemolymph at approximately equal a c t i v i t i e s . I t also seems that metamorphosis could p o s s i b l y be a f f e c t i n g the uptake and storage of c e r t a i n or a l l cardenolides from a natural food source. The l e v e l s of r a d i o a c t i v i t y (Table VI) i n the mid-dorsal abdominal glands were determined by c o l l e c t i n g micro-volumes of f l u i d with adequate accuracy to ensure that i t could be determined whether the f l u i d contained higher l e v e l s of cardenolide than the haemolymph, but not with s u f f i c i e n t pre-c i s i o n to make comparisons between gland f l u i d to haemolymph a c t i v i t y r a t i o s v a l i d or to compare to the equivalent r a t i o s determined f o r the 1 day-old and 1 week-old i n s e c t s . Large Standard Errors of the Mean were derived from the data (Table V I ) . This can be explained by the f a c t that only 3-5 samples were taken f o r each i n d i v i d u a l determination. Also, i f fluxes of the cardenolides were a c t u a l l y occurring as i s suggested i n the curve f o r digitoxin-5H (Figure 6) between the 5th i n s t a r and 1 week-old adults, more accurately timed samples would have to be taken; animals f o r these determinations were only estimated as to t h e i r age within ± 12 hours. 68 Figure 6 also suggests that the uptake of cardiac glycosides"is not e f f i c i e n t , since animals fed on the labelled seed of 440 d.p.m./mgm. and 2800 d.p.m./mgm. of ouabain-% and digitoxin- H respectively at the 1 week adult stage have only sequestered about 12700 d.p.m./animal for both isotopes (approximately 24-0 d.p.m./mgm). This shows ;that ouabain-^H i s absorbed by the insect 2.5 times more e f f i c i e n t l y than digitoxin-^H. Efficiency of uptake of the isotope, p a r t i c u l a r l y ^ ouabain,is much less than 54% calculated on a 1:1 relationship between growth (weight increase) and the amount of seed eaten. Beck et a l . (1958) reports Oncopeltus 5th instar nymph eats 9 mgms. of seed a day. This value would at least lower the uptake of the isotopes and l i k e l y the natural glycosides to about a 5% efficiency of uptake l e v e l . From natural seeds 415 micrograms/gram of seed (0.97 migrograms/seed) of cardenolide i s available, the adult 1-3 week old insect has sequestered only 73-111 micrograms of cardenolide. (b) The selective sequestering a b i l i t y of the dorso-late r a l meso- and metathoracic glands. The results to this point have indicated (Figures >3 and 6, Tables V and VI) that Oncopeltus while sequestering cardiac glycosides, also d i f f e r e n t i a l l y concentrates these compounds i n i t s glands with reference to the polarity of the •z. 7. glycosides studied, namely ouabain--^H, digitoxin- H and the natural cardiac glycosides of the seeds of Asclepias syriaca. No proof has been provided dir e c t l y that the insect's glands 69 are able to remove these compounds from the haemolymph and/ or maintain i t s preference f o r polar glycosides. In t h i s experiment ouabain or d i g i t o x i n l a b e l , d i l u t e d with c o l d ouabain, were i n j e c t e d into separate t e s t animals' haemolymph, and the r a t i o s of the gland a c t i v i t y to that of the haemolymph followed over several hours. The l e v e l of c o l d ouabain was 0.1 micrograms i n 1 m i c r o l i t e r of i n j e c t i o n f l u i d , which when d i l u t e d by the volume of haemolymph, approximately 3-5 m i c r o l i t e r s / a d u l t , would not be excessively above the l e v e l s of natural glycoside i n the haemolymph, i f at a l l , but c e r t a i n l y f a r below the normal l e v e l s of glycoside occurring i n the gland f l u i d (35 micrograms/microliter). The r e s u l t s show (Figure 7) that the i n s e c t can remove cardiac glycoside from the haemolymph and store i t i n i t s d o r s o - l a t e r a l thoracic glands with a d e f i n i t e preference f o r the more polar ouabain. Also Table VII in d i c a t e s that i t can remove the l a b e l from the haemolymph and store the l a b e l i n a concentrated s o l u t i o n i n the v e n t r a l metathoracic glands of the adult. There i s no comparable uptake of digxtoxxn- H i n the presence of ouabaxn- H and c o l d ouabaxn, further f o r t i f y i n g the evidence that high l e v e l s of a polar glycoside impede the entrance of a more l i p i d soluble cardenolide. (c) The f a t e of non-polar glycosides i n the body The above experiments show that l i p i d soluble glycosides are not accumulated i n the d o r s o - l a t e r a l glands s i g n i f i c a n t l y above the l e v e l s present i n the haemolymph. Thin Figure 7: The uptake of ouabain-^H and d i g i t o x i n - ^ H from the haemolymph int o the d o r s o - l a t e r a l meso- and meta thoracic gland of the adult Oncopeltus f a s c i a t u s . 7. • The uptake of ouabain- H. A The uptake of d i g i t o x i n - H. a The anal f l u i d (urine) activity/haemolymph a c t i v i t y . • The a c t i v i t y of the d o r s o - l a t e r a l abdominal gland fluid/haemolymph a c t i v i t y . Male and female inse c t s were i n j e c t e d with the i n d i c a t e d amount of l a b e l (into the haemplymph); the d i g i t o x i n isotope was d i l u t e d with c o l d ouabain; see experiment c i n the Methods. A l l points are based on at l e a s t three samples; one sample being a c o l l e c t i o n of the gland f l u i d s and haemolymph from one i n s e c t . The error i s expressed as the Standard Error of the Mean. Note that r a t i o s (G/H) achieved f o r o u a b a i n - % are very high on t h e i r upper l i m i t s compared to the values attained i n Tables VI and IX; the time i n t e r v a l to a t t a i n these r a t i o s i s much shorter. This argues f o r the mass e f f e c t since the l a b e l i s c a r r i e d with much higher concentrations of c o l d ouabain (.1 microgram/ l y ) , than most of the other experiments, except i n Figure 10 where very high d.p.m. l e v e l s were employed. Gland f l u i d / haemolymph r a t i o o f d.p.m./x. ro CO H ro OJ o o o o H 4 CO ^ P. c+ Hj 13- C+ CD O H H-C-J. CD O O Hj c+ H-O O P 5» cr o SB Hj ro OD o >d ro L O D.p.m./x. o f g l a n d f l u i d x 100 H Q w 71 layer chromatograms of the faeces collected from f i l t e r paper l e f t i n the insect colonies also indicated that a l l the glycosides present i n the extracts of seeds were present i n these excretions. However, the l a t t e r points do not indicate i n any manner whether the glycosides entering the haemolymph remain there permanently while the action of the dorso-lateral glands prevents the accumulation of the polar glycosides. They may enter the haemolymph and pass hack across the gut and then be excreted with the faeces, or most of the glycosides taken up may be passed directly down the gut and voided. Thin layer chromatograms of the haemolymph indicate that l i p i d glycosides are entering this f l u i d selectively, compared to the polar glycosides. The haemolymph has more l i p i d soluble glycoside represented than the glandular f l u i d s . This indicates that significant levels of glycoside are not inhibited from entering the haemolymph by the gut ( as i n Figure 11). Analysis of anal f l u i d by chromatography, which also showed the presence of cardiac glycoside, did not clearly differentiate between the urine containing cardenolides and cardenolides resulting from faeces. To test whether cardiac glycosides could be removed from the haemolymph v i a the urine insects which had been injected with labelled glycoside (into the haemolymph; experiment c) were sampled for their urine several hours after injection. x The fact that digitoxin- H enters the haemolymph i n levels x roughly equal to ouabain- H but i s not concentrated i n the TABLE VII: Distribution of cardenolide label i n Oncopeltus  fasciatus i l l u s t r a t i n g the concentration of cardenolides by the various secretory glands. These values are derived from experiments i n which Oncopeltus was fed upon a glycoside labelled crushed seed preparation (see Figure 6) or i n which isotope was injected into the haemolymph (see Figure 10). Each value was determined from at least three samples. The error expressed i s the Standard Error of the Mean. These values are not directly comparable; they serve merely to provide evidence that the various glands are capable of concentrating cardiac glycosides as evidence i n Table VIII for the natural cardiac glycosides. The values marked * were dervied from the injection experi-ments and are not equivalent to the other values derived from the feeding experiment. The anal f l u i d i s composed of both faeces and urine. d.p.m./microliter haemolymph Ratio of d o r s o - l a t e r a l meso-metathoracic gland f l u i d to haemolymph Ratio of d o r s o - l a t e r a l abdominal gland f l u i d to haemolymph Ratio of mid-dorsal abdominal gland f l u i d to haemolymph Ratio of v e n t r a l metathoracic gland f l u i d to haemolymph Ratio of anal f l u i d to haemolymph 5th Instar Nymph Adult Ouabain D i g i t o x i n Ouabain D i g i t o x i n 352 + 61 662 + 305 3425 + 2605 2003 + 245 glands absent glands absent 20.7 + 1.1 1.8 + 0.19 glands absent glands absent approx. 21 not done 1.9 + 1.2 19.5 +. 10.1 glands not fun c t i o n a l glands not fun c t i o n a l gland absent gland absent approx. 32* not done 2.3 not done approx. 21* approx. 1.5' 7 3 gland supplies evidence that p o s s i b l y the more l i p i d soluble cardiac glycosides of Asclepias also enter the haemolymph but are not absorbed by the glands and thus pass out i n the faeces. Table VII i n d i c a t e s that glycoside l e v e l s i n the urine (anal f l u i d ) can exceed that of the haemolymph. Figure 3 and Table V also showed that the faeces possessed detectable l e v e l s of a l l cardenolides. Figure 7 shows the urine to haemolymph a c t i v i t y to be a r a t i o of almost 2 0 . This r a t i o i s much higher than the l e v e l s accumulated i n the glands per uni t volume over the samev period of time which thus shows a more r a p i d movement of l a b e l i n t o the urine than i n t o the glands. These r a t i o s are not d i r e c t l y comparable since the l a b e l i n the gland f l u i d i s concentrated i n a small volume ( 0 . 5 m i c r o l i t e r s / g l a n d ) ; whereas, 1 m i c r o l i t e r of anal f l u i d was e a s i l y c o l l e c t e d pointing out that l e s s of a concentration f a c t o r i s involved. PARAMETERS OF GLYCOSIDE ACCUMULATION IN ONCOPELTUS FASCIATUS The major l i m i t i n g f a c t o r i n the sequestering of cardiac glycosides from a food source by Oncopeltus has been the f i n d i n g that the d o r s o - l a t e r a l complex of glands seem to be able to sequester only the more polar glycosides. This could be a very r e s t r i c t i n g p h y s i o l o g i c a l feature of the animal! However, there must be other parameters involved i n the uptake of cardenolides into the body of t h i s i n s e c t which could influence i t s p a l a t a b i l i t y . 74-Some of these other parameters that were examined are: (a) The uptake and carry-over of natural cardenolides i n the l i f e cycle. (b) The retention of specific cardiac glycosides. (c) Glycoside dependent retention. (d) The uptake capability of adults. (a) The uptake and carry-over of cardenolides i n the l i f e cycle. It has been shown that Oncopeltus fasciatus sequesters preferentially the more polar cardenolides from a food source and that this accumulation of cardenolides i s subject to changes i n i t s kinetics during metamorphosis (Figure 8 and Table VIII). However, these results were based only upon the uptake of isotopic cardiac glycosides and not upon the measurements of uptake of the natural cardenolides present i n seeds of Asclepias syriaca. It was necessary to determine i f the natural cardenolides ( a l l 1 5 measured together) were subject to changes i n concentration of insect during metamorphosis. Table VIII outlines the colorimetrically determined amounts of natural glycoside present (on a total insect basis) based on three different cardiac glycoside standards; the values were based on three different standards to reflect a probable range of glycoside concentration. Figure 8 i l l u s t r a t e s the amount of glycosides taken up and carried over throughout the l i f e cycle of Oncopeltus on a micrograms/ TABLE VIII: Cardiac glycoside concentrations i n various stages of development of Oncopeltus f a s c i a t u s grown on seeds of Asclepias s y r i a c a , as determined by colorimetry with reference to ouabain, d i g i t o x i n , and strophanthin K as standard cardiac glycosides. The animals used i n these determinations were derived from laboratory cultures i n large aquaria; they had been fed e x c l u s i v e l y on milkweed seed. The c o l o r i m e t r i c determinations were based on extracts of groups of whole animals. Male and female adults (determined separately) were 1-3 weeks o l d . The teneral i n s e c t s were both males and females. Error i s expressed as the Standard Error of the Mean. The microgram concentrations/insect were c a l c u l a t e d by determining the micrograms/mgm. wet weight and mul t i p l y i n g t h i s by the average weight f o r that i n s t a r or type. Insect Mgm. Weight No. Insects/ Micrograms of Cardiac Glycoside/ Stage of Sample Stage Determined by Standard Samples Ouabain Strophanthin K D i g i t o x i n Instars 1 0.63 +_ 0.10 4 13 — 17 0.06 + 0.07 0.09 + 0.08 0.15 + 0.11 2 1.36 +, 0.32 5 7 - 16 0.15 + 0.05 0.21 + 0.06 0.38 + 0.08 3 ' 4.38 +, 0.50 8 4 - 8 4.20 + 0.16 5.93 + 0.25 10.6 + 0.25 , 4 10.5 +_ 2.40 10 4 - 8 17.4 ± 0.27 24.6 +, 0.43 44.6 ± 0.43 5 40.4 ±_ 6.20 10 2 - 6 57.4 + 0.52 80.9 + 0.83 147 + 0.83 Adults Teneral 45-5 ±^ 2.16 12 1 - 2 78.3 + 0.42 78.3 + 0.76 201 ± 0.67 1-3 weeks 53.0 +_ 2.76 7 2 - 3 111 ;> 0.71 156 + 0.76 284 ^ 1.14 male 38.6 +_ 1.66 7 2 - 3 73.0 + 0.93 103 + 1.00 187 + 1.49 female 55.6 +, 2.69 7 2 - 3 120 + 1.57 169 + 1.68 307 + 2.51 Egg 0.33 _+ 0.001 3 11 - 31 1.02 + 0.20 1.14 + 0.25 2.61 + 0.37 7 6 Figure 8: The uptake of the natural cardiac glycosides of the seeds of Asclepias s y r i a c a by Oncopeltus f a s c i a t u s . as determined by colorimetry with reference to ouabain as a standard cardenolide. • Migrograms of cardenolide/insect stage. • Micrograms of cardenolide/milligrams of wet body weight. The animals used i n these determinations were derived from laboratory cultures i n large aquaria; they had been fed e x c l u s i v e l y on milkweed seed. The c o l o r i m e t r i c determinations were based on extracts of groups of whole animals.^ See Table VIII f o r f u r t h e r d e t a i l s . This graph demonstrates that the uptake of cardiac glycosides from the seed food i s not d i r e c t l y r e l a t e d to growth as measured by an increase i n weight. Adults r a i s e d from eggs on Sunflower seeds acquired only l i m i t e d amounts of cardenolide i f placed upon milkweed seeds f o r a week. Females' have a higher glycoside content / i n s e c t ) than males because of the cardenolides i n eggs. This d i f f e r e n c e i s s t a t i s t i c a l l y s i g n i f i c a n t , though adult males are not s t a t i s t i c a l l y s i g n i f i c a n t from 5th i n s t a r nymphs i n t h e i r t o t a l glycoside content. o •6 CD w ' ,Q -p; o CD CQ a •H \ CD T3 •H H O £ M CD a3 •a •a a3 o -P . w o a3 , to CQ 03 w •H o a3 M .Q o a3 •H s O 120 r-- 3 - 2 - 1 10 20 4th i n s t a r _l J . 3rd i n s t a r ^nd i n s t a r 1 s t i n s t a r 5th i n s t a r 1 day a d u l t 1 week a d u l t egg 3 CD cr P CQ CD P i O o p P CQ P O O ct- TO 4 P CQ O O P 4 P i P CD cr 3 O H H-P CD \ CQ 3 ct- • p 2 o P i Hj p 4 s! P J CD c+ cr o p ^ • H 00 M i l l i g r a m w e i g h t o f the v a r i o u s . i n s t a r s 77 milligram wet weight b a s i s . . Unfortunately, no comparison was possible between these l e v e l s and the l e v e l s that a c t u a l l y occur i n the animals that have fed upon Asclepias i n the w i l d . Figure 8 i n d i c a t e s that the uptake of these compounds i s sigmoidal i n nature. There i s , however, a s i g n i f i c a n t drop i n micrograms/mgm. of wet weight i n the t r a n s i t i o n from the f o u r t h to the f i f t h i n s t a r (1.66 +_ 0.082 to 1.42 £ 0.016 micrograms/mgm.); whereas, i n Table VIII i t i s observed that from the fourth to the f i f t h i n s t a r i n terms of t o t a l body content there i s an increase of cardiac glycoside concentration from 17.4 +_ 0.52 to 57-4- +. 0.52 micrograms per nymphal i n s t a r . When the i n s e c t has reached adulthood (1-3 weeks o l d of adult stage) the l e v e l s have r i s e n to 2.09 0.098 micrograms/mgm. wet weight or 111 +_ 0.71 micrograms per adult. These r e s u l t s i n d i c a t e that growth as measured by weight i s not d i r e c t l y r e l a t e d to the t o t a l l e v e l s of sequestered cardiac glycoside. Table VIII and Figure 8 i n d i c a t e that the. l a r g e s t percentage of the sequestered (taken up and carried-over) cardiac glycoside i s acquired during the growth from the f i r s t to the f o u r t h i n s t a r on a microgram/mgm. ba s i s , and from the t h i r d to the f i f t h nymphal i n s t a r on a t o t a l microgram body content. No l o s s occurred during the moult into an adult. This suggests that Oncopeltus i n the environment i s obliged to associate with Asclepias during most of i t s l i f e to accumulate large stores of cardenolides. These large 78 stores are aff e c t e d l i t t l e by metamorphosis; but, nothing i s known about the e f f e c t of metamorphosis on the i n d i v i d u a l glycoside. From Figure 8 i t appears that the adult does not s i g n i f i c a n t l y continue to concentrate.cardenolides once i t has moulted from the 5th i n s t a r (micrograms/mgm.). Table VIII shows that teneral adults are not s i g n i f i c a n t l y d i f f e r e n t from males alone (which were 1-3 weeks old) i n t h e i r l e v e l s of sequestered cardiac glycosides, suggesting that adults, at l e a s t males do not have the a b i l i t y to sequester cardiac glycosides. Female adults (Table VIII) have s i g n i f i c a n t l y higher l e v e l s of cardiac glycosides than males or tenerals (both males and females assayed); however, eggs were demonstrated to contain very high l e v e l s of cardiac glycosides and t h i s may account f o r the d i f f e r e n c e between males and females, rather than the female alone having the a b i l i t y to continue to sequester cardiac glycosides i n i t s defensive glands. Eggs were shown (Table VIII) to contain approximately 1.02 micrograms of cardenolide per egg, or 3-09 micrograms/ milligram of egg. I t was also observed (Figure 3) that the eggs had more l i p i d soluble glycosides present than either the gland f l u i d s or the haemolymph, but s t i l l l i t t l e or none of the most l i p i d soluble cardenolides present i n the seeds. Unfortunately, the, quan t i t a t i v e c o l o r i m e t r i c method i s r e l a t i v e l y crude since i n d i v i d u a l cardenolides from the 79 seeds or i n s e c t have not been i s o l a t e d and the concentration determined at every growth stage i n d i c a t e d i n Figures 6 and 8. The uptake curve f o r the natural cardenolides cannot be considered to be f u l l y i l l u s t r a t i v e of what i s a c t u a l l y occurring i n the experimental Oncopeltus. However, t h i s does point out that i n such studies both the monitoring of each glycoside and a l l glycosides together are important parameters. (b) The r e t e n t i o n of s p e c i f i c cardiac glycosides Since i t has been shown that Oncopeltus sequesters cardiac glycosides and concentrates them i n various glands with preference to t h e i r p o l a r i t y , these questions were asked: (1) whether the r e t e n t i o n of polar and non-polar glycosides was dependent upon a continued source of glycosides,, and (2) whether or not d i f f e r e n t stages of growth showed varying a b i l i t y to r e t a i n both polar and non-polar cardiac glycosides? : Figures 9 a and 9b show the r e s u l t s of the experi-ments undertaken to answer the above questions. The Standard Errors of the Means are very large i n these experiments because the manner i n which the l a b e l l e d seed was prepared f o r the insect (described i n Methods, Experiment ,1>) was not as e f f e c t i v e i n producing equally l a b e l l e d animals. Insects pulsed with o u a b a i n - % u n t i l t h e i r t h i r d i n s t a r and then a f t e r t h i s period allowed to develop to 1 week-ol d adults on unlabelled whole milkweed seeds showed that there was a |L| r e l a t i v e l o s s of l a b e l by the 1 week-old adult stage, and that t h i s l o s s i s gradual over the developmental period spanned. The values were obtained from whole i n s e c t extracts; the l o s s of l a b e l from the d o r s o - l a t e r a l glands or the haemolymph was not determined. The r e t e n t i o n curve i s presented i n Figure 9a. Figure 9b shows that there i s a 1.8 r e l a t i v e l o s s of d i g i t o x i n - ^ H from the t h i r d i n s t a r nymph to a 1 week-old x adult, reared exactly as f o r the ouabain- H pulse, and that an abrupt l o s s of l a b e l occurred during the transformation into an adult as was suggested i n Figure 6 but which i s not s t a t i s t i c a l l y v e r i f i a b l e ; also, the values i n Table VI suggest t h i s l o s s i s also occurring on a mgm. body content basis during normal growth because i n the t r a n s i t i o n from a f i f t h nymphal i n s t a r the l e v e l s dropped from 262 +_ 28.8 to 209 +, 28.3 d.p.m. per mgm. of body weight,-despite the continual x presence of cardiac glycoside l a b e l i n the food. Ouabain- H di d not show a s i m i l a r drop (Table VI) over the same moulting periods. D i g i t o x i n was being handled i n a d i f f e r e n t manner than ouabain; however, the Standard Errors of the Means are too large f o r a l l values to permit any other i n t e r p r e t a t i o n than ouabain and d i g i t o x i n are p a r t i a l l y retained once absorbed i n t o the body even though those p a r t i c u l a r glycosides are not i n the food and other natural glycosides of Asclepias  s y r i a c a are d i l u t i n g them. 81 Figure 9a: The re t e n t i o n of ouabain-^H by Oncopeltus f a s c i a t u s when feeding on foods with and without cardiac glycosides. Figure 9b: The re t e n t i o n of d i g i t o x i n - H by Oncopeltus f a s c i a t u s when feeding on foods with and without cardiac glycosides. Insects were pulsed with eit h e r l a b e l applied to whole seeds of Asclepias s y r i a c a by permitting them to feed upon t h i s seed t i l l t h e i r t h i r d i n s t a r ; whence, they were tran s f e r r e d to whole unlabelled milkweed seeds or to sunflower seeds (no cardiac g l y c o s i d e s ) . See experiment - b i n the Methods. The s p e c i f i c a c t i v i t y of o u a b a i n - i s 11 .7 curies/mM; whereas, d i g i t o x i n - ^ H i s 4 .5 curies/mM. Therefore, the r e l a t i v e l o s s axis r e f l e c t s the t o t a l l o s s of ouabain ( t r i t i u m isotope plus c o l d d i l u t a n t ) as opposed to that of d i g i t o x i n . . The :.c.p.m../instar r e f l e c t s the apparent loss of isotope. • Fed on sunflower seeds from the t h i r d i n s t a r and measured f o r l a b e l at the 5th i n s t a r . Fed on sunflower seeds from the f i f t h i n s t a r and c o l l e c t e d at the 1 day-old and 1 week-old adult stages. A Fed on whole unlab e l l e d milkweed seeds from t h i r d i n s t a r and measured f o r l a b e l at every stage Indicated i n the graph. • 100% percent r e t e n t i o n . Each point i s based on at l e a s t three samples; one sample being the extract of one whole i n s e c t measured f o r l a b e l . The error i s expressed as the Standard Error of the Mean. M i l l i g r a m weight o f i n s t a r s 20 30 50 70 1_ I I I I I I 1 5 1 _L • _L 0 0.4 0.8 1.2 1.6 2.0 2v4 3 r d 4 t h . 5 t h l d a y 1 week", i n s t a r i n s t a r i n s t a r . . a d u l t . a d u l t Time a f t e r l a b e l l e d p u l s e F i g u r e 9A. O O O H X !x) CD o3. H -P JB c+ £ H-<! 0 \ H •a O CO P. co • O . 5 3 M i l l i g r a m w e i g h t ^of i n s t a r s 20 30 50 70 i I I ' ' ' ' • - 1 O J l . O _L _L _L Ixf CD P5 c+ 2 . 0 re 3 . 0 o CO co 4 . 0 3 r d . 4 t h 5 t h 1 day 1 week i n s t a r i n s t a r i n s t a r a d u l t a d u l t Time a f t e r l a b e l l e d p u l s e F i g u r e 9.B. 82 (c) Glycoside dependent r e t e n t i o n I t was also of i n t e r e s t to examine the retention of these isotopes when insects were placed on a non-glycoside containing food source. Figures 9a and 9b show that 3rd i n s t a r nymphs when reared to the 5th nymphal i n s t a r on an excess of sunflower seeds l o s t d i s s i m i l a r amounts of ouabain (isotope plus d i l u t a n t ) and d i g i t o x i n ; that i s the r e l a t i v e l o s s was 0*4 and 3«2 f o r ouabain and d i g i t o x i n , r e s p e c t i v e l y . Thus, on a food source with no glycosides present u n t i l the 5th nymphal i n s t a r , Oncopeltus retained both cardenolides with u n e q u a l e f f i c i e n c y . However, on a food source with glycosides i t appears that d i g i t o x i n i s not retained as e f f i c i e n t l y as ouabain. In nymphs, the r e t e n t i o n of d i g i t o x i n i s most susceptible to the presence of natural glycosides. When 5th i n s t a r nymphs, that had been pulsed u n t i l t h e i r t h i r d i n s t a r and t r a n s f e r r e d to whole milkweed seeds (unlabelled), are transferred to sunflower seeds, large losses of l a b e l also occur. In^the adult, the l o s s of d i g i t o x i n appears to be independent of the food source. The r e t e n t i o n of the polar isotope seems to require the presence of a d d i t i o n a l cardenolides. The f a c t that ouabain and d i g i t o x i n a r e not f u l l y r e t a i n e d even i n the presence of a d d i t i o n a l glycosides suggests there i s a f l u x i n g pool, .since isotopes are concentrated i n these glands. However, c o l o r i m e t r i c determination of the l o s s of natural glycosides from 83 Oncopeltus reared on an excess of sunflower seeds from the 3rd i n s t a r a f t e r t r a n s f e r from Asclepias seeds showed no s i g n i f i c a n t l o s s of cardenolides at the 5th i n s t a r , 1-day o l d and 1-week ol d i n t e r v a l s . This suggests that the natural glycosides of Asclepias seeds are more e f f i c i e n t l y retained than the a r t i f i c i a l glycosides, ouabain and d i g i t o x i n . Therefore, the r e t e n t i o n of the natural glycosides of Asclepias s y r i a c a seeds are almost independent of the food source, while a r t i f i c i a l cardiac glycosides are dependent upon external f a c t o r s i n f l u e n c i n g t h e i r concentration i n the body (glands), p a r t i c u l a r l y the more polar cardenolides. Analysis showed that these isotopes maintained t h e i r chromatographic i d e n t i e s a f t e r extended periods i n the body of Oncopeltus; the animal does not noticeably metabolizing them. A s t r a i n of milkweed bug that would feed on sunflower seeds (crushed) with glycoside additives could not be derived. Sunflower seeds, moreover, contain some component(s) which i n t e r f e r e with the c o l o r i m e t r i c procedure. (d) The uptake c a p a b i l i t y of the adult. I t appears that adult Oncopeltus i s not able to sequester cardiac glycosides i n i t s d o r s o - l a t e r a l thoracic glands or e l i c i t a preference f o r the accumulation of the more polar cardenolides. I t was found that the l e v e l s of cardiac glycoside per animal were not s i g n i f i c a n t l y d i f f e r e n t when the t e n e r a l values and the 1-3 week o l d male values were compared. I t was considered possible that the 84 p r e f e r e n t i a l s e l e c t i o n of the more polar glycoside might be a phenomenon r e s t r i c t e d to nymphal i n s t a r s . Table IX gives the r e s u l t s of an experiment where adult i n s e c t s of 10 days or older were allowed to feed f o r three days on a h i g h l y s p e c i f i c a l l y active seeds of e i t h e r l a b e l . Adults absorbed the cardiac glycosides, and sequestered them according to t h e i r p o l a r i t y (3-day feed). A f t e r three days of feeding on t h i s Asclepias seed source they had sequestered 747 ± 350 d.p.m./microliter of ouabain-^H i n the gland f l u i d compared to the 108 +_ 30 d.p.m./microliter of d i g i t o x i n - H, even though the l a t t e r was 2.5 times as concentrated as the former i n a s i m i l a r seed preparation. The values presented i n Table VI f o r the uptake of ouabain and d i g i t o x i n isotopes from the crushed milkweed seed preparation (a) i n d i c a t e d that adults up to 1 week o l d were harbouring l a r g e r q u a n t i t i e s of l a b e l than adults only 1-day o l d . Comparison of the data derived from the three" day feeding experiment with that of the l i f e c y c l e uptake of glycoside l a b e l s (experiment a) suggest that the concentration of the glycoside i n the food i s a f a c t o r i n the rate of uptake into the glands, since the l e v e l s of incorporated l a b e l from the three day feeding experiment almost equalled that of the uptake of a 1 day-old and 1 week-old adult that had been fed from the f i r s t i n s t a r on a low s p e c i f i c a c t i v i t y food (Table IX). A f t e r roughly 3 weeks of feeding on a seed source 85 Figure 10: The uptake of ouabain- H from the seeds of Asclepias s y r i a c a i n t o the d o r s o - l a t e r a l thoracic glands of 1 week and older adults of Oncopeltus f a s c i a t u s . • Gland f l u i d activity/haemolymph a c t i v i t y r a t i o determined from d.p.m./microliter of f l u i d , f o r d o r s o - l a t e r a l thoracic glands. A D.p.m./microliter of gland f l u i d . • Gland f l u i d activity/haemolymph a c t i v i t y r a t i o determined from d.p.m./microliter of f l u i d f o r the d o r s o - l a t e r a l abdominal glands. • D.p.m./microliter of f l u i d from the dorso-l a t e r a l abdominal glands. Adult males and females (at l e a s t ! week old) were i permitted to feed from 1-10 days upon whole seeds of Asclepias s y r i a c a to which had been applied ,7. approximately 1 m i l l i o n d.p.m. of ouabam-'H. Each point i s based on three samples; one sample being a c o l l e c t i o n of a volume of the various f l u i d s . The values c i t e d f o r the d o r s o - l a t e r a l abdominal glands are comparable to the other values since they were c o l l e c t e d from the same insects used to determine the other values. The error i s expressed as the Standard E r r o r of the Mean. Number o f days f e d on l a b e l l e d seeds 86 TABLE IX: The p o l a r i t y e f f e c t on the uptake of cardiac glycosides from l a b e l l e d seeds of Asclepias s y r i a c a i n t o the d o r s o - l a t e r a l meso- and metathoracic glands of Oncopeltus f a s c i a t u s . Dorso-lateral meso-and metathoracic gland f l u i d a c t i v i t y d.p.m./ m i c r o l i t e r Ratio of do r s o - l a t e r a l meso- and metathoracic gland a c t i v i t y / a c t i v i t y i n haemolymph  d.p.m./micro-Samples l i t e r 'ratio (G/H) Ouabain 3 day feed 1 day uptake 1 week uptake 747 + 350 3425 + 2605 2006 + 548 3 3 3 2.9 + 1.0 2.6 + 1.1 20.7'.+ 8.3 Di g i t o x i n 3 day feed 1 day uptake 1 week uptake 108 + 30 2003 + 245 708 + 145 3 5 3 0.76 + 0.16 2.3 ± 1.3 1.8 + 0.19 The 3 day feed represents adult i n s e c t s at l e a s t 1 week o l d that were fed on A. sy r i a c a seeds with approximately 3 x 10 d.p.m./seed of d i g i t o x i n and 0.5 x 10 d.p.m./seed of ouabain f o r three days. The above a c t i v i t y values are not corrected f o r the 2.5 times concentration d i f f e r e n c e of d i g i t o x i n compared to the s i m i l a r preparation f o r ouabain. The 1 day-feed and 1 week feed values were derived from the gland f l u i d a c t i v i t i e s presented i n Table VI depicting the uptake of these two isotopes from a crushed seed preparation. < The s p e c i f i c a c t i v i t y of these Seeds were: 440 d.p.m./mgm. and 2800 d.p.m./mgm. of seed f o r o u a b a i n - % and digitoxin-^H r e s p e c t i v e l y . One seed weighs approximately 4.3 milligrams. O O •H -p o3 J-r o CD o3 •Xi xi •H H Hours a f t e r commencement Of d r i n k i n g l a b e l l e d H^O • .Ouabain- H as taken.up from gut i n a s o l u t i o n o f 1,000 d.p.m:/v. w i t h 0.02 jj". o f oua'bain/x. . Average d.p.m./ o f g l a n d f l u i d f o r o u a b a i n - H. 1 hr.' 250 2 h r s . 213 3 h r s . 398 . 4 h r s . 1,153 Each p o i n t c o n s i s t s o f a t l e a s t 3 samples. E r r o r i s e x p r e s s e d as the S t a n d a r d E r r o r o f t h e Mean. F i g u r e 11. The movement o f c a r d i a c g l y c o s i d e s ( o u a b a i n - H ) from the g u t t o t h e d o r s o - l a t e r a l t h o r a c i c g l a n d s o f Oncopeltus f a s c i a t u s a f t e r d r i n k i n g l a b e l l e d water. 00 -o 88 of 440 d.p.m./mgm. seeds and 2800 d.p.m./mgm. of seed of ouabain and d i g i t o x i n . isotopes only 3425 +. 2605 d.p.m./ micro-l i t e r of gland f l u i d of o u a b a i n - % and 2003 +_ 245 d.p.m./ x m i c r o l i t e r of d i g i t o x i n - H had been incorporated into the glands of 1 day o l d adults; whereas, on a seed source of 6 6 3 x 10 and 0.5 x 10 d.p.m./whole seed (each seed weighed 4.3 milligrams) i n three days 747 +, 350 d i s i n t e g r a t i o n s of -x x ouabain- H and 108 +_ 30 of d i g i t o x i n - H had been incorporated into 1 m i c r o l i t e r of glandular f l u i d . Thus, besides concentration of glycosides i n the food, temporal association with the plant i s an obvious f a c t o r i n determining the l e v e l of uptake of cardiac glycosides. Further evidence of the a b i l i t y of the adult to take up cardiac glycosides was obtained from an experiment i n which l a b e l l e d glycosides were added to d r i n k i n g water. Adult animals were f i r s t starved and held without water f o r 1 day p r i o r to the experiment. Figure 11 shows that the glycosides were present i n the haemolymph and thus must have been taken up v i a the gut. Figure 11 provides a d d i t i o n a l evidence that t h i s i nsect takes up cardiac glycosides i n the absence of other nutrients from the lumen of the gut. Figure 10 also i n d i c a t e s that t h i s uptake from the gut i n t o the haemolymph i s r a p i d since i n 3-4- hours ah/approximately equal amount of ouabain-^H was concentrated i n the gland. Since the l e v e l of l a b e l acquired by the gland from 89 the 3 day feeding experiment (Table IX) did not exceed the levels acquired by an animal feeding on a labelled food with a much lower specific activity i t was questioned whether ouabain might be reaching saturating levels i n the animal since i t i s not a compound normally encountered by the insect. Figure 10 shows that i f Oncopeltus adults which are at least 1 week old are placed on a food source with approximately 1 million d.p.m./seed, the animal over a ten day period w i l l accumulate much higher levels of cardiac glycoside i n i t s glandular contents than demonstrated i n any of the previous experiments. It i s seen from this figure that ratios (G/H) greater than 20 are reached after two days (compare this to the value of 20.7 for a 1 week old adult i n Table VI and IX) and that at 10 days the ratio has exceeded 60. Uptake of the label from these seeds appears to be uniform throughout the ten days indicating that no saturation i s occurring. The levels of label i n the gland i t s e l f were higher for the various intervals than observed before. Figure 10 also shows that the dorso-lateral abdominal glands were also accumulating large levels of label. PREDATION EXPERIMENTS Since Oncopeltus fasciatus, a brightly coloured Lygaeid, has been shown clearly to be able to sequester appreciable amounts of cardiac glycoside i t i s important to determine i f these two.things are related. That i s , does the bright colouration serve as a warning colouration to predators. 90 Oncopeltus f a s c i a t u s , regardless of the medium upon which i t i s grown, i s t o t a l l y palatable to frogs and toads. Both the toads and frogs would accept r e a d i l y at l e a s t 5 Oncopeltus adults reared on milkweed seed with no r e j e c t i o n behaviour i n a period of several minutes, and even minutes l a t e r would, without h e s i t a t i o n , repeat t h i s i n d i f f e r e n c e to the cardiac glycosides present i n the animal. S u r p r i s i n g l y , even Tenebrio adults which had had t h e i r e l y t r a t h i n l y smeared with petroleum j e l l y and 5-10 mgms. of the glycoside mixture applied were accepted without h e s i t a t i o n repeatedly. No abnormal behaviour was observed a f t e r eating several of these Tenebrio. The frogs would even consume l e t h a l amounts of the pure glycoside with apparent i n d i f f e r e n c e . Therefore, Oncopeltus  f a s c i a t u s r a i s e d on seeds of A. s y r i a c a i s not unpalatable or aposematic to these species of toads or frogs. A l l forms i n Table X were eaten with equal a v i d i t y . However, the green anole (Table X) r e j e c t e d the Oncopeltus fed on sunflower seeds and milkweed seeds merely by sight, which suggests that they have been ( F l o r i d a area) conditioned to r e j e c t such aposematic forms. The observations on the two a l l i g a t o r l i z a r d s presented with Oncopeltus also showed r e j e c t i o n and thus these l i z a r d s seem to have had some experience i n the environment of coa s t a l B r i t i s h Columbia with comparable i n s e c t s (Hemiptera). Two anoles were presented adult Tenebrio onto which the body:fluids of Oncopeltus reared on milkweed seeds had been smeared; the beetles were eaten but TABLE X: Predation r e s u l t s with frogs (Rans p i p i e n s ) , toads (Bufo b o r e a l i s ) and Chamelions (Anolis c a r o l i h e r i s i s ) as predators to determine the p a l a t a b i l i t y of Oncopeltus f a s c i a t u s . Test Food Predators Frogs (4) Toads (4) Chamelions (5) Presented Accepted Presented Accepted Presented Accepted Tenebrio larvae Tenebrio adults Oncopeltus sunflower reared Oncopeltus milkweed reared Tenebrio plus glycoside' 7 7 7 7 100 100 100 100 100 7 7 7 7 100 100 100 100 100 7 7 7 7 100 100 0 0 not done For sequence of presentation to predators see Predation Methods * Toads and frogs would w i l l i n g l y accept 5 Tenebrio adults coated with a t h i n f i l m of v a s e l i n e and approximately 5-10 mgs. of cardiac glycoside (Strophanthin K: Ouabain: D i g i t o x i n , 2:1:1) applied to vaseline. The frogs would engulf enough cardenolide by t h i s method to k i l l themselves. TABLE XI: Predation r e s u l t s with t u r t l e s (Pseudemys s c r i p t s ) as predators to determine the p a l a t a b i l i t y of Oncopeltus f a s c i a t u s . Experiment Food Type Predators and Response to Prey T u r t l e 1 T u r t l e 2 T u r t l e 3 T u r t l e 4 0 A R O A R O A R O A R 1 2 3 Tenebrio adults Tenebrio control Tenebrio plus sunflower reared 0. f a s c i a t u s extract Tenebrio plus milkweed reared 0. f a s c i a t u s extract Tenebrio plus glycoside (larvae) Oncopeltus fasciatus reared on sunflower 7 7 0 7 7 0 7 7 0 7 7 0 7 7 0 7 7 0 7 7 0 7 7 0 7 6 1 7 7 0 7 7 0 7 6 1 7 6 1 7 7 0 7 7 0 7 6 1 7 6 1 7 6 1 7 5 2 7 5 2 7 0 7 7 0 7 7 0 7 7 0 7 0 =« number animals offered; 1 each 2nd day A = numbers accepted R = numbers rej e c t e d 93 the animals showed mouth wiping procedures not normally observed i n the feeding on palatable forms. This aspect was not studied f u r t h e r . I t appears then that Oncopeltus f a s c i a t u s was avoided by these green anoles only on the basis of a warning v i s u a l stimulus. I t i s doubtful i f the scent glands of the bug were secreting enough f l u i d to d i s i n t e r e s t the predator over a distance of 30 cm. Whether the scent glands' contents would cause r e j e c t i o n by the predator by application of t h e i r odours to other foods was not examined. The t u r t l e experiments (Table XI were not extended to f u l l s i g n i f i c a n c e as outl i n e d by Cole (1962) since more than 7 feedings of one t e s t food type was required. Since the experiment was done i n the winter and the animals were a l l feeding at low l e v e l s i t was desired that a l l animals receive the same amount of food and as l i t t l e of one t e s t food type as pos s i b l e . In Table XI, experiments 1-5 show that the four t u r t l e s ate Tenebrio regardless of the presence of cardiac glycosides from Oncopeltus or of the addition of pure cardenolides as used i n the toad experiments. One beetle contained the equivalent of 1 Oncopeltus extract f o r both the sunflower and milkweed preparations. The Tenebrio larvae contained 100 micrograms of the pure glycoside mixture, t h i s quantity considered to be representative of the amount of cardenolide present i n the animal (see Table V I I I ) . The few cases of r e j e c t i o n , p a r t i c u l a r l y f o r t u r t l e s 3 and 4- i n 9 4 experiments 4 and 5 are more l i k e l y a result of dissatisfaction with the often pulpy nature of the test food type, rather than a reaction to the amount of cardiac glycoside present. Most significant i n Table XII i s the fact that the four turtles rejected Oncopeltus reared on sunflower seeds, which indicates that the rejections, tasting and spitting out during their f i r s t associations, were due to the ventral metathoracic stink glands, since experiment 3 showed that these animals reared on sunflower seeds were otherwise palatable. Thus, for Pseudemys, Oncopeltus i s an aposematic and distaste-f u l insect because of i t s scent glands rather than the presence of cardiac glycosides. Three young chickens were employed to test the palatability of Oncopeltus reared on milkweed seeds to a s t i l l higher grade of predator. Chickens were found to be very f a c i l e animals to work with because of their inquisitiveness, insatiety, and lack of timidity during feeding times. Animals could be handled without disturbing any experimental design. In Table XII Oncopeltus fasciatus (Os) i s shown to be significantly rejected by the chickens. On their f i r s t exposures a l l chickens pecked at the sunflower insect but quickly learned to reject i t on sight alone. Even i f the animals were starved for a day and a half, they were reluctant to eat this animal (part 5) which suggests that the reinforce-ment i s strong enough to overcome an attack behaviour. The TOm experiment was designed to show that extracts 95 TABLE XII: Predation r e s u l t s with chicks (Gallus) as predators to determine the p a l a t a b i l i t y of Oncopeltus f a s c i a t u s . Experiment Test Pood Predators and Response to Prey Chick 1 Chick 2 Chick 3 A R P A R P A R P 1 T 7 0 0 7 0 0 7 0 0 Os 0 5 2 0 1 6 0 2 5 L 7 0 0 7 0 0 7 0 0 2 L 7 0 0 7 0 0 7 0 0 TOm 7 0 0 7 0 0 7 0 0 L 7 0 0 7 0 0 7 0 0 3 L 7 0 0 7 0 0 7 0 0 Os-ve 7 0 0 7 0 0 7 0 0 L 7 0 0 7 0 0 7 0 0 4 L 7 0 0 7 0 0 7 0 0 Om-ve 7 0 0 7 0 0 7 0 0 L 7 0 0 7 0 0 7 0 0 5 L 5 0 0 5 0 0 5 0 0 (starved) Os 0 4 1 3 1 0 4 1 L 5 0 0 5 0 0 5 0 0 A = Accepted immediately T = Tenebrio adults Os = Oncopeltus reared on sunflower seeds andpresented a l i v e to chicks Om-ve = as Os-ve but animal reared on milkweed seeds. R = Rejected on sight L = Tenebrio larvae P = Pecked at but rejec t e d Os-ve j Oncopeltus reared on sun-flower seeds but with v e n t r a l metathoracic glands seared out and whole dead animal painted black. TOm = Tenebrio adults with Oncopeltus-, r e ar ed-on-Milkwe ed-seed extract:-,' i n j e c t e d into beetle husk The f i r s t two symbols (food type) were randomized i n presentation to the chicks i n each experiment. The l a s t symbol was presented a f t e r each randomized p a i r to ensure continuance of i n s a t i e t y . In experiment 5 the animals were deprived of food f o r Vk days and then presented during 12 hours 5 Os animals. 96 of Oncopeltus fed on Milkweed seed, might cause r e j e c t i o n of a food to which the equivalent of one such Oncopeltus was added. These r e s u l t s show that t h i s t e s t food type was t o t a l l y palatable; but i t was f e l t that the albumin of the egg might be masking the e f f e c t of the cardiac glycosides or that there was p o t e n t i a l l y an error i n the extraction procedures and that the making and i n j e c t i o n of the albumin mixture into the beetle husk r e s u l t e d i n lower l e v e l s of cardenolides than were a c t u a l l y present i n the l i v e animal. Therefore, the experiments with sunflower or milkweed bred Oncopeltus, with the v e n t r a l metathoracic s t i n k glands seared away were c a r r i e d out. These experiments show that i f the v e n t r a l metathoracic st i n k glands are seared out and the whole animal painted black with a c r y l i c paint, both the Os-ve andOm-ve t e s t food types are f u l l y p a l a t a b l e . This therefore shows that Oncopeltus  fa s c i a t u s reared on seeds of Asclepias s y r i a c a i s unpalatable because of the v o l a t i l e secretions of the v e n t r a l metathoracic glands and not because of the presence of cardiac glycosides. F l u i d (3 m i c r o l i t e r s ) from the d o r s o - l a t e r a l thoracic glands of milkweed-reared bugs was applied to the tongues of two chickens, and no obvious sense of r e v u l s i o n was observed i n the chickens. Tenebrio beetles could be coated with the same mixture of cardiac glycosides as used f o r the frogs, and the chickens would accept them r e a d i l y and repeatedly. Force feeding of g e l a t i n capsules with approximately 50 mgms. of 97 the cardenolide mixture caused no emetic response. This was repeated several times with three chickens. A l l t h i s evidence suggests that the domestic chicken does not tas t e cardiac glycosides, but does react to the a l k y l v o l a t i l e s i n the v e n t r a l scent glands of Oncopeltusv In summary, Oncopeltus f a s c i a t u s has been shown to sequester large quantities of cardiac glycosides from the seeds °f Asclepias s y r i a c a and store the most polar cardiac glycosides i n a complex of d o r s o - l a t e r a l thoracic and abdominal glands. However, i t has been shown that these sequestered cardiac glycosides are not a s i g n i f i c a n t f a c t o r i n the r e j e c t i o n response of the f i v e vertebrate predators employed (frogs, toads, t u r t l e s , l i z a r d s , and chickens). Oncopeltus f a s c i a t u s i n these experiments was shown to be t o t a l l y palatable to toads and frogs; unacceptable by sight to green anoles; and unpalatable to t u r t l e s and chickens because of the secretions of the ve n t r a l metathoracic glands. 98 DISCUSSION The a s s o c i a t i o n of the "brightly coloured Lygaeine, Oncopeltus f a s c i a t u s with c e r t a i n Asclepiads and Apocynads as food-hosts i s considered to he an important f a c t i n the presentation of the argument that Oncopeltus might be present-ing i t s e l f to predators as an aposematic species which i s unpalatable to c e r t a i n or a l l predators. The s i g n i f i c a n c e of the Asclepiads and Apocynads (Apocynales) i n the environment i n our present epoch i s that they are p o t e n t i a l l y a source of powerful drugs f o r feeding i n s e c t s , presenting as t h e i r p r i n c i p l e drugs the cardiac glycosides (cardenolides) as i n the case of Asclepias, Apocynum, Calotr o p i s , Gomphocarpus, Nerium and Strophanthus (Bower, 1969; Hoch, 1961). Renowned f o r t h e i r potency (Moe et a l . , 1965; S t o l l , 1953; Wilbrandt, 1963), these steroids have r e c e n t l y been considered to be the chemical basis f o r u n p a l a t a b i l i t y amongst various aposematic Lepidoptera i n the Americas (Brower et a l . , 1964; Brower, 1969; Parsons, 1965). The key to the su r v i v a l of the aposematic Monarch b u t t e r f l y , Danaus plexippus (contrasting orange and dark brown colouration on wings), i n North America i s that i t feeds upon the poisonous Asclepiads, p a r t i c u l a r l y A. curassavica (Brower, 1969), i n i t s l a r v a l stages.deriving from the plant s u f f i c i e n t q u a ntities of emetic cardiac glycosides that are c a r r i e d over to the adult stage (Brower et a l . , 1968). 99 With the knowledge that Oncopeltus f a s c i a t u s , an eye-catching Hemipteran, i s associated throughout i t s l i f e with Asclepias s y r i a c a (Blatchley, 1924; Weiss et al,., 1921), Nerium  oleander (Hussey, 1952; Packchanian, 1957) and Apocynum (Proeshner, 1944), a l l plants of the Apocynales shown to contain cardiac glycosides (Bauer et a l . , 1964; Duffey, 1970, Hoch, 1961, Singh et a l . , 1970), the argument that Oncopeltus  fa s c i a t u s i s perhaps unpalatable and therefore aposematic i s ci r c u m s t a n t i a l l y well founded. Oncopeltus f a s c i a t u s can be reared i n the laboratory on several seed d i e t s (Beck et a l . , 1958; Prings et a l . , 1957; Sweet, I960) with or without the presence of cardiac glycosides, and i s thus an advantageous animal to study. The r e l a t i o n s h i p between a plant sucking Hemipteran and i t s p o t e n t i a l l y poisonous host plant, Asclepias  s y r i a c a , can thus be studied experimentally to determine i f cardiac glycosides assume any r o l e i n the p a l a t a b i l i t y of the inse c t , p o s s i b l y making i t aposematic. Since Oncopeltus i s more e a s i l y reared than the milkweed-feeding Monarch b u t t e r f l y , t h i s Hemipteran also provides an excellent means to study how such animals might handle such potent drugs. I n i t i a l l y i t had to be shown that the seeds of Asclepias s y r i a c a and other Asclepiads contained cardiac glycosides, and whether these compounds could be detected i n s i g n i f i c a n t concentrations i n the adult and nymphal stages of Oncopeltus f a s c i a t u s . C r i t i c a l p h y s i c a l , chemical, and b i o l o g i c a l evidence has been provided that cardenolides occur i n the eighteen species of Asclepias studied and that the adult 100 and larval forms of Oncopeltus do acquire cardiac glycosides from the seeds of Asclepias syriaca (Tables I, IV and V, and Figure 3)« Therefore, both adults and nymphs have the potential chemical basis to be unpalatable and aposematic. Experiments with Oncopeltus fasciatus showed that i t could sequester cardenolides from a number of Apocynales (Table IV); however, no attempt was made to delineate the effects of the varied glycoside compositions upon uptake. From this i t appears that i n the environment Oncopeltus may not be obliged to associate solely with Asclepias syriaca as a source of cardenolides. It would be more advantageous for such an animal to be able to feed upon a large range of poisonous plants. The 18 species of Asclepias and one of Acerates from diverse regions of North America examined showed that a l l these contained cardenolides, but not a l l of the same composition (Table I and I I ) . These plants then provided a spectrum of food which Oncopeltus could feed upon, possibly acquiring various degrees of unpalatability. A f u l l survey of other Asclepiads (Woodson, 1954) and related genera would be meaningful, depicting the extent of potentially noxious plants available to insects such as Oncopeltus. Oncopeltus sequesters the cardenolides from a food source without chemically altering these molecules: therefore, i t i s concluded that Oncopeltus i s unable to synthesize the cardenolide molecule (Table IV and Figure 3)« It remains an academic procedure to show by the introduction of the appropriate steroidal precursors that this conclusion i s v a l i d . A similar 101 lack of synthesis on the part of the ins e c t also appears to occur i n the Monarch b u t t e r f l y (Brower et a l . , 1964) and the Orthopteran, P. bufonis (v. Euw et a l . , 1967; Rothschild, 1966). The p o t e n t i a l of cardiac glycosides to re p e l predators has been a t t r i b u t e d to t h e i r a b i l i t y to induce emesis and cause mouth-wiping behaviour reminiscent of f o u l tastes (Brower, 1969; Brower et a l . , 1968; Brower, 1958a, 1958b). There are, however, thresholds (amount of cardenolide/kg. of predator) required to induce emesis (Hoch, 1961); therefore, i t was ess e n t i a l that some estimation of the quantity of cardiac glycosides contained i n one adult Oncopeltus be undertaken. Hoch (1961) c i t e s various emetic values ( L . D . ^ Q , M.E .D . ) f o r ouabain on animals such as frogs and pigeons (0.05 -0.3 mgm./kg.) and these are within the range of the determined l e v e l s of cardiac glycoside present i n one adult Oncopeltus (73-111' micrograms). Thus i t i s f e a s i b l e to consider Oncopeltus as an animal with the p o t e n t i a l to be unpalatable. Although such large q u a n t i t i e s of cardenolide are found to be present i n one i n d i v i d u a l of Oncopeltus a study of the d i s t r i b u t i o n within the animal shows that i t i s not i n the haemolymph, or the anal f l u i d (urine and faeces) (Tables V and V I I I ) . I t seemed improbable that the muscle t i s s u e or associated structures of the ins e c t could be containing i n t r a -c e l l u l a r l y such large quantities of these poisonous drugs. A dramatic morphological or biochemical means of d e t o x i f i c a t i o n 102 would have to be evolved by the animal to accomodate approximately 100 micrograms (0.19 x 10~5'M./insect) i n the small amount of muscle present. Besides, i n the newly moulted adult Oncopeltus the f l i g h t muscles are not f u l l y developed (Hewson, 1968). The insect also would not be l i k e l y to store these drugs i n the f a t body, a major s i t e of metabolism, because of the diverse and powerful e f f e c t s of cardiac glycosides on various aspects of metabolism (Lee et a l . , 1970; Vuilliemoz et a l . , 1968). In vertebrates l e v e l s of cardiac glycosides from 10~®~ 10""^ M. have toxic a c t i v i t y (Frank etal . , 1 9 6 7 ; Wilbrandt, 1963), though -4- -3 higher q u a n t i t i e s , 10 - 10 -H. appear to be necessary to cause metabolic i n h i b i t i o n i n insects (I r v i n e et a l . , 1970; Treherne, 1966). Even i f one assumes the adults of Oncopeltus to have a volume of 1 ml. the molarity achieved with 100 micrograms i n -3 t h i s volume i s l a r g e , 1.6 x 10 M. (.59 x 10 M, i n g l a n d f l u i d ) . Because a s i g n i f i c a n t amount of cardenolide was not found i n any of the main body components investigated, i t was questioned whether the glycosides might be l o c a l i z e d i n some sp e c i a l and s p e c i f i c compartment of the i n s e c t . One such group of compartments are the adult v e n t r a l metathoracic and the nymphal mid-dorsal abdominal s t i n k glands (see Figures 4- and 5) c h a r a c t e r i s t i c of many Hemipterans (Calam et a l . , 1968; Johansson, 1953; Remold, 1963; Rothschild, 1961; Youdeowei et a l . , 1969); i t was suspected that they might be able to harbour t h i s glycoside content. Table V shows that these glands not only contain a l k y l compounds but also concentrate cardiac 103 glycosides at l e v e l s considerably more concentrated than the haemolymph. However, the volumes a v a i l a b l e f o r c o l l e c t i o n i n d i c a t e that these glands and t h e i r contents are not responsible f o r the large amount of cardenolide present i n Oncopeltus, p a r t i c u l a r l y i n the adults. However, an addit i o n a l complex of previously undescribed glands i n the adults of Oncopeltus was discovered and shown to contain high concentrations of polar glycosides (Table V): the concentration and/or the volume accounted f o r the t o t a l body content of glycosides (Tables V and VIII, and Figure 3). These glands, the d o r s o - l a t e r a l complex, show defensive c a p a b i l i t y f o r they secrete small droplets of f l u i d when the insect i s f o r c i b l y handled. There i s a d e f i n i t e concentration gradient of cardiac glycosides between these glands and the .haemolymph. A crude estimate suggests that the d o r s o - l a t e r a l gland f l u i d i s 300-500 times more concentrated on a u n i t volume basis than the haemolymph; on a t o t a l volume basis i t i s a f a c t o r of 1000. These values are within equivalence f o r the concentration of cardiac glycosides of Asclepias curassavica sequestered i n the thoracic gland of the Orthopteran P. bufonis (v. Euw et a l . , 196?; they state a 1000 times f a c t o r ) . The discovery that the glycosides i n the d o r s o - l a t e r a l glands were decidedly polar suggested that there, was an unequal sequestering of glycosides. The study of the sequestering of cardiac glycosides i n Oncopeltus (Figure 8 ) , as resolved by colorimetry, was very l i m i t e d . Therefore, ouabain- H and d i g i t o x i n - H were employed to acquire more d e t a i l e d information on the uptake, retention, d i s t r i b u t i o n and e l i c i t i n g of the p o l a r i t y e f f e c t i n Oncopeltus f a s c i a t u s feeding upon the seeds of Asclepias s y r i a c a (Figure 6). However, i t i s important to r e a l i z e that uptake of each isotope from the seed source does not represent the uptake of a s i n g l e isotope but the uptake i n the presence of much greater q u a n t i t i e s of the natural glycosides of the seeds (415 micrograms/gm.). The r e s u l t s of Figure 6 i n d i c a t e the manner of the sequestering of the i n d i v i d u a l isotopes throughout the l i f e cycle demonstrating that t h i s i n s e c t does not accumulate •z 3 ouabain- H i n a s i m i l a r manner to d i g i t o x i n - H and that also these sequestering k i n e t i c s are d i f f e r e n t from that of the pattern f o r t h e n a t u r a l glycosides of seeds of Asclepias s y r i a c a . Figure 6 also shows that there i s a d e f i n i t e p r e f e r e n t i a l storage of the more polar isotope, thus s p e c i f i c a l l y v e r i f y i n g the p o l a r i t y e f f e c t . The k i n e t i c s of the uptake and storage of ouabain- H i s s i m i l a r to that of the natural glycosides, more so than the l i p i d isotope, d i g i t o x i n - ^ H . This, therefore, brings to question both the v a l i d i t y of the c o l o r i m e t r i c procedures and the use of some isotopes f o r determining such k i n e t i c s since the three curves (Figure 6) are d i f f e r e n t . The pattern of sequestering of cardiac glycosides as observed by the use of these isotopes may not be v a l i d unless cardenolides are used which are natural to the food-plant upon which, the i n s e c t normally feeds. I t can he argued that the curves i n Figure 6 represent a r t i f a c t s since the two ( i s o t o p i c ) glycosides have not been reported to be present i n Asclepias  s y r i a c a or i n the species of Asclepias (Hoch, 1961; Singh et a l . , 1970)• Ouabain i s present i n species of Acokantheria and Strophanthus (Hoch, 1961), genera of the Apocynales; the i n s e c t ' s tolerance and mode of handling of s p e c i f i c glycosides may be species s p e c i f i c rather than at a generic or family l e v e l . D i g i t o x i n i s t y p i c a l of the species of D i g i t a l i s (Eoch, 1961),a plant .upon w h i c h . Oncopeltus has not been reported to feed. Despite t h i s c r i t i c i s m , cardenolides of equal p o l a r i t y and greater l i p i d i t y were present i n the seeds of Asclepias 7. 7. s y r i a c a which argues f o r the use of ouabain- H and d i g i t o x i n - H 7. as the extremes of p o l a r i t y i n these experiments. D i g i t o x i n - H a digitoxose t r i o s i d e , was chosen as the l i p i d cardenolide representative rather than an equally l i p i d aglycone to avoid the c r i t i c i s m that any p o l a r i t y e f f e c t observed might be due to the absence of sugar residues on the s t e r o i d a l nucleus rather than to the s o l u b i l i t y properties of the molecule. Ouabain i s a rhamnose monoside; i t i s assumed that the d i f f e r e n c e i n the sugar moieties i s not e l i c i t i n g the p o l a r i t y e f f e c t observed i n Figure 6, through s e l e c t i v i t y of various sugars by Oncopeltus. I t i s generally considered that the presence of sugar residues makes the cardenolide more polar (Hoch, 1961). The only f a c t o r s which w i l l be s i g n i f i c a n t l y a f f e c t i n g p o l a r i t y are the number of hydroxyl groups or polar functions substituted on the s t e r o i d ; ouabain and d i g i t o x i n d i f f e r i n t h i s respect; (see Hoch, 1961). . ' 106 The uptake of aglycon.es was not examined i n Oncopeltus though i t would appear that t h i s i n s e c t i s not capable of maintaining large concentrations of these supposedly more non-polar molecules (Figure 3). ' The d o r s o - l a t e r a l thoracic and abdominal glands are highly f u n c t i o n a l as a defense mechanism i f the cardiac glycosides contained i n the glandular f l u i d a c t u a l l y r e s u l t i n the r e j e c t i o n of a predator with or without the complementary action of the ve n t r a l metathoracic glands. When Oncopeltus i s f o r c i b l y handled the various glands exude viscous droplets which remain o b t r u s i v e l y at the o r i f i c e of the: :glands. The thrashing of the legs of t h i s insect during seizure f a c i l i t a t e s the spreading of these drop-l e t s of f l u i d ; t h i s has been reported to occur i n other Lygaeids which spread the secretions from t h e i r v e n t r a l metathoracic glands (Remold, 1963). The external morphology of these dorso-l a t e r a l glands i s unique when compared to the t y p i c a l structures observed from the v e n t r a l metathoracic glands .and nymphal mid-dorsal glands (Crossley et a l . , 1969; Remold, 1963). There i s no evaporatorium or mushroom-body area; instead, erect and miniscule b r i s t l e s are present at the o r i f i c e s of the l a t e r a l metathoracic and l a t e r a l abdominal o r i f i c e s which appear to be adapted to maintain the s t a b i l i t y of a protruding drop of l i q u i d . Such s t r u c t u r a l d i fferences e x t e r n a l l y (and the mere presence of these glands) implies that b i o l o g i c a l l y they must be s i g n i f i c a n t ; structures i n animals do not appear and p e r s i s t without functioning i n the biology of the animal (Cain, 1968). 107 In order to demonstrate the responses of various predators to Oncopeltus f a s c i a t u s to both the cardiac glycoside cpntaining d o r s o - l a t e r a l glands and the v e n t r a l metathoracic glands, four gross grades of predators were chosen; Amphibians (frogs and toads), Chelonians ( t u r t l e s ) , L a c e r t i l i a n s (anoles and a l l i g a t o r l i z a r d s ) and Avians (chickens and s t a r l i n g s ) . I t was considered the Amphibians represented the most unselective predators o r i e n t i n g t h e i r attack with reference to perception of motion (Kaess et a l . , I960). The l i z a r d s and t u r t l e s were assumed to be a stage above t h i s perceiving prey both by apparent motion and judging prey by o l f a c t i o n (Jacobson^s organ; Moncton, 1967) and p o s s i b l y t a s t e . The avian predators represented the most so p h i s t i c a t e d predator r e l y i n g h e a v i l y on sight and taste, and p o s s i b l y o l f a c t i o n (Brower, I960; Duncan, I960; Rothschild, 1966, 1964; Yang et a l . , 1968). Toads and frogs were found to be unable to d i s t i n g u i s h between any form of Oncopeltus presented to i t , with or without scent and/or cardenolides (Table X). I t was considered Oncopeltus might not have enough glycoside to cause an emetic response i n the frogs, but addition of l a r g e r amounts of glycosides to Tenebrio adults d i d not cause any r e j e c t i o n response but death of the amphibian d i d ensue (see Hoch, 1961 f o r l e t h a l and emetic values of various cardiac g l y c o s i d e s ) . The author f e e l s that toads and frogs may lack the appropriate receptors to permit them to taste cardiac glycosides, or to taste or smell the v o l a t i l e secretions of the v e n t r a l meta-thoracic gland of Oncopeltus. Thus to these two amphibian 108 predators Oncopeltus i s not an aposematic i n s e c t and i t s association with Asclepias i s of no b e n e f i t . However, being t e r r e s t r i a l they may be of no consequence as predators to the a e r i a l populations of Oncopeltus. VanDoesburg (1968) mentions that i n the West Indies a tree f r o g , Hyla s e p t c e n t r i o n a l i s . does eat Dysdercus andraae, an animal which Darlington (1938) found to be r e j e c t e d on sight by Anolis s a g r e i . Whether t h i s i s a s i g n i f i c a n t predator of Oncopeltus i n the Caribbean i s not known. The reactions of predators such as toads and frogs i n the w i l d may be d i f f e r e n t as may be the p a l a t a b i l i t y of Oncopeltus r a i s e d i n the w i l d . The green anoles (arboreal), Anolis c a r o l i n e n s i s , r ejected Oncopeltus f a s c i a t u s on sight; these r e s u l t s (Table X) agree with Darlington's (1938) and Sexton's (1969) observations with the p r e v i o u s l y mentioned species of A n o l i s . Obviously some prey has conditioned these predators to regard the consummation of such a prey as an unpleasant experience. The l i z a r d s used i n t h i s author's experiment were supposedly derived from the F l o r i d a area. Therefore, to Anolis c a r o l i n e n s i s , Oncopeltus i s wamingly coloured, and i s subsequently avoided. Lizards are suspected of being the major i n s e c t i v o r e predators i n the t r o p i c s (Darlington, 1938); regardless of the p a l a t a b i l i t y of Oncopeltus e i t h e r by smell or by cardiac glycosides i t i s protected by some unknown model. A t e r r e s t r i a l l i z a r d , Gerrhonotius coeruleus p r i n c i p i s , the northern a l l i g a t o r l i z a r d r e j e c t e d Oncopeltus on s i g h t . 109 I t i s possible the l i z a r d d i d smell t h i s i n s e c t but most probably the predators had had previous experiences with l o c a l Hemiptera which r e i n f o r c e d the animal to r e j e c t b r i g h t l y coloured forms. These l i z a r d s were c o l l e c t e d at Vancouver, B r i t i s h Columbia. T u r t l e s , though not l i k e l y a predator of insects such as Oncopeltus« were chosen i n an attempt to present f u r t h e r evidence that carnivorous animals of. approximately the same evolutionary scale as l i z a r d s could discriminate against Oncopeltus by taste and/or smell. I t was found that the t u r t l e s only r e j e c t e d Oncopeltus on the basis of i t s v o l a t i l e and odorous defensive secretions and not because i t s glycoside (Table XI). Therefore, t u r t l e s are l i k e frogs and toads i n that they appear to be unresponsive to the l e v e l s of cardiac glycoside presented to them i n the t e s t foods, but d i f f e r e n t i n that they, l i k e the l i z a r d s , seem to be able to discriminate against Oncopeltus by o l f a c t i o n . S t a t i s t i c a l l y v a l i d experiments were not c a r r i e d out but a p p l i c a t i o n of the f l u i d s from a crushed Oncopeltus fed on Milkweed seeds to a Tenebrio adult which was presented to Anolis c a r o l i n e n s i s caused obvious mouth wiping behaviour; these were not observed i n a s i m i l a r t e s t with the two a l l i g a t o r l i z a r d s . From these predation experiments i t i s revealed that the responses of various lower vertebrate predators to Oncopeltus i s varied; Oncopeltus i s aposematic to only some of them. Most of the predation studies on unpalatable insects 11.0 and t h e i r mimics has been done with the Jay, Cyanocitta c r i s t a t a  bromia (Brower, 1969). Another species of Jay, Cyanocitta coerulescens  coerulescens (Brower, 1958) and Silverbeak Tanagers, Ramphocelus  carbo magnirostris (Brower et a l . , 1964) have been used i n studying u n p a l a t a b i l i t y amongst t r o p i c a l b u t t e r f l i e s . L i n s l e y et a l . , (1961) employed the Jay, Cyanocitta s t e l l e r i , to study Lycid and Cerambycid mimicry complexes. These have been very benign predators i n that they have given confirmative r e s u l t s f o r p a l a t a b i l i t y complexes. No range of avian predators was employed to see i f one form of insect i s equally palatable to a l l types of b i r d s . Rothschild (1964) stresses the importance of avian i n t e r - s p e c i f i c v a r i a t i o n i n taste; although she reported crows were strongly r e i n f o r c e d by aposematic forms. Undescribed experiments by the author i n d i c a t e that s t a r l i n g s and robins, indigenous to Vancouver do not respond emetically to cardenolides i n Oncopeltus. Domestic chickens were also employed as a predator of Oncopeltus; ..birds are often used i n t h e o r e t i c a l studies of feeding preferences (Rabinowitch, 1968; Schmidt, 1958). The experiments (Table XII) were designed to r e a d i l y and simply d i s t i n g u i s h between the p o t e n t i a l r e p e l l a n t e f f e c t of the ventral, gland secretions and the cardiac glycosides i n the d o r s o - l a t e r a l glands. The experimental procedure i n v o l v i n g the i n j e c t i o n of cardenolides into Tenebrio husks i n an egg albumin-starch s l u r r y , to.show the e f f e c t of the glycosides alone from Oncopeltus, was considered p o s s i b l y misleading since cardiac l i ' X glycosides have been reported to bind to proteins and have t h e i r t o x i c i t y lessened by such behaviour (Farah, 194-5; Fawaz et a l . , 1944). A l t e r n a t i v e experiments, were; c a r r i e d . out. The predation studies with the chickens indicated that these predators are not responding to the cardiac glycosides i n Oncopeltus but rather to the defensive secretions of the v e n t r a l metathoracic defense glands. Even high quantities of glycosides force fed to these chickens, which according to Hoch (1961) would cause emesis i n pigeons, had no e f f e c t on the chickens. This i s strong evidence f o r v a r i a b i l i t y i n the taste response of various predators to the a c r i d taste and inducement of emesis. However* Brower (I960) showed chickens responsive to the b i t t e r taste of quinine and S t a r l i n g s responsive to quinine dihydrochloride. Duncan (i960) has shown f e r a l pigeons respond to a v a r i e t y of taste s t i m u l i . The predation experiments show that not a l l predators feeding on a prey suspected of being aposematic behave i n the same manner to the i n s e c t ' s defense mechanism; the predators used are not natural predators i n the environment but the r e s u l t s of the reactions of these various predators are h e u r i s t i c i n the f a c t that they point to a deeper consideration of the predator spectrum as well as that of the food-plant and the insect p a l a t a b i l i t y . Since Oncopeltus seems to be able to sequester only the more polar glycosides from Asclepias s y r i a c a , i t i s i n t e r e s t i n g to speculate that the p a l a t a b i l i t y spectrum 112 observed by Brower (1969) may be i n part explained by the inherent uptake physiology of the Monarch b u t t e r f l y rather than by the emetic q u a l i t y of the plant; however both f a c t o r s are important i n the a b i l i t y of an i n s e c t to be unpalatable. The author considers the physiology of Oncopeltus to be a very important f a c t o r i n explaining why t h i s i n s e c t i s palatable. The d e t a i l e d experiments with glycoside isotopes revealed that the p o l a r i t y e f f e c t i s a c t u a l l y e l i c i t e d at the d o r s o - l a t e r a l gland -complex l e v e l rather than across the gut or i n the haemolymph (Figures 7, 10 & 11). Thus these s p e c i a l l y evolved glands are themselves r e s t r i c t i n g the a b i l i t y of Oncopeltus f a s c i a t u s to sequester the more non-polar glycosides reputed to be emetic. The binding c a p a c i t i e s of the insects haemolymph p r o t e i n may be a c r i t i c a l f a c t o r i n t h i s p h y s i o l o g i c a l l i m i t (Farah, 1945; Fawaz, et a l . , 1944). The combination of gland showing d i s t i n c t i v e sequestering c a p a b i l i t i e s and a protein associated with glycoside movement, make the antagonistic action of d i f f e r e n t glycosides at d i f f e r e n t concentrations i n the food source and the haemolymph seem more tangible. To breed Oncopeltus f a s c i a t u s upon Asclepias curassavica, which contains more l i p i d glycosides (and emetic p r i n c i p l e s , see Brower, 1969), could g r e a t l y elucidate the e f f e c t of d i f f e r e n t glycosides on the glands: a b i l i t y to sequester. Many other fac t o r s inherent i n the behaviour and physiology of the i n s e c t remain to be examined i n terms of sequestering glycosides since i t also appears that the r e l a t i o n s h i p s between temporal association with the food-plant, the s p e c i f i c a c t i v i t y of the food and the d i s t r i b u t i o n of the glycosides i n the food source are not simple (Figures 6 and 10 and Table IX). In f a c t a p o l a r i t y e f f e c t exerted by the d i f f e r e n t i a l s o l u b i l i t y of the glycosides i n the insect's s a l i v a may be the f i r s t p h y s i o l o g i c a l f a c t o r complicating sequestering. Non^polar' glycosides were l o s t from the body independently of the food source but polar glycosides were even l e s s s u b s t a n t i a l l y l o s t when glycosides were i n the food (Figures 9a and 9b). Mass e f f e c t s of the natural glycosides or even one glycoside present i n a much higher concentration might play a s i g n i f i c a n t r o l e i n determining what glycoside and how much of i t are sequestered into the d o r s o - l a t e r a l glands. This means that Oncopeltus t h e o r e t i c a l l y could feed upon a plant with high l e v e l s of a polar r e l a t i v e l y non-emetic glycoside, and that the concentrations of t h i s glycoside i n the plant could cause the elimination from the gland of much lower concentrations of a potent emetic p r i n c i p l e : the ins e c t would then remain palatable ( r e f e r to Figures 7 & 10). From the above predation experiments i t i s not possible to postulate that Oncopeltus i s an unpalatable model because of i t s a s s o c i a t i o n with species of Asc l e p i a s : i t could serve as a model because of i t s v o l a t i l e secretions. However, the evolution of the d o r s o - l a t e r a l gland complex s t r i c t l y f o r d e t o x i f i c a t i o n purposes i s harder to envisage than that these glands are being employed as a defensive mechanism. What then i s the actual status of Oncopeltus a b r i g h t l y coloured i n s e c t containing a considerable l e v e l of cardiac glycosides, and abnoxiously odorous? Is Oncopeltus a Batesian or M u l l e r i a n mimic, or both? Is i t involved i n a mimicry complex based on smell f o r i t s u n p a l a t a b i l i t y (Rothschild, 1961) and/or i n one based on cardiac glycosides? The p o t e n t i a l to be both a model and/or a mimic i s c l e a r when one r e l a t e s t h i s i n s e c t to i t s geographic p o s i t i o n and the plant i t i s feeding upon, the associated i n s e c t fauna, and the type of predator i t v i e s with ( e s p e c i a l l y migratory b i r d s ) . Oncopeltus occurs with other b r i g h t l y coloured i n s e c t s . In Table I I I i t i s shown that Oncopeltus sandarachatus, Lygaeus  kalmii k a l m i i , L.k. angustomarginatus, Tetraopes oregonensis, and T. tetraophthalmus a l l contain cardiac glycosides. T_. tetraophthamus i s reported to feed upon the leaves of Asclepias s y r i a c a , A. pulchra and A. v i r i d i f l o r a and upon the roots of A. cor n u t i as a larvae (Weiss et a l . , 1921), Lygaeus  kalmii and L. t u r i c u s upon A. syriaca^ A^ tuberosa and A. speciosa (Sl a t e r et a l . , 1969; Weiss et a l . , 1921). The r e s u l t s of Table I I I provide chemical v a l i d i t y to the observations of Jones (1937, 1934, 1932) f o r he considered several of the above in s e c t s to be warningly coloured, since they were not eaten by bi r d s and extracts of them were r e j e c t e d by ants. However, the author considers Jones' experiments do not meet the more rigorous requirements of predation studies and predator behavioural responses employed 115 by Brower (1958), Brower et a l . , (1964), de Ruiter (1952), and Schmidt, (1958). They only give a suggestion of the presence of a mimicry complex whose chemical basis i s cardiac glycosides. I f cardiac glycosides are not simply synonymous with u n p a l a t a b i l i t y then the function of the t o t a l glycoside concentration and/or l e v e l s of s p e c i f i c glycosides may be important. Two i n d i v i d u a l i n s e c t s of one species feeding on d i f f e r e n t species of plants may have d i f f e r e n t p a l a t a b i l i t i e s because of plant differences (Table I I ) . A l t e r n a t e l y , i n d i v i d u a l insects of two d i f f e r e n t species feeding on the same plant, or parts thereof, may have d i f f e r e n t p a l a t a b i l i t i e s because of the i n s e c t s ' p h y s i o l o g i c a l d i f f e r e n c e s . A p o l a r i t y spectrum as discussed by Brower (1969) and Brower et a l . (1968), may exist but i t may have more than a simple b a s i s . The Monarch b u t t e r f l y when reared on northern species of Asclepias was found to be palatable and supposed to be free of cardenolides because they were s a i d to be lacking i n the food-host (Brower, 1969); on the contrary these insects were found to contain cardenolides (Duffey, 1970; Table I I I ) as were several species of northern Asclepias (Table I ) . The fa c t that these b u t t e r f l i e s were palatable again points out that u n p a l a t a b i l i t y cannot be a simple matter of glycosides or no glycosides. An i n d i v i d u a l plant may permit the existence of a p a l a t a b i l i t y spectrum i n one or several species of insects; i t has been shown that leaves and pods of Asclepias have fewer 116 and d i f f e r e n t cardenolides from those present i n seeds of the s a m e L p l a n t (Table I I ) . Thus an i n s e c t l i k e Oncopeltus could acquire d i f f e r e n t (amounts of) glycosides from a l e a f eating species l i k e Tetraopes. The manner of sampling ( P o l l a r d , 1969) the plant t i s s u e w i l l also be a c r i t i c a l f a c t o r i n the a c q u i s i t i o n of cardenolides. The physiology of Tetraopes i s unknown. The presence of threshold l e v e l s of c e r t a i n or a l l glycosides i n the plant food may be a key f a c t o r , as well as r a t i o s of c e r t a i n glycoside components (mass e f f e c t or antagonism i n the process of sequestering). None of these f a c t o r s have been examined i n t h i s t h e s i s or i n Brower's work (1969). I t has been shown that various species of Asclepias do appear to have d i f f e r e n t glycosides i n t h e i r t i s s u e s (Table I I ) . Van Doesburg (1968) mentions that b i r d s i n Trinidad have been reported to feed upon Dysderous species and that cotton stainers- have been found i n t h e i r gut samples. Yet, Dysdercus has powerful scent glands containing a l k y l compounds l i k e hex-2-enol (Remold, 1963; Youdeowei et a l . , 1969; van Doesburg, 1968). Obviously before p a l a t a b i l i t y amongst various i n s e c t s r e l a t i v e to a group of predators can be defined some basic comparative work on the taste c a p a b i l i t i e s and drug tolerances of various (avian) predators has to be undertaken, and the natural predators of the insects must be ascertained. The lack of r e a c t i o n by toads and frogs to cardenolides demonstrates a diff e r e n c e to Brower's Jays (Brower et a l . , 1968). Not u n t i l 11.7 t h i s i s c a r r i e d out w i l l Brower's p a l a t a b i l i t y spectrum (Brower et a l . , 1968; Brower, 1969) and the Oncopeltus colouration become e c o l o g i c a l l y meaningful. The s i m i l a r shape and c o l o u r a t i o n of the various Coleoptera and Hemiptera on Asclepias suggest that they are involved i n a complex of e i t h e r Batesian or M u l l e r i a n mimics. This t h e s i s shows c l e a r l y that the mimicry cannot be based on the occurrence of cardiac glycosides i n a simple manner. Further i t shows that any mimicry complex based on cardiac glycosides i s complicated by the f a c t that many forms l i k e Oncopeltus possess stink glands as defense mechanisms. 118 SUMMARY 1 The Large Milkweed Bug, Oncopeltus f a s c i a t u s , a brightly-coloured a t y p i c a l Lygaeid, because of i t s association with Asclepiad plants, was suspected of containing cardiac glycosides derived from i t s food; these chemicals could provide the chemical basis f o r u n p a l a t a b i l i t y . 2 In order to study the pl a n t - i n s e c t system, techniques have been devised which permit the r a p i d i s o l a t i o n , p u r i f i c a t i o n , chromatography, and q u a n t i f i c a t i o n of cardiac glycosides from i n s e c t and seed t i s s u e . 3 C r i t i c a l p h y s i c a l , chemical, and b i o l o g i c a l evidence i s provided that a l l stages of development of Oncopeltus sequester cardiac glycosides from various t i s s u e s of Asclepiad p l a n t s . 4 Almost a l l the cardenolides sequestered by Oncopeltus are concentrated i n a new complex of previously undescribed d o r s o - l a t e r a l glands. 5 These glands sequester approximately 35 micrograms of cardenolide per m i c r o l i t e r of glandular f l u i d (111 micro-grams/adult animal); these glands show a marked preference f o r more polar glycosides. 6 Ouabain- H i s p r e f e r e n t i a l l y sequestered i n these glands (compared to digitoxin-^H) eith e r because of the 119 l i m i t a t i o n s of these d o r s o - l a t e r a l glands or because of some other p h y s i o l o g i c a l parameter such as the glycoside;'; c a r r i e r p r o t e ins. 7 The uptake of isotopes from a food source represents the uptake of these minimal amounts of i s o t o p i c glycoside i n the presence of much la r g e r concentrations of natural cardenolides, and because of t h i s these isotopes are susceptible to mass e f f e c t s . This could be a c r i t i c a l feature i n determining u n p a l a t a b i l i t y when low concentrations of an emetic glycoside must be sequestered i n the presence of large concentrations of l e s s or non-emetic cardenolides. 8 Eighteen species of Asclepias from diverse points about North America have been shown to contain cardenolides, which are a v a i l a b l e f o r feeding i n s e c t s . 9 Lygaeus k a l m i i k a l m i i and L.k. angustomarginatus, Oncopeltus sandarachatus, Tetraopes tetraophthalmus, T. oregonensis have been shown to contain cardiac glycosides. Theseabove b r i g h t l y coloured i n s e c t s also have "the chemical basis to be unpalatable l i k e 0. f a s c i a t u s and to function as a model or Batesiam mimic i n mimicry complexes continental i n s i z e . 10 Besides various Asclepiads forming a p a l a t a b i l i t y specrum, one plant can a f f o r d a range of p a l a t a b i l i t y , thus the above i n s e c t s could feed on one species but not have equal p a l a t a b i l i t i e s . 12 0 11 Mimicry complexes i n Hemiptera (based on cardenolides) are complicated by the f a c t that Oncopeltus has v o l a t i l e secretions. Many other Hemiptera l i k e Dysdercus which do not contain cardenolides, but have v o l a t i l e secretions, are. coincident with Oncopeltus throughout North America. 12 Predation studies with Oncopeltus have shown that i t i s not a model because of i t s glycoside content, but can be so because of i t s a l k y l compounds. Thus, the presence of cardenolides i s not synonymous with u n p a l a t a b i l i t y . 13 Mimicry complexes are not equivalent to a l l grades of predators, nor are the responses of a l l predators to cardenolides equivalent. 14 In the uptake of cardiac glycosides from a food source by Oncopeltus other f a c t o r s l i k e temporal association with the food, the s p e c i f i c a c t i v i t y and d i s t r i b u t i o n of glycosides within the food, mass e f f e c t s , and a b i l i t y to r e t a i n cardenolides independently of the presence of a continued source of glycosides i n the food, are also important f a c t o r s i n determining an insect's p a l a t a b i l i t y . 15 Mimicry complexes which have a chemical basis are not as simple as previously described. 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