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The biosynthesis of defensive chemicals by millipedes : parrallelism with plant biosynthetic pathways Duffey, Sean Stephen 1974

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I THE BIOSYNTHESIS OF DEFENSIVE CHEMICALS -BY MILLIPEDES: PARALLELISM WITH PLANT BIOSYNTHETIC PATHWAYS by SEAN STEPHEN DUFFEY ( Zool. ), University of B r i t i s h Columbia,1968 ( Zool. ), University of B r i t i s h Columbia,1970 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Botany We accept t h i s thesis as conforming to the required standards. B.Sc. M.Sc. THE UNIVERSITY OF BRITISH COLUMBIA October, 1974 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 a n 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 b e g r a n t e d b y t h e H e a d o f my D e p a r t m e n t o r b y 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 b e 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 . D e p a r t m e n t o f Botany  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 Date October 30, 1974 ABSTRACT The biogenesis of hydrogen cyanide. ( HCN ) and benzalde-hyde has been studied i n two polydesmid millipedes, Harpaphe haydeniana and Oxidus g r a c i l i s . I t was shown by feeding Oxidus . 1 4 g r a c i l i s D, L-phenylalanme- C l a b e l l e d i n the 1,2,3, or r i n g carbons that the r i n g and carbon-3 were u t i l i z e d to make benz-aldehyde, and carbon-2 to make the cyanide carbon. I t i s post-ulated that this occurs by the catabolism of phenylalanine to mandelonitrile. I t was found that Harpaphe haydeniana was also capable of 14 incorporating D,L-phenylalanme- . C s p e c i f i c a l l y i n t o HCN and benzaldehyde, as d i d O. g r a c i l i s . H. haydeniana was shown to store D-(R)-mandelonitrile as a droplet i n the storage v e s t i -bule of each cyanogenic gland. 14 3 Using C and H l a b e l l e d compounds i t was shown that H. haydeniana synthesizes HCN v i a a mechanism s i m i l a r to that of plants. The pathway i n the millipede appears to be: phenyl-alanine > N-hydroxyphenylalanine ( phenylpyruvic acid oxime ? ) phenylacetaldoxime phenylaceto-n i t r i l e and/or 2-hydroxyphenylacetaldoxime mandelonitrile. 14 From feeding experiments using D,L-phenylalanme-ring- C,_D,L-14 14 phenylalanine-2- C and p h e n y l a c e t o n i t r i l e - 1 - C i t was possible to show the natural occurrence of four intermediates known to occur i n plants; the oxime, the n i t r i l e , mandelonitrile, and a glycoside of mandelonitrile. Two enzymes have also been i s o l a t e d from H. haydeniana which are a part of t h i s biosynthetic pathway; the p-glycosidase and the a-hydroxynitrile lyase. Both enzymes are associated with the cyanogenic gland. The pH optima for the enzymes are 4.2 and 6.0 r e s p e c t i v e l y . The fate of HCN and benzaldehyde from decomposed mandelo-n i t r i l e was determined i n H. haydeniana. I t was found that 14 14 H CN was converted to (3-cyanoalanme- C and subsequently 14 asparagine- C, as i s known to occur i n plants. Benzaldehyde 14 . 14 C was converted to p-hydroxybenzoic a c i d - C. The t o x i c i t y of HCN to two polydesmid millipedes, one p a r a j u l i d millipede, one hemipteran, and one orthopteran was compared, and on a weight basis the millipedes were more r e s i s t a n t to HCN. The hypothesis for the mechanism of HCN production i n polydesmid millipedes i s appraised by new biochemical evidence. Employing biochemical and morphological data from t h i s thesis and from the accepted hypothesis, a more de t a i l e d chemical and physical explanation i s given for the production of HCN. The occurrence of HCN was demonstrated i n s i x other polydesmid millipedes; Boraria s t r i c t a , Caraibodesmus sp., Pseudopolydesmus branneri, Polydesmus angustus, Nearctodesmus  cerasinus, and Scytonotus insulanus. The nature of the ketone or aldehyde was only determined i n H. haydeniana. I t has been shown that Boraria s t r i c t a , Cherokia georgiana, Harpaphe hay- deniana, Oxidus g r a c i l i s , Polydesmus angustus, Nearctodesmus  cerasinus, and Scytonotus insulanus store a droplet of o i l i n each storage vestibule of the cyanogenic gland. This droplet was shown to be mandelonitrile i n H. haydeniana and O. g r a c i l i s . Three p-benzoquinones have been i d e n t i f i e d i n a Jamaican millipede, Rhinocricus holomelanus; benzoquinone, toluquinone, and 2-methyl-3-methoxy-benzoquinone. The biosynthesis of these chemicals appeared to be based on the catabolism of phenyl-14 . 1 4 . alanine- C and tyrosine- C. The aromatic r i n g of these ammo acids gave r i s e to the nucleus of the quinone. The presence of these three benzoquinones has been de-tected i n nine other millipedes of diverse f a m i l i e s ; Bollman-iulus sp., Eurhinocricus sp. nr. sabulosus, E. sp. nr. bruesi, i v Leptogoniulus naresi, Qrthoporus ornatus, Rhinocricus holo-melanus, Saiulus sp. nov. nr. s e t i f e r , Trigoniulus lumbricinus, and Tuniulus h e w i t t i . No chemotaxonomic value can be ascribed to the occurrence of these benzoquinones i n these species. V ACKNOWLEDGEMENTS Premier thanks are extended to Prof. G.H.N. Towers as a supervisor for his a i d i n choosing the topic of th i s t h e s i s . Much gratitude i s f e l t for h i s generous support from h i s Nat-ion a l Research Council grant. I also wish to thank him for much food and drink for both thought and pleasure. His academic and aesthetic i n t e r e s t i n millipedes has made t h i s thesis a reward-ing experience. His c r i t i c i s m s , questions and suggestions lead one h e u r i s t i c a l l y to v a l i d decisions. His friendship i s also appreciated. Several other people have been invaluable i n t h e i r w i l -lingness to discuss c e r t a i n aspects of t h i s thesis and other to p i c s : Prof. B.A.Bohm, Prof. G.G.E.Scudder, and Dr. J . Maze. To these people the author i s indebted. To the members of my committee I extend thanks for t h e i r c r i t i c a l examination of my thesis, namely; Prof. B.A.Bohm, Prof. K. Graham, Dr. J . Maze, Prof. G.G.E. Scudder, Dr. I.E.P. Taylor, and Prof. G.H.N. Towers. I am g r a t e f u l to Prof. K. Graham for his suggestion many years ago to persue the study of insects from the zoologic a l aspect. Without the expert advice of the following people much of t h i s thesis would have been impossible; Dr. R.J. Bose, Dept. of F i s h e r i e s , Goverment Canada, Vancouver; Dr. F. Curzon, Dept. of Physics, U.B.C.;Prof. D. Hayward, Dept. of Chemistry, U.B.C.; Dr. R.L. Hoffman, Dept. Biology, Radford College, Radford, Va., U.S.A.; Prof. N.L. Paddock, Dept. of Chemistry, U.B.C.; and Mr. L. Veto, Dept. of Botany, U.B.C. The generosity of Dr. E.W, Underh i l l and Dr. M. Chisholm i s appreciated for t h e i r donation of many i s o t o p i c precursors. v i Lastly, I wish to thank the H.R. MacMillan family for a graduate student fellowship. v i i TABLE OF CONTENTS Page ABSTRACT i i ACKNOWLEDGEMENTS v TABLE OF CONTENTS v i i LIST OF TABLES x LIST OF FIGURES x i PART I I- INTRODUCTION 1 I I - BIOLOGICAL ASPECTS 1 11.1- GENERAL CONSIDERATION OF MILLIPEDES 7 11.2- GENERAL CONSIDERATION OF HARPAPHE HAYDENIANA 8 I I I - MATERIALS AND METHODS 11 111.1- ANIMALS 11 111.2- ACKNOWLEDGEMENTS FOR MATERIALS AND METHODS 11 111.1.1— Isotopes 12 111.1.2- Isotope feeding experiments 12 111.3- ISOTOPIC MATERIALS 12 111.4- DETERMINATION OF THE SURFACE TENSION AND 15 VISCOSITY OF MANDELONITRILE 111.5- ADMINISTRATION OF LABELLED COMPOUNDS 16 111.6- HCN TOXICITY STUDIES 17 111.7- ISOLATION OF RADIOACTIVE NATURAL BIOSYNTHETIC 18 INTERMEDIATES 111.8- DETERMINATION OF AUTONOMY OF THE CYANOGENIC 20 GLAND 111.9- COLLECTION OF HCN AND BENZALDEHYDE 21 111.10- COLLECTION OF HCN AND BENZALDEHYDE IN 22 PRECURSOR FEEDING STUDIES 111.11- RADIOACTIVITY DETERMINATIONS 25 111.12- ISOLATION OF MANDELONITRILE AND MANDELIC 26 ACID III.13 CIRCULAR DICHROISM OF MANDELONITRILE AND 29 MANDELIC ACID v i i i 111.14- ISOLATION OF p-CYANOALANINE 31 111.15- ISOLATION OF p-HYDROXYBENZOIC ACID 31 111.16- ISOLATION OF CYANOGENIC GLYCOSIDES 32 111.17- EXTRACTION OF NITRILE LYASE AND p-GLYCO-SIDASE 33 111.18- ASSAY CONDITIONS FOR LYASE AND GLYCOSIDASE ACTIVITY 34 111.19- HISTOLOGY OF THE CYANOGENIC GLAND 35 111.20- ELECTRON-MICROGRAPHIC SCANNING OF HARPAPHE  HAYDENIANA 36 IV- RESULTS 36 IV.1- IDENTIFICATION OF BENZALDEHYDE AND HCN AND THEIR BIOSYNTHESIS IN OXIDUS GRACILIS 36 IV.2- CHARACTERIZATION OF HCN AND BENZALDEHYDE PRODUCED BY HARPAPHE HAYDENIANA 37 IV.3 THE IDENTIFICATION OF HCN IN OTHER POLY-DESMID MILLIPEDES 37 IV.4- MANDELONITRILE AS THE STORED CYANOGEN 40 14 IV.5- RESULTS OF FEEDING C-PRECURSORS TO WHOLE LIVE HARPAPHE HAYDENIANA 44 IV.6- ISOLATION OF INTERMEDIATES IN THE BIOSYN-THESIS OF HCN 51 IV.7- EXPERIMENTS TO DETERMINE THE AUTONOMY OF THE GLAND 53 IV.8- THE METABOLIC FATE OF H 1 4CN 54 14 IV.9- THE METABOLIC FATE OF BENZALDEHYDE- C AND BENZOIC ACID- 1 4C 55 IV.10- HCN TOXICITY STUDIES 57 IV.11. EVIDENCE FOR g-GLYCOSIDASE AND NITRILE LYASE ACTIVITY 57 IV.12- GROSS MORPHOLOGY AND MICROANATOMY OF THE CYANOGENIC GLAND 61 IV.13 LIGHT-MICROSCOPIC STUDIES OF THE CYANOGENIC GLAND 68 V - DISCUSSION 72 PART II P a g e VI - INTRODUCTION 102 VII- MATERIALS AND METHODS 105 V I I . l - CHEMICALS AND MATERIALS . 105 VII.2- DERIVATIVES OF VARIOUSLY SUBSTITUTED BENZOQUINONES 107 VII.3- EXTRACTION AND PURIFICATION OF MILLIPEDE QUINONES 109 14 VII.4- ADMINISTRATION OF C-PRECURSORS 110 VII. 5- COLLECTION OF MILLIPEDES 110 VIII- RESULTS 110 VIII. 1- THE STRUCTURAL IDENTIFICATION OF BENZO-QUINONES OF RHINOCRICUS HOLOMELANUS 110 VIII.2- THE IDENTIFICATION OF BENZOQUINONES IN OTHER MILLIPEDES 120 VIII.3- THE INCORPORATION OF 14C-PRECURSORS INTO THE BENZOQUINONES OF RHINOCRICUS HOLOMELANUS 120 IX- DISCUSSION -123 X- APPENDIX:PERSPECTIVE 126 X . l - INTRODUCTION 126 X.2- PHYSICAL ADAPTATIONS 127 X.3- BEHAVIOURAL ADAPTATIONS 130 X.4- CHEMICAL ADAPTATIONS 133 X.4.1- Chemical adaptations;physical 135 X.4.2- Chemical adaptations;behavioural 138 X.4.3- Chemical adaptations;pharmacological 140 X.5- ENVIRONMENTAL IMPACT OF CHEMICALS 149 X.6- DETOXICATION OF CHEMICALS 157 X.7- MORPHOLOGICAL ASPECTS ' 159 X.8- ORIGIN OF DEFENSIVE CHEMICALS 165 X.9- BIOSYNTHESIS OF DEFENSIVE CHEMICALS 172 XI- SUMMARY 172 XII- REFERENCES : ADDENDUM 196 X LIST OF TABLES TABLE page 1 4 I The incorporation of va r i o u s l y C-labelled phenylalanine i n t o HCN and benzaldehyde i n the defensive secretion of Oxidus g r a c i l i s . 3 8 I I The presence of HCN i n various polydesmid millipedes. 4 1 I I I Incorporation of l a b e l l e d compounds int o HCN and benzaldehyde of the millipede Harpaphe  haydeniana. 4 8 1 4 I V Data showing the incorporation of C-precursors into the natural intermediates i n the biosyn-thesis of HCN and benzaldehyde. 52 V The metabolic fate of HCN and benzaldehyde i n Harpaphe haydeniana. 56 V I Substrate r e a c t i v i t y and Q J Q data for (3-D-glyco-sidase and a-hydroxynitrile lyase a c t i v i t y i n Harpaphe haydeniana. 6 0 V I I The occurrence of simple benzoquinones i n arthro-pods. 1 0 5 V I I I Melting point data for some p-benzoquinones of arthropod o r i g i n . 1 1 2 I X Retention values of benzoquinones and derivatives of benzoquinones of Rhinocricus holomelanus compared to standards by gas l i q u i d chromat-ography. 1 1 4 X Spectroscopic data used to i d e n t i f y benzoquinones present i n Rhinocricus holomelanus. - ^ 5 X I The presence of benzoquinones i n various m i l l i -pedes. 1 2 1 1 4 X I I Incorporation of C-compounds int o benzoquinones of Rhinocricus holomelanus. 122 X I I I Some chemicals sequestered from plants by i n s e c t s . 1 6 7 x i LIST OF FIGURES FIGURE page IA The proposed biosynthetic pathway for cyanogens i n plants. 3 IB Examples of n a t u r a l l y occurring cyanogens and cyanogenic glycosides from plants and millipedes. 3 2 Proposed mechanism for the production of HCN and benzaldehyde i n polydesmid m i l l i -pedes. 5 3 The polydesmid millipede, Harpaphe haydeniana, and the external aspects of i t s cyanogenic glandular apparatus. 6 4 Diagram of the c o l l e c t i o n apparatus for HCN and benzaldehyde. 13 , 1 4 5 Combustion apparatus for c o l l e c t i o n of CC^ from 2,4-DNP d e r i v a t i v e s . 2 4 6 Biosynthetic pathway for the production of HCN and benzaldehyde from dietary phenyl-alanine i n Oxidus g r a c i l i s . 39 7 C i r c u l a r dichroism spectra of enantiomeric mandelic acids and of mandelonitrile from Harpaphe haydeniana. 45 8 The diagrammatic representation of the c h i r a l -i t y of L- and D-enantiomers of mandelonitrile-(3-O-D-glucoside and mandelonitrile. 46 9 The proposed biosynthetic sequence for the production of a cyanogen i n plants and mand-e l o n i t r i l e i n Harpaphe haydeniana leading to HCN production. 49 10 The metabolic fate of HCN and benzaldehyde r e s u l t i n g from the decomposition of mandelo-n i t r i l e i n Harpaphe haydeniana. 58 Graphical representation of the collapse of several insects and millipedes exposed to HCN vapours. The e f f e c t s of pH upon the a c t i v i t y of p-glycosidase and a-hydroxynitrile lyase from Harpaphe haydeniana. Scanning electronmicrographs of the phlange of Harpaphe haydeniana. Diagrammatic representation of the cyanogenic gland of Harpaphe haydeniana. Photographs of the cyanogenic glandular apparatus of Harpaphe haydeniana. Light microscopic analysis of the cyanogenic apparatus of Harpaphe haydeniana. Pathway for the biosynthesis of HCN and benzaldehyde i n Harpaphe haydeniana. Proposed morphological and biochemical model of the cyanogenic glandular apparatus of a polydesmid millipede, Harpaphe haydeniana. Proposed ph y s i c a l model for the production of HCN and benzaldehyde i n the polydesmid millipede Harpaphe haydeniana. Theoretical pathways for the biosynthesis of arthropod benzoquinones. Some n a t u r a l l y occurring quinones of plant, fungal, and animal o r i g i n . Analysis of mass spectrum data of benzo-quinones of Rhinocricus holomelanus. Some representative defensive chemicals i s o l a t e d from t e r r e s t r i a l arthropods. Diagrammatic representation of three types of defensive glands and t h e i r products. x i i i F I G U R E p a g e 25 Theoretical pathways for the biosynthesis of arthropod benzoquinones. 186 26 Theoretical pathways for phenolic metabol-ism i n in s e c t s . 189 THOUGHTS ON SCIENCE LEST WE BE VAIN. He had been eight years upon a project for extract-ing sunbeams out of cucumbers, which were to be put i n v i a l s hermetically sealed, and l e t out to warm the a i r i n raw inclement summers. He t o l d me that he d i d not doubt that i n eight years more he should be able to sup-ply the governor's gardens with sunshine at a reasonable rate; There was a man born b l i n d , who had several apprent-ices i n his own condition. Their employment was to mix colors for painters, which t h e i r master taught them to d i s t i n g u i s h by f e e l i n g and smelling. I t was indeed my misfortune to f i n d them a l l at that time not very perfect i n t h e i r lessons, and the professor himself happened to be generally mistaken. ....he showed me vast numbers of f l i e s most beauti-f u l l y colored, wherewith he fed h i s spiders, assuring us that the webs would take the t i n c t u r e from them; and as he had them of a l l hues, he hopes to f i t everybody's fancy, as soon as he could f i n d proper food f o r the f l i e s , of cer-t a i n gums, o i l s , and other glutinous matter, to give strength and consistence to the threads. i n A VOYAGE TO LAPUTA, from GULLIVER'S TRAVELS, by J . SWIFT. PART 1 1 I- INTRODUCTION This dissertation i s p r i m a r i l y concerned with the bio-, genesis of defensive chemicals i n mil l i p e d e s . This topic re-, presents one academic corner of a very broad f i e l d of study termed " Chemical Ecology ". This area considers the i n t e r -action of organisms at the chemical as well as b i o l o g i c a l l e v e l ( Sondheimer and Simeone, 1970; Whittaker and Feeny,1971), In order that the reader may r e l a t e the s p e c i a l topic of t h i s thesis to the general f i e l d of chemical ecology, namely defen-= sive mechanisms i n arthropods, a review i s presented i n the appendix of thi s t h e s i s . The main aim of the review was to discuss the functions and biogenesis of defensive chemicals i n arthropods. The biogenesis of plant cyanogens has been postulated to occur by a s.eries of reactions s t a r t i n g with the corres-ponding amino acid ( Eyjolfsson, 1970; Tapper et a l . , 1971,1972), This involves the conversion of the amino a c i d to the N^hydroxy= amino acid, and then po s s i b l y v i a the a-oximino ac i d before decarboxylation to the 1-oxime, and then v i a the 1 - n i t r i l e and/or the 2-hydroxy-oxime to the 2-hydroxy-nitrile. The n i t r i l e i s subsequently glucosylated to form the p-O-D-glucoside as outlined i n Figure IA. The cyanogenic glycosides are storage products i n some plants which release t o x i c HCN and an aldehyde or ketone a f t e r tissue i n j u r y . Some structures of n a t u r a l l y occurring plant cyanogens are i l l u s t r a t e d i n Figure l B f Benz-aldehyde i s a common product of cyanogenesis, and may be de-tected along with HCN i n almond, apple, plum, and peach p i t s , or the crushed leaves of the che r r y - l a u r e l ( Prunus laurocer-asus L. ). 2 The production of HCN by polydesmid millipedes has been known for approximately ninety years ( Guildensteeden-Egeling, 1882; Wheeler, 1890 ). This process has been reported to occur i n many millipedes for example, Pachydesmus crassicatus ( Blum and Woodring, 1962 ), Apheloria corrugata, Pseudopolydesmus  serratus, Cherokia georgiana, Oxidus g r a c i l i s , and a Nannaria sp. ( Eisner H. et a l . , 1963 ), Polydesmus c o l l a r i s c o l l a r i s ( Casnati et a l . , 1963 ), Gomphodesmus pavani ( Barbetta et a l . , 1 9 6 6 ) and Polydesmus vicinus ( Pallares, 1946 ). HCN i s also produced by a v a r i e t y of unrelated arthropods; by a geophilid centipede Pachymerium ferrugineum ( Schildknecht et a l . , 1968a), by a chrysomelid beetle Paropsis atomaria ( Moore, 1967 ), and by the zygaenid moths Zygaena f i l i p e n d u l a e and Procris geryon ( Jones et a l . , 1962 ). The hypothesis that millipedes produce HCN i n a manner p a r a l l e l to that of plants has several observations i n i t s favour. I t i s known ( 1 ) that benzaldehyde, i n most cases, accompanies the formation of HCN. I t i s known ( 2 ) that m i l l i -pedes contain mandelonitrile ( Barbetta et a l . , 1963 ), and ( 3 ) that they contain glucosides of the n i t r i l e ( p-isopropyl-mandelonitrile glucoside, Figure IB; P a l l a r e s , 1 9 4 6 ) . However, the remaining evidence for the existence of e i t h e r mandelo-n i t r i l e or i t s glycoside i n other millipedes i s only chromato-graphic ( Blum and Woodring, 1962; Eisner H. et a l . , 1963; Eisner et a l . , 1963a; Moore, 1967;Woodring and Blum, 1 9 6 3 ) . None of these authors have c l e a r l y indicated i n which form the millipede stores the cyanogen p r i o r to release of HCN. In order to understand completely the degree of parallelism between plants and millipedes i t i s e s s e n t i a l to know whether the n i t r i l e and/or the glycoside or some other product i s the storage form i n millipedes. FIGURE IB. Examples of naturally occurring cyanogens glycosides from plants and millipedes. and cyanogenic * ** * Cyanogen Amino Acid Family (Ref. ) Triglochinin L-3,4-dihydroxyphenylalanine (446) Juncaginaceae 172 Sambunigrin L-Phenylalanine Caprifoliaceae 61 . Taxiphyllin L-Tyrosine (35) Taxaceae 469 Gynocardin L-2-Cyclopentene-l-glycine Flacourtiaceae 469 Linimarin L-Valine (87) Leguminosae 256 A cyanolipid Polydesmus glycoside L-2-Amino-3, 5^dihydroxy-4-methylene-valenc acid L-p-Isopropylphenylalanine Sapindaceae Polydesmidae 330 364 Millipede cyanogen L-Phenylalanine Polydesmidae 96 Millipede glycoside L-Phenylalanine Xystodesmidae 153,55 * For comprehensive reviews of cyanogen occurrence and biosynthesis refer to Conn, 1974,and Eyjolfsson, 1970; the taxonomic distribution here is not comprehensive. ** Amino acid from which cyanogen is ( supposedly ) derived; reference to tracer evidence is given in brackets. FIGURE 5 A . The proposed biosynthetic pathway for cyanogens i n plants. /COOH .C-C R?' NNH2 Amino acid fts V /COOH .C-C R sNHOH • ^ c 'H Rf' *N0H N-Hydroxy amino ac id Aldoxime H R''C S Nitrile \ R" PH  N r - r RT *NOH rf-Hydroxyateloxime H> O H <»-Hydroxynitrite 4 D » O - G l c Glycoside CEN HOCH2 HC-0 / ° *\ T r i g l o c h i n i n • S a m b u n i g r i n T a x i p h y l l i n OH C y n o c a r d i n HOCH2 CEN -C-CH, HOX { ^ 3 OH L l n i m a r i n 0 CH ( C 2 ) a c i d } - C 0 - C H - C - C H - C H N I 0 I ( C a a c i d ) a C y a n o l i p i d H0CH2 H O C s N ° ^ .OHC CH 3 H C ' V C H 3 l>olyde«ras glycoside 0 C=N O H C K i l l i p e d e c y a n o g e n I O H C G l y c ' K i l l i p e d e g l y c o s i d e FIGURE- 5 B . Examples of n a t u r a l l y occurring cyanogens and cyanogenic glycosides from plants and millipedes. 4 A model of HCN production i n a millipede has been pro-posed by Eisner and Meinwald ( 1966 )( see Figure 2 ). This model postulates that mandelonitrile i s the storage form of HCN; whereas, Woodring and Blum ( 1962 ) suggested that the glycoside might be the storage product. Neither of these two arguments are conclusive, nor have they been subjected to biochemical scrutiny. This thesis examines the v a l i d i t y of the model for HCN production ( Figure 2 ), as well as exam-in i n g the hypothesis that a millipede, Harpaphe haydeniana, biosynthesizes HCN and benzaldehyde i n a manner s i m i l a r to green plants. The study of cyanogenesis i n the polydesmid millipede Harpaphe haydeniana ( Figure 3 ) i n t h i s thesis represents a consideration of comparative biochemistry i n r e l a t i o n to form and function. Thus, from biochemical, anatomical, and p h y s i c a l data an elaborated model of HCN production i s proposed. m u s c l e ( INSIDE OF ANIMAL ) secretory c e l l s CN HOH mandelonitrile ( OUTSIDE OF ANIMAL ) benzaldehyde STORAGE ( FLEXIBLE SAC ) REACTION ( RIGID CHAMBER ) FIGURE 2. Proposed mechanism for the production of HCN and benzaldehyde i n polydesmid millipedes. ( according to Eisner and Meinwald, 1966) FIGURE 3. The polydesmid millipede, Harpaphe haydeniana, and the external aspects of i t s cyanogenic glandular apparatus. Frame A: A l a t e r a l view showing the p o s i t i o n of the pores of the gland through which HCN e x i t s . The pores (p) are shown. Frame B: A l a t e r a l view showing one of the b i l a t e r a l phlanges. The cyanogenic glands are situated beneath the yellow patches. The animal i s approx-imately 40 mm. x 6 mm. Frame C; A cross-sectional view i n d i c a t i n g the the r e l a t i v e s i z e of the phlange to that of the body. 6b 7 I I - BIOLOGICAL ASPECTS II.1- GENERAL CONSIDERATIONS OF MILLIPEDES Cloudsley-Thompson ( 1968 ) gives an excellent discussion of the biology of millipedes. Most millipedes are c i r c u l a r i n cross-section with elongated bodies composed of 7-many segments. The shape of the body and of the vaulted diplosegment i s an evolutionary adaptation for a burrowing s t y l e of l i f e ( Manton, 1954 ). The a b i l i t y to burrow through l e a f layers and r o t t i n g logs i s helped by the strongly pushing leg system. The pushing force i s achieved by a metachronal g a i t i n which the backstroke i s of a longer duration than the forward stroke. Rather than f l e e i n g to escape danger most millipedes c u r l i n t o a s p i r a l with the head at the centre of the s p i r a l . This means of defence i s e f f e c t i v e because many millipedes have hard s l i p p e r y c u t i c l e s which can make grasping d i f f i c u l t . The hard c u t i c l e also serves to d e f l e c t p o t e n t i a l l y penetrating b i r d beaks ( Eisner, 1970, 1968, 1967 ). This c u t i c u l a r strength i s more pec u l i a r t o . s p i r o b o l i d and j u l i d type millipedes, and not often to the more f r a g i l e polydesmid forms. The toughness of the c u t i c l e i s obtained not only by c a l c i f i c a t i o n of the c u t i c l e ( Cloudsley-Thompson, 1968; Krishnan, 1968 ), but also by tanning. The presence of calcium ions i n forest l e a f l i t t e r has been shown to make leaves more a t t r a c t i v e as a food for some millipedes. I t has also been suggested that the a v a i l -a b i l i t y of calcium ions to millipedes might be an important factor i n determining the permeability of the integument to water ( Cloudsley-Thompson, 1968; Lydford, 1943 ). Millipedes are very susceptible to desiccation and gen-e r a l l y are found i n an environment with very high humidity ( Cloudsley-Thompson, 1968; Crawford, 1972; Toye, 1966a,b,c ) / although the c u t i c l e with i t s thick l i p i d layer provides some r e s i s t a n c e t o w a t e r l o s s . T h e h i g h h u m i d i t y r e q u i r e m e n t i s m e t b y s e v e r a l b e h a v i o u r a l f e a t u r e s ; o n e i s t h e i r r e s i d e n c e i n m o i s t l i t t e r o f f o r e s t f l o o r s . A n o t h e r i s t h a t m i l l i p e d e s t e n d t o b e n e g a t i v e l y p h o t o t a c t i c a n d n o c t u r n a l . M i l l i p e d e s p r e f e r t o e a t s o f t , o f t e n r o t t i n g p l a n t m a t -e r i a l ; t h e i r w e a k m o u t h p a r t s l i m i t t h e n a t u r e o f t h e i r d i e t . I n a d d i t i o n t o a p r e f e r e n c e f o r c a l c i u m i o n s , m i l l i p e d e s , s u c h a s O x i d u s g r a c i l i s a r e k n o w n t o h a v e p r e f e r e n c e s f o r s u c r o s e a n d g l u c o s e ( C l o u d s l e y - T h o m p s o n , 1968 ) . S o m e m i l l i p e d e s c a n b e c o m e s e r i o u s p e s t s o f c r o p s l i k e s u g a r b e e t s . T h e s p o t t e d s n a k e m i l l i p e d e , B l a n i u l u s g u t t u l a t u s , a n d O x i d u s g r a c i l i s a r e o f t e n p e s t s o f g r e e n h o u s e s . T h e r o l e o f m i l l i p e d e s i n a f o r e s t e n v i r o n m e n t i s p r o b a b l y e q u i v a l e n t t o t h a t o f w o r m s , f o r i n t h e f i e l d i t i s n o t e d t h a t v a s t a m o u n t s o f p l a n t l i t t e r c a n b e r e -d u c e d t o f e c a l p e l l e t s . M i l l i p e d e s , d e s p i t e t h e i r l i b e r a t i o n o f H C N o r q u i n o n e s a s s u p p o s e d d e t e r r e n t s t o p r e d a t o r s ( E i s n e r a n d E i s n e r , 1965; W o o d r i n g a n d B l u m , 1963 ) , a r e e a t e n b y a n t s , f i s h , t o a d s , r e p t i l e s , b i r d s a n d m a m m a l s . O f t h e s e t h e t o a d s a n d s t a r l i n g s a r e t h e m o s t i n d u l g e n t . T h e A m e r i c a n t o a d , B u f o l e n t i g i n o s u s , h a s b e e n c i t e d t o i n c l u d e m i l l i p e d e s t o t h e e x t e n t o f 10% i n i t s d i e t ( C l o u d s l e y - T h o m p s o n , 1968 ). I I . 2 - G E N E R A L C O N S I D E R A T I O N S O F H A R P A P H E H A Y D E N I A N A T h e g e n u s H a r p a p h e i s c o m p r i s e d o f 9 p r o v i s i o n a l s p e c i e s ( C h a m b e r l i n a n d H o f f m a n , 1958 ) , w h i c h a r e l o c a t e d o n t h e w e s t c o a s t o f N o r t h A m e r i c a f r o m C a l i f o r n i a t o B r i t i s h C o l u m b i a . H a r p a p h e h a y d e n i a n a a p p e a r s t o b e d i s t r i b u t e d i n O r e g o n , W a s h -i n g t o n , a n d B r i t i s h C o l u m b i a . H a r p a p h e h a y d e n i a n a i s a b r i g h t l y c o l o u r e d m i l l i p e d e 9 with black glossy c u t i c l e highly contrasting with the b r i l -l i a n t ye'llow protruding b i l a t e r a l phlanges ( Figure 3 ) . The defensive glands are situated on the metazonites of segments V /VII #IX /X /XIII /XV /XVI /XVII / and XVIII. Large adults have approximately 20 segemnts and a t t a i n a s i z e of 50 mm. x 7 mm. The yellow colouration of the phfenge, beneath which l i e s the cyanogenic gland, i s replaced by red pigmentation i n approx-imately 0.5 % of the population. No other colour phenotypes were observed. The anterior of the millipede shown i n Figure 3B i s on the r i g h t . A close-up of the phlange ( Figure 3A ) reveals that the small pore ( p ) of the cyanogenic gland i s approximately 50 u i n diameter. The o r i f i c e of the pore protrudes as a c o l l a r into a shallow p i t approximately 200 u i n diameter. The extent of the protrusion of the phlanges ( keels or paranota ) i s shown i n Figure 3C where the p o s i t i o n of the pore ( p ) i s indicated, as well as the approximate i n t e r n a l p o s i t i o n of the gland. This segment ( Figure 3C ) shows the male g e n i t a l i a positioned v e n t r a l l y on the prozonite of the segment. Young millipedes and adults of both sexes secrete HCN and benzaldehyde. During the release of these two chemicals f l u i d can be seen to exude from the pores and remain for a short time within the p i t just outside the c o l l a r . This containment gives a slow release of HCN i n addition to the i n i t i a l burst. Extensive manual stimulation of the millipede depletes the store of defensive secretion. The millipedes examined i n t h i s thesis were c o l l e c t e d p r imarily on the Endowment Lands of the University of B r i t i s h Columbia. This s i t e i s a mixed conifer-deciduous forest bord-ering the b l u f f s of Point Grey. The common plants on t h i s s i t e were Alnus rubra Pursh, Sambueus racemosa var. arborescens 10 ( T. & G. ) Gray, Pseudotsuga menziesii var. menziesii ( Mirbel ) Franco., Thuja p l i c a t a Bonn., Rubus spp., U r t i c a d i o i c a L., Dicentra formosa ( Andr. )Walp., and Polystichum spp.. These plants provided a r i c h l e a f l i t t e r with numerous decaying branches. The millipedes both i n c a p t i v i t y and i n s i t u fed on t h i s decaying plant debris. In the spring and summer they wandered over and through t h i s debris, p a r t i c u l a r i l y i n locations where the deciduous l e a f l i t t e r was abundant compared to coniferous l i t t e r . In the f a l l they receded to lower layers of the l i t t e r and the top few inches of s o i l . Even when the top 3 inches of s o i l was frozen the millipedes were found only several inches below the frozen s o i l . In t h i s cold they were s t i l l somewhat active f o r when disturbed they c o i l e d i n defence. I f brought into the lab they revived within a few hours, and could produce HCN copiously. During the winter females were gravid, and the eggs were probably l a i d i n the spring since at that time the greatest number of larvae were seen. In mid-summer large numbers of Harpaphe haydeniana were ava i l a b l e . I t was estimated from c o l l e c t i o n s that about 2000 individu a l s populated approximately one acre. Several other species of millipedes were present i n t h i s s i t e . Three polydes-mids, Polydesmus angustus Lantzel, Nearctodesmus cerasinus ( Wood ), Scytonotus insulanus Attems,and three species of p a r a j u l i d millipedes, Tuniulus hewitti ( Chamberlin ), B o l l -maniulus sp. , and Sailus sp. nov. nr. s e t i f e r Chamberlin. The above millipedes occurred approximately i n the following r a t i o s respectively; 100:10:1:5,000:1:100. Other arthropods abundant i n the l e a f l i t t e r were several species of t e r r e s t r i a l isopods; Oniscus asellus L., P o r c e l l i o  scaber L a t r e i l l e , and Armadillidium vulgare ( L a t r e i l l e ); several u n i d e n t i f i e d species of beetles, many apterygote insects 11 and many arachnids , one species of which clung to the e x t e r i o r of Harpaphe haydeniana. The Western Red-backed salamander, Plethedon vehiculum ( Cooper ) i s common i n the l e a f l i t t e r of southern B r i t i s h Columbia. Two such animals i n c a p t i v i t y , eager to attack meal worm larvae rejected small Harpaphe haydeniana. I t i s assumed that the salamanders had learned to r e j e c t t h i s millipede i n the f i e l d . I l l - MATERIALS AND METHODS 111.1- ANIMALS The millipedes were c o l l e c t e d during the summer and early f a l l and stored i n large wooden screen-topped cages ( 1.5 x 0.5 x 0.02 m. ) f i l l e d p a r t i a l l y with moist peat moss and l e a f l i t t e r . This treatment permitted the animals to l i v e for several months, although they were seldom kept for more than 2 weeks before use. The cages were maintained at room temp-erature and l i g h t regime. A translucent p l a s t i c top was kept over the cages to maintain a high humidity. For the radioactive feeding experiments animals were placed, a f t e r i n j e c t i o n of ~ the isotope, into c i r c u l a r p l a s t i c containers ( 10 x 30 cm. ) with 2 cm. of moist peat moss and several large decaying leaves. During the 24 hour period a f t e r i n j e c t i o n they were placed under a low i n t e n s i t y fluorescent l i g h t . A translucent p l a s t i c • l i d confined the millipedes. 111.2- ACKNOWLEDGEMENTS FOR MATERIALS AND METHODS The author must d i s t i n g u i s h between his work and that >v of others. The contribution of others i s stated below. 12 III.2.1- Isotopes 14 The following C-labelled compounds were provided by Dr. E.W. Underhill and Dr. M. Chisholm; D,L- N-hydroxyphenyl-14 . . . 14 alanine-2- C, phenylpyruvic acid oxime-2- C, phenylacetald-14 14 oxime-1- C, phenylacetamide-1- C, phenylacethydroxamic a c i d -14 . 1 4 1- C and phenylethylamine-1- C. The author synthesized a l l the unlabelled chemicals and the following i s o t o p i c compounds; 14 . 3 phenylacetonitrile-1- C and 2-hydroxyphenylacetaldoxime-2a- H. III.2.2- Isotope feeding experiments Professor G.H.N. Towers fed the l a b e l l e d amino acids to Oxidus g r a c i l i s , and i s o l a t e d both HCN and benzaldehyde. I analyzed the samples for r a d i o a c t i v i t y . III.3- ISOTOPIC MATERIALS 14 D,L-phenylalanine, l a b e l l e d with C e i t h e r i n the r i n g , carbon-1, carbon-2, or carbon-3 of the side chain, was obtained from New England Nuclear Co. D i l u t i o n s of the isotope to lower s p e c i f i c a c t i v i t i e s were with L-phenylalanine. 14 D,L-N-Hydroxyphenylalanine-2- C was prepared from D,L-14 phenylalanine-2- C as described by K i n d l and U n d e r h i l l ( 1968b ), 14 Phenylpyruvic acid oxime-2- C was synthesized as described by Barry and Hartung ( 1946 ). Unlabelled phenylpyruvic acid oxime was made on a larger scale by t r e a t i n g the sodium s a l t of phenylpyruvic acid i n ethanol with sodium acetate and hydroxyl-amine hydrochloride ( Vogel, 1967 ). The reaction mixture was d i l u t e d with i c e cold water, s l i g h t l y a c i d i f i e d , and extracted with d i e t h y l ether. The ether extract was dried with anhydrous Na2SO^, and the sample taken to dryness i n vacuo. The product was r e c r y s t a l l i z e d twice from petroleum ether ( pet. ether ) ( b.r. 65-110°C. ):ether ( 2:1, v/v ), and washed with pet. ether 13 ( b.r. 30-60°C. ). M.P.= 165-166 °C. ( L i t . 166-167°C, syn-isomer ). 14 Phenylacetaldoxime-1- C was prepared from L-phenyl-14 alanine-2- C ( Tapper and Butler, 1971 ) and unlabelled phenylacetaldoxime was synthesized on a larger scale from phenylacetaldehyde treated with hydroxylamine hydrochloride ( Vogel, 1967 ). The oxime was c r y s t a l l i z e d from d i e t h y l ether: n-hexane ( 1:5, v/v ) twice, washed with pet. ether ( b.r.30-60°C. )and r e c r y s t a l l i z e d from toluene: pet. ether ( b.r. 60-110°C. )( 1:2, v/v ). M.P.= 94-96 ° C , L i t . 96-98°C. . 3 2-Hydroxyphenylacetaldoxime-2-a- H was prepared from phenylglyaldoxime as outlined by Tapper et a l . ( 1972 ). P r i o r to use phenylglyaldoxime was c r y s t a l l i z e d from pet. ether (b.r.60-110°C): ether ( 10:1, v/v ) and then chromatographed repeatedly on S i l i c a gel G t h i n layer plates with benzene:methanol ( 20;3, v/v )( Tapper et a l . , 1972 ). A f t e r c r y s t a l l i z a t i o n from toluene: pet. ether ( b.r. 60-110°C. )( 1:20, v/v ) , the t r i t i a t e d 2-hydroxy-oxime was p u r i f i e d by the above chromatographic system. Exchangeability of t r i t i u m was tested ( Tapper et a l . , 3 3 1972 ) to ascertain the r a t i o of 2a- H/ 2-0 H. To furthur v e r i -3 . . fy the presence of 2a- H an aliquot of the l a b e l l e d oxime, d i s -solved i n ethanol, was added to an excess of o x i d i z i n g s o l u t i o n ( K 2Cr 20 7:H 2S0 4:H 20- 5:100:400, w/v/v ) containing an excess of 2,4-dini trophenylhydrazine. A f t e r standing for 1 hour the 2,4-dinitrophenylhydrazone was i s o l a t e d and combusted as described below. Unlabelled phenylacetaldoxime was prepared i n a s i m i l a r manner. M.P.= 90°C, Lit.,89-90°C. 14 14 Phenylacetonitrile-1- C was prepared from K CN ( New England Nuclear ) and f r e s h l y d i s t i l l e d benzaldehyde as d e s c r i -bed by Tapper and Butler ( 1971 ). I t was extracted from the reaction mixture with methylene chloride, and p u r i f i e d on a 14 S i l i c a g el G thin layer plate developed with benzene:methanol ( 20:3, v/v )( Rf= 0.65 ). The phenylacetontrile was eluted from the plate with acetone and the solvent removed i n vacuo at 25 °C. I t was stored i n 0.5 ml. of methylene chloride over anhydrous Na^O^ at -20°C. When preparing the i n j e c t i o n f l u i d the solvent was allowed to evaporate before d i l u t i o n with the i n j e c t i o n solvent. Unlabelled compound was made s i m i l a r l y , Unlabelled phenylacetamide ( A l d r i c h Chemicals ) was p u r i f i e d by two washings i n large volumes of 5 % aqueous ethan-o l for three hours. The compound was c r y s t a l l i z e d from aqueous acetone. M.P= 153-155°C, L i t . 157°C. Unlabelled phenylacethydroxamic ac i d was prepared by treatment of ethylphenylacetate ( Gilman et a l . , 1951 ), with hydroxylamine hydrochloride as outlined by Renfrow and Hauser ( 1937 ). After standing for four days the reaction mixture was di l u t e d with an excess of a c i d i f i e d H20, and the so l u t i o n ex-tracted with d i e t h y l ether. The ether f r a c t i o n was dried with anhydrous Na2S0^, and taken to dryness i n vacuo. The c r y s t a l s were dissolved i n a minimal volume of d i e t h y l ether and c r y s t -a l l i z e d by the addition of pet. ether ( b.r= 65-110°C.). M.P= 142-145°C. Unlabelled phenylethylamine hydrochloride was produced by d i s t i l l i n g 2-phenylethylamine ( B.P.= 198°C ), d i s s o l v i n g the amine i n acetone, and adding dropwise an excess of concen-trated HCl. The c r y s t a l s were f i l t e r e d and washed with acetone and d i e t h y l ether. M.P= 215°C, Lit.214°C. The synthesis of racemic mandelonitrile was c a r r i e d out according to Tapper and Butler ( 1971 ) using a K^WPO^ buffer, f r e s h l y d i s t i l l e d benzaldehyde, and an excess of KCN. Af t e r a reaction time of 1 hour at 0°C. the n i t r i l e was allowed 1 5 t o s e p a r a t e f r o m t h e a q u e o u s p h a s e . I t w a s d e c a n t e d , d i s s o l v e d i n d i e t h y l e t h e r : p e t . e t h e r ( b . r . = 3 0 - 6 0 ° C . ) ( 3 : 1 , v / v ) a n d w a s h e d s e v e r a l t i m e s w i t h e q u a l v o l u m e s o f b u f f e r . T h e o r g a n i c p h a s e w a s t h e n d r i e d w i t h a n h y d r o u s N a 2 S 0 4 , a n d t h e s o l v e n t r e m o v e d i n v a c u o a t 4 0 ° C . T h e o i l w a s t h e n w a s h e d f o u r t i m e s w i t h e q u a l v o l u m e s o f p e t . e t h e r ( b . r . 6 0 - 1 1 0 ° C . ) . T h e w a s h -i n g s w e r e d i s c a r d e d , a n d t h e n i t r i l e f r e e d o f s o l v e n t i n v a c u o . T h e n i t r i l e w a s k e p t u n d e r r e d u c e d p r e s s u r e f o r 1 h o u r t o r e -m o v e b e n z a l d e h y d e . T h e n i t r i l e w a s s t o r e d i n a s c r e w - c a p p e d c e n t r i f u g a t i o n t u b e o v e r a n h y d r o u s N a 2 S 0 4 a n d u n d e r N 2 a t - 2 0 ° C . I t w a s c e n t r i f u g e d b e f o r e u s e i f t u r b i d . T h e a b o v e p r o c e d u r e w a s m o d i f i e d t o p r e p a r e p - h y d r o x y -m a n d e l o n i t r i l e . p - H y d r o x y b e n z a l d e h y d e w a s p u r i f i e d b e f o r e u s e b y d e c o l o u r i z a t i o n w i t h c h a r c o a l i n h o t x y l e n e , a n d r e c r y s t a l l i z a t i o n s e v e r a l t i m e s f r o m e t h a n o l b y t h e a d d i t i o n o f x y l e n e ( a p p r o x . 1 : 5 , v / v ) . T h e n i t r i l e w a s a f t e r w a r d s t r e a t e d a s d e s c r i b e d f o r m a n d e l o n i t r i l e . I I I . 4 - D E T E R M I N A T I O N O F T H E S U R F A C E T E N S I O N A N D V I S C O S I T Y OF M A N D E L O N I T R I L E . • F o r t h e s y n t h e s i s o f m a n d e l o n i t r i l e , b e n z a l d e h y d e w a s f i r s t p u r i f i e d b y m e a n s o f a b i s u l p h i t e c o m p l e x ( V o g e l , 1 9 6 7 ) ; t h i s p r o c e d u r e p e r m i t t e d t h e s y n t h e s i s o f a p u r e r n i t r i l e . O t h e r -w i s e t h e m a n d e l o n i t r i l e w a s s y n t h e s i z e d a n d t r e a t e d a s a b o v e p r i o r t o p h y s i c a l d a t a d e t e r m i n a t i o n s . T h e s u r f a c e t e n s i o n o f m a n d e l o n i t r i l e w a s d e t e r m i n e d b y m e a s u r i n g t h e p r e s s u r e r e q u i r e d t o f o r m a n d r e l e a s e a b u b b l e o f a i r f r o m a s u b m e r g e d s m a l l p o r e o f k n o w n d i a m e t e r ( S t o k e s ' m e t h o d ) . T h e v i s c o s i t y o f m a n d e l o n i t r i l e w a s d e t e r m i n e d w i t h a C a n n o n - F e n s k e v i s c o m e t e r , a n d t h e c e n t i p o i s e v a l u e d e t e r m i n e d b y c o m p a r i s o n o f t h e f l o w r a t e o f t h e n i t r i l e p e r t i m e u n i t w i t h t h a t o f w a t e r ( a k n o w n p a r a m e t e r ) . 16 III.5- THE ADMINISTRATION OF LABELLED COMPOUNDS . 1 4 Oxidus g r a c i l i s were given C-precursors i n four p e t r i dishes ( 9.5 x 1.5 cm. ) each containing a disk of Whatman No.l f i l t e r paper ( 4.25 cm. diameter ). On each disk 1.7 pCi of 14 C-precursor m o . l ml. of O.IN HCl along with 0.2 ml. of 2% sucrose. Both males and females were placed i n the p e t r i dishes which were covered with paper to give a d i f f u s e l i g h t . The f i l t e r paper was kept moist by the addition of water. The millipedes began feeding almost as soon as they contacted the sweetened paper. The experiments were terminated a f t e r 45-50 hours. With Harpaphe haydeniana an i n j e c t i o n technique was used . . 14 to administer the C-precursors. The tolerance l e v e l of each precursor i n vivo was determined. Phenylacetaldoxime and phenyl-a c e t o n i t r i l e were found to be the most to x i c to the millipe d e s . The i n j e c t i o n of 0.30 umoles of each compound i n 2 p i . of P.W.A. ( polyethylene g l y c o l : H 20: acetone- 2:2:1, v/v/v ) was non-to x i c . Stock i n j e c t i o n solutions were made by adding 14 the molar equivalent of 2 mgm. of phenylalanine of each C-precursor i n 100 p i . of P.W.A., unless otherwise s t i p u l a t e d . S o l u b i l i t y problems with phenylalanine required that i t be d i s -solved i n 200 p i . of P.W.A.. This solvent d i d not i n t e r f e r e with spectrophotometry determinations of s p e c i f i c a c t i v i t y . To i n j e c t the precursors, the animals were bent gently between the fore-finger and the thumb and pierced through a dorsal i n t e r s c l e r i t e with a 10 p i . syringe. Two p i . of each stock s o l u t i o n wexe inj e c t e d i n t o each animal ( 10 animals per experiment ). 14 The administration of H CN to Harpaphe haydeniana was car r i e d out i n a 50 ml. beaker sealed with a parafilm sheet. 17 14 . The K CN was placed i n a small p l a s t i c v i a l l i d suspended 14 at the top of the jar by adhesive tape. The H CN was released by the i n j e c t i o n of 0.5 ml. of 6N HCl i n t o the cap. The hole i n the parafilm was sealed with adhesive tape. The animals 14 were permitted to metabolize the H CN for 30 minutes before extraction of p-cyanoalanine. An a d d i t i o n a l feeding experiment 14 with H CN was c a r r i e d out wherein the millipedes were exposed to the gas for 45 minutes, then placed i n fresh a i r and allowed 14 to metabolize the H CN for a further 2 hours before extraction. 14 . . Benzaldehyde-carbonyl- C was administered to the m i l l i -14 pedes by placing them i n a 50 ml. beaker, applying the C-benzaldehyde to the upper walls of the beaker, and sea l i n g i t with parafilm. The animals were subjected to the fumes f o r 30 minutes, given a further 2 hours metabolic i n t e r v a l , and then extracted for phenolic and aromatic acids. 14 14 Benzoic a c i d - carboxyl- C and benzoic a c i d - r i n g - C were injected i n t o the millipedes as were the other precursors. After a metabolic period of several hours the phenolic and aromatic acids were extracted. III.6- HCN TOXICITY STUDIES A q u a l i t a t i v e t e s t to determine the t o x i c i t y of HCN to Harpaphe haydeniana, Polydesmus angustus, Tuniulus h e w i t t i , Oncopeltus fasciatus, and Schistocerca gregaria ph. gregaria was c a r r i e d out. Known quantities of HCN were generated,as 14 described i n the K CN experiments; the amount of gas generated was assumed to 100 % of the equivalent of KCN i n the gas chamber. Large beakers were used ( 500 ml. ) to avoid the reduction of HCN concentration by absorption i n the animals. Three animals of a kind were used for each HON concentration l e v e l . The ef-fectiveness of the gas as a toxicant was measured by counting 18 the elapsed time from exposure to the vapours u n t i l the time of collapse. Collapse was defined a r b i t r a r i l y as the loss of mobility before a 20 minute time l i m i t . Lack of e f f e c t i n t h i s time counted as a negative r e s u l t . In some cases p r o s t r a t i o n was observed rather than convulsions. III.7- ISOLATION OF RADIOACTIVE NATURAL BIOSYNTHETIC INTERMEDIATES  Experiments were designed to detect p h e n y l a c e t o n i t r i l e and phenylacetaldoxime i n Harpahe haydeniana. This was done . . . . . . . . 14 by i n j e c t i n g the millipedes with D,L-phenylalanine-ring- C, and extracting 2 millipedes at 2,4,6,8,10 and 24 hours a f t e r i n j e c t i o n . The 12 millipedes were extracted together i n b o i l i n g ethanol, the extract f i l t e r e d through a pad of C e l i t e , the f i l t r a t e d i l u t e d with an equal volume of water and washed with pet. ether (b.r. 60-110°C. ). The aqueous ethanol was then d i l u t e d further with water, a c i d i f i e d with HCl, and a f t e r the addition of 2 mgm. each of phenylacetaldoxime and phenylaceto-n i t r i l e extracted with d i e t h y l ether. The ethereal f r a c t i o n was dried with anhydrous Na2SO^, f i l t e r e d , and taken to dryness i n vacuo at 40°C. The aqueous ethanolic f r a c t i o n was neutralized with NaOH and added to other extracts i n an attempt to detect a glycoside of mandelonitrile. This attempt i s described l a t e r . Chromatography of p h e n y l a c e t o n i t r i l e and phenylacetald-oxime from the millipede was done 2 - d i r e c t i o n a l l y on S i l i c a g e l G t h i n layer plates using benzene:ethyl acetate-7:l ( v/v ) ( Kindl and Underhill, 1968b ), and benzene:methanol-20:3 ( v/v) ( Tapper et a l . , 1972 ). The zones corresponding to the two above compounds were eluted from the g e l with acetone, and the solvent removed i n vacuo at 40°C. Phenylacetonitrile was hydrolyzed to phenylacetic a c i d 19 by r e f l u x i n g for 8 hours i n 12N H^SO^. The hydrolysate was di l u t e d with water , extracted with d i e t h y l ether, the ether phase dried with anhydrous Na^O^, f i l t e r e d , and evaporated to dryness i n vacuo , The phenylacetic acid was chromatographed on Whatman 3MM paper with 2 % formic acid, and then benzene: acetic acid:I^O-10:7:3 ( v/v/v ). The chromatographic zones corresponding to the aci d were eluted from the paper with 2 % formic acid. The eluate was a c i d i f i e d with HCl and the aci d re-extracted with d i e t h y l ether. The phenylacetic a c i d was next repeatedly chromatographed on S i l i c a g e l G plates with pet. ether ( b.r.30-60°C. ):pyridine-50:1( v/v ) and the acid eluted from the gel with acetone. The acetone was removed i n vacuo and the c r y s t a l s dissolved i n d i e t h y l ether p r i o r to determination • of r a d i o a c t i v i t y . The presence of acetone caused serious quench-ing. Phenylacetaldoxime was oxidized to phenylacetic a c i d by di s s o l v i n g the oxime i n 1 ml. of acetone and adding 10 ml. of the chromate o x i d i z i n g s o l u t i o n ( from the method used to de-3 grade the 2a- H- oxime ) . The reaction mixture was allowed to stand at room temperature over-night i n a sealed v i a l . The acid d e r i v a t i v e was extracted from the oxidation s o l u t i o n with d i e t h y l ether and p u r i f i e d by the same chromatographic systems used to p u r i f y phenylacetic aci d . The pyridine-pet. ether solvent separated the phenylacetic a c i d from the benzoic aci d . The l a t t e r was a contaminant arising from the decomposition of mandelonitrile. The oxime, the n i t r i l e , and the aromatic acids were detected on the s i l i c a gel by spraying with an aqueous s o l u t i o n of 5 % KMn04. To i s o l a t e a glycoside of mandelonitrile, millipedes . 1 4 were injected with eit h e r D,L-phenylalanine-ring- C, glucose-14 14 C, or phenylacetonitrile-1- C. They were permitted.to meta-bolize the precursors for 2-10 hour periods. As i n the case of the two previous intermediates the 12 millipedes were extracted together i n b o i l i n g ethanol. The extract was f i l t e r -ed, d i l u t e d with water, a c i d i f i e d with HCl and washed with pet ether ( b.r. 60-110°C.): ether -1:1 ( v/v ). The aqueous phase was neutralized with NaOH and taken to near dryness, d i l u t e d with water and extracted continuously with e t h y l acetate for 8 hours. The ethyl acetate f r a c t i o n was dried with anhydrous Na^SO^, and the solvent removed i n vacuo. This f r a c t i o n was chromatographed as described under the i s o l a t i o n of cyanogenic glycosides. III.8- DETERMINATION OF THE AUTONOMY OF THE CYANOGENIC GLAND To eliminate the p o s s i b i l i t y that alimentary symbionts of H. haydeniana were responsible for HCN biosynthesis, two kinds of experiments were undertaken. F i r s t , D,L-phenylalanine-14 . C i n sol u t i o n was used to f i l l the degutted body c a v i t y ( 0.1 uCi, 300 pCi/mMole, i n 0.2 M. Na 2HP0 4 buffer, pH 7 ). This preparation was allowed to stand for 2 hours, a f t e r which i t was extracted with b o i l i n g ethanol to which had been added unlabelled mandelonitrile. The extracted mandelonitrile was p u r i f i e d by chromatography, eluted from the g e l with acetone, -and the solvent removed i n vacuo at 20°C. The n i t r i l e was de-composed by heating, and the resulting, benzaldehyde c o l l e c t e d by sublimation on a cold glass finger. The semicarbazone of benzaldehyde was made and i t s r a d i o a c t i v i t y determined.-Second, i n v i t r o preparations of i s o l a t e d glands were prepared. The cyanogenic gland attached to approximately 3 mm. of c u t i c l e was dissected from the phlange. Without disrupting the i n t e g r i t y of the gland,as much as possible of the adherent tissue was teased away from the two chambers. Several such isp. lated glands were placed i n a c i t r a t e buffer ( enzyme assay ) 21 containing D,L-phenylalanine-2- C ( 8,200 uCi/mM. ) at a concentration of 1 x 10^ d.p.m./ 100 p i . . A f t e r three hours ethanol was added, the sample d i l u t e d with water, made basic 14 with NaOH, and then a c i d i f i e d with HCl. The H CN was c o l l e c t e d 14 i n 0,1 N NaOH as previously described, and converted to Ag CN before determination of r a d i o a c t i v i t y . III.9- COLLECTION OF BENZALDEHYDE AND HCN To i d e n t i f y the v o l a t i l e components of the secretions of O. g r a c i l i s , a stream of a i r was passed for 1 hour over 300 adults i n a fl a s k heated on a water bath,and the HCN and benz-aldehyde c o l l e c t e d i n a series of traps ( Figure 4 ). The f i r s t trap contained a saturated sol u t i o n of 2,4-dinitrophenylhydrazine i n 6 N HCl for c o l l e c t i n g the benzaldehyde as the hydrazone. The second trap contained a so l u t i o n of 1 % AgNO^ i n 3.4 N HNO^ for c o l l e c t i n g CN as AgCN. The l i b e r a t i o n of HCN from the millipedes and from acid treated AgCN was detected by t e s t i n g with sodium p i c r a t e , or with copper acetate-benzidine reagents ( F e i g l , 1958 ). The same series of traps was used to c o l l e c t the HCN and benzaldehyde produced from radioactive m i l l i p e d e s . The 2,4-dinitrophenylhydrazone of benzaldehyde was pur-f i e d by c r y s t a l l i z a t i o n from acetone. The millipede sample was compared to a known sample by means of melting point, chromatographic behaviour ( R u f f i n i , 1965 ), and i n f r a r e d s p e ctral a n a l y s i s . 14 In experiments employing C-precursors with 0. g r a c i l i s , the millipedes were transferred to a flask, the fl a s k heated, and the HCN and benzaldehyde c o l l e c t e d as described above. The pr e c i p i t a t e d AgCN was f i l t e r e d on a micro-sintered glass funnel, dried, weighed, and stored i n the dark before determination of r a d i o a c t i v i t y . The crude benzaldehyde hydrazone was c o l l e c t e d 22 by f i l t r a t i o n , weighed, and r e c r y s t a l l i z e d from acetone. The derivative was combusted to determine the r a d i o a c t i v i t y . III.10- COLLECTION OF HCN AND BENZALDEHYDE IN PRECURSOR FEEDING STUDIES  Twenty-four hours a f t e r the i n j e c t i o n of the l a b e l l e d precursors H. haydeniana were r e f r i g e r a t e d at 4°C. for one ha l f hour, and then anaesthesized with CO^. They were c a r e f u l l y placed i n a two-armed d i s t i l l a t i o n f l a s k ( Figure 4 ) through which a slow stream of a i r passed leading f i r s t to the benz-aldehyde trap ( 6 N HCL saturated with 2,4-dinitrophenylhydra-zine ) and then to the second trap for ~CN ( IO ml. of 0.2 N NaOH ). Sintered glass a i r bubblers were used i n both traps. To remove a l l the benzaldehyde and HCN from the millipedes the flask containing the animals was heated at 85°C. for 1 hour. Benzaldehyde 2,4-dinitrophenylhydrazone was f i l t e r e d from the trapping s o l u t i o n by means of a sintered glass funnel. The c r y s t a l s were thoroughly washed with 6 N HCl followed by water. The derivative was dissolved i n acetone, and chromato-graphed on S i l i c a g el G (1.5 mm. thick ) plates with benzene: g l a c i a l a c e t i c a c i d - 5:1 ( v/v ), eluted, and rechromatographed xyl e n e : g l a c i a l a c e t i c a c i d - 5:1 (v/v ). The hydrazone was eluted from the g e l and i t s concentration determined spectrophotometri-c a l l y i n acetone at 380 nm.. I t was combusted, and the amount of r a d i o a c t i v i t y determined ( Figure 5 ). However, the benzaldehyde derived from the millipedes 3 fed 2-hydroxyphenylacetaldoxime-2a- H was treated i n a d i f f e r e n t manner. This benzaldehyde 2,4-dinitrophenylhydrazone was com-busted i n the presence of 5 ml. of 1 N NaOH, and the combustion chamber allowed to stand for one h a l f hour. A f t e r t h i s i n t e r v a l the chamber was rotated on i t s side to c o l l e c t a l l the conden-FIGURE 4. Diagram of the c o l l e c t i o n apparatus for HCN and benzaldehyde. TRAP 1 TRAP 2 a - N a O H TRAP 1 - (j)CHO 2 - H C N B u b b l e r FIGURE 5. Combustion apparatus for c o l l e c t i o n of CO from 2,4-DNP de r i v a t i v e s . 2,4-DNP = 2,4-dinitrophenylhydrazone d e r i v a t i v e s . Sy = 25 ml. syringe and needle. P = r o l l e d f i l t e r paper impregnated with 2,4-DNP derivative; f i l t e r paper wrapped with Kleenex to aid i g n i t i o n . B = balloon to r e l i e v e pressure during combustion. V"i = one-way valve to con t r o l flow of gas. J = detatchable j o i n t allowing i n t r o d u c t i o n of 0 2 at J . S = rubber stopper. Co = 25 ml. pipette f i l l e d with glass beads. PEA = a-phenylethylamine and H^O ( 3:1 ) i n 5 ml. test-tube. The combustion chamber containing 10 ml. o f 2N NaOH i s saturated with high grade O . V 2 i s closed and V, i s opened before applying current across terminals. The current i s maintained during burning of sample. A f t e r combustion the sample i s allowed to cool f o r 20 min. The in t e r n a l side of the f l a s k i s washed with the base by gentle r o t a t i o n of the f l a s k . A i r i s introduced into the flask by opening V 2 ( not yet attached to the C0 2 trap ). The glass stopper of the combustion f l a s k i s replaced with S as shown i n diagram. The C0 2 trap containing PEA i s connected at J and V. i s closed, and V~ opened. A i r i s passed through the whole system such that PEA i n the trapping chamber r i s e s into the exchange column ( Co ), and the bubbling e f f e c t i s moderate. Then the H-^ SO^  i s injected slowly into the NaOH so l u t i o n r e l e a s i n g the C0 2 which is blown into the PEA. The a i r i s passed over the a c i d i f i e d medium for 15 min. a f t e r which the PEA i s re-moved from the trap. The column i s washed with a small volume of H20; t h i s volume i s added to the former PEA. The PEA solution i s d i l u t e d to a knownvolume with H 20 and an aliquot removed for s c i n t i l l a t i o n counting. F I G U R E 5. C o m b u s t i o n a p p a r a t u s f o r c o l l e c t i o n o f C C ^ f r o m 2,4 -DNP d e r i v a t i v e s . 25 sation. A 1 ml. aliquot was taken for determination of radio-a c t i v i t y . This aliquot was added d i r e c t l y to the s c i n t i l l a t i o n f l u i d . III.11- RADIOACTIVITY DETERMINATIONS 14 In the C-precursor experiments using O. g r a c i l i s the 14 r a d i o a c t i v i t y of the Ag CN was determined by suspending, these c r y s t a l s i n Cab-o-sil and counting i n a l i q u i d s c i n t i l l a t i o n spectrometer. Radioactivity i n the benzaldehyde of O. g r a c i l i s and H. haydeniana was determined by combustion of the 2,4-di-nitrophenylhydrazone i n a sealed f l a s k containing 0 2« The C0 2 produced was c o l l e c t e d i n 1 N NaOH and subsequently l i b e r a t e d from the base by the addition of 4 N H 2S0 4. The C0 2 was flushed from the flask by means of a stream of a i r . The C0 2 was c o l l e c t e d by passing the stream of a i r through a fin e glass b e a d - f i l l e d column containg 2-phenylethylamine. The so l u t i o n of 2-phenyl-ethylamine carbonate was mixed with Aquasol and counted i n a l i q u i d s c i n t i l l a t i o n spectrometer. See Figure 5 for more d e t a i l s of the combustion apparatus. 14 . . . In the C-precursor experiments using H. haydeniana the s c i n t i l l a t i o n f l u i d consisted of 6 gm. of PPO, 0.4 gm. of POPOP dissolved i n 125 ml. of toluene / and 275 ml. of ethanol. To measure the r a d i o a c t i v i t y i n the HCN from H. hayden-iana the following method was used. To determine the concen-traction of HCN i n the 0.2 N NaOH trap s o l u t i o n a modification of the method of Jacobs ( 1967 ) was employed. F i f t y u l . of the trap s o l u t i o n was d i l u t e d to 1.5 ml. with 0.2 N NaOH ( larger samples were d i l u t e d to the same volume ). The pH was adjusted to 6-7 with 0.1 ml. of 25 % ac e t i c acid, followed by the addi-t i o n of 0.02 ml. of 1 % aqueous chloramine-T. A f t e r 1-2 minutes, 1 ml. of the pyridine-pyrazolone reagent was added. The sample 26 was d i l u t e d to a t o t a l volume of 3 ml. with d i s t i l l e d water and read at 625 nm., a f t e r allowing 14 minutes for development of colour. 14 To determine the s p e c i f i c a c t i v i t y of the H CN from the millipede, the t o t a l trap s o l u t i o n was d i l u t e d with 50 mgm. of unlabelled KCN, and made to a volume of approximately 20 ml. with a s o l u t i o n of 4 % AgNO^ i n 3.4 N HNO^. From t h i s point on, the AgCN p r e c i p i t a t e was protected from l i g h t as much as p o s s i -ble. The AgCN was allowed to f l o c c u l a t e for 5 minutes, the pre-c i p i t a t e centrifuged, the supernatant discarded and the p e l l e t resuspended and washed with 3.4 N HNO^. The AgCN was r e c e n t r i -fuged and s i m i l a r i l y washed with water, followed by resuspen-sion and washing with acetone. The washed p e l l e t was then dried at 110°C. for 1 hour, placed i n an agate c r u c i b l e and ground vigorously i n Nujol o i l to a homogeneous paste. Scint-i l l a t i o n f l u i d , 1.5 ml., was added to the c r u c i b l e . The mixture was s t i r r e d u n t i l the Nujol dissolved i n the f l u o r and the AgCN could s e t t l e as a fine powder. The f l u o r and the AgCN were trans-ferred to a s c i n t i l l a t i o n v i a l to which was added approximately 4 ml. of Cab-o-sil and a further volume of f l u o r ( approximate-l y 6 ml. ) to give a stable emulsion on vigorous shaking. This method was found to be 70 % e f f i c i e n t based on determinations 14 . . . with a standard s o l u t i o n of K CN. The r a d i o a c t i v i t y i n t h i s standard AgCN was determined as follows. Approximately 5 mgm. of 14 Ag CN was dissolved i n 2 ml. of concentrated H^SO^, and 10-50 p i . of t h i s s o l u t i o n d i l u t e d with 2 ml. of 0.2 N NaOH to which was added 10. ml. of s c i n t i l l a t i o n f l u i d . III.12- ISOLATION OF MANDELONITRILE AND MANDELIC ACID Adults of H. haydeniana were c h i l l e d to immobility at 4°C. and then frozen on dry ice p r i o r to i s o l a t i o n of mandelonitrile. 27 The animals were then crushed with sand i n b o i l i n g ethanol. The extract was f i l t e r e d with the a i d of C e l i t e and the residue re-extracted with hot ethanol. The combined extracts were reduced i n volume at 0°C. i n vacuo, d i l u t e d with water and extracted with d i e t h y l ether. The ether f r a c t i o n was dried with anhydrous Na 2S0 4, taken to dryness i n vacuo, dissolved i n acetone and applied to a S i l i c a gel G pla t e . The f i r s t chromatography was with pet. ether ( b.r.30-60°C. ), followed by pet. ether:methanol: CCl^- 125:50:50 ( v/v/v ). The impure mandelonitrile had a Rf value=0.5 i n the second solvent system. The presence of mandelo-n i t r i l e was detected i n three ways: ( 1) The TLC plate was sprayed with 2% p i c r i c acid ( w/v ) i n aqueous 1 % Na 2C0 3, and over-sprayed with 5% NaOH. The HCN released formed a dark brown complex with the p i c r i c a c i d . ( 2 ) The plate was sprayed with 5% NaOH followed by an over-spray of a saturated s o l u t i o n of 2,4-dinitrophenylhydrazine i n 6 N HCl. An orange spot on a yellow background indicated the presence of an aldehyde or ketone.( 3 ) The plate was sprayed with emulsin, which also contains n i t r i l e lyase ( Haisman and Knight, 1967 ). The re-lease of HCN was detected by the p i c r i c a c i d reagent described i n ( 1 )'. Mandelonitrile was also eluted from the gel with ethanol and i t s UV spectrum compared to that of a synthetic sample. The above extraction procedure was also followed to deter-14 mine i f the C-precursors had been incorporated into mandelo-n i t r i l e ; but, unlabelled mandelonitrile was also added to the i n i t i a l ethanolic extract. The chromatographically p u r i f i e d mandelonitrile was eluted from the gel with d i e t h y l ether, the ether removed i n vacuo, and the n i t r i l e transferred to a small flask connected to a HCN trap ( Figure 4 ). The mandelonitrile was gently heated i n the flask for 1 hour. The AgCN p r e c i p i t a t e f i l t e r e d , washed with HNO^ and ethanol, and assayed for radio-28 a c t i v i t y . The conversion of mandelonitrile from H. haydeniana to mandelic acid was achieved i n the following way. Anaesthe-sized animals were extracted with b o i l i n g ethanol and the ex-t r a c t f i l t e r e d . A n equal volume of concentrated HCl was added to the f i l t r a t e , and heated on a steam bath bath for 8 hours.The extract was then taken to dryness i n vacuo, the residue r e d i s -solved i n d i l u t e HCl and extracted twice with equal volumes of d i e t h y l ether. The ether f r a c t i o n was taken to dryness i n  vacuo, banded on a TLC plate ( A v i c e l : S i l i c a g e l G - l : l , v / v ) and defatted with pet. ether ( b. r. 30-60C0). The mandelic a c i d was eluted with acetone and banded on a 1 mm. thick plate ( above support ) and developed with 2% formic a c i d . The region corrseponding to the mandelic a c i d was eluted with acetone and chromatographed 2 - d i r e c t i o n a l l y on Whatman 3MM paper with 2% formic a c i d followed by benzene:acetic aci d : H20-19:7:3 ( v/v/v ). The area corresponding to mandelic a c i d was eluted with acetone. The a c i d was detected by spraying with bromcresol green ( Stahl, 1969 ). To determine the s p e c i f i c a c t i v i t y of the mandelic a c i d 14 . C a known amount of chromatographically pure, r e c r y s t a l l i z e d D,L-mandelic ac i d ( approximately 0 . 5 gm. ) was added to the millipede product. This sample was c r y s t a l l i z e d from xylene: ethanol:n-hexane- 5 : 1 : 1 0 ( v/v/v ) u n t i l s p e c i f i c a c t i v i t i e s were found to be consistent, 14 Mandelic a c i d - C was decarboxylated by p l a c i n g the c r y s t a l s i n an excess of the o x i d i z i n g s o l u t i o n s i m i l a r to that 3 used to degrade the 2-hydroxy-oxime-2a- H . The C0 2 evolved was c o l l e c t e d i n 2-phenylethylamine. The decarboxylation of 14 synthetic mandelic a c i d - C produced l a b e l l e d phenylethylamine carbonate. The benzaldehyde produced from the decarboxylation 29 of the millipede sample was oxidized to benzoic a c i d . This was c o l l e c t e d by f i l t r a t i o n , and r e c r y s t a l l i z a t i o n from aqueous acetone. I t was i d e n t i f i e d by comparison of IR, UV, M.P. and chromatographic properties to a known. In order to ascertain the presence or absence of a glyc-oside of mandelonitrile, 50 anesthesized H. haydeniana were ex-tracted i n b o i l i n g ethanol. The ethanolic extract was reduced i n vacuo to near dryness, d i l u t e d with water, defatted by wash-ing with pet. ether ( b.r.30-60°C. ), and the aqueous phase continually extracted with ethyl acetate. The e t h y l acetate f r a c t i o n was taken to dryness i n vacuo, and the residue chromatogrpahed 2 - d i r e c t i o n a l l y on an A v i c e l TLC plate with 2% formic acid followed by benzene:acetic acid:water-10:7:3 ( v/v/v). To detect a glycoside the developed plate was sprayed with a 1% s o l u t i o n of emulsin i n pH 7 t r i s buffer. A s t r i p of f i l t e r paper moistened with p i c r i c a c i d reagent was placed over the TLC plate, and the paper covered by a glass p l a t e . Using t h i s procedure i t was possible to detect microgram amounts of prunasin. III.13- CIRCULAR DICHROISM OF MANDELONITRILE AND MANDELIC ACID  To determine whether the mandelonitrile of H. haydeniana was o p t i c a l l y active ( L*or D-enantiomer ) c i r c u l a r dichroism ( CD ) was employed. This technique permits the establishment of the absolute configuration about the a-carbon, i n d i c a t i n g whether the molecule exhibits R- ( rectus ) or S- ( s i n i s t e r ) c h i r a l i t y . The mandelonitrile was acquired from c h i l l e d , CO^ anaes-thetized large adults of H. haydeniana. The relaxed animal was decapitated, the p o s t e r i o r few segments removed and the gut 30 c a r e f u l l y withdrawn using fine forceps. This procedure avoided the expulsion of the n i t r i l e from the glands by the alarmed animal. The glands were exposed by d i s s e c t i n g i n a sa l i n e bath ( 0.9% NaCl ); t h i s involved c a r e f u l l y teasing away the sur-rounding tissues without tearing the storage vestibule away from the reaction chamber. Storage vestibules containing a large droplet of mandelonitrile were plucked away from the reaction chamber by the neck, rinsed b r i e f l y i n fresh s a l i n e and dipped i n a known volume of d i s t i l l e d ethanol. No gland tissue was allowed to contaminate the ethanol. Between 6 and 10 storage sacs were necessary to give enough n i t r i l e f or UV and CD analyses. A f t e r c o l l e c t i o n of the mandelonitrile i n ethanol _4 ( approximately 1.1 x 10 Molar ) the CD and UV spectra were run as soon as possible, and monitored for loss of o p t i c a l a c t i v i t y f o r one h a l f hour. A f t e r t h i s time 10 yil. of 5% aqueous NaOH was added, and a new CD and UV spectra taken. The addition of NaOH caused the decay of mandelonitrile to HCN and benzalde-hyde. A f t e r obtaining a UV spectrum of the degradation product(s). of mandelonitrile, NaBH^ was added to the cuvette, and several minutes l a t t e r a new UV spectrum obtained The CD spectrum of mandelic a c i d of H. haydeniana was compared to both D- and L- mandelic acids i n order that the sign of the absorption of the millipede's n i t r i l e and derived mandelic acid could be compared to a series of knowa c h i r a l i t y . Mandelic derived from the millipede's mandelonitrile was prepared by d i s -solving the droplets of n i t r i l e i n 50% ethanolic HCl and heating the mixture on a steam bath for 4 hours. The ethanol and HCl were removed i n vacuo and the c r y s t a l s dissolved i n a known volume of d i s t i l l e d ethanol p r i o r to UV and CD s p e c t r a l det-erminations. 31 111.14- ISOLATION OF 3-CYANOALANINE P-Cyanoalanine was i s o l a t e d from H.haydeniana by the procedure of Blumenthal et al.(1968 ). A reference standard of p-cyanoalanine was provided by Prof. E.E. Conn ( Univ. C a l i f . , Davis, C a l i f o r n i a ). The amino acids which were eluted from the re s i n were chromatographed on Whatman 3MM paper with solvent A ( see below ). The band corresponding to p-cyanoalanine was dil u t e d with 0.1 N HCl, and chromatographed i n three aliquots using three solvent systems, A,B and C: solvent A ( ethanol: t-butanol:H 20: 15N NH^OH- 12:4:3:1, v/v/v/v ); solvent B ( n-butanol:H 20:15N NH^OH- 12:3:5, v/v/v ); solvent C ( 80% phenol i n H 20 ). The chromatograms were developed i n each case for 24 hours, and then scanned for r a d i o a c t i v i t y . The regions corres-ponding to p-cyanoalanine were cut out as narrow bands to reduce contamination from other amino acids; thus, the determination of r a d i o a c t i v i t y i n p-cyanoalanine was not quantitative. The p-cyanoalanine was eluted from the three chromatograms, and each eluate adjusted to 6 N HCl, and subsequently hydrolyzed to aspartic acid at 110°C. for 24 hours i n a closed t e s t tube. Each hydrolysate was dried i n vacuo and each separately chro-matographed with another of the above solvent systems, a f t e r which aspartic acid was eluted from the chromatograms with 0.1 N HCl, and the r a d i o a c t i v i t y determined, 111.15- ISOLATION OF p-HYDROXYBENZOIC ACID 14 Millipedes used i n C-precursor studies or unlabelled animals were anaesthesized with C0 2 p r i o r to extraction i n b o i l i n g ethanol. The hot ethanolic extract was f i l t e r e d through a pad of C e l i t e , and the crushed residue re-extracted with hot ethanol, f i l t e r e d and pooled with the o r i g i n a l extract. The combined extracts were reduced i n vacuo to a quarter of the volume, d i l u t e d with an equal volume of water, washed with 32 pet. ether ( b.r. 60-110°C. ), and the aqueous portion taken to near dryness i n vacuo. The aqueous extract was then d i l u t e d with 1% NaHCO.j/ washed with d i e t h y l ether ( ether discarded ), a c i d i f i e d with HCl, and re-extracted with d i e t h y l ether. The ether f r a c t i o n was dried with anhydrous Na 2S0 4, and the solvent removed i n vacuo. The extract was chromatographed 2-direction-a l l y on Whatmann 3 MM paper with 2% formic a c i d and benzene: acetic acid:H 20-10:7:3 (v/v/v ). Radioactive compounds were detected by autoradiography. The spot corresponding to p-hydroxybenzoic a c i d was eluted with acetone and rechromato-graphed with solvent A ( pg. 31, III.14 ). and 2% formic a c i d . The p-hydrobenzoic acid was eluted from the paper,the solvent removed i n vacuo, and the c r y s t a l s sublimed on a cold glass finger. The sublimed chemical from the millipede was d i l u t e d with p u r i f i e d p-hydroxybenzoic acid, and c r y s t a l l i z e d to a constant s p e c i f i c a c t i v i t y from toluene:ethanol-8:1 ( v/v ). Chromatographic comparisons were made to various other phenolic standards ( Ibrahim and Towers, 1960 ). P-Hydroxybenzoic a c i d and other simple phenolic and aromatic acids were detectable on chromatograms by spraying with bromcresol green reagent for adds and with diazotized p - n i t r o a n i l i n e reagent for phenolics ( Stahl, 1969 ). III.16- ISOLATION OF CYANOGENIC GLYCOSIDES Prunasin ( D-mandelonitrile-|3-0-D-glucopyranoside ) and t a x i p h y l l i n ( D-p-hydroxymandelonitrile-$-0-D-glucopyranoside ) were avail a b l e as standards, previously i s o l a t e d by Prof. G.H.N. Towers following the method outlined by Towers et a l . ( 1964 ). Prunasin was extracted from Prunus laurocerasus L. and t a x i -• p h y l l i n from Taxus canadensis Marsh.. A crude preparation 33 of sambunigrin ( L-mandelonitrile-(3-0-D-glucopyranoside ) was extracted from Sambueus racemosa var. arborescens ( T.&G.) Gray as outlined by Towers et a l . ( 1964 ) except that the e t h y l acetate f r a c t i o n was used d i r e c t l y for chromatography. The cyanogens from both plant and animal extracts were f i r s t chromatographed on Whatman 3MM paper with benzene:acetic acid:H 20- 10:7:3 ( v/v/v ) for 24 hours; the cyanogens remain at the o r i g i n . Further p u r i f i c a t i o n involved chromatography with the following solvents: ( 1 ) 2 % formic acid;(2 ) n-butanol: ac e t i c acid:H 20- 20:1:4 (v/v/v ); ( 3)ethanol:t-butanol?H 20:15N NH^OH-12:4:3:1 ( v/v/v/v ); and benzene: a c e t i c a c i d - 5 : l (v/v ). The cyanogen was detected on the chromatogram by the emulsin-p i c r i c a c i d t e s t . A f t e r each chromatography the cyanogen was eluted from the paper with 2% formic a c i d . Acetate derivatives of the cyanogens were made by d i s -solvingthem i n a c e t i c anhydride with c a t a l y t i c amounts of pyr-idine for 24 hours at room temperature. The acetates were pre-c i p i t a t e d from the reaction mixture by the addition of crushed i c e . The p r e c i p i t a t e was f i l t e r e d , washed with water, and re-c r y s t a l l i z e d several times from aqueous acetone, and f i n a l l y from xylene by the slow addition of pet. ether ( b.r. 60-110°C). III.17- EXTRACTION OF NITRILE LYASE AND B-GLYCOSIDASE Pr i o r to the extraction of the enzymes of H, haydeniana, the animals were c h i l l e d for several hours at 50C., and just before use anaesthesized with C0 2. They were degutted (first cut-t i n g o f f the head and the l a s t few abdominal segments) by withdraw-ing the gut by. means of fine forceps; 10-20 animals were used per enzyme preparation. The degutted millipedes were crushed with a mortar and pestle with fine powdered acid-washed glass i n a c i t r a t e - N a 2 P 0 4 buffer (0.05 M. ) at pH 5.4, at 0°C. The 34 enzyme extract was f i l t e r e d through two layers of cheese-cloth and the p e l l e t washed with a small volume of buffer such that the t o t a l volume of buffer was 20 ml.. Sodium isoascorbate ( 10 mgm. ) was added to t h i s f i l t r a t e , the sample centrifuged at 8,000 g. for 20 minutes. The supernatant was made 80% satur-ated with (NH^) 2 S°4/ a n < ^ ^ n e mixture centrifuged at 8,000 g. for 30 minutes. The supernatant was discarded and the p e l l e t dissolved i n 5 ml. of the extraction buffer. I t was dialyzed for 24 hours against two changes of 250 ml. of buffer at 0 ° C V each change containing 50 mgm. of sodium isoascorbate. . The enzyme was e i t h e r used immediately or stored at -20°C i n 5 ml. of buffer with several mgm. of sodium isoascorbate. III.18- ASSAY CONDITIONS FOR LYASE AND GLYCOSIDASE ACTIVITY The a c t i v i t y of e i t h e r (3-glycosidase ( EC 3.2.1.21 ) or a-hydroxynitrile lyase ( EC 4.1.2.10 ) was assayed i n the citrate-Na 2HP0 4 buffer at pH 5.4 at 30°C. unless otherwise stated. The t o t a l assay volume was 3 ml. containing 0.05 ml.-0.3 ml. of enzyme preparation and 1-50 p i . of substrate; the remainder was buffer. To assay for (3-glycosidase a c t i v i t y , prunasin, sambuni-grin, and t a x i p h y l l i n were used as substrates.Stock solutions of these substrates were prepared i n the buffer. The hydrolysis of prunasin and sambunigrin was detected by measuring the form-ation of benzaldehyde at 249 nm.. The formation of p-hydroxy-benzaldehyde from the hydrolysis.of t a x i p h y l l i n was measured at 285 nm. . D-Glucose ( O.l M. ) was used to i n h i b i t (3-glyco-sidase a c t i v i t y . D,L-mandelonitrile was synthesized by the method of Tapper and Butler ( 1971 ), and stored under N 0 with Na SO. 35 at -20 WC. u n t i l use. It was centrifuged before use i f t u r b i d . For the assay a 10 p i . aliquot of mandelonitrile was added to 1 ml. of buffer and shaken vigorously so as to form a suspen-sion. This s o l u t i o n of mandelonitrile was replaced every 5 minutes because of the decay of the n i t r i l e . M i c r o l i t e r sized aliquots of t h i s suspension were used for the enzyme assays. The rate of decomposition of mandelonitrile was measured for each d i f f e r e n t assay condition to d i s t i n g u i s h t h i s rate from that of the enzyme catalyzed reaction. p-Hydroxymandelonitrile was treated i n a smilar manner for enzyme assays. To determine the e f f e c t of pH on both p-glycosidase and a-hydroxynitrile lyase a c t i v i t y a citrate-Na 2HP04 buffer was used from pH 2.6-7.0, and a Tris-HCl buffer ( 0.05 M. ) at pH 9.0. Assays were also c a r r i e d out at pH 5.4 to determine the e f f e c t of temperature,increased substrate, and increased ' enzyme concentrations on the rate of reaction of both enzymes. Protein concentration i n the assay medium was determined by the method of Lowry et a l . ( 1951 ). III.19- HISTOLOGY OF THE CYANOGENIC GLAND In order to obtain glands for h i s t o l o g i c a l studies i n -d i v i d u a l segments were removed from the body and cut i n h a l f . The tissues about the glands were teased away, and t h i s h a l f segment f i x e d i n F.A.A. ( H 20:100% ethanol:formalin: a c e t i c a c i d -225:225:30:20, v/v/v/v) for 24 hours i n vacuo. The a c e t i c a c i d d e c a l c i f i e d the c u t i c l e making i t p l i a b l e and cuttable. The fixed dissections were then imbedded i n paraffin, sectioned at a thickness of 15 p., stained with Mayer's haematoxylin, and counter-stained with Eosin Y. The sections were mounted with Pro-tex Mounting Medium ( from S c i e n t i f i c Products ). 36 III.20- ELECTRON-MICROGRAPHIC SCANNING OF H.HAYDENIANA Specimens of H. haydeniana were anaesthesized, frozen and then l y o p h i l i z e d . The dried specimens were gently broken into i n d i v i d u a l segments. Some of these segments were coated with elemental gold i n vacuo. The e i c t r o n micrographic analysis was done by Mr. L. Veto, Dept. of Botany, U.B.C.e IV- RESULTS IV.1- IDENTIFICATION OF BENZALDEHYDE AND HCN AND THEIR BIO-SYNTHESIS IN OXIDUS GRACILIS It was confirmed that O. g r a c i l i s produces HCN and benz-aldehyde. The i d e n t i t y of the benzaldehyde was established by i t s chromatographic behaviour, melting point, and IR spectrum, which were i d e n t i c a l to a synthetic standard of the 2,4-dinitro-phenylhydrazone of benzaldehyde. A p o s i t i v e reaction with sodium p i c r a t e ( brown spot ) and a p o s i t i v e t e s t with the copper ace-tate-benzidine reagent ( blue colour ) indicated the presence of HCN. F a i l u r e to obtain HCN by emulsin or aci d hydrolysis of concentrates of ethanolic extracts indicates that mandelo-n i t r i l e glucoside i s not the storage product. Chromatography of ethanolic extracts of t h i s millipede showed the presence of a chemical i d e n t i c a l to mandelonitrile. Dissections showed that each storage vestibule of 0. g r a c i l i s contained a large o i l y droplet which was shown chromatographically to be mandelonitrile. 14 . . . The r e s u l t s of the C-precursor studies with O. g r a c i l i s ( Table I and Figure 6 ) indicate that the production of HCN and benzaldehyde a r i ses from the degradation of phenylalanine. In-37 corporations of D,L-phenylalanine- C were low; however,in each case the s p e c i f i c a l l y l a b e l l e d precursors gave r i s e to HCN and benzaldehyde without scrambling of the l a b e l . This 14 14 organism did not use D,L-tyrosine-2- C to produce H CN. The low incorporations are not the r e s u l t of poor eating by the millipedes, because these animals accumulated at l e a s t 100,000 d.p.m. per experiment. IV.2- CHARACTERIZATION OF HCN AND BENZALDEHYDE PRODUCED BY HARPAPHE HAYDENIANA H. haydeniana, when alarmed, secretes HCN and benzalde-hyde. HCN was i d e n t i f i e d by simple chemical means; i t gives a p o s i t i v e blue colour with benzidine copper-acetate, a p o s i -t i v e reaction for the Prussian blue test, a p o s i t i v e reaction with the pyrazolone reagent, and a p o s i t i v e reaction with p i c r i c acid ( see Jacobs, 1967, for the above tests ). The gas forms a white p r e c i p i t a t e when bubbled through a AgNO^ so l u t i o n . This AgCN was l i g h t sensitive,and yielded HCN on digestion with hot K 2Cr 20 7:H 2S0 4:H 20- 5:100:5 ( w/v/v ). The presence of benzaldehyde i n the millipede was est-ablished by comparison of the M.P. ( 236-238°C. ), UV and IR spectra, and chromatographic properties of the 2,4-dinitro-phenylhydrazone with those of a synthetic sample. Comparison of the semicarbazone d e r i v a t i v e of benzaldehyde to that of a synthetic known showed i d e n t i c a l M.P. ( 222-223°C. ), and UV spectrum. Retention values on GLC analyses of the animal alde-hyde and that of a synthetic standard were i d e n t i c a l . No other major chemicals were detectable by GLC. IV.3- THE IDENTIFICATION OF HCN IN OTHER POLYDESMID MILLIPEDES 14 TABLE I. The incorporation of variously C-labelled phenylalanine into HCN and and benzaldehyde i n the defensive secretion of Oxidus g r a c i l i s . Precursor i n food 14 amount of C precursor fed mCi/mMole t o t a l pCi ° f ^ l l i p e d e s Total a c t i v i t y A c t i v i t y i n i n ETOH extract A c t i v i t y i n HCN benzaldehyde pCJ pMoles pCi pMoles 14 D,L-Phenylalanine-1- C 4.8 6.7 1.0 x i o " 1 0 52 0 56 14 D,L~Phenylalanine-2- C 8.2 6.7 1.7 x i o " 1 l.Q x IO" 4 21 0 43 8.2 6.7 2.5 x i o " 1 5.4 X io" 3 62 0 22 8.2 6.7 1.0 x i o - 1 2.4 X i o " 2 51 ; 0 92 14 D,L-Phenylalanine-3- C 12 6.7 5.0 x i o " 2 0 35 2.4 x i o - 4 73 12 6.7 1.2 x i o " 1 0 72 2.3 x i o " 3 65 D,L-Phenylalanine-l-ring-14 C 2.1 6.7 1.6 x i o - 1 6 15 2.0 x IO" 3 54 14 D /L-Tyrosme-2- C 50 6.7 1.7 x i o "1 0 53 0 50 HCN c o l l e c t e d as AgCN; benzaldehyde c o l l e c t e d as the 2,4-dinitrophenylhydrazone as combusted. The data i s corrected for the assumption that only the L-enantiomer i s u t i l i z e d . 00 1 C 0 2 1 C O O H / 2 ' l k + H C ^ N 2 C H N H 2 2 C = N 3 9 H2 3 C H O H -> > » DL-Phenylalanine Mandelonitrile Benzaldehyde F I G U R E 6 Biosynthetic pathway for the production of HCN and benzaldehyde from dietary phenylalanine i n Oxidus  g r a c i l i s . 40 Other polydesmid millipedes were examined for the pro-duction of HCN ( p i c r i c a c i d t e s t ). These tests show ( Table II ) that seven previously unexamined millipedes of diverse families release HCN. The nature of the aldehyde or ketone was only determined i n the case of H. haydeniana. Sphaerio-desmus did not appear to release HCN when tested i n the f i e l d . IV.4- MANDELONITRILE AS THE STORED CYANOGEN H. Haydeniana stores mandelonitrile rather than a gly-coside of mandelonitrile. The stored material has the same chromatographic c h a r a c t e r i s t i c s , colour, and UV absorption maximum as mandelonitrile. I t y i e l d s KCN and benzaldehyde upon spraying with 5%Na0H. On acid hydrolysis mandelic a c i d was ob-tained and i d e n t i f i e d by i t s melting point ( 127-128°C. ), and UV and IR spectra a f t e r p u r i f i c a t i o n by 2 - d i r e c t i o n a l chrom-atography. The product of a c i d hydrolysis yielded upon decarboxy-l a t i o n benzaldehyde ( hydrazone obtained ) and C0 2. Upon fur-ther oxidation of benzaldehyde, benzoic a c i d was obtained. This characterization was based on UV data, M.P. ( 120-122°C. ), and chromatographic properties. Chromatography of the ethanolic extract of 50 millipedes f a i l e d to indicate any chemical repre-sentative of a glycoside. The amount of mandelonitrile stored by H. haydeniana was determined i n three ways, no account being taken for the weight of the animals: ( 1 ) by c o l l e c t i o n of benzaldehyde from d i s t -i l l e d animals ( 0.51 umoles/ animal ), ( 2 ) by c o l l e c t i o n of HCN from d i s t i l l e d animals ( 0.41 umoles/animal ), and (3 ) by conversion of the n i t r i l e to mandelic acid ( 0.20 umoles/animal ). The low y i e l d of the t h i r d method i s probably because of the loss of mandelonitrile and mandelic acid during i s o l a t i o n . MILLIPEDE • LOCATION HCN OIL REPT'D Subclass: Helminthomorpha Order: Polydesmida Family: Chelodesmidae Caraibodesmus sp, Sphaerodesmidae Sphaeriodesmus sp. nov. nr. brueai Chamb. Paradoxostcmatidae Orthomorpha coarctata ( DeSauss. ) Oxidus g r a c i l i s ( Kock ) Xystodesmidae Boraria s t r i c t a ( Brolemann ) Cherokia georgiana ( Bollman ) Harpaphe haydeniana ( Wood ) Polydesmidae Polydesmus angustus Latzel Pseudopolydesmus branneri ( Bollman ) Scytonotus insulanus Attems Nearctodesmidae Nearctodesmus cerasinus ( Wood ) Runaway Bay, Jamaica. Windsor Caves, Jamaica. ? Runaway Bay, Jamaica. I I H Radford, Va., USA. Midland, Ont., Canada. + ' + Vancouver, B.C., Canada. + + Hornby Is,, B.C., Canada. + Vancouver, B.C., Canada. + + + ' + + + + + HCN = hydrogen cyanide produced; OIL «= millipede has o i l y droplet i n storage vestibule; REPT'D of HCN previously documented. , • presence TABLE I I . The presence o f HCN i n va r i o u s polydesmid m i l l i p e d e s * 42 Further evidence that mandelonitrile i s the storage pro-duct i n H. haydeniana was obtained by i n j e c t i n g e i t h e r D,L-14 14 phenylalanine-2- C or D,L-phenylalanine-3- C ( approximately 2 uCi/5 animals ), and subsequently i s o l a t i n g the crude mand-e l o n i t r i l e ( d i l u t e d with unlabelled n i t r i l e ). The mandelo-n i t r i l e was degraded and i t was found that the r e s u l t i n g HCN ( 600 d.p.m. ) and benzaldehyde ( 1,000 d.p.m. ) were radio-a c t i v e . Other evidence that mandelonitrile i s the storage pro-duct was obtained by the i s o l a t i o n of 2 mgm. of radioactive 14 . mandelic a c i d - C. This chemical was i s o l a t e d a f t e r feeding 14 14 D,L-phenylalanine-2- C and D,L-phenylalanine-3- to 20 animals, and the mandelic acid d i l u t e d futher with unlabelled mandelic acid from 222 animals (. n i t r i l e hydrolyzed to ac i d . ). The p u r i f i e d acid was d i l u t e d with a known q a u n t i t i y of D,L-mand-e l i c acid, and r e c r y s t a l l i z e d to a constant s p e c i f i c a c t i v i t y ( 54 d.p.m./mgm. of d i l u t e d sample). I t was found by c a l c u l a t i o n that the undiluted mandelic a c i d from the millipedes had a t o t a l count of 24,300 d.p.m.. The undiluted millipede mandelic acid had a melting point suggestive of a non-racemic mixture ( M.P.= 127-128°C: racemate M.P.= 118°C ). Also, the UV and IR spectra were i d e n t i c a l to those of a p u r i f i e d L-mandelic a c i d ( not to a D,L- mixture ). Morphological evidence i s also presented i n Figure 15 that H. haydeniana stores mandelonitrile. A droplet of mandelo-n i t r i l e i s observed ( d ) within the storage vestibule ( sv ) of each cyanogenic gland. The droplet of mandelonitrile appeared i n other dissections to be r e s t i n g i n a c l e a r f l u i d within the storage ves t i b u l e . In millipedes which had not been disturbed for weeks the droplet of mandelonitrile occupied most of the volume of the vesti b u l e . Several droplets of n i t r i l e were often observed i n one gland, but these were considered to be the re-4 3 s u i t of fragmentation of the one droplet during d i s s e c t i o n . I t was observed that the following polydesmid millipedes also contained a large droplet of o i l i n each of t h e i r storage vestibules; Boraria s t r i c t a , Cherokia georgiana,Oxidus g r a c i l i s , Polydesmus angustus, Nearctodesmus cerasinus, and Scytonotus  insulanus. The nature of the o i l was only determined i n H. hay-deniana and O. g r a c i l i s , but i t i s assumed, since a l l the above millipedes produce HCN ( Table II ), that the o i l i s a n i t r i l e . By c a r e f u l removal of the droplets of o i l from the glands of H. haydeniana i t was possible to show t h i s substance gave r i s e to benzaldehyde and HCN. A UV spectrum of t h i s o i l indicated an absorption maximum s i m i l a r to that of synthetic mandelonitrile ( 215 nm. ); subsequent d i s s o c i a t i o n of the chemical gave r i s e to a c h a r a c t e r i s t i c spectrum of benzaldehyde. Exposure of t h i s carbonyl degradation product to NaBH^ eliminated the carbonyl absorption, leaving the phenyl absorption peak. C i r c u l a r dichroism spectra of mandelonitrile ( Figure 7B ) i s o l a t e d from the storage vestibule of H. haydeniana, and of mandelic a c i d derived by a c i d hydrolysis of glandular mandelo-n i t r i l e , showed that both these compounds were o p t i c a l l y a c t i v e . Comparison of the CD spectrum of synthetic L- and D-mandelic acids ( Figure 7A ) showed that the L-enantiomer ( S - c h i r a l i t y ) gave a + CD absorption for the phenyl region; whereas, the D-enantiomer ( R - c h i r a l i t y ) gave a -CD absorption i n the phenyl region. The mandelic acid derived from the millipede showed a -CD absorption i n the phenyl region i n d i c a t i n g that the animal synthesizes the D-(R)-mandelonitrile. The millipede's mandelo-n i t r i l e was o p t i c a l l y active (+CD),although of an opposite sign to that of the a c i d d e r i v a t i v e . Synthetic mandelic enantiomers were used to ascribe the r e l a t i o n s h i p between the sign of the CD absorption and the c h i r a l i t y , because no c h i r a l synthetic 44 mandelonitrile was a v a i l a b l e . That the o p t i c a l a c t i v i t y of the mandelonitrile and the mandelic acid derived from the millipede i s ascribable to only those chemicals i s shown by two observations. F i r s t , i n Figure 7B, the CD spectrum of the millipedes mandelonitrile ( Mn-H ) shows a negative dispersion. However, when t h i s n i t r i l e i s treated with NaOH the CD dispersion gradually vanishes ( be-comes the baseline, Mn-b ), u n t i l a l l the o p t i c a l a c t i v i t y i s destroyed. Benzaldehyde i s a c h i r a l and therefore i s not detect-ed by a CD spectrophotometer. Second, when comparing the CD spectrum of a known molar s o l u t i o n of standard L-mandelic acid ( S 2 ) to the spectrum of the millipede's mandelic a c i d ( approximately the same molarity ), i t i s observed that the CD spectra are nearly of the same i n t e n s i t y but of opposite sign. Also, i t was found that the = +13 for L-mandelic -4 acid ( 1.5 x 10 m o l e s / l i t . ) and that of the millipede ( 1.1 x 10 m o l e s / l i t . ) was ^ 1^,^= -11. Therefore, the contribution of contaminants to the o p t i c a l a c t i v i t y of the millipede's mandelonitrile i s n e g l i b i b l e . That H. haydeniana produces a c h i r a l mandelonitrile i s i n agreement with the melting point data and IR data, that suggested a non-racemate. The s t r u c t u r a l basis of the c h i r a l i t y i s portrayed i n Figure 8. 14 IV.5- RESULTS OF FEEDING C-PRECURSORS TO WHOLE LIVE HARPAPHE HAYDENIANA Table III gives the r e s u l t s of the C-precursor feeding experiments with H, haydeniana. The experiments are i n three groups, (1) low s p e c i f i c a c t i v i t y feeding ( expts. 1-9 ), F I G U R E 7 . C i r c u l a r dichroism spectra of enantiomeric mandelic acids and of mandelonitrile from Harpaphe haydeniana. s = S i n i s t e r configuration for D-mandelic a c i d . R= Rectus configuration for L-mandelic a c i d . Frame A: comparison of S- and R-mandelic acids' CD spectra ( 0.0022 Molar ). Frame B; comparison of CD spectra of S-mandelic acid, millipede mandelonitrile ( Mn-H ), and millipede mandelic acid ( Md-H ). -4 S„= S-mandelic acid at 1.5 x IQ Molar 2 at a scale of A A= 6 x 10 ^ cm. Mn-H= millipede mandelonitrile at an un-known concentration but approximately that of S j . Mn-b= baseline of Mn-H a f t e r a d d i t i o n of NaOH. Md-H= millipede mandelic a c i d at a con-centration of 1.1 x 10~ 4Molar with A £ w ^ n n = -11. TheA£ values for L-mand-e l i c acid ( frame A ) are i n agree-ment with Legrande and Viennet ( 1966 for the three small peaks. They s i t e the phenyl absorption value as + 1.3. ETOH= ethanolic baseline for Md-H, and S^ and S2« 45b F I G U R E 7 . C i r c u l a r d i c h r o i s m s p e c t r a o f e n a n t i o m e r i c m a n d e l i c a c i d s a n d o f m a n d e l o n i t r i l e f r o m H a r p a p h e  h a y d e n i a n a . 4 6 L - I s o m e r S-Chirality Sambunigrin D - I s o m e r R - C h i r a l i t y H I HOCH2 0 ' ° \ H G * 0 H B OH Prunasin Mandelonitrile F I G U R E 8 . The diagrammatic representation of the c h i r a l i t y of L- and D-enantiomers of mandelonitrile-[3-0-D-glucoside and mandelonitrile. 47 (2)competitive feedings ( expts. 10-12 ), and (3) high s p e c i f i c a c t i v i t y feedings ( expts. 13-21 ). From Table III ( expts. 1-6 ) i t i s observed that the u t l i z a t i o n of phenylalanine was s l i g h t ( 0.002% ); a d i l u t i o n value of 8,900 demonstrates t h i s poor u t i l i z a t i o n when compared to the d i l u t i o n values bf the other precursors used. The r i n g carbons and carbon-3 of phenylalanine gave r i s e to benzaldehyde and carbon-2 of the side chain of phenylalanine yielded the cyanide carbon. Since HCN and benzaldehyde ( Table I I I ) were obtained i n approximately a 1:1 r a t i o the metabolic conversion of phenyl-alanine as a C^:C_ unit to a C-:C_ ( mandelonitrile ) i s most b 3 o 2. probable. The precursors ( 1-6 and 15-17 ) are arranged i n the order that they have been shown to occur i n the plant pathway ( Figure 9 ). The u t i l i z a t i o n of the other isotopes compared to phenylalanine was much greater i n terms of percentage i n -corporations and d i l u t i o n values.These r e s u l t s c l e a r l y demon-strate the a b i l i t y of precursors 2-6 and 14-17 to function as suitable substrates for the biosynthesis of HCN and benzaldehyde i n t h i s m i l l i p e d e . The s p e c i f i c a c t i v i t y or dose ( jomoles ) at which the isotopes were fed g r e a t l y affected the incorporation 14 values. Two precursors, phenylacetaldoxime-1- C and D,L-2-hy-3 droxyphenylacetaldoxime-2a- H, when fed at a high dosage ( low s p e c i f i c a c t i v i t y ), gave r i s e to d i l u t i o n values many times lower than the high s p e c i f i c a c t i v i t y feeding counterparts ( expts. 14 15 and 17 ). The incorporation of ph e n y l a c e t o n i t r i l e - 1 - C i n terms of d i l u t i o n was only s l i g h t l y a l t e r e d by a 5.2 f o l d i n - -crease i n dose. These observations i l l u s t r a t e the s u i t a b i l i t y of the l a t t e r l a b e l l e d compounds as precursors for HCN. 48 a TABLE III Incorporation of l a b e l l e d compounds int o benzaldehyde and HCN of the millipede Harpaphe haydeniana. The incoporation data for D,L-enantiomers i s cor-rected on the assumption that only the L-isomer i s u t l i z e d in. the production of HCN and benzalde-hyde. The isotopes were injected i n t o the m i l l i -pedes. The length of each experiment was 24 hours. HCN = hydrogen cyanide c o l l e c t e d as AgCN. j2fCHO= benzaldehyde c o l l e c t e d as the 2,4-dinitrophenylhydrazone.The radio-a c t i v i t y was estimated by combustion of t h i s derivative PRECURSORS FED TO MILLIPEDES RADIOACTIVITY IW PRODUCTS ISOLATED FROM MILLIPEDES ( 10 a n i m . T l s / p x r t . ) E x p e r i m e n t number P r e c u r s o r la b 2 3 4 5 6 7 8 9 10 11 12 13a b c 14 15 16 17 18 19 20 21 S p e c i f i c A c t i v i t y ( u C i / m M o l e ) Amount l n j . / A n i m a l ( umoles ) D L - P h e n y l a l a n i n e - 2 ' • 1 4 c 1 4 , D L - P h e n y l a l a n i n e - 3 - C D L - P h e n y l s l a n i n e - r i n g - 1 4 , D L - N - H y d r o x y p h e n y l a l a n i n e - 2 . • 1 4 C 14 . P h e n y l p y r u v i c a c i d o x i m e - 2 - C • 1 4 C 1 4 , p h e n y l a c e t a l d o x i m e - l Pheny l a c o t o n l t r i l e - 1 . D L - 2 - H y d r o x y p h e n y l a c e t a l d o x i m e - 2 a -14 P h e n y l a c c t a m i d e - 1 - C 14 P h e n y l e t h y l a m l n e - 1 - C 14 P h e n y l a c e t h y d r o x a m i c a c i d - 1 - C 14 D L - N - H y d r o x y p h e n y l a l a n i n e - 2 - C + p h e n y l p y r u v i c a c i d oxime P h e n y l p y r u v i c a c i d ox ime-2 . + p h e n y l a c e t a l d o x i m e Pheny l a c e t a l d o x i m e - l - ^ C + p h e n y l a c e t o n i t r i l e 14„ D L - P h e n y l a l a n i n e - 2 . 14 , P h e n y l p y r u v i c aritl o x i m o - Z r ^ ^ C P h e n y ] a c c t o l r l o x i m o - 1 - ' ' ' 4 C P h e n y l . - ) c e t o n i t r i l o - l - * 4 C D L - 2 - H y d r o x y p h e n y l a c e t a l d o x i m e - 2 o - 3 H Pheny l a c e t a m i d e - 1 . 14, P h e n y l e t h y l a m i n e - l - 1 4 C P h e n y l a c e t h y d r o x a m i c a c i d - l - 1 4 C D L - S h i k i m a t e - G - 14 , Amount i s o l a t e d ( uMoles ) IZiCHO HCN S p e c i f i c a c t i v i t y Precursor 's D i l u t i o n (pCi /mMole) % o f tiCHO HCN c o n v e r s i o n r a d i o a c t i v i t y 89 0 .15 3 .2 2 .7 0 0 . 01 0 .002 8 ,900 82 91 0 .31 0 . 1 9 4 . 0 3 .0 4 . 6 2 .3 0 .01 0 0 0 o.ooz 0 9 ,000 82 0 .17 2 .3 0 .01 0 .002 8, 200 92 0 .14 3 .4 2 .3 0 0 .77 2 . 8 . 60 100 0 .19 5 .5 5 .0 0 0 .03 0 .08 3 ,200 62 0.22 3 .4 3 .8 0 1.6 4 . 3 39 51 0 .33 5 .4 3 .8 0 0 .72 1.5 74 34 6.24 7.5 3.8 1.0 0 9 .7 34 125 0 .18 3.8 4 . 2 0 0 0 83 0 .22 7 .1 4 . 6 0 0 0 50 0 .22 7 .4 5 .0 0 0 0 92 0 .16 + 0 .22 5 .8 7 .6 0 0 .01 0 .65 8 ,400 100 0 . 1 9 + 0 .22 7 .4 9 .3 0 0 .03 0 .16 3 ,100 62 0 .32 + 0 .22 7.2 7 . 2 . 0 0 .15 0 . 8 0 4 ,000 890 890 2600 0 .09 0 .09 0 .02 6 . 0 4 . 1 5 .0 2 .8 1.9 1.9 0 0 0 0 .07 0 .21 0 .43 0 . 0 5 0 . 1 0 0 .14 12 ,400 4 ,100 6 , 0 0 0 215 0 .12 0 .4 D.O 0 0 .19 0.-10 .1.100 440 0 .03 4 .3 4 .6 0 1.5 6 .3 300 210 0 .06 4 . 3 5.8 0 4 .4 12 •in 545 0 .01 5.3 5 .0 4 . 5 0 4 . 0 120 1300 0 .06 3 .8 6 . 9 0 0 .01 0 .05 9 8 , 0 0 0 190 0 . 0 9 5 .7 4 . 6 0 0 .005 0 . 0 1 38 ,000 130 0 .13 2 .3 5 .0 0 0 .04 0 .12 4 ,400 6800 0 .05 9 .2 8 .5 0 0 0 T A B L E I I I . I n c o r p o r a t i o n o f l a b e l l e d c o m p o u n d s i n t o HCN a n d b e n z a l d e h y d e o f t h e m i l l i p e d e H a r p a p h e h a y d e n i a n a . 4^> CO :9a FIGURE 9 . The proposed biosynthetic sequence for the production of a cyanogen in plants and mandelonitrile in Harpaphe haydeniana lead-ing to the production of HCN. (1 ) Phenylalanine (2) N-hydroxyphenylalanine (3 Phenylpyruvic acid oxime (4) Phenylacetaldoxime (5 ) Phenylacetonitrile (6) 2-Hydroxyphenylacetaldoxime ( 7 ) Mandelonitrile (8) Hydrogen cyanide (9) Benzaldehyde ( 1 0 ) Mandelonitrile-p-O-D-glucoside CCOH »C=NOH • CH2 900H •/ HOHN*CH / •CH2 /• ec-Oximino acid / ® C02 oc-Hydroxytamine 2-OH-Oxime © HCN ® + H?C-0 Aldehyde L^Glucose Glucoside The proposed biosynthetic sequence for the production of a cyanogen i n plants and mandelonitrile i n Harpaphe haydeniana leading to HCN production. 50 However, the d i l u t i o n value for phenylpyruvic a c i d 14 oxime-2- C was increased 3 f o l d by a marginal increase m dose ( expts. 3 and 14 ). These experiments do not elucidate the role of phenylpyruvic a c i d oxime as a possible precursor. Also, 14 although both phenylacetonitrile-1- C and D,L-2-hydroxyphenyl-3 acetaldox.ime-2a- H were both e f f i c i e n t l y incorporated i n t o HCN these experiments do not d i s t i n g u i s h between these compounds as alternate or obligatory intermediates i n the biosynthetic pathway ( Figure 9 ). Evidence for the possible r o l e of phenylpyruvic a c i d oxime as a precursor i n cyanogen biosynthesis i n millipedes was observed ( Table III ) i n the competitive feeding experiments ( 10 and 11 ). I f the acid oxime were not a precursor i n the pathway one would not expect t h i s a c i d to cause a large increase 14 i n the d i l u t i o n value of D /L-N-hydroxyphenylalanine-2- C (140 f o l d ) and a decrease i n percent incorporation ( 43 f o l d ). Likewise, i t would not be expected that phenylpyruvic a c i d 14 oxime-2- C ( expt. 11 ) incorporation values would be al t e r e d by the presence of equimolar amounts of phenylacetaldoxime unless the entry point for the former molecule occurred before the l a t -14 ter i n the pathway. Since p h e n y l a c e t o n i t n l e - 1 - C could also 14 i n h i b i t the incorporation of phenylacetaldoxime-1- C ( expt. 12 ) thi s i s considered as evidence that these two compounds are functional precursors i n cyanogen biosynthesis. The millipede was capable of u t i l i z i n g very small amounts 14 . 14 of phenylacetamide-1- C, phenylethylamme-1- C and phenyl-14 acethydroxamic acid-1- C when fed at low doses ( expts. 18-20 ). These are not considered as l i k e l y intermediates i n the pathway, because when they were given at higher doses ( expts. 7-9 ) no incorporation was obtained. 51 D,L-Shikimic acid-G-^C was not incorporated in t o benzaldehyde ( expt. 21 ). This i s an i n d i c a t i o n that the an i -mal i s incapable of synthesizing i t s own amino acid. IV.6- ISOLATION OF INTERMEDIATES IN THE BIOSYNTHESIS OF HCN In order to present futher evidence that several of the precursors used by H. haydeniana ( Table I I I , expts. 1-5 )for the biosynthesis of HCN were natural products, experiments were c a r r i e d out to i s o l a t e radioactive intermediates. The i s o l a t i o n of phenylacetaldoxime and phe n y l a c e t o n i t r i l e was at-tempted by i n j e c t i n g high s p e c i f i c a c t i v i t y D,L-phenylalanine-14 . . rin g - C, Both phenylacetaldoxime and phe n y l a c e t o n i t r i l e were i s o l a t e d ( Table IV ) as radioactive components of H. hayden-iana. Phenylacetaldoxime was i s o l a t e d twice with an a c t i v i t y of 300 and 400 d.p.m., and phe n y l a c e t o n i t r i l e once containing 6,500 d.p.m.. The r a d i o a c t i v i t y was shown to be present i n phenylac e t o n i t r i l e by GLC on a Carbowax 1540 ( 5%) at 140°C. with He 2 as the c a r r i e r gas ( 60 ml./min. ). Table IV also shows evidence from l a b e l l e d precursor experiments confirming the existence of mandelonitrile as a natural product of the biosynthetic pathway. These data confirm 14 that D^-phenylalanine- C i s incorporated s p e c i f i c a l l y . This was also shown by the s p e c i f i c incorporation of D,L-phenylalanine 14 C int o the HCN and benzaldehyde of O. g r a c i l i s ( Table I and Figure 6 ) . As i n O. g r a c i l i s , the presence of an emulsin s e n s i t i v e glycoside could not be detected i n H. haydeniana. Attempts were made to i s o l a t e a glycoside from H. haydeniana using a v a r i e t y 14 of C-precursors ( Table IV ). I t was possible to i s o l a t e a a radioactive f r a c t i o n which had chromatographic properties PRECURSOR INJECTED ISOLATED INTERMEDIATE DERIVATIVE OF ISOLATED INTERMEDIATE • # Compound u C i / p C i / animals Name and s t r u c t u r e D e r i v a t i v e T o t a l d.p.m.s animal mMole (hrs.of expt.) 14, 14, 14 DL-Phenylalanine-3- C 1 4 DL-Phenylalanine-2- C 1 4 DL-Phenylalanine-2- C Phenylace t o n i t r i l e - 1 - 1 4 C 14, 14 D-Glucose-U- C 1 . 2 260 1 2 : (6*) Phenylacetaldoxime | P h e n y l a c e t i c a c i d 400 1 . 2 260 1 2 : (6) , <t>CH2CH=N0H . P h e n y l a c e t o n i t r i l e (j) C H 2 C = N 300 1 . 2 260 1 2 : (6*) 1 P h e n y l a c e t i c a c i d 6 , 5 0 0 0.8 600 7: (24) M a n d e l o n i t r i l e Benzoic a c i d 600 1. 2500 2 0 : (24) , H (DC-C=N T 0 H Mandelic a c i d 2 4 , 3 0 0 0 . 1 2500 1 0 : ( 2 4 ) AgCN X000 0.4 0 . 4 212 212 2 : (6) 1 0 : ( 6 ) t S t a b i l i z e d M a n d e l o n i t r i l e ( P - D - o - g l y c o s i d e ?) AgCN ( emulsin.) ( HCl ) 510 760 0.2 260 2: (6) H C O -Glyc Benzoic a c i d 700 1 ? 4 : (5) <j> gives r a d i o a c t i v e compound(s) with same chromatographic m o b i l i t y as prunasin; approx. 800 d.p.m. . 14 . TABLE IV. Data showing the incorporation of C-precufsors into the natural intermediates i n the biosynthesis of HCN in Harpaphe haydeniana. 53 i d e n t i c a l to that of sambunigrin and prunasin. Digestion of 14 14 this fractions with emulsin yielded H CN ( as Ag CN, 510 14 d.p.m. ). Acid hydrolysis of t h i s f r a c t i o n also yielded H CN 14 ( 760 d.p.m. ), as well as benzoic a c i d - C ( 700 d.p.m. ). However, the r a d i o a c t i v i t y derived from various digestions re-presented only 1% of the t o t a l a c t i v i t y of that f r a c t i o n . 14 H. haydeniana incorporated ( Table IV ) D-glucose- C into the chromatographic f r a c t i o n corresponding to prunasin and sambunigrin. This suggested the existence of a glycoside, but hydrolysis of t h i s f r a c t i o n was not c a r r i e d out because the resul t s would have been ambiguous. I t would not have been pos-s i b l e with the techniques and the amount of material a v a i l a b l e 14 to show whether the D-glucose- C was incorporated s p e c i f i c a l l y , into the a-hydroxy p o s i t i o n of mandelonitrile as a p-glucoside. The existence of other glycosides with i d e n t i c a l chromatographic behaviour as contaminants could not be ruled out. Experiments involving the trapping of a s t a b i l i z e d 14 form of mandelonitrile ( f e e d i n g p h e n y l a c e t o n i t r i l e - 1 - C at high s p e c i f i c a c t i v i t y ), and repeated c r y s t a l l i z a t i o n of the tetra-acetate from various solvents gave a product which contained no a c t i v i t y . Thus, the s t a b i l i z e d mandelonitrile does not appear to be prunasin, nor can i t be sambunigrin, since the millipede's n i t r i l e has the R - c h i r a l i t y . Its i d e n t i t y at present remains unknown. III.7- EXPERIMENTS TO DETERMINE THE AUTONOMY OF THE GLAND To eliminate the p o s s i b i l i t y that the biosynthesis of HCN and benzaldehyde i s the ( p a r t i a l ) r e s u l t . o f symbio'tic alimentary micro-organisms, dissected glands were placed i n a 54 buffer containing D,L-phenylalanine-2- C. Af t e r several hours HCN was i s o l a t e d as AgCN ( 300 d.p.m. ) showing the glands are able to synthesize HCN independently. In another experiment the body of a whole degutted m i l l i -14 pede was f i l l e d with a sol u t i o n of D,L-phenylalanine-3- C for several hours. I t was possible to i s o l a t e radioactive benz-aldehyde ( 250 d.p.m. ) a f t e r TLC p u r i f i c a t i o n and degradation of mandelonitrile. This also indicates autonomy of the millipede i n i t s biosynthesis of HCN. 14 IV.8-THE METABOLIC FATE OF H CN 14 When several H. haydeniana were subjected to H CN for one h a l f hour, i t was possible to i s o l a t e p-cyanoalanine ( Table V ). Hydrolysis of the millipede p-cyanoalanine with 6N HCl yielded a compound with chromatographic properties i d e n t i c a l 14 to aspartic a c i d . The incorporation value of the C-precursor into p-cyanoalaine was low; the method of cut t i n g the chromato-graphic bands out t h i n l y reduced the y i e l d . Nevertheless, t h i s i s evidence that the millipede d e t o x i f i e s HCN by converting i t to p-cyanoalanine. E l u t i o n of the chromatographic band cor-responding to asparagine showed i t to be radioactive ( 400 d.p.m.) which suggests that the millipede further converts HCN to asp-aragine v i a p-cyanoalanine. Acid hydrolysis of radioactive asp-aragine from the millipede yielded radioactive a s p a r t i c a c i d . In a second experiment ( Table V ) the millipedes were 14 exposed to H CN vapours for 45 minutes, removed to fresh a i r and k i l l e d a f t e r 2 hours further.metabolism. In t h i s case p -cyanoalanine was found to contain 600 d.p.m. and the asparagine to contain 500 d.p.m.. This i s consistent with the above pathway of detoxication of HCN .( Figure 10 ). -55 14 IV.9- THE METABOLIC FATE OF BENZALDEHYDE- C AND BENZOIC ACID- 1 4C The fate of benzaldehyde produced by the decomposition of mandelonitrile was studied by exposing H. haydeniana to 14 14 vapours of benzaldehyde-carbonyl- C. Benzaldehyde- C was 14 converted to p-hydroxybenzoic acid- C ( Figure 10 and Table 14 V ). The p-hydroxybenzoic acid- C from the millipede was d i l u t e d with unlabelled acid and c r y s t a l l i z e d to a constant s p e c i f i c a c t i v i t y of 35 d.p.m./mgm. ( t o t a l a c t i v i t y 37,000 d.p.m. ). Benzoic a c i d i s o l a t e d at the same time from the millipede was found to contain 125,000 d.p.m.. I t i s assumed that benzoic acid-_1 4C had arisen a b j d o g i c a l l y by the oxidation of benzaldehyde 14 - C. 14 In separate experiments benzoic a c i d - r i n g - C and benz-14 oic acid-carboxyl- C were inj e c t e d i n t o H, haydeniana to de-termine i f benzoic acid could function as an intermediate i n the hydroxylation and oxidation of benzaldehyde to p-hydroxy-benzoic a c i d . I t i s noted ( Table X ) that the millipedes were capable of converting benzoic acid to p-hydroxybenzoic a c i d . However, no attempts were made to determine i f p-hydroxybenz-aldehyde resulted from the hydroxylation of benzaldehyde. I t was possible to i s o l a t e p-hydroxybenzoic a c i d i n microgram quantities from unlabelled i n d i v i d u a l s , suggesting that the 14 . . . conversion of the C-precursors to p-hydroxybenzoic ac i d i s not a forced detoxication. The detection of p-hydroxybenzoic a c i d as a natural product suggests that the degradation of mandelonitrile within the body of the millipede i s a continual process, for also when 14 D,L- phenylalanine- C was fed, benzoic acid and p-hydroxy-benzoic acid were found to be radioactive. The i d e n t i f i c a t i o n of PRECURSORS ADMINISTERED Compound # uCi/ uCi/ # hours animal mMole animals/ prec. expt. fed METABOLIC FATE OF PRECURSORS (or derivatives) P-Cyanoalanine Asparagine p-OH-Benzoic acid ( radioactivity i n sample i n dpm. ) H 1 4CN ( fed as vapour ) 20 15 1000 1000 15 5 3/4 (2 rest) 4000 (Asp.) 600 400 500 ( Asp.) 14 Benzaldehyde- CHO ( fed as vapour ) 2500 (2 rest) 37,000* ( 125 , 000in benzoic) 14 Benzoic- COOH acid 0.2 ( K"1" salt injected ) Q 4 14 DL-Phenylalanine-rmg- C 1 ( injected ) 2500 500 '260 2 24 24 radioactive spot 16,000 radioactive spot ( also benzoic ) * Crystallized to a constant specific a c t i v i t y . TABLE V. The metabolic fate of HCN and benzaldehyde i n Harpaphe haydeniana. cr. these two acids i n t h i s case was based soley on chromato-graphic behaviour and reactions with spray reagents. No ex-periments were undertaken to determine i f the natural decomp-o s i t i o n of mandelonitrile gave r i s e to 3-cyanoalanine. IV.10- HCN TOXICITY STUDIES Since i t was found that H. haydeniana was capable of detoxifying HCN, comparative t o x i c i t i e s of HCN to four other arthropods were investigated ( Figure 11 ). By use of the loss of mobility or coordination as a c r i t e r i o n of HCN t o x i c i t y i t was found that both the locust and the Hemipteran were approx-imately 30 times more s e n s i t i v e to HCN than the two polydesmids The p a r a j u l i d millipede was the most r e s i s t a n t . No tests were undertaken to determine the modes of resistance i n these other millipedes. IV.11-EVIDENCE FOR B-C-LYCOSIDASE AND NITRILE LYASE ACTIVITY Table VI shows that both (3-glycosidase and a-hydroxy-n i t r i l e lyase were detected i n enzyme preparation of degutted H. haydeniana. The 0-glycosidase could be shown, by removal of the phlange from frozen animals to he l o c a l i z e d within the phlange. The n i t r i l e lyase was also detected i n the l i q u i d contents of the reaction chamber by assaying dissected reaction chambers. The p-glycosidase Of H. haydeniana catalyzed the degrad-ation of prunasin, sambunigrin, and t a x i p h y l l i n . The rate of breakdown of t a x i p h y l l i n was the slowest. Addition of D-glucose caused i n h i b i t i o n of the hydrolysis of prunasin by B-glycosidas< of the milli p e d e . The rate of hydrolysis of prunasin was approx-imately 9 nmole/hour/mgm. of protein. The rate of degradation •C=N HCOH NH2 • C ^ N / ^ CH2 . a . CH2 CHNH2 Cysteine I I y or CHNH2 COOH Serine 0 * i COOH Asparagine p-Cyanoalanine cc-hydroxy-nitrile lyase t D - M a n d e l o n i t r i l e B e n z a l d e h y d e FIGURE 10. The metabolic fate of HCN and benzaldehyde r e s u l t i n g from the decomposition of mandelonitrile i n Harpaphe haydeniana 59 FIGURE 11. Graphical representation of the collapse of several insects and millipedes exposed to HCN vapours. The superscript of the i n i t i a l indicates the average weight i n mgm. of the l i v e animal. 44 H = Harpaphe haydeniana 3200 , « . L = Schistocerca gregaria ph. gregaria Oncopeltus fasciatus Polydesmus angustus Tuniulus hewitti FIGURE 11. Graphical representation of the collapse of several insects and millipedes exposed to HCN vapours. Collapse Time (min) 60 a TABLE VI. Substrate r e a c t i v i t y and Q 1 0 data for B-O-D-glycosidase and a-hydroxynitrile a c t i v i t y i n Harpaphe haydeniana The assays were c a r r i e d out at pH 5.5 i n a citrate-Na-PO^ buffer, with prunasin at a cone, of 260 pM. and a mandelonitrile cone, of 140 uM., with 2 mgm. of crude enzyme i n each assay. The degradation of prunasin was measured at 249 nm., that of t a x i p h y l l i n at 279 nm., and that of mandelonitrile at 249 nm. + = p o s i t i v e reaction - = negative reaction BREAKDOWN OF SUBSTRATES WITH ENZYMES Q l o ' S 0 F B R E A K D 0 W N OF MILLIPEDE Enzyme Substrate A c t i v i t y 10-20°C. 20-30°C. 30-39°C. P-Glycosidase + Prunasin Prunasin 1.7 0 2.6 0 a-Hydroxynitrile lyase + Mandelonitrile + Mandelonitrile + ( racemic ) 1.1 1.4 2.6 1.8 P-Glycosidase + Taxiphyllin + 1 Sambunigrin + a-Hydroxynitrile lyase p-OH-Mandelo- + r nxtrixe TABLE VI. Substrate .reactivity and QIQ data for p-D-glycosidase and c t-hydroxynitrile lyase a c t i v i t y i n Harpaphe haydeniana. . 61 of mandelonitrile by the lyase was much faster, approximately 4.5 pmoles/hour/mgm. of protein. These reaction rates show that the B-glycosidase a c t i v i t y i n v i t r o cannot account quant-i t a t i v e l y for the release of several micrograms of HCN secreted by H. haydeniana when alarmed. The n i t r i l e lyase can account quantitatively for t h i s rapid release of HCN since the enzyme i n v i t r o , the amount of enzyme i n l/o of an animal/could produce several micrograms of HCN from mandelonitrile i n a few seconds. Table VI shows the r e a c t i v i t y of the enzymes to various substrates,and Figure 12 the pH optimum of both enzymes. At the pH optimum for the n i t r i l e lyase a c t i v i t y the decay of mandelonitrile i s n e g l i g i b l e compared to the degradation caused by the enzyme. Boiled enzyme preparations showed no a c t i v i t y to-wards various substrates. Both enzymes showed an increase i n r e a c t i v i t y with i n -creasing substrate concentrations or increasing enzyme concen-t r a t i o n s . TheB-glycosidase and n i t r i l e lyase a c t i v i t i e s showed Q^0 values expected of enzymic systems ( Table VI ); whereas, a physical system such as the decomposition of mandelontrile showed lower Q,n values. 10 IV.12-GR0SS MORPHOLOGY AND MICROANATOMY OF THE CYANOGENIC GLAND Some external morphological aspects have been previously discussed on pg. 8-9. The pore of the cyanogenic gland i s surrounded by a s l i g h t l y raised c o l l a r inside a large p i t ( Figure 3A ). This can be more c l e a r l y seen by the electron-micrographic scans of a phlange ( Figure 13A ). The pore ( approximately 25 u i n diameter ) i s situated i n a p i t approx-FIGURE 12. The e f f e c t of pH upon the a c t i v i t y of (3-gly-cosidase and a-hydroxynitrile lyase from Harpaphe haydeniana. 2 3 4 5 6 7 8 9 10 pH units Reactions c a r r i e d out at 30 WC. i n a citrate-Na2PC" 4 buffer ( o.05 M. pH 2-7 ), and i n a Tris-HCl buffer ( 0.1 M., pH 9 ), t o t a l volume 3 ml. Hydrolysis of prunasin ( 260 pM. + 0.6 mgm. protein ) measured at 249 nm.. Lysis of mandelonitrile ( 70 pM. +0.3 mgm. protein ) measured at 2 79 nm.. a-hydroxynitrile lyase a c t i v i t y o = g-glycosidase a c t i v i t y • = mandelonitrile decay The v a r i a t i o n indicated at each point represents the extremes of three r e p l i c a t e s . imately 100 p i n diameter. A closer view of the pore ( Figure 133 ) with i t s c o l l a r shows that the pore channel ( approxi-mately 30-40 p i n diameter ) extends in t o the reaction chamber. At the bottom of the pore channel the lumen of the reaction chamber begins as the sharp t r a n s i t i o n between the smooth wall of the pore channel and the fuzzy p r e c i p i t a t e within the chambe A clearer picture of the p i t or trough surrounding the c o l l a r i s seen i n Figure 13C. The sculpturing of the c u t i c l e ( Figure 13C ) makes the surface of the p i t a poor trapping area which i s not l i k e l y to be wet by many l i q u i d s . The double chambered nature of the cyanogenic gland i s diagrammed i n Figure 14. The large r e s i l i e n t and f l e x i b l e storage vestibule ( sv ) narrows to a neck ( nk ) on which i s attached "a muscle c o n t r o l l i n g the c o n s t r i c t i o n i n the neck ( cs ). The neck i n s e r t s i t s e l f into the reaction chamber ( rc as a nozzle ( nz ). In t h i s region both the wrinkled neck and the point of attachment, the nozzle, are r i g i d . The wall of the reaction chamber i s thicker than that of the storage vest-i b u l e . I t was observed that both chambers contained a f l u i d . The storage vestibule contained mandelonitrile ( droplet 200-300 p i n diameter ). The r i g i d reaction chamber narrows to a short neck or pore channel, which leads to the external pore ( Pp). This pore channel and reaction chamber have no c o n s t r i c t i o n s or valves preventing f l u i d from moving to the e x t e r i o r . The sides of the pore channel ( Figure 13B ) are r e l a t i v e l y smooth except for s l i g h t undulations i n the c u t i c l e and the small depressions where dermal gland ducts e x i t . The reaction chamber i n fr e s h l y s a c r i f i c e d animals was was found to be nearly f u l l of a cl e a r f l u i d which wet the FIGURE 13. Scanning electron-micrographs of the phlange of Harpaphe haydeniana. Frame A: The phlange of the metazonite points up-wards. The pore of the cyanogenic gland i s situated i n the bottom of a p i t . The pore i s surrounded by a low c o l l a r . The crest of the c o l l a r i s lower than the crest of the p i t w a l l . The t i p of the phlange points towards the rear of the mill i p e d e . Frame B: This expanded view of the pore shows more c l e a r l y the c o l l a r that borders the pore. On the r i g h t , the wall of the pore channel extends inward to the reaction chamber. The area of t r a n s i t i o n between the pore channel and the reaction chamber i s seen as a d i s t i n c t v i s u a l demarcation between the smooth wall of the pore channel and a fuzzy amorphous material, which i s probably dehydrated enzyme. On the wall of the pore channel small depressions are seen. These are assumed to be the o r i f i c e s of gland c e l l ducts ( see F i g . 16 ). Frame C: This expanded view of the pore delineates c l e a r l y the shape of the c o l l a r i n perspect-ive with the p i t and the wall of the trough. The lack of prominent sculpturing of the c u t i c l e i n the p i t area and on the c o l l a r are apparent. 66 Mt = metazonite M = muscle m = point of attachment of muscle Nz = nozzle Nk = neck Pp = pore and p i t area Pr = prozonite Rc = reaction chmaber SV = storage vestibule FIGURE 14* Diagrammatic representation of the cyano-genic gland of Harpaphe haydeniana. 6 7 surface of the reaction chamber. I f the animals were anaes-thesized before d i s s e c t i o n i t was noted that the meniscus began just where the pore channel started. But, i f the animals were disturbed manually and then dissected , the raensicus was lower down the wall of the reaction chamber ( about 20% loss ). By the addition of droplets of water, benzaldehyde or mandelo-n i t r i l e to the p i t area of the gland, i t was found that t h i s area was non-wettable. On the other hand, the reaction chamber, when dissected from the animal and cleared of i t s contents, was found to be wettable by these three l i q u i d s . Also, the pore channel served as a c a p i l l a r y tube for these three l i q u i d s . Chemical studies have shown that H. haydeniana stores mandelonitrile. Morphological evidence shows (Figure 15)that t h i s millipede stores mandelonitrile. In t h i s p icture droplets of mandelonitrile can be seen i n the storage v e s t i b u l e . Each vestibule has one droplet i n properly dissected m i l l i p e d e s . This droplet does not wet the sides of the vestibule, but, the neck of the storage vestibule acts as a c a p i l l a r y tube f o r the n i t r i l e . In d i s s e c t i o n the droplet of n i t r i l e i s always found to be held i n the sac nearest to the neck. Even by probing with a f i n e glass poker i t i s d i f f i c u l t to dislodge the droplet. A-dissected two-chambered gland of H. haydeniana i s shown i n Figure 15A. The reaction chamber ( rc ) has been broken away from the pore at the end of the neck. The reaction chamber can be seen to be p a r t i a l l y surrounded by the yellow tissue ( yt ) of the phlange. This yellow tissue also covers from view the area of attachment of the storage vestibule ( sv ). The other two pictures ( Figure 15B and 15C ) more c l e a r l y delineate the storage vestibule with i t s droplets of mandelonitrile ( d ). Picture B shows the r e l a t i v e s i z e of the two chambers. The reaction chamber i s approximately 150 p. long including the neck 68 and about 150 u wide. I t i s not p e r f e c t l y round as indicated i n Figure 14. The storage vestibule i s approximately 1200 u by 500 u i f expanded. The words " vestibule" and "chamber" are used interchangeably. Figure 15C shows that about both chambers are c l o s e l y appressed tissue ( yt and at ) of unknown function. The major-i t y of tissues surrounding the gland have been teased away. IV.13-LIGHT-MICRQSCOPIC STUDIES OF THE CYANOGENIC GLAND The results of a l i g h t microscopic analysis of the cyanogenic gland of Harpaphe haydeniana are shown i n Figure 16. An oblique cross-section of the reaction chamber ( rc )( F i g -ure 16A shows the wrinkled neck (nk) a r i s i n g from the t h i n wall of the storage vestibule ( sv ). This neck protrudes as a nozzle ( nz ) i n t o the lumen of the reaction chamber ( rc ). The rounded reaction chamber ( 200 ja i n diameter ) has a-much thicker wall ( 10 u ) than the storage vestibule ( 1 u ). The wall of the reaction chamber on the haemolymph side i s surround-ed by various tissues ( Figure 16A and 16B ), namely the yellow tissue ( yt ) of the phlange, and the dark s t a i n i n g "attached tissue" ( at ) adhering to what can be considered the epidermis ( ep , see Figure 16C ). The r e l a t i o n s h i p of these tissues i s not understood. The reaction chamber ( Figure 16B ) seems to be the re-s u l t of the invagination of the c u t i c l e ( cu ). The epidermis ( ep ) l i n i n g the inside wall of the c u t i c l e i s continuous over the c u t i c u l a r invagination; although, the overlying yellow tiss u e ( yt ) obscures t h i s . In both Figure 16A and 16B, an amorphous material i s seen within the lumen of the reaction chamber. This FIGURE 15. photographs of the dissected cyanogenic glandular apparatus of Harpaphe haydeniana. Frame A: the reaction chamber ( rc ) surrounded by the yellow tiss u e of the phlange ( yt ). A droplet ( d ) of mandelonitrile i s i n the storage vestibule ( sv ). Frame B: the droplet of mandelonitrile ( d ) shown more c l e a r l y . The broken neck of the reaction chamber ( rc ) leads to the pore on the phlange. Frame C: showing more c l e a r l y the adherence of the yellow tissue ( yt ) and the "attached tissue" ( at ) to the two chambers. 69* d X B F i g u r e FIGURE 16. Light microscopic analysis of the cyanogenic glandular apparatus of Harpaphe haydeniana. rc= reaction chamber yt= yellow t i s s u e nz= nozzle of rc ep= epidermis nk= neck of sv p= pore or rc at= attached tissue cu= c u t i c l e cs= c o n s t r i c t i o n of nk ex= sculpturing on cu n= nucleus w= wall of sv m= muscle from cu to cs t= tissu e about sv gd= gland c e l l duct dp= pore of gd sv= storage vestibule co= c o l l a r Frame A/ cross-section of rc showing attachment to sv, and nk and nz of sv. B/ cross-section of rc showing p of phlange. C/ cross-section of re's p showing co. D/ cross-section of rc showing attachment of m to nk and cs. E/ cross-section of w of sv showing n and in f o l d i n g s of cu. F/ cross-section of w of rc showing ep from which arises the gd e x i t i n g as a dp i n the lumen of the r c . G/ cross-section of nk showing attachment of m to cs. H/ shows o r i g i n and i n s e r t i o n of m. 1 / cross-section of w of sv i n d i c a t i n g n. 71 material i s considered to be the p r e c i p i t a t e d enzyme. The morphology of the pore area i s c l e a r l y shown i n Figure 16B and 16C. The pore ( p; 30 y i n diameter ) opens to the e x t e r i o r v i a an unblocked channel terminating i n a small c o l l a r ( co ) of c u t i c u l a r material ( ex )( 15 u high ). This c o l l a r i s situated i n a larger concavity ( 150 y i n diameter ), somewhat akin to an evaporatorium, but with l i t t l e external sculpturing ( indicated at ex ). Another s a l i e n t feature of the c u t i c l e i s exhibited i n Figure 16C. This i s the presence of gland c e l l ducts ( gd ) that arise from the epidermal area ( ep ). Inclusions from the epidermal area are seen i n the gland c e l l ducts of the cu-t i c l e . The nature of the c e l l s giving r i s e to these inc l u s i o n s was not investigated. These gland c e l l ducts penetrate the c u t i c l e to the e p i c u t i c l e ( observe at co ). The modification, of these gland ducts within the wall of the reaction chamber ( Figure 16F ) i s considered to be associated with the secre-t i o n of enzyme int o the reaction chamber. The gland ducts ( gd ) of the reaction chamber are empty of the i n c l u s i o n s found i n s i m i l a r ducts of the exoskeletal region ( Figure 16C ). These ducts also arise from the epidermal area ( ep ) of the reaction chamber, each ending as a small open pore ( dp; 1.5 u i n d i a -meter ) i n the lumen of the chamber. The wall of the storage vestibule ( sv; Figure 16 ) i s tough and f l e x i b l e . The luminal side of the wall ( w ) appears to have a wrinkled inner l i n i n g which i s probably c u t i c u l a r (cu). The tissues on the serosal side of the wall are nucleated ( n ). The presence of n u c l e i i n the secretory c e l l s of the storage vestibule i s more c l e a r l y seen i n Figure 161 . This view i s possible because the sectioned wall f e l l on i t s side during 72 staining and mounting. Shown i n Figure 16D i s the muscle that originates from nearby c u t i c l e ( cu; see Figure 14 ) and i n s e r t s on the neck of the storage vestibule just behind the nozzle ( nz ) of the reaction chamber. The nature of the c o n s t r i c t i o n ( cs ) i s more c l e a r l y shown i n Figure 16G, where the muscle ( m ) i s attached to a h e e l - l i k e structure of the neck ( nk ). The o r i -gin and i n s e r t i o n of the muscle are also demonstrated i n . Figure 16H. Note the p o s i t i o n of t h i s muscle i n Figure 14 i n respect to the whole diplosegment. V- DISCUSSION Cyanogenesis i s not an i s o l a t e d phenomenon i n the plant world, i t occurs i n about 25 families of plants that are not taxonomically r e l a t e d ( Eyjolfsson, 1970; Mahadevan, 1973 ). Its presence a f f e c t s man to some extent because of the high concentrations of cyanogens i n c e r t a i n plant-foods. The t i p s of immature bamboo roots have been reported to contain as much as 800 mgm. of HCN per 100 gm. of shoots. Other products such as tapioca, b i t t e r almonds, and lima beans contain 10 - 250 mgm. of HCN per 100 gm,of tissue ( Montgomery, 1969 ). Cases of human poisoning and deaths from HCN have been reported as a r e s u l t of eating apricot seeds, almond seeds, and bamboo shoots ( Mont-gomery, 1969; Sayre and Kaymakcalans, 1964 ). The p r i n c i p a l cyanogens encountered by man are amygdalin, l i n i m a r i n and dhurrin. Although small d a i l y doses are d e t o x i f i e d ( L D ^ Q of HCN o r a l l y i s 0.5-3.5 mgm./kg. of body weight .), cyanide i s suspected of causing chronic poisoning p a r t i c u l a r i l y i n A f r i c a where cyanogenic plants are staple foods. These chronic devast-ations, some of which are the r e s u l t of smoking tobacco, are discussed by Montgomery ( 1969 ). The b r i g h t l y coloured polydesmid millipede Harpaphe hay-deniana contains 27 mgm. of HCN per gm. of millipede ( 12 /tf. animal ). How an animal can store t h i s quantity of HCN and not suff e r from HCN poisoning poses an i n t e r e s t i n g problem. This question l e d b i o l o g i s t s to discover that millipedes store cyan-ide i n chemical forms s i m i l a r to those known to occur i n plants. The exact nature of t h i s biochemical p a r a l l e l has not been previously studied i n d e t a i l . The following discussion expands the understanding of t h i s p a r a l l e l between millipedes and plants . . . . 1 4 Experiments on the u t i l i z a t i o n of phenylalanine- C as precursors for HCN biosynthesis i n another polydesmid millipede, Oxidus g r a c i l i s , indicated that the carbons of the benzene r i n g and carbon-3 of the side chain were incorporated i n t o benzajde-14 hyde- C, and that carbon-2 of the side chain produced the 14 C-cyanide carbon ( Table I and Figure 6 ). These observations prompted further exploration of the hypothesis that millipedes elaborate HCN and benzaldehyde v i a a pathway s i m i l a r to that described i n plants ( Tapper and Butler, 1971 ). In the more detailed experiments with H. haydeniana a v a r i e t y of precursors known to function as precursors for cyanogenesis i n plants, plus several other phenylethane homologues were used ( Table III ). The r e s u l t s with H. haydeniana c l e a r l y e s t a b l i s h that this animal has the enzymatic a b i l i t y to u t i l i z e each of the precursors ( 1-6 and 13-17 i n Table III ) as a substrate for the biosynthesis of HCN and benzaldheyde. I t i s most u n l i k e l y that the incorporation values obtained for these i s o t o p i c precursors over a 24 hour feeding period could be explained by dispropor-74 ti o n a t i o n ( 1 mole of the N-hydroxy-amino ac i d degrading to h mole of the amino acid and % mole of the oxime ), or by the conversion i n aqueous medium of phenylacetaldoxime to phenyl-a c e t o n i t r i l e ( Ahmad and Spenser, 1961; Kindl and U n d e r h i l l , 1968b ). This l a s t reaction i s slow, and does not give r i s e to the h y d r o x y n i t r i l e . It was also observed ( Table III ) that the precursors ( 1-6 and 13-17 ) were u t i l i z e d d i f f e r e n t i a l l y by the millipede; that is, a trend i s exhibited for an increase i n percent incor-poration, and a decrease i n the d i l u t i o n value the closer the precursor was to the end of the biochemical sequence ( Figure 17 ), Using t h i s as a suggestive index of order i n the biochem-i c a l pathway, the r e s u l t s show that the millipede u t i l i z e d the isotopes i n a manner s i m i l a r to plants, but with some d i f f e r -ences to be discussed below. I t has been established i n plants ( B l e i c h e r t et a l p , 1966; Butler and Conn, 1964; Underhill and Chisholm, 1964;Uribe and Conn, 1966 ) that the amino nitrogen i s retained i n the bio-synthesis of cyanogenic glycosides. This needs to be determined for H. haydeniana. I t has also been shown during cyanogen and glucosinolate biosynthesis i n plants that the Cg-C2~N unit re-mains i n t a c t (Bleichert et al.,1966; Butler and Conn, 1964; Underhill and Chisholm, 1964; Uribe and Conn, 1966 ). I t i s as-sumed that t h i s i s the case i n H. haydeniana rather than the s i t u a t i o n observed i n ephedrine biosythesis i n Ephedra . In t h i s plant phenylalanine i s f i r s t converted to benzoic acid, and the carbon-2 and the nitrogen added to the Cg-C-^ unit v i a an unknown C 2-N unit to form a C6-C3-N compound ( Yamasaki et al.,1973 ). Comparing the millipede feeding experiments to the s i t -uation i n plants ( Ben-Yehoshua and Conn, 1964; Tapper et a l . , 1972 1972; Tapper and Butler, 1971 ), the most obvious difference COOH H 2 N * C H • C H , COOH I HOHN*CH / • C H 2 / • / Amino acid t-Hydroxylamine 2-OH-Oxime HC»N ® + H ? C - 0 Aldehyde C'N H t f C H ^ 2-OH-Nitri le L-»Glucose »C-N GlucoseO?CH 0 Glucoside FIGURE 17. Pathway for the biosynthesis of HCN and benzaldehyde i n Harpaphe haydeniana. 76 1 4 i s the low incorporation of D, L-phenylalanme- C ( at high and low doses ) by the m i l l i p e d e . This low incorporation was also observed to be the case for 0. g r a c i l i s ( Table I ). The i n e f -f i c i e n c y of u t i l i z a t i o n i s probably the r e s u l t of permeability r e s t r i c t i o n s and metabolic diversions. The formation of an N-hydroxy-amino acid, N-hydroxy-glycine, from glycine leading to the production of a c y c l i c ' hydroxamic acid, hadacidin ( Stevens and Emery, 1966 ), the incorporation of S-N-hydroxyornithine i n t o ferrichrome ( Em-ery, 1966 ), and the requirement for NADPH i n aromatic amino acid N-hydroxylation ( Kindl, 1968; Kindl and U n d e r h i l l , 1968a ) document the presence of N-hydroxylating systems i n organisms. I t seems reasonable from the l a b e l l i n g studies i n plants ( Hahl-brock and Conn, 1970; Kindl and Und e r h i l l , 1968b; Tapper et a l . , 1972 )to suggest that the order of the f i r s t steps i n the bio-synthesis of glucosinolates and cyanogens i s from the analogous amino ac i d v i a the N-hydroxy-amino acid . In Table III the low 14 d i l u t i o n value ( 60 ) for N-hydroxyphenylalanine-2- C shows i t to be a more e f f i c i e n t precursor for HCN biosynthesis than 14 i s D,L-phenylalanme- C. This suggests that the millipede uses the N-hydroxylation process i n incorporating the amino ac i d into benzaldehyde and HCN. Conjecture about phenylpyruvic acid oxime being an i n t e r -mediate remains i n respect to plants ( Kindl and Underhill,1968b; Tapper and Butler, 1971 ). From our studies i t was not possible to v e r i f y i t s function. In both feeding experiments ( 3 and 14 ) d i l u t i o n values were high, although less than with phenylalanine. The low incorporations may be the r e s u l t of t o x i c i t y , since i n experiments to f i n d unlabelled dose tolerances the oximes proved to be quite t o x i c . The t o x i c i t y of phenylpyruvic acid 7 7 o x i m e h a s a l s o b e e n s u s p e c t e d o f r e d u c i n g i n c o r p o r a t i o n ( K i n d l a n d U n d e r h i l l 1 9 6 8 b ; T a p p e r a n d B u t l e r , 1 9 7 1 ) . H o w e v e r , s i n c e p h e n y l p y r u v i c a c i d o x i m e i n t h e p r e s e n c e o f o f D , L - N - h y d r o x y -1 4 p h e n y l a l a n i n e - 2 - C ( e x p t . 1 0 ) r e d u c e d t h e i n c o r p o r a t i o n o f t h e l a t t e r c h e m i c a l a n d i n c r e a s e d t h e d i l u t i o n s 1 4 0 f o l d , t h e f o r m e r m a y a c t u a l l y b e a f u n c t i o n a l i n t e r m e d i a t e . P h e n y l a c e t a l d -o x i m e a l s o g r e a t l y r e d u c e d t h e u t i l i z a t i o n o f p h e n y l p y r u v i c 1 4 a c i d o x i m e - 2 - C ( e x p t . 1 1 ) . T h e s e d a t a s u p p o r t t h e p r o p o s e d o r d e r i n F i g u r e 1 7 . H o w e v e r , t h e y d o n o t e l i m i n a t e t h e p o s s i -b i l i t y t h a t p h e n y l p y r u v i c a c i d o x i m e e n t e r s t h e p a t h w a y ( r a t h e r t h a n b e i n g p a r t o f i t ) b e t w e e n N - h y d r o x y p h e n y l a l a n i n e a n d p h e n y l a c e t a l d o x i m e . T h e i n c o r p o r a t i o n o f t h e o t h e r i s o t o p e s , p h e n y l a c e t a l d -1 4 3 o x i m e - 1 - C , a n d 2 - h y d r o x y p h e n y l a c e t a l d o x i m e - 2 a - H w a s s u f f i c i e n t t o w a r r a n t t h e i r c o n s i d e r a t i o n a s i n t e r m e d i a t e s a s d e p i c t e d i n F i g u r e 1 7 . I t i s n o t e d b y c o m p a r i n g e x p e r i m e n t s 1 - 6 w i t h 1 3 - 1 7 t h a t t h e e f f e c t o f d o s e i s a f a c t o r w h i c h g r e a t l y a l t e r s d i l u t i o n v a l u e s . T h e r e i s a n e x c e l l e n t c o r r e l a t i o n f o r t h i s p a t h -w a y b e t w e e n t h e o r d e r t h a t m u s t o c c u r c h e m i c a l l y a n d t h e o r d e r . d e r i v e d f r o m e x p e r i m e n t s 1 3 - 1 7 . H o w e v e r , t h e s e s t u d i e s d o n o t a n s w e r w h e t h e r p h e n y l a c e t o n i t r i l e a n d / o r 2 - h y d r o x y p h e n y l a c e t a l d -o x i m e i s t h e b i o l o g i c a l p r e c u r s o r f o r H C N . T h e g r e a t e r c o n v e r s i o n 3 o f t h e H - o x i m e i s m o r e s a f e l y c o n s i d e r e d t o b e t h e r e s u l t o f o f i t s b e t t e r s o l u b i l i t y a n d l e s s e r t o x i c i t y . 1 4 E x p e r i m e n t s 7 - 9 i n d i c a t e t h a t p h e n y l a c e t a m i d e - 1 - C , 1 4 1 4 p h e n y l e t h y l a m i n e - 1 - C , a n d p h e n y l a c e t h y d r o x a m i c a c i d - 1 - C a r e p r o b a b l y n o t p r e c u r s o r s o n t h e b i o g e n i c p a t h w a y . H o w e v e r , t h e s m a l l i n c o r p o r a t i o n s ( e x p t s . 1 9 - 2 1 ) m a y r e f l e c t t h e a n i m a l ' s a b i l i t y t o u t i l i z e , i n m i n o r a m o u n t s , s t r u c t u r a l l y s i m i l a r m o l -c u l e s a c q u i r e d f r o m i t s f o o d . 78 H. haydeniana was not able to u t i l i z e D,L-shikimate-14 • . G- C as a precursor for mandelonitrile biosynthesis. This suggests that the animal r e l i e s on an exogenous source or sym-b i o t i c ( gut ) associations for i t s aromatic precursors, and therefore does not possess the shikimate pathway. The evidence for the proposed intermediates i n plants i s not documented solely by l a b e l l i n g patterns i n end products from i n vivo tracer studies. P a r t i a l evidence for the i s o l a t i o n of intermediates i n l i n i m a r i n biosynthesis i s given by Tapper et a l . ( 1972 ). Underhill ( 1967 ) has i s o l a t e d a radioactive product 14 considered to be phenylacetaldoxime- C during biosynthetic stud-ies on glucosinolates. The cyanogenic pathway and glucosinolate pathway have i n common the f i r s t three intermediates outlined i n Figure 17. Studying the biosynthesis of churrin Farnden et a l . ( 1973 ) have shown p-hydroxyphenylacetaldoxime to be a radio-active intermediate. For a more d e t a i l e d discussion of the e v i -dence for the existence of these pathways r e f e r to Conn's review ( 1974 ). Enzymic evidence also exists for the presence of these pathways i n plants. An i n v i t r o enzyme system has been i s o l a t e d by Kindl and U n d e r h i l l ( 1968b ) which converts phenylalanine to phenylacetaldoxime. Other studies on a l i p h a t i c and aromatic cyanogens have shown the presence of glucosyl transferases ( Conn, 1974; Hahlbrock and Conn, 1970 ), and the existence of an a-hydroxynitrile lyase ( Bove and Conn, 1961; Conn, 1974; Haisman and Knight, 1967; Seeley et a l . , 1966 ). With respect to H. haydeniana the v a l i d i t y of postulating that t h i s arthropod synthesizes HCN i n a manner s i m i l a r to that of plants i s substantiated not only by the incorporation data from the precursor feeding experiments ( Table III ) but also 79 by enzymic and intermediate-isolation data. I t has been shown that phenylacetaldoxime, phenylacetonitrile, mandelonitrile and a glycoside of mandelonitrile could be i s o l a t e d as radio-14 active natural products a r i s i n g from C-precursor studies ( Table IV ).Although, the incorporation of D,L-phenylalanine-14 C into these intermediates was low ( less than 0.01 % ) i t was s t i l l greater than the incorporation obtained i n HCN re-recovered from mandelonitrile ( Table I I I , expts. 1 and 13 ). The existence of large quantities of mandelonitrile i n H. haydeniana i s an important fact i n assessing the model of HCN production. Morphological evidence ( Figure 15 ) shows that that t h i s chemical i s stored i n the form of a large droplet i n the storage vestibule of the gland. In unalarmed animals t h i s droplet can occupy most of the storage sac. Several types of chemical evidence v e r i f i e d that the storage product i s mandelo-n i t r i l e : ( 1 ) i t s UV spectrum i s i d e n t i c a l to a synthetic sample, and ( 2 ) upon decomposition i t gave r i s e to benzaldehyde and HCN. Both these chemicals ( 3 ) can be i s o l a t e d containing radio-14 a c t i v i t y i f the millipede i s fed D,L-phenylalanine- C. The l a b e l l i n g of the n i t r i l e i s s p e c i f i c . The n i t r i l e ( 4 ) can be converted to mandelic acid and shown to have c h i r a l i t y ; thus the millipedes n i t r i l e was found to be D-(R)-mandelonitrile. Barbetta et a l . ( 1966 ) have previously shown the occurrence of D-(+)-mandelonitrile i n a millipede, but did not ascribe an absolute configuration. The importance of mandelonitrile as a storage product w i l l be discussed l a t e r . The existence of a cyanogenic glycoside, isopropyl-mandelonitrile glucoside ( Figure IB )has been demonstrated ( Pallares, 1946 ). The benzoyl ester of mandelonitrile has been i s o l a t e d from Polydesmus c o l l a r i s c o l l a r i s ( Casnati et a l . , 1963 ). Circumstantial evidence for the existence of glycosides 18. Proposed morphological and biochemical model of the cyanogenic glandular appar-atus of a polydesmid millipede, Harpaphe haydeniana. Md = droplet of mandelonitrile Ep = epidermis Yt = yellow tissue Gd = gland c e l l duct M = muscle Enz - P-glycosidase i n the storage chamber and a-hydroxynitrile lyase i n the reaction chamber. 80 b FUGURE 18. Proposed morphological and biochemical model of the cyanogenic apparatus of a polydesmid millipede, Harpaphe haydeniana. 81 has been based on the i d e n t i f i c a t i o n of glucose and dissach-arides i n the secretions which produce HCN ( Eisner et a l . , 1963a;Moore, 1967; Woodring and Blum, 1963 ), as well as being based on the i s o l a t i o n of mandelonitrile ( Barbetta et a l . , 1966; Eisner et a l . , 1963a ). These data are unclear i n demonstrating whether a n i t r i l e and/or a cyanogenic glycoside constitute the major storage product. In the model of HCN production i n a polydesmid millipede ( Figure 2; Eisner and Eisner, 1965; Eisner et a l . , 1963a; Eisner and Meinwald, 1966 ) mandelonitrile i s postulated to be the compound preceeding HCN formation. This postulation was based on an extractable chemical with an Rf value s i m i l a r to mandelonitrile from which benzaldehyde and HCN were formed. I t has also been postulated that Pachydesmus  crassicatus might store the glycoside ( Blum and Woodring, 1962; Woodring and Blum, 1963 ). Ethanolic extracts of H. haydeniana analyzed by chromato-graphy and reagent detection systems f a i l e d to show the presence of a mandelonitrile glycoside. However, experiments employing 14 14 D,L-phenylalanine-ring- C, D, L-phenylalanme-2- C and phenyl-14 a c e t o n t r i l e - 1 - C as precursors ( Table IV ) showed that a s t a -b i l i z e d form of mandelonitrile could be i s o l a t e d . This chemical when digested by B-glycosidase or HCl yielded radioactive HCN and benzoic acid ( Table IV ). Although t h i s stable form of the n i t r i l e was i d e n t i c a l to sambunigrin and prunasin chromatograph-i c a l l y , i t was not chemically i d e n t i c a l , I t also appeared to 14 be contaminated with other compounds since the y i e l d of H CN was less than 1% of the t o t a l r a d i o a c t i v i t y i n that chromato-graphic f r a c t i o n . These contaminants might be glycosides, and 14 thus /the incorporation of D-glucose- C into the s t a b i l i z e d compound must be considered ambiguous. These experiments do not i d e n t i f y the exact chemical nature of the s t a b i l i z e d mand-82 e l o n i t r i l e , but indicate that a (3-glycosidase s e n s i t i v e form i s present i n very low concentrations. This glycoside i s pro-bably quickly hydrolyzed to the n i t r i l e preparatory for storage. The millipede's glycoside does not appear to be prunasin ( (3-0-D-glucoside , see Figure IA and 8 ), because the t e t r a -acetate d e r i v a t i v e of prunasin was net c o - c r y s t a l l i z a b l e with the radioactive millipede glycoside. I t appears that other arthropods may also make compounds such as amygdalin ( a (3-0-D-l-6-diglucoside of mandelonitrile ). A dissacharide,as well as glucose, e x i s t i n the l i b e r a t e d defensive secretion of beetles and millipedes ( Moore, 1967; Woodring and Blum, 1963 ). Cursory examination of the secretion of H. haydeniana f a i l e d to show the presence of any sugars. According to the model ( Figure 18) two enzymes must be present, a (3-glycosidase ( EC 3.2.1.21 ) and an a-hydroxynitrile lyase ( EC 4.1.2.10 ).. Evidence for the l a t t e r enzyme ( Eisner et a l . , 1963a ) was based on the mixing of the contents of the storage vestibule and the reaction chamber. This mixture produced HCN ; whereas, neither chamber by i t s e l f was capable of t h i s . This approach i s ambiguous. However, i t was possible to i s o l a t e from degutted H. haydeniana ( or i t s phlanges ) a p a r t i a l l y p u r i f i e d enzyme which catalyzed the degradation of mandelonitrile, as well as p-hydroxymandelonitrile. This enzyme was shown to be contained i n the reaction chamber. The enzymic a c t i v i t y present i n extracts of phlanges was equivalent to the degutted body extracts. Thus, a l l the a c t i v i t y i n the degutted body extracts was assumed to come from the gland alone. The a c t i v i t y of the enzyme can account q u a n t i t a t i v e l y for the re-lease of HCN i n a l i v e animal. No attempts were made to p u r i f y t h i s lyase beyond the f i r s t ammonium sulphate p r e c i p i t a t i o n . 83 No evidence has been obtained previously for the ex-istence of a [3-glycosidase connected with the formation of mandelonitrile. (3-glycosidase a c t i v i t y has been demonstrated histochemically by Happ ( 1968 ) i n the tenebrionid beetle Eleodes l o n g i c o l l i s , which produces benzoquinones, and the corresponding hydroquinones and glycosides. The enzyme a c t i v i t y 1 was shown to reside i n the wall of the secretory t i s s u e of the gland. A complicating factor i n evidencing (3-glycosidase i n H. haydeniana i s that t h i s enzyme along with a-glycosidase and other sugar digesting enzymes exists i n the gut of many arthro-pods ( Evans and Payne, 1964; Ito and Tanaka, 1959; Retief and Hewitt, 1973a,b ). These enzymes can also be detected i n the f a t body, and enzymes such as a-glycosidase have been detected i n sensory h a i r s which are associated with feeding behaviour ( K i -jima et a l . , 1973 ). One can also suspect the existence of 3-glycosidases directed at the hydrolysis of aromatic glycosides i n arthropod guts such as known i n rats ( p h l o r i d z i n hydrolase; Colombo et a l . , 1974 ). The great v a r i e t y of glycosidases and t h e i r d i s t r i b u t i o n within an animal make the determination of a [3-glycosidase a c t i v i t y i n extracts of degutted animals ambig-uous . The (3-glycosidase derived from H. haydeniana showed the classical enzyme responses to substrate and temperature as did the lyase. This (3-glycosidase was capapb]e of hydrolyzing prunasin, sambunigrin, and t a x i p h y l l i n . The hydrolysis of prunasin was i n -h i b i t e d by D-glucose,a known i n h i b i t o r ( Barman, 1969 )( Table VI ) . Although the (3-glycosidase a c t i v i t y could be l o c a l i z e d to the phlanges, these experiments do not rule out the p o s s i b i l i t y that t h i s enzyme originated from the tissue adjacent to the gland rather than from the walls of the storage vestibule. Attempts were made to show the enzymic conversion of phenylacetaldoxime to p h e n y l a c e t o n i t r i l e , but the a b i o l o g i c a l 84 conversion of the oxime to the n i t r i l e ( Ahmad and Spenser, 1961 ) occurred as r a p i d l y as i n the presence of an enzyme preparation. A basic question remains to be asked. Is the biosynthe-s i s of HCN the r e s u l t of the millipede's biochemistry, or can micro-organisms be implicated i n t h i s process? Studies with 14 D,L-phenylalanine- C showed that the excised glands of H. haydeniana devoid of most adherent tissue were capable of pro-14 14 ducmg H CN and benzaldehyde- C. This suggests that the m i l l i -pede i s biochemically autonomous when the appropriate amino aci d precursor i s supplied, for t h i s millipede could not u t i l -14 iz e D,L-shikimic a c i d - C ( Table III ). The r o l e of alimentary micro-organisms i n the synthesis of e s s e n t i a l amino acids for a v a r i e t y of arthropods i s well documented ( Dadd, 1973 ). Another p o s s i b i l i t y i s that i n t r a c e l l u l a r symbionts are involved i n the biosynthesis of HCN, such as has been shown for the biosynthesis of ascorbic acid by the cockroach Leucophaea  maderae ( Pierre, 1962 ). H i s t o l o g i c a l studies can r e a d i l y show the presence of symbionts ( Buchner, 1965; G r i f f i t h s and Beck, 1973; Hinde, 1973 ) i n ants, aphids and a v a r i e t y of other organisms. These organisms are very c l e a r l y seen i n electron-micrographs. However electron-micrographic studies of the de-fensive glands of insects show no such symbiotic inclusions ( Crossley and Waterhouse, 1968a,b; Eisner et a l . , 1964; Happ et a l . , 1966; Noirot and Quennedey, 1974; Tschinkel, 1969, 1972). No symbionts were seen i n the t h i n sections of the cyan-ogenic gland of H. haydeniana viewed by oil-immersion micro-scopy ( Figure 16 ). I t i s assumed that t h i s millipede i s auto-nomous. In respect to the gland, possibly the c e l l s of, or ad-jacent to the storage vestibule are wholely responsible for the 85 synthesis of HCN. Woodring and Blum ( 1963 ) showed h i s t o l o -g i c a l evidence for large secretory c e l l s i n ce r t a i n regions of the storage vestibule. Most authors concerned with the biosynthesis of defensive chemicals are remiss because they do not allow for the presence of symbionts. They usually do not even name a tissue responsible for the biogenesis of the de-fensive chemical(s). An animal which stores l a b i l e mandelonitrile would have to contend with the continual release of tox i c HCN and benzal-dehyde. The metabolic fate of both HCN and benzaldehyde were studied i n H. haydeniana. 14 When H. haydeniana were exposed to H CN, the compound 14 14 was converted to 0-cyanoalanine- C and asparagine- C ( Table V and Figure 10 ). No other l a b e l l e d compounds were noted i n the amino acid f r a c t i o n . Other fract i o n s were not monitored for . 1 4 . 1 4 r a d i o a c t i v i t y . The conversion of H CN to B-cyanoalanine- C was small ( less than 1%; Table V ), but t h i s can be accounted for i f one assumes that the millipede possesses a l t e r n a t i v e methods of metabolizing HCN, such as the conversion to thiocy-anate v i a the enzyme rhodanese. The conversion of HCN to a protein amino acid, asparagine, i s a means of avoiding carbon-nitrogen loss during the continual breakdown of mandelonitrile and i t s resynthesis. Experiments 14 employing D,L-phenylalanine- C showed that the incorporation 14 into H CN was maximal at 24 hours. A f t e r 48 hours of metabol-. 1 4 14 l z i n g D,L-phenylalanme- C, less H CN was obtained from the animal suggesting a rapid turnover of the n i t r i l e . 14 14 Most plants convert H CN to B - cyanoalanine- C ( Blum-enthal et a l . , 1963; Floss et a l . , 1965; Hendrickson and Conn, 86 1969; Oaks and Johnson, 1972; Mead and Segal, 1973 ), and sub-sequently convert t h i s n i t r i l e into asparagine ( Oaks and Johnson, 1972 ). Some legumes a l t e r n a t i v e l y ( V i c i a ) convert the p-cyanoalanine to ^ -glutamyl-p-cyanoalanine ( Blumenthal et a l . , 1968 ) . The a b i l i t y to condense HCN with either serine or cysteine to produce p-cyanoalanine i s widely d i s t r i b u t e d , occurring i n E. c o l i , Sorghum vulgare, C h l o r e l l a , and a v a r i e t y of legumes ( Eyjolfsson, 1970 ). Rhodanese i s an a l t e r n a t i v e means of detoxifying HCN, since the product, thiocyanate, i s r e l a t i v e l y harmless. Rhod-anese exi t s i n cyanophoric plants( Chew and Boey, 1972; E y j o l f -sson, 1972; Mahadevan, 1973 ), as well as i n mammals ( Dixon and Web, 1964; Montgomery, 1969 ). I t occurs i n a b u t t e r f l y , Polyommatus icarus, and i n several moths, Zygaena f i l i p e n d u l a e and Z. lonicerae that feed on Lotus corniculatus ( Parsons and Rothschild, 1964 ). Diptera and Hymenoptera that p a r a t i s i z e cyanogenic Zygaena spp. also use the l a t t e r method of detoxica-t i o n ( Jones et a l . , 1962 ). However, several other insects do appear to detoxify HCN by rhodanese ( Rothschild, 1973 ) and no s a t i s f a c t o r y a l t e r n a t i v e has been proposed. I t may be that the production of p-cyanoalanine i s the primary detoxication mech-anism i n these i n s e c t s . Cysteine can also react with HCN to form 2-amino-4-thiazoline-carboxylic a c i d ( Montgomery, 1969; Wood and Cooley, 1956 ). Neither of these p o s s i b i l i t i e s has been examined i n H. haydeniana. The a b i l i t y of H. haydeniana to metabolize HCN accounts p a r t i a l l y for the greater resistance of millipedes to HCN than of insects observed i n these studies ( Figure 11 ). Similar observations have been reported by H a l l et a l . ( 1969,1971 ). In polydesmid millipedes t h i s type of detoxication may obviate 87 the need for non-iron haemes i n r e s p i r a t i o n , or s p e c i a l o x i -dative by-passes as has been argued for millipedes ( H a l l et a l . , 1971 ), for insects ( Yust and Sheldon, 1952 ), and f o r plants ( Antonini et a l . , 1971; Bonner and Bonner, 1971; Schon-baum et a l . , 1971 ). Another aspect of t h i s millipede's adaptation for s t o r i n g mandelonitrile i s i t s a b i l i t y to detoxify benzal-dehyde. I t has been shown that H. haydeniana ( Table V and 14 Figure 10 ) converts benzaldehyde- C to p-hydroxybenzoic a c i d -14 C. The formation of p-hydroxybenzoic ac i d represents an eco-nomical way of obtaining a major precursor for ubiquinone bio-synthesis. The hydroxylation of benzoic ac i d to the p-hydroxy-acid and subsequent conversion to protocatechuic a c i d i s a common phenomenon i n fungi ( Brown and Swinburne, 1971, 1973; Towers and Subba Rao, 1972 ). whether p-hydroxybenzaldehyde i s an intermediate i n H. haydeniana i s not discernable by these experiments. The millipede was capable of hydroxylating benzoic ac i d . p-Hydroxybenzoic acid was found to occur i n microgram quantities i n H. haydeniana, suggesting that the metabolism of introduced benzaldehyde and benzoic a c i d i s not a forced detox-i c a t i o n reaction. The ultimate fate of p-hydroxybenzoic ac i d and benzoic ac i d i n t h i s millipede i s not known. The l i t e r a t u r e on detoxication mechanisms i n arthropods says that aromatic compounds can be hydroxylated, sulphated, glycosylated, phos-phorylated, or form conjugates with glycine, agmatine and other amino componds ( Binning et a l . , 1967; Hitchcock and Smith, 1966; Raymond et a l . , 1972; Smith, 1962 ). The gross morphology ( Figure 14 and 16 ) of the cyano-genic glands of H. haydeniana was found to be s i m i l a r to that of Pachydesmus crassicatus ( Woodring and Blum, 1963 ). However, 88 the electron-micrographic scans of the glandular o r i f i c e show this area to be more complex than previously described ( Figure 13 ). The pore ( 25 u i n diameter ) i s surrounded by a s l i g h t c o l l a r , and to the outside of the c o l l a r a c i r c u l a r p i t whose wall r i s e s above the l e v e l of the pore. The possible advantage of t h i s o r i f i c i a l structure w i l l be discussed below. The microanatomy of the cyanogenic gland ( Figure 16 ) i s s i m i l a r to that of Pachydesmus crassicatus ( Woodring and Blum, 1963 ). The studies with H. haydeniana more c l e a r l y show the anatomy of the pore area ( c o l l a r et cetera ), and also the dermal gland ducts passing through the wall of the reaction chamber. These ducts end as small pores that open to the lumen of the reaction chamber. Through these ducts the n i t r i l e lyase i s supposedly secretedqjthough no evidence has been given f o r t h i s . I t was not determined i n H. haydeniana whether there were s p e c i a l secretory c e l l s of the storage ves t i b u l e as shown fo r P. crassicatus . I t i s now possible to assess the model of HCN production i n view of the biochemical and morphological data on H. hay-deniana. A more de t a i l e d diagram of t h i s model incorporating the biochemical data i s shown i n Figure 18. These data confirm the o r i g i n a l hypothesis ( r e f e r to Figure 2 ). Is mandelonitrile the storage product i n millipedes? In H. haydeniana D-(R)-mandelonitrile i s stored as a large droplet ( Figure 15 ) within the sto rage vestibule of each gland. The d i s s e c t i o n of the following millipedes revealed the presence of a large o i l y droplet i n the storage chamber; Boraria s t r i c t a , Cherokia georgiana, Oxidus g r a c i l i s , Polydesmus angustus, Nearc- todesmus cerasinus, and Scytonotus insulanus. The chemical nature of these droplets was not determined; they are probably n i t r i l e s . However, 0. g r a c i l i s stores mandelonitrile. 8 9 What i s the role of the glycoside, and of the 0-glyco-sidase? The following can be hypothesized. The glycoside, a water soluble relatively non-toxic stable chemical, i s synthe-size d within the c e l l s of the storage chamber and/or the adjacent t i s s u e s . I t i s then transported from the secretory c e l l s i n some form which i s passed to the lumen of the storage chamber and converted to the n i t r i l e . I t i s not known i f the hydrolysis of the glycoside by p-glycosidase occurs i n the se-cretory c e l l s , on t h e i r luminal surfaces, or within the storage chamber. The n i t r i l e accumulates as a droplet within the storage chamber. This l a b i l e n i t r i l e can be r e a d i l y converted to HCN and benzaldehyde when i t comes i n contact with the n i t r i l e lyase. The disadvantages of st o r i n g the glycoside of mandelo-n i t r i l e are several. The glycoside ( 1 ) i s a s o l i d , and there-fore to be e f f e c t i v e as a lat e n t source of HCN would need to be stored i n an excellent solvent to approximate the storage capacity of a droplet of n i t r i l e . The storage ( 2 ) of a gl y -coside would require two enzymatic steps to l i b e r a t e HCN. Mechanistically, a two step process could be less rapid. F i n a l l y , ( 3 ) the i n v i t r o B-glycosidase a c t i v i t y of H. haydeniana ( Figure 12 ) cannot account q u a n t i t a t i v e l y f o r the release of several micrograms of HCN i n a few seconds. When alarmed H. haydeniana c u r l s into a s p i r a l ; t h i s precedes the l i b e r a t i o n of HCN. This defensive c o i l i n g i s coupled with the increase i n haemocoelic pressure that squeezes the f l e x i b l e storage sac. I f the l a s t few abdominal segments o f H. haydeniana are quickly severed while i t i s walking, the animal i s incapable of l i b e r a t i n g HCN. This confirms that pressure required to force the n i t r i l e i n t o the reaction chamber i s pro-duced by the haemocoele. Attempts to measure the haemocoelic 90 pressure during cyanide l i b e r a t i o n were unsucessful. Is there a n i t r i l e lyase i n the reaction chamber? I t has been shown that a-hydroxynitrile lyase could be i s o l a t e d from the reaction chamber of H. haydeniana. The a c t i v i t y of the a-hydroxynitrile lyase i n v i t r o can account q u a n t i t a t i v e l y for the rapid evolution of HCN i n vivo. Several problems remain to be i s o l a t e d concerning the nature of the cyanogenic gland; (1) i s phenylalanine the f i r s t precursor or i s another phenylalkyl d e r i v a t i v e more e f f e c t i v e ; (2) how i s the synthesis of mandelonitrile regulated; (3) how i s the droplet of mandelonitrile formed and s t a b i l i z e d ; (4) what con t r o l does the millipede have over the amount of mand-e l o n i t r i l e released, and i s there any r e l a t i o n s h i p between the amount of enzyme av a i l a b l e and the amount of substrate stored? In respect to point "3", mandelonitrile i s more stable i n a c i d conditions. I t i s i n t e r e s t i n g to note that Polydesmus c o l l a r i s  c o l l a r i s ( Casnati et a l . , 19^ 63 ) produces considerable quant-i t i e s of i s o v a l e r i c , formic , and a c e t i c acids i n i t s HCN secretory process. I f these acids were associated with mandelo-n i t i l e i n the storage vestibule, they would retard the decay of the n i t r i l e , and create an a c i d i c environment for the n i t r i l e lyase. Note that the n i t r i l e lyase of H. haydeniana has an a c i d -i c pH optimum ( Figure 12 ), as do other lyases ( Barman, 1969 ). The presence of compounds such as phenol ( i n Oxidus g r a c i l i s ; Blum et a l . , 1973a ) could provide a c i d i t y , and act as a s t a -b i l i z i n g agent, besides being a predator deterrent. From -flis more d e t a i l e d biochemical d e s c r i p t i o n of cyano-genesis i n H. haydeniana i t i s possible to postulate a simple phyaical model of how the gland works . The sequences leading to HCN production are depicted i n Figure 19. One H. haydeniana 91 produces on the average 0.45 p M . ( 12^) of HCN. By c a l c u l a t i o n s based on the size of the storage chamber ( Figure 15 ), taking the diameter of an average droplet as 300 u , i t can be c a l -culated that each gland w i l l contain approximately 0.03 p M . ( 0 . 9 y ) . Since each adult has approximately 20 glands, the t o t a l content of the millipede would be 0.7 p M . ( 18y ). The determined content (12 y) and the t h e o r e t i c a l amount ( 18 y) are i n reasonable agreement considering the assumptions made. These quantities of HCN are representative of the lower values reported to occur i n the polydesmid Apheloria corrugata (Eisner, 1970) In f r e s h l y dissected H. haydeniana i t was noted that the reaction chamber was.almost f u l l of the aqueous enzyme soluti o n . The d i r e c t i o n of the meniscus of t h i s enzyme so l u t i o n showed that i t wets the wall of the reaction chamber. This i s shown i n sequence 1 of Figure 19. The wall of the pore channel was found to be wettable by t h i s aqueous phase. This means that with pore channel approximately 25 p i n diameter the l i q u i d i n the reaction has the p o t e n t i a l to r i s e nearly 8 cm., i f t h i s channel were a long c a p i l l a r y tube ( see formula below ). height of c a p i l l a r y i n cm. surface tension of n i t r i l e ; i t i s 32 dynes/cm., H»0 i s 70. radius of c a p i l l a r y i n cm. density of n i t r i l e ; i t i s 1.11 gm./ml., H 20 i s 1.0 g r a v i t a t i o n a l acceleration, 981 cm./sec.^o However, regardless of the p o s i t i o n of the millipede the enzyme solut i o n i s not l o s t from the animal, nor does the p o s i t i o n of the meniscus change i n response to g r a v i t y . The reasons for these conditions are several. ( 1 ) The reaction h T r a FIGURE 19. Proposed physical model for the production of HCN i n the polydesmid mill i p e d e Harpaphe  haydeniana. CO = c o l l a r CP = pore channel RC = reaction chmaber W = wall of RC SV = storage vestibule N = mandelonitrile M = muscle HCN = hydrogen cyanide P = pore 93 chamber i s sealed at the other end ( c o n s t r i c t i o n ) allowing no outward movement of l i q u i d . ( 2 ) The s i z e of the chamber ( approximately 200 u i n diameter ) permits the adhesive forces of the l i q u i d to the wall of the reaction chamber to overcome the force of gravity. By t h i s mechanism the millipede conserves i t s enzyme instead having i t leak out because of c a p i l l a r i t y , or flow out by permitting a i r into the chamber. Even though the neck of the storage chamber, where the muscle attaches, i s a long c a p i l l a r y ( approximately 150ylong by 30 p i n diameter ) the mandelontrile does not r i s e up the cap-i l l a r y because of the presence of the c o n s t r i c t i o n , which i s necessary to overcome the upward pressure of the mandelonitrile. Mandelonitrile wets the neck region of the storage vestibule; thus, c a p i l l a r i t y keeps the n i t r i l e poised i n the neck of the reaction chamber, rather than having i t f l o a t i n g f r e e l y within the storage vestibule. This p o s i t i o n i n g ensures that mandelo-n i t r i l e , and not the c l e a r l i q u i d also present i n the storage vestibule, i s exuded into the reaction chamber. From the above formula i t can be calculated that the n i t r i l e has the p o t e n t i a l to r i s e approximately 4 cm. up the neck, i f i t were continuous. However, t h i s r i s e would be slow. Instead of r e l y i n g upon cap-i l l a r i t y H. haydeniana resorts to haemocoelic pressure to expel the n i t r i l e into the reaction chamber. From the formula below i t can be calculated the the millipede must exert approximately 2 3,000 dynes/cm. to expel the n i t r i l e from the storage a r e a . i t i s assumed i n t h i s c a l c u l a t i o n that the column of n i t r i l e 130 u — 6 long by 30 p i n diameter i s expelled i n 1 second ( 7.1 x 10~ ml. per sec. ) . I f the rate i n r e a l i t y i s faster, i t follows that more pressure w i l l be required to push the viscous n i t r i l e . At-mospheric pressure i s approximately 10° dynes/ cm. ( 78 cm. of 94 Hg ). This pressure exerted by the millipede i s rather small, and the p o s s i b i l i t y of higher pressures i s not unfeasible. P = pressure i n dynes/cm? t = radius of c a p i l l a r y i n cm. j"fV^r » = length of c a p i l l a r y i n cm. •| = v i s c o s i t y of the n i t r i l e i n poises; the n i t r i l e i s 8.2 cp, and H2O i s 1 cp. volume displaced i n ml./sec. The a p p l i c a t i o n of pressuie ( sequence 2 ) w i l l drive a b u l l e t of mandelonitrile i n t o the reaction chamber quickly. Since the n i t r i l e i s immiscible with water i t w i l l r e t a i n i t s i n t e g r i t y as i t passes i n t o the enzyme so l u t i o n . The nozzle of the reaction chamber ( see Figure 16D ) introduces the n i t r i l e i n t o the centre of the chamber with a d e f i n i t e shape. Because of the h e a r t - l i k e shape of the reaction chamber and because of the f r i c t i o n a l drag between the moving n i t r i l e and the enzyme so l u t i o n one might expect some t o r o i d a l c i r c u l a t i o n of the enzyme so l u t i o n ( indicated by the arrows ). This c i r -cular motion of the aqueous f l u i d would f a c i l i t a t e the laminar flow of the n i t r i l e through the aqueous medium. By a p p l i c a t i o n of Reynold's formula ( below ) i t can be determined that the flow of the n i t r i l e under an extreme range of i n i t i a l v e l o c i t i e s w i l l be non-turbulent ( laminar ). Thus, the enzyme w i l l be par-t i a l l y conserved by the non-mixing of the two-phases. However, some enzyme i s displaced to the e x t e r i o r upon the introduction of a volume of mandelonitrile ( bulging f l u i d from pore i n se-quence 2 ). The water wettable pore channel does not hinder t h i s displacement, but rather aids i t . I f the pore channel were non-wettable to the enzyme so l u t i o n extra pressure would be required 95 to drive the l i q u i d out. This displaced enzyme flows over the f l - 1 — = i f <, 1,000 turbulent flow if>1,000 laminar flow p = density of n i t r i l e , i t i s , 1.1 gm./ml. A* ^ ~£ 10 /V = v e l o c i t y of flow i n cm./sec. f = radius of channel i n cm. ft = v i s c o s i t y of n i t r i l e , i t i s *" 8. 2 cp. c o l l a r of the o r i f i c e and i s trapped i n the non-wettable sur-face of the p i t . An i n t e r e s t i n g feature of t h i s model ( sequence 2 ) i s that the " b u l l e t " of mandelonitrile, despite the angle at which the millipede resides, w i l l behave as a moving s o l i d . I t w i l l not bend through i t s length i n response to gravity, but w i l l continue i n the d i r e c t i o n of the pore. A f t e r a c e r t a i n volume of mandelonitrile has been in j e c t e d i n t o the reaction chamber, the muscle can relax. This closes the c o n s t r i c t i o n i n the neck of the storage vestibule preventing the entry of more mandelo-n i t i l e ( sequence 3 ). This e f f e c t i v e l y allows the b u i l d i n g pressure,that has arisen from the evolution of HCN, to escape only to the ext e r i o r v i a the unblocked pore. In sequence 3 i t i s postulated that the " b u l l e t " of n i -t r i l e i s expelled from the reaction chamber and the pore chan-nel on a cushion of expanding HCN. This gas i s r a p i d l y produced at the immiscible i n t e r f a c e between the n i t r i l e and the enzyme solution. The l i q u i d benzaldehyde produced at th i s i n t e r f a c e i s much more soluble i n the n i t r i l e than i n the enzyme solution, so i t would not be expected to a l t e r the i n t e r f a c e . The HCN w i l l tend to remain i n a gaseous form rather than i n aqueous sol u t i o n because the enzyme so l u t i o n should be a c i d i c , t t was 96 determined that the pH optimum for the n i t r i l e lyase was 4.2 ( Figure 12 ). In sequence 3, the escaping HCN causes a break i n the continuity of the enzyme solut i o n which r e s u l t s i n some of the enzyme i n the pore channel being pushed to the outside where i t i s trapped i n the p i t area. The surface of t h i s p i t ( trough ) i s non-wettable with water, so the s o l u t i o n beads f a c i l i t a t i n g i t s confinement i n the trough. The electron-micrographic scan of the p i t area ( Figure 13C ) shows no c u t i c u l a r structures which f a c i l i t a t e wetting, as has been des-cribed for hemipteran evaporatoria ( F i l s h i e and Waterhouse, 1969; Remold, 1963 ). In sequence 4, the n i t r i l e has been driven from the reaction chamber as a " b u l l e t " . A s l i g h t loss of the enzyme i s shown; i n dissections t h i s was observed to be approximately 20%. The expulsion of the n i t r i l e by a gas "explosion" i s a feature which would conserve the amount of enzyme so l u t i o n . Woodring and Blum ( 1963 ) have also observed the s l i g h t loss of l i q u i d from the reaction chamber of Pachydesmus crassicatus, and postulated that the HCN and benzaldehyde are blown from the chamber. The fate of the undissociated mandelonitrile i s indicated i n sequence 4. I t i s trapped i n the p i t about the c o l l a r ; neither mandelonitrile or benzaldehyde wet the p i t . The n i t r i l e , by i n t e r a c t i o n with the enzyme i n the p i t as well as by natural decomposition, degrades gradually to HCN and benzaldehyde. Thus, the animal has an i n i t i a l burst of HCN de-ri v e d from the f i r s t contact of the n i t r i l e with the enzyme, as well as a second delayed release of HCN from the p i t . This delayed release of HCN as a prolonged defence has been describ-ed by Eisner ( 1970 ). The c o l l a r surrounding the pore probab-97 l y prevents the return of the mandelontrile-enzyme mixture to the reaction chamber. HCN i s toxic so i t would be bene-f i c i a l to have as much of the HCN release occur outside the body as possible. The p i t not only stops the reaction mixtures re-turn, but also prevents the spread of these f l u i d s over the rest of the body. Under a binocular microscope the droplet of enzyme-nitrile mixture can be seen to exude from the pore and remain within the p i t u n t i l i t evaporates completely. This mode of release of HCN i n H. haydeniana i s s l i g h t l y d i f f e r e n t from that of plants. The millipede requires a fast action mechanism independant of tissue damage to deter pred-ators. This requirement i s met by the storage of mandelonitrile i n t e r a c t i n g with a highly active n i t r i l e lyase. Plants, on the other hand, have selected a release process i n which damage to the l e a f exposes the glycoside to stored B-glycosidase. The li b e r a t e d n i t r i l e then in t e r a c t s with the n i t r i l e lyase to form a ketone or aldehyde plus HCN. No glandular structures are involved i n cyanogenesis i n plants, although some compart-mentalization must be present. In cases where i t i s known that a millipede produces a glycoside of a n i t r i l e , t h i s glycoside i s u n l i k e l y to be the pre-cursor for HCN i f the millipede possesses a glandular system l i k e H. haydeniana, because mechanistic problems a r i s e . The water soluble glycosides are sparingly soluble i n b i o l o g i c a l organic solvents ( e.g., alkanes, a l i p h a t i c a c i d s ) . I f the glycoside were stored as an aqueous so l u t i o n and i t were introduced into the reaction chamber mixing ( non-laminar flow ) would occur, unless one of the aqueous phases were very viscous. From t h i s mixing process the mandelonitrile would tend to form a disper-sion rather than a"bullet" The formation of a dispersion would make i t d i f f i c u l t to eject the glycoside as a " b u l l e t " . The 98 HCN that would form from such a dispersion would tend to blow the enzyme out of the chamber, rather than conserve i t . D i l i g e n t observation of other millipedes should show that they store the n i t r i l e rather than the glycoside. This has been shown to be the case for 7 millipedes mentioned ear-l i e r i n t h i s t h e s i s . However, the production of the benzoyl ester of mandelonitrile by Polydesmus c o l l a r i s c o l l a r i s ( Cas-n a t i et a l . , 1963 ) i s not inconsistent with my proposal. This ester i s an o i l , and may behave i n an analogous manner to mandelonitrile. One needs evidence for the existence of an esterase and lyase within the reaction chamber, i f the ester i s the storage product. These authors have not proved i t i s the storage product. In respect to H. haydeniana, i t can be seen that t h i s millipede i s e f f i c i e n t l y designed biochemically, morphologically and behaviourally for the production of HCN. F i n a l l y , the reader might ask how such a biochemical and morphological apparatus could develop as a p a r a l l e l process i n c e r t a i n millipedes and plants. Certain aspects of t h i s can be imagined, but others are unanswerable enigmata of evolution. In H. haydeniana the cyanogenic gland appears to be of ectodermal o r i g i n as indicated by l i g h t microscopy ( Figure 16). I f one can make an analogy between insect integument ( epider-mal c e l l s ) and that of diplopods, i t can be stated that the epidermal layer represents a group of c e l l s with diverse bio-chemical c a p a b i l i t i e s . This statement i s supported by the f a c t that these c e l l s are considered responsible for the biosynthe-s i s of c u t i c l e phenolics ( Andersen, 1971; Brunet, 1967 ), for the deposition and tanning of the c u t i c l e ( Brunet, 1967;Chap-man, 1969 ), f o r the biosynthesis of c u t i c u l a r l i p i d s ( Conrad 99 and Jackson, 1971; Jackson and Baker, 1970; Lambremont, 1972; Oudejans and Zandee, 1973 ), for the biosynthesis of proto-catechuic acid i n the cockroach ootheca ( Shaaya and Sekeris, 1970 ), for the production of the maturation hormone i n the locust ( Strong, 1970 ), and for the biosynthesis of a v a r i -ety of other chemicals. Some of these biosyntheses include the vast array of pheromonal and defensive chemicals known to exude from ectodermal gland tissue ( r e f e r to Blum, 1969, 1970, 1971; Jacobson and Crosby, 1971; Noirot and Quennedey, 1974; Weatherston and Percy, 1970 ). The evolutionary development of the glandular structure of H. haydeniana is d i f f i c u l t to explain i n simple terms. Suf-f i c e i t to say that the invagination of the c u t i c l e , which already contained dermal gland c e l l s could give r i s e to the two chambers. I f one examines the c u t i c l e of H. haydeniana i t i s noted that the t o t a l area i s r i d d l e d with large ducts resembling those which appear to be leponsible for the secretion of the n i -t r i l e lyase into the reaction chamber. These large exoskeletal ducts resemble gland duct c e l l s , and may have had diverse bio-chemical c a p a b i l i t i e s i n ihe evolutionary past. The evolutionary development of the biochemical pathway for cyanogenesis i s im-possible to explain without a large number of u n j u s t i f i a b l e assumptions. In summary, the b r i g h t l y coloured polydesmid millipede Harpaphe haydeniana has evolved a mechanism to autonomously 14 synthesize HCN and benzaldehyde. The incorporation of C-pre-cursors, the i s o l a t i o n of several biosynthetic intermediates, and elementary enzymic evidence substantiate the hypothesis that t h i s biosynthesis i s s i m i l a r to that known to occur i n plants. Other biochemical p a r a l l e l s with non-animal organisms are the formation of B-cyanoalanine from HCN, and the formation of 100 p-hydrobenzoic acid from benzaldehyde. Using biochemical and morphological data on H. haydeniana a model of the mechanism of HCN production explains the appropriateness of mandelo-n i t r i l e as a l a b i l e precursor of HCN. PART II THE IDENTIFICATION AND BIOSYNTHESIS OF BENZOQUINONES IN A MILLIPEDE, RHINOCRICUS HOLOMELANUS 102 VI- INTRODUCTION Simple benzoquinones are produced by a v a r i e t y of organ-isms: fungi, plants, arthropods, and echinoderms ( Thomson,1971). The structures of some of these simple benzoquinones are de-picted i n Figure 21, along with some examples of other natur-a l l y occurring benzoquinones. The chemical i d e n t i f i c a t i o n of benzoquinones i n arthropods has received considerable attention ( Thomson, 1971; Weatherston and Percy, 1970; see Table 21, #3 ). Considerably less biosynthetic work has been done with arthro-pods . The o r i g i n a l aim of t h i s thesis was to study the biosyn-thesis of simple benzoquinones i n a Jamaican millipede Rhino-cricus holomelanus. This project was i n t e r e s t i n g from the view-point that benzoquinones of millipedes, benzoquinone, toluquin-one, and 2-methyl-3-methoxy-benzoquinone are s i m i l a r to fungal benzoquinones ( Figure 21, #1,3,and 6).' A su b s t a n t i a l l i t e r a t u r e i s a v a i l a b l e on the biosynthesis of simple benzoquinones ( Thomson, 1971 ). By asking s i m i l a r questions as those posed i n the study of Harpaphe haydeniana, i t was hoped to determine the pathways by which these quinones are biosynthesized ( see Figure 20). A discussion of the possible pathways i s given i n the appendix, pp.184- 187. A f t e r two years' labour, no substantial information was acquired concerning the biosynthesis of the benzoquinones i n R. holomelanus. No matter what experimental approach was taken the millipedes d i d not incorporate any of the isotopes e f f i c i e n t l y i n t o the quinones. I t was decided to choose another problem; That problem was the biosynthesis of HCN i n H. haydeniana. 1 0 3 nu 1 T 11 1 P r o p i o n y l - C o A 3 M a l o n y l - C o A 3 M a l o n y l - C o A ' 1 A c e t y l CoA OH Chorismic a c i d 6-Methyl | S a l i c y l i c a c i d Plants Phenylalanine OH OH OH 6-Ethyl m-Ethylcresol Ethylquinol 0 Ethylquinone 0 m-cresol OH j ^ ^ C H 2C00H OH v i a cinnamic a c i d or phenylpy-r u v i c a c i d Homogentisic a c i d I P-OH-phenyl-\ P-° H-Pnenylpyruvic a c i d p l a n t s P v r u v i c a c i d V ' mCiU animals ? UUUrl _P-OH-Cinnamic C O O H ' (| j ' ' a c i d 0 p-OH-Phenyl- j ^ N ^ « - - - l a c t i c a c i d T y O H O H X t ^ / S ^ C O O H 0 3-Methoxy-toluquinone 0 Benzoic a c i d bacteria C 0 0 H HOOC 1 OH CH2 Chorismic a c i d FIGURE 20. Theoretical pathways for the biosynthesis of arthropod benzoquinones. PRESENCE IN ORGANISMS* QUINONE OR PHENOLIC Fungi Plants Arthropods 1 Benzoquinone + + + 2 2,3-dimethylbenzoquinone + 3 2-methyl-3-methoxybenzoquinone + 4 Thymoqu i non e + 5 Lawsone + 6 Toluquinone + + 7 2,3,5-trimethylbenzoquinone + 8 Me thoxybenzoquinone 9 Gentisylquinone + 10 Ethylbenzoquinone + 11 2-methyl-3-hydroxybenzoquinone + 12 2,6-dimethoxybenzoquinone + 13 Arbutin + + 14 Emodin + * Often the chemical i s found to occur as the hydroquinone. For comprehensive l i t e r a t u r e re t h i s table r e f e r to Thomson ( 1 9 7 1 ) and Weatherston and Percy ( 1 9 7 0 ) . FIGURE 21. Some n a t u r a l l y occurring quinones of plant, fungal, and animal o r i g i n 104b FIGURE 21. Some n a t u r a l l y occurring quinones of plant, fungal, and animal o r i g i n . 1 0 5 ARTHROPOD REFERENCE # Phalangids Beetles Roaches True bugs Termites Earwigs Grasshoppers Millipedes 1 5 8 , 4 6 3 , 1 7 7 5 , 4 6 , 66,101,245,295,341,399,47^501 3 9 8 , 4 0 0 3 9 7 3 1 2 , 3 3 7 1 5 1 , 4 6 3 1 5 6 2 7 0 , 3 3 5 , 4 6 3 , 4 9 3 , 4 9 5 TABLE VII. The occurrence of simple benzoquinones i n arthropods. VII- MATERIALS AND METHODS V I I . l - CHEMICAL MATERIALS p-Benzoquinone was obtained commercially from Eastman. I t was p u r i f i e d by vacuum sublimation for spectrophotometry purposes. Hydroquinone was obtained commercially from A l d r i c h . I t was decolourized with charcoal i n b o i l i n g toluene, and re-c r y s t a l l i z e d from that solvent several times p r i o r to use. p-Toluhydroquinone was obtained commercially from Fluka A.G,. I t was p u r i f i e d i n the same manner as hydroquinone. Tolu-106 quinone was synthesized from toluhydroquinone by oxidation with chromic anhydride and g l a c i a l a c e t i c acid ( Vogel, 1967). The c r y s t a l s of toluquinone were f i l t e r e d , washed with cold water, dissolved i n d i e t h y l ether and dried with anhydrous Na 2S0 4. The ether was removed i n vacuo at 5 ° C , the cr y s t a l s redissolved i n a minimal amount of d i e t h y l ether: pet. ether ( b.r.60-110°C. )- 1:12 ( v/y ), and recry.stallized at 0°C. The 2,3; 2,5; and 2,6-dimethyl derivatives of benzoquin-one were synthesized from the corresponding dimethyl phenols, which were converted to the p-amino-phenols v i a diazotized p-amino-sulphanilic ac i d . The p-amino-phenols were oxidized to the quinones with F e C l 2 and HCl as described by Fieser and Ar-dao ( 1956 ) and Fieser and Fieser ( 1935 ). A f t e r d i s t i l l a t i o n , the quinones were dissolved i n pet. ether ( b.r. 30-60°C o ) and washed with water saturated with NaCl. The organic phase was dried with anhydrous Na^O^, the solvent removed i n vacuo at 5°C. to near dryness, and pet. ether ( b.r. 60-110°C. ) added to p r e c i p i t a t e the quinones. The p r e c i p i t a t e was f i l t e r e d , a i r dried i n the dark, and then sublimed i n vacuo to remove the non-volatile o-quinones. 2,3,5-Trimethyl-benzoquinone was synthesized from the corresponding hydroquinone ( Fluka A.G. ) using NaNC>2 i n g l a -c i a l a c e t i c acid as described by Fieser and Peters ( 19 31 ) i n Fieser and Fieser (1967). The hydroquinone p r i o r to oxidation was decolourized i n a manner s i m i l a r to that for hydroquinone.The quinone was f i l t e r e d from the reaction mixture, washed with cold water, dissolved i n pet. ether ( b.r. 30-60°C ), dried with anhydrous Na^O^, and the solvent removed i n vacuo at 0°C.. The cry s t a l s were a i r dried i n the dark, and then sublimed i n vacuo. 107 The synthesis of 2-methyl-3-methoxy-benzoquinone as described by F l a i g et a l . ( 1958 ) was unsucessful. VII.2- DERIVATIVES OF VARIOUSLY SUBSTITUTED BENZOQUINONES The hydroquinone was produced by the reduction of the corresponding benzoquinone with NaBH^ i n d i e t h y l ether with a small amount of methanol. The hydroquinone was extracted with ad d i t i o n a l d i e t h y l ether a f t e r d i l u t i o n of the reaction mixture with d i l u t e HCl. The ether f r a c t i o n was dried with anhydrous Na2$0^ and taken to dryness i n vacuo. The diacetate derivatives of the various quinones were produced by placing the hydroquinone i n ac e t i c anhydride with a c a t a l y t i c amount of pyridine for 24 hours. The diacetate was pr e c i p i t a t e d by the addition of crushed i c e to the reaction mixture, f i l t e r e d , and washed with water. Subsequent p u r i f i c a -t i o n of the diacetate involved r e c r y s t a l l i z a t i o n from aqueous ethanol,and from benzene by the addition of pet. ether ( b.r. 60-llO°C). The t r i a c e t a t e s of the benzoquinones were synthesized using the Thiele-Winter reaction ( Vogel, 1967 ). The t r i a c e -tates were p r e c i p i t a t e d by the addition of crushed i c e to the reaction mixture. The c r y s t a l s were f i l t e r e d , washed with water, dissolved i n acetone, and r e c r y s t a l l i z e d by the addition of water. The a i r dried c r y s t a l s were then p r e c i p i t a t e d from xylene. Attempts were also made to prepare deriavtives of benzo-quinones which might be more stable than the quinones and also more separable by thin-layer chromatography. These were the (3-thiopropionic acid derivatives ( Blackhall and Thomson, 1953; 108 Fieser and Fieser, 1956); the cyano- derivatives ( Thiele and Meisenheimer, 1900 ); and the semicarbazone derivatives ( Vogel, 1967 ). These attempts were unsuccessful. A table of chemical data of benzoquinones i s provided ( Table VIII ). The values therein are complied from the l i t -erature and t h i s t h e s i s . UV,IR, and NMR spectra of known quinones and quinones from R. holomelanus were compared to the c i t e d data ( Thomson, 1971 gives a compilation ). Mass sp e c t r a l ( MS ) data were compared to those reported by Bowie et a l . ( 1966 ) and Thomson ( 1971 ). The benzoquinones were i d e n t i f i e d by TLC ( Harrison and Weatherston, 1967; Petterson, 1963 ). The hydroquinones were separated by 2-dire c t i o n a l TLC on A v i c e l with the following solvents; benzene:acetic acid;H2O-10:7:3 ( v/v/v ), 2% formic acid, and benzene:acetic acid - 9:1 ( v/v ). They were detected with diazotized p - n i t r o - a n i l i n e as a spray reagent ( Stahl, 196 9 ). Hydroquinones turned green, and quickly faded to a l i g h t brown when sprayed with t h i s reagent. Gas l i q u i d chromatography of the benzoquinones was ac-complished by a Perkin Elmer 680 gas chromatograph with a flame i o n i z a t i o n detector. Various derivatives of benzoquinones were also separated by th i s method. The support phases, l i q u i d phases, and conditions under which the various derivatives were separated are indicated i n Table IX. The benzoquinones from R. holomelanus were p u r i f i e d for NMR and MS analyses by extraction and sublimation ( described below ) and GLC separation on 5% BDS ( conditions as above for 109 10% BDS ). The i n d i v i d u a l quinones were c o l l e c t e d at the e x i t port i n a U-shaped expansion tube. This tube was c h i l l e d i n a mixture of dry-ice and acetone during the trapping process. The quinone was removed from the U-tube with CDCl^ and used d i r e c t l y NMR. Samples for MS analysis were c o l l e c t e d i n glass c a p i l l a r y tubes c h i l l e d with dry-ice. Teflon c o l l a r s were used to attach the c a p i l l a r y to the e x i t port. VII.3- EXTRACTION AND PURIFICATION OF MILLIPEDE QUINONES R. holomelanus, whether radioactive or non-radioactive, were extracted i n the following way to i s o l a t e benzoquinones. They were anaesthesized with CO,,, decapitated and the l a s t few posteri o r segments cut o f f . They were then degutted by gently p u l l i n g out the alimentary canal with a fine p a i r of forceps,, Each animal was then placed i n a test-tube containing enough pet. ether ( b.r. 30-60°C. ) to cover the carcass, and about 200 mgm. of NaCl. A glass rod was then inserted i n t o the animal ( replacing the gut ) and the glass rod rotated so that the animal was rubbed i n a c i r c u l a r fashion against the wall of the test-tube. The s h e l l was discarded a f t e r the orange colour of the pet. ether remained the same i n t e n s i t y . An aqueous so l u t i o n of NaHCO^ was added to the tube, and the mixture agitated with a vortex s t i r r e r . The organic phase was removed, dried with anhydrous Na2SO^, and the solvent removed i n vacuo at 0°C. i n a sublimation thimble. This thimble was then attached to i t s finger and the benzoquinones sublimed i n vacuo at 50°C. on the cold glass finger. The quinones were eluted from the finger with d i e t h y l ether and stored i n t h i s solvent over Na2SO^ u n t i l further use. 110 14 VII.4- ADMINISTRATION OF THE C- PRECURSORS 14 . . 1 4 Sodium acetate- C, mevalonic a c i d - C, sodium propion-14 14 ate- C, sodium shikimate- C were dissolved i n Na2HPO^ ( 0.02 M. ) buffer. Other aromatic precursors were dissolved i n P.W.A, ( p. 16 ) i f they were used for i n j e c t i o n experiments. For each experiment with R. holomelanus the i n j e c t i o n volume was 5-10 p i . The duration of the feeding experiments ranged from 1 week to several hours. The millipedes were milked of some of 14 t h e i r quinones before i n j e c t i o n of the C-precursors. VII.5- COLLECTION OF MILLIPEDES The millipedes used i n t h i s portion of the thesis were mainly c o l l e c t e d at Runaway Bay, Jamaica, West Indies. The Caribbean animals were av a i l a b l e at a l l times of the year. Other millipedes, as indicated i n Table XI, were c o l l e c t e d l o -c a l l y . The animals were kept as described for H. haydeniana. R. holomelanus l i v e d for 6-8 months i n laboratory conditions. XIII- RESULTS XIII.1- THE STRUCTURAL IDENTIFICATION OF BENZOQUINONES OF RHINOCRICUS HOLOMELANUS The presence of benzoquinone, toluquinone, and 2-methyl-3-methoxy-benzoquinone was confirmed i n R. holomelanus as well as i n a number of other r h i n o c r i c i d , p a r a j u l i d , s p i r o s t r e p t i d , and pachybolid millipedes. P a r t i a l i d e n t i f i c a t i o n of the quinones of R. holomelanus i s based on the GLC retention values compared to that of knowns I l l ( Table IX ). The underlined values show coincidence of re-tention times of the n a t u r a l l y occurring quinones with that of the known quinones. The agreement of rentention time data on three counts ( quinones, diacetates, and Thiele derivatives ) i s strong evidence for the occurrence of these three quinones i n the defensive secretions of thi s m i l l i p e d e . Melting point data for the synthetic quinones are shown i n Tables 8 and D. The melting points derived for the n a t u r a l l y occurring quinones of R. holomelanus are i n good agreement ( Table 10 ). UV and IR analyses ( Table X ) of the natural compounds aided i n t h e i r i d e n t i f i c a t i o n . The millipede's benzo-quinone exhibited UV and IR absorption values c h a r a c t e r i s t i c of synthetic benzoquinone. Likewise, the millipede's toluquinone showed sp e c t r a l agreement with the synthetic standard. The m i l l i -pedes 2-methyl-3-methoxy-benzoquinone d i d not r e l a t e to the expected UV and IR values. The l a s t compound was shown to be a 2,3-substituted benzoquinone by use of benzene:acetic a c i d - 5 : l ( v/v ) as a s p e c t r a l solvent. UV analyses of synthetic benzoquinones showed that the chemicals that were un-, mono-, 2,5-,or 2,6-substituted had one absorption maximum ( 278 nm. ); whereas, the 2,3-substituted benzoquinone had two absorption maxima ( 278 and 333 nm. for the dimethyl-quinone ). S i m i l a r i l y , the millipede's 2-methyl-3-methoxy-benzoquinone showed two absorp-t i o n maxima i n t h i s solvent ( Table X ). R. holomelanus contained only trace amounts of benzo-quinone, so i t was not possible to obtain enough of thi s chem-i c a l for NMR anal y s i s . The NMR spectrum of synthetic toluquinone was i d e n t i c a l Parent Quinone o Melting Point Of Derivatives of Parent Quinones i n C  Quinone Hydroquinone Diacetate Triacetate Unsubstituted 2-methyl-2,3-dimethyl-2.5- dimethyl-2.6- dimethyl-2,3,5-trimethyl-2-methyl,3-methoxy-115 69 55 123-125 68-71 29 28-29 116 124-125 221-226 219-221 145-151 170-173 110-136 121-124 43-44 105-106 133-135 85-95 109-110 96-97 105-107 103-105 101-103 100 TABLE VIII. Melting point data of some p-benzoquinones of arthropod o r i g i n . i—• to TABLE IX. Retention times of benzoquinones and deriva-tives of benzoquinones of Rhinocricus holo-melanus compared to standards by gas l i q u i d chromatography. He^ was the c a r r i e r gas i n a l l cases. Carbo W = Carbowax 1540, 5% on Chromsorb W ( 60-80 mesh), column 150 cm, at 120°C, gas flow 65 ml./min.. Poly-m0 = Poly-meta-phenylether- ( 5 r i n g ), 10% on Chromsorb W, column 150 cm. at 155°C, gas flow 75 ml./min.. BDS = Butanediolsuccinate, 10% on Chrom-sorb W ( 60-80 mesh ), column 180 cm. at 155°C, gas flow 50 ml./min.. OV-l = OV-l, 1% on Chromsorb W ( 60-80 mesh ), column 300 cm. at 155°C. for diacetates and at 170°C. f o r Thiele der., gas flow 60 ml./min. for both der.. SE-30 = SE-30 ( s i l i c o n e gum ), 5% on Chromsorb W ( 60-80 mesh ), column 180 cm. at 165°C ( gas flow 120 ml./ min. for diacetates ) and at 180°C. ( gas flow 70 ml./min. for Thiele der. ). * No chemical standard ava i l a b l e , but Trigoniulus  lumbricinus contains t h i s chemical ( Thomson, 1971 )so i t was considered the standard. Retention time of quinones and derivatives by G.L.C Quinone Diacetate Thiele deriv, Compound p-benzoquinones) Carbo W Poly-m0 BDS OV-l SE-30 OV-1 SE-30 Parent 3.8 2.0 2.2 2.5 2.5 4.0 4.5 2-CH3 4.6 3.0 3.0 3.0 3.2 5.1 5.9 2 /3-di-CH 3 5.6 4.6 3.8 3.9 4.0 6.2 6.8 2 /5-di-CH 3 5.6 4.6 3.8 3.9 4.0 6.2 6.8 2 / 6 - d i - C H 3 6.0 4.7 4.0 3.9 4.0 - ' -2, 3,5-tri-CH.j 8.3 7.5 5.5 5.6 6.0 - - . 2-CH 3 /3-OCH 3* 10.7 6.5 .6.0 5.0 4.6 8.1 9.9 (underlined values i- represent presence i n millipede sample) TABLE IX. Retention values of benzoquinones and derivatives of benzoquinones of Rhinocricus holomelanus compared to standards by gas l i q u i d chromatography. 115a TABLE X. Spectroscopic data used to i d e n t i f y the benzo-quinones present i n Rhinocricus holomelanus Obs. = observed Exp, Min. = minimum Max, TMS = trimethylsilane m = mult i p l e t s d = doublet m.w, = expected = maximum s i n g l e t molecular weight Solvents a = b = c = chloroform cabon t e t r a c h l o r i d e benzene: g l a c i a l a c e t i c a c i d - 5 : l Chemical.and spectrometric data used i n I d e n t i f i c a t i o n of benzoquinones Quinone Mel t i n g Point (C°) UV S p e c t r a l data(mu) IR Data NMR Data ( 100 KHz ) Structure Benzoquinone ( mv.= 108 ) Toluquinone ( mw.= 122 ) 2-CH3,3-OCH3-benzoquinone ( mw.- 152 ) Obs. 114-116 116 27-28 29 Obs. Exp. ( i n CC1 4 ) Values (S=o r e l a t i v e to TMS) ( i n CDC1 3 j Exp. Max. Min. Max. Min. 6 5 3 2 3 2 Obs. Exp. H ^ N . H' C H 3 246 278 65-66 68-69 249 254 C278 255 262. _278 288 7 315 435 374 374 376 246 288 439 1669 1669 1651 1653 1603 249 255 5278 315 436 1674 1659 1669 1653 254 386 2,3-subst. cmds give double abs. ? ? . • ? ( not enough sample ) 1661 1661 6.65 (m) 6.58 (m) 2.03 (d> 6.59 (rn) 1.95 (s) 4.00 (s) TABLE X. Spectroscopic data used to i d e n t i f y the benzoquinones present i n ; Rhinocricus holomelanus. . H H 116 to that of toluquinone i s o l a t e d from the millipede.'An un-resolved multiplet i s observed at S = 6.65 a r i s i n g from the non-equivalent protons at positions 5 and 6. A multip l e t i s also observed at 8 = 6.58 which represents the coupling of the proton at p o s i t i o n 3 with the 2-methyl group. This l a t t e r group i s evidenced by the appearance of a doublet at &=2.03 int e g r a t i n g for 3 protons. The integration of the other protons i n t h i s molecule i s consistent with the in t e r p r e t a t i o n that the molecule i s toluquinone. No synthetic 2-methyl-3-methoxy-benzoquinine was a v a i l -able for comparison to the millipede's chemical. NMR analysis of the l a t t e r compound showed an unresolved m u l t i p l e t at <5=6.59 a r i s i n g from the coupling of the non-equivalent protons at po-s i t i o n s 5 and 6 ( Table X ). No proton coupling with the 2-methyl group was observed; instead, a s i n g l e t at 8 = 1.95 integrat i n g for 3 protons was detected i n d i c a t i n g that the molecule was 2,3-substituted. The s u b s t i t u t i o n at p o s i t i o n 3 i s a methoxy group that gives a s i n g l e t at £ = 4.00 and does not couple with the 2-methyl group . This s i n g l e t at £ = 4.00 integrated for 3 protons. These data are consistent with the interpretation that the chemical i s 2-methyl-3-methoxy-benzo-quinone. No sample of benzoquinone from R_. holomelanus was c o l -l e c t a b l e for MS analysis; however the other two quinones were analyzed. The re s u l t s of these analyses are depicted i n Figure 22. The millipede's toluquinone gave a base peak of 122, which corresponds with the parent ion. The subsequent loss of produces a fragment ( m/e =94 ). A p a r t i c l e ( m/e = 40 ) corresponds to the most highly substituted acetylene ( see Bowie et a l . , 1966 ) and another fragment ( m/e=82) represents 117 the other h a l f o f t h i s cleavage. These observations are consistent with previous interpretations that t h i s chemical i s toluquinone. The MS analysis of the millipede's 2-methyl-3-methoxy- . benzoquinone shows a base peak of m/e = 152, as well as a prominent peak of m/e = 122 which arises through the loss of formaldehyde ( CH 20 ). The peak of m/e = 122 represents the presence of toluquinone as a fragment, and i t i s noted that fragments corresponding to those previously described for toluquinone-decay occur i n t h i s a n a l y s i s . The loss of CO from the di-substituted quinone gives r i s e to a fragment ( m/e = 124 ). A p a r t i c l e ( m/e = 109 ) corresponds to the loss of the methyl group from the methoxy function of t h i s fragment ( m/e = 124 ). The loss of the methoxy methyl group from the most highly substituted acetylene i s observed i n the fragment ( m/e = 53 ). The formation of a fragment ( m/e = 83; 0=C-C=0 indicates that the molecule i s 2,3-substituted. These C H3 observations are consistent with the i n t e r p r e t a t i o n that the quinone i s 2-methyl-3-methoxy-benzoquinone. Reduction of the benzoquinones of R. holomelanus, and chromatography of the r e s u l t i n g hydroquinones v e r i f i e d that i d e n t i t y of these quinones by comparison to synthetic standards. Both natural and synthetic chemicals gave i d e n t i c a l responses to diazotized p - n i t r o - a n i l i n e over-sprayed with 5% NaOH. Chrom-atography of the fr e s h l y secreted defensive l i q u i d of R.holo-melanus revealed the presence of the three hydroquinones cor-responding to the quinones. 118 FIGURE 22. Analysis of Mass Spectrum data of the benzo-quinones of Rhinocricus holomelanus. The structure at the top of the opposing page i s that of toluquinone ( m.w.= 122 ) and at the bottom that of 2-methyl-3-methoxy-benzoquinone ( m.w.= 152 ). The molecular fragments of these compounds are indicated i n the m/e column. In brackets are the r e l a t i v e i n t e n s i t i e s of the frag-ments in.respect to the base peak 100 M. The decomposition of benzoquinones i s char-acterized by the loss of CO, and also by the loss of the most highly substituted acetylene. C H 2 0 - J v 0 < 124 82 m / e 1 2 2 ( 1 0 0 M ) 9 4 ( 56 ) 82 ( 6 5 ) 6 8 ( 4 0 ) 5 4 ( 7 9 ) 4 0 ( 4 3 ) 1 5 2 [ 1 0 0 M 1 2 4 [ 1 0 1 2 2 ( 5 5 1 0 9 ( 4 9 9 4 [ 1 4 8 3 ( 4 2 8 2 [ 4 0 5 4 ( 5 0 5 3 ( 6 5 4 0 ( 2 7 FIGURE 22. Analysis of mass spectrum data of benzo-quinones of Rhinocricus holomelanus. 120 XIII.2- THE IDENTIFICATION OF BENZOQUINONES IN OTHER MILLIPEDES Nine other taxonomically unrelated millipedes have been shown to contain traces of benzoquinone, and larger quantities of toluquinone and 2-methyl-3-methoxy-benzoquinone. The d i f -ferences i n the r a t i o s of the chemicals i n a millipede compared to those.of the other millipedes are i n s u f f i c i e n t to warrant the consideration of these benzoquinones as taxonomic char-acters ( Table XI ). 14 XIII.3- THE INCORPORATION OF C-PRECURSORS INTO THE BENZO-QUINONES OF RHINOCRICUS HOLOMELANUS Table XII shows the incorporation data of various i n -14 jected C-precursors i n t o the benzoquinones of R. holomelanus. Under a v a r i e t y of feeding regimes, e.g., varied dose ( high and low s p e c i f i c a c t i v i t y ) and varied duration of the experi-14 ment, i t was found that only D,L-phenylalanine- and D,L-tyr-14 . . osine- C were incorporated into the three benzoquinones. Since 14 a l l C-precursors were r i n g - l a b e l l e d i t i s assumed that the nucleus of the aromatic acid was incorporated d i r e c t l y i n t o the nucleus of the quinone. Combustion or degradation of the quin-ones was not ca r r i e d out , nor were any attempts made to det-ermine the s p e c i f i c a c t i v i t y of the quinones. The amount of quinone that can be i s o l a t e d from d i f f e r e n t millipedes varies 14 so greatly that d i l u t i o n values of the C-precursors would be meaningless. Also the very low incorporation ( less than 0.01 %) would have made s p e c i f i c a c t i v i t y determinations f r u i t l e s s . Experiments using whole i s o l a t e d glands or whole degut-MILLIPEDE LOCALITY p-BENZOQUINONES Subclass: Helminthomorpha Order: Julida Family: Parajulidae Tuniulus hewitti ( Chamberlin ) Bollmaniulus Saiulus sp. nov. nr. s e t i f e r Chamberlin Spirostreptida Spirostreptidae Orthoporus ornatus ( Girard ) Vancouver, B.C., Canada Hornby Is., " " Austin, Texas, U.S.A. BZ TOL 2,3-MTOL + + + ++++ ++++ ++++ ++++ ++++ Spirobolida Pachybolidae Leptoqoniulus naresi ( Pocock ) Runaway Bay, Jamaica + ++++ ++ Triqoniulus lumbricinus ( Gerst&cker) Oulaloa Dump, Hawaii . + ++++ ++++ Rhinocricidae Eurhinocricus sp. 1 nr. sabulosus Runaway Bay, Jamaica + ++++ +-H-Eurhinocricus sp. 2 nr. bruesi ? ti •i it. _ + ++++ +++ Rhinocricus holomelanus Pocock ti it n + ++++ ++ Rhinocricus monilicornis ( Porath ) it it II + ++++ ++ TABLE XI. The presence of benzoquinones i n various millipedes TABLE XIII. Incorpora • holomelanus. t i o n of 1 4 C - compounds into benzoquinones of Rhinocricus RADIOACTIVITY ADMINISTERED RADIOACTIVITY IN ISOLATED BENZOQUINONES. Precursor D.L- Phenylalanine- ring- C D.L- Phenylalanine- ring- C 14 D.L- Phenylalanine- U- C 14 D,L- Phenylalanine- U- C 14 D,L- Tyrosine- U- C „ 14„ D,L- Tyrosine- U- C Sodium acetate-1' Sodium acetate-1' Sodium acetate-1 - 1 4 c - 1 4 c - 1 4 c 14, Sodium acetate-1- C •, 14„ Sodium acetate-1- • C 14„ Sodium acetate-1- C 14 Benzoic acid- U- C 14 Ma Ionic acid -2- CH 14 D,L- Shikimic acid- U- C amount fed/ 2 animals duration of expt. i n hours uCi mCi/mMole # animals used/ expt., Radioactivity i n dpm BZ TOL 2,3-BZ 3 0.013 12 1' 0 0 0 3 0.013 18 1 100 30 350 1 0.013 72 1 0 300 0 0.4 0.013 1 week 2 0 0 0 0.1 1.5 36 1 0 0 0 1 1.5 1 week 1 250 500 100 o. l 250 2 1 0 0 0 0.2 250 2 1 0 0 0 3 , 250 18 1 0 0 ' 0 0.3 250 36 1 0 0 0 4 250 72 1' 0 0 0 0.1 360 1 week 2 0 0 0 0.3 45 1 week ' 2 0 0 50 0.2 • 1.7 1 week 2 0 0 0 1 5 1 week 2 0 0 0 123 ted animals bathed i n a buffer containing """^ C- precursors were unsucessful. In the whole animals the incorporation of 14 the C-precursors was not affected by the previous milking 14 of the quinones. The incorporation of the C- precursors d i d not appear to be improved by dietary presentation, as opposed to i n j e c t i o n . The duration of time permitted to metabolize the 14 C-precursors seemed to have no e f f e c t upon incorporation, nor did i t have any bearing on the incorporation .Thus, these metabolic studies with R. holomelanus suggest that the biosynthesis of the benzoquinones i s a slow process, such that 14 administered C-precursors meet other fates before they can be incorporated e f f i c i e n t l y i n t o the benzoquinones. IX- DISCUSSION The Jamaican millipede, Rhinocricus holomelanus, produces three simple benzoquinones, benzoquinone, toluquinone, and 2-methyl-3-methoxy-benzoquinone . The i d e n t i t y of these quinones has been confirmed by gas chromatography, UV, IR, NMR, and MS analyses ( Tables IX and X ). Also the benzoquinones of nine other Helminthomorphan millipedes has been shown to be the same as R. holomelanus. ( Table XI ) . The d i s t r i b u t i o n of these quinones i n these millipedes provides no basis for chemotaxonomy. I t i s worth noting that Uroblaniulus canadensis produces 2,3-dimeth-oxy-benzoquinone ( Weatherston and Percy, 1969 ). Thomson ( 1971 ) has compiled the reports of the occur-rence of benzoquinones i n diplopods and many other arthropods. It can be surmised from t h i s compilation that there i s a s i m i l a r lack of chemotaxonomic characters. Of the millipedes i n v e s t i g a t -ed ( Table XI ) only Trigoniulus lumbricinus had been previously studied. 124 Attempts to determine by what pathway R. holomel- anus synthesizes i t s benzoquinones have been unsuccessful 14 ( Table XII ). The incorporation of a v a r i e t y of C- pre-cursors was poor. The observation that t h i s millipede can con-14 14 vert D,L-phenylalanine- C and D,L-tyrosine- C into the three benzoquinones suggests that the nucleus of the aromatic a c i d was incorporated into the nucleus of the quinone. These re s u l t s are ambiguous i n that the d i s t r i b u t i o n of the l a b e l within the quinone was not determined. The i n e f f i c i e n c y of the incorporation would have made t h i s d i f f i c u l t . Obviously, 14 . . experiments i n which one i n j e c t s C-precursors or u t i l i z e s 14 whole i s o l a t e d glands bathed i n buffer containing C-precursors, are not applicable to R. holomelanus. What was simple with Harpaphe haydeniana i s not f e a s i b l e with R. holomelanus. The reason for t h i s may be that the biosynthesis of the benzoquin-ones of the l a t t e r millipede i s such a slow process and grad-14 ual process that C-precursors are u t i l i z e d by other metabolic processes before high incorporation can be achieved. I t was ob-served i n the lab that millipedes which had been depleted of t h e i r quinone took several weeks to replenish the supply such that the glands were f u l l of quinones. I f one assumes that the incorporation into the benzo-14 . 1 4 quinones of the r i n g of phenylalanine- C and tyrosine- C i s by d i r e c t degradation of the side chain of these aromatic acids, and not by degradation and reincorporation of a'fragment of these acids, then the biosynthesis of benzoquinones by R, holomelanus i s c e r t a i n l y d i f f e r e n t from that reported for the beetle Eleodes l o n g i c o l l i s ( Meinwald et a l . , 1966 ). In t h i s beetle benzoquinone arises from the degradation of e i t h e r phenylalanine or tyrosine with the nucleus of the amino ac i d 125 giving r i s e to the nucleus of the quinone. Toluquinone i s syn-thesized from acetate and malonate, and ethyl-benzoquinone i s synthesized from propionate and malonate. Further experiments with millipedes t r y i n g to elucidate the biochemical pathway by which they synthesize benzoquinones should be rewarding i n terms of comparative biochemistry. 1 2 6 X - A P P E N D I X : P E R S P E C T I V E X . l - I N T R O D U C T I O N C o n t r a r y t o s u r v i v a l i s t h e r e l e n t l e s s a t t a c k b y p r e d -a t o r s . D e f e n s i v e o r p r o t e c t i v e m e c h a n i s m s a g a i n s t s u c h d e p r e -d a t i o n s m a y o c c u p y a n o t e w o r t h y p o s i t i o n i n t h e a n i m a l ' s b i o -l o g y / i n v o l v i n g m a j o r b o d i l y c o m m i t m e n t s f o r t h e p r o d u c t i o n o f m o r p h o l o g i c a l , b i o c h e m i c a l a n d / o r b e h a v i o u r a l t r a i t s . T h i s t h e s i s c o n s i d e r s t h e r o l e o f t h e s e t r a i t s i n t h e r e l e a s e o f a d e f e n s i v e s e c r e t i o n , H C N , b y a p o l y d e s m i d m i l l i p e d e . H o w e v e r , t o s h o w t h e p e r t i n e n c e o f t h i s d i s s e r t a t i o n t o t h e g e n e r a l f i e l d o f C h e m i c a l E c o l o g y a n e l e m e n t a r y r e v i e w i s g i v e n b e l o w . T h e d e f e n s i v e m e c h a n i s m s o f a r t h r o p o d s w i l l b e d i s c u s s e d u n d e r t h e f o l l o w i n g h e a d i n g , w i t h p a r t i c u l a r e m p h a s i s o n t h e c h e m i c a l a d a p t a t i o n s . X . 2 - P H Y S I C A L A D A P T A T I O N S : C r y p s i s S o n i c s S t r u c t u r e W a r n i n g d i s p l a y X . 3 - B E H A V I O U R A L A D A P T A T I O N S : I m m o t i l i t y M o t i l i t y M i m i c r y X.4- C H E M I C A L A D A P T A T I O N S : P h y s i c a l B e h a v i o u r a l P h a r m a c o l o g i c a l 127 X.2- PHYSICAL ADAPTATIONS Physical adaptations are gross anatomical features, par-t i c u l a r l y architecture and pigmentation, which lend s u r v i v a l value. Pigmentation i s c l a s s i f i e d as a p h y s i c a l a t t r i b u t e be-cause the observer perceives colour by a wave phenomenon rather than by pharmacological means. Reviews on pigmentation i n i n -sects are avai l a b l e ( Cromartie, 1959; Fuzeau-Braesch, 1972 ), but t h i s aspect w i l l not be discussed here. Capture i s often avoided by an arthropod resembling i t s background. Such c r y p t i c t a c t i c s can be accomplished by many means. Two such means of camouflage, countershading and d i s -ruptive colouration, involve the a l t e r a t i o n of the appearance of the animal as a s o l i d body. Countershading reduces the shadow cast by the animal through the use of h o r i z o n t a l l y or v e r t i c a l l y graded pigmentation ( l i g h t to dark ). The l i g h t side of the organisms i s turned away from the source of l i g h t and the dark side faces the d i r e c t i o n of l i g h t . Thus, i n optimal natural l i g h t i n g conditions a l l portions of the body w i l l re-f l e c t l i g h t equally ( Cott, 1950; Ruiter, 1955 ). This pheno-menon reduces the distinctness of shape and thereby lessens the chance of being seen by a predator. Disruptive colouration ut-i l z e s random blotches of variously coloured pigmentation. This disrupts the outline of the animal's body such that i t can blend into the ground, surface vegetation, shadows of leaves, or moss and l i c h e n speckled bark. Excellent discussion of both these phenomena are given by Cott ( 1950 ) and Ruiter ( 1955 ). This l a t t e r adaptation i s seen i n many moths that vanish from sight when rest i n g on bark. This type of adaptation has been well studied i n i n d u s t r i a l melanism ( Kettlewell, 1955; Kettlewell and Berry, 1969 ). 128 Many more s t r i k i n g examples of im i t a t i v e crypsis are known. A v a r i e t y of insects resemble twigs, s t i p u l e s , buds, thorns, b i r d droppings, t u f t s of lichens, flowers, s t i c k s , and many other natural objects ( Carrick, 1936; Cott, 1950; Edmunds, 1972; Robinson, 1969; Ruiter, 1952, 1955; Wickler, 1968 ). These ploys can be e f f e c t i v e as primary defensive mechanisms. Frequently i f these i l l u s i o n s f a i l the prey then r e l i e s upon secondary protective mechanisms for protection ( sonics, warn-ing displays, stdjrctural deterrents, and chemical defences). A l l the above defences may function e x c l u s i v e l y of each other. Upon seizure by a predator, many prey s o n i c a l l y attempt to s t a r t l e the predator. The production of sound may r e s u l t from the rubbing of body parts g i v i n g r i s e to audible c l i c k i n g , buzzing, chirping, flapping or even u l t r a - s o n i c sounds ( Blest, 1964; Claridge, 1968; Edmunds, 1972 ). Hissing often accompan-ied by the ej e c t i o n of f r o t h ( Carpenter, 1938; Eisner et a l . , 1971a; von Euw et a l . , 1971 ) i s the r e s u l t of the rapid e j e c t -ion of a i r from the tracheae. During the i n i t i a l contact with the predator many simple external morphological features can repel further attacks. Insects with thick, uneasily crushed c u t i c l e s can prevent sucess-f u l attcks by predators as i n the case of p i l l millipedes of the genus Sphaerotherium. These millipedes form t i g h t b a l l s that are capable of d e f l e c t i n g birds beaks. These b a l l s also can not be grasped by birds such as the jay,Cyanocitta c r i s t a t a ( Eisner, 1967, 1968 ). At the other extreme, f l e x i b l e , e l a s t i c and compressible bodies can help r e s i s t capture. Cott ( 1950) mentions several A f r i c a n b u t t e r f l i e s ( e.g., Pais decora and Eusemia euphemia ) which can be mauled and compressed with con-siderable force, and yet escape and l i v e . Cockroaches and the 129 Monarch b u t t e r f l y , Danaus plexippus, show t h i s considerable resistance to bodily d i s t o r t i o n , which gives them the oppor-tunity to escape while the predator handles them. The presence of barbs or spines on the legs and thorax may serve to i n f l i c t pain upon c e r t a i n predators. Many l e p i d -opterous larvae and adults possess highly s p e c i a l i z e d t u f t s or complete coverings of epidermal hairs and barbs. These project-ions often contain venoms and poisons which can cause severe dermatitis or u r t i c a r i a ( Beard, 1963; Bucherl et a l . , 1971; Rothschild et a l . , 1970b). A p i e r c i n g and p a i n f u l counter-attack may also r e s u l t from the venomous stings and bites of some arthropods ( Brand et a l . , 1972; Bucherl et a l . , 1971; Haber-mann, 1972 ). Pigmentation patterns on wings, such as eye-spots, create the i l l u s i o n of structure, and displace the focus of the pred-ator's attack to these non-vital areas, rather than at the body proper ( Blest, 1957; Cott, 1950; Wickler, 1968 ). This means of deterrence has often been considered a warning display, although i t should be considered a d e f l e c t i v e t a c t i c . The best documented kind of s t a r t l e or warning display i s the sudden exposure of previously camouflaged b r i g h t l y co-loured hind-wings ( Blest, 1964; Cott, 1950; Edmunds, 1972 ). This and other defensive displays such as boxing i n mantids, extrusion of b r i g h t l y coloured fleshy processes and hairs may cause the foe to hesitate just long enough to allow t h e prey to escape ( Blest, 1964; Edmunds, 1972 ), or for other defensive resources to be readied. These s t a r t l e displays are not e f f e c t -ive against a l l predators ( Robinson, 1969 ) and may i n fact a t t r a c t c e r t a i n foes. 130 Many of the above adaptations e n t a i l behavioural co-ordination and sophisticated mechanical design for t h e i r ef-fectiveness. A more d e t a i l e d discussion of mechanical and be-havioural sequencing i s presented by Blest ( 1964 ), Edmunds ( 1972 ) and Robinson ( 1969 ). X.3- BEHAVIOURAL ADAPTATIONS Some prey escape predation as a r e s u l t of c e r t a i n behav-i o u r a l adaptations. Often these adaptations are enhanced by chemical defences which augment the si g n a l received by the predator. Cryptic adaptations are functional deterrents only i f ce r t a i n behavioural prerequisites are met, namely that the prey i s diurnal, and that under threat of attack the prey remains motionless. Carrick's studies ( 1936 ) demonstrated that l e p i d -opterous larvae camouflaged as twigs were only protected from avian predators i f they remained motionless. This behavioural t r a i t i s as important as t h e i r guise. Even in non-cryptic species 1 immotility reduces predator success. Lack of movement i n animals i n many instances represents pantomine or feigning death. This phenomenon i s observed i n grasshoppers, phasmids, and mantids ( Edmunds, 1972; Robinson, 1969 ). One a t t r i b u t e of remaining motionless i s the lack of auditory cues for the predators. Pantomime i s p a r t i c u l a r l y ad-vantageous against predators that respond to movement and active escape t a c t i c s . Several milkweed bugs ( Lygaeus spp.) and milk-weed beetles ( Tetraopes spp.) when approached drop from the plant and lay motionless on the ground. I f they are subsequent-l y mauled the bugs resort to chemical defences and the beetles to sonic defence. 131 Anyone attempting to capture cockroaches, grasshoppers, or centipedes w i l l immediately appreciate the value of t h e i r instantaneous f l e e i n g behaviour. Such adaptations may be l i n k -ed with s t a r t l e displays. These however are diurnal processes. One remarkable adaptation seen i n some nocturnal moths i s the diving response to the u l t r a - s o n i c searching signals of bats ( Roeder, 1964,1966 ). In t h i s case the moths have auditory s p e c i a l i z a t i o n s as well as behavioural ones which permit them to plunge r a p i d l y away from the sonic beam of the bat. Some moths even produce warning sonics to advertize t h e i r distastefulnees to the bats ( Eisner, 1970 ). Rather than rapid escape as a means of protection, many animals resort to f l a i l i n g of legs, r o t a t i o n of the abdomen or s p e c i a l l y evolved processes, and r o l l i n g and twisting of the body ( Cott, 1950; Edmunds, 1972; Robinson, 1969 ). These are probably most e f f e c t i v e against s i m i l a r l y s i z e predators. Many millipedes a f t e r secreting noxious quinones, often abandon t h e i r c o i l e d posture and rotate t h e i r bodies by s p i r a l l y twisting, thereby enhancing the d i s t r i b u t i o n of the ooze ( e.g., Rhino-cricus holomelanus and Orthoporus ornatus ). A s i m i l a r adapt-ation i s seen i n many Hemiptera that secrete odiferous chemicals. These insects thrash t h e i r legs upon seizure and e f f e c t i v e l y d i s t r i b u t e these chemicals from t h e i r thorax ( Remold, 1963 ). A chrysomelid beetle larva, Cassida rubiqinosa combines a unique behavioural and s t r u c t u r a l adaptation. I t has a forked abdominal appendage which has become a repository for f e c a l material and exoskeletons of i t s prey ( Eisner et a l . , 1967 ). This detri t u s i s held on the fork over the body l i k e a s h i e l d , and upon attack the chrysomelid rotates the s h i e l d to parry the attacker. Animals such as ants are often l e f t with a mouth-132 f u l of waste, while the prey escapes. Mimicry, p a t i c u l a r l y Batesian and Mullerian ( Cott, 1950; Rettenmeyer, 1970; Wickler, 1968 ), i s an excellent example of the complex inte g r a t i o n of physical, behavioural, and chemical adaptations employed by many d i f f e r e n t insects ( Brower and Brower, ±964; E h r l i c h and Raven, 1964 ). Since p h y s i c a l and behavioural adaptations play such a major role role i n t h i s manner of defence i t i s most convenient to discuss i t under a behavioural category. Mimicry can be defined as the close resemblance of org-anisms to each other i n size , form and behaviour such that some of the time some predators.are confused i n t o thinking that a l l the organisms are i d e n t i c a l ( Rettenmeyer, 1970 ). Batesian and Mullerian mimicry r e f e r to the resemblance of b r i g h t l y co-loured insects to each other i n which some of the members of the mimcry complex are palatable ( Batesian mimic ) while others are unpalatable, or i n which a l l the members of the complex are unpalatable to some degree ( Mullerian mimic ). The source of the distastefulness arises e i t h e r from chemicals produced by the insect ( Eisner et a l . , 1970,1971b; Rothschi3d et a l . , 1970a, 1972 ), or sequestered from food plants ( Brower and Brower, 1964; Rothschild , 1972, 1973 ). The resemblance of these insects when on the wing i s close enough i n f l i g h t be-haviour, colouration, and shape that predatory birds generally become confused as to which are the palatable and unpalatable forms. This mimcry need not be v i s u a l l y perfect to be e f f e c t i v e ( Brower et a l . , 1971; Schmidt, 1958; Rothschild, 1971 ).A s i g n i f i c a n t b i o l o g i c a l feature of Batesian-Mullerian mimicry i s that t h i s means of defence i s i n s t r u c t i v e for the predator, i n that by continual exposure to s t a t i s t i c a l l y greater numbers of unpalatable b r i g h t l y coloured insects ( models ) than to 133 the smaller numbers of palatable mimics, the predator gen-e r a l i z e s that the bright colouration represents d i s t a s t e f u l -ness. Such learning by a predator affords protection for a l l species involved i n the mimcry complex ( Brower, 1958a,b,c; Brower et a l . , 1968; Brower, 1970 ). This world wide pheno-menon i s e s p e c i a l l y prominent i n the t r o p i c s . It has also ' evolved i n the Hemiptera, the Coleoptera, and Hymenoptera ( Rettenmeyer, 1970; Wickler, 1968 ). This discussion gives a very l i m i t e d view of p h y s i c a l and behavioural adaptations for the purposes of defence. Comp-arable s p e c i a l i z a t i o n s are also found amongst birds, f i s h , mammals, l i z a r d s , frogs, and others. These s p e c i a l i z a t i o n s need not be only adaptive for defence, for the same mechanism can enhance a predator"s success at attack by f o o l i n g the prey. The monographs by Cott (1950 ) and Wickler ( 1968 ) give many examples of the above phenomena. X.4- CHEMICAL ADAPTATIONS The use of chemicals by organisms against predators i s only one aspect of the growing awarness of the ecology of nat-u r a l chemicals i n the environment ( Sondheimer and simeone, 1970; Whittaker and Eeeny, 1971 ). Inter- and i n t r a s p e c i f i c behavioural and p h y s i o l o g i c a l modifications induced by chemicals e x t r i n s i c to an i n d i v i d u a l are observed i n most phyla. An e f f e c t i v e nomenclature describing the multifarious ef f e c t s of chemicals i n the environment i s not a v a i l a b l e . Many terms have been coined to enhance our understanding of the chem-i c a l i n t e r a c t i o n between organisms - hormones, ectohormones, ectomones, pheromones, kairomones and allomones ( Brown et a l . . 134 1970; Karlson and Butendandt, 1959; Law and Regnier, 1971; Regnier and Law, 1968; Whittaker and Feeny, 1971 ). Many of these neologies are inadeqajite to describe a complicated con-tinuum of in t e r a c t i o n s . But three of these terms at present provide a s i m p l i s t i c frame of reference for categorizing chemical communication - 1/ hormone: within a c e l l or between c e l l s of an i n i d i v i d u a l , 2/ pheromone: between members of the same species, and 3/ allomone: between members of d i f f e r e n t species ( Wilson, 1970 ). This thesis i s p r i m a r i l y concerned with allomones, a l -though many of the chemicals serving as allomones are often used by the same or d i f f e r e n t animal as pheromones ( Blum, 1969,1970; Jacobson, 1972; Regnier and Law, 1968; Wilson and Regnier, 1971 ). The study of allomones considers such diverse topics as the chemical interactions between micro-organisms and plants ( Goodman et a l . , 1967 ), between plants ( Muller, 1968; Went, 1970; Whittaker, 1970 ), between herbivores and plants ( Beck, 1965; Dethier, 1970; Dodson et a l . , 1969; Jacob-son and Crosby, 1971 ) and between animals and t h e i r foes ( Eisner, 1970; Karlsson, 1973; Mebs, 1973 ). The l a s t of these interactions i n respect to arthropods i s the focus.of t h i s discussion. Defensive chemical adaptations involve the use of a prey's chemical(s) which upon contact with the receiver r e s u l t ( s ) i n the cessation or diminuation of attack. This deterrent qual-i t y may be the r e s u l t of the chemical's p h y s i c a l and/or pharm-acol o g i c a l properties. The phys i c a l properties of chemicals represent c h a r a c t e r i s t i c s such as v o l a t i l i t y , p a r t i t i o n coef-f i c i e n t , chemical r e a c t i v i t y , v i s c o s i t y , and other c o l l i g a t i v e features which are independant of the mode of perception. The 135 pharmacological properties of the chemicals e n t a i l t h e i r ab-i l i t y to a l t e r behavioural and p h y s i o l o g i c a l homeostasis i n the receiver. Defensive chemicals have an antagonistic p o t e n t i a l a-gainst other organisms.This p o t e n t i a l can be r e a l i z e d i n two ways which are analogous to the e f f e c t of pheromones. F i r s t l y , the defensive chemical may have an immediate behaviourally modifying e f f e c t on the attacker; thus, the chemical has i n a sense functioned as a "releaser" ( Regnier and Law, 1968 ) i n pheromonal terminology. Secondly, the chemical may have a delayed e f f e c t on other organisms by modifying t h e i r p h y s i o l -ogy such as would occur i f the chemical had a n t i b i o t i c proper-t i e s . In t h i s l a t t e r sense the defensive chemical can be con-sidered to have functioned i n an analogous fashion to a "primer" pheromone ( Regnier and Law, 1968 ). Many defensive chemicals seem to function i n both manners. X.4.1- Chemical adaptations: physical The deterrent e f f e c t s of a defensive chemical need not be based on i t s repulsiveness. Resistance to attack can be ac-quired by employing the p h y s i c a l properties of a chemical, such as s t i c k i n e s s , sliminess, or i t s a b i l i t y to c r y s t a l l i z e or poly-merize. Onychophorans, c e r t a i n l i t h o b i d centipedes, and a m i l l i -pede discharge a viscous l i q u i d which e f f e c t i v e l y entangles small predators. This secretion i n the Onycophorans ( Onycho;-phora spp.) and i n the millipede Glomeris marginatus quickly becomes gummy on exposure to a i r . Entangled predators eventual-l y die i f trapped i n t h i s gum ( Eisner, 1970 ). These s t i c k y defensive secretions that polymerize on exposure to a i r often 136 contain toxic chemicals, as i n the cases of the millipede Glomeris marginata ( quinazolinone a l k a l o i d , Meinwald et a l . , 1966b; Schildknecht et a l . , 1967b ) and the s o l d i e r termite Mastotermes darwiniensis ( quinones , Moore, 1968 ). An i n t e r e s t i n g use of a physio-chemical phenomenon c a l - ' led supercooling has been suggested to be a defensive mechanism i n aphids, e.g., Aphis fabae ( Dixon, 1958; Edwards, 1966 ). This e n t a i l s the secretions of supercooled droplets of l i p i d s ( mainly myristic acid t r i g l y c e r i d e s ; Strong, 1967 ) from the - aphid's c o r n i c l e s . These droplets remain i n a lijuid state at the tip s of the cornicles u n t i l touched; whereupon, they c r y s t a l -l i z e having the p o t e n t i a l to immobilize small predators ( Ed-wards, 1966 ). Kislow and Edwards ( 1972 ) report that these cornicular secretions of . Myzus persicae also have odours that are repellent. The exact role of these supercooled droplets as a defence i s disputable. They appear to serve p r i m a r i l y as an excretory process compensating for high carbohydrate ( phlo-em ) diets ( Wynn and Bourdeaux, 1972 ). However, cornicular secretions probably serve many purposes for the l i q u i d has been shown to contain the glyceride sorbodimyristin ( Bowie and Cameron, 1965; Callow et a l . , 1973 )which i s based on an antifungal agent sorbic acid ( hexadienoic acid ) ( Shimizu, 1971 ). The production of heat coupled with the synthesis of an e f f e c t i v e chemical deterrent becomes a devastating weapon i n the bombardier beetles, Brachinus spp. ( Aneshansley et a l . , 1969; Eisner, 1970 ). Under attack the beetle d i r e c t s i t s post-e r i o r toward the foe, and triggers a cannon-like device which oxidizes r e l a t i v e l y i n e r t hydroquinones i n the presence of 137 hydrogen peroxide and enzymes to noxious benzoquinones. This exothermic reaction i s c a r r i e d out i n a r i g i d and r e s i s t a n t chamber inside the insect, from which the the hot quinones are expelled at the adversary ( p o t e n t i a l l y 100°C. ). The solvent properties of a defensive secretion , apart from the pharmacological e f f e c t s , often enhance the e f f e c t i v e -ness as a deterrent. The i r r i t a n t p r i n c i p l e s ( quinones, phenols, acids, terpenes and alkaloids ) are often associated with an abundance of "non-noxious" chemicals that serve as c a r r i e r s . L i p o p h i l i c organic solvents such as n-nonyl acetate ( Eisner, 1970 ), or n-tridecane ( Bernardi e_t a l . , 1967; Waterhouse e_t a l . , 1961 ) are examples of c a r r i e r substances. The enhancement of the i r r i t a n c y i s ascribed to the a b i l i t y of these c a r r i e r s to a i d i n penetration and spreading of the t o t a l components in t o the c u t i c l e or skin ( Eisner, 1970; Remold, 1963 ). These c a r r i e r substances might also f a c i l i t a t e the stor-age of noxious chemicals by serving as a p r e f e r e n t i a l solvent over the aqueous i n t e r n a l environment. They could also aid i n the deployment of the noxious chemicals by reducing the v i s c o -s i t y of the defensive secretion making the expulsion through small glandular o r i f i c e s more f a c i l e . C a r r i e r s also appear to aid i n retarding the rapid loss of very v o l a t i l e components. Chemicals such as water also serve as c a r r i e r substances pro-viding a medium i n which acids such as formic, a c e t i c and butyric can ionize and i r r i t a t e . In fact, many defensive secretions are biphasic ( aqueous and organic ).The presence of a l i p o p h i l i c phase such as n-tridecane can i n t e n s i f y and accelerate the i r -r i t a n c y of these chemicals by acting as a t i s s u e solvent ( Berg-strftm and L&fqvist, 1970; Blum, 1969, 1970; Remold, 1963 ). 138 The role of c a r r i e r substances i s not well defined i n many cases; i t i s d i f f i c u l t to explain why an insect would possess a series of n-altenes £.g.,Cg-C]_3 i f they do not appear to have d e f i n i t e pheromonal or allomonal properties. One i n -t r i g u i n g suggestion made by Blum and Brand ( 1972 ) i s that these hydrocarbons upon contact with a predator 1s antennal re- r ceptors "jam" the chemo-receptive s i t e s . The predator can then no longer decipher chemical messages normally and may have i t s at-tack behaviour disrupted so as to make the prey o l f a c t o r i l y imperceivable. X.4.2- Chemical adaptations: behavioural Certain aspects of chemical defence lessen the d i s t i n c -t i o n between allomones and pheromones. This i s evident when de-fensive chemicals have profound e f f e c t s as modifiers of s o c i a l behaviour. S o c i a l ants and termites use a v a r i e t y of compounds termed "alarm pheromones" which modify t h e i r own behaviour when disturbed or attacked. Low concentrations of an alarm pheromone orient some insects so that they congregate at the source of the pheromone. On exposure to higher concentrations of the same chemical they exh i b i t frenzy and attack behaviour ( Blum, 1969; Regnier and Law, 1968 ). In c e r t a i n instances alarm pheromones have been shown to have i n t e r s p e c i f i c defensive c a p a b i l i t i e s . The ant Acanthomyops clav i g e r produces the terpenes c i t r o n e l l a l and c i t r a l i n i t s mandibular glands; both these chemicals have allomonal and pheromonal properties ( Blum, .1969 ). The a l l o -monal properties of c i t r o n e l l a l are p a r t i c u l a r l y e f f e c t i v e be-cause i t acts as a solvent and wetting agent for the formic acid. C i t r o n e l l a l i s also a skin i r r i t a n t ( discussed i n Blum, 1969 re Ghent, 1961 ). In the genus of ants, Atta, c i t r a l only 139 functions for defence while another class of chemicals ( e.g., 4-methyl-3-heptanone ) acquires the pheromonal role ( Blum et a l . , 1968a ). In Lasius umbratus c i t r o n e l l a l i s the alarm pheromone while c i t r o n e l l o l serves as the defensive chemical ( Blum et a l . , 1968a ). C i t r a l produced by the sti n g l e s s robber bee Lestrimel-i t t a limao acquires the ro l e of chemically disguising the bee's obligatory theft from the nests of other s t i n g l e s s bees, T r i -gona spp. ( Blum et a l . , 1970 ). The l a t t e r species u t i l i z e c i t r a l ( neral and geranial ) as a t r a i l pheromone while har-vesting food. However, L. limao has become an opportunist with i t s a b i l i t y to copiously produce c i t r a l . These robbers locate a nest of Trigona probably by means of scouts which are muti-lated at the nest entrance. Their death l i b e r a t e s copious quan-t i t i e s of c i t r a l which a t t r a c t s distant robbers. The c i t r a l meanwhile permeates through the nest causing disruption of s o c i a l order and defensive behaviour. This disorganization permits a multitude of robber bees to sack the nest for food with impunity ( Blum et a l . , 1970). A similar, phenomenon i s ob-served i n ants which r a i d other colonies to acquire slaves; the sucessful r a i d and capture i s effected by the aggressive use of i n t e r s p e c i f i c pheromones ( Bergstr&m and Lbfqvist, 1968 ). The acme of physical, behvioural and chemical mimicry i s exhibited by mymecophilous beetles ( Hblldobler, 1971 )'. Some adult and l a r v a l beetles are p a r a s i t i c i n that they mimic the behaviour of ants both i n attitudes and i n chemical secretions, thereby obtaining regurgitated food. The guise i s so e f f e c t i v e that the ants even groom and maintain the beetle larvae with t h e i r own. This permits the beetles to l i v e with r e l a t i v e im-punity i n the ant colonies. When an ant approaches, the beetle 140 flexes i t s abdomen upwards for p a l p i t a t i o n by the ant; i t ap-pears that t h i s gesture /mediated by the release of a chemical from the beetle,appeases the ant by mimicking i t s recognition sig n a l s . In.some ant-beetle in t e r a c t i o n s , the beetles, P e l l a  laponicus and P. comes , have been shown to produce a chemical s i m i l a r to c i t r o n e l l a l which i s the alarm pheromone of the host ant, Lasius spatheus ( Kistner and Blum, 1971 ). The release of th i s chemical occurs when the i n i t i a l recognition signals f a i l and the ant begins to aggressively handle the beetle. The i m i t a t -ive release of a c i t r o n e l l a l - l i k e chemical causes the ant to turn away as i n a normal alarm response. Studies on the defen-sive secretions of such beetles ( D r u s i l l a caniculata and Lo-me chus a strumosa show a complicated array of chemicals such as benzoquinones and n-tridecane ( Blum et a l . , 1971; Brand et a l . , 1973 ). Holldobler ( 1971 ) gives an excellent discussion of the d i v e r s i t y of strategies and degrees of s o p h i s t i c a t i o n i n behav-i o u r a l and chemical mime i n myrmecophilous beetles. X.4.3- Chemical adaptations: pharmacological The pharmacological properties of a defensive secretion are often an indispensible feature of e f f e c t i v e deterrence. In a broad sense, these properties involve the a b i l i t y of the chemical to stimulate o l f a c t o r y and gustatory organs, to i r r i t a t e sensi-t i v e tissues, and to induce biochemical and p h s y i o l o g i c a l changes i n another organism. The p a i n f u l stings and b i t e s of a v a r i e t y of arthropods are not merely the r e s u l t of punctured f l e s h . The e f f e c t s are the r e s u l t of the i n j e c t i o n of potent venoms, poisons or toxins which are comprised of polypeptides, proteins, a l k a l o i d s , simple acids, or a v a r i e t y of other chemicals. The proteinaceous sub-141 stances include kinins, proteinases, phospholipases, hyaluron-idases, hemolytic agents and neurotoxins. The smaller molecular weight compounds also have a v a r i e t y of e f f e c t s : ( 1 ) histamine causing anaphyllaxis, (2) choline and other biogenic amines i n t e r f e r r i n g with nervous functioning, and (3) substances l i k e formic acid causing l o c a l i z e d i r r i t a t i o n . These chemicals often 1 are highly functional for the capture of prey, and t h e i r use as a defence i s only secondary i n these cases. The l i t e r a t u r e i n t h i s f i e l d i s vast ( Beard, 1963; Baslow, 1971; Bucherl et a l . , 1971;Habermann, 1972; Karlsson, 1973; Mebs, 1973 ). The reduviid bug Platymeris rhadamantus ( Edwards, 1960 ) has evolved the a b i l i t y to repeatedly and accurately s p i t s a l i v a over considerable distances. This l i q u i d contains i r r i t a t i n g enzymes ( phospholipase, and hyaluronidase ). I t has been sug-gested that t h i s could a f f o r d protection against larger pred-ators such as monkeys that have s e n s i t i v e eyes and mouth parts. On a d i f f e r e n t scale insects are known to have various blood components which are t o x i c to larger predators and pro-bably i n t e r n a l p a r a s i t e s . The presence of phenol oxidases i n the blood of insects, although involved i n tanning, may be bacterio-s t a t i c or vermicidal ( Maier, 1973; Z l o t k i n et a l . , 1973 ). Other proteinaceous factors which have been i s o l a t e d from insect blood, e.g., pederin ( Brega et a l . , 1968; Frings et a l . , 1948; Hsiao and Fraenkel, 1969 ), are toxic to foreign micro-organisms. I t i s also f e a s i b l e to view blood contained substances such as HCN ( Jones et a l . , 1962 ), and cardenolides ( von Euw et a l . , 1971 ) as i n t e r n a l b i o s t a t i c agents, although they are e f f e c t i v e externally against predators. Cantharidin serves as a predator deterrent for meloid beetles ( Epicauta spp. ). The chemical i s 142 e f f e c t i v e l y released to the e x t e r i o r by r e f l e x bleeding ( C a r r e l and Eisner, 1974 ) . Many defensive chemicals employed by arthropods are re-p e l l e n t of t h e i r own accord i n apparently simple manners. This obnoxiousness often e n t a i l s bad smells or tastes. The chemical basis of Batesian and Mullerian mimicry i s dependent upon the d i s a g r e e a b i l i t y to predators of c e r t a i n chemicals contained within the animals, e.g., b u t t e r f l i e s . In one case of mimicry invo l v i n g the Monarch b u t t e r f l y Danaus plexippus ( model ) and other North American Lepidoptera ( models and mimics )( Brower, 1958a,b,c ) i t has been shown that the chemical basis i s the possesion of cardiac glycosides i n some of the b u t t e r f l i e s . These chemicals are derived by l a r v a l feeding upon members of the Apocynales ( Brower and Brower, 1964; Brower et a l . , 1968; Brower, 1969, 1970 ). When the b i r d eats the b u t t e r f l y the presence of cardiac glycosides i n the prey usually causes the b i r d to vomit within several minutes of swallowing. The employment of cardiac glycosides as an anti-pred-ator device i s seen on a world wide basis occurring i n other insects such as the grasshopper Poekilocerus bufonis, and i n mimetic Hemiptera ( e.g., Lygaeus spp.)( von Euw et a l . , 1967; Jones, 1932, 1934;Scudder and Duffey, 1972; Rothschild, 1972 ). Other compounds that deter predators because of the taste, smell, or p h y s i o l o g i c a l a f t e r e f f e c t s also may occur i n the same or d i f f e r e n t species involved i n a mimicry complex. This creates a complicated dimension of chemical s t i m u l i with which predators must cope: a r i s t o l o c h i c acids, B-hydroxybutyric acid, HCN, h i s t -amine, a c r y l y l c h o l i n e , and others. Discussions r e l a t i n g t h i s diverse defensive array to taxonomy and to the value of mimcry are given by Brower and Brower ( 1964), E h r l i c h and 143 and Raven ( 1964 ) and Rothschild ( 1972 ). The aposematic grasshopper Poekilocerus bufonis contains sequestered cardiac glycosides ( von Euw et a l . , 1967; Roth-s c h i l d , 1966 ), but does not s a c r i f i c e i t s e l f l i k e some mimetic b u t t e r f l i e s for the benefit of the population. Rather, i t e-jects the b i t t e r cardenolides i n a foam, which enables the pre-dator to taste before swallowing. The foam, also with a high histamine content, has the a b i l i t y to sicken the predator, and even make i t vomit. B r i g h t l y coloured hind wings advertise i t s undesireable q u a l i t i e s . The bad smell of a prey can deter a predator p r i o r to determined attack. The p a p i l i o n i d b u t t e r f l y P a p i l i o aegus pro-duces both i s o b u t y r i c and B-hydroxybutyric a c i d which supposed-l y deter ichneumid wasps because of t h e i r smell ( Seligman and Doy, 1971 ). The phenols and alkaloids i n the d y t i s c i d water beetle Ilybius fenestratus, on the other hand, present a b i t t e r deterrent to small f i s h and aquatic vertebrates ( Schildknecht, 1971; Schildknecht and Tacheci, 1971 ). The b i t t e r a l k a l o i d s present i n a v a r i e t y of c o c c i n e l i d beetles are e f f e c t i v e against ants and quail ( Pasteels et a l . , 1973 ). Most e f f e c t i v e as i r -r i t a n t s are the benzoquinones which are produced by a v a r i e t y of arthropods ( Thomson, 1971 ). The vapours of these chemicals are i r r i t a t i n g to the eyes and the nose, and contact with these tissues can cause severe pain. They are apparently capable of blin d i n g chickens ( Burtt, 1947; Roth and Eisner, 1962 ). The presence of ad d i t i o n a l defensive components can magnify the re-pulsiveness of the major chemical(s) for the appropriate pre-dators. Like crypsis, mimicry and other defensive adaptations for protection are not always perfect. 144 The types of defensive chemicals used by arthropods are diverse. Figure 23 shows some 35 chemical structures of the several hundred chemicals i d e n t i f i e d i n arthropods. Defensive secretions of a given species have been found usually to be chemically complex conataining several members of: a s t r u c t u r a l family plus large or trace quantities of unrelated chemicals. A grasshopper, Poekilocerus bufonis contains h i s t -amine and cardenolides ( von Euw et a l . , 1967 ); a moth,Zygaena  f i l i p e n d u l a e contains HCN and histamine ( Jones et a l . , 1962 ); a hemipteran, Dyscercus intermedius produces a l k y l aldehydes and ketones plus long chain alkanes ( Calam and Youdeowei, 1968); a millipede, Rhinocricus insulatus produces several benzoquinones and 2-dodecenal ( Wheeler et a l . , 1964 ); a lacewing, Chrysopa  oculata produces n-tridecene and skatole ( Blum et a l . , 1973b ); a quinoline a l k a l o i d and several steroids are secreted by water beetles, e.g., Ilybius fenestratus ( Schildknecht, 1971 ); a l -kanes, alkenes, alkanols, and terpenes are produced by ants ( Bergstrom and Lofqvist, .1972, 1973; Morgan and Wadhams, 1972); and HCN, benzaldehyde, and phenol are secreted by a millipede, Oxidus g r a c i l i s ( Blum et a l . , 1973a ). Chemical d i v e r s i t y amongst the arthropods i s not l i m i t e d to one gland producing a mixture of substances. Many insects, p a r t i c u l a r l y beetles, and s o c i a l bees and ants have several glands secreting d i s s i m i l a r compounds. Morphological and chem-i c a l d i v e r s i t y of t h i s kind i s well studied i n myrmecine and formicine ants ( Herout, 1969;'Regnier and Wilson, 1968; Wilson, 1970; Wilson and Regnier, 1971 ). Ants of these groups have three glands functioning as an alarm-defence, system: (1) the abdominal Dufour's gland, which produces several n-alkanes and a few alkanones which serve not only as alarm substances 145 a D E F E N S I V E C H E M I C A L S T N V A R I O U S A R T H R O P O D " C O M P O U N D TYPE COMMON NAME R E F E R E N C E NUMBER 1 NITRILE Hydrogen cyanide 50,55, 96, 152, 153, 252b, 338, 364, 398, 422, 496, 507 2 PHENOLIC o-Cresol 27, 341, 432 3 BENZOQUINONE Toluquinone 46, 66,101, 224, 245, 270, 312,335, 337, 34L 399,400,423, 463,4743,475,495 4 NAPHTHOQUINONE 6-Methylnaphthoquinone 475 5 KONOTERPENE Cantharidin 94, 98, 99, 329, 429 6 POLYACETYLENE Dihydromatricaria acid 324 7 ALDEHYDE Benzaldehyde 50, 52, 55, 96, 153, 216, 298, 338, 364, 496 8 PHENOLIC Protocatechuic a c i d 417 9 PHENOLIC Hydroquinone 158, 224, 417, 421 10 PHENOLIC Homogentisyl methylester 417 11 AMINE Histamine 33, 83, 167, 217, 402 12 ALKENONE trans-4-oxo-hex-2-en-1-a1 89, 190, 199 13 PIPERIDINE ALKALOID Solenopsin A 67, 301 14 MONOTERPENE Anisomorphal 98, 99, 149b, 225, 226, 322, 325 15 ALIPHATIC ACID 0-Hydroxybutyric a c i d 440,441 16 PHENANTHRENE A r i s t o l o c h i c acid I 170, 4o2, 406 17 CARDENOLIDE Cardiac glycoside type 73, 75, 139, 167, 168, 402, 403, 405, 407 18 MONOTERPENE Geraniol 53, 282, 384, 502, 503 19 n-ALKANE Undecane 29, 66, 243, 385 20 QUINOLINE ALKALOID 8-Hydroxyquinoline-2-carboxy ester 417, 4 1 8 ( 4 1 9 < 4 2 6 21 QUINAZOLINE ALKALOID Glomerine 327a, 417, 427, 428 22 ALKYL DISULPHIDE Dimethylsulphide 96, 314, 492 23 PREGNANE STEROID A pregnadiene s t e r o i d 418, 426, 471 24 SESQUITERPENE P-Silenene 157 25 GLUCOSINOLATE S i n i g r i n 402 26 ALIPHATIC ACID T i g l i c a c i d 25, 341, 423 27 ALKYLAZOXY GLUCOSIDE Cycasin 461 28 AROMATIC ESTER Phenylethyl i s o b u t y r i c ester 45 29 COCCINELLIN ALKALOID Prococcinelline II 367 30 MONOTERPENE a-Pinene 337, 339 31 NORSESQUITERPENE A sesquiterpene keto allene 323 32 PHENOLIC 2, 5-Dichlorophenol 156 33 NITRILE Prunasin ( ? ) 55, 153, 507 34 PHENOLIC Marginalin 417 35 PYRROLIZIDINE ALKALOID P y r r o l i z i d i n e type 11, 402 F I G U R E 23. Some r e p r e s e n t a t i v e defensive chemicals i s o l a t e d from t e r r e s t r i a l arthropods. 1 FIGURE 23. * Some representative defensive chemicals i s o l a t e d from t e r r e s t r i a l arthropods. 146 but also as solvents and spreaders for formic acid produced by the (2) abdominal poison gland and (3) the mandibular glands that produce several . monoterpenes, e.g., c i t r o n e l l a l . A chemical, morphological and functional comparison of these glands as an alarm-defence system i s discussed i n an evolution-ary context by Wilson and Regnier ( 1971 ). Holldobler ( 1971 ) describes the function i n beetles of the abdominal adoption and appeasement glands which a i d i n mimicking the ants pheromonal recognition signals.Unfortunately the chemistry of these myrme-cophilous beetles has not been studied i n d e t a i l . A l e a f cutting ant Atta sexdans, besides producing i t s compliment of pheromones such as 4-methyl-3-heptanone (Blum, 1969 ), secretes from i t s metathoracic glands L— B-hydroxy-decanoic acid, phenylacetic acid, and i n d o l y l a c e t i c a c i d . These three substances a i d the fungal-ant symbiosis by promoting the growth of the fungi ( Schildknecht et a l . , 1973 )'. I t has been shown that phenylacetic acid and L-p-hydroxydecanoic acid are e f f e c t i v e as a n t i b i o t i c s against organsims such as E. c o l i , Staphlococcus aureus, and P e n i c i l l i u m glaucum. This a n t i b i o s i s e f f e c t i v e l y maintains pure cultures of the fungus for the ant ( Maschwitz et a l . , 1970; Schildknecht, 1971 ). The presence of i n d o l y l a c e t i c acid stimulates the growth of the fungi through an auxin-like e f f e c t ( Schildknecht et a l . , 1973 ). See addendum. In non-social animals such as d y t i s c i d water beetles ( e.g., Ilybius fenestratus and Dytiscus marginalis ) several glands e x i s t . A given beetle stores within i t s p y g i d i a l glands ( i n the po s t e r i o r of the abdomen ) a mixture of aromatic acids ( e.g., Figure 23,#6 ), and i n i t s prothoracic glands a v a r i e t y of pregnane steroids ( e.g., Figure 23, #23 ). The former secre-tions, i n conjunction with a glycoprotein,,functions to destroy 147 micro-organisms growing on the c u t i c l e which would render the animal hydrophilic. These s t e r o i d a l secretions also function as a deterrent to various predators ( Schildknecht, 1971; Schildknecht et a l . , 1967a ). The v a l i d i t y of these chemicals as a n t i b i o t i c s needs more rigorous i n v e s t i g a t i o n . The odiferous scent glands of Hemiptera ( Haviland-Brindley, 1930; Hepburn and Yonke, 1971; Waterhouse and Gilby, 1964; Remold, 1963; Tsuyuki et a l . # 1965 ) produce mainly short chain aldehydes and ketones ( e.g., oct-2-enal ), although alkanes ( e.g., n-tridecane ) and alkylacetates ( e.g., dodec-enyl acetate ) have been i s o l a t e d (Blum, 1961; Blum et a l . , 1961; Games and Staddon, 1973; Gilby and Waterhouse, 1967; G i l c h r i s t et a l . , 1966; McCullough, 1967; Pinder and Staddon, 1965; Waterhouse et a l . , 1961 ). trans-Hex-2-e'nal, a common component i n many of these Hemiptera, also has been shown to occur i n the secretions cf roaches, Eurycotis spp. ( Dateo and Roth, 1966; Eisner et a l . , 1959 ) and ants, Crematogaster spp. ( Crewe et a l . , 1972). These compounds have a reputed value as deterrents against a va r i e t y of predators; for large predators r e j e c t i o n seems to have an o l f a c t o r y basis. These compounds also possibly function as a n t i - b a c t e r i a l agents ( Major et a l . , 1960). This type of chemical defence i n d u l l coloured Hemiptera i s simple compared to the more complex defence i n mimetic or warningly coloured Hemiptera. Some b r i g h t l y coloured Hemiptera, e.g., Lygaeids such as Oncopletus spp. and Lygaeus spp., have not only metathoracic glands ( Johansson, 1957; Remold, 1963 ) which produce alkanones ( Games and Staddon, 1973; Waterhouse et a l . , 1961 ), but also have glands located d o r s o - l a t e r a l l y i n the the thorax and abdomen which store cardenolides ( Duffey, 1970; Scudder and Duffey, 1972 ). Thus, such animals may have two p o t e n t i a l l y d i f f e r e n t defensive mechanisms aimed at pre-148 dators' o l f a c t o r y and gustatory a b i l i t i e s . Experiments to de-termine the p a l a t a b i l i t y of c e r t a i n b r i g h t l y coloured Lygaeids have been ambiguous i n that they do not determine the unpala-table p r i n c i p l e ( F e i r and Suen, 1971; Gelperin, 1968; Sexton, 1964, 1966 ). Depending upon the predator's discriminatory c a p a b i l i t i e s , the prey w i l l or w i l l not be a Batesian-Mullerian mimic. Marshall ( 1902 ) a f t e r studying b r i g h t l y coloured and d i s t a s t e f u l insects suggested that many of the s i m i l a r l y s i z e d and coloured beetles, ants, bugs and roaches were involved i n mimicry complex. We now know more about the chemical bases of mimicry, and r e a l i z e that many d i f f e r e n t chemicals can function simultaneously as bases for mimicry complexes. Studies indicate that a chemically diverse Batesian-Mullerian mimicry complex exists i n North America. Some members are associated with the milkweeds, Asclepias spp. ( Duffey and Scudder, 1972; Jones, 1937; S l a t e r and Knop, 1969 ), as i s the case with c e r t a i n Lygaeids ( Lygaeus spp. ). Associated with these Hemiptera are b r i g h t l y coloured beetles ( e.g., Tetraopes spp., and Labido- mera c l i v i c o l l i s ) which feed on milkweeds and sequester card-enolides ( Duffey and Scudder, 1972 ), b r i g h t l y coloured beetles producing t h e i r own poisons ( cantharidin i n Epicauta spp.; C a r r e l and Eisner, 1974; dihydromatricaria a c i d i n Chauliogna-thus l e c o n t e i ; Meinwald et a l . , 1968b; see Figure 23, #4 and 6 ), and a v a r i e t y of mimetic beetles producing unknown chemicals ( Linsley, 1959, 1960,1961; L i n s l e y et a l . , 1961 )'. Mingled with t h i s complex are other mimetic Hemiptera ( e.g., Lygaeus spp. Dysdercus spp. ) that produce alkanones. This type of large complex was intimated by Darlington ( 1938 ) and Doesburg (1968). Other mimetic insects are also present that produce phenolics, quinones, and simple acids ( r e f e r to Eisner, 1970 ). The i n f e r -149 ence i s that a l l these mimetic insects share a wealth of weapons under the scrutiny of a v a r i e t y of predators. The complexity of defence i n the envirnoment i s f i n a l l y magnified by the discrimination of the p r e d a t o r s . l t has been shown ( Yang and Kare, 1968 ) that common defensive chemicals ( e.g., quinones, terpenes, phenolics and simple organic acids ) are d i f f e r e n t i a l l y e f f e c t i v e as a taste deterrent to q u a i l . One cannot assume that a l l predators have the same t a s t i n g or smelling a b i l i t i e s , or that the predator w i l l not a l t e r temp-o r a l l y i t s eating h a b i t s . Because of t h i s , colouration and/or distatefulness i s only meaningful i n terms of a perceptive predator. X.5- ENVIRONMENTAL IMPACT OF CHEMICALS The number of arthropod species approximates 1.5 m i l l i o n ( Borror and DeLong, 1971 ) of which there are stated to be 80,000 of poisonous q u a l i t y ( Pavan and Dazzini, 1971 ). Just over 200 chemicals have been i d e n t i f i e d from members of t h i s phylum. Many reviews on defensive chemicals are a v a i l a b l e ( Baslow, 1 9 7 1 ; Blum, 1969; Bucherl et a l . , 1 9 7 1 ; Eisner and Roth, 1962; Eisner, 1 9 7 0 ; Habermann, 1 9 7 2 ; Karlsson, 1 9 7 3 ; Mebs, 1 9 7 3 ; Pavan and Dazzini, 1 9 7 1 ; Roth and Eisner, 1 9 6 2 ; Weatherston, 1967; Weatherston and Percy, 1 9 7 0 ; Whittaker and Feeny, 1971 ) . The chemical structures of 35 defensive chemicals are depicted i n Figure 23. Other aspects of defence which are germane to a general understanding of chemical ecology are: a) the various roles a chemical has i n various organisms, and i t s spectrum of biolog-i c a l a c t i v i t y , b) the mode of storing and deploying the defen-150 sive chemical, and c) the o r i g i n of the chemical. These facets w i l l be considered below. HCN ( Figure 23, #1 ) i s produced not only by some plants, fungi, and micro-orgahsims ( Eyjolfsson, 1971; Montgomery, 1969), but also by millipedes, e.g., Apheloria corrugata ( Eisner H. et a l . , 1963 ), by a centipede Pachymerium ferrugineum ( S c h i l d -knecht et a l . , 1968a ),by moths, Zygaena spp. ( Jones et a l . , 1962 ), and by a beetle Paropsis atomaria ( Moore, 1967 ). The production of benzaldehyde ( Figure 23, #7 ) i s often concommitant with HCN production; i t should a r i s e from a common precursor, e.g., prunasin ( Figure 23, #33 ), as occurs i n plants ( Conn, 1974; Eyjolfsson, 1970 ). In arthropods that pro-duce HCN and benzaldehyde i t has been shown that these two chem-i c a l s serve as a defensive mechanism against predators( Blum et a l . , 1973a; Eisner and Eisner, 1965 ). Benzaldehyde might serve another function i n animals l i k e m i l l i p e d e s . I t quickly oxidizes i n a i r to benzoic acid; benzoic a c i d i s claimed to be a f u n g i s t a t i c agent for the apple and aspen trees ( Brown and Swinburne, 1973; Hubbes, 1969 ). The presence of phenol along with benzaldehyde i n Oxidus g r a c i l i s ( Blum et a l . , 1973a ) may enhance a n t i b i o s i s . A c l o s e l y r e l a t e d substance, benzyl alcohol, i s implicated i n the resistance of barley to the aphid Schizaphis gramihum ( Juneja et a l . , 1972 ). In other insects the role of a n t i b i o s i s has also been suggested for phenolic compounds ( Gilmour, 1965; Schildknecht, 1971 ). Benzaldehyde has been shown to occur i n the ant Veromessor pergandei ( no HCN produced ) as a defensive secretion ( Blum et a l . , 1969 ). In the noctuid moths Leucania impura, Pseudaletia separata, and P. unipunctata t h i s chemical has been selected as a sex phero-mone ( A p l i n and Birch, 1970; Clearwater, 1972; Grant et a l . , 1972 ). Benzaldehyde also functions as an alarm pheromone i n 151 conjunction with alkanols and alkanones i n the s t i n g l e s s bees Trigona mexicana and T. p e c t o r a l i s ( Luby et a l . , 1973 ). Phenolic acids such as protocatechuic acid ( Figure 23, #8 ) have been implicated not only i n resistance to disease i n onions ( Cruickshank and Perrin, 1964 ), but also i n a l l e l o -pathy amongst green plants ( Levin, 1971; Went, 1970; Whittaker, 1970 ). They may function likewise i n beetles which have evolved the a b i l i t y to secrete these compounds ( Schildknecht, 1971 ). Protocatechuic acid along with other compounds i s a growth factor for the s i l k moth Bombyx mori ( Hamamura, 1970 ). These chemi-cals are deposited i n the c u t i c l e of the insect ( Andersen, 1971; Brunet, 1967 ). Benzoquinones ( Figure 23, #3 ) and the corresponding hydroquinones ( Figure 23, #9 ) are produced by a v a r i e t y of fungi, green plants, and arthropods ( Thomson, 1971 ). The de-terrent q u a l i t i e s of benzoquinones are well documented ( Eisner, 1960; Eisner, 1970 ). Norris ( 1969,1970 ) showed that benzo-quinones act as a deterrent to insect feeding. I t appears that benzoquinones also have a n t i b i o t i c properties as was shown by F i e s e r and Ardao ( 1956 ) for the quinones of the arachnid Gony-lepti d e sp.. Hydroquinone ( Figure 23, #9 ) and i t s glycoside arbutin are related to resistance against b l i g h t i n pear (Thomson, 1971). Hydroquinone aid banzoguinone i s o l a t e d from the fungus Nectaria  c o r y l i also have a n t i b a c t e r i a l properties ( Nair and Anchel, 1972). These observations suggest that the possession of these chem-i c a l s by arthropods, which l i v e i n moist s o i l and l e a f l i t t e r , could be an e f f e c t i v e weapon against parasites and disease. Simple naphthoquinones occur i n green plants, fungi, and echinoderms ( Thomson, 1971 ). Various naphthoquinones have been 1 5 2 implicated i n resistance to disease i n a v a r i e t y of plants ( Goodman pt a l . , 1967 ). An a n t i b i o t i c , 6-methyl-naphthoquinone ( Figure 2 3, #4 ) has recently been i s o l a t e d from a fungus, Marasimus graminum ( Thomson, 1971 ). This naphthoquinone, along with simple benzoquinones, has been shown to occur i n the ten-ebrionid beetle Agroporis alutacea as a defensive secretion ( Tschinkel, 1972 ). Another simple naphthoquinone, juglone, ( Figure 21, #5 ) which occurs i n hickory, Carya ovata, has been shown to play an important role i n the host s e l e c t i o n pro-cess i n two wood beetles. The beetle Scolytus muMstriatus, feed-on Ulmus americana as a host plant, perceives juglone as a feeding deterrent. The beetle Scolytus quadrispinosus normally feeds on hickory and perceives juglone as a phagostimulant. Inclusion of the quinone i n an elm based d i e t prevents normal feeding by S. m u l t i s t r i a t u s ( G i l b e r t and Norris, 1968; Norris et a l . , 1970 ). Polyacetylenes such as dihydromatricaria acid ( Figure 23, # 6 ) i s o l a t e d from the beetle Chauliognathus lecontei ( Meinwald et a l . , 1968b ) are well known components of many plants, espec-i a l l y composites and umbellifers ( Bohlmann et a l . , 1968). Some polyacetylenes ( e.g., c i s - and trans-dihydromatricaria ester ) are e f f e c t i v e as antifungal agents ( Drake and Lam, 1974 ). Their convulsant properties (Quill lam and Sharpies, 1 9 6 9 ) may be e f f e c t i v e against herbivores and carnivores. Alkanones and alkenones are important chemicals for arthro-pods. The functions of hex-2-en-l-al ( 2-hexenal ) as a defensive agent i n Hemiptera has already been discussed. For phytophagous insects 2-hexenal functions e i t h e r as an attractant or repellent agent giving the insect i n i t i a l cues as to the nature of the food ( Beck, 1965; Dethier, 1970; Major, 1967; Murray et a l . , 1972 ). 153 Alkanones and alkenones are common constituents of plants ( Kemp et a l . / 1973; Meigh et a l . , 1973; Nicholas, 1973 ). Their roles i n resistance against insects and pathogens ( Major et a l . , 1960 ) are poorly understood. Certain alkanones and alk-enones have been selected by s o c i a l hymenoptera as alarm-de-fence pheromones (Blum, 1969,1970; Law and Regnier, 1971 ). trans-2-Hexenal i s a prominent constituent of oak leaves, and a Polyphemus moth, Antheaea polyphemus has adapted i t s sexual behaviour to depend upon the presence of t h i s chemical ( R i d d i f o r d , 1967 ). When the female moth feeds upon the leaves, 2-hexenal i s li b e r a t e d ; t h i s chemical then tr i g g e r s the release of her sex pheromone. The female moth i s highly adapted to the chemical constituents of oak, for i f she feeds upon another plant the v a r i a t i o n i n v o l a t i l e constituents, even i f 2-hexenal i s present, i s s u f f i c i e n t to prevent her from releasing her pheromone ( Riddiford, 1967 ). Iridoid s such as anisomorphal ( Figure 23. #14 ) are produced mainly by ants, e.g., Iridomymex spp. and Dolichoderus spp. ( C a v i l l , 1969; C a v i l l and Clark, 1971 ). These chemicals function as defensive secretions f o r insects such as the phasmid Anisomorpha buprestoides ( Meinwald et a l . , 1962, 1966a ). They also have i n s e c t i c i d a l properties ( McGurk et a l . , 1968 ). The active p r i n c i p l e of Catnip, Nepeta, i s an i r i d o i d that i s repellent to feeding insects ( Eisner, 1964 ). n-Undecane ( Figure 23,#9 ) occurs as an alarm-defence pheromone i n formicine ants ( Law and Regnier, 1971 ), and acts both as a behaviour modifier and as a solvent. However, the use-fulness of t h i s chemical i s not r e s t r i c t e d to just pheromonal tasks. I t i s a common component of the c u t i c l e of both insects 154 and plants ( Kolattukudy, 1970; Jackson and Baker, 1970; Tulloch, 1970 ). Alkanes of the c u t i c l e ( e p i c u t i c l e ) of i n -sects have been shown to reside as an oriented layer of molecules which i s responsible for rendering the c u t i c l e hydro-phobic, thus preventing water loss ( Chapman, 1969; Locke, 1961 ). Their function i n plants i s analogous, although not morphologically i d e n t i c a l i n t h e i r arrangement ( Eglinton and Hamilton, 1967; Kolattukudy, 1970 ). The role of cardiac glycosides ( Figure 23,#17 ) has already been discussed i n context with mimicry i n i n s e c t s . Homologues of these chemicals, the bufadienolides, are synthe-sized by the toad Bufo marinus ( Chen and Osuch, 1969; Tschesche 1972 ),and serve as e f f e c t i v e anti-predator devices. Sulphated cardenolides are found i n echinoderms and holothurans but t h e i r function i s not understood. Cardenolides have a wide occurrence i n plants ( Hoch, 1961; Singh and Rastogi, 1970 ). Cows avoid plants containing these substances probably because of t h e i r bitterness and emetic properties. The presence of these chemi-cals can deter feeding i n insects such as grasshoppers ( Dadd, 1963 i n reference to S c i l l a ). Cardenolides are also capable of inh i b i t i n g the growth of micro-organsims because of t h e i r c y t o t o x i c i t y and interference with membrane i n t e g r i t y ( Kupchan et a l . , 1964; Goodman and Gilman, 1970 ). Other polyhydroxylated steroids from plants have profound hormonal e f f e c t s i n i n s e c t s . These are the ponasterones which mimic the e f f e c t of the moulting hormone, ecdysone ( Horn, 1971; Rees, 1971; Williams, 1970 ). The abundance of steroids i n plants ( phytosterols, cardenolides, saponins, et cetera ) i s d i f f i c u l t to explain from our present Ikiowledge. I t i s pos-155 "sible that many of the phytosterols and cardenolides i n t e r f e r with the uptake and metabolism of e s s e n t i a l steroids by i n -sects. A discussion of s t e r o i d u t i l i z a t i o n by various organ-isms i s given by Clayton ( 1964,1970 ). Some simple dietary steroids provide gustatory cues for feeding insects ( Hama-mura, 1970; Schoohoven, 1972 ). Saponins also provide p o s i -t i v e and negative feeding s t i m u l i f or several insects ( Beck, 1965; Herout, 1969; Schoonhoven, 1972 ) as well as being t o x i c to feeding insects ( Applebaum et a l . , 1965; Basu and Rastogi, 1967; Birk, 1969; Harley and Thorsteinson, 1967; Schoonhoven, 1972; Schrieber, 1959 ) and mammals ( Birk, 1969 ). Several reviews ( Clayton, 1970; Dodson, 1970; Herout, 1969; Wiliiams, 1970 ) discuss the impact of isoprenoids i n the environment with p a r t i c u l a r reference to in s e c t s . The e f f e c t of simple monoterpenes such as c i t r a l and c i t r o n e l l a l has been discussed. Geraniol ( Figure 23, #18 ), i n s o c i a l formicine ants, and i n the bee Apis mellifera/functions as an alarm-defence chemical ( Law and Regnier, 1971 ) but also functions i n the bee as a recruitment pheromone. C i t r a l and s i m i l a r substances have been c i t e d as feeding stimulants f o r the s i l k moth Bombyx mori which feeds on the mulberry ( Morus )( Hamamura, 1970; Schoon-hoven, 1972 ) . The c y c l i c monoterpene liminone has been shown to be an alarm-defence chemical i n termites, Drepanotermes spp. ( see Moore, 1968 ), as well as a feeding attractant for bark beetles ( Schoonhoven, 1972 ). A very subtle role for a-pinene and other monoterpenes i s t h e i r e f f e c t upon sexual maturation i n desert locuts, Schistocerca gregaria ( C a r l i s l e et a l . , 1965 ). The locust gears i t s egg production with the rains by responding to the release of these chemicals from the buds of a myrrh shrub ( Commiphora spp.). The role of 156 a- and p-pinene i n bark beetles ( Ips spp., Scolytus spp., and Dendroctonus spp. ) i s complicated because these terp-enes,along with geraniol and others, are the attractants used for host detection ( Rudinsky, 1966 ). Upon ingestion both a-and p-pinene are oxidized by Dendroctonus f r o n t a l i s to i t s aggre-gation pheromones ( Renwick et a l . , 1973 ) . These oxidation pro-ducts a t t r a c t others of the species to the trees ( Bedard et a l . , 1969; Law and Regnier, 1971; Renwick et a l . , 1973 ). A predacious beetle, Thanasimus dubius,has become an opportunist through a precise olfactory locatory mechanism. I t u t i l i z e s the sex pheromones produced from a- and p-pinene to locate i t s prey, the southern.pine beetle D. f r o n t a l i s ( V i t e and Williamson, 1970 ). The mustard o i l s ( glucosinolates; Figure 23, #25 ) are toxic to c a t t l e ( VanEttem, 1969 ). Certain glucosinolates, such as s i n i g r i n and glucocapparin, have been shown to be ef-f e c t i v e stimulants, deterrents, as well as o v i p o s i t i o n a l stim-ulants for several insects ( David and Gardner, 1966a,b; Schoon-hoven, 1972; Thorsteinson, 1953, 1960 ). Matsumoto ( 1970 ) discusses the role of glucosinolates, isothiocyanates, and other n a t u r a l l y occurring sulphur compounds i n i n f l u e n c i n g attack by herbivores. Some lepidoptera, e.g., P i e r i s brassicae and P. rapae, sequester some glucosinolates for defence ( Rothschild, 1972 ). In order for arthropod? to avoid being poisoned by chem-i c a l s acquired from t h e i r food or v i a t h e i r own defensive bio-chemistry they must have adequate detoxication and/or storage mechanisms. 157 X.6- DETOXICATION OF CHEMICALS The arthropod has evolved i n a v a r i e t y of ways to res-pond to the injurious chemicals encountered i n i t s food. A morphological study determining the evolutionary impact of d i f f e r e n t amounts of s i l i c o n i n various species of grasses upon various species of grasshoppers revealed that the orthopt-erans that fed upon the high s i l i c o n grasses had mandibles which were more extensively s c l e r o t i z e d and adapted for crush-ing ( Isley, 1944 ). I t i s easy to surmise that insects which suck plants face d i f f e r e n t mechanical problems a r i s i n g from l i g n i f i c a t i o n than do insects that chew. Plants can provide s i g n i f i c a n t morphological b a r r i e r s to feeding because of h a i r s , spines, c r y s t a l s , and other structures ( based on non-toxic use of chemicals ( Beck, 1965; G i l b e r t , 1971; Breedlove and E h r l i c h , 1968 ). Some insect herbivores can avoid a p o t e n t i a l l y toxic plant because of the presence of s p e c i f i c chemicals that i n t e r f e r with feeding behaviour. This has been mentioned with reference to naphthoquinones,glucosinolates, and s t e r o i d a l compounds. For d e t a i l e d discussions of t h i s aspect of host-plant recognition see Beck ( 1965 ), Dethier ( 1970 ), Herout ( 1969 ), Schoonhoven ( 1972 ) and Wood et a l . ( 1970 ) . I f an arthropod feeds upon a plant with t o x i c chemicals i t must i n some manner avoid these chemicals. This can be ac-complished by s e l e c t i v e feeding habits. Rothschild et a l . ( 1970a) have shown that the aphid Aphis n e r i i when feeding on t o x i c oleander, Nerium oleander, avoids many of the cardenolides i n the plant by i n s e r t i n g i t s s t y l e t s i n t o the phloem. They also mention a sphingid moth, D e i l e p h i l i a n e r i i feeds upon the same plant and avoids cardenolides by passing them d i r e c t l y through the gut. There i s a s i m i l a r adaptation i n the tobacco hornworm 158 larva, a sphingid Protocarpe sexta,that feeds upon tobacco. This moth avoids the nicotine ( Yang and Guthrie, 1969 ) by-possessing an e f f i c i e n t excretory system such that the nico-tine, even though i t enters the haemolymph, never reaches t o x i c l e v e l s ( S e l f et a l . , 1964a,b ). Some other insects that feed upon tobacco avoid t o x i c a t i o n by o x i d i z i n g nicotine to non-tox i c cotinine and other products ( Schmeltz, 1971; S e l f et a l . , 1964a,b ). The a b i l i t y of insects to enzymatically detoxify foreign substances, be they of natural or synthetic origin, i s well documented ( Hitchcock and Smith, 1966; Krieger et a l . , 1971; Menzie, 1972; Raymond et a l . , 1972; Smith, 1962 ). T o l -erances to noxious chemicals,either because of p h y s i o l o g i c a l i n s e n s i t i v i t y or enzymic detoxication processes, are important for host-herbivore relationships i n the evolutionary time scale ( Bock, 1972; Krieger et a l . , 1971 ), as well as the l i f e - c y c l e scale. Tolerances to plant poisons among insects have led to some important defensive mechansims against predators. Asclep-iadaceae and Apocynaceae contain t o x i c cardenolideswhich are sequestered by various insects. The Aristolochiaceae provides p a p i l i o n i d b u t t e r f l i e s ( Pachlioptera a r i s t o l o c h i a e , Battus and B. polydamus with a r i s t o l o c h i c acids, Figure 23,#16 ) ( Rothschild, 1972 ). The a b i l i t y of these insects to tole r a t e such chemicals i n part l i e s i n t h e i r a b i l i t y to l o c a l i z e the chemicals i n gland-like structures or within r e l a t i v e l y i n e r t wings ( Parsons, 1965; Scudder and Duffey, 1972 ). This l o c a l -i z a t i o n of defensive chemicals i n glands maximizes t h e i r de-ployment. When a predator seizes the prey, the glandular con-tents are released as an ooze or spray, where rotations of the body , thrashings of legs, and/or movement of the wings can spread the chemicals with greater e f f i c i e n c y . 159 Predators can avoid the undesireable q u a l i t i e s of t h e i r prey by s e l e c t i v e l y eating choice body parts. Birds, such as jays, avoid the offensive regurgitation of grasshoppers ( e.g., Romalea microptera ) by tearing o f f the head and eating a l l but the head and crop ( Eisner, 1970 ). Mice avoid the caudal discharges of quinone producing beetle ( Eleodes spp. ) by holding the prey so i t discharges a l l of i t s offensive ware into the ground ( Eisner, 1970 ). A mouse opossum, Marmosa  demararae, attacks the terpene producing phasmid Anisomorpha  buprestoides by f i r s t holding i t with i t s jaws and then i t s paws u n t i l a l l the chemical of the insect i s v a i n l y used ( Eisner, 1970 ). In terms of predatory reduviids and penta-tomids, defensive chemicals confined i n the prey's gland(s) may not be e f f e c t i v e as a deterrent. I f the predator acheives an ef-fe c t i v e grasp, the prey could discharge i t s t o t a l arsenal i n e f f e c t u a l l y while the predator sucks on the body contents which are i s o l a t e d from the poisons. However, even .the cantharid beetle Epicauta immaculata, which i s immune to attack from a number of predators,such as the beetle Calosoma prominens, f a l l s prey to mantids ( Stagomantis spp.) and reduviids ( Ap-iomeris spp. )( C a r r e l and Eisner 1974; Eisner, 1970 ). X.7- MORPHOLOGICAL ASPECTS OF DEFENCE The evolution of chemical defences usually demands the coincident s e l e c t i o n of morphological and behavioural adapta-tions. Most arthropod defences are b u i l t on several simple plans: a) e n t e r i c or haemocoelic discharges involving no s p e c i a l glands as compared to (b) f l e x i b l e or r i g i d glandular chambers contained i n the body, and (c) external processes such as hairs, barbs, and scales. The chemicals, i n most cases, are 160 presented to the attacker by the combined action of haemo-c o e l i c pressure ( hydrostatic ), and muscles which co n t r o l gland c o n s t r i c t i o n s , opercula, or the return of eversible sacs. A few examples w i l l be given; Eisner (1970 ) s i t e s many examples with p a r t i c u l a r reference to chemistry and function. Several examples have been been described; the aphid c o r n i c l e s , the bombardier beetle, and the hemipteran scent gland. The enteric regurgitation of the locust Locusta migra-o r i a i s i r r i t a t i n g and p o t e n t i a l l y t o x i c to mammals.( Freeman, 1968 ), as well as unacceptable to ants and birds ( Eisner, 1970 ). This method of defence l i k e defecation involves only the alimentary canal, rather than s p e c i a l i z e d storage struc-tures. Reflex bleeding as described i n cantharid and meloid beetles ( Ca r r e l and Eisner, 1974; Eisner, 1970 ) ar i s e s from the breakage of inherently weak c u t i c l e at l eg j o i n t s . In meloid beetles the expressed f l u i d contains cantharidin ( Spanish F l y ) which has the a b i l i t y to deter predators. Another simple means of expelling defensive chemicals i s by the use of eversible sacs, such as has been described for the beetle Agroporis alutacea ( Tschinkel, 1972 ) and for butter-f l i e s , e.g., P a p i l i o machaon•( Eisner and Meinwald, 1965 ). The b u t t e r f l y larva everts the gland by means of hydrostatic pres-sure. I t i s able to control which side of the b i l a t e r a l gland i t extrudes and the extent to which i t i s extruded. This control can r e s t r i c t the evaporative loss of i s o b u t y r i c and 2-methyl-butyric acids. The eversible abdominal glands of the tenebrion-i d beetle Agroporis alutacea ( secretes benzo- and naphthoquin-ones ) are extruded by an increase, i n haemocoelic pressure. Upon extrusion the movement of the legs e f f e c t i v e l y spreads the 161 quinones over i t s body. In the event that not a l l the secre-t i o n i s used, the glands are able to r e t r a c t with the remain-der ( Tschinkel, 1972 ). In these eversible glands the means of r e t r a c t i o n i s eit h e r caused by muscle and/or the drop i n haemocoelic pressure. Glands need not be eversible i n order to expel t h e i r contents. The structure of the metathoracic glands of Hemipt-era i s c l e a r l y outlined by Remold ( 1963), Johansson ( 1957 ), Waterhouse and Gilby (1964)and Hepburn and Yonke ( 1971 ). In most cases these glands consist of paired i n t e r n a l e l a s t i c reservoirs with or without accessory glands. The e l a s t i c s tor-age reservoirs lead, v i a ducts with complex valve apparatus, to b i l a t e r a l o r i f i c e s onto an evaporatorium ( the evaporative area consists of microscopic mushroom bodies; F i l s h i e and Waterhouse, 1969; Remold, 1963 ). Upon alarm, hydrostatic pres-sure compresses the storage reservoirs permitting the f l u i d to flow out past the opened valves onto the evaporator-ium. The f l u i d spreads over t h i s mushroom bodied area, but doos not leave i t , because these bodies trap and confine the f l u i d . This not only avoids s e l f - t o x i c a t i o n but s i m p l i f i e s the task of presenting the f l u i d to a predator through the action of thrashing legs ( Remold, 1963 ). A carabid beetle, Calosoma prominens ( salicylaldehyde ) and a phasmid , Anisomorpha buprestoides ( anisomorphal ) hav<~; f l e x i b l e i n t e r n a l glands which extrude t h e i r contents by the contraction of associated muscles ( Eisner, 1965, 1970; Eisner et al.,1963b,c ), rather than by haemostatic pressure. Another means of ejecting defensive f l u i d i s by loading the tracheal system with i t and using a i r pressure to blow out the l i q u i d . This mechanism i s employed by the cockroach Diploptera punct?>.):•'-* 162 which produces quinones ( Roth and Stay, 1958 ). The adult grasshopper Poekilocerus bufonis, which sequesters cardeno-l i d e s , releases these chemicals from a f l e x i b l e gland such that the defensive f l u i d flows down the body to the abdominal tracheae where expelled a i r causes frothing ( von Euw et a l . , 1967; Rothschild, 1966 ). The morphology and mechanism involved i n the produc-t i o n of benzoquinones by a s p i r o b o l i d millipede has been comp-ared to that of other millipedes, including HCN producing poly-desmids ( Woodring and Blum, 1963 ). These are discussed below. The s p i r o b o l i d type gland i s composed of one sac ( Figure 24A ) that secretes quinones from a surrounding layer of c e l l s i n t o the lumen of the sac. The stored quinones are exuded by pressure exerted from the body wall muscles i n which the gland i s embedded. Pressuie can be b u i l t up i n the sac by the delayed release of the muscles attached to the neck of the gland. These muscles control the c o n s t r i c t i o n and tongue near the o r i f i c e of the gland. Millipedes of t h i s type can e i t h e r l e t the f l u i d ooze out or eject i t as a j e t . Om species studied by me could spray a narrow j e t of defensive f l u i d a meter ( Rhinocricus  holomelanus). For more d e t a i l s see Weatherston and Percy ( 1969 ) and Woodring and Blum (1963 ). The polydesmid type of gland which produces HCN and benz-aldehyde i s more complicated i n that i t consists of two chambers ( Woodring and Blum, 1963 ). One chamber i s c u t i c u l a r but f l e x -i b l e , and the other c u t i c u l a r and r i g i d ( Figure 24C ). The f l e x -i b l e chamber stores a n i t r i l e , which when squeezed into the r i g i d chamber by haemocoelic pressure ( the muscle retracts c o n s t r i c -t i o n ), interacts with an enzyme that breaks the n i t r i l e apart 163 F I G U R E 2 4 . D i a m r a m m a t i c r e p r e s e n t a t i o n o f t h r e e t y p e s o f d e f e n s i v e g l a n d s a n d t h e i r p r o d u c t s . 164 into HCN and benzaldehyde. These two products ooze from the o r i f i c e i n comparison to the ejection process of the s p i r i o -b o l i d type. The reaction chamber of a polydesmid millipede does not house an explosive reaction l i k e that encountered i n some quinone producing beetles ( Aneshansley et a l . , 1969; Eisner, 1970; Schildknecht and Holoubek,1961; Schildknecht et a l . , 1970 ). Figure 24B shows the two chambered glandular system of a Bomb-ardie r beetle composed of a storage chamber with an adjoining accessory gland and a reaction chamber. The storage chamber houses a mixture of hydroquinones and hydrogen peroxide ( Ti^O^ ) . When these compounds are squeezed in t o the reaction chamber and mixing occurs between the contents of the two chambers, an exo-thermic explosion occurs. The heat i s produced by the oxidation of the quinones mediated by peroxidase and catalase; the pro-duced by the action of catalase on H202 drives the hot mixture of quinones through the unblocked pore of the reaction chamber. The connection between the two chambers during the explosion i s blocked. A more complicated arrangement of the two-chambered secretory c e l l system i s seen i n some tenebrionid beetles, where-i n a multitude of two-chambered units empty t h e i r quinones- i n t o a common chamber for discharge. For more d e t a i l s of t h i s type ref e r to Eisner et a l . ( 1964 ) and Happ ( 1968 ). A modern review ( Noirot and Quennedey, 1974 ) describes the f i n e structure of insect epidermal glands, which includes s a l i v a r y , pheromonal, and defensive glands. However, c e r t a i n exocrine glands i n s o c i a l Hymenoptera are not of ectodermal or-i g i n . Blum ( 1970 ) discusses these allomonal and pheromonal glands. Dermal glands are secretory structures ( ectodermal ) 165 lying within the epidermis of the cuticle. The epidermis is a unicellular layer of cells lining the internal portions of the cuticle ( Borror and DeLong, 1971; Chapman, 1969; Maloeuf, 1938 ). In most instances these glands are lined with cuticle on the luminal side. This configuration can be seen in Figure 24A. In this case the glandular structures represent an i n -vagination of the cuticle, so that the previously external cuticle now faces the lumen of the gland, and the epidermis becomes the layer of secretory c e l l s . The fine structure of defensive and pheromonal glands are described in the following references ( Crossley and Waterhouse, 1969a,b; Eisner et a l . , 1964; Fletcher, 1969; Happ et a l . , 1966; Lai-Fook, 1970, 1972, 1973; Noirot and Quennedey, 1974; Tschinkel, 1969, 1972 ). X.8- ORIGIN OF DEFENSIVE CHEMICALS The structures of the chemicals depicted in Figure 23 represent natural secondary products that occur in a variety of organisms ( bacteria, fungi, plants and animals ). The oc-currence of these kinds of chemicals particularly in angiosperms and t e r r e s t r i a l arthropods raises the questions as to whether these chemicals are of plant or animal origin. Several possibil-i t i e s exist within these two categories, a) that the chemicals are sequestered from the food source, b) that the chemicals are sequestered from the food source and subsequently modified chemically by the arthropod, or by the arthropod's symbionts, c) that the chemicals are synthesized by the organism's own meta-bolic capabilities, or d) that the molecules are p a r t i a l l y or completely constructed by alimentary, i n t e r s t i t i a l , or i n t r a c e l l -ular symbionts with or without the arthropod 1s biochemistry. These aspects w i l l be discussed below. 166 Because arthropods feed upon plant and animal material not only to acquire food but also e s s e n t i a l chemicals ( v i t a -mins, polyunsaturated fats, s t e r o l s , amino acids, metals and other growth factors; Dadd, 1973 ) they w i l l also acquire po-t e n t i a l l y dangerous chemicals ( see Table XIII ). These may be s e l e c t i v e l y excreted, made metabolically i n e r t , or sequest- '• ered. This l a s t a l t e r n a t i v e endows c e r t a i n arthropods with un-ique chemicals for defensive purposes ( Table XIII ). S l a t e r ( 1877 ) proposed that b r i g h t l y coloured butter-f l y larvae acquired t h e i r distastefulness from t h e i r odorous, d i s t a s t e f u l or poisonous host plants. Hasse ( 1896 i n Brower and Brower 1964 ) showed a c o r r e l a t i o n between l a r v a l feeding upon obnoxious plants and d i s t a s t e f u l n e s s . A hiatus existed for many years concerning the r o l e of secondary plant compounds i n the bidLogy of insects, u n t i l the middle of the 1900's when bio-l o g i s t s became concerned with the r o l e of plant chemicals i n terms of host s e l e c t i o n mechanisms ( Dethier, 1941, 1954; Fraen-k e l , 1959; Thorsteinson, 1958 ), and the evolution of plant insect r e l a t i o n s h i p s with reference to mimicry and unpalatab-i l i t y ( Brower and Brower, 1964; Ehrlich' and Raven, 1964 ). Then came proof that mimetic distasteful insects, e.g., the Monarch b u t t e r f l y Danaus plexippus, were able to sequester chemicals from plants, and transfer these chemicals from the larvae to the adult ( Brower, 1970; Brower et a l . , 1967 )„ The cardiac glycosides sequestered by the Monarch butter-f l y are found to be l o c a l i z e d i n the wings and other portions of the body ( Parsons, 1964 ). This means that these chemicals have crossed gut and haemolymph. This process i s unique perhaps only i n that we consider these devastating drugs when contacted i n high concentrations. However, the deposition of plant f l a v -C H E M I C A L S S E Q U E S T E R E D I N S E C T T Y P E U S E R E F E R E N C E # F L A V O N O I D S B u t t e r f l i e s C o l o u r a t i o n 3 4 4 , 4 6 3 C A R O T E N E S B u t t e r f l i e s , m o t h s C o l o u r a t i o n 1 0 8 , 2 2 7 C A R D E N O L I D E S B u t t e r f l i e s , g r a s s h o p p e r s , b u g s D e f e n c e 4 0 2 P Y R R O L I Z I D I N E A L K A L O I D S M o t h s D e f e n c e 1 1 , 3 4 8 , 4 0 2 C Y C A S I N M o t h D e t o x i c a t i o n 4 6 1 A M A R Y L L I D A C E O U S A L K A L O I D S M o t h D e f e n c e 4 0 2 H Y P E R I C I N B e e t l e D e f e n c e 4 0 2 G L U C O S I N O L A T E S B u t t e r f l i e s D e f e n c e 4 0 2 N O R S E S Q U I T E R P E N E G r a s s h o p p e r D e f e n c e 3 2 3 A R I S T O L O C H I C A C I D S B u t t e r f l i e s D e f e n c e 1 7 0 , 4 0 2 , 4 0 6 C H L O R O - I N S E C T I C I D E S G r a s s h o p p e r D e f e n c e 1 5 6 M O N O T E R P E N E S B e e s C o m m u n i c a t i o n 1 3 6 a , b T A B L E X I I I . Some c h e m i c a l s s e q u e s t e r e d f r o m p l a n t s b y i n s e c t s 16 8 anoids i n wings of Lepidoptera ( Morris and Thomson,1964 ), and the metabolism and deposition of plant carotenes in t o the cu-t i c l e and fat body of Lepidoptera ( Clark,1971; F e l t w e l l and Valadon, 1972; Harashima et a l . , 1972; Mummery and Valadon, 1974 ) seems less dramatic. P y r r o l i z i d i n e a l kaloids from Senecio spp. are to x i c ( Mattocks, 1968 ) to c a t t l e , but these chemicals may be of no consequence to the unpalatable moth larvae of Callimorpha  jacobeae that feed upon tansy ragworts ( A p l i n et a l . , 1968; Rothschild, 1972 ). The a b i l i t y of moths of Zygaena spp. to tolerat e HCN l i b e r a t e d from t h e i r tissues when wounded ( Jones et a l . , 1962 )is s u r p r i z i n g i f one considers the r e a c t i v i t y of mammalian haemoglobins and cytochromes.with ~CN. But i f one considers that these moths probably lack haemoglobins, do not have high r e s p i r a t i o n a l rates, and also possess rhodanese to convert the HCN to thiocyanate ( SCN ) (Jones et a l . , 1962 ), i t i s not remarkable that they are tol e r a n t . The formation of SCN i s common i n plants and mammals (Chew and Boey, 1972; Dixon and Webb,1964; Montgomery, 1969 ). Sp e c i a l i z a t i o n s can be based on the optimization of common b i o l o g i c a l features rather that evolution of s t r i k i n g adaptations. Rothschild has reviewed ( 1972 ) the presence of second-ary chemicals sequestered from plants by insects i n r e l a t i o n to defence and mimicry. Some examples of plant chemicals sequest-ered mainly for defensive purposes are given i n Table XIII. In many cases these chemicals seem to be stored i n the body i n glands, wings or unknown pools without metabolic a l t e r a t i o n . In ce r t a i n cases the insects are able to metabolize the plant con-s t i t u e n t s . The moth S e i r i a r c t i a echo ( Teas, 1967 ) which feeds 16 9 on.Cycas containing the poisonous glucoside, cycasin ( Figure 23, #27 ), avoids t o x i c a t i o n from the carcinogenic aglycone { Smith, 1966; Yang and Mickelson, 1969 ) as follows. The glycoside i s i n i t i a l l y hydrolyzed by action of p-glycosidases i n the plant or gut of the insect. The aglycone i s then con-verted back to the non-toxic glycoside within the various t i s -ues of the insect. Glycosylation i s a common method of detox-i c a t i o n i n insects ( Smith, 1962 ). The occurrence of several defensive chemicals ( mammal-ian steroids, salicylaldhyde, skatole, a norsesquiterpene, and a dichlorophenol for example. ) raises i n t e r e s t i n g questions concerning t h e i r o r i g i n . Studies of these chemicals have not revealed the r o l e of the plant or the insect i n t h e i r production. The presence of pregnanes and oestrones i n the defensive secretions of d y t i s c i d water beetles, e.g., Ilybius fenestratus ( Figure 23, #23; Schildknecht, 1971; M i l l e r and Mumma, 1973 ), indicates an e x t r i n s i c o r i g i n for these molecules since insects have been shown to be incapable of synthesizing steroids ( Good-fellow and Liu, 1972 ). These beetles are predatory and thus must derive t h e i r steroids from insects which have fed upon . plants containing oestrones or pregnanes, or have metabolized them from basic cholestanes or other phytosterols. Plants are known to contain steroids such as testosterone and oestrone ( Dean et a l . , 1971; Gawienowski and Gibbs, 1969 ). However, Schildknecht et a l . have shown cholesterol to be aprecursDr for such stem ids in Dytiscids* . Insects have the a b i l i t y to metabolize a v a r i e t y of phytosterols ( Clayton, 1970; Heftmann, 1968 ). The inform-ation concerning dietary steroids implies t h i s a b i l i t y to metabolize steroids. I t has recently been shown that the is o l a t e d abdomen of the s i l k moth Bombyx mori can synthesize 14 . . a- and p-ecdysones from c h o l e s t e r o l - C ( Nakanishi et a l . , 1972 ). * Schildknecht et a l . 1970 , Ang. Chemie 9:1. 170 Salicylaldehyde occurs i n a number of insects, e.g., Chrysomela interrupta and Calosoma prominens as a defensive secretion ( Blum et a l . , 1972; Eisner et a l . , 1963b; Wain,1943; Wallace and Blum, 1969 ). A related compound,salicyl alcohol, occurs i n a number of plants ( S a l i x ) as the O-glucoside ( Harborne, 1964 ). I t has not been c l a r i f i e d whether s a l i c y l -aldehyde originates i n these insects by hydrolysis of the glyc-oside with subsequent oxidation of the alcohol, or v i a the hy-droxylation of compounds l i k e benzaldehyde and benzoic ac i d . I t i s possible that symbiotic micro-organisms produce s a l i c y l a l d e -hyde, for i t i s known to a r i s e from isochorismic acid i n bac-t e r i a ( Towers, 1964 ). A s i m i l a r s i t u a t i o n exists with the presence of skatole i n a lacewing Chrysopa oculata ( Blum et a l . , 1973b ). I t i s not known i f t h i s aquatic larva acquires the skatole from micro-organismal a c t i v i t y i n the environment or v i a t h e i r own metabolism of tryptophan. Salicylaldehyde Skatole B-Ionone Locustol Meinwald et a l . ( 1968b ) suggest that the norsesquiter-penoid ( Figure 23, #31 )found i n the grasshopper Romalea  microptera originates by the ingestion and metabolism of xantho-phy l l s from plants;-But perhaps the plant c a r r i e s out t h i s ox-i d a t i o n since s i m i l a r compounds are known to a r i s e during " f e r -mentation" of tea and tobacco ( Aasen et a l . , 1972; Sanderson, 1972 ), i n which p-ionone derivatives are formed by the oxid -171 ation of carotenes and xanthophylls. A pheromone recently i d e n t i f i e d i n the frass of the locust Locusta migratoria migratorioides ( Nolte et a l . , 1973 ) bears a s t r i k i n g resemblance to the c o n i f e r y l derivatives of plants. Those authors suggest the p o s s i b i l i t y that the insect degrades plant phenolics to produce t h i s pheromone. The modern use of i n s e c t i c i d e s has had i t s impact even on the defensive secretions of arthropods. Eisner et a l . ( 1971a ) reported the existence 2 y5-dichloro-phenol ( Figure 23. #32) i n the defensive f l u i d of the grasshopper Romalea  microptera. They suggest that t h i s chemical arose from the metabolism of i n s e c t i c i d e s by plants. The s i t u a t i o n i s doubt-f u l l y that simple. 2,4-Dichloro-phenol has been i d e n t i f i e d as i n s o i l as a fungal natural product ( Ando and Kato, 1970 ). Menzie ( 1972 ) reviews the derivation of many chlorinated phenols from the biodegradation of i n s e c t i c i d e s such as DDT and y-BHC. The presence of 2,4-dichloro-phenol i n Romalea may be the r e s u l t of previously extensive metabolism by micro-or-ganisms and plants, and by photo-oxidation ( Matsumura et a l . , 1972 ). A complicating factor i n the study of the o r i g i n of defen-sive chemicals i n arthropods i s the a b i l i t y to d i s t i n g u i s h un-ambiguously between the metabolism of the insect and that of symbionts. The function of micro-organisms i n the synthesis of e s s e n t i a l amino acids for insects i s well documented ( Dadd, 1973 ). The production of the pheromones of the southern pine beetle Dendroctonus f r o n t a l i s by the ingestion of a-and 3-pinenes , and the release of these pheromones i n the faeces ( Renwick et a l . , 1973 ),is suggestive that metabolism of these 172 p i n e n e s i s a c c o m p l i s h e d b y o r g a n i s m s s u c h as pseudomonads. Pseudomonads a r e c a p a b l e o f o x i d i z i n g p - p i n e n e t o t h e s e x pheromones o f t h e s e b e e t l e s i n v i t r o ( F o n k e n and J o h n s o n , 1 9 7 2 ) . The b i o s y n t h e s i s o f a s c o r b i c a c i d f r o m s u g a r s i n t h e c o c k r o a c h L e u c o p h a e a maderae i s t h e r e s u l t o f i n t r a c e l l u l a r s y m b i o n t s ( P i e r r e , 1962 ) . The s y m b i o n t s must be i s o l a t e d and t h e i r met-a b o l i s m s t u d i e d a n d compared t o t h a t o f a p o s y m b i o t i c h o s t s i n o r d e r t o a c c o u n t f o r t h e c a p a b i l i t e s o f t h e h o s t . X.9- BIOSYNTHESIS OF DEFENSIVE CHEMICALS Some a r t h r o p o d d e f e n c e s a r e n o t o f p l a n t o r i g i n , b e c a u s e t h e y a r e p r o d u c e d r e g a r d l e s s o f t h e n a t u r e o f t h e f o o d . A s p r e -v i o u s l y m e n t i o n e d t h e s t r u c t u r e s o f t h e c h e m i c a l s d e p i c t e d i n F i g u r e 2 3 a r e r e m i n i s c e n t o f p l a n t a n d / o r f u n g a l p r o d u c t s . I t i s t h e r e f o r e l o g i c a l t o a s k i f t h e a r t h r o p o d p r o d u c e s t h e s e c h e m i c a l s i n a manner s i m i l a r t o m i c r o - o r g a n i s m s , f u n g i , o r 14 p l a n t s .There are few b i o c h e m x c a l e x p e r i m e n t s e m p l o y i n g C - p r e -cursors t o show t h a t i n s e c t s a r e r e s p o n s i b l e f o r t h e i r own b i o -s y n t h e s e s , a n d f e w e r t h a t e l u c i d a t e t h e b i o s y n t h e t i c p a t h w a y s o f d e f e n s i v e c h e m i c a l s i n a r t h r o p o d s . I n one o f t h e f i r s t i n v e s t i g a t i o n s t o d e t e r m i n e t h e o r -g i n o f i n s e c t d e f e n s i v e s e c r e t i o n s ( G o r d o n e t a l . , 1963 ), t h e b i o s y n t h e s i s o f t h e s c e n t c o n s t i t u e n t s o f t h e p e n t a t o m i d bug . . . . 14 N e z a r a v i r i d u l a was i n v e s t i g a t e d . T h ey f o u n d t h a t a c e t a t e - 1 -was i n c o r p o r a t e d w i t h low e f f i c i e n c y i n t o h e x - 2 - e n a l , d e c - 2 - e n a l , and n - t r i d e c a n e . They c o n c l u d e d t h a t t h e a n i m a l makes i t s own s c e n t components f r o m a c e t a t e . A l t h o u g h t h i s e x p e r i m e n t p a r t i a l l y e s t a b l i s h e s t h e b i o c h e m i c a l autonomy o f t h e i n s e c t i t c o n t r i b u t e s l i t t l e i n f o r m a t i o n as t o t h e p a t hway i n v o l v e d and a s c r i b e s no t i s s u e o f o r i g i n . T h e s e r e s u l t s aus ambiguous b e c a u s e i t i s 173 possible that a f a t t y acid might be made f i r s t and then broken 14 down to form hex-2-enal. The incorporation of l m o l e n i c acid - C into this aldehyde i n the presence of O2 has been documented i n Ginkgo ( Major and Thomas, 1972; Major et a l . , 1972 ). Along the same l i n e of reasoning, the o r i g i n of the poly-acetylenic acid, dihydromatricaria a c i d ( Figure 23, #6 ) may be v i a the oxidation of f a t t y acid precursors such as l i n o l e i c or o l e i c acids; t h i s appears to be the pathway employed by plants ( Bohlmann et a l . , 1973; Geissman and Crout, 1969 ). On the other hand the beetle Chauliognathus leco n t e i ( Meinwald et a l . , 1968b ) may sequester t h i s chemical from a composite or umbellifer. trans-2-Hex-2-enal Dendrolasin A furanoterpenoid, dendrolasin, i s a component of the mandibular gland defences of the ant Lasius fuliginosus ( Bern-a r d i et a l . , 1967 ) L a b e l l i n g studies ( Waldner et a l . , 1969 ) 14 14 14 with acetate-1- C, mevalonate-2- C, and glucose- C showed that the f i r s t two precursors were incorporated into dendrolasin according to theory ( r e f e r to Goodwin, 1971; Pridham, 1967 ) 14 into the whole molecule. Degradations of the C-product con-firmed t h i s . Unfortunately, other terpene derivatives were not administered to test the hypothesis that the animal's pathway i s i d e n t i c a l to that of the plants. These experiments did not account for symbionts or the tissue of o r i g i n . A s i m i l a r study by Meinwald et a l . ( 1966a ) showed 174 that the monoterpene, anisomorphal ( Figure 23, #14 ), of the phasmid Anisomorpha buprestoides was synthesized by that i n -14 14 14 sect from acetate-1- C, acetate-2- C, and mevalonate-2- C. Degradation of anisomorphal were not undertaken to determine the p o s i t i o n of the l a b e l . This experiment did not allow for sym-bionts or the tissue of o r i g i n . There i s excellent evidence for the existence of the mevalbnate pathway i n insects leading to the production of terpenes. A recent report on the terpenoid composition of the defensive secretions of several s t a p h y l i n i d beetles, e.g., Eulissus orthodoxus ( Bellas et a l . , 1974 ), showed the presence of four chemicals suggestive of the biochemical pathway leading to the production of a c t i n i d i n e . Dolichoderine ants also produce i r i d o i d a l monoterpenes as well as a c y c l i c monoterpenes such as c i t r o n e l l a l . These terpenes have the appropriate functional group and stereochemistry to be intermediates i n a f e a s i b l e biosynthetic pathway ( for d e t a i l s see C a v i l l and Clark, 1971; Weatherston and Percy, 1970 ) . iso-Valeraldehyde C i t r o n e l l a l I r i d o i d i a l A c t i n i d i n e Much of the evidence for the existence of the mevalonate pathway i n insects comes from the biosynthetic studies attempting to determine the reason for the i n a b i l i t y of insects to synthe-size s t e r o i d s . Several early studies had shown insects incapable 14 of making the s t e r o i d nucleus from acetate- C ( Bloch et a l . , 175 1956; Clark and Bloch, 1959 ). The nature of the metabolic de-f i c i t was not understood. Recently Goodfellow 1s group has l o c -ated the biochemical blockage more p r e c i s e l y . They showed that the f l e s h f l y . Sarcophaga b u l l a t a had the a b i l i t y to form ger-14 anylgeraniol from mevalonate-2- C, as well as from geraniol, farnesol, and n e r o l i d o l ( Goodfellow et a l . , 1972 ), i n d i c a t i n g that the blockage l i e s beyond the formation of geranylgeranyl-pyrophosphate. The presence of squalene i n t h i s f l y appears to be the r e s u l t of dietary a c q u i s i t i o n ( Goodfellow et a l . , 1973b), 14 although the a p p l i c a t i o n of squalene- C gave r i s e to to a com-pound s i m i l a r to squalene-2,4-epoxide. The formation of lano-s t e r o l or other s t e r o i d a l compounds ( Goodfellow and Liu, 1972 ) was not observed. Their interpretation was that the oxidation of squalene to the epoxide represents the r e s i d u a l a c t i v i t y of a once existent system ( Goodfellow and Liu, 1972 ). Other studies with aposymbiotic organisms have indicated that these above intermediates are not the r e s u l t of i n t e s t i n a l f l o r a ( Good-fellow and Barnes, 1971; Goodfellow et a l . , 1972,1973a ). From these biochemicallines of evidence i t i s easy to account f o r the presence of other terpenoids i n insects such as P-pinene, p-silenene ( Figure 23, #24 and 30 ), as well as many pheromonal terpenoids already discussed. C i t r a l and c i t r o -n e l l a l produced by the mandibular glands of the ant Acantho-14 myops clav i g e r have been shown to a r i s e from acetate-1 and -2- C, 14 and mevalonate-2- C ( Happ and Meinwald, 1965 ). Again, t h i s study does not indicate where the chemicals are manufactured nor by what organism(s). Experiments by Metzler et a l . ( 1971 ) that attempted to show that juvenile hormone of the giant s i l k moth Hyalophora 176 cecropia was synthesized by the mevalonate pathway met with f a i l u r e . Many insect products which appear to be terpenoid i n structure can a r i s e fron non-terpenoid precursors such as amino acids. I t was suggested ( Metzler et a l . , 1971 ) that a branched amino ac i d such as isoleucine may be involved i n the biosynthesis of juvenile hormone. Juvenile Hormone CHO C i t r o n e l l a l CHO j ^ - C H O C i t r a l Isobutyric acid has been shown to occur i n beetles ( e.g., Broscus cephalotus;Schildknecht et a l . , 1968b ) and b u t t e r f l i e s ( e.g., P a p i l i o aegus; Eisner et a l . , 1970; Eisner and Meinwald 1965; Seligman and Doy, 1972 ). In the l a t t e r organism 3-hydroxy-buty r i c acid occurs alone ( Figure 23, #15 ) as an osmetrial secretion of the ultimate larvae. . Biosynthetic studies with P. aegus ( Seligman and Doy, 1973 ) have shown that 14. i s o b u t y r i c acid arises from v a l i n e - C , and that p-hydroxybu-t y r i c acid arises from a c e t a t e . 1 4 C and l e u c i n e - 1 4 c . The close resemblance of i s o b u t y r i c acid to the isoprene unit might make - ^ O O H Isobutyric acid ' N H2 J COOH ^ N - COOH Valine Leucine COOH p-OH-butyric acid y Isoprene S » 0 COOH Acetic acid Isovaleraldehyde 177 one think i t to be derived from mevalonate. Further precursor studies were not done to help elucidate the pathway, but studies were undertaken which showed that the osmetria were metabolically responsible for the formation of these two acids ( Seligman and Doy, 1973 ) . Isovaleraldehyde, which was previously mentioned as a defensive chemical i n staphylind beetles ( Bellas et a l . , 1974), occurs i n tomatoes. Metabolic studies with t h i s plant have shown 14 that i t a r i s e s from L-leucine- C ( Yu et a l . , 1968 ), and thus the chemical i n beetles may not be b i o g e n i c a l l y r e l a t e d to the terpenoids. The biosynthesis of many defensive chemicals w i l l not always succumb e a s i l y to armchair a n a l y s i s . For example, the cockroaches, P l a t y z o s t e r i a spp., produce 2-methylenebutanal ( Waterhouse and Wallbank,- 1967 ), which looks very much l i k e a terpene. 2-Methylbutyric acid, a compound with the same carbon 14 skeleton, i s however synthesized from propionate- C and ace-14 tate- C ( v i a methylmalonate ) i n the p a r a s i t i c worm Ascaris  lumbricoides ( Saz and Weil, 1960 ). Does 2-methylenebutanal arise via isopentyl pyrophosphate, from acetate-propionate, or from an amino acid? This remains to be determined. COOH /COOH w r\ 2-Methylenebutanal 2-Methylbutyric acid The acrylates, t i g l i c a cid and angelic acid, occur i n many beetles ( Moore and Wallbank, 1968; Schilknecht et a l . , 1968b )( Figure 23, #26 ). These two chemicals also resemble the isoprene unit. Also methacrylic and e t h a c r y l i c acids have 178 been identif ied in the carabid beetle Carabus taedatus ( Benn 14 et a l . , 1973 ). Preliminary C-precursor studies with this 14 beetle showed that D,L-val ine-4- C was incorporated into methacrylic acid. The posit ion of the label in the acrylate was not determined. These results suggest that the beetles synthesize acrylates in an anlogous fashion to that of angio-sperms. This biosynthesis involves the degradation of isoleucine, via 2-methylbutyric acid to either angelic or t i g l i c ac id . The lat ter two acids are interconvertible by means of an isomerase ( Basey and Wooley, 1973a,b; Boyle and Fowden, 1971; Fowden and Mazelis, 1971; Leete, 1973 ). Note that 2-methylbutyric acid arises dif ferently in the plant than i n the paras i t ic worm. 2-Methylbutyric acid is a common defensive chemical in p a p i l i -onid butterf l ies ( Lopez and Quesnel, 1970 ). COOH ^ \—COOH Valine Methacrylic acid COOH T i g l i c acid H 2 N ^ C 0 0 H COOH Isoleucine 2-Methylbutyric acid COOH A n g e l i c a c i d The vesicant, cantharidin ( Figure 23, #5 )/ produced by the meloid beetle Lytta vesicatoria arises from acetate-1-"1'4C 1 7 9 14 and mevalonate-2- C ( Schlatter et a l . , 1968 ). Their data . . 14 derived from p a r t i a l degradation of cantharidm- C suggest a head to head condensation of two isoprene u n i t s . Some biosynthetic schemes are more obvious than those just described. The biogenesis of the quinazolinine ( quinazo-l i n e ) a l k a l o i d , glomerin, from the p i l l millipede Glomeris marginata ( Figure 23, #21; Meinwald et a l . , 1966c ) would be expected to involve a n t h r a n i l i c acid. This has been shown by Schildknecht and Wenneis ( 1967 ) using a n t h r a n i l i c a c i d carbox-14 y l - C. The formation of qumazolme alk a l o i d s m plants has been shown to involve a n t h r a n i l i c acid, a C 2 unit ( p-carboline position) and an amino acid such as a s p a r t i c acid ( L i l j e g r e n , 1971; Luckner, 1972 ) as has been shown for vascine. N H 2 . CY O O H - C 2 • ir ^ N H 2 ° 2 k , C O O H A n t h r a n i l i c acid Aspartic a c i d Vascine Likewise, one would expect that the quinoline a l k a l o i d , 8-hydroxy-2-carboxy-quinoline methyl ester ( Figure 2 3, #20), produced by the beetle Ilybius fenestratus ( Schildknecht, 1971 ) i s derived from a n t h r a n i l i c acid and a 3-carbon unit ( see Geissman and Crout, 1969 ). This hypothesis remains to be tested. An a l t e r n a t i v e route would involve quinaldic acid, a tryptophan degradation product, which has been detected i n the feces of cockroaches ( Mullins and Cockran, 1973 ). 180 N^COOH Quinaldic acid Beetle Quinoline Biosynthetic studies of the d i a l k y l p i p e r i d i n e a l k a l o i d s ( Figure 23, #13 ) have not been attempted i n the f i r e ants, Solenopsis spp.. I t i s l o g i c a l however, to suspect that they are elaborated i n a fashion analogous to the mechanisms now understood to ex i s t i n Solanaceae ( Korzan and Gilbertson, 1974; Leete and Chedekel, 1972 ). In these plants l y s i n e i s converted to 5-amino-pentanal and the l a t t e r to a piperideinium nucleus followed by a l k y l a t i o n . Tobacco alkaloids C ^ N B 2 ^ N H 2 Lysine a 0 ^NH 2 5-Amino-pentanal Piperideinium nucleus Piperidine a l k a l o i d The biosynthesis of alkanes and alkanones i n arthropods and plants i s poorly understood. To explain the der i v a t i o n of chemicals such as n-decane, and trans- 4-oxo-hex-2-en-l-al ( Figure 23, #12 and 19 ) i s only p a r t l y p o s s i b l e . I t has been known for some time that arthropods are capable of synthes i z i n g t h e i r own f a t t y acids and neutral l i p i d s ( alkanes )( Bade, 1964; Gilby, 1965; Nelson, 1969; piek, 1964 ), but the biosyn-t h e t i c r e l a t i o n s h i p between them i s not s e t t l e d , nor i s the contribution of blood c e l l s ( oenocytes ), the fat body and the 181 integument f u l l y understood. Some studies show that insects can absorb dietary alkanes and incorporate them into c u t i c u l a r l i p i d s . Insects are also able to u t i l i z e a v a r i e t y of alkenoic-14 1- C precursors ( C„-C„ 0) to produce longer chain-length alkanes, alkenes, alkanols, and alkanones ( C^^ and longer ) ( Conrad and Jackson, 1971; Bloomquist and Jackson, 1973 ). These observations indicate that insects possess an elongation decarboxylation mechanism as described for plants ( Kolattukudy, 14 1968, 1970 ) . I t was .also observed that propionate-1- C was incorporated into 3-methyl-branched alkanes which i s consistent with an elongation-decarboxylation method ( Conrad and Jackson, 1971 ). Lambremont ( 1972 ) has reported that insects convert 14 f a t t y acids-1- C xnto the corresponding alcohols, although i t was not determined i f t h i s involved a ketonic intermediate. o o C24 acid A<-H / C26 acid r ( 1-14C ) ^ M / CASH C 2 5 alkane o / V A A A ^ V <=• C29 alkane, branched C3Q acid, branched Elongation-decarboxylation Apparently i n the millipede Graphidostreptus tumuliporus the elongation-decarboxylation process exists as a route to long chain c u t i c u l a r l i p i d s ( Oudejans and Zandee, 1973 ). Pal-14 m i t i c acid-1- C was incorporated into alk-l-enes ( C - C ^ ) , 182 branched and unbranched ). This suggests a a-oxidation process for the formation of aldehydes, ketones, and 1-enes. Labelled precursors were not incorporated i n the alkanes, suggesting the lack of an alkene reducing enzyme. The alkanes would therefore be of dietary o r i g i n . I f other arthropods u t i l i z e t h i s mechan-ism of elongation-decarboxylation using eit h e r formate, acetatei or propionate, most l i p i d substances i n the c u t i c l e can be accounted for, as well as many defensive chemicals. Coupling these observations with Lambremont's ( 1972 ) discovery of the reduction of the carboxyl groups of fats to an alcohol function, the d e r i v a t i o n of l i p i d s seems less complex. Thus, i f these mechanisms apply to the biosyntheses of short chain f a t t y acids ( C-.-C 0 i , the biosynthesis of many defensive and pheromonal aldehydes and ketones can be explained. •* , . a-oxid. . ,,. (even) (odd) > ii \ (even) R / V V ^ o — ^ R / ^ ^ c H 2 O H —> ( as above ) This accounts only for the production of carbon-1 mod-i f i c a t i o n s . However, evidence from the biosynthesis of second-ary alcohols and ketones i n Brassica olearacea suggest the following relationships for the production of i n t e r n a l ketones and alcohols ( Kolattukudy and Liu, 1970 ). OH O 183 Applying the previous scheme to short chain f a t t y acids i t i s possible to derive i n t e r n a l ketones, alcohols and -enes. Fungi have been reported to synthesize 2-heptanone from ca-p r y l i c acid ( Gehrig and Knight, 1961 ). Capry l i c acid occurs i n an arachnid, Mastigoproctus giganteus ( Eisner et a l . , 1961 ) and i n the beetle Eleodes l o n g i c o l l i s ( Hurst et a l . , 1968 ). 2-Heptanone also occurs i n s o c i a l ants as a pheromone ( Blum et a l . , 1968a ). Caprylyl-CoA w i l l a r i s e during the biosynthe-s i s of free f a t t y acids. - C O O H 0 n Caprylic a c i d 2-Heptanone The biosynthesis of exte r n a l l y branched l i p i d s ( i s o -and anteiso- ) probably a r i s e v i a the terminal condensation of valine and isol e u c i n e * t o f a t t y acids, as has been reported i n rats and micro-organisms ( Grigor et a l . , 1970a,b; Lennarz, 1961 ). Internal branching seems to a r i s e by the incorporation of propionate during elongation ( Lederer, 1964; Conrad and Jack-son, 1971; Oudejans and Zandee, 1973 ). anteiso- 1 S O -Isoleucine Valine (even) X Leucine ( odd ) 184 Simple benzoquinones ( Figure 23, #3 ) are common de-fensive agents i n beetles, millipedes, earwigs, and a v a r i e t y of insects ( see Table VII ). Some elementary biosynthetic studies with the beetle Eleodes l o n q i c o l l i s have shown that the 14 14 rin g of tyrosine-U- C and of phenylalanine-rmg- C are i n -corporated into benzoquinone. The a l k y l benzoquinones, t o l u -14' quinone and ethyl-benzoquinone, were derived from acetate- C, 14 14 malpnate- C, and propionate- C ( Meinwald et al.,1966b). These studies did not show the beetles to be wholely responsible for t h i s synthesis, nor i n what tissues the chemicals were made. Hydroquinone Benzoquinone Toluquinone Ethylquinone Arbutin These benzoquinones are reminiscent of s i m i l a r products found i n various fungi and angiosperms ( Thomson, 1971 ). Unless the biosynthesis of these insect products i s studied with emph-asis on intermediates rather than the i n i t i a l precursors the biogenic derivation of these chemicals w i l l remain ambiguous. I t i s proposed that beetles form benzoquinone from the oxidation of hydroquinone. The glycoside of hydroquinone, ar-butin, i s also present i n beetles. Arbutin occurs i n some mem-bers of the Rosaceae, Ericaceae, and Compositae ( Harborne, 1964 ) . The glycoside i s formed by the catabolism of phenylala-14 14 nine-ring- C and cinnamic a c i d - r i n g - C ( Grisdale and Towers, 14 1960 ). Tyrosine-U- C was a poor precursor m the pear plant. 14 The pear was able to u t i l i z e shikimate-U- C but not benzoic 185 a c i d - r i n g - C. This indicates that shikimic acid was incorp-orated v i a phenyalanine. The beetle was able to u t i l i z e both 14 14 tyrosine- C and phenylalanine- C ( Meinwald et a l . , 1966b ). 14 They did not use a v a r i e t y of C precursors to determine the pathway by which the beetle produces i t s benzoquinones. The biosynthetic p o s s i b i l i t i e s for the production of benzoquin-ones are numerous i f one considers some of the pathways i n plants, bacteria and fungi ( see Figure 25 ). From the view-point of the t h e o r e t i c a l pathways i n Figure 25, i t i s seen that the biochemical studies with E. l o n g i c o l l i s are ambiguous. This i s even more the case when one considers the r e s u l t s obtained for the biosynthesis of benzoquinones i n Rhinocricus holomelanus ( t h i s thesis ). For a d e t a i l e d discussion of quinone biosyn-theses r e f e r to Rudney ( 1971 ), T h r e l f a l l and Whistance ( 1971), and Zenk and Leistner ( 1968 ). The dual t h e o r e t i c a l o r i g i n of toluquinone ( Figure 24 ) also allows for the production i n insects of homogentisic acid derivatives ( Schildknecht, 1971 ), and m-methyl-cresol ( Eisner et a l . , 1963b; Schildknecht et a l . , 1968b ). The presence of 6-methyl-salicylic acid has been documented many times as a pro-duct of fungi ( Gatenbeck and Lonnroth, 1962; Thomson, 1971; Towers, 1964 ). The r o l e of 6-methyl-salicylic acid as an i n t e r -mediate i n the formation of hydroxylated quinones i s refuted; however, i n some cases i t can be hydroxylated to form o r s e l -l i n i c acid, or decarboxylated to form m-cresol ( Packter, 1965; Packter and Steward, 1967; Pettersen, 1966a,b ). Via these path-ways i t i s easy to t h e o r e t i c a l l y devise the o r i g i n of 2-hydroxy-6-methyl-benzaldehyde ( Moore and Brown , 1972 ) which i s a defensive chemical i n the beetle Phorocantha semipunctata. 186 FIGURE 25. T h e o r e t i c a l pathways for t h e b i o s y n t h e s i s of a r t h r o p o d b enzoquinones. OH 0 1 Propionyl-CoA- / L, I T \ ' OH OH OH Y 0 3 Malonyl-CoA J 6-Ethyl m-Ethylcresol E t h y l q u i n o l Ethylquinone 3 Malonyl-CoA^ S a l i c y l i c a c i d , ^ OH 1 A c e t y l CoA ^ Chorismic a c i d 6-Methyl 8 S a l i c y l i c a c i d PT t s COOH Phenylalanine ' ^ f ^ C O O H ) / OH OH m-cresol OH j^j-CH 2COOH OH T o l u q u i n o l Via cinnamic a c i d or phenylpy-r u v i c a c i d p-OH-phenyl- \ P-OH-Phenylpyruvic a c i d p y r u v i c a c i d Homogen t i s i c a c i d p l a n t s animals ? COOH COOH i animals ? CHOH p-OH-Cinnamic a c i d 6H2 1H QC00H OH G e n t i s i c a c i d 0 3-Methoxy-toluquinone 0 0 Benzoquinone p-OH—Phenyl-' - - l a c t i c a c i d OH OH i b a c t e r i a C 0 0 H HOOC ' p-OH-benzoic a c i d OH Hydroquinone Benxoic a c i d OH 0 -C-COOH OH CH2 . Clioiismic acid 187 The biosynthetic o r i g i n of benzoquinones i n arthropods i s an open question, e s p e c i a l l y the dimethyl and trimethyl benzoquinones ( Eisner et a l . , 1971c; Fi e s e r and Ardao, 1956). 2,3-Dimethoxy rbenzoquinone occcurs i n the millipede Uorblan-iulu s canadensis ( Weatherston and Percy, 1969 ). This quinone may ar i s e by the catabolism of ingested cinnamic ac i d deriva-t i v e s . The co-occurrence of benzoquinones and alkyl-naphtho -quinones ( Figure 23, #4 ) i n the beetle Agroporis alutacea ( Tschinkel, 1972 ) raises an i n t e r e s t i n g biochemical problem. Similar naphthoquinones are know to a r i s e a) from the poly-acetate pathway, b) from the shikimate pathway with the con-t r i b u t i o n of a 3-carbon fragment, a 4-carbon fragment, or an isoprene unit, and c) from the catabolism of tyrosine with the addition of an isoprene unit ( Thomson, 1971; Zenk and Leistner, 1968 ). Some of the simpler phenolics used by insects for defen-sive purposes are s t r u c t u r a l l y depicted below. I f insects COOH 2,3-dimethoxy-benzoquinone 0 0 COOH. COOH COOH OH I I I III IV V VI VII 188 are found to possess phenylalanine ammonia lyase ( see Figure 25 ) they could derive by B-oxidation either benzoic acid (I) or p-hydroxybenzoic acid (II) from phenylalanine. The alcohol or aldehyde could then be formed by reduction. However, no evidence exists that arthropods possess t h i s pathway, which has been documented i n higher plants and fungi ( Camm and Towers, 1973 ). The derivation of aromatic compounds ( C^-C^ and C^) through the conversion of p-hydroxyphenylacetic acid to p- coum-arate, or to p-hydroxyphenylpropionic acid ( M i l l s and Lake, 1971 ) i s biochemically unprecedented. I t i s easier to search for more probable pathways. A hypothesis put forward over a decade ago ( Aerts et a l . , 1960; Figure 26 ) deriving many of the above phenolics has received l i t t l e attention. Its f e a s i -b i l i t y l i e s i n the fact that many of these compounds have been i d e n t i f i e d i n insect c u t i c l e . This pathway e s s e n t i a l l y involves the conversion of tyrosine to DOPA or tyramine with subsequent a-oxidation to the mandelic a c i d d e r i v a t i v e . This d e r i v a t i v e which i s e a s i l y deaminated, to give the aldehyde and by o x i -dation the acid, i s also e a s i l y decarboxylated once the a c i d i s formed. By t h i s scheme i t i s easy to postulate the derivation of benzaldehyde ( without HCN ), benzoic acid ( I ), p-hydroxy-benzaldehyde ( III ), p-hydroxy-benzoic a c i d ( II ), and proto-catechuic acid ( IV ). From t h i s scheme phenylacetic acid ( VII ) also a r i s e s . The exact order of the pathway may not be i d e n t i c a l to the scheme, but the concept i s valuable. The structures shown below are those of chemicals that have been i s o l a t e d from insect c u t i c l e . By examination of Figure 26 t h e i r biosynthesis i s explainable ( Aerts et a l . . 1960; An-dersen, 1971; Brunet, 1967; Lipke, 1971; M i l l s et al.,1967; Towers and Subba Rao, 1972 ). It should be noted that the pro-1 8 8 a R 3 = COOH = CHO 1 8 9 o n CH CCOOH p. hydroxyphenyl -11 pyrj vie odd CHjCHOHCOOH dthydroxyptitnyllaciLc acH CH COOH OH OH p hydroxy • p hydroxyphenyl phen ylac 11 aldehyde COCH VOH OH prcioccie chuic acid ocelic ocid 0 II CCOOW CH OH dihydroiyphenyl-ocelic ocd CHOHCOOH OH OH dihydroxymandelic acid OH dihydroxyphenyi - * -kno-ocelic acid FIGURE 26. Theoretical pathways for phenolic metabolism i n i n s e c t s . 190 ducts formed from tyrosine catabolism i n insects are i n many cases i d e n t i c a l to the catecholamines of vertebrates ( Molinoff and Axelrod, 1971 ). I t would seem that a-oxidation of the phenylethanoid and phenylpropanoid units i s the means by which animals degrade aromatic acids; whereas, plants and fungi use ammonia lyases to produce cinnamic a c i d derivatives . Protocatechuic acid 4-0-(3-gIucoside and the r e l a t e d a l -cohol glucoside occur i n the cockroach ootheca ( Blaberus  d i s c o i d a l i s and Periplaneta americana)( Brunet, 1967; Pau and Acheson, 1968; Shaaya and Bodenstein, 1970; Shaaya and Sekeris, 1969 ). Biosynthetic studies have shown that protocatechuic 14 . 14 acid was derived from both glucose- C and tyrosine-G- C 14 ( Brunet, 1963 ). The incorporation of the glucose- C i s a l i k e l y contribution of micro-organsims since arthropods sup-?-posedly lack the shikimate pathway. I t has also been reported. the cockroach P. americana produces benzoic acid acid from 14 phenylpyruvic acid-U- C ( Murdock et a l . , 1970 ). The conversion of e i t h e r benzoic a c i d or p-hydroxybenzoic a c i d to protocate-chuic a c i d by fungi i s well documented ( Brown and Swinburne, 1973; Moore et a 1 ., 1968; Towers and Subba Rao, 1972 ). However, protocatechuic a c i d can also be derived from an intermediate i n the shikimate acid pathway,(dehydroshikimate; Harborne, 1964) with or without passing through aromatic intermediates. Such a multitude of biogenic o r i g i n s makes i t e s s e n t i a l for biosynthetic studies on arthropod defensive secretions to be c a r r i e d out c r i t i c a l l y . Such an approach w i l l make any attempts at e l u c i d a t i o n of pathways or chemotaxonomy more meaningful. 191 a c e t i c a c i d F i n a l l y , two defensive secretions remain to be consider-ed, benzaldehyde and HCN ( Figure 23, #1 and 7 ). The former i s produced by bees, moths, ants, beetles, millipedes, and centipedes. In some cases the production of benzaldehyde i s accompanied by the release of HCN. The production of benzalde-hyde alone might be the r e s u l t of a-or [3- oxidation of phenyl-alanine; whereas, the production of i t with HCN i s reminiscent of the method of cyanogenesis i n plants ( Conn, 1964 ). This l a t t e r point i s the subject of t h i s t h e s i s , 192 XI- SUMMARY The aim of t h i s d i s s e r t a i o n was to compare the bio-synthesis of HCN and benzaldehyde i n a polydesmid millipede Harpaphe haydeniana to the known method of biosynthesis i n angiosperms. Harpaphe haydeniana and Oxidus g r a c i l i s were shown to produce HCN and benzaldehyde from mandelonitrile. Some other polydesmids of diverse families namely; Boraria s t r i c t a , Caraibodesmus sp., Polydesmus angus-tus, Pseudoplydesmus branneri, Nearctodesmus cerasinus and Scytonotus insulanus, were shown to produce HCN, but the aldehyde or ketone was not characterized. Oxidus g r a c i l i s was shown to incorporate dietary 14 . 14 phenylalanme-ring- C and phenylalanine-3- C spe-14 c i f i c a l l y into benzaldehyde, and phenylalanme-2- C i n the cyanide carbon. I t was postulated that t h i s occured v i a the synthesis of mandelonitrile as i s known to occur i n plants. This hypothesis was tested i n Harpaphe haydeniana . . . . 1 4 which also s p e c i f i c a l l y incorporated phenylalanine- C, It was also shown i n t h i s millipede by the i n j e c t i o n 14 3 of C- and H-precursors that HCN was synthesized i n a manner p a r a l l e l to that of plants. The presence of th i s biochemical p a r a l l e l was further evidenced by the 14 . 14 . i s o l a t i o n of the C-oxime, the C - n i t r i l e , the 14 14 C-mandelonitnle, and a C-glycoside. Also, two enzymes involved i n t h i s biosynthetic pathway were iso l a t e d , 3-glycosidase and a-hydroxynitrile lyase. 193 These enzymes were shown to be l o c a l i z e d i n the cyanogenic gland. D-(R)-Mandelonitrile was shown to be the storage form of HCN i n H. haydeniana, and mandelonitrile i n O. g r a c i l i s . The n i t r i l e was held as a large droplet within the storage vestibule of the cyanogenic gland. The following millipedes were also shown to contain a droplet of o i l i n t h e i r storage vestibules; Boraria  s t r i c t a , Cherokia georgiana, Polydesmus angustus, Nearctodesmus cerasinus, and Scytonotus insulanus. The amount of glycoside of mandelonitrile i n H. haydeniana i s n e g l i g i b l e . Mandelonitrile degrades i n vivo; thus, H. haydeniana d e t o x i f i e s HCN by converting i t f i r s t to p-cyanoala-nine and then to asparagine. I t d e t o x i f i e s benzalde-hyde by converting i t to p-hydroxybenzoic aci d . Benz-o i c a c i d i s also converted to p-hydroxybenzoic a c i d . Based on morphological and biochemical data from H. haydeniana,a model employing basic p h y s i c a l concepts i s proposed to explain the mechanism of hydrogen cyanide production. This model j u s t i f i e s the n i t r i l e as the storage product, and extends the current hypo-thesis of the mechanism of HCN production. Light microscopic and electron-micrographic analyses of the cyanogenic gland of H. haydeniana were undertaken. Three benzoquinones were shown to.be the major defen-sive products of the following millipedes: Bollman-i u l u s sp., Eurhinocricus sp. nr. sabulosus, E. sp. nr. 1 9 4 b r u e s i , Leptogoniulus naresi, Orthoporus ornatus, Rhinocricus holomelanus, R. monilicornis, Sailus sp. nov. nr. s e t i f e r , and Trigoniulus lumbricinus. These compounds were benzoquinone, toluquinone, and 2-methyl-3-methoxy-benzoquinone. These quinones i n these millipedes provide no useful characters for chemotaxonomy. 1 4 Preliminary experiments with C-precursors i n R. holomelanus indicate that these three above benzo-quinones a r i s e from the catabolism of both tyrosine and phenylalanine with the nucleus of the aromatic acid being incorporated into the nucleus of the quinone. These re s u l t s suggest that the biosynthesis of benzoquinones i n millipedes i s d i f f e r e n t from that i n beetles.-195 REFERENCES :ADDENDUM Aasen, A.J., Kimland, B., Almqvist, S-0, E n z e l l , C.R. 1972. Tobacco chemistry 15. New tobacco c o n s t i t u -ents - The structures of f i v e isomeric megasti-matrienones. Acta. Chem. Scand. 26: 2573-2576. Abou-Donia, S.A., Fish, L.J., Pattenden, G. 1971. I r i -doidial from the odiferous glands of Staphylinus  olens ( Coleoptera: Staphylinidae ). Tetrahedron Lett. 43: 4037-4038. Aerts, F., Vercauteren, R., Decleir, W. 1960. Enzymes i n the metabolism of phenolic acids i n in s e c t s . . XI Int. Congr. Entomol. V7ien 3: 177-199. Ahmad, A., Spenser, I.D. 1961. The conversion of a-keto-acid oximes to n i t r i l e s i n aqueous so l u t i o n . Can. J. Chem. 39: 1340-1359. Alexander, P., Barton, D.H.R. 1943. The excretion of ethylquinone by the f l o u r beetle. Biochem. J . 37: 463-465. Andersen, S.O., 1971. Phenolic compounds i s o l a t e d from insect hard c u t i c l e and t h e i r r e a l t i o n s h i p to the s c l e r o t i z a t i o n process. Insect Biochem. 1: 157-170. Ando, K., Kato,A. 1970. Is o l a t i o n of 2,4-di.chlorophenol from a s o i l fungus and i t s b i o l o g i c a l s i g n i f i c a n c e . Biochem. Biophys. Res. Commun. 39- 1104-1107. Aneshanseley, D., Eisner, T., Widom, J.M., Widom, B. 1969. Biochemistry at 100°C.: the explosive discharge of bombardier beetles ( Brachinus ). Science Wash. 165: 61-63. Antonini, E., Brunori, M., Greenwood, C., Malmstrom, B. G., R o t i l l i o , G.C. 1971. The i n t e r a c t i o n of cyan-ide with cytochrome oxidase. Eur. -J. Biochem. 23: 396-400. Aplin, R.T., Birch, M.C. 1970. I d e n t i f i c a t i o n of odor-ous compounds from male lepidoptera. Experientia 26: 1193-1194. 196 11 Aplin, R.T., Benn, M.H., Rothschild, M. 1968. Poisonous alkaloids i n the body tissues of the cinnibar moth ( Callimorpha jacobaeae L. ). Nature Lond. 219: 747-748. 12 Applebaum, S.W., Gestetner, B., Birk, Y. 1965. Physio-l o g i c a l aspects of host s p e c i f i c i t y i n the Bruchi-dae - VI. Developmental incompatability of Soybeans for Callosobruchus. J . Insect Physiol. 11: 611-616. 13 Bade, M. 1964. Biosynthe/is of f a t t y acids i n the roach Eurycotis f l o r i d a . J . Insect Physiol. 10:333-341. 14a Barbetta, M., Casnati,G., Pavan, M. 1966. The presence of D (+) -mandelonitrile i n the defensive secretion of the myriapode Gomphodesmus pavani. Mem. Soc. Ent. Ital-. 45: 169-176. 14b Barman, T.E., 1969. i n Enzyme Handbook Vol. I & I I , Springer-Verlag, New York. 15 Barry, R.H., Hartung,W.H. 1946. a-Oximino acid interme- . diates for the synthesis of a-amino acids. J . org. Chem. 12: 460-467. 16 Basey, K., Woolley, J.G. 1973a. Biosynthesis of the t i -g l o y l esters of Datura: c i s - t r a n s isomerism. Phyto-chem. 12: 2883-2886. 17 Basey, K., Woolley, J.G. 1973b. Biosynthesis of the t i -g l o y l esters i n Datura: the r o l e of 2-methybutyric acid . Phytochem. 12: 2197-2201. 18 Baslow, M.H. 1971. Marine toxins. Ann Rev. Pharmacol. 11: 447-454. 19 Basu, N., Rastogi, R.P. 1967. Triterpenoid saponins and sapogenins. Phytochem. 6: 1249-1270. 20 Beard, R.L. 1963. Insect toxins and venoms. Ann. Rev. Entomol. 8: 1-18. 21 Beck, S.D. 1965. Resistance of plants to i n s e c t s . Ann. Rev. Entomol. 10: 207-232. 197 22 Bedard, W.D., Tilden, P.E., Wood, D BL., S i l v e r s t e i n , R.M., Brownlee, R.G., Roden, J.O. 1969. Western pine b e e t l e : F i e l d response to i t s sex pheromone and a s y n e r g i s t i c host terpene, myrcene. Science Wash. 164: 1284-1285. 23 Bellas, T.E., Brown, W.V., Moore, B.P. 1974. The alka-l o i d a c t i n i d i n e and p l a u s i b l e precursors i n defen-sive secretions of rove beetles. J . Insect Physiol. 20: 277-280. 24. Bendall, D.S., Bonner, W.D.Jr. 1971. Cyanide-insensi-t i v e r e s p i r a t i o n i n plant mitochondria. Plant Phy-s i o l . 47: 236-241. 25 Benn, M.H., Lencucha, A., Maxie, S., Telang, S.A., 1973. The p y g i d i a l defensive secretion of Carabus taed-atus. J. Insect Physiol. 19: 2173-2176. 26 Ben-Yehoshua, S., Conn, E.E. 1964. Biosynthesis of prunasin, the cyanogenic glycoside of peach. Plant Physiol. 39: 331-333. 27a Bergstrom, G., Lofqvist, J . 1973. Chemical congruence of the complex odiferous secretions from Dufour's gland i n three species of ants of the genus Formica. J . Insect Physiol. 19: 877-907. 27b Bergstrom, G., Lofqvist, J . 1972. S i m i l a r i t i e s between the Dufour secretions of the ants Camponotus l i g n i -perda ( Latr. ) and Camponotus herculeanus ( L. ) ( Hym. ). Entomol. Scand. 3: 225-238. 28 Bergstrom, G., Lbfqvist, J . 1970. Chemical basis for odour communication i n four species of Lasius ants. J. Insect Physiol. 16: 2353-2375. 29 Bergstrom, G., Lofqvist, J. 1968. Odour s i m i l a r i t i e s be-tween the slave-keeping ants Formica sanguinea and Polyergus rufescens and t h e i r slaves Formica fusca and Formica r u f i b a r b i s . J . Insect Physiol. 14:995-1011. 30 Bernardi, R., Cardani, C., G h i r i n g h e l l i , D., Selva, A., Baggini, A., Pavan, M. 1967. On the components of the secretion of mandibular glands of the ant Lasius ( Dendrolasius ) f u l i g i n o s u s . Tetrahedron Lett. 40: 3893-3896. LEAF 198 OMITTED IN PAGE NUMBERING. 199 & 200 31 Binning, A., Darby, F.J.,•Heenan, M.P., Smith, J.N. 1967. The conjugation of phenols with phosphate i n grass grubs and f l i e s . Biochem. J . 103: 42-48. 32 Birk, Y. 1969. Saponins ( Chpt. 7 ), i n Toxic Constit-uents of Plant Foodstuffs, ed. I.E., Liener, Aca-demic Press, New York. 33 Bisset, G.W., Frazier,J.F.D., Rothschild, M., Schachter, M. 1960. A pharmacologically active choline ester and other substances i n the t i g e r moth, A r c t i a caja. Proc. R. Soc. ( B ) 152: 255-262. 34 Blackhall, A., Thomson, R.H. 1953. Quinones. Part I I . The addition of mercapto-acids to benzoquinones and 1:4-naphthoquinones. J. Chem.Soc. 1138-1143. 35 B l e i c h e r t , E.F., Neish, A.C., Towers, G.H.N. 1966. Bio-synthesis of t a x i p h y l l i n i n Taxus. Proc. 2nd Mtg. Fed. Eur. Biochem. Soc. 3: 119-127. 36 Blest, A.D. 1964, Protective d i s p l a y and sound production i n some new world a r c t i i d and ctenuchid moths. Zoologica 49: 161-181. 37 Blest, A.D. 1957. The evolution of protective displays i n the Saturnoideae and Sphingidae ( Lepidoptera ). Behaviour 6: 257-309. 38 Bloch, K., Langdon, R.G., Clark, A., Fraenkel, G. 1956. \ Impaired s t e r o i d biogenesis i n insect larvae. Bio-chim. Biophys. Acta. 21: 176- 183. 39 Blomquist, G.J., Jackson, L.L. 1973. Incorporation of l a b e l l e d dietary n-alkanes into c u t i c u l a r l i p i d s of the grasshopper Melanoplus sanquinLpes . j . Insect Physiol. 19: 1639-1647. 40 Blum, M.S. 1971. Arthropod defences. Proc. 2nd Int. IUPAC Congr., ed. A.S., Tahori, Tel-Aviv, I s r a e l , i n Chemical Releasers i n Insects, Vol 3: 163-176. 41 Blum, M.S. 1970. The chemical basis of insect s o c i a l i t y , i n Chemicals C o n t r o l l i n g Insect Behaviour, ed., M. Beroza, Academic Press, New York, pg. 61-94. 200 Blum, M.S. 1969. Alarm Pheromones. Ann. Rev. Ento-mol. 14: 57-80. Blum, M.S. 1961. The presence of 2-hexenal i n the scent of the pentatomid Brochymena quadrapustulata. Ann. Entomol. Soc. Amer. 54: 410-412. Blum, M.S., Brand,J.M. 1972. S o c i a l insect pheromones: ' th e i r chemistry and function. Amer. Zool. 12: 533-676. Blum, M.S., Brand, J.M., Wallace, J.B., Fales,' H.M. 1972. Chemical characterization of the defensive secretion of a chrysomelid larvae. L i f e Sciences 11 ( II ): 525-531. Blum, M.S., Crain, R.D. 1961. The occurrence of para-quinones i n the abdominal secretion of Eleodes  h i s p i l a b r i s ( Coleoptera: Tenebrionidae ). Ann. Entomol. Soc. Amer. 54: 474-477. Blum, M. S. , Crain, R.D., Chidester, J.B. 1961. Trans-2-hexenol i n the scent gland of the hemipteran Acanthocephala femorata. Nature Lond. 189: 245-246. Blum, M.S., Crewe, R.M., Pasteels, J.M. 1971. Defensive secretion of Lomechusa strumosa a myrmecophilous beetle. Ann. Entomol. Soc. Amer. 64: 975-976. Blum, M.S., Crewe, R.M./Kerr, W.E., Keith, L.H., Gar-ri s o n , A.W., Walker, M.M. 1970. C i t r a l i n s t i n t l e s s bees: I s o l a t i o n and functions i n t r a i l - l a y i n g and robbing. J . Insect Physiol. 16: 1637- 1648. Blum, M.S., MacConnell, J.G., Brand, J.M., D u f f i e l d , R.M., Fales, H.M. 1973a. Phenol and benzaldehyde i n the defensive secretion of a strongylosomid m i l l i p d e . Ann. Entomol. Soc. Amer. 65. 2 35. Blum, M.S., Wallace, J.B., Fales, H.M. 1973b. Skatole and tridecene: i d e n t i f i c a t i o n and possible role i n a chrysopid secretion. Insect Biochem. 3: 353-357. Blum, M.S., Padovani, F., Curley, A., Hawk, R.E. 1969. Benzaldehyde: defensive secretion of a harvester ant. Comp. Biochem. Physiol. 29: 461-465. 201 Blum, M.S., Padovani, F., Amante, E. 1968a. Alkan-ones and terpenesin the mandibular glands of Atta species ( Hymenoptera: Formicidae ). Comp. Biochem. Physiol. 26: 291- 299. Blum, M.S., Padovani, F., Hermann, H.R.Jr., Kannowski, P.B. 1968b. Chemical releasers of s o c i a l behaviour. XI. Terpenes i n the mandibular glands of Lasius  umbratus. Ann. Entomol. Soc. Amer. 61: 1354-1359. Blum, M.S., Woodring, J.P. 1962. Secretion of benzalde-hyde and hydrogen cyanide by the millipede Pachy-desmus crassicatus. Science Wash. 138: 512-513. Blumenthal-Goldschmidt, S., Butler, G,W., Conn, E.E. 196 3. Incorporation of hydrocyanic acid l a b e l l e d with carbon--'--'into asparagine i n seedlings. Nature Lond. 197: 718-719. Blumenthal, S.G., Hendrickson, H.R., Abrol, Y.P., Conn, E.E. 1968. Cyanide metabolism i n higher plants. J. B i o l . Chem. 243: 5302-5307. Bock, W.J. 1972. Species interactions and macroevolu-ti o n , ( Chpt. 1 ), i n Evolutionary Biology Vol.5., ed., T., Dobzhansky, M.K., Hecht, W.C., Steere., Appelton-Century-Crofts, New York. Bohlmann, F., Burkhardt, T., Zdero,C. .1973. i n Naturally  Occurring Acetylenes, Academic Press, New York. Borror, D.J., DeLong, D.M. 1971. i n An Introduction to the Study of Insects, 3rd ed., Holt, Rhlnehart, and Winston, New York. Bourquelot, E., Danjou, E., 1905. Preparation du gluco-side cyanohydrique du surreau a l ' e t a t c r i s t a l l i s e . J. Pharmacol. Chim. 22: 219-222. Bove, C , Conn, E.E. 1961. Metabolism of aromatic com-pounds i n higher plants. II P u r i f i c a t i o n and pro-perties of the o x y n i t r i l a s e of Sorghum vulgare. J. B i o l . Chem. 236: 207-210. 202 63 Bowie, J.H., Cameron, D.W., G i l e s , R.G.F., Williams, D.H. 1966. Studies i n mass spectrometry. Part V. Mass spectra of benzoquinones. J. Chem. Soc,(B) 335-339. 64 Bowie, J.H., Cameron, D.W. 1965. Colour matters of the Aphididae. Part XXV. A comparison of aphid c o n s t i -tuents v/ith those of t h e i r hosts. A glyceride of sorbic a c i d . J . Chem. Soc. 5651-5657. 65 Boyle, J.E., Fowden, L. 1971. Biosynthesis of 2-amino-4-methylhex-4-enoic acid i n Aesculus c a l i f o r n i c a : the route from isoleucine and the role of amino-transferases. Phytochem. 10: 2671-2678. 66 Brand, M., Blum, M.S., Fales, M.H., Pasteels, J.M. 1973. The chemistry of the defensive secretion of the beetle, D r u s i l l a c a n a l i c u l a t a . J . Insect Physiol. 19: 369-382. 67 Brand, J.M., Blum, M.S., Fales, H.M., MacConnell, J.G. 1972. F i r e ant venoms: comparative analyses of a l -k a l o i d a l components. Toxicon 10: 259-2 71. 68 Breedlove, D.E., E h r l i c h , P.R. 1968. Plant-herbivore coevolution: lupines and lycaenids. Science Wash. 162: 671-672. 69 Brega, A., Falaschi, A., deCarl, L., Pavan, M. 1968. Studies on the mechanisms of action of pederine. J. C e l l B i o l . 36: 485-496. / 70 Brower, J.V.Z. 1958a. Experimental studies of mimicry i n some North American b u t t e r f l i e s . Part I. The Monarch, Danaus plexippus, and Viceroy, Limenitis archippus  archippus. Evolution 12: 32-47. 71 Brower, J.V.Z. 1958b. Experimental studies of mimicry i n some North American b u t t e r f l i e s . Part I I . Battus  philenor and P a p i l i o t r o i l u s , P. polyxenes, and P. glaucus. Evolution 12: 123-136. 72 Brower, J.V.Z. 1958c. Experimental studies of mimicry i n some North American b u t t e r f l i e s . Part I I I . Danaus  gil i p p u s berenice and Limenitis archippus f l o r i d e n s i s , Evolution 12:273-285. 203 Brower, L.P., 1970. Plant poisons i n a t e r r e s t r i a l food chain and implications for mimicry, i n Bio-chemical Coevolution, ed., K.L. Chambers, Proc. 29th Ann. B i o l . C o l l . , pp. 69-82. Brower, L.P., Alcock, J., Brower, J.V.Z. 1971. Avian feeding behaviour and the s e l e c t i v e advantage of i n c i p i e n t mimicry, i n E c o l o g i c a l Genetics and  Evolution ( Chpt. 12'), ed. R. Creed, Oxford:Black-wells. Brower, L.P., Brower, J.V.Z., Corvino, J.M. 1967. Plant poisons i n a t e r r e s t r i a l food chain. Proc. Nat. Acad. S c i . 57; 893-898. Brower, L.P., Brower, J.V.Z., 1964. Birds, b u t t e r f l i e s , and plant poisons: a study i n e c o l o g i c a l chemistry. Zoologica 49: 137-159. Brower, L.P. Ryerson, W.N., Coppinger, L.L., G l a z i e r , S.C. 1968. E c o l o g i c a l chemistry and the palatab-i l i t y spectrum. Science Wash. 1349-1351. Brown, A.E., Swinburne, T.R. 1973. Degradation of benzoi acid by Nectaria galligena Bres. i n v i t r o and i n vivo. Physiol, plant P h y s i o l . 3: 453-459. Brown, A.E. Swinburne, T.R. 1971. Benzoic a c i d : an antifungal compound formed i n Bramley 1s seedling apple f r u i t s following i n f e c t i o n by Nectaria  galligena Bres.. Physiol. Plant Physiol. 1: 469-475. « Brown, W.L. J r . , Eisner, T., Whittaker, R.H. 1970. Allomones and kairomones: Transspecific chemical messengers. Bioscience 20: 21-22. Brunet, P.C.G. 1967. S c l e r o t i n s . Endeavour 26: 68-74. Brunet, P.C.G. 1963. Tyrosine metabolism i n i n s e c t s . Nature Lond. 199: 492-493. Bucherl, W. 1971. Venomous chilopods and centipedes., i n Venomous Animals and t h e i r Venoms, Vol. I I I . ( Chpt. 50 ), ed., W. Bucherl , E. Buckley, V. Deulofeu , Academic Press, New York. 204 84 Bucherl, W., Buckley, E., Deulofeu, V., ed. 1971. i n Venomous Animals and t h e i r Venoms, Vol. I I I . , Academic Pres.';, New York. 85 Burtt, E. 1947. Exudate from millipedes with p a r t i c u l a r reference to i t s injurious e f f e c t s . Trop. Dis. Bull, 44:7-12. 86 Butler, C.G. 1967. Insect pheromones. B i o l Rev. 42: 42-87. 87 Butler, G.W., Butler, B.G. 1960. Biosynthesis of l i n -amarin and l o t a u s t r a l i n i n white clover. Nature Lond. 187: 780-781. 88 Butler, G.W., Conn, E.E. 1964. Biosynthesis of the cyanogenic glucosides l i n i m a r i n and l o t a u s t r a l i n . I. L a b e l l i n g studies i n vivo with Linum u s i t a t i s -simum. J . B i o l . Chem. 239: 1647- 1679. 89 Calam, D.H., Youdeowei, A. 1968. I d e n t i f i c a t i o n and functions of the secretions from the p o s t e r i o r scent gland of fifth i n s t a r s of the bug, Dysdercus  intermedius ( Pyrrhocoridae ). J . Insect Physiol. 14: 1147- 1156. 90 Callow, R.K., Greenway, A.R., G r i f f i t h s , D.C. 1973. Chemistry of the secretions from the c o r n i c l e s of various species of aphids. J . Insect Physiol. 19: 737-748. 91 Camm, E.L., Towers, G,H,N. 1973. Phenylalanine ammonia lyase. phytochem., 12: 961-973. 92 C a r l i s l e , D.B., E l l i s , P.E., Betts, E. 1965. The i n f l u -ence of aromatic shrubs on sexual maturation i n the desert locust Schistocerca gregaria. J . Insect Physiol. 11: 1541-1558. 93 Carpenter, G.D.H. 1938. Audible emission of defensive frot h by insects with an appendix on the abdominal structures concerned i n a moth by H. Eltringham. Proc. Zool. Soc. ( London )(A)108: 243-252. 205 94 C a r r e l , J.E., Eisner, T. 1974. Cantharidin; Potent feeding deterrent to i n s e c t s . Science Wash. 183: 755-757. 95 Carrick, R. 1936. Experiments to test the e f f i c i e n c y of protective adaptations i n i n s e c t s . Trans. R. Ent. Soc. ( London ) 85: 131-140. 96 Casnati, G., Nencini, G., Q u i l i c o , A., Pavan, M., Ricca, A., S a l v a t o r i , T. 1963. The secretion of the myria-pod, Polydesmus c o l l a r i s c o l l a r i s . Experientia 19: 409-411. 97 Casnati, G., Ricca, A., Pavan,M. 1967. Defensive secre-t i o n of the mandibular glands of Paltothyreus  tarsatus ( Fabr. ). Chim. Ind. Milan 49: 57-61. 98 C a v i l l , G.W.K. 1969. Insect terpenoids and nepeta-lactone., i n Cyclopentanoid Terpene DerivativesQhpt. 3) ed., W.I. Taylor, A.R. Battersby, Marcel Dekker, New York. 99 C a v i l l , G.W.K., Clark, D.V. 1971. Ant secretions and cantharidin., i n Naturally Occurring i n s e c t i c i d e s ( Chpt. 7 ), ed., M. Jacobson, D.G. Crosby, Marcel Dekker, New York. 100 C a v i l l , G.W.K., Williams, P.J., W h i t f i e l d , F.B. 1967. a-Farnesene, Dufour' s gland secretion i n the ant Aphaenogaster longiceps. Tetrahedron 2201-2205. 101 Chadha, M.S., Eisner, T., Meinwald, J . 1961. Defence mechanisms of arthropods- IV. Para-benzoquinones i n the secretion of Eleodes l o n g i c o l l i s Lec. ( Cole-optera: Tenebrionidae ). J . Insect Physiol. 7: 46-50. 102 Chamberlin, R.V., Hoffman, R.L. 1958. Checklist of the millipedes of North America. U.S. Nat. Museum B u l l . 212: 1-236. 103 Chapman, R.F. 1969. i n The Insects: Structure and Function, American E l s e v i e r Publ Co., New York. 104 Chen, C., Osuch, M.V. 1969. Biosynthesis of bufadieno-l i d e s - 3 B-hydroxycholanates as precursors i n Bufo marinus bufdienolide synthesis. Biochem Pharm. 18: 1797-1802. 206 105 106 107 108 109 110 112 113 114 115 Chew, M.Y., Boey, C.G. 1972. Rhodanese of tapioca l e a f . Phytochem. 11: 167-169. Claridge, L.C. 1968. Sound production i n species of Rhychaenus ( = Orchestes ) ( Coleoptera: Curcul-• ionidae ). Trans. Ent Soc. Lond. 120: 287-295. Clark, A.J., Bloch, K. 1959. The absence of s t e r o l biosynthesis i n in s e c t s . J. B i o l . Chem. 234: 2578-2582. Clark, R.M. 1971. Pigmentation of Hyalophora cecropia larvae fed a r t i f i c i a l d i e ts containing carotenoid ad d i t i v e s . J . Insect Physiol. 19: 1593-1598. Clayton, R.B. 1970. The chemistry of nonhormonal i n t e r -actions: terpenoid compounds i n ecology,,in Chemical Ecology ( Chpt. 10 ), ed. E. Sondheimer, J.B. Simeone, Academic Press, New York. Clayton, R.B. 1964. The u t i l i z a t i o n of sterols by insects. J . L i p i d Res. 5: 3-19. I l l Clearwater, J.R. 1972. Chemistry and function of a pheromone produced by the male of the southern armyworm Pseudaletia separata. J . Insect Physiol. 18: 781-789. Cloudsley-Thompson, J.L. 1968. Mil l i p e d e s . , i n Spiders, Scorpions, Centipedes, and Mites ( Chpt. II ), Pergamon Press , Oxford. Colombo, V., Lorenz-Meyer, H., Semenza, T. 1973, Small i n t e s t i n a l p h l o r i z i n hydrolase: the "3-glycosidase complex ". Biochim. Biophys. Acta. 327: 412-424, Conn, E.E. 1974. Biosynthesis of cyanogenic glycosides. Biochem.Soc. Symp. 38: 277-302. Conrad, C.W., Jackson, L.L. 1971. Hydrocarbon biosynthe-s i s i n Periplaneta americana. J. Insect Physiol. 17: 1907-1916. 116 Cott, H.B. 1950. i n Adaptive Coloration i n Animals., Methuen & Co. Ltd. London. 2 0 7 117 Crawford, C.S.. 1972. Water rektiors i n a desert m i l l i -pede Orthoporus ornatus ( Girard )( Spirostrept-idae ). Comp Biochem. Physiol. 42A: 521- 535. 118' Crewe, R.M., Blum, M.S., Collingwood, C.A. 1972. Comp-arative analysis of alarm pheromones i n the ant genus Crematogaster. Comp. Biochem. Physiol. 43B: 703-716. 119 Cromartie, R.I.T. 1959. Insect pigments. Ann. Rev. Entomol. 4: 59-76. 120 Crossley , A.C., Waterhouse,D.F. 1969a. The u l t r a s t r u c -ture of the osmetrium and the nature of i t s secre-t i o n i n P a p i l i o larvae ( Lepidoptera ). Tissue & C e l l 1: 525- 554. 121 Crossley, A.C., Waterhouse, D.F. 1969b. The u l t r a s t r u c -ture of a pheromone-secreting gland i n the male scorpion-fly Harpobittacus a u s t r a l i s ( Bittacidae: Mecoptera ). Tissue & C e l l 1: 273-294. 122 Crout, D.H., Benn, M.H., Imaseki, H., Geissman, T.A. 1966. The biosynthesis of s e n e c i p h y l l i c a c i d . Phytochem. 5: 1-21. 123 Cruickshank, I.A.M., Perrin, D.R. 1964. Pathological function of phenolic compounds i n plants., i n Biochemistry of Phenolic Compounds ( Chpt. 14 ), ed., J.B. Harborne, Academic Press, New York. 124 Dadd, R.H. 1973. Insect n u t r i t i o n : Current develop-ments and metabolic implications. Ann. Rev. Ento-mol. 18: 381-420. 125 Dadd, R.H. 1963. Feeding behaviour and n u t r i t i o n i n grasshoppers and locusts., In Advances i n Insect Physiology, Vol. I: 47-109., ed., J.W.L. Beament, J.E. Treherne, V.B. Wigglesworth, Academic Press, New York. 1 2 6 Darlington, P.J. 1 9 3 8 . Experiments i n mimicry i n Cuba with suggestions for further study, Trans. R. Ent. Soc. ( Lond. ) 8 7 : 6 8 1 - 6 9 5 . 208 127 Dateo, G.P., Roth, L.M. 1967. Occurrence of gluconic acid and 2-hexenal i n the defensive secretions of three species of Eurycotis ( B l a t t a r i a : B l a t t i d a e : P o l y z o s t e r i i r a e ) . Ann. Entomol. Soc. Amer. 60: 1025-1030. 128 David, W.A.L., Gardiner, B.O.C. 1966a. Mustard o i l gly-cosides as feeding stimulants for P i e r i s brassicae larvae i n a semi-synthetic d i e t . Entomol. Exp. Appl, 9: 248-255. 129 David, W.A.L., Gardiner, B.O.C. 1966b. The e f f e c t of s i n i g r i n on the feeding of P i e r i s brassicae L. transferred from various d i e t s . Entomol. Exp. Appl. 248: 95-98. 130 Dean, P.D.G., Exley, D., Goodwin, T.W. 1971. Steroid oestrogens i n plants: re-estimation of oestrone i n pomegranite seeds. Phytochem. 10: 2215-2216 131 Dethier, V.G. 1970. Chemical interactions between plants and insects., i n Chemical Ecology, ( Chapt. 5 ), ed. E. Sondheimer, J . B . Simeone, Academic Press, New York. 132 Dethier, V.G. 1954. Evolution of feeding prefences i n phytophagous i n s e c t s . Evolution 8: 33-54. 133 Dethier, V.G. 1941. Chemical factors determining the choice of food plants by P a p i l i o larvae. Amer. Nat. 75: 61-73. 134 Dixon, A.F.G. 1968. The protective function of the siphunculi of the n e t t l e aphid, Microlophium  evansi ( Theob.)( Hem.:Aphididae ). Entomol. Mon. Mag. 94: 8-11. 135 Dixon, M., Webb, E.C. 1964. i n Enzymes, 2nd ed., pg. 627. 136a Dodson, C.H. 1970. The role of chemical attractants i n orchid p o l l i n a t i o n . , i n Biochemical Coevolution, ed., K.L. Chambers, Proc. 29 th Ann. B i o l . C o l l . pp. 83-107. 136b Dodson, C.H. , Dressier, R, L. , Hills, H.G., Adams, R.M. , Williams, N.H. 1969. B i o l o g i c a l l y active compounds i n orchid fragrances. Science Wash. 164:1243-1249. 209 137 Doesburg, P.H. van 1968. A r e v i s i o n of the new world species of Dysdercus Guerin Meneville ( Heteropt-era, Pyrrhocoridae ). Zool. Verh. Rijksmus Natuur. Hist. Leiden 97: 1-215. 138 Drake, D., Lam, J . 1974. Polyacetylenes of Artemisia v u l g a r i s . Phytochem. 13: 455-457. 139 Duffey, S.S. 1970. Cardiac glycosides and d i s t a s t e f u l -ness: Some observations on the p a l a t a b i l i t y spectrum of b u t t e r f l i e s . Science Wash. 169: 78-79. 140 Duffey, S.S., Scudder, G.G.E. 1972. Cardiac glycosides i n North American Asclepiadaceae, a basis for un-palatability i n b r i g h t l y coloured Hemiptera and Coleoptera. J. Insect Physiol. 18: 63-78. 141 Edmunds, M. 1972. Defensive behaviour i n Ghanaian praying mantids. Zool. J. Linn. Soc. 51: 1-32. 142 Edwards, J.S. 1966. Defensive smear: Supercooling i n the c o r n i c l e wax of aphids. Nature Lond. 211: 73-74. 143 Edwards, J.S. 1960. S p i t t i n g as a defensive mechanism i n a predatory reduviid. XI Int. Congr. Entomol. Wien 3: 259-263. 144 Eglinton, G., Hamilton, R.J. 1967. Leaf e p i c u t i c u l a r waxes. Science Wash. 156: 1322-1335. 145 E h r l i c h , P.R., Raven, P.H. 1964. B u t t e r f l i e s and plants: A study i n coevolution. Evolution 18: 586-608. 146 Eisner, H.E., Eisner, T., Hurst, J . J . 1963. Hydrogen cyanide and benzaldehyde produced by millipedes. Chem. Ind. 124-125. 147 Eisner, T. 1970. Arthropod defences., i n Chemical E c o l -ogy, ( Chpt. 8 ), ed. E. Sondheimer, J.B. Simeone, Academic Press, New York. 148 Eisner, T. 1968. Mongoose and M i l l i p e d e . Science 156:1367. 149a Eisner, T., 1967. Mongoose throwing and smashing m i l l i -pedes. Science Wash. 155: 577-579. Eisner, T. 1965. Defensive spray of a phasmid insect. Science Wash. 148: 966-988. 149b 210 150 Eisner, T. 1964. C a t n i p , i t s raison d 1 etre. Science Wash. 146: 1318-1320. 151 Eisner, T. 1960. D e f e n c e mechanisms of arthropods. I I . The chemical and mechanical weapons of an earwig. Psyche 67: 62-69. 152 Eisner, T., Eisner,H.E. 1965. Mystery of a millipede. Nat, H i s t . 74 (3): 30-35. 153 Eisner, T., Eisner, H.E., Hurst, J . J . , Kafatos, F.C., Meinwald, J. 1963a. Cyanogenic glandular apparatus of a milli p e d e . Science Wash. 139: 1218-1220. 154 Eisner, T., Hurst, J . J . , Meinwald, J . 1963b. Defence mechanisms of arthropods. XI. The structure and function of phenolic secretions of the glands of a choreumoid millipede and a carabid beetle. Pysche 70: 94-116. 155 Eisner, T., Swithenbank, C., Meinwald, J . 1963c. Defence mechanisms of arthropods. VIII. Secretion of s a l i -cylaldehyde by a carabid beetle. Ann. Entomol. Soc. Amer. 56: 37-41. 156 Eisner, T., Hendry, L.B., Peakall, D.B., Meinwald, J . 1971a. 2,5-dichlorophenol ( from ingested h e r b i -cide ? ) i n defensive secretion of grasshopper. Science Wash. 172: 277-278. 157 Eisner, T., Kluge, A.F., Ikeda, M.I., Meinwald, Y.C., Meinwald, J . 1971b. Sesquiterpenes i n the osmetrial secretion of a p a p i l i o n i d b u t t e r f l y , Battus poly- damas. J . Insect Physiol. 17:245-250 158 Eisner, T., Kluge, A.F., Ca r r e l , J.E., Meinwald, J . 1971c. Defence of phalangid: L i q u i d r e p e l l e n t ad-ministered by leg dabbing. Science Wash. 173: 650-651. 159 Eisner, T., McHenry, F., Salpeter, M.M. 1964. Defence mechanisms of arthropods. XV. Morphology of the quinone-producing glands of a tenebrionid beetle ( Eleodes l o n g i c o l l i s L e e ) . J . Morph. 115: 355-400. 211 160 Eisner, T., Meinwald, J. 1966. Defensive secretions of arthropods. Science Wash. 153: 1341-1350. 161 Eisner, T., Meinwald, Y.C., 1965. Defensive secretion of a c a t e r p i l l a r ( P a p i l i o ). Science Wash. 150: 173-175. 162 Eisner, T., Meinwald, J., Monro, A., Ghent, R. 1961. Defence mecahnisms of arthropods. I. The composi-t i o n and function of the spray of the whipscorpion Mastigoproctus giganteus ( Lucas ) ( Arachnida: Pedipalpida ). J . Insect Physiol. 6: 272-298. 163 Eisner, T., P l i s k e , T.E., Ikeda, M., Owen, D.F., Vazquez, L., Perez, H., Franclemont, J.G., Meinwald, J . 1970. Defence mechanisms of arthropods. XXVII. Osmetrial secretions of p a p i l i o n i d c a t e r p i l l a r s ( Baronia, P a p i l i o , Eurytides ). Ann. Entomol. Soc. Amer. 63: 914-915. 164 Eisner,T., McKittrick, F., Payne, R. 1959. Defence sprays of roaches. Pest. Control 27: 11-12. 165 Eisner, T, T a s s e l l , van E., Carrel , J.E. 1967. Defensive use of a " Feceal Shield " by a beetle l a r v a . Science Wash. 158: 1471-1473. 166 Emery, T.F. 1966. I n i t i a l steps i n the biosynthesis of ferrichrome. Incorporation of &-N-hydroxyornithine. Biochem. 5: 2694-2701. 167 von Euw, J., Fishelson, J . , Parsons, J.A., Reichstein, T. 1967. Cardenolides ( heart poisons ) i n a grass-hopper feeding on milkweeds. Nature Lond. 214: 35-39, 168 von Euw, J., Reichstein, T., Rothschild, M. 1971. Heart poisons ( cardiac glycosides ) i n the lygaeid bugs Caenocoris n e r i i and Spilostethus pandurus. Insect Biochem. 1: 373-384. 170 von Euw, J . , Reichstein, Y., Rothschild, M. 1968. A r i s t o l o c h i c acid-I i n the swallowtail b u t t e r f l y Pachlioptera a r i s t o l o c h i a e ( Fabr, )( Papilionidae). I s r a e l j . Chem. 6: 659-760. 212 171 Evans, W.A.L., Payne, D.W. 1964. Carbohydrases of the alimentary t r a c t of the desert locust, Schisto-gregaria Forsk.. J. Insect Physiol. 10: 657-674. 172 Eyjolfsson, R. 1970. Recent advances i n the chemistry of cyanogenic glycosides. Prog. Chem. Org. Nat. Prod-ucts. 28: 74-108. 173 Farnden, K.J.F 0 / Rosen, M.A., L i l i j e g r e n , D.R. 1973. Aldoximes and n i t r i l e s a s intermediates i n the bio-synthesis of cyanogenic glycosides. Phytochem. 12: 2673- 2677. 174 F e i g l , F. 1958. i n Spot Tests i n Organic Chemistry, 5th ed., translated by R.E. Oesper, E l s e v i e r Publ. Co,, New York, pg. 276. 175 F e i r , D., Suen, J-S. 1971. Cardenolides i n the milkweed plant and feeding by the milkweed bug. Ann. Ento-mol. Amer. 64. 1173-1174. 176 F e l t w e l l , J.S.E. Valadon, L.R.G. 1972. Carotenoids of P i e r i s brassicae and of i t s food plant. J. Insect Physiol. 18: 2203-2215. 177 Fie s e r , L.F., Ardao, M,I. 1956. Investigation of the chemical nature of gonyleptidine. J . Amer, Chem. Soc. 78: 774-781. 178 181 Fies e r , L.F., Fies e r , M. 1967. i n Reagents for Organic Synthesis , John Wiley and Sons, New York. 179 Fieser, L.F., Fies e r , M. 1935. The reduction p o t e n t i a l of various naphthoquinones. J . Amer. Chem Soc. 57: 491-494. 180 F i l s h i e , B.K., Waterhouse, D.F., 1969. The structure and development of a surface pattern on the c u t i c l e of the green bug Nezara v i r i d u l a . Tissue & C e l l 1: 367-385. F l a i g , von W., S a l f e l d , J-C., Baume, E. 1958. UV- Spek-tren und Konstituion von p-Benzochinon. Annalen 618: 117-139. 213 182 Fletcher, B.S. 1969. The structure and function of the sex pheromone glands of the male Queensland f r u i t f l y , Dacus t y r o n i . J. Insect Physiol. 15: 1309-1322. 183 Floss, H.G.,Hardwiger, L., Conn, E.E. 1965. Enzymatic formation of B-cyanoalanine. Nature London 208: 1207-1208. 184 Fonken, G.S., Johnson, R.A. , ed. 1972. i n Chemical Oxidations with Micro-organisms. Marcel Dekker, New York. 185 Fowden, L., Mazelis, M. 1971. Biosynthesis of 2-amino-4-methylhexenoic acid i n Aescuius c a l i f o r n i c a : the precursor r o l e of i s o l e u c i n e . Phytochem. 10: 359-365. 186 Fraenkel, G.S. 1959. The raison d'etre of secondary plant substances. Science Wash. 129: 1466-1470. 187 Freeman, M.A. 1968. Pharmacological properties of the regurgitated crop f l u i d of the A f r i c a n migratory locust, Locusta migratoria L.. Comp. Biochem. Physiol. 26: 1041-1049. 188 Frings, H., Goldberg, E., Arentzen, J.C. 1948. A n t i -b a c t e r i a l action of the blood of the large milk-weed bug. Science 108: 689-690. 189 Fuzeau-Braesch, S. 1972. Pigments and colour changes. Ann. Rev. Entomol. 17: 403-424. 190 Games, D.E., Staddon, B.W. 1973. Composition of scents from the larva of the milkweed bug, Oncopeltus  fasciatus , J . Insect Physiol. 19: 1527-1532. 191 Gatenbeck, S., Lonnroth, I. 1962. The biosynthesis of g e n t i s i c a c i d . Acta Chem. Scand. 16: 2298-2299. 192 Gawienowski, A.M., Gibbs, C.C. 1969. The i s o l a t i o n of oestrone from apples. Phytochem. 8: 685-686. 193a Gehrig, R.F., Knight, S.G. 1961. Formation of 2-heptan-one from c a p r y l i c acid by spores of various f i l a -mentous fungi. Nature Lond. 192: 1185. 214 193b Geissman, T.A., Crout, D.H.G., 1969. i n Organic Chemistry of Secondary Plant Metabolism, Academic Press, New York. 194 Gelperin, A. 1968. Feeding behaviour of the preying mantis: a learned modification. Nature London 219: 399-400. 195 Ghent, R.L. 1961. Adaptive refinements i n the chemical defence mechanisms of ce r t a i n Formicinae., PhD. thesis, Cornell University, Ithaca, New York, 88P. 196 G i l b e r t , B.L., Norris, D.M. 1968. A chemical basis for bark beetle ( Scolytus ) d i s t i n c t i o n between host an and non-host trees. J. Insect Physiol. 14: 1063-1068. 197 G i l b e r t , L.E. 1971. Bu t t e r f l y - p l a n t coevolution: Has P a s s i f l o r a adenopoda won the s e l e c t i o n a l race with Heliconiine b u t t e r f l i e s ? Science Wash. 172: 585-586. 198 200 201 Gilby, A.R. 1965. Lipids and t h e i r metabolism i n ins e c t s . Ann. Rev. Entomol. 10: 141-160. 199 Gilby, A.R., Waterhouse, D.F. 1967. Secretions from the l a t e r a l scent gland of the green vegetable bug Nezara viridula.Nature Lond.216: 90-91. Gilman, H., B l a t t , A.H. 1951. i n Organic Syntheses C o l l , V ol. I: 270-271., 2nd ed., John Wiley and Sons, New York. Gilmour, D. 1965. i n The Metabolism of Insects, Oli v e r and Boyd, Edinburgh. 202 Goodfellow, R.D., Barnes, F.J. 1971. Mevalonate .kinase from the larva of the f l e s h f l y , Sarcophaga b u l -l a t a . Insect Biochem. 1: 271-282. 203 Goodfellow, R.D., Huang, Y-S., Radtke, H.E. 1972. Iso-prenol biosynthesis i n the f l e s h f l y , Sarcophaga  b u l l a t a . Insect Biochem. 2: 467-475. 204 Goodfellow, R.D., Liu, G.C.K. 1972. Squalene metabolism i n the larva of the f l y , Sarcophaga b u l l a t a . J . Insect Physiol. 18: 95-103. 215 205 Goodfellow, R.D., Liu, G.C.K., Stein, J.P., Harker, K. 1973a. Mevalonate biosynthesis i n the f l y , Sarcpphaga b u l l a t a . Insect Biochem. 3: 113-122. 206 Goodfellow, R.D., Radtke, H.E., Huang, Y.S., Liu, G.C.K. 1973b. Studies on squalene biosynthesis i n Sarcophaga b u l l a t a larvae. Insect Biochem. 3:61-65. 207 Goodman, L.S., Gilman, A. ed. 1970. i n The Pharmacolo-g i c a l Basis of Therapeutics, 4 ed., MacMillan, Toronto. 208 Goodman, R.W., Kivaly, Z., Z a i t l i n , M., ed. 1967. i n The Biochemistry and Physiology of Infectious  Plant Diseases, Van Nostrand Comp. Inc., Toronto. 209 Goodwin, T.W., ed. 1971. i n Aspects of Terpenoid Bio-chemistry, Academic Press, New York. 210 Gordon, H.T., Waterhouse, D.F., Gilby, A.R. 1963. In-corporation of 14c-acetate into scent constituents by the green vegetable bug. Nature Lond. 197: 818. 211 Grant, G.G., Brady, J.M. 1972. Male armyworm scent brush secretion: I d e n t i f i c a t i o n and electroantennogram study of major components. Ann, Entomol. Soc. Amer. 65: 1224-1227. 212 G r i f f i t h , G.W. , Beck, S.D. 1973. I n t r a c e l l u l a r sym-bionts of the pea aphid. J. Insect Physiol. 19: 75-84. 213 Grigor, M.R., Dunckley, G.G., Purves, H.D. 1970a. The branched chain f a t t y acids of rat faecal l i p i d s . The contribution of the i n t e s t i n a l micro-organisms. Biochim. Biophys. Acta 218: 400-406. 214 Grigor, M.R.,Dunckley, G.G., Purves, H. D. 1970b. The synthesis of branched-chain f a t t y acids of r a t skin surface l i p i d . Biochim. Biophys. Acta 218: 389-399. 215 Grisdale, S.K., Towers, G.H.N. 1960. Biosynthesis of arbutin from some phenylpropanoid compounds i n Pyrus communis. Nature Lond. 18: 1130-1131. 216 216 Guldensteeden-Egeling, C. 1882. Ueber Bildung von Cyanwasserstoffsaure bei einem Myriapoden. Arch. Ges. Physiol. 28: 576-577. 217 Habermann, E. 1972. Bee and wasp venoms. Science Wash. 177: 314-322. 218 Hahlbrock, K. , Conn, E.E. 1970. The biosynthesis of cyanogenic glycosides i n higher plants. I. P u r i -f i c a t i o n and properties of a uridine diphosphate glucose-ketone cyanohydrin &-glucosyltransferase from Linum usitatissimum. J . B i o l . Chem. 245: 917-922. 220 Haisman, D.R., Knight, D.J. 1967. The enzymic hydrolysis of amygdalin. Biochem. J . 103: 528-534. 221 H a l l , F.R., Hollingworth, R.M., Shankland, D.L. 1971. Cyanide tolerance i n millipedes: the biochemical basis. Comp. Biochem. Physiol. 38B: 723-737. 222 H a l l , F.R. # Hollingworth, R.M., Shankland, D.L. 1969. Cyanide tolerance i n millipedes: comparison of r e s p i r a t i o n i n millipedes and i n s e c t s . Entomol. News 80: 277-282. 22 3 Hamamura, Y. 1970. The substances that control the feed-ing behaviour and the growth of the silkworm Bombyx  mori., i n Control of Insect Behaviour by Natural  Products, ed., D.L. Wood, R.M. S i l v e r s t e i n , M. Nakajima, Academic Press, New York, pp. 55-80. 224 Happ, G.M. 1968. Quinone and hydrocarbon production i n the defensive glands of Eleodes l o n g i c o l l i s and Tribolium castaneum ( Coleoptera:Tenebrionidae ). J. Insect Physiol. 14: 1821-1837. 225 Happ, G.M., Meinwald, J. 1965. Biosynthesis of arthro-pod secretions. I. Monoterpene synthesis i n an ant ( Acanthomyops, cl a v i g e r ). J . Amer. Chem. Soc. 87: 2507. 226 Happ, G.M. , Strandberg, J.D., Happ, C M . 1966. The terpene-producing glands of a phasmid insect. C e l l morphology and histochemistry. J. Morph. 119: 143-159. 217 227 Harashima, K., Ohno, T., Sawachika, T., Hidaka, T., Ohnishi, E. 1972. Carotenoids i n orange pupae of the swallowtail, P a p i l i o xuthus. Insect Biochem. 2: 29-48. 228 Harborne, J.B., ed. 1964. i n Biochemistry of Phenolic Compounds, Academic Press, London. 229 Harley, K.L.S., Thorsteinson, A.J. 1967. The influence of plant chemicals on the feeding behaviour, devel-opment and s u r v i v a l of the two-striped grasshopper, Melanoplus b i v i t t a t u s ( Say ), Acrididae: Orthop-tera. Can. J. Zool. 4 5 : 305-319. 230 Harrison, V.J., Weatherston, J . 1967. Thin-layer chrom-atography of simple n a t u r a l l y occurring benzoquin-ones. J. Chromat. 31: 258-259. 231 Haviland-Brindley, M.D. 1930. On the metathoracic scent-glands of c e r t a i n Heteroptera. Trans. R. Entomol. Soc. Lond. 78: 199-208. 232 Heftmann, E. 1968. Biosynthesis of plant s t e r o i d s . Lloydia 31: 293-317 233 Hendricksoh, H.R., Conn, E.E. 1969. Cyanide metabolism i n higher plants. IV. P u r i f i c a t i o n and properties of B-cyanoalanine synthetase of blue lupine. J . B i o l . Chem. 244: 2632-2640. 234 Hepburn, H.R., Yonke, T.R. 1971. The metathoracic scent glands of Coreid Heteroptera. J . Kansas Entomol. Soc. 44: 18-210. 235 Herout, V. 1969. Some re l a t i o n s between plants, insects and t h e i r isoprenoids., i n Prog, i n Phytochem., ( Chapt. 2 ), ed., L. Reinhold, Y. Liwschitz, John Wiley Interscience Publications, London. 236 Hinde, R. 1971. The control of the mycetome symbionts of the aphids Brevicoryne brassicae, Myzus persicae and Macrosiphum rosae . J . Insect Physiol. 17: 1791-1800. 237 Hitchcock, M., Smith., J.N., Comparative detoxication. The detoxication of aromatic acids by invertebrates: 218 detection of agmatine derivatives i n scorpions. Biochem. J. 98: 736-741. Hoch, J.H. 1961. i n A Survey of Cardiac Glycosides and  Genins, University of South Carolina Press. Hblldobler, B. 1971. Communication between ants and t h e i r guests. S c i . Amer. 224(3): 86-93. Horn, D.H.S. 1971. The ecdysones., i n Naturally Occur-rin g Insecticides ( Chpt. 9 ), ed. M. Jacobson, D.G. Crosby, Marcel Dekker Inc., New York. Hsiao, T.H., Fraenkel, G. 1969. Properties of leptino^-t a r s i n , a toxic haeomolymph protein from the Colorado potato beetle. Toxicon 7: 119-130. Hubbes, M. 1966. Benzoic and s a l i c y l i c acids i s o l a t e d from a glycoside of aspen bark and t h e i r e f f e c t on Hypoxylon pruinatum. Can J . Bot. 47: 1295-1301. Hurst, J . J . , Meinwald, J . ; Eisner, T. 1964. Defence mechanisms of arthropods. XII. Glucose and hydrocarbons i n the quinone-containing secretion of Eleodes l o n g i c o l l i s . Ann. Entomol. Soc. Amer. 57: 44-57. Ibrahim, R.K., Towers, G.H.N. 1960. The i d e n t i f i c a t i o n by chromatography of plant phenolic acids. Arch. Biochim. Biophys. 87: 125-128. Ikan , R., Cohen, E., Shulov, A. 1970. Benzo- and hydro-quinones i n the defence secretions of Blaps sulcata and Blaps wiedemanni . J . Insect Physiol. 16: 2201-2206. Isley, F.B. 1944. Correlation between mandibular morph-ology and food s p e c i f i c i t i e s on grasshoppers. Ann. Entomol. Soc. Amer. 37: 47-67. Ito, T., Tanaka, M. 1959. Beta-glucosidase of the mid-gut of the silkworm Bombyx mori. B i o l . B u l l . 116: 95-105. Jackson, L.L., Baker, G.L. 1970. Cuticular l i p i d s . Lipids 5: 239-246. 219 249 Jacobs, M.B. 1967. i n The A n a l y t i c a l Toxicology of I n d u s t r i a l Inorganic Poisons, Chemical Analyses Vol. 22: 721-735., Interscience Publishers. 250 Jacobson, M. 1972. i n Sex Pheromones, Acadmeic Press, New York, 251 Jacobson, M., Crosby, D.G., ed. 1971. i n Naturally Occurring Insecticides, Marcel Dekker Inc., New York. 252a Johansson, A.S. 1957. The functional anatomy of the metathoracic scent glands of the milkweed bug Oncopeltus fasciatus ( Heteroptera:Lygaeidae ). Norsk. Entomol. T i d s s k r i f t . 10: 95-109. 252b Jones, D.A., Parsons, J., Rothschild,M. 1962. Release of hydrocyanic acid from crushed tissues of a l l stages i n the l i f e - c y c l e of species of the Zygaenininae ( Lepidoptera ). Nature Lond. 193: 52-53. 253 Jones, F.M. 1937. Relative a c c e p t a b i l i t y and poisonous food plants. Proc. R. Entomol. Soc. Lond. ( A ) 12: 74-80. 254 Jones, F.M. 1934. Further experiments on colouration and r e l a t i v e a c c e p t a b i l i t y of insects to b i r d s . Trans. R. Entomol. Soc. Lond. 82: 443-453. 255 Jones, F.M. 1932. Insect colouration and r e l a t i v e a c c e p t a b i l i t y of insects to b i r d s . Trans. R. Entomol. Soc. Lond. 80: 345-385. 256 Jorissen, A., Hairs, E. 1891. Nouveau glucoside, fourn-issant de l'acide cyanohydrique par dedoublement et r e t i r e Linum ustitatissimum. B u l l . Acad. R. S c i . Beiges 21: 529-539. 257 Juneja, P.S., Gholson, R.K., Burton, R.L., Starks, K.J. 1972. The chemical basis for greenbug resistance i n small grains. 1. Benzyl alcohol as a possible resistance factor. Ann. Entomol. Soc. Amer. 65: 961-964. 220 258 Karlsson, E. 1973. Chemistry of some potent animal toxins. Experientia 29: 1319-1327. 259 Karlson, P., Butendandt, A . 1959. Pheromones ( Ecto-hormones ) i n ins e c t s . Ann. Rev. Entomol. 4: 39-58. 260 Kemp, T.R., Knavel, D.E., S t o l t z , L.P. 1973. V o l a t i l e Cucumis melo components: I d e n t i f i c a t i o n of addi-t i o n a l compounds and e f f e c t of storage conditions. Phytochem. 12: 2921-2924. 261 Kettlewell, H;B.D., Berry, R.J. 1969. Gene flow i n a c l i n e . Amathes glareosa Esp. ( Lepidoptera, Caradrinidae ) and i t s melanic f. edda Staud. i n Shetland. Heredity 24: 1-14. 262 Kettlewell, H.B.D. 1955. Selection experiments on i n d u s t r i a l melanism i n the Lepidoptera. Heredity 9: 323-342. 263 Kijima, H., Koizumi, 0., Morita, H. 1973, a-Glycosidase at the t i p of the contact chemosensory seta of the blowfly, Phormia regina. J . Insect Physiol. 19: 1351-1362. 264 Kindl, H. 1968. Oxydasen und Oxygenasen i n hoheren Pflanzen, I. Uber das Vorkomen von Indoly l - ( 3 ) -acetaldoxim und seine Bildung aus L-Tryptophan. Hoppe-Seyler"s Z. Physiol. Chem. 349: 519-520. 265 Kindl, H., Un d e r h i l l , E.W. 1968a. The enzymatic trans-formation of amino acids to aldehyde oximes. Abstract of a paper presented before the 5th Fed. Eur. Biochem, Soc. Mtg. Prague,Czechoslovakia, July, 1968. 266 Kindl, H., Underhill, E.W. 1968b. Biosynthesis of mus-tard o i l glucosides: N-hydroxyphenylalanine, a precursor of glucotropaeolin and a substrate for the enzymatic and non-enzymatic formation of phenyl-acetaldoxime. Phytochem. 7: 745-756. 267 Kislow, C.J., Edwards, L.J. 1972. Repellent odours i n aphids. Nature Lond. 235: 108-109. 268 Kistner, D.H., Blum, M.S. 1971. Alarm pheromone of 2 2 1 L a s i u s ( D e n d r o l a s i u s ) spatheus ( Hymenoptera: F o r m i c i d a e ) and i t s p o s s i b l e m i m i c r y by two s p e c i e s o f P e l l a ( C o l e o p t e r a : S t a p h y l i n i d a e ). Ann. Entomol. Soc. Amer. 6 4 : 5 8 9 - 5 9 4 . 2 6 9 K j a e r , A. 1 9 6 3 . IsothLocyanates o f n a t u r a l d e r i v a t i o n . Pure A p p l . Chem. 7 : 2 2 9 - 2 4 5 . 2 7 0 K l u g e , A.F., E i s n e r , T. 1 9 7 1 . Defence mechanisms o f a r t h r o p o d s . X X V I I I . A quinone and a p h e n o l i n t h e d e f e n s i v e s e c r e t i o n o f a p a r a j u l i d m i l l i p e d e . Ann. E n tomol. Soc. Amer. 6 4 : 3 1 4 - 3 1 5 . 2 7 1 K o l a t t u k u d y , P . E . 1 9 7 0 . P l a n t waxes. L i p i d s 5 : 2 5 9 - 2 7 5 . 2 7 2 K o l a t t u k u d y , P . E . 1 9 6 8 . B i o s y n t h e i s o f s u r f a c e l i p i d s . S c i e n c e W a s h . 1 5 9 : 4 9 8 - 5 0 5 . 2 7 3 K o l a t t u k u d y , P . E . , L i u , T-J. 1 9 7 0 . D i r e c t e v i d e n c e f o r b i o s y n t h e t i c r e a l t i o n s h i p s among h y d r o c a r b o n s , s e c o n d a r y a l c o h o l s and k e t o n e s i n B r a s s i c a  o l e a r e a c e a . Biochem. B i o p h y s . Res. Commun. 4 1 : 1 3 6 9 - 1 3 7 4 . 2 7 4 K o r z a n , P., G i l b e r t s o n , T.J. 1 9 7 4 . B i o s y n t h e i s o f t h e p i p e r i d i n e n u c l e u s : m e t a b o l i s m o f D , L - 6 -N-methyl-H - l y s i n e - 2 - 1 4 c by Sedum a c r e . Phytochem. 1 3 : 4 3 5 -4 3 7 . 2 7 5 K r i e g e r , R . I . , Feeny, P.P., W i l k i n s o n , C.F. 1 9 7 1 . D e t o c i c a t i o n enzymes i n g u t s o f c a t e r p i l l e r s : An e v o l u t i o n a r y answer t o p l a n t d e f e n c e s . S c i e n c e Wash. 1 7 2 : 5 7 9 - 5 8 1 . 2 7 6 K r i s h n a n , G. 1 9 6 8 . The m i l l i p e d e Thyropogus w i t h s p e c i a l r e f e r e n c e t o I n d i a n s p e c i e s . CSIR Z o o l . Memoirs on I n d i a n A n i m a l t y p e s 1 : 1 - 8 4 2 7 7 Kupchan, S.M., Knox, J.R., K e l s e y , J . E . 1 9 6 4 . C a l o t r o p i n , a c y t o t o x i c p r i n c i p l e i s o l a t e d from A s c l e p i a s  c u r a s s a v i c a . S c i e n c e Wash. 1 4 6 1 6 8 5 . 2 7 8 L a i - F o o k , J . 1 9 7 3 . The f i n e s t r u c t u r e o f Ve r s o n ' s g l a n d s i n m o l t i n g l a r v a e o f Calpod e s e t h l i u s ( H e s p e r i i d a e , L e p i d o p t e r a ). Can J . Z o o l 5 1 : 1 2 0 1 - 1 2 1 0 . 222 2 79 Lai-Fook, J . 1972. A comparison between the dermal glands i n two insects Rhodnius prolixus ( Hemip-tera ) and Calpodes e t h l i u s ( Lepidoptera ). J. Morph. 136: 495-504. 28° Lai-Fook, J . 1970. The fine structure of developing type "B" glands i n Rhodnius pr o l i x u s . Tissue & C e l l 2: 119-138. 281 Lambremont, E.N. 1972. L i p i d metabolism of insects: interconversion of f a t t y acids and f a t t y alcohols. Insect Biochem. 2: 197-202. 282 Law, J.H., Regnier, F.E. 1971. Pheromones. Ann. Rev. Biochem. 40: 533-548. 283 Lederer, E. 1964. The o r i g i n and function of some methyl groups i n branched-chain f a t t y acids, plant s t e r o l s and quinones. Biochem. J . 93: 449-468. 284 Leete, E. 1973. Biosynthetic conversion of a-methyl-but y r i c acid to t i g l i c acid i n Datura meteloides. Phytochem. 12: 2203-2205. 205 Leete, E., Chedekel, M.*R. 1972. The abberant formation of (-)-N-methylanabasine from N-methyl-^-piperi-deinium chloride i n Nicotiana tabacum and N. glauca. Phytochem. 11:2751-2756. 285 Legrand, M., Viennet, R. 1966. Dichroism c i r c u l a i r e optique. XXI.- 'Etude du chromphore benzenique. B u l l . Soc.Chim. 9: 2798-2801. 286 Lennarz, W.J. 1961. The role of isoleucine i n the bio-synthesis of branched chain f a t t y acids by Micro-coccus l y s o d e i k t i c u s . Biochem Biophys. Res. Cornmun. 6: 112-116. 287 Levin, D.A. 1971. Plant phenolics: an e c o l o g i c a l perspective. Amer. Nat. 105: 157-181. 288 L i l j e g r e n , D.R. 1971. Biosynthesis of quinazoline alkaloids of Peganum harmala. Phytochem. 10: 2661-2669. 223 289 Linsley, E.G. 1961. L y c i d - l i k e Cerambycidae ( Coleop-tera ). Ann. Entomol. Soc. Amer. 54: 628- 635. 290 Linsley, E.G. 1960. Mimetic assemblages of s i b l i n g species of l y c i d beetles. Ann. Entomol. Soc. Amer. 52: 125-131. 291 Linsley, E.G. 1959. Mimetic form and co l o r a t i o n i n the Cerambycidae ( Coleoptera ). Ann. Ent. Soc. Amer. 52: 125-131. 292 Linsley, E.G., Eisner, T. , Klots, A.B. 1961. Mimetic assemblage of s i b l i n g species of l y c i d beetles. Evolution 15: 15-29. 293 Lipke, H. 1971. Conjugates of glucosamine i n coackroach c u t i c l e . Insect Biochem. 1: 189-198. 294 Locke, M. 1961. Pore canals and related structures i n insect c u t i c l e . J. Biophys. Biochem. Cytol. 10: 589-618. 295 Loconti, J.D., Roth, L.M. 1953. Composition of the odorous secretion of Tribolium castaneum. Ann. Entomol. Soc. Amer. 46: 281-289. 296 Lopez, A., Quesnel, V.C. 1970. Defensive secretions of some p a p i l i o n i d c a t e r p i l l a r s . CarLb . J . S c i . 10: 5-7. 297 Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. 1951. Protein measurement with F o l i n phenol reagent. J. B i o l . Chem. 19: 265-275. 298 Luby, J.M., Regnier F.E., Clarke, E.T., Weaver, E.C., Weaver, N. 1973. V o l a t i l e cephalic substances of the s t i n g l e s s bees, Trigona mexicana and Trigona  p e c t o r a l i s . J . Insect Physiol. 19: 1111-1127. 299 Lucknqr, M. 1972. i n Secondary Metabolism i n Plants and and Animals. Academic Press, New York. 300 Lyford, W.H., J r . 1943. The p a l a t a b i l i t y of f r e s h l y f a l l e n forest tree leaves to millipe d e s . Ecology 24: 252-261. 224 301 MacConnell, J.G., Blum, M.S. 1971. The chemistry of f i r e ant venom. Tetrahedron 26: 1129-1139. 302 Machlis, L. 1962. The coming age of sex pheromones i n plants. Mycologia 64: 235-247. 303 Mahadevan, S. 1973. Role of oximes i n nitrogen metabolism i n plants. Ann. Rev. Plant Physiol. 24: 69-80. 304 Maier, W.A. 1973. Die Phenoloxydase von Chironomous thumni und Ihre Beeinflussung durch Parasitare Me-mithiden. J. Insect Physiol. 19: 85-95. 305 Major, R.T. 1967. The ginkgo, the most ancient l i v i n g tree. Science Wash. 157:1270-1273 306 Major, R.T., C o l l i n s , O.D., Marchini, P., Schnabel, H.W. 1972. Formation of 2-hexenal by leaves. Phytochem. 11: 607-610. 307 Major, R.T., Marchini, P., Sproston, T. 1960. I s o l a t i o n from Ginkgo biloba L. of an i n h i b i t o r of fungus growth. J. B i o l . Chem. 235: 3298-3299. 308 Major, R.T., Thomas, M. 1972. Formation of 2-hexenal from l i n o l e i c acid by macerated Ginkgo leaves. Phytochem. 11: 611-617. 309 Maloeuf, N.S.R. 1938. Secretions from ectodermal glands of arthropods. Quart. Rev. B i o l . 13: 169-195. 310 Manton, S.M. 1954. The evolution of arthropodan locomot-ory mechanisms- Part 4. The structure , habits and evolution of Diplopoda. J. Linn Soc. ( Zool. ) 42: 299-368. 311 Marshall, G.A.K. 1902. Five years observations and ex-periments ( 1896-1901 ) on the bionomics of South A f r i c a insects c h i e f l y directed to the i n v e s t i g a t -ion of mimicry and warning colours. Trans. Entomol. Soc. Lond. 287-584. 312 Maschwitz, U., Jander, R., Burkhardt, D. 1972. Wehrsub-tanzen und Wehrvehalten der Termite Macrotermes  carbonarius. J. Insect Physiol. 18: 1715-1720. 313 Maschwitz, U., Koob, K., Schildknecht, H. 1970. Ein 225 Beitrag zur Funktion de Metathoracaldruse der Ameisen. J . Insect Physiol. 16: 387-404. 314 Matsumoto, Y. 1970. V o l a t i l e organic s u l f u r compounds as insect attractants with s p e c i a l reference to host sel e c t i o n . , i n Control of Insect Behaviour by  Natural Products,ed.,D.L.Wood, R.M. S i l v e r s t e i n , M. Nakajima, Academic Press, New York, pp. 133-160.. 315 Matsumura, F.,Boush, G.M.,Misato, T., ed. 1972. i n Environmental Toxiciology of Pesticides, Academic Press, New York. 316 Mattocks, A.R. 1968. T o x i c i t y of p y r r o l i z i d i n e alka-l o i d s . Nature Lond. 217: 723-728. 317 McCullough, T. 1967. Quantitative determination of trans-2-hexenal i n the defensive scent f l u i d of Acanthocephala d e c l i v i s and A. franulosa ( Hemiptera: Coreidae ). Ann. Entomol. Soc. Amer. 60: 862. 318 McGurk, D.J., Frost, J., Waller, G.R., Eisenbraun, E.J., Vick, E., Drew, W.A., Young, J . 1968. I r i d o d i a l v a r i a t i o n i n dolichoderine ants. J, Insect Physiol. 14: 841-845. 319 Mead, R.J., Segal, W. 1973. Formation of p-cyanolalanine and pyruvate by Acacia georginae. Phytochem. 12: 1977-1981. 320 Mebs, D. 1973. Chemistry of animal venoms, poisons and toxins. Experientia 29: 1328-1334. 321 Meigh, F,D., Firmer, A.A.E., S e l f , R. 1973. Growth-in-h i b i t o r y v o l a t i l e aromatic compounds produced by Solanum tuberosum tubers. Phytochem. 12: 987-993. 322 Meinwald, J., Chadha, M.S., Hurst, J . J . , Eisner, T. 1962. Defence mechanisms of arthropods. IX. Aniso-morphal, the secretion of a phasmid in s e c t . Tetrahedron Lett. 1: 29-33. 323 Meinwald, J., Erickson, K., Hartshorn, M., Meinwald, Y.C., Eisner, T. 1968 a. Defensive Mechanisms of arthropods. XXIII. An a l l e n i c sesquiterpenoid from the grasshopper Romalea microptera. Tetrahedron Lett. 25: 2959-2962. 226 324 Meinwald, J . , Meinwald, Y.C., Chalmers, A.M. 1968b. Dihydromatricaria a c i d : acetylenic acid secreted by s o l d i e r beetle. Science Wash. 160: 890-892. 325 Meinwald, J . , Happ, G.M., Lapows, J . , Eisner, T. 1966a. Cyclopentanoid terpene biosyntheis i n a phasmid insect and i n catmint. Science Wash. 151: 79-80. 326 Meinwald, j . , Koch, K.F., Rogers, J.E.Jr., Eisner, T. 1966b. Biosynthesis of arthropod secretions. I I I . Synthe/is of some simple p-benzoquinones i n a beetle ( Eleodes l o n g i c o l l i s ). J . Amer. Chem. Soc. 88: 1590- 1592. 327a Meinwald, Y.C., Meinwald, J., Eisner, T. 1966c. 1,2-Dialkyl-4(3H)-quinazolinones i n the defensive secretion of a millipede ( Glomeris marginata ). Science Wash. 154: 390-391. 327b Menzie, C.M. 1972. Fate of pes t i c i d e s i n the environ-ment. Ann. Rev. Entomol. 17: 199-222. 328 Metzler, M., Dahm, K.H„, Meyer, D., Roll e r , H. 1971. On the biosynthesis of Juvenile Hormone i n the adult cecropia moth . Z e i t . Naturforsch. 266: 1270-1276. 329x Meyer, D., Schlatter, Ch., Schlatter-Lanz, H., Bovey, P. 1968. Die Zucht von Lytta vesicatoria im Laboratorium und Nachweis der Cantharidinsynthese i n Larven. Experientia 24: 995-998. 330 Mikolajczak, K.L., Smithe, R., Tjark, L.W. 1971. Cyanolipids of Kroelreuteria paniculata Laxm. seed o i l . Lipids 5: 672-677. 331 M i l l e r , J.R., Mumma, R.O. 1973. Defensive agents of the american water beetles Agabus seriatus and Grapho- derus l i b e r u s . J . Insect Phsyiol. 19: 917-925. 332 M i l l s , R.R. Lake, R.C., 1971. Metabolism of tyrosine p-hydroxyphenylpropionic acid and to p-hydroxy-phenylacetic acid by the haemolymph of the american cockroach. Insect Biochem. 1: 264-270. 227 333 M i l l s , R.R., Lake, C.R., Alworth, W.L. 1967. Bio-synthesis of N-acetyl-dopamine by the american cockroach. J. Insect Physiol. 13: 1539-1546. 334 Molinoff, P.B., Axelrod, J . 1971. Biochemistry of catecholamines. Ann. Rev. Biochem. 40: 465-500. 335 Monro, A., Chadha, M.S., Meinwald, J., Eisner, T. 1962. Defence mechanisms of arthropods. VI. Para-benzo-quinones i n the secretion of f i v e species of millipedes. Ann. Entomol. Soc. Amer. 55: 261-262. 336 Montogomery, R.D. 1969. Cyanogens., i n Toxic Constitu-' ents of Plants Foodstuffs ( Chpt. 5 ), ed., I.E. Liener, Academic Press, New York. 337 Moore, B.P. 1968. Studies on the chemical composition and function of the cephalic gland secretion i n au s t r a l i a n termites. J . Insect Physiol. 14: 33-39. 338 Moore, B.P., 1967. Hydrogen cyanide i n the defensive secretions of l a r v a l ParopsLni ( Coleoptera: Chrysomelidae ) J . A u s t r a l . Entomol. 6: 36-38. . 339 Moore, B.P., 1964. V o l a t i l e terpenes from Nausitermes so l d i e r s ( Isoptera; Termitidae ). J . Insect Phsyiol. 10: 371-375. 340 Moore, B.P., Brown, W.V., 1972. Thechemistry of the metasternal secretions of the common eucalypt longhorn, Phorocantha semipunctata ( Coleoptera; Cerambycidae ). Aust. J . Chem. 25: 591-598. 341 Moore, B.P., Wallbank, B.E. 1968. Chemical composition of the defensive secretion i n carabid beetles and i t s importance as a taxonomic character. Proc. R. Entomol. Soc. Lond. ( B) 37: 62-72. 342 Moore, Koy Subba Rao, P.V., Towers, G.H.N. 1968. Degra-dation of phenylalanine and tyrosine by Sporobo-myces roseus. Biochem. J . 106: 507-514. 343 Morgan, E.D., Wadhams, L.J. 1972. Chemical constituents of Dufour's gland i n the ant, Myrmica rubra. J. Insect Physiol. 18: 1125-1135. 228 344 Morris, S.J., Thomson. R.H. 1964. The flavonoid pigments of the small heath b u t t e r f l y , Coeno-nympha pamphilus L.. J . Insect Physiol. 10: 377-383. 345 Moser, J.C. 1970. Pheromones of s o c i a l i n s e c t s . , i n Control of Insect Behaviour by Natural Products, ed., D.L. Wood, R.M. Silverstein, M. Nakajima, Academic Press, New York, pp. 131-178. 346 Muller, C.H. 1968. The r o l e of alle l o p a t h y i n the evolution of vegetation., i n Biochemical Coevol- ution, ed., K.L. Chambers, Proc 29th Ann. B i o l . C o l l . , Oregon State University Press, pp. 13-31. 347 Mullins, D.E., Cochran , D.G. 1973. Nitrogenous excre-tory materials from the american cockroach. J . Insect Physiol. 19: 1007-1018. 348 Mummery, R.S., Valadon, L.R.G. 1974. Carotenoids of the l i l y beetle ( L i l i c o c e r i s l i l i i " ) and i t s food plant ( Lilium hansonii ). J . Insect Physiol. 20: 429-433. 349 Murdock, L.L., Hopkins, T.L., Wirtz, R.A. 1970. Phenylalanine metabolism i n cockroaches, P e r i - planeta americana; tyrosine and benzoyl - B-glu-coside biosynthesis. Comp. Biochem. Physiol. 36; 535-545. 350 Murray, R.D.H., Martin, A., St r i d e , G.O. 1972. Identi-f i c a t i o n of the v o l a t i l e phagostimulants i n Solanum campylacanthum for Epitanchna fulvosignata. J . Insect Physiol. 18: 2369-2373. 351 Nair, M.S.R., Anchel, M. 1972. An a n t i b a c t e r i a l quinone hydroquinone p a i r from the Ascomycete, Nectaria  c o r y l i . Tetrahedron Letters 9: 795-796. 352 Nakanishi, K., Moriyama, H., Okauchi, T., Fujioka, S., Koreeda, M. 1972. Biosynthesis of a- and B -ecdysones from cholesteroloutside the prothoracic gland i n Bombyx mori. Science Wash. 176: 51-52. 353 Nelson, D.R. 1969. Hydrocarbon synthesis i n the american cockroach. Nature Lond. 221: 854-855. 229 354 Nicholas, H.J., 1973. Miscellaneous v o l a t i l e plant products., i n Phytochemistry Vol. I I . Organic  Metabolites, ed. L.P. M i l l e r , Van Nostran Rheinhold, NewYork, pp. 381-399. 355 Noirot, C., Quennedey, A. 1974. Fine structure of insect epidermal glands. Ann. Rev. Entomol. 19: 61-80. 356 Nolte, D.J., Eggers, S.H., May, I.R. 1973. A locust pheromone l o c u s t o l . J . Insect Physiol. 19: 1547-1554. 357 Norris, D.M. 1970. Quinol stimulation and quinone deter-rency on gustation by Scolytus m u l t i s t r i a t u s ( Coleoptera: Scolytidae ). Ann. Entomol. Soc. Amer. 83: 476-478. 358 Norris, D.M. 1969. Transduction mechanism i n o l f a c t i o n and gustation. Nature Lond. 222: 1263-1264. 359 Norris, D.M., Ferkovich, S.M., Rozental, J.M. 1970. Energy transduction : I n h i b i t i o n of cockroach feeding by naphthoquinone. Science Wah. 170: 754-755. 360 Oaks, A., Johnson, F.J. 1972. Cyanide as an asparagine prescursor i n corn roots. Phytochem. 11: 3465-3471. 361 Oudejans, R.C.H.M., Zandee, D.I. 1973. The biosynthesis of the hydrocarbons i n males and females of the millipede Graphidostreptus tumuliperus. J . Insect Physiol. 19: 2245-2253. 362 Packter, N.M. 1965. Studies on the biosynthesis of quinones i n Fungi. Incorportaion of 6-methylsali-c y l i c acid into fumigatin and related compounds i n A spergillus fumigatus I.M.I. 89353. Biochem. 97: 321-332. 363 Packter, N.M., Steward,M.W. 1967 . Studies on the biosyn-thesis of phenols _ i n Fungi. Biosynthesis of 3,4-di-methoxy-6-methyl-toluquinone and g l i o r o s e i n i n Gliocladium roseum. I.M.I. 93065. Biochem. J . 102: 122-132. 230 364 Pallares, E. S.1946. Note on the poison produced by the Polydesmus ( Fontaria) vicinus L i n . . Arch. Biochem. Biophys. 9:105-108. 365 Parsons, J.A., Rothschild, M. 1964. Rhodanese i n the larva and pupa of the Common Blue b u t t e r f l y ( Polyommatus icarus ( Roff. ))( Lepidoptera ). Entomol. Gaz. 15: 58-59. 366 Parsons. J.A. 1965. A d i g i t a l i s - l i k e toxin i n the Monarch b u t t e r f l y , Danaus plexippus. J . Physiol. 178: 290-304., 367 Pasteels, J.M., Deroe, C., Tursch., B. , Braekman, J.C., Daloze, D., Hootele,„C. 1973. D i s t r i b u t i o n et a c t i v i t e s des alcaloides defensifs de Coccinelidae. J. Insect Physiol. 19: 1771-1784. 368 Pau, R.N., Acheson, R.M. 1968. The i d e n t i f i c a t i o n of 3-hydroxy-4-0-B-D-glycosido-benzyl alcohol i n the l e f t c o l l a t e r a l gland of Blaberus d i s c o i d a l i s . Biochim. Biophys. Acta 158: 206-211. 369 Pavan, M. 1956. Studi sui Formicidae. II S u l l ' origine s i g n i f i c a t o b i o l o g i c o e isolamento d e l l a dendro-la s i n a . Ric. S c i . 26: 144-150. 370 Pavan, M. 1952. " Iridiomyrmecin" as an i n s e c t i c i d e . IX th Int r . Congr. Entomol. Amsterdam 1: 321-327. 371 Pavan, M., Dazzini, M.V. 1971. Toxicology and pharma-cology- Arthropods Chemical Zool. 6:365-409. 372 Pettersson, G. 1966a.On the role of 6-methylsalicylic acid i n the biosynthesis of fungal benzoquinones. Acta Chem. Scand. 20: 151-158. 373 Pettersson, G.1966b. New metabolites from Lentinus degener. Acta Chem Scand. 20: 45-50. 374 Piek, T. 1964. Synthesis of wax i n the honey bee ( Apis m e l l i f e r a L.). J. Insect Physiol. 10: 563-572. 375 Pierre, L.L. 1962. Synthesis of ascorbic acid by the normal fat body of the cockroach , Leucophaea  maderae, and by i t s symbionts. Nature Lond. 193: 904-905. 231 376 Pinder, A.R., Staddon, B.W. 1965. The odiferous secretion of the water bug Sigara f a l l e n i ( F r i b ). J . Chem. Soc. 530: 2955-2958. 377 Power, F.B., Gornall, F.H. 1904. Gynocardin, a new cyanogenic glucoside. Preliminary note. Proc. Chem. Soc Lond. 20: 137. 378 Pridham, J.B., ed. 1967. i n The Terpenoids i n Plants, Aca-demic Press, New York. 379 Q u i l i c o , A., P i o z z i , F, Pavan, M. 1957. The structure of dendrolasin. Tetrahedron 1: 177-185. 380 Qulliam, J.P., Stables, R. 1969. Convulsant e f f e c t s of cunaniol, a polyacetylenic alcohol i s o l a t e d from the plant Clibadium sylvestre, on frogs and mice. Pharmacol. Res. Commun. 1: 7-14. 381 Raper, J.R. 1970. Chemical ecology among lower plants., i n Chemical Ecology ( Chpt. 2 ), Ed., E. Sondhei-mer, J.B. Simeone, Academic Press, New York. 382 Raymond, S.H., Yang,S.H., Wilkinson, C.F. 1972. Enzymic sulphation of p-nitro-phenol and steroids by l a r v a l gut tissues of the southern armyworm ( Prodenia  eridania ). Biochem. j . 130: 487-493. 383 Rees, H.H. 1971. Ecdysone., i n Aspects of Terpenoid Chemistry and Biochemistry, ( Chpt. 7. ), ed., T.W. Goodwin, Academic Press, New York. 384 Regnier, F.E., Law, J.H. 1968. Insect Pheromones. J. L i p i d Res. 9: 541-550. 385 Regnier, F.E., Wilson, E.O. 1968. The alarm-defence system of the ant Ac a n thornyop s c l a v i g e r . J . Insect Physiol. 14: 955-970. 386 Regnier, F.E., Wilson, E.O. 1969. The alarm-defence system of the ant Lasius alienus. J . Insect Physiol. 15: 893-898. 387 Remold, H. 1963. Scent-glands of land bugs, t h e i r physiology and biological function. Nature Lond. 198: 764-768. 232 388 Renfrow, W.B.Jr. , Mauser, C.F. 1937. The r e l a t i v e rates . of decomposition of potassium s a l t s of c e r t a i n para and meta substituted dibenzhydroxamic acids. Study of the Lossen Rearrangement. J. Amer. Chem. Soc. 59: 2308-2315. 389 Renwick, J.A.A., Hughes, P.R., Tanletin, DeJ. T.Y. 1 9 7 3 . Oxidation products of pinene i n the bark beetle, Dendroctonus f r o n t a l i s . J . Insect Physiol. 19: 1735 -1740. 390 Retief, L.W., Hewitt, P.H. 1973a. Digestive carbohydrases of the harvester termite Hodotermes mossambicus: a-glycosidases. J. Insect Physiol. 19: 105-113. 391 Retief, L.W., Hewitt, P.H. 1973b. Digestive (3-glycosid-ases of the harvester termite, Hodotermes mossam-bicus ; properties and d i s t r i b u t i o n . J . Insect Physiol. 19: 1837-1847. 392 Rettenmeyer, C.W. 1970. Insect mimicry. Ann. Rev. Ento-mol. 15: 43-74. 393 Riddiford, L . M . 1967. trans-2-Hexenal: mating stimulant for Polyphemus moths. Science Wash. 158: 139-141. 394 Robinson, M . H . 1969. The defensive behaviour of some orthopteroid insects from Panama. Trans. R. Ent. Soc. Lond. 121: 281-303. 395 Roeder, K.D. 1966. Acoustic s e n s i t i v i t y of the noctuid tympanic organ and i t s range for the c r i e s of bats. J. Insect Physiol. 12: 859. 396 Roeder, K.D. 1964..Aspects of the noctuid tympanic nerve response having s i g n i f i c a n c e i n the avoidance of bats. J. Insect Physiol. 10: 529-546. 397 Roth, L . M . 1961. A study of the odiferous glands of Scaptocoris divergens ( Hemiptera:Cydnidae ) Ann. Entomol. Soc. Amer. 54: 900-911. 398 Roth, L . M . , Eisner, T. 1962. Chemical defences of Arthropods. Ann. Rev. Entomol. 7: 107-136. Roth, L.M. , Rowland, R.B. 1941. S t u d i e s on t h e gaseous s e c r e t i o n o f T r i b e I i u m confusum D u v a l . Ann. E n t o -mol. Soc. Amer. 34: 151-175. Roth, L.M., S t a y , B. 1958. The o c c u r r e n c e o f p a r a -q u i n o n e s i n some a r t h r o p o d s w i t h emphasis on t h e q u i n o n e - s e c r e t i n g t r a c h e a l g l a n d s o f D i p l o p t e r a  p u n c t a t a . J . I n s e c t P h y s i o l . 1: 305-318. R o t h s c h i l d , M. 1973. S p e c u l a t i o n s on m i m i c r y w i t h Henry F o r d . , i n E c o l o g i c a l G e n e t i c s and E v o l u t i o n , ( Chpt. 10 ), ed., R. C r e e d , O x f o r d : B l a c k w e l l s . R o t h s c h i l d , M. 1972. Secondary p l a n t s u b s t a n c e s and w a r n i n g c o l o u r a t i o n i n i n s e c t s . , i n I n s e c t / P l a n t R e l a t i o n s h i p s ,ed., H.F. van Emden, Symp. R. E n t o -mol. Soc. Lond. 6: 59-83, B l a c k w e l l S c i e n t i f i c P u b l i c a t i o n . O x f o r d . R o t h s c h i l d , M. 1966. E x p e r i m e n t s w i t h c a p t i v e p r e d a t o r s and t h e p o i s o n o u s g r a s s h o p p e r P o e k i l o c e r u s b u f o n i s ( K i u g ). Proc.R. E n t o m o l . Soc Lond. (C) 31-32. R o t h s c h i l d , M. 1961. D e f e n s i v e odours and M u l l e r i a n m i m i c r y among i n s e c t s . T r a n s . R. E n t o m o l . Soc. Lond. 113: 101-131. R o t h s c h i l d , M., von Euw, J., R e i c h s t e i n , T. 1973. C a r d i a c g l y c o s i d e s ( h e a r t p o i s o n s ) i n t h e p o l k a -d o t moth Syntomeida e p i l a i s V7alk. ( C t e n u c h i d a e : Lep„ ) w i t h some o b s e r v a t i o n s on t h e t o x i c q u a l i t i e s o f Amata (= Syntomis ) phegea ( L . ) . P r o c . R. Soc. Lond. (B) 183: 227-247. R o t h s c h i l d , M., von Euw, J., R e i c h s t e i n , T. 1972. A r i s t o l o c h i c a c i d s s t o r e d by Z e r y n t h i a p o l y x e n a ( L e p i d o p t e r a ). I n s e c t Biochem. 2: 334-343. R o t h s c h i l d , M . , von Euw, J . , R e i c h s t e i n , T. 1970a. C a r d i a c g l y c o s i d e s i n t h e o l e a n d e r a p h i d A p h i s n e r i i . J . I n s e c t P h y s i o l . 16: 1141-1145. R o t h s c h i l . d , M., R e i c h s t e i n , T., von Euw, j . , A p l i n , R., Harman, R.R.M. 1970b. T o x i c L e p i d o p t e r a . T o x i c o n 0: 293-299 R u d i n s k y , J.A. 1966. S c o l y t i d b e e t l e s a s s o c i a t e d w i t h Douglas f i r : r e s p onse t o t e r p e n e s . S c i e n c e Wash. 152: 218-219. Rudney, H. 1971. The b i o s y n t h e s i s o f t e r p e n o i d quinone i n N a t u r a l l y O c c u r r i n g Quinones, ed., R.H. Thomson 2 nd ed., Academic P r e s s , New York, pp. 89-103. R u f f i n i , G. 1965. T h i n - l a y e r chromatogarphy o f 2,4-d i n i t r o p h e n y l h y d r a z o n e s o f a r o m a t i c a l d e h y d e s and k e t o n e s . J . Chromatog. 17: 483-489. R u i t e r , L., de. 1955. C o u n t e r s h a d i n g i n c a t e r p i l l a r s . A r c h . N e e r l . Z o o l . 11: 1-57. R u i t e r , L., de. 1952. Some e x p e r i m e n t s on t h e camou-f l a g e o f s t i c k c a t e r p i l l a r s . B e h a v i o u r 4: 222-232. c o n t i n u e d on n e x t page 235 414 Sanderson, G.W. 1972. The c h e m i s t r y o f t e a and t e a m a n u f a c t u r i n g . , i n S t r u c t u r a l and F u n c t i o n a l  A s p e c t s o f P h y t o c h e m i s t r y , VOL.5,, ed., V.C. R u n e c k l e s , T.C. Tso, Academic P r e s s , New Y o r k , pp. 247-316. 415 S a y r e , J.W., Kaymakaca.lans, S. 1964. C y a n i d e p o s i o n i n g from a p r i c o t seeds among c h i l d r e n i n c e n t r a l T urkey. New E n g l . J . Med. 270: 1113-1115. 416 S a z , H.J., W e i l A. 1960. The mechanism o f f o r m a t i o n o f a - m e t h y l b u t y r a t e from c a r b o h y d r a t e by A s c a r i s  l u m b r i c o i d e s m u s c l e . J . B i o l . Chem. 235: 914- 918. 417 S c h i l d k n e c h t , H. 1971. E v o l u t i o n a r y peaks i n t h e de-f e n s i v e c h e m i s t r y o f i n s e c t s . Endeavour 30: 136-141. 418 S c h i l d k n e c h t , H., B i r r i n g e r , H., M a s c h w i t z , U . 1967a. T e s t o s t e r o n e as a p r o t e c t i v e a gent o f t h e w a t e r b e e t l e I l y b i u s . Angew. Chem. Intern.. Ed. E n g l i s h 6: 558-559. 419 S c h i l d k n e c h t , H., M a s c h w i t z , U . , Wenneis, W.F. 1967b. Neue S t o f f e aus dem W e h r s e k r e t d e r D i p l o p o d e n e n -g a t t u n g G l o m e r i s . uber A r t h r o p o d e n - A b w e h r s t o f f . XXIV. N a t u r w i s s e n s c h a f t e n 54: 195-197. 420 S c h i l d k n e c h t , H., Holoubek, K. 1961. D i e B o m b a r d i e r k a f e r und i h r e E x p l o s i o n s c h e m i e . V. M i t t e i l u n g u ber I n s e k t e n - A b w e h r s t o f f e . Angew. Chem. 73:1-7. 421 S c h i l d k n e c h t , H., Kramer, H. 1962. Zum Nachweis von Hyd r o c h i n o n e n neben Chinone i n den A b w e h r b l a s e n von A r t h r o p o d e n - XV. M i t t e i l u n g tiber I n s e k t e n -Abwehrs t o f f e . Z. N a t u r f , 176: 701-702. 236 422 S c h i l d k n e c h t , H., M a s c h w i t z , U., K r a u s s , D, 1968a. B l a u s a u r e im W e h r s e k r e t des E r d l a u f e r s Pachymerium  f e r r u g i n e u m . N a t u r w i s s e n s c h a f t e n 55: 230. 423 S c h i l d k n e c h t , H., M a s c h w i t z , U., W i n k l e r , H. 1968b. Zur E v o l u t i o n d e r C a r a b i d e n d - W e h r d r u s e s e n s e k r e t e . Uber A r t h r o p o d e n - A b w e h r s t o f f e X X X I I . N a t u r w i s s e n -s c h a f t e n 55: 112-117. 424 S c h i l d k n e c h t , H., M a s c h w i t z , E, M a s c h w i t z , U. 1970. D i e E x p l o s i o n s c h e m i e d e r B o m b a r d i e r k a f e r S t r u k t u r und E i g e n s c h a f t e n der Brennkammerenzyme. J . I n s e c t P h y s i o l . 16: 749-789. 425 S c h i l d k n e c h t , H., Reed, P.B., Reed, F.D., Koob, k. 1973. A u x i n a c t i v i t y i n t h e s y m b i o s i s o f l e a f - c u t t i n g a n t s and t h e i r f u n g us. I n s e c t Biochem. 3: 439-442. 426 S c h i l d k n e c h t , H., T a c h e c i , H. 1971. C o l y m b e t i n , a new d e f e n s i v e s u b s t a n c e o f t h e w a t e r b e e t l e , Colymbetes  f u s c u s , t h a t l o w e r s b l o o d p r e s s u r e - L I I . J , I n s e c t P h y s i o l . 17: 1889-1896. 427 S c h i l d k n e c h t , H., Wenneis, W.F. 1967. Uber A r t h r o p o d e n -A b w e h r s t o f f e XXV. A n t h r a n i l s a u r e a l s P r e c u r s o r d e r A r t h r o p o d e n - A l k a l o i d e G l o m e r i n un Homoglomerin, T e t r a h e d r o n L e t t e r s 19: 1815-1818. 428 S c h i l d k n e c h t , H, Wenneis, W.F., Weis, K.H., M a s c h w i t z , U, 1966. G l o m e r i n , e i n neues A r t h r o p o d e n - A l k a l o i d . N a t u r f o r s c h . 21B: 121-127. 429 S c h l a t t e r , Ch. Waldner, E.E., Schmid, H. 1968. Zur B i o s y n t h e s e des C a n t h a r i d i n s . E x p e r i e n t i a 24: 994-995. 430 S c h m e l t z , I . 1971 N i c o t i n e and o t h e r t o b a c c o a l k a l o i d s . , i n N a t u r a l l y O c c u r r i n g I n s e c t i c i d e s , ( Chpt. 3 ) , ed., M. J a c o b s o n , D,G, C r o s b y , M a r c e l Dekker, New Y o r k . 431 Schmidt, R.S. 1958. B e h a v i o u r a l e v i d e n c e on t h e e v o l u t i o n o f B a t e s i a n m i m i c r y . B e h a v i o u r 6: 129-138. 432 Schonbaum, G.R., Bonner, W.D.Jr., S t o r e y , B.T., Bahr, J . T. 1971. S p e c i f i c i n h i b i t i o n o f t h e c y a n i d e - s e n -s i t i v e r e s p i r a t o r y pathway i n p l a n t m i t o c h o n d r i a by hydroxamic a c i d s . P l a n t P h y s i o l . 47: 124-128. Schoonhoven, L.M. 1972. Secondary p l a n t s u b s t a n c e s and i n s e c t s . , i n Recent Advances Phytochem. 5: 197 - 2 2 4 . Schoohoven, L.M. 1969. G u s t a t i o n and f o o d p l a n t s e l e c -t i o n i n some l e p i d o p t e r o u s l a r v a e . E n t o m o l o g i a 12: 555-564. S c h r e i b e r , K. 1959. Uber e i n g e I n h a l s t o f f e d e r S o l a n a c e e n und i h r e Bedeutung f u r d i e K a r t o f f c e k a f e r r e s i s t e n z . E n t omol. E xp. A p p l . 1: 28-37. Scudder, G.G.E., D u f f e y , S.S. 1972. C a r d i a c g l y c o s i d e s i n t h e L y g a e i n a e ( H e m i p t e r a : L y g a e i d a e ). Can. J . Z o o l . 50: 35-52.' S e e l e y , M.K., C r i d d l e , R.S., Conn, E.E. 1966. The meta-b o l i s m o f a r o m a t i c compounds i n h i g h e r p l a n t . V I I I . On t h e r e q u i r e m e n t o f h y d r o x y n i t r i l e l y a s e f o r f l a v i n . J . B i o l . Chem. 241: 4457-4462. S e l f , L.S., G u t h r i e , F.E., Hodgson. E. 1964a. M e t a b o l i s m o f n i c o t i n e by t a b a c c o - f e e d i n g i n s e c t s . N a t u r e Lond. 204; 300-301. S e l f , L.S., G u t h r i e , F.E., Hodgson, E. 1964b. A d a p t a -t i o n o f t o b a c c o hornworms t o t h e i n g e s t i o n o f n i c o t i n e . J . I n s e c t P h y s i o l . 10: 907-914. S e l i g m a n , I.M., Doy, F.A. 1973. B i o s y n t h e s i s o f d e f e n -s i v e s e c r e t i o n s i n P a p i l i o aegus . I n s e c t Biochem. 3: 205-215. S e l i g m a n , I.M. , Doy, F.A. 1972. B-Hydroxy--n-butyric a c i d i n t h e d e f e n s i v e s e c r e t i o n o f P a p i l i o aegus. Comp. Biochem. P h y s i o l . 42B: 341-342. S e x t o n , O.J. 1966. A n o l i s c a r o l i n e n s i s : E f f e c t s o f f e e d i n g on r e a c t i o n t o a p o s e m a t i c p r e y . S c i e n c e Wash. 15 3: 1140. 238 443 S e x t o n , O.J. 1964. D i f f e r e n t i a l p r e d a t i o n by t h e l i z a r d A n o l i s c a r o l i n e n s i s upon u n i c o l o u r e d and p o l y -c o l o u r e d i n s e c t s a f t e r an i n t e r v a l o f no c o n t a c t . A n i m a l B e h a v i o u r 7 : 101-110. 444 Shaaya; E., B o d e n s t e i n , D. 1969. The f u n c t i o n o f t h e a c c e s s o r y sex g l a n d i n P e r i p l a n e t a americana ( L . ) . I I . The r o l e o f j u v e n i l e hormone i n t h e synthesis o f p r o t e i n and p r o t o c a t e c h u i c a c i d g l u c o s i d e . J . Exp. B i o l . 1 7 0 : 281-292. 445 Shaaya, E., S e k e r i s , C.E. 1970. The f o r m a t i o n o f p r o t o -c a t e c h u i c a c i d - 4 - 0 - ( 3 - g l u c o s i d e i n P e r i p l a n e t a  a m e r i c a n a and t h e p o s s i b l e r o l e o f j u v e n i l e hormone. J . I n s e c t P h y s i o l . 16: 323-330. 446 S h a r p i e s , D., S p r i n g , M.S., S t o k e r , J.R. 1972. B i o -s y n t h e s i s o f the major c y a n o g e n i c g l y c o s i d e o f T h a l i c t r u m a q u i l e g i f o l i u m . Phytochem. 11: 2999-3QD2. 447 S h i m i z u , Y. 1971. A n t i f u n g a l s o r b i c a c i d c o n t a i n i n g g l y c e r i d e i n a p h i d s . N a t u r w i s s e n s c h a f t e n 58: 366-367. 448 449 S horey, H.H. 1973. B e h a v i o u r a l r e s p o n s e s t o i n s e c t pheromohes. Ann Rev. Entomol. 18: 349-380. S i n g h , B., R a s t o g i , R.P. 1970. C a r d e n o l i d e s - G l y c o s i d e s and g e n i n s . Fhytochem. 9: 315-331. 450 S l a t e r , J.A., Knop, N.F. 1969. G e o g r a p h i c v a r i a t i o n s i n t h e N o r t h A m e r i c a n milkweed bugs o f t h e Lygaeus  k a l m i i complex. Ann. Entomol. Soc. Amer. 62: 1221-1232. 451 S l a t e r , J.W., 1877. On t h e f o o d o f g a i l y - c o l o u r e d c a t e r p i l l a r s . T r a n s . R. Entomol. Soc. Lond. (1811) 205-209. 452 S m i t h , D.S 0E C 1966. M u t a g e n i c i t y o f c y c a s i n a g l y c o n e ( m e t h y l a z o x y m e t h a n o l ), a n a t u r a l l y o c c u r r i n g c a r c i n o g e n . S c i e n c e Wash. 152: 1273-1274. 453 S m i t h , J.N. 1962. D e t o x i c a t i o n mechanisms. Ann. Rev. Entomol. 7 : 465-480. 239 454 Sondheimer, E. , Simeone, J.B., ed. 1970. i n C h e m i c a l E c o l o g y , Academic P r e s s , New Y o r k . 455 S t a h l , E. 1969. i n T h i n L a y e r Chromatography: a L a b o r -a t o r y Handbook, 2nd. ed., S p r i n g e r - V e r l a g . 456 459 460 464 466 S t e v e n s , R.L., Emery, T.F. 1966. The b i o s y n t h e s i s o f h a d a c i d i n . Biochem. 5: 74-81. 457 S t r o n g , F.E. 1967. O b s e r v a t i o n s on a p h i d c o r n i c l e s e c r e t i o n s . Ann. Entomol. Soc. Amer. 60: 668-673. 458 S t r o n g , L. 1971. I n t r a c e l l u l a r d u c t s i n t h e e p i d e r m i s o f t h e male d e s e r t l o c u s t . J . I n s e c t P h y s i o l . 17: 1823-1831. Tapper, B.A., B u t l e r , G.W. 1971. Oximes, n i t r i l e s and 2 - h y d r o x y n i t r i l e s as p r e c u r s o r s i n t h e b i o s y n t h e -s i s o f c y a n o g e n i c g l u c o s i d e s . Biochem. J . 124: 935-941. Tapper, B.A., Z i l g , H., Conn, E.E. 1972. 2-Hydroxy-a l d o x i m e s as p o s s i b l e p r e c u r s o r s i n t h e b i o s y n -t h e s i s o f c y a n o g e n i c g l u c o s i d e s . Phytochem. 11: 1047-1053. 461 Teas, H.J. 1967. C y c a s i n s y n t h e s i s i n S e i r a r c t i a echo ( L e p i d o p t e r a ) l a r v a e f e d m e t h y l a z o x y m e t h a n o l . Biochem. B i o p h y s . Res. Commun. 26: 686-690. 462 T h i e l e , J . , M e i s e n h e i m e r , J . 1900. Ueber d i e A d d i t i o n von B l a u s a u r e an C h i n o n . Chem. Ges. B e r . B e r l i n 33: 675-676. 463 Thomson, R.H. 1971. i n N a t u r a l l y O c c u r r i n g Quinones, 2nd ed., Academic P r e s s , New Y o r k . T h o r s t e i n s o n , A . J . 1960. Host s e l e c t i o n i n phytophagous i n s e c t s . Ann. Rev. Entomol. 5: 193-218. 465 T h o r s t e i n s o n , A . J . 1958. A c c e p t a b i l i t y o f p l a n t s f o r phy(pphagous i n s e c t s . P r o c . 1 0 t h I n t . Ccngr. E n t . M o n t r e a l 2: 599-602. T h o r s t e i n s o n , A . J . 1953. The c h e m o t a c t i c r e s p o n s e s 240 t h a t d e t e r m i n e h o s t s p e c i f i c i t y i n an o l i g o -phagous i n s e c t P l u t e l l a m a c u l i p e n n i s . Can. J . Z o o l . 31: 52-57. T h r e l f a l l , D.R., W h i s t a n c e , G.R. 1971. B i o s y n t h e s i s o f i s o p r e n o i d benzoquinones and chromanols., i n A s p e c t s o f T e r p e n o i d C h e m i s t r y and B i o c h e m i s t r y , ( Chapt. 1 2 ) , ed. T.W. Goodwin, Academic P r e s s , New Y o r k . Towers, G.H.N. 1964. M e t a b o l i s m o f p h e n o l i c compounds i n p l a n t s . , i n B i o c h e m i s t r y o f P h e n o l i c Compounds, ( Chpt. 7 ), ed., J.B. Harborne, Academic P r e s s , New Y o r k . Towers, G.H.N., M c l n n e s , A.G., N e i s h , A.C. 1964. The a b s o l u t e c o n f i g u r a t i o n o f t h e p h e n o l i c c y a n o g e n i c g l u c o s i d e s t a x i p h y l l i n and d h u r r i n . T e t r a h e d r o n 20: 71-77. Towers, G.H.N., Subba Roa, P.V. 1972. D e g r a d a t i v e m e t a b o l i s m o f p h e n y l a l a n i n e , T y r o s i n e and DOPA., i n Recent Advances i n P h y t o c h e m i s t r y t ( c h p t . 1 ), P r o c . 9 t h Ann. Symp. Phytochem. Soc. N.A., A p p l e t o n - C e n t u r y - C r o f t s , New Y o r k . Toye, S.A. 1966a. The r e a c t i o n s o f t h r e e s p e c i e s o f N i g e r i a n m i l l i p e d e s ( S p i r o s t r e p t u s a s s i n i e n s i s , Oxydesmus sp., and Habrodesmus f a l x ) t o l i g h t , h u m i d i t y , and t e m p e r a t u r e . Entomol. Exp. A p p l . 9: 468-484. Toye, S.A. 1966b. The e f f e c t o f d e s i c c a t i o n on t h e b e h a v i o u r o f t h r e e s p e c i e s o f N i g e r i a n m i l l i p e d e s : S p i r o s t r e p t u s a s s i n i e n s i s , Oyxdesmus sp., and Habrodesmus f a l x . Entomol. .Exp. A p p l . 9: 378-384. Toye,S.A. 1966c. S t u d i e s on t h e l o c o m o t o r y a c t i v i t y o f t h r e e s p e c i e s o f N i g e r i a n m i l l i p e d e s : S p i r o -s t r e p t u s a s s i n i e n s i s , Oxydesmus sp,, and Habro-desmus f a l x . Entomol. Exp. A p p l . 9: 369-377. Tschesche, R. 1972. B i o s y n t h e s i s o f c a r d e n o l i d e s , b u f a -d i e n o l i d e s and s t e r o i d s a p o g e n i n s . P r o c . R. Soc. Lond. (B) 180: 187-202. T s c h i n k e l , W.R. 1972. 6 - A l k y l - l , 4 - n a p h t h o q u i n o n e s from the d e f e n s i v e s e c r e t i o n o f t h e t e n e b r i o n i d b e e t l e 241 A g r o p o r i s a l u t a c e a . j . I n s e c t P h y s i o l . 18: 711-722 4 7 5 T s c h i n k e l , W.R. 1969. Phenols and quinones from t h e d e f e n s i v e s e c r e t i o n s o f t h e t e n e b r i o n i d b e e t l e , Zophobas r u g i p e s . J . I n s e c t P h y s i o l . 15: 191-200. 476 T s u y u k i , T., Ogata, Y., Yamamoto, I . , S h i n i , K. 1965. S t i n k bug a l d e h y d e s . A g r i c . B i o l . Chem. 29: 412-427. 477 T u l l o c h , A.P. 1970. The c o m p o s i t i o n o f beeswax and o t h e r waxes s e c r e t e d by a p h i d s . L i p i d s 5: 247-258. 478 U n d e r h i l l , E.W. 1967. B i o s y n t h e s i s o f must a r d o i l s : c o n v e r s i o n o f p h e n y l a c e t a l d e h y d e oxime and 3-p h e n y l p r o p i o n a l d e h y d e oxime t o g l o c o t r o p a e o l i n and g l u c o n a s t u r t i i n . E u r . J . Biochem. B i o p h y s . Res. Commun. 14: 425-430. 479 U n d e r h i l l , E.W., C h i s h o l m , M.D. ,1964. B i o s y n t h e s i s o f mustard o i l s g l u c o s i d e s . I I I . F o r m a t i o n o f g l u c o t r o p a e o l i n from L - p h e n y l a l a n i n e - c l 4 _ N l 5 # Biochem. B i o p h y s . Res. Commun. 14: 425-430. 480 U r i b e , E.G., Conn, E.E. 1966. The m e t a b o l i s m o f a r o -m a t i c compounds i n h i g h e r p l a n t s . V I I . The o r i g i n o f t h e n i t r i l e n i t r o g e n atom o f d h u r r i n ( 3-D-g l u c o p y r a n o s y l o x y - L - p - h y d r o x y m a n d e l o n i t r i l e ). J . B i o l . Chem. 241: 92-94. 481 V a n E t t e n , C.H. 1969. G o i t r o g e n s . , i n T o x i c C o n s t i t u e n t s o f P l a n t F o o d s t u f f s , ( Chapt. 4 ), ed., I.E. L i e n e r , Academic P r e s s , New Yo r k . 482 V i t e , J.P., W i l l i a m s o n , D.L. 1970. Thanasimus d u b i u s : P r e y p e r c e p t i o n . J . I n s e c t P h y s i o l . 16: 233-239. 483 V o g e l , A . I . 1967. i n A Textbook o f Pra±ical O r g a n i c C h e m i s t r y , 3 r d ed., Longmans and Co. L t d . , London. 484 Wain, R. 1943. The s e c r e t i o n o f s a l i c y l a l d e h y d e by t h e l a r v a e o f the b r a s s y w i l l o w b e e t l e ( P h y l l o d e c t a  v i t e l i n a e ). Rept. A g r i c . Res. S t n . U n i v , B r i s t o l 108-110. 242 485 W a l l b a n k , B.E., Waterhouse, D.F. 1970. The d e f e n s i v e s e c r e t i o n s o f P o l y z o s t e r i a and r e l a t e d c o c k r o a c h e s . J . I n s e c t P h y s i o l . 16: 2081-2096. 486 Waldner, E.E., S c h l a t t e r , Ch., Schmid, H. 1969. Zur B i o s y n t h e s e des D e n d r o l a s i n s , e i n e s , I n h a l t s -s t o f f e s d e r Ameise L a s i u s f u l i g i n o s u s L a t r . . H e l v . Chim. A c t a 52: 15-24. 487 W a l l a c e , J.B., Blum, M.S. 1969. R e f i n e d d e f e n s i v e mechansim i n Chrysomela s c r i p t a . Ann. Entomol. Soc. Amer. 62: 503-506. 488 Waterhouse, D.F., W a l l b a n k , B.E. 1967. 2-Methylene- . b u t a n a l and related compounds i n t h e d e f e n s i v e s c e n t o f P l a t y z o s t e r i a c o c k r o a c h e s ( B l a t t i d a e : P o l y -z o s t e r i i n a e ). J . I n s e c t P h y s i o l . 13: 1657-1669. 489 Waterhouse, D.F., F o r s s , D.A., Hackman, R.H. 1961. C h a r a c t e r i s t i c odour components o f t h e s c e n t on s t i n k bugs. J . I n s e c t P h y s i o l . 6: 113-121. 490 Waterhouse, D.F., G i l b y , A.R. 1964. The a d u l t s c e n t g l a n d s and s c e n t o f n i n e bugs o f s u p e r f a m i l y C o r e o i d e a . J . I n s e c t P h y s i o l . 10: 977-987. 491 W e a t h e r s t o n , J . 1967. The c h e m i s t r y o f a r t h r o p o d d e f e n s i v e s u b s t a n c e s . Q u a r t . Rev. Chem. Soc. Lond. 21: 287-313. 492 W e a t h e r s t o n , J . , P e r c y , J.E. 1970. A r t h r o p o d d e f e n s i v e s e c r e t i o n s . , i n C h e m i c a l s C o n t r o l l i n g I n s e c t  B e h a v i o u r , ed., M. B e r o z a , Academic P r e s s , New Y o r k , pp. 95-144. 493 W e a t h e r s t o n , J . , P e r c y , J.E. 1969. S t u d i e s o f p h y s i o l -o g i c a l l y a c t i v e a r t h r o p o d s e c r e t i o n s . I I I . C h e m i c a l , m o r p h o l o g i c a l , and h i s t o l o g i c a l s t u d i e s o f t h e d e f e n c e mechanism o f U r o b l a n i u l u s c a n a d e n s i s ( Say ) ( D i p l o p o d a : J u l i d a ). Can. J . Z o o l . 47: 1389-1394. 494 Went, F.W. 1970. P l a n t s and t h e c h e m i c a l e n v i r o n m e n t . , i n C h e m i c a l E c o l o g y , ( Chpt. 4 ), ed., E. Sondheimer, J.B. Simeone, Academic P r e s s , New Y o r k . 243 495 Wheeler, J.W., Me i n w a l d , J . , H u r s t , J . J . , E i s n e r , T. 1964. t r a n s - 2 - D o d e c e n a l and 2 - m e t h y l - l , 4 - b e n z o -quinone p r o d u c e d by a m i l l i p e d e . S c i e n c e Wash, 144: 540-541. 496 Wheeler, W.M. 1890. H y d r o c y a n i c a c i d s e c r e t e d by Polydesmus v i r g i n i e n s i s D r u r y . Psyche 5: 422. 497 W h i t t a k e r , R.H. 1970. The b i o c h e m i c a l e c o l o g y o f h i g h e r p l a n t s . , i n C h e m i c a l E c o l o g y , ( Chpt. 3 ), ed., E. Sondheimer. J.B. Simeone, Acadaemic P r e e s , New Y o r k . 498 W h i t t a k e r , R.H., Feeny, P.P. 1971. A l l e l o c h e m i c s : chem-i c a l i n t e r a c t i o n s between o r g a n i s m s . S c i e n c e Wash. 171: 757-770. 499 W i c k l e r , W. 1968. i n M i m i c r y i n P l a n t s and A n i m a l s , t r a n s l a t e d from German by R.D. M a r t i n , W e i d e n f e l d and N i c o l s o n , London. 500 W i l l i a m s , C.M. 1970. Hormonal i n t e r a c t i o n s between p l a n t s and i n s e c t s . , i n C h e m i c a l E c o l o g y , ( Chpt. 6 ), ed., E. Sondheimer, J.B. Simeone, Academic P r e s s , New Y o r k . 501 W i l l i s t o n , S.W. 1884. P r o t e c t i v e s e c r e t i o n s o f s p e c i e s o f E l e o d e s . Psyche 4: 168-169. 502 W i l s o n , E.O. 1970. C h e m i c a l communication w i t h i n a n i m a l s p e c i e s . , i n C h e m i c a l E c o l o g y , ( Chpt. 7 ), ed., E. Sondheimer, J.B. Simeone, Academic P r e s s , New y o r k . 503 W i l s o n , E.O., B o s s e r t , W.H. 1963. C h e m i c a l communications among a n i m a l s . Recent P r o g r . Horm. Res. 19: 673-716. 504 W i l s o n , E.O., R e g n i e r , F . E . , J r . 1971. The e v o l u t i o n o f t h e a l a r m - d e f e n c e system i n t h e f o r m i c i n e a n t s . Amer. Nat. 105: 279-289. 505 Wood, D.L., S i l v e r t e i n , R.M., N a k a j i m a , M. 1970. i n C o n t r o l o f I n s e c t B e h a v i o u r by N a t u r a l P r o d u c t s , Academic P r e s s . 244 506 Wood, J . I . , C o o l e y , S.L. 1956. D e t o x i c a t i o n o f c y a n i d e by c y s t e i n e . J . B i o l . Chem 218: 449-457. 507 Woodring, J.P., Blum, M.S. 1965. The anatomy and comp-a r a t i v e a s p e c t s o f t h e r e p u g n a t o r i a l g l a n d s o f O r t h o c r i c u s a r b o r e u s ( D i p l o p o d a : S p i r o b i l i d a ). J . Morph. 116: 99-108. 508 Woodring, J.P., Blum, M.S. 1963. The anatomy and p h y s i o -l o g y o f t h e r e p u g n a t o r i a l g l a n d s o f Pachydemus  c r a s s i c a t u s ( D i p l o p o d a ). Ann. Entomol. Soc. Amer. 56: 448-453. 509 Wynn,G.G., Boudreaux, H.B. 1972. S t r u c t u r e and f u n c t i o n o f a p h i d c o r n i c l e s . Ann. Entomol. Soc. Amer. 65: 157-166. 510 Yamasaki, K., Tamaki, T., Uzawa, S., Sanakawa, U., S h i b a t a , S. 1973. P a r t i c i p a t i o n o f Cg-C^ u n i t i n t h e b i o s y n t h e s i s o f e p h e d r i n e i n Ephedra. Phytochem. 12: 287-282. 511 Yang, M.G., M i c k e l s o n , O. 1969. Cycads., i n T o x i c C o n s t i t u e n t s o f P l a n t F o o d s t u f f s , ( Chpt. 6 ), ed., I.E. L i e n e r , Academic P r e s s , New York. 512 Yang , R.S.H., G u t h r i e , F.E. 1969. P h y s i o l o g i c a l r e s p o n s e s o f i n s e c t s t o N i c o t i n e . Ann. Entomol. Soc. Amer. 62: 141-146. 513 Yang, R.S.H., K a r e , M.R. 1968. T a s t e r e s p o n s e o f a b i r d t o c o n s t i t u e n t s o f a r t h r o p o d d e f e n s i v e s e c r e t i o n s . Ann. Entomol. Soc. Amer. 61: 781-782. 514 Yu, M.H., S a l u n k h e , D.K., O l s o n , L.E. 1968. P r o d u c t i o n o f 3 - m e t h y l b u t a n a l from L - l e u c i n e by tomato e x t r a c t s . P l a n t . C e l l . P h y s i o l . , Tokyo 9: 633-638. 515 Y u s t , H.R., S h e l d e n , F.F. 1952. A s t u d y o f t h e p h y s i o -l o g y o f r e s i s t a n c e to h y d r o c y a n i c a c i d i n t h e C a l i f o r n i a r e d s c a l e . Ann. Entomol. Soc. Amer. 45: 220-228. 516 Z l o l t k i n , E., G u r e v i t z , M., S h u l o v , A. 1973. The t o x i c e f f e c t s o f t h e haemolymph o f t e n e b r i o n i d b e e t l e s . J . I n s e c t P h y s i o l . 19: 1057-1065. 245 517 Zenk, M.H., L e i s t n e r , E. 1968. B i o s y n t h e s i s o f q u i n o n e s . L l o y d i a 31: 275-292. ADDENDUM From page 146, p a r a g r a p h 2, i t s h o u l d be n o t e d t h a t t h e p u t a t i v e r o l e o f t h e m e t a p l e u r a l g l a n d s e c r e t i o n s i s f a r from b e i n g e s t a b l i s h e d i n t h e m i l i e u o f t h e c o l o n y . S c h i l d k n e c h t ' s work demonstrates t h e s e compounds a r e s t i m u l a t o r y t o f u n g a l g r o w t h b u t does v a l i d l y p r o v e t h a t t h e s e c h e m i c a l s have a n t i b i o t i c v a l u e a g a i n s t f o r e i g n f u n g i . F o r a n o t h e r v i e w p o i n t o f a n t -fungus s y m b i o s i s one s h o u l d r e f r t o work o f M a r t i n ( c i t e d b e l o w ) where c o n t r a d i c t o r y o b s e r v a t i o n s a r e o b t a i n e d . 518 M a r t i n , M. M., 1970. The b i o c h e m i c a l b a s i s o f t h e f u n g u s - a t t i n e a n t s y m b i o s i s . S c i e n c e 169:.16-20. 519 M a r t i n , M. M. , MacConnelL, J.G., G a l e , G.R. 1969. The c h e m i c a l b a s i s f o r t h e a t t i n e - a n t - f u n g u s s y m b i o s i s . Absence o f a n t i b i o t i c s . Ann. Entomol. Soc. Amer. 62: 286-388. 520 M a r t i n M.M., M a r t i n . J . S . 1970. The b i o c h e m i c a l b a s i s f o r t h e symbiosis betv reen t h e ant. A t t a Colombia  t o n s i p e s , and i t s f o o d fungus. J . I n s e c t P h y s i o l . 16: 109-119. 

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