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Insect antifeedant and growth regulating activity of phytochemicals and extracts from the plant family… Champagne, Donald Edmond 1989

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INSECT ANTIFEEDANT AND GROWTH REGULATING ACTIVITY OF PHYTOCHEMICALS AND EXTRACTS FROM THE PLANT FAMILY MELIACEAE By DONALD EDMOND CHAMPAGNE B.Sc., The University of Ottawa, 1981 M.Sc, The Ottawa-Carleton Center for Graduate Research, 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (BIOLOGY) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1989 © Donald Edmond Champagne, 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of QOTAMY The University of British Columbia Vancouver, Canada Date OCT- /a. (1%*) DE-6 (2/88) Abstract This thesis represents studies on aspects of the defenses against insect herbivores in species of the plant family Meliaceae, particularly with regard to phytochemicals. Methanolic extracts of foliage from thirty species in twenty-two genera were bioassayed for toxicity and growth inhibitory activity against the variegated cutworm, Peridroma saucia f and for feeding inhibition against the migratory grasshopper, Melanoplus sanguinipes. A l l but three species were inhibitory to £. saucia, members of the tribe Melieae being most inhibitory. Members of the subfamily Melioideae were more active than members of the Swieteniodeae. Newly identified species with activity comparable to neem (Azadirachta indica) foliage extracts included Aglaia odorata and Turreae h o l s t i i . Deciduous species produced extracts which were significantly more active than evergreen species, indicating a greater reliance on phytochemical-based defenses. Evidence i s also presented to suggest that the leaves of evergreen species are tougher than deciduous species, and that there i s a negative correlation between leaf toughness factors (physical defenses) and phytochemical-based defenses. These results are in agreement with predictions of the resource avai l a b i l i t y hypothesis. The phytochemistry of Aglaia odorata, A. odoratissima. and A. argentia was examined in detail. Compounds i i i i d e n t i f i e d included the dammaranes, aglaiondiol and a g l a i t r i o l , and the bis-amides (S,S)-odorine, (S,R)-odorine (a new natural product), (S,S)-odorinol, and (S,R)-odorincT. Three dihydroflavanones were i d e n t i f i e d from the Meliaceae for the f i r s t time: 3-hydroxy-5,7,4'-trimethoxyflavanone (a new natural product), 5,7,4'-trimethoxyflavanone, and 5-hydroxy-7,4'-dimethoxyflavanone. A l l compounds were inactive against E« saucia. The i n h i b i t o r y a c t i v i t y of A.. odorata appeared to be due to a compound, t e n t a t i v e l y i d e n t i f i e d as a limonoid, which may be acting i n conjunction with a synergist. This compound i n h i b i t s E- saucia l a r v a l growth i n the absence of antifeedant a c t i v i t y . The toxicology of limonoids, representing the major biosynthetic classes, was examined against E> saucia and the large milkweed bug, Oncopeltus f a s c i a t u s . Cedrelone and anthothecol i n h i b i t e d E« saucia growth by 90%, but not feeding, when applied i n d i e t at 0.5 /imol/g fwt. Cedrelone also i n h i b i t e d O. fasciatus molting, with an MD 5 0 of 12.2 ^g/nymph. In contrast, anthothecol, with an acetoxy function at C - l l , was inactive against O. fasciat u s . The D-seco compound gedunin, and the A,D-seco limonoids obacunone, nomilin, and pedonin were inactive i n these assays; harrisonin i n i t i a l l y i n h i b i t e d feeding by neonate P. saucia but produced no long-term e f f e c t s on growth rate. Bussein i n h i b i t e d growth by 35% but entandrophragmin had no e f f e c t . Azadirachtin was the most t o x i c compound examined i n t h i s study. Peridroma saucia growth ( E C 5 0 =0.4 nmol/g d i e t i v fwt), survivorship ( L C 5 0 =5.2 nmol/g), pupation, pupal weight, and adult emergence were decreased i n a dose-dependent manner. Chemosensory antifeedant a c t i v i t y was implicated i n neonates but was much less marked with t h i r d i n s t a r larvae. Azadirachtin decreased r e l a t i v e growth and consumption rates at doses lower than those a f f e c t i n g n u t r i t i o n a l e f f i c i e n c y , or feeding i n the choice t e s t s . This suggests an action d i r e c t l y on the gut or on the neural regulation of feeding. B i o a c t i v i t y of other limonoids d i d not correlate with measures of s k e l e t a l oxidation or rearrangement, although these are dominant themes i n the evolution of the limonoids. Melanoplus sanauinipes lacked an antifeedant response to azadirachtin, up to concentrations of 500 ppm. However, subsequent molting was markedly effected. Application of azadirachtin o r a l l y , t o p i c a l l y , or by i n j e c t i o n , allowed determination of the r o l e of the gut and integument i n l i m i t i n g the b i o a v a i l a b i l i t y of t h i s compound to putative target s i t e ( s ) within the insect. The ora l MD50, 10.8 ng/g insect fwt, was s i g n i f i c a n t l y higher than the injected MD50, 3.01 pg/g, i n d i c a t i n g a b a r r i e r to b i o a v a i l a b i l i t y i n the gut. The ora l a c t i v i t y of azadirachtin was synergised by coadministration of piperonyl butoxide, i n d i c a t i n g that the b a r r i e r i s due lar g e l y to oxidative metabolism. There was no s i g n i f i c a n t difference between t o p i c a l (3.8 ng/g) and injected a c t i v i t y , i n d i c a t i n g that the integument does not pose a b a r r i e r to b i o a v a i l a b i l i t y . Azadirachtin decreased V growth and consumption at doses which did not a f f e c t n u t r i t i o n a l e f f i c i e n c y , again i n d i c a t i n g an e f f e c t on the gut or neural regulation of feeding. No difference was seen i n n u t r i t i o n a l indices of nymphs treated with azadirachtin at 10 and 15 pq/q, although these doses produced markedly d i f f e r e n t e f f e c t s on molting. This observation suggested that e f f e c t s on endocrine events are not d i r e c t l y related to n u t r i t i o n a l e f f e c t s . The e f f e c t s of azadirachtin treatment were not a l l e v i a t e d by dietary supplementation with ch o l e s t e r o l , and azadirachtin did not a f f e c t the hemolymph transport or metabolism of 1 4 C - B - s i t o s t e r o l , i n d i c a t i n g that s t e r o l metabolism i s not the target f o r azadirachtin a c t i v i t y . Azadirachtin also did not form adducts with cysteine, suggesting that non-specific binding to s u l f h y d r y l - r i c h protein i s also u n l i k e l y as a mechanism of action. v i Table of Contents Page Abstract i i Table of Contents v i Lis t of Tables xi Lis t of Figures x i i i L i s t of Abbreviations xiv Acknowledgements xv Chapter 1: General Introduction 1 Literature Review 2 The Insect Response 10 Coevolution. 12 Evolution of Deterrent Responses 14 Phytochemistry of the Meliaceae 16 Antifeedant and Insecticidal Activity of Azadirachtin.. 26 Regulation of Molting 33 Selection of Test Insects 39 Objectives of the Thesis 42 Chapter 2: Insecticidal and Growth-Reducing Activity of Foliar  Extracts from the Meliaceae Introduction . 44 Materials and Methods 54 Results 61 A) Growth inhibition studies with Peridroma saucia 61 B) Antifeedant studies with Melanoplus sanauinipes 68 v i i C) Bioassays f o r a n t i b i o t i c and phototoxic a c t i v i t y . 71 D Leaf Toughness 71 E- Defensive C h a r a c t e r i s t i c s of Deciduous and Evergreen Meliaceae 72 Discussion 79 A) Crude Extract Screening .. 79 B) Resource A v a i l a b i l i t y Hypothesis 83 Chapter 3: Phytochemical Investigation of Aglaia Species Introduction 89 Materials and Methods 91 A) Sources of Plant Material 91 B) I s o l a t i o n and i d e n t i f i c a t i o n of secondary metabolites i n Aglaia f o l i a g e 91 Bl) Solvent p a r t i t i o n i n g . 91 B2) Normal-Phase Chromatography 92 B3) HPLC 93 C) Qualitative and quantitative analyses 94 D) Bioassay 95 Results 97 A) MeOH Extract Screening 97 B) Solvent P a r t i t i o n i n g 97 C) Chromatography 97 D) Phy tochemi s t r y 106 E) Q u a l i t a t i v e and quantitative comparison of Aglaia species 125 F) Insect Bioassays 141 Discussion 149 v i i i A) Phytochemistry .149 B) I n s e c t i c i d a l A c t i v i t y 152 Chapter 4 : E f f e c t s of Limonoids from the Rutales on  Peridroma saucia and Oncopeltus fasciatus Introduction 156 Materials and Methods 175 A) Sources of Chemicals 175 B) Insects .178 C) Growth Studies 179 D) Feeding Assays 180 E) N u t r i t i o n a l Analyses 179 F) Molt I n h i b i t i o n Assays 182 G) Correlation Between Evolutionary Advancement and A c t i v i t y Against Insects 183 Results 184 A) Growth and Feeding Studies: Limonoids Other than Azadirachtin 184 B) Growth Studies with Azadirachtin 186 C) Feeding Choice Tests with Azadirachtin 190 D) Diet U t i l i z a t i o n Experiments 192 E) Molt I n h i b i t i o n Assays 196 F) Relationship of Anti-insect A c t i v i t y to Oxidation and Skeletal Rearrangement 201 Discussion 205 A) Group 2 Limonoids 205 B) D-seco Limonoids 207 i x C) A,D-seco Limonoids 208 D) B,D-seco Limonoids 210 E) C-seco Limonoids (Azadirachtin). 211 F) N u t r i t i o n a l Indices 213 G) Limonoid Evolution and Structure-Activity Relationships 215 H) Correlation of Phytochemistry with Methanolic Extract Screening 217 I) Comparison of I n s e c t i c i d a l and Cytotoxic A c t i v i t y 219 Chapter 5; E f f e c t s of azadirachtin on the n u t r i t i o n and development of the migratory grasshopper, Melanoplus sanguinipes Fab. Introduction 221 Materials and Methods 223 A) Experimental Insects 223 B) Source of Chemicals. 223 C) Antifeedant A c t i v i t y Assays 224 D) Dietary U t i l i z a t i o n Experiments 224 E) Molt I n h i b i t i o n Assays 225 F) Piperonyl Butoxide Synergism Assay 226 G) Fecundity Experiment 227 H) E f f e c t of Dietary Sterols 228 I) Sterol Transport Experiment 228 J) In v i t r o assay f o r the formation of adducts....229 Results 231 A) Antifeedant assays 231 X B) Growth and Dietary U t i l i z a t i o n 231 C) Molt I n h i b i t i o n Studies... 233 D) Synergism by piperonyl butoxide 238 E) Fecundity Experiment 245 F) Sterol supplementation assays 245 G) In v i t r o assay for adduct formation 250 Discussion 252 A) Antifeedant and n u t r i t i o n a l e f f e c t s . 252 B) Oral, t o p i c a l , and i n j e c t i o n experiments 254 C) Fecundity Experiment 257 D) Sterol studies... ..258 E) In v i t r o formation of adducts 261 F) A g r i c u l t u r a l implications 262 Chapter 6 : General Summary 265 References: 275 Appendix 1: 3/3 Appendix 2: 3/5 x i L i s t of Tables Table 2-1. Sources, c o l l e c t o r s , and c o l l e c t i o n dates of plant material used i n t h i s study.... 55 Table 2-2. Growth i n h i b i t o r y a c t i v i t y of meliaceous leaf extracts on neonate P. saucia 64 Table 2-3. Extraction y i e l d (mg MeOH extract/g leaf dwt), leaf toughness (N/cm2), leaf pubescence (lower surface only) (glab=glabrous, axil=hairs i n a x i l s of main veins, pub= pubescent), and "leaf habit" (deciduous [D] or evergreen [E]) for species of Meliaceae included i n t h i s study 74 Table 2-4. Comparison of MeOH extract t o x i c i t y (as E C 5 0 to Peridroma saucia [% of natural concentration]) and toughness (N/cm2) between deciduous and evergreen species of Meliaceae 78 Table 3-1. Typical r e s u l t s of f l a s h column chromatography (Si g e l , 240-400 mesh) of A., odorata (Et 20 soluble phase) 103 Table 3-2. 1H-NMR spectral data of compounds 3, 4, and 5...110 Table 3-3. Aglaia odorata compounds: chromatographic behavior and colour reactions with Ehr l i c h s reagent and v a n i l l i n reagent 127 Table 3-4. Qualitative TLC analysis of Aglaia samples 128 Table 3-5. HPLC retention times (min) of Aglaia odorata compounds 129 Table 3-6. Concentration (nq/q leaf dwt) of flavanones and bis-amides i n Aglaia species, determined by a n a l y t i c a l HPLC 140 Table 3-7. Aglaia odorata compounds: concentration bioassayed, and resultant p_. saucia growth and survivorship (as % of Control) 143 Table 3-8. E f f e c t of combinations of phytochemicals from Aglaia odorata on the growth of neonate Peridroma saucia (as % of Control) 147 Table 3-9. E f f e c t of Compound 6 on d i e t choice by neonate Peridroma saucia 148 Table 4-1. Eff e c t s of limonoids on insect feeding and growth 159 x i i Table 4-2. E f f e c t of limonoids on growth and d i e t choice of neonate Peridroma saucia ....185 Table 4 - 3 . E f f e c t of azadirachtin on Peridroma saucia pupation and adult emergence 189 Table 4 - 4 . E f f e c t of azadirachtin on d i e t choice by neonate and t h i r d i n s t a r Peridroma saucia 191 Table 4-5. E f f e c t of azadirachtin on t h i r d i n s t a r Peridroma saucia growth and n u t r i t i o n 193 Table 4-6. Comparison of E C 5 0 values of crude extracts of Meliaceae with predictions of a c t i v i t y based on classes of limonoids reported to occur i n the genera examined .218 Table 5-1. E f f e c t of azadirachtin on Melanoplus sanguinipes growth and n u t r i t i o n 232 Table 5-2. Radioladelled s t e r o l composition of control and azadirachtin-treated Melanoplus sanguinipes fed 4 - 1 4 C - 8 - s i t o s t e r o l 251 x i i i Figure 1-1. Figure 1-2, Figure 1-3. Figure 1-4, Figure 2-1, Figure 2-2, Li s t of Figures Biosynthetic pathway leading to the formation of an apo-euphol type limonoid (modified from Siddiqui e_t al., 1988) 19 Biosynthetic pathway leading to the formation of a C-seco limonoid (modified from Siddiqui et a l . , 1988) 21 Biosynthetic pathway leading to the formation of a C-seco limonoid, according to Jones et a l . (1988) 23 Relationship of neurosecretory and neurohemal organs involved i n the endocrine regulation of molting i n insects 53 Graphical depiction of the assumed r e l a t i v e cost of maintaining a chemical or ph y s i c a l l y -based defense against herbivores. 52 Growth (as % of control) of neonate Peridroma  saucia fed a r t i f i c i a l d i e t treated with a MeOH extract of fo l i a g e of Azadirachta i n d i c a , Melia toosenden, or Melia azedirach at 1, 2, or 3% of natural concentration 62 Figure 2-3, Figure 2-4, Figure 3-1. Figure 3-2. Figure 3-3. Figure 3-4, Consumption of g l a s s - f i b r e d i s c s , treated with 10% aqueous sucrose and MeOH extracts of Azadirachta i n d i c a , Melia azedirach, Turreae  h o l s t i i , and Aglaia odorata at 1, 2.5, and 5 times natural concentration (on a wt/wt b a s i s ) , by f i f t h i n s t a r nymphs of Melanoplus  sanguinipes 69 Relationship between leaf toughness (i n N/cm2) and b i o a c t i v i t y of the MeOH extract of f o l i a g e , calculated as 100-EC 5 0 • 76 Ef f e c t of f o l i a r MeOH extracts of Aglaia odorata, A., odoratissima, and A., argentia on the growth of neonate Peridroma saucia 98 Growth and survivorship of neonate Peridroma  saucia fed a r t i f i c i a l d i e t containing solvent extracts of Aglaia odorata (Hawaiian sample)...100 Preparative HPLC chromatogram of a growth i n h i b i t o r y f r a c t i o n from Aglaia odorata 104 Structures of dammarane triterpenes i s o l a t e d from Hawaiian samples of Aglaia odorata 108 xiv Figure 3-5. Structures of flavanones i s o l a t e d from Hawaiian samples of Aglaia odorata 113 Figure 3 - 6 . Mass spectrum of 3-hydroxy-5,7,4' -trimethoxyf lavanone 115 Figure 3-7. Mass spectrum fragments from 3'-hydroxy-5,7,4'-trimethoxy dihydroflavanone 117 Figure 3 - 8 . Structures of bis-amides i s o l a t e d from Hawaiian samples of Aglaia odorata 121 Figure 3 - 9 . HPLC trace of E t 2 0 soluble f r a c t i o n from Aglaia odorata (Hawaiian sample) 130 Figure 3-10 HPLC trace of E t o O soluble f r a c t i o n from Aglaia odorata (Thailand sample) 132 Figure 3-11. HPLC trace of E t 2 0 soluble f r a c t i o n from Aglaia odorata (Taiwan sample) 134 Figure 3-12. HPLC trace of the E t 2 0 soluble f r a c t i o n from Aglaia odoratissima 136 Figure 3-13. HPLC trace of the E t 2 0 soluble f r a c t i o n from Aglaia argentia 138 Figure 3-14. E f f e c t of Compound 6 on the growth and survivorship of neonate Peridroma saucia 144 Figure 4-1. Structures of limonoids included i n Table 4-1..168 Figure 4-2. Major biosynthetic routes of limonoids i n the Meliaceae 173 Figure 4 - 3 . Structures of limonoids examined i n t h i s study.176 Figure 4 - 4 . E f f e c t of dietary azadirachtin on growth and survivorship of Peridroma saucia neonates 187 Figure 4-5. Plot of RGR against RCR for larvae of Peridroma  saucia fed d i e t containing various concentrations of azadirachtin 194 Figure 4 - 6 . E f f e c t of cedrelone on molting success i n Oncopeltus fasciatus 196 Figure 4-7. E f f e c t of azadirachtin on molting success i n Oncopeltus fasciatus 199 Figure 4 - 8 . Comparison of insect growth i n h i b i t i n g a c t i v i t y of limonoids with measurements of oxidation and sk e l e t a l rearrangement 203 X V Figure 5-1. Morphogenic effects of orally administered azadirachtin on fifth-instar nymphs of Melanoplus sanguinipes 234 Figure 5-2. Effect of orally administered azadirachtin on molting success of fifth-instar nymphs of Melanoplus sanguinipes 236 Figure 5-3. Effect of injected azadirachtin on molting success of f i f t h instar nymphs of Melanoplus  sanguinipes 239 Figure 5-4. Effect of topically applied azadirachtin on molting success of fifth-in s t a r nymphs of Melanoplus sanguinipes 241 Figure 5-5. Effect of co-administered piperonyl butoxide (PBO) on the molt inhibitory activity of orally administered azadirachtin 243 Figure 5-6. Effect of orally administered azadirachtin on Melanoplus sanguinipes fecundity 246 Figure 5-7. Pharmacokinetics of radiolabelled sterols in the hemolymph of control and azadirachtin treated nymphs 248 L i s t of Abbreviations ACHN: A c e t o n i t r i l e . AD: Approximate D i g e s t a b i l i t y . ANOVA: Analysis of variance. CH 2 C1 2 : Dichloromethane. CHCI3: Chloroform. E C 5 0 : E f f e c t i v e concentration for 50% e f f e c t ( i e growth i n h i b i t i o n ) . ECD: E f f i c i e n c y of conversion of digested food. ECI: E f f i c i e n c y of conversion of ingested food. ED 5 0: E f f e c t i v e dose f o r 50% e f f e c t (ie growth i n h i b i t i o n ) . E t 2 0 : Ethyl ether. EtOAc: Ethyl acetate. EtOH: Ethanol (ethyl a l c o h o l ) . HPLC: High pressure l i q u i d chromatography. IGR: Insect growth regulator. JH: Juvenile hormone. L C 5 0 : Lethal concentration for 50% of treated population. LD 5 0: Lethal dose f o r 50% of treated population. MC 5 0: Concentration which i n h i b i t s molting i n 50% of the treated insects. MD50: Dose which i n h i b i t s molting i n 50% of the treated insects. MeOH: Methanol (methyl alcohol). 1H-NMR: Proton nuclear magnetic resonance spectroscopy. P.E.: Petroleum ether, b.p. . PTTH: Prothoracicotrophic hormone. Relative consumption rate Relative growth rate. Thin layer chromatography x v i i i Acknowledgements F i r s t l y I thank my wife, Christy, for a l l her love, support, and patience. This thesis, and a l l that I do, i s dedicated to her. I thank my supervisor, Dr. G.H.N. Towers, f o r his support, encouragement, and enthusiasm. Dr. M.B. Isman has also been a de facto supervisor of t h i s project, and gave generously of his time and knowledge, not to mention support and lab space. Without his assistance t h i s thesis would not have been possible. I would also l i k e to thank my committee members, Drs. I.E.P. Taylor and M. Shaw, for a l l t h e i r advice. Numerous people have contributed materially to the research reported here, by providing plant material or pure compounds for bioassay. In p a r t i c u l a r I would l i k e to thank Dr. Kelsey Downum and Lee Swain, of F l o r i d a International University, Miami, for t h e i r generous assistance and h o s p i t a l i t y . Plant material was provided by: Tim Flynn, P a c i f i c Tropical Gardens, Maui; Dr. S. Dossagi, Kenyan National Museum, Nairobi; Dr. Zhun Jun, Kunming In s t i t u t e of Botany, China; Dr. G.B.S. Straley, U.B.C.; Dr. J.T. Arnason, University of Ottawa; and Kanti Patel, U.B.C. Dr. J.C. Maxwell assisted Dr. Towers i n the f i e l d i n Thailand. Samples of limonoids were provided by: Dr. A. Hassanali, I.C.I.P.E., Nairobi; Dr. I. Kubo, Berkley; Dr. J.T. Arnason, University of Ottawa; and Dr. J . Kaminski, University of xix Ottawa. Dr, Hector Barrisos-Lopez synthesized the odorine isomers which allowed confident i d e n t i f i c a t i o n of the plant consituents. Felipe Balza provided invaluble assistance with the i d e n t i f i c a t i o n of the flavanones. Dr. B. Bhom generously allowed me access to his computerized l i b r a r y of the flavonoid l i t e r a t u r e . F i n a l l y , I wish to acknowledge the contribution of my fellow graduate students, p a r t i c u l a r l y Paul Spencer, Murray Webb, C r i s Guppy, Greg Salloom, Sue Dreier, Shona E l l i s , Cathy McDougall, and many others, for much pleasant conversation, coffee, and p a r t i c u l a r l y for helping me keep a sense of perspective and humour about the l a s t few years. 1 Chapter 1: General Introduction Interactions between phytophagous insects and t h e i r host plants constitute perhaps the largest class of i n t e r s p e c i f i c interactions i n the t e r r e s t r i a l biosphere. Study of the factors which serve to regulate these interactions has become one of the most dynamic f i e l d s i n ecology. Many of the interactions are of d i r e c t relevance to Man, e s p e c i a l l y when the plants involved are of economic importance. Chemical ecology (the study of biochemically mediated interactions between species) therefore has two facets: advances i n theory may y i e l d novel pest management strategies (Raffa, 1986), and the study of applied a g r i c u l t u r a l problems may increase our understanding of problems ranging from the structure of communities to the nature of evolutionary processes. This thesis reports a serie s of investigations of the e f f e c t of phytochemicals from members of the t r o p i c a l plant family Meliaceae on herbivorous insects. This family was chosen f o r investi g a t i o n because, although some species such as the neem tree, Azadirachta i n d i c a , are known to produce i n s e c t i c i d a l phytochemicals, most members of the family have not yet been examined f o r t h i s property. The phytochemistry of the family i s f a i r l y well known, which provides a convenient basis f o r such studies. F i n a l l y , the toxicology and mode of action of several known i n s e c t i c i d a l compounds requires c l a i r i f i c a t i o n . 2 Li t e r a t u r e Review The potential impact of insect herbivory on plant populations i s well i l l u s t r a t e d by examples from the b i o l o g i c a l control of weeds (Crawley, 1989; Krischik and Denno, 1983). For instance, klamath weed (Hypericum  perforatum) (Hypericaceae) populations i n western North America were reduced by over 95% following the introduction of the beetle Chrysolina quadrigemina (Chrysomelidae) (Holloway, 1964). At present klamath weed i s l a r g e l y confined to shaded habitats where C_. quadrigemina prefers not to o v i p o s i t . Opuntia s t r i c t a (Cactaceae) was introduced into A u s t r a l i a i n 1839. By 1920 t h i s cactus had spread to cover 24 X 10 6 ha, about half of which was infested by stands so dense as to be impenetrable (Holloway, 1964). The Opuntia-feedina moth Cactoblastis cactorum was introduced i n 1925, and by 1930 most areas of Opuntia had been k i l l e d . Opuntia i s presently r e s t r i c t e d to small i s o l a t e d populations. Insect exclusion studies, using i n s e c t i c i d e s or exclusion cages, provide the strongest evidence that insect herbivory can a f f e c t the structure of plant communities (Crawley, 1989). Despite potential methodological problems, including phytotoxic or stimulatory e f f e c t s of i n s e c t i c i d e s and herbivore rebound i n the absence of natural enemies, over half of the studies indicate changes i n species composition when herbivorous insects are excluded from plant communities (Crawley, 1989). In some cases, even a singl e 3 herbivore species can exclude a plant from pot e n t i a l habitats. When caged and uncaged Machaeranthera canescans (Asteraceae) were transplanted to areas populated by the grasshopper Hesperotettix v i r i d i s , the uncaged plants were completely d e f o l i a t e d a f t e r an average of 7.4 days, but 77% of the caged plants survived to flowering (Parker and Root, 1981). Despite t h i s p o t e n t i a l impact on plants, the t e r r e s t r i a l environment remains green. Indeed, the e f f e c t s of herbivory are r a r e l y evident i n the f i e l d , and plant population dynamics have generally been thought to be regulated by resource l i m i t a t i o n s ( i . e . Slobodkin et, a l . , 1967) or competition (Hairston et a l . , 1960). According to t h i s view insect populations are regulated at low densities by natural enemies, p a r t i c u l a r l y predators, parasites, and diseases, and so cannot have a major impact on plant populations (Hassell and Anderson, 1984; Strong et, a l . , 1984; Bernays and Graham, 1988). However, i t also has been r e a l i z e d that most plants possess resistance mechanisms that l i m i t both the range of herbivore species which can i n f l i c t damage and the rate at which the damage can occur. Consequently, each plant species i s susceptible to herbivory by only a small percentage of the phytophagous insects to which i t i s exposed. For example, i n a Costa Rican dry f o r e s t each plant species supports an average of four to eight lepidopteran species; the greatest herbivore load supported 4 by any one plant i s 17 out of the 3140 species of c a t e r p i l l a r s known from t h i s habitat (Janzen, 1988). Over 50% of the c a t e r p i l l a r s are monophagous, and v i r t u a l l y a l l of the r e s t feed on less than f i v e related plant species; fewer than 10% of the lepidopteran species can be considered polyphagous. Tabulations from temperate l o c a l i t i e s indicate a s i m i l a r pattern (Strong et a l . , 1984; Thorsteinson, 1960; Scott, 1988). Even acceptable host plants may provide less than optimal substrates for growth. Black cutworms, Aarotis  i p s i l o n . fed corn (Zea mays) r a preferred host plant, grew at only 15% the rate of s i b l i n g s reared on an a r t i f i c i a l d i e t (Reese and F i e l d , 1986). Low-quality food may also increase the effectiveness of natural enemies and maintain herbivore populations at low densities (Lawton and McNeil, 1979). Although differences i n water (Scriber, 1979; Scriber and Slansky, 1981) and nitrogen content (Mattson, 1980; Scriber, 1984) may a f f e c t insects, most plants appear to contain s u f f i c i e n t nutrients to support insect growth (Fraenkel, 1959). Resistance to herbivory i s to a large extent expressed at the stage of host plant s e l e c t i o n and r e s u l t s from chemical and t a c t i l e s t i m u l i received by the insect. Plant architecture, p r o f i l e , colour, and odor are perceived by mobile phytophagous insects and often have a r o l e i n locating p o t e n t i a l host plants ( M i l l e r and S t r i c k l e r , 1984). However, the primary factor governing 5 host plant acceptance or r e j e c t i o n appears to be the p r o f i l e of "secondary metabolites" present i n the plant (Whittaker and Feeny, 1971; Feeny, 1976; Rhoades and Cates, 1976; Bernays and Chapman, 1977, 1978; Rosenthal and Janzen, 1979). The term "secondary metabolite" was f i r s t used by the German chemist A. Kossel i n 1891; i n 1896 he defined t h i s term ( c i t e d i n Mothes, 1980; Schneider, 1988): "The search and description of those atomic complexes, which are the essence of l i f e are the foundation f o r the investigation of the l i f e processes. I propose to c a l l the es s e n t i a l components of the c e l l primary and those that are not found i n a l l the c e l l s that have the capacity to develop, secondary. The decision whether a substance i s a primary or a secondary one i s i n some cases d i f f i c u l t . " The d i v e r s i t y of the secondary metabolites i s astonishing: Swain (1977) estimated that 100,000-400,000 such natural products may e x i s t . Each plant contains from a few to hundreds of these compounds; t y p i c a l l y they vary q u a l i t a t i v e l y and qu a n t i t a t i v e l y between organs of the same i n d i v i d u a l , between tissues of d i f f e r e n t ages, and between indi v i d u a l s and populations within a species (McKey, 1979). Although roles i n "primary" metabolism have been i d e n t i f i e d or postulated f o r a few secondary metabolites (Seigler and Price, 1976), f o r the most part they seem to function as signals mediating i n t e r s p e c i f i c interactions (Swain, 1977). S p e c i f i c terms have been developed to describe the s i g n a l l i n g r o l e of secondary metabolites (Shorey, 1977; Nordlund, 1981). Semiochemicals, chemicals which mediate 6 interactions between organisms, can be divided into pheromones, which mediate i n t r a s p e c i f i c i n teractions, and allelochemics, which mediate i n t e r s p e c i f i c interactions (Nordlund, 1981). Kairomones are compounds which, when released from the emitting organism, benefit the receiving organism. Synomones benefit both the emitter and the receiver, and function i n mutualistic i n t e r a c t i o n s . Allomones are deleterious to the receiving organism, and include repellants and toxins used f o r defense. As implied from the above d e f i n i t i o n s , secondary metabolites may a f f e c t both the behavior and the physiology of phytophagous insects. The range of tox i c (physiological) e f f e c t s of plant allelochemicals i s impressive. Non-protein amino acids may be incorporated into proteins, r e s u l t i n g i n non-functional enzymes or s t r u c t u r a l protein (Rosenthal and B e l l , 1979). Many alkaloids are neurotoxic to vertebrate and insect herbivores (Robinson, 1979), and one, nicotine, found use as an i n s e c t i c i d e (Schmeltz, 1971; Jacobson and Crosby, 1971). Other potent neurotoxins include the pyrethrins (Matsui and Yamamoto, 1971; Mabry and G i l l , 1979), isobutylamides (Jacobson, 1971; Miyakado et a l . , 1989), some a l i p h a t i c acetylenes including c i c u t i t o x i n (Towers and Wat, 1978; Robinson, 1980), and some mono- and diterpenes (Ryan and Byrne, 1988). Sesquiterpene lactones (Mabry and G i l l , 1979; Pieman et a l . , 1979), drimane sesquiterpenes (Ma, 1975; D'Ischia et a l . , 1982; Asakawa et a l . , 1988) and some hydroxamic acids (Niemeyer et a l . , 1982) 7 react with amines or sulfhydryl amino acids to alkylate proteins. Saponins (Applebaum and Birk, 1979) and the s t e r o i d a l a l k a l o i d tomatine (Duffey and Bloem, 1987; Bloem et a l . , 1989) complex with dietary s t e r o l s and reduce t h e i r a v a i l a b i l i t y to the insect. Some compounds require near-UV l i g h t f o r t o x i c i t y : these include the l i n e a r and angular furanocoumarins, B-carboline a l k a l o i d s , and isoquinoline a l k a l o i d s , which photobind to DNA (Berenbaum, 1978, 1983; Berenbaum and Feeny, 1981; Towers and Champagne, 1987), and many a l i p h a t i c and phenyl acetylenes and thiophenes, which disrupt membranes (Downum et a l . , 1984; Champagne et a l . , 1986). Some natural products disrupt hormonal regulation of physiological processes i n insects. Juvenile hormone t i t r e s are reduced by the a l l a t i c i d a l precocenes (Bowers, 1983); other compounds are juvenile hormone analogues and produce non-reproductive supernumerary ins t a r s i n s e n s i t i v e insects (Slama, 1979; Bowers, 1983). The protective r o l e of the phytoecdysones i s controversial (Jones, 1983): while these molting hormone analogues are highly t o x i c when injected into the hemocoel, they may be much less active following o r a l administration, perhaps due to rapid metabolism and excretion (Feyereisen et. a l . , 1976). As w i l l be discussed i n d e t a i l l a t e r , the limonoid azadirachtin disrupts both juvenile hormone and ecdysone t i t r e s , r e s u l t i n g i n molt f a i l u r e or chemosterilization of adults. 8 Many secondary metabolites deter feeding or ov i p o s i t i o n by insects. Detection of such compounds i s accomplished v i a chemoreceptors located on the maxillary palps, labrum, i n the epipharyngeal cavity, and frequently on the t a r s i and ovipositors (Chapman and Blaney, 1979; Chapman, 1982; Hanson, 1983, 1987). Sampling of poten t i a l host plants i s usually accomplished by stereotypic behaviors which maximize contact of the chemoreceptors with the plant surface (Frazier, 1986; Chapman and Bernays, 1989). B u t t e r f l i e s sample pote n t i a l o v i p o s i t i o n s i t e s by r a p i d l y drumming t h e i r t a r s i against or scratching on the leaf surface (Rothschild, 1987; Renwick, 1989). Locusts i n i t i a l l y touch a leaf with t h e i r antennae, then bring the labrum into contact, and ra p i d l y touch the t i p s of the maxillary p a l p i to the leaf (Williams, 1954). F i n a l l y , a t e s t b i t e may be taken, i n i t i a l l y expressing leaf sap without removing t i s s u e . In grasshoppers a s i m i l a r sequence i s seen but the antennae play a lesser r o l e (Mulkern, 1969). Further examples are given by Chapman and Bernays (1989). Contact chemoreceptor morphology and physiology have recently been reviewed (Stadler 1984; Fra z i e r , 1986; Hanson, 1987). In b r i e f , chemoreceptors (sensory hairs or s e n s i l l a styloconica) are characterized by a single a p i c a l pore which admits to a cavi t y f i l l e d with a "sensillum l i q u o r " bathing dendrites. Compounds are detected as they bind to s p e c i f i c receptors or "transducing proteins" (Norris, 1988), e l i c i t i n g an action p o t e n t i a l . C l a s s i c a l l y four types of 9 chemosensory c e l l s have been recognized, based on the substances to which they show the maximum response. These are the sugar, s a l t , water, and anion (or second s a l t ) c e l l s ; however, each c e l l also has other functions which include the detection of feeding deterrents. A l t e r n a t i v e l y to stimulating a deterrent c e l l , some secondary metabolites i n h i b i t the a c t i v i t y of the sugar receptor c e l l (Ma, 1977; M i t c h e l l and S u t c l i f f , 1984). In a few monophagous insects one receptor has become sp e c i a l i z e d f o r the detection of s p e c i f i c secondary metabolites ("sign stimuli") c h a r a c t e r i s t i c of t h e i r host plant. Examples include the glucosinolate receptor i n P i e r i s brassicae (Nielsen et a l . , 1979) and the hypericin receptor i n Chrysolina brunsvicensis (Rees, 1969). Only i n a few cases do secondary metabolites mediate an "all-or-none" response by a c t i v a t i n g a s p e c i f i c neural pathway, termed a " l a b e l l e d l i n e " . D i a b r o t i c i t e beetles are stimulated to feed on any substrate containing cucurbitacins, triterpenes c h a r a c t e r i s t i c of t h e i r normal host plant (Metcalf and Lampman, 1989). The desert locust, S. gregaria, w i l l starve to death rather than consume food treated with azadirachtin at concentrations as low as 4 ng/cm2 (Blaney, 1980). However, i n most cases the insect response i s more l a b i l e , and may be affected by the combination of deterrents and stimulants present and the ph y s i o l o g i c a l state of the insect (Dethier, 1982; M i l l e r and S t r i c k l e r , 1984; Reese and Schmidt, 1986). A plant which i s 10 unacceptable to a satiated insect may well be consumed, a l b e i t i n small meals, by a hungry one. Processing of sensory information appears to occur at the l e v e l of the central nervous system rather than at the peripheral s e n s i l l a , probably v i a a c r o s s - f i b r e patterning (Dethier, 1982). Schoonhoven and Blom (1988) suggested a simple model for neural processing i n P i e r i s brassicae i n which phagostimulant and deterrent signals are simply summed, with each deterrent action p o t e n t i a l equivalent to 2.5 stimulant action p o t e n t i a l s . The Insect Response Exposure to allelochemicals i n host plant tissues can se l e c t f o r resistance mechanisms i n phytophagous insects; t h i s s e l e c t i o n may involve behavioral, p h y s i o l o g i c a l , or biochemical resistance. Ingested phytochemicals may be excreted i n t a c t , presumably due to non-absorption; examples include cocaine i n E t o r i a noyesi feeding on Erythroxylum  coca (Blum et a l . , 1981), cardenolides i n Schistocerca  gregaria (Scudder and Meredeth, 1982), and thiophenes i n Melanoplus sanguinipes (Smirle, Champagne, and Isman, unpublished data). Once a compound penetrates the mucosal membrane of the gut i t may be confronted by a v a r i e t y of physiological or biochemical resistance mechanisms (Brattsten, 1988, 1986, 1979; Ahmad, 1986; Ahmad et a l . . 1986). The major enzyme systems involved are the cytochrome P450-based microsomal mixed-function oxidases (MFOs), 11 recently renamed polysubstrate monooxygenases (PSMOs) (Brattsten, 1988), associated with the smooth endoplasmic reticulum. The MFOs are a family of monooxygenases with overlapping, broad substrate a f f i n i t i e s , whose net e f f e c t i s to oxidize l i p o p h i l i c compounds to more water-soluble forms and thereby f a c i l i t a t e excretion. Other enzyme systems involved i n d e t o x i f i c a t i o n and excretion include the glutathione S transferases (GSTs), which conjugate compounds with an e l e c t r o p h i l i c center to glutathione. Unlike the MFOs, the GSTs are not membrane bound and can attack water-soluble allelochemicals. Hexose transferases conjugate allelochemicals to glucose or other sugars. Although they are known to be involved i n the excretion of i n s e c t i c i d e s , t h e i r importance i n plant-insect interactions has not been extensively studied (Brattsten, 1988). These enzyme systems are present i n high concentrations p a r t i c u l a r l y i n the midgut and f a t body but may also occur i n other tissues including the Malpighian tubules. The MFOs i n p a r t i c u l a r may be r a p i d l y induced by exposure to c e r t a i n secondary metabolites (Terriere, 1984; Yu, 1983, 1986). Resistance may also involve t a r g e t - s i t e i n s e n s i t i v i t y (Berenbaum, 1986; Brattsten, 1988). Examples include the i n s e n s i t i v i t y of N a + / K + ATPases from milkweed bugs and monarch b u t t e r f l i e s to i n a c t i v a t i o n by cardenolides (Vaughn and Jungreis, 1977; Moore and Scudder, 1985). Studies with synthetic i n s e c t i c i d e s suggest that t a r g e t - s i t e i n s e n s i t i v i t y appears a f t e r metabolism-based resistance 12 (Brattsten, 1986; Berenbaum, 1986). I t may occur when se l e c t i o n pressure comes from extremely t o x i c allelochemicals, or when an allelochemical i s sequestered for defensive uses. Coevolution The tendency f o r p a r t i c u l a r herbivores to associate with p a r t i c u l a r host plants has long been noted (Brues, 1924) and was at t r i b u t e d to coevolution by E h r l i c h and Raven (1964). According to t h e i r model, the appearance, by mutation or recombination, of a novel allelochemical i n a plant w i l l r e s u l t i n a decrease i n herbivore pressure, leading to evolutionary r a d i a t i o n . The a v a i l a b i l i t y of competitor-free space w i l l then s e l e c t f o r r e s i s t a n t populations among the herbivore fauna. Once able to e x p l o i t t h i s resource, the herbivores w i l l also undergo evolutionary r a d i a t i o n . Eventually the r e s i s t a n t herbivores w i l l again s e l e c t f o r further novel allelochemicals, which w i l l continue the s e l e c t i o n f o r resistance. This pattern of r e c i p r o c a l evolution may lead to progressively t i g h t e r associations between host plants and t h e i r adapted insect fauna, i n part at l e a s t due to the cost of maintaining metabolic defenses against the en t i r e range of allelochemicals to which a generalist i s p o t e n t i a l l y exposed. However, Neal (1985) was unable to show any metabolic cost associated with the induction of nine-fold higher MFO t i t r e s i n the generalist c a t e r p i l l a r H e l i o t h i s 13 zea, suggesting that other factors are involved i n the evolution of a l i m i t e d host-plant range. Although there i s no doubt that evolution of plant-insect associations has occurred, few examples are known where there i s evidence of the re c i p r o c a l s e l e c t i o n pressures e s s e n t i a l to coevolutionary theory (Janzen, 1980; Thompson, 1 982 ) . Possibly the most convincing case i s the association of a s p e c i a l i z e d insect fauna, p a r t i c u l a r l y p a p i l i o n i d b u t t e r f l i e s , with plants which contain l i n e a r and angular furanocoumarins (Berenbaum, 1978, 1981, 1983 ; Berenbaum and Feeny, 1 981 ) . In t h i s case the plant elaborates not only the to x i c furanocoumarins but also a se r i e s of synergists, the methylenedioxyphenyl compounds m y r i s t i c i n , s a f r o l e , and i s o s a f r o l e (Berenbaum and Neal, 1987; Neal, 1 989 ) . However, the insect mixed-function oxidases are not only r e s i s t a n t to the synergists (Neal and Berenbaum, 1989) but are s p e c i f i c a l l y induced by the furanocoumarins (Cohen et a i . , 1 989 ) . More commonly herbivore s e l e c t i o n pressure r e s u l t s from a diverse fauna, which may also i n t e r a c t as competitors; coevolution i n such a case i s said to be " d i f f u s e " (Fox, 1982, 1988; Fox and Morrow, 1 981 ) . In such a s i t u a t i o n the evolution of t i g h t associations between host plants and t h e i r insect fauna i s less l i k e l y ; rather, any change i n se l e c t i o n pressure due to a change i n any of the partic i p a n t s w i l l a f f e c t a l l members of the association. 14 Evolution of Deterrent Responses According to the p r e v a i l i n g view, the presence of allelochemicals i n a plant can s e l e c t f o r physiological resistance or the a b i l i t y to detect and avoid those allelochemicals (feeding and o v i p o s i t i o n deterrence) (Scriber, 1984; Berenbaum, 1986). I t i s generally thought that, due to the adaptive nature of insect behavior, antifeedants which are not accompanied by t o x i c i t y w i l l be quickly overcome i n evolutionary time. Most or a l l antifeedants, therefore, should be t o x i c i f ingested, or i n v a r i a b l y be associated with toxins. Recently Bernays and Chapman (1987) (also Bernays and Graham, 1988) have challenged t h i s view; they point to several studies i n which non-host plants were shown to support normal l a r v a l growth, to studies which have f a i l e d to f i n d t o x i c i t y associated with antifeedant a c t i v i t y , and to studies of o v i p o s i t i o n behavior of b u t t e r f l i e s i n which plants which were rejected could sustain l a r v a l development. They suggest that factors other than physiological s p e c i a l i z a t i o n and the avoidance of t o x i c i t y may mediate host plant s p e c i a l i z a t i o n ; i n p a r t i c u l a r that g e n e r a l i s t insects are subject to higher rates of predation from generalist predators, and that s p e c i a l i s t herbivores enjoy a reduced predation rate. In support of t h i s concept, Bernays (1988) has shown that generalist herbivores are more l i k e l y to be found and eaten by the generalist wasp Mischocyttarus f l a v i t a r s u s than are s p e c i a l i s t s when both are exposed to the predator on the 15 same host plant. Polyphagous c a t e r p i l l a r s are also more palatable than aposematic or c r y p t i c s p e c i a l i s t s to the generalist predator Iridomyrmex humilis (Bernays and Cornelius, 1989). C r u c i a l to the r e j e c t i o n of plant chemistry as an important primary factor d r i v i n g the evolution of host-plant s p e c i f i c i t y i s the conclusion that most deterrents are "harmless", that i s , that they are not i n themselves t o x i c , nor are they usually associated with toxins (Bernays and Graham, 1988). However, as noted by Schultz (1988), t h i s i s argued l a r g e l y from "absence of evidence" rather than "evidence of absence". The limonoids are a case i n point: due to t h e i r b i t t e r taste to vertebrates, they have been considered to be primarily feeding deterrents. As a r e s u l t , the majority of published assays of these compounds have been designed to detect only antifeedant a c t i v i t y (reviewed i n Chapter 4), creating the impression that t h i s i s the only b i o l o g i c a l a c t i v i t y these compounds possess (Taylor, 1987). Limonoids, i n common with most other known antifeedants, need re-evaluation with bioassays capable of detecting b i o l o g i c a l a c t i v i t y other than feeding i n h i b i t i o n ; t h i s i s one focus of Chapter 4. 16 Phybochemistry of the Meliaceae Phytochemically, the family Meliaceae i s characterized by the oxidized triterpenoids known variously as limonoids, meliacins, or tetranortriterpenoids (Taylor, 1981). Limonoids, named f o r limonin, the f i r s t such structure to be elucidated (Arigoni et a l . , 1960), possess a C 2 2 4,4,8-trimethyl s t e r o i d nucleus with a furan r i n g attached at C-17, and may be extensively oxidized and rearranged (Taylor, 1981, 1983; Connolly, 1983). For the most part, biosynthetic studies with radiotracers are lacking, but known compounds may be arranged i n a reasonable biosynthetic sequence which also derives support from i n v i t r o synthetic studies (Buchanan and Halsal, 1970; Lavie and Levy, 1971; Siddiqui et a l . , 1988). To form the protolimonoids, euphanol (20BH) or t i r u c a l l o l (20crH) derivatives give r i s e to a A 7 derivative such as butyrospermol, which i s subsequently epoxidized at C7-8 (Figure 1-1). Opening of the 7B, 8B-epoxide induces a euphol-apo-euphol (or t i r u c a l l o l - a p o - t i r u c a l l o l ) rearrangement, with formation of a C-7 OH, migration of the C-14 CH 3 to C-8, and formation of a double bond at C-14,15, r e s u l t i n g i n the c h a r a c t e r i s t i c limonoid nucleus. The existence of protolimonoids with a C 8 a l i p h a t i c side chain, including m e l i a n t r i o l , suggests that t h i s rearrangement precedes the c y c l i z a t i o n of the side chain. The furan r i n g i s formed by c y c l i z a t i o n of the C 8 chain to a c y c l i c hemiacetal, followed by the loss of four carbons (hence tet r a n o r t r i t e r p e n o i d ) . 17 Subsequent oxidations and rearrangements give r i s e to a d i v e r s i t y of structures; more than 300 have been described to date (Taylor, 1987). The major biosynthetic pathways have been described by Das et a l . (1984, 1987). The most s t r u c t u r a l l y diverse pathway begins with oxidation of the D r i n g to an epoxylactone (see Figure 4-2). The parent apoeuphol, with a double bond at C-14,15, undergoes a l l y l i c oxidation to a A 1 4 , 1 6-ketone, which i s oxidized to a 14,15-epoxy-16-keto d e r i v a t i v e . B a e y e r - V i l l i g e r oxidation then y i e l d s the epoxylactone. This sequence i s exemplified by the series azadirone-azadiradione-epoxyazadiradione-gedunin, i s o l a t e d from Azadirachta indica (Kraus, 1981; Schwinger et a l . , 1983) (see Figure 4-1). Subsequent Baeye r - V i l l i g e r reactions may lead to oxidation of e i t h e r the A r i n g ( r i n g -A,D seco limonoids) or the B r i n g (ring-B,D seco limonoids). This pathway i s p a r t i c u l a r l y c h a r a c t e r i s t i c of the subfamily Swietenioideae (Taylor, 1981; Das ejfe a i . , 1984). In the subfamily Meliodeae, two additional pathways are also expressed. The A r i n g may be oxidized to a lactone; r a r e l y t h i s may be followed by oxidative opening of the B r i n g . F i n a l l y , the i n t a c t precursor may undergo ring-C opening and rearrangement (Figure 1-2). Radiotracer experiments have established that, contrary to the generally accepted scheme fo r limonoid biosynthesis, the isomers of 3H-euphol, t i r u c a l l o l , and butyrospermol were more e f f i c i e n t l y used than were the A 7-isomers i n the biosynthesis of the ring-C-seco limonoid nimbolide (Ekong et 18 a l . , 1971; Ekong and Ibiyemi, 1985). This suggested that a 8ott9 tx—epoxide gives r i s e to a A 7 ' 9 ( H ) - d i e n e ; the A 7 function leads to the 7B-0H, and the A 9 ^ 1 1 ^ function would activate C-12, leading to oxidative f i s s i o n of the C r i n g . Also (perhaps simultaneously), the 7<x-0H could bind to C-15 of the D r i n g to form a new furan r i n g connecting rings B and D (Siddiqui et a l . , 1988). Further addition of substituents and oxygen bridges gives r i s e to a d i v e r s i t y of highly oxidized compounds including azadirachtin. An a l t e r n a t i v e pathway, based on i n v i t r o chemical transformations, has the C-seco limonoids a r i s i n g from an i n t a c t A 1 4 apo-euphol type limonoid with a carbonyl function at C-12 (Figure 1-3) (Jones et a i . , 1988). The pathway to ring-C seco limonoids i s confined to the t r i b e Melieae, consisting of the two genera Azadirachta and Melia (Das et a i . , 1984). The Meliaceae appear to be remarkable for the paucity of phytochemical constituents other than limonoids; i n t h i s respect they contrast strongly with t h e i r sister-group, the Rutaceae. Known coumarins include only the 6,7-oxygenated compounds aesculetin, scopoletin, isoscopoletin, and scoparon, common to many taxa, and the more unusual ekersenin from Ekebergia seneaalensis and s i d e r i n from Toona  c i l i a t a (Gray, 1983). The only chromone known i s rohitukine, which has a very unusual 8-(3-hydroxy-l-Figure 1-1. Biosynthetic pathway leading to the formation of an apo-euphol type limonoid (modified from Siddiqui et. a l . . 1988). 20 21 Figure 1-2. Biosynthetic pathway leading to the formation of a C-seco limonoid (modified from Siddiqui e t aj,., 1988). 22 23 Figure 1 -3 . Biosynthetic pathway leading to the formation of a C-seco limonoid, according to Jones gt a i . ( 1 988 ) . 2k 25 methylpiperid-4-yl) substituent, i s o l a t e d from Amoora  rohituka (Harmon et a l . , 1979). Amoora i s now considered a junior synonym of Aglaia (Pennington and Styles, 1975). The only furanocoumarin described to date i s bergapten from Toona c i l i a t a seeds (Chatterjee et a l . , 1971). The d i v e r s i t y of alkaloids i s equally l i m i t e d . Xylocarpus granatum produces the angular pyranoquinoline N-methylflindersine and a benzo[c]phenanthride (Chou et a l . , 1977), and Ekeberaia cylindricum produces the simple 3-hydroxylpyridine (Menster, 1983). Aglaia odorata and A.. roxburghiana leaves contain bis-amides of 2-aroinopyrrolidine, odorine and odorinol (Shiengthong et a l . f 1979; Purushothaman gt aJL., 1979). The c l o s e l y r e l a t e d compound p i r i f e r i n e occurs i n f o l i a g e of A., p i r i f e r a (Saifah et a l . , 1988). An u n i d e n t i f i e d Aglaia species contained tiglamide (Johns and Lamberton, 1969). Recently a s e r i e s of cardioactive 1-phenylethylisoquinoline a l k a l o i d s were is o l a t e d from the f o l i a g e of the t r a d i t i o n a l medicinal plant Dysoxylum l e n t i c e l l a r e (Aladesanmi and Ilesanmi, 1987); they co-occur with the m o l l u s c i c i d a l a l k a l o i d l e n t i c e l l a r i n e (Aladesanmi et a l . , 1988). Few flavonoids have been i d e n t i f i e d i n the Meliaceae; most common are glycosides of quercetin and kaempferol, including 3-galactosides, arabinosides, rhamnosides, rutinosides, and glucosides (Harborne, 1983). Azadirachta  indica produces, as well, the rare myricetin-3'-arabinoside. Aglycones include only nimbaflavone (5,7-hydroxy-4'-methoxy-26 8,3'-di-C-prenyl flavanone) from A., indica (Garg and Bhakuni, 1984). Based on the re s u l t s of a preliminary survey of 12 species (W. Crins and D. Champagne, unpublished data), the d i v e r s i t y of flavonoids i n the Meliaceae i s probably under-represented i n the l i t e r a t u r e . Diterpenes include s u g i o l , nimbiol, nimosone, nimbosone, methyl nimbiol, methyl nimbionone, nimbionone, and nimbionol from A., indica bark (Hegenauer, 1969; Ara et a l . , 1988; Siddiqui et a l . , 1988) and eperu-13-en-8B,15-diol from Aphanamixus polystachya (Chandrasekharan and Chakrabortty, 1968). Dysoxylum l e n t i c e l l a r e has recently yielded two diterpenes, phyllocladene and fi-hydroxysandaracopimarene (Aladesanmi et a l . , 1986). Sesquiterpenes, known from Lansium anamalayanum, include (-)-K-gurjunene, (-)-<x-trans-bergamotene, and (-)-<x-bisabolene (Krishnappa and Dev, 1973). Pentacyclic triterpenes include b e t u l i n , b e t u l i n i c acid, katonic and i n d i c i c acids, walsurenol, onoceradienone, and l a n s i c acid (Hegnauer, 1983). Aphanamixus polystachya seeds contain a saponin, stigmastienol diglycoside (Bhatt gt a i . , 1981). Uncharacterized monoterpenes occur i n the leaf glands, and a few species produce 2,6-dimethoxy-benzoquinone (Hegnauer, 1983). Antifeedant and I n s e c t i c i d a l A c t i v i t y of Azadirachtin Leaves and f r u i t s of the neem tree, Azadirachta indica A. Juss., have long been used i n t r a d i t i o n a l medicine and 27 agriculture i n India; Sanskrit writings from 2,000 B.C. describe the preparation of water extracts for use against locust plagues (Pradhan gt a l . , 1962). Pradhan and coworkers (1962) confirmed the locust repellent a c t i v i t y of neem extracts. As well, neem leaves are s t i l l used, mixed with grain or placed i n woolen clothing, to protect against insect damage (Saxena, 1989). Work i n I s r a e l resulted i n the description of the protolimonoid m e l i a n t r i o l as a locust feeding deterrent from f o l i a g e of A. indica and the c l o s e l y related chinaberry, Melia azedirach L. (Lavie et a l . , 1967). A year l a t e r Butterworth and Morgan (1968) i s o l a t e d a mic r o c r y s t a l l i n e compound, which they named azadirachtin, as a potent locust antifeedant from neem seed. The structure of azadirachtin (see Figure 3-3) proved elusive: a p a r t i a l structure (Butterworth and Morgan, 1971; Butterworth gt a l . , 1972), l a t e r completed by Zanno gt a l . (1975), was subsequently revised based on 13C-NMR (Kraus et a l . , 1985) and X-ray d i f f r a c t i o n studies of d e t i g l o y l azadirachtin ( B i l t o n gt a l . , 1985; Broughton gt a i . , 1986). The phytochemistry of the neem tree i s complex. Jones et a l . (1988) l i s t 53 limonoids and two protolimonoids, most of which have been i s o l a t e d from the seed o i l . Some of these are very s i m i l a r to azadirachtin, and indeed have been designated "azadirachtins A-G" by Rembold (1987, 1988). Several neem limonoids are active against insects, although 28 none are as active as azadirachtin i t s e l f (reviewed i n Chapter 4). Azadirachtin completely i n h i b i t s feeding by the desert locust, Schistocerca gregaria, at concentrations as low as 70 ng/1 (= 4 ng/cm2 leaf disc) (Butterworth and Morgan, 1968, 1971). S. gregaria i s remarkably s e n s i t i v e , as i t does not feed on cabbage treated with a 0.001% aqueous soluti o n of neem kernel extract (Pradhan gt a l . , 1962). A ten-fold higher concentration was needed to deter feeding by L. migratoria. Subsequently e i t h e r pure azadirachtin or neem o i l preparations have been shown to i n h i b i t feeding or growth i n nearly two hundred species of phytophagous insects (Warthen, 1979; Jacobson, 1986; Saxena, 1989). The neurophysiology underlying the antifeedant response has been examined by several authors (Blaney, 1980, 1981; Schoonhoven and Jenny, 1977; Simmonds and Blaney, 1983). A receptor other than the sugar c e l l responds to azadirachtin; i n some insects i n t e r a c t i o n occurs between these c e l l s at the peripheral l e v e l , reducing the frequency of action po t e n t i a l s , but i n Mamestra brassicae and Spodoptera exempta no i n t e r a c t i o n occurs and the c o n f l i c t between signals from the deterrent and sugar receptors are resolved i n the central nervous system (Simmonds and Blaney, 1983). Habituation to azadirachtin can occur, as larvae exposed to the compound fo r two days showed a markedly reduced neurophysiological response (Simmonds and Blaney, 1983). 29 Soon a f t e r the discovery of the antifeedant a c t i v i t y of azadirachtin, i t was noted that t h i s compound also delayed or i n h i b i t e d the molting of a var i e t y of insects, r e s u l t i n g i n death or the malformation of adults (Ruscoe, 1972; Steets, 1975; Meisner et a l . , 1976; Ladd et a l . , 1978). (Readers unfamiliar with the endocrine regulation of molting i n insects are referred to the review of t h i s t o p i c given on pp 32- 3<r of t h i s Introduction). Injection of azadirachtin was followed by an immediate rapid decline i n juvenile hormone (JH) t i t r e s i n Locusta miaratoria (Sieber and Rembold, 1983) and G a l l e r i a mellonella (Malczewska et a l . , 1988). The appearance of ecdysone peaks was delayed i n a dose-dependent manner i n Locusta migratoria (Sieber and Rembold, 1983; Mordue et a l . , 1986; Mordue and Evans, 1987), Oncopeltus fasciatus (Redfern et a l . , 1982), Manduca sexta (Schluter et a l . , 1985; Pener g£ a l . , 1988), O s t r i n i a  f u r n a c a l i s (Min-Li and Shin-Foon, 1987), Calliphora v i c i n a (Koolman et a l . , 1988), and G a l l e r i a mellonella (Malczewska et a l . , 1988). In most of these cases ecdysone t i t r e s were also decreased, but i n some ( i . e . Schluter et a l . , 1985; Malczewska et a l . , 1988) the delayed ecdysone peak was ac t u a l l y higher than the controls, and appeared to f a l l more gradually. Azadirachtin also i n h i b i t s oogenesis i n Locusta  migratoria (Rembold and Sieber, 1981), Oncopeltus fasciatus (Dorn et a l . , 1986), and Dysdercus k o e n i g i i (Koul, 1984). Similar e f f e c t s have been produced by exposure to neem 30 extracts i n Epilachna v a r i v e s t i s (Steets and Schmutterer, 1975) and Leptinotarsa decemlineata (Steets, 1976; Schmutterer, 1986). Again t h i s e f f e c t i s related to i n h i b i t i o n of JH production and ovarian ecdysteroid t i t r e s (Rembold and Sieber, 1981). Curiously, l a s t - i n s t a r nymphs treated with s u f f i c i e n t azadirachtin to completely i n h i b i t molting can show development of the ovaries (Dorn et ai., 1986a,b; Shalom et al., 1988). V i t e l l o g e n i n concentrations i n over-age L- migratoria nymphs were 6-7 times the l e v e l s i n control adult females (Shalom g£ ai., 1988). The mechanism by which azadirachtin i n t e r f e r e s with molting and the production of neurohormones remains unclear. The prothoracic glands are not d i r e c t l y affected, as these glands remain able to synthesize ecdysone and respond to prothoracicotrophic hormone (PTTH) i n v i t r o (Koul et al., 1987; Pener et a i - , 1988), although Koolman gt ai. (1988) found i n h i b i t i o n of release but not synthesis of ecdysone i n the brain-ring gland complex of Calliphora v i c i n a . Indeed prothoracic glands of azadirachtin-treated insects remained i n t a c t and able to secrete ecdysone long a f t e r the glands degenerated i n control insects (Pener et a l . , 1988). Brains of azadirachtin-treated Manduca sexta contained as much PTTH as did control animals (Pener et al., 1988), but Subrahmanyam et al- (1989) found that azadirachtin reduced the rate of incorporation and turnover of 3 5 S - l a b e l l e d cysteine i n neurosecretory material. This evidence points 31 to a delay i n , and i n some cases complete i n h i b i t i o n of, the release of PTTH from the corpora cardiaca. That azadirachtin t o x i c i t y involves more than simple i n h i b i t i o n of the release of ecdysone or JH i s indicated by the f a i l u r e of subsequently applied hormones to reverse t o x i c i t y i n Manduca sexta (Schluter gt a i . , 1985). C h i l l i n g induces the production of supernumerary inst a r s i n G a l l e r i a  mellonella larvae; t h i s e f f e c t was abolished by azadirachtin (due to marked i n h i b i t i o n of JH synthesis) and the i n h i b i t i o n was not reversed by application of the JH analogue ZR512 (Malczewska et a l . , 1988). However, azadirachtin-induced i n h i b i t i o n of molting was reversed i n Rhodnius prolixus by o r a l a p p l i c a t i o n of ecdysone or t o p i c a l a p p l i c a t i o n of a JH analogue (Garcia and Rembold, 1984). Azadirachtin does not i n h i b i t binding of ecdysteroids to t h e i r receptors (Koolman gt a i - , 1988), apparently because of a d i f f e r e n t conformation of the A and B rings (Rembold, 1988). In addition to i n h i b i t i o n of molting and reproduction, azadirachtin i n h i b i t s gut p e r i s t a l s i s i n v i t r o (Mordue gt a i . , 1985; Mordue and Evans, 1987). This e f f e c t i s related to a generalized i n h i b i t i o n of proctolin-induced muscle contraction (Mordue and Evans, 1987; Mordue gt a l . , 1989). Mordue has suggested that the observed endocrine e f f e c t s of azadirachtin could be due to disruption of normal gut functioning, which i s involved i n feedback loops regulating the timing of some endocrine events (Nijhout, 1981). To 32 that end she has shown that azadirachtin can i n h i b i t molting even i f applied a f t e r the ecdysteroid peak and at a time when JH t i t r e s should be low. In t h i s case azadirachtin i n h i b i t s the air-swallowing behavior necessary to s p l i t the old c u t i c l e and may i n h i b i t the release of bursicon (Mordue et a l . , 1985). The only other physiological e f f e c t reported to date which can be attr i b u t e d to azadirachtin i s a marked decrease i n the rate of synthesis of RNA and subsequently DNA i n a suspension culture of the p r o t i s t Tetrahymena  thermophila (Fritzsche and CTeffman, 1987). In s p i t e of the confusion surrounding the molecular-mode of action of azadirachtin, t h i s compound and neem preparations have been applied to a wide v a r i e t y of a g r i c u l t u r a l problems (Schmutterer, 1988). Such applications r e l y on neem o i l , cake, or semipurified extracts due to the p r o h i b i t i v e expense of pure azadirachtin (Schmutterer and Hellpap, 1988). Promising a c t i v i t y has been found against pests of vegetables and f r u i t trees (Schmutterer and Hellpap, 1988), r i c e (Saxena, 1989), stored grains (Saxena et a l . , 1988), and ornamental crops (Larew, 1988). Various household pests and disease vectors may also be controlled by neem extracts (Ascher and Meisner, 1988; Rembold et a l . , 1989). A neem o i l preparation, Marigosan 0, has now been registered for use on ornamental crops i n the United States (Larson, 1988). Toxicological t e s t i n g has indicated that azadirachtin i t s e l f i s not t o x i c to mammals, birds, or f i s h (Schmutterer, 33 1988; Jacobson, 1986;, Okpanyi and Ezeukwu, 1981). However, neem o i l has a large range of pharmacological e f f e c t s including a n t i p y r e t i c and d i u r e t i c a c t i v i t y , promoting smooth muscle contraction, and i n h i b i t i o n of fungal skin parasites, scabies, and eczema (Jacobson, 1988). Neem o i l also inactivated the potato-X v i r u s (Singh, 1971), and has spermicidal and a n t i f e r t i l i t y a c t i v i t y i n various mammals (Jacobson, 1988). However, the p r i n c i p l e s responsible f o r these a c t i v i t i e s were nimbin and nimbidin and not azadirachtin. A further favorable aspect of neem i s i t s systemic a c t i v i t y i n a va r i e t y of plants including corn and r i c e (Saxena et a l . , 1983). Azadirachtin apparently translocates into and protects new fo l i a g e or f r u i t s which appear a f t e r the application of the neem extract. Regulation of Molting To f a c i l i t a t e discussion of the mode of action of azadirachtin, a review of the endocrine regulation of molting i s given here. Figure 1-4 i l l u s t r a t e s the rel a t i o n s h i p of the various secretory and neurohaemal organs i n the insect. Molting i s a two-step process: apolysis, the secretion of new c u t i c l e and p a r t i a l resorption of the endocuticle, i s followed by ecdysis, the process of act u a l l y shedding the old c u t i c l e . Apolysis i s regulated by the prohormone B-ecdysone (ecdysterone), produced i n the prothoracic glands i n 34 response to the neuropeptide prothoracicotrophic hormone (PTTH). In Manduca sexta, PTTH i s synthesized i n the pars i n t e r c e r e b r a l i s , each hemisphere having a single prothoracicotropic c e l l located i n the l a t e r a l protocerebrum (Gilbert gt a l (1981). Although the corpus cardiacum (CC) has generally been considered the s i t e of PTTH release (Gilber t and King, 1973; G i l l o t t , 1982), i n Manduca the prothoracicotropes are connected v i a axons to the corpus allatum (CA) and t h i s i s the s i t e of PTTH release to the hemolymph. Two forms of PTTH, termed "big" and "small", with molecular weights of about 22,000 and 7,000 respectively, are present (Bollenbacher g t a l . , 1984). Corpora Cardiacum Figure 1-4. Neuroendocrine structures discussed i n the text. Modified from G i l l o t t , 1980. 36 The factors which t r i g g e r PTTH release are poorly understood but are related to i n t e r n a l measurements of factors correlated with feeding, s i z e , and photoperiod (Nijhout, 1981). In Rhodnius prolixus molting follows feeding a f t e r a c h a r a c t e r i s t i c period of time. N u t r i t i o n a l factors are not involved as a series of small blood meals do not provoke molting but a single large meal (>100 mg) does (Wigglesworth, 1934). Wigglesworth suggested that nervous impulses from abdominal str e t c h receptors were required, and showed that severing the ventral nerve cord between the head and the thorax prevented the response to a blood meal. Beckel and Friend (1964) subsequently showed that a non-n u t r i t i v e s a l i n e meal could i n i t i a t e molting. A s i m i l a r system i n i t i a t e s molting i n Oncopeltus fasciatus r as molting may be triggered by i n f l a t i n g the abdomen with s a l i n e or a i r (Nijhout, 1979). The required degree of stretching i s associated with a sharply defined c r i t i c a l weight, which i s usually attained within the f i r s t 24 h of the i n s t a r (Nijhout, 1979; Blakley and Goodner, 1978). The c r i t i c a l weight depends on some factor or structure whose dimensions are determined at the previous molt, as there are p o s i t i v e l i n e a r c o r relations between the c r i t i c a l weight and dimensions of s c l e r o t i z e d structures such as femur length. In Manduca sexta the release of PTTH i s regulated by both a c r i t i c a l weight (5 g i n the f i n a l instar) (Nijhout and Williams, 1974) and a photoperiod-controlled "gating" (Truman, 1972; Truman and Riddiford, 1974; Riddiford and 37 C u r t i s , 1978). In the 5th i n s t a r the gate opens shortly a f t e r l i g h t s - o f f and closes at the beginning of the next l i g h t phase. Larvae must a t t a i n c r i t i c a l weight at l e a s t 24 h before a p a r t i c u l a r gate closes; otherwise that gate i s bypassed and PTTH i s not secreted u n t i l the subsequent gate (Nijhout, 1981). A physiological event associated with the attainment of c r i t i c a l weight i s the cessation of juvenile hormone (JH) synthesis: the 24 h latent period represents the time necessary to c l e a r JH from the hemolymph. In the l a s t i n s t a r the presence of JH i s s u f f i c i e n t to i n h i b i t PTTH secretion, but t h i s mechanism does not operate during e a r l i e r l a r v a l - l a r v a l molts. In the s a t u r n i i d moth Samia  cynthia, photoperiodic control of molting i s regulated by an endogenous circadian clock located i n the prothoracic glands (Mizoguchi and I s h i z a k i , 1982). In the prothoracic glands the PTTH i s thought to bind to c e l l - s u r f a c e receptors associated with a C a 2 + channel, leading to an i n f l u x of e x t r a c e l l u l a r C a 2 + (Smith and G i l b e r t , 1986). Subsequent stimulation of a C a 2 + s e n s i t i v e adenylate cyclase r e s u l t s i n an increase i n i n t r a c e l l u l a r cAMP; calmodulin may mediate t h i s stimulation as Ca 2 +/calmodulin s e n s i t i v e adenylate cyclases have been i d e n t i f i e d i n other insect tissues (Combest et a l . , 1985). Ad d i t i o n a l l y , some receptors are associated d i r e c t l y with the adenylate cyclase. The cAMP may then activate a cAMP-dependent protein kinase responsible f o r phosphorylating a r a t e - l i m i t i n g enzyme(s) involved i n ecdysone synthesis. 38 Although PTTH has been considered the p r i n c i p a l hormone regulating ecdysone production, recent evidence suggests that other factors may modulate prothoracic gland a c t i v i t y . In Manduca sexta JH was shown to have a prothoracicotropic action on ecdysone synthesis (Gruetzmacher gt a_l., 1984a). Subsequently, t h i s was shown to be an i n d i r e c t e f f e c t : JH stimulates the production of a 30 kD hemolymph factor from the f a t body (Gruetzmacher et a l . , 1984b; Watson et a l . , 1985, 1988). Stimulation of ecdysone synthesis occurred even i n the presence of saturating t i t r e s of PTTH. The factor may be a hemolymph c a r r i e r protein which transports a s t e r o l substrate used by the glands i n the production of ecdysone. In Bombyx mori t h i s function may be f u l f i l l e d by high molecular weight lipoproteins (>200 kD) which transport cholesterol to the prothoracic glands (Chino et a l . , 1974). In H e l i o t h i s zea, diapause i s terminated by an increase i n ecdysone synthesis, mediated by a temperature-dependent humoral factor (Meola and Gray, 1984). In the Lepidoptera, ecdysis i s triggered by the release of eclosion hormone (EC) i n response to f a l l i n g ecdysteroid t i t e r s (Truman, 1981; Reynolds and Truman, 1983). EC i s apparently released from the ventral ganglion at l a r v a l -l a r v a l and larval-pupal molts, and from the CC at adult eclosion. Injection of exogenous ecdysterone i n h i b i t s both the release of EC and s e n s i t i v i t y to exogenously applied EC, and so r e s u l t s i n a dose-dependent delay of eclosion. In larvae, high doses of ecdysterone can permanently i n h i b i t 39 ecdysis. In adults, where ecdysis i s gated by photoperiod, eclosion may be delayed u n t i l a gate several days subsequent to the ecdysterone dose. EC release t r i g g e r s a ser i e s of stereotyped behaviors which serve to release the insect from the old c u t i c l e , including swallowing a i r to expand the body and rhythmic p e r i s t a l t i c muscular contractions. These actions apparently r e s u l t from EC binding to receptors located on each of the abdominal ganglia. The presence of EC i n non-lepidopteran insects has not been unequivocally demonstrated. However, locusts and other insects also have stereotyped behaviors associated with ecdysis, and the onset of these behaviors can be delayed by exogenous ecdysterone i n Locusta (Rembold, 198 ). Truman et a l . (1981) found that extracts from the nervous system of insects from f i v e orders (other than Lepidoptera) i n i t i a t e d ecdysis when applied to Manduca pupae, suggesting the presence of an EC-like hormone. Selection of Test Insects Factors to be considered i n the s e l e c t i o n of an appropriate insect species f o r bioassay have been discussed by Berenbaum (1986). If the goal of a study i s the development of a control strategy f o r a p a r t i c u l a r t e s t , the pest nat u r a l l y becomes the bioassay target. I f questions of an evolutionary or eco l o g i c a l nature are addressed, the choice becomes more r e s t r i c t i v e . Oligophagous and monophagous insects usually require s p e c i f i c "sign s t i m u l i " , secondary 40 metabolites c h a r a c t e r i s t i c of t h e i r host plants, before feeding w i l l be i n i t i a t e d (Dethier, 1941). Such insects are also usually highly s e n s i t i v e to the presence of secondary metabolites foreign to t h e i r normal food plants, and so tend to show an exaggerated antifeedant response. Further, these insects appear to elaborate a l i m i t e d range of MFO's, and are possibly more susceptible to i n t o x i c a t i o n by allelochemicals found i n non-host plants (Kreiger et a l . , 1971). A more conservative bioassay i s provided by polyphagous insects: host-plant choice i n these insects i s constrained by the presence of antifeedants rather than the absence of phagostimulants (Bernays, 1983) and, as they elaborate a wider range of MFOs (Kreiger et a i . , 1971), they are less l i k e l y to show an exaggerated response to the i n t r i n s i c t o x i c i t y of a given allelochemical. Most of the bioassays reported i n t h i s thesis involve the variegated cutworm, Peridroma saucia (Hubner). This i s a highly polyphagous species whose known host-plant range includes species from over twenty plant families (Appendix 1); some of the species attacked, including wild onion, are generally considered quite t o x i c to insects (Bierne, 1971). Both herbs and deciduous and coniferous trees are included; P. saucia generally attacks the f o l i a g e but may also consume f r u i t s (Bierne, 1971). The only species on i t s host plant l i s t known to contain limonoids are some species of C i t r u s , but as these are recent introductions to North America i t seems u n l i k e l y that exposure to these compounds could have 41 influenced the evolution of deterrent or physiological responses i n E- saucia. As E . saucia i s a constant background pest which occasionally outbreaks to major pest status (Bierne, 1971), i t s use i s j u s t i f i e d to develop novel pest-control strategies relevant to Canadian a g r i c u l t u r e . Although E- saucia does not o r d i n a r i l y encounter limonoids, i t s use as an evolutionary or e c o l o g i c a l model may also be j u s t i f i e d , as i t s response to these compounds may be taken as representative of unadapted, polyphagous herbivores encountering limonoid-containing plants f o r the f i r s t time. Such unadapted herbivores are generally assumed to be the primary target of allelochemical-based defenses (Feeny, 1976; Rhoades and Cates, 1976; Coley et a l . , 1985). Similar arguments may be made for the use of the migratory grasshopper, Melanoplus sanguinipes. This polyphagous insect i s considered the fourth most serious pest i n Canadian agricu l t u r e (Bierne, 1971). Further advantages to the use of t h i s insect are discussed i n Chapter 5. In b r i e f , the lack of an antifeedant response to azadirachtin makes t h i s a good model insect i n which to study the physiological e f f e c t s of that compound. In some assays, I used the milkweed bug Oncopeltus  fas c i a t u s . This species was used as a model to compare the r e l a t i v e m o l t - i n h i b i t i n g a c t i v i t y of several limonoids, as i t i s known to be s e n s i t i v e to t o p i c a l l y applied IGR compounds, including azadirachtin (Dorn, 1983). 42 Objectives of the Thesis The objectives of the work reported i n t h i s thesis were to examine aspects of the e f f i c a c y and mode of action of phytochemicals from species of the plant family Meliaceae for the control of phytophagous insects. In the f i r s t i n v e s tigation, f o l i a r extracts from t h i r t y species of Meliaceae were screened f o r growth-inhibiting a c t i v i t y and t o x i c i t y against the variegated cutworm, Peridroma saucia. In addition, r e s u l t s of the screening assays and measurements of leaf toughness from f i f t e e n species were used to assess aspects of the resource a v a i l a b i l i t y hypothesis of Coley e£ a l (1985). As extracts of Aglaia  odorata were highly active i n the crude extract screening, the phytochemistry of three species of the genus Aglaia were examined, and are reported i n Chapter 3. In Chapter 4, I describe the comparison of ten limonoids, representing the major pathways of limonoid biosynthesis, f o r i n h i b i t i o n of feeding and growth i n p_. saucia f and f o r i n h i b i t i o n of molting and reproduction i n the milkweed bug, Oncopeltus fascia t u s . The toxicology of one of these compounds, azadirachtin, was examined i n d e t a i l i n these species. Results are discussed i n terms of mechanism of action, structure/function r e l a t i o n s h i p s , and the s i g n i f i c a n c e of i n s e c t i c i d a l a c t i v i t y i n the evolution of limonoids. In Chapter 5 I reexamined the reported resistance of the migratory grasshopper, Melanoplus  sanguinipes. to azadirachtin. I found that t h i s compound 43 lacked antifeedant a c t i v i t y against M.. sanguinipes. but, once ingested, i t produced a va r i e t y of physiological e f f e c t s . The toxicology of azadirachtin and factors involved i n i t s b i o a v a i l a b i l i t y i n t h i s insect were examined i n d e t a i l . F i n a l l y , I proposed and tested two hypotheses as to the mode of action of azadirachtin. 44 Chapter 2: I n s e c t i c i d a l and Growth-Reducing A c t i v i t y of F o l i a r Extracts from the Meliaceae Introduction P r i o r to the advent of synthetic i n s e c t i c i d e s , pest-control strategies r e l i e d l a r g e l y on plant-derived extracts and preparations (Jacobson and Crosby, 1971). In ce r t a i n regions, members of the Meliaceae figured prominently i n t h i s respect. In p a r t i c u l a r , the neem tree, Azadirachta  indica A. Juss., has long been noted f o r i t s effectiveness i n protecting crops, clothing, and stored grains from attack by insects. Sanskrit writings over 2,000 years o ld d e t a i l proceedures f o r preparing water extracts of the f o l i a g e to protect grain f i e l d s from locusts (Radwanski, 1977). A s i m i l a r ancient h i s t o r y of use pertains to Melia azedirach i n the Middle East (Lavie gt a l . , 1967). Subsequent work has confirmed the potent antifeedant and i n s e c t i c i d a l a c t i v i t y of these preparations and has led to the i s o l a t i o n of a var i e t y of t r i t e r p e n o i d constituents, exclusively of the limonoid or protolimonoid c l a s s , as the active p r i n c i p l e s (reviewed i n Chapter 4 of t h i s Thesis). The use of plant extracts declined as inexpensive synthetic i n s e c t i c i d e s became available f o r t y years ago (Jacobson and Crosby, 1971). However, intensive use of synthetic compounds has resulted i n numerous environmental problems including impact on non-target organisms (including 45 humans), contamination of water and s o i l with persistant residues ( Z i t t e r , 1985), and the appearance of resistance i n over 400 species of economically important pest insect species (Luck et a l . , 1977). As well, the cost of synthetic i n s e c t i c i d e s has escalated (Kinoshita, 1985) and such compounds are now less economically a t t r a c t i v e , p a r t i c u l a r l y i n the Third World. As a r e s u l t , i n t e r e s t i n botanical i n s e c t i c i d e s has been rekindled i n recent years (Balandrin et a l . , 1985; Hedin, 1982). Plants may provide useful compounds or extracts d i r e c t l y , or i n s e c t i c i d a l and antifeedant phytochemicals may provide leads f o r the synthesis of new compounds. Given the remarkable i n s e c t i c i d a l a c t i v i t y of extracts of neem and chinaberry, I decided to bioassay methanolic extracts of a diverse assemblage of species of the Meliaceae, with the aim of i d e n t i f y i n g further species of pot e n t i a l i n t e r e s t . To t h i s end a c o l l e c t i o n of f o l i a g e samples of thirty-one species, out of about 500 i n the family (Pennington and Styles, 1975), i n twenty-two genera (out of 51) was assembled and bioassayed against the variegated cutworm, Peridroma saucia. Extracts which showed the strongest a c t i v i t y against E . saucia were also assayed for feeding i n h i b i t i o n against the migratory grasshopper, Melanoplus sanguinipes. A primary goal i n the study of plant-herbivore interactions i s to explain why plants d i f f e r i n t h e i r commitment to defenses and consequently i n t h e i r 46 s u s c e p t a b i l i t y to herbivores (Coley et a l . , 1985). In the f i r s t attempt at such a synthesis, Feeny (1976) and Rhoades and Cates (1976) independently proposed that the p r o b a b i l i t y of discovery by herbivores, termed plant apparency by Feeny, governs the evolution of defensive strategy. According to the hypothesis, apparent plants, which are generally l a t e -successional perennials, are d i s t r i b u t e d predictably i n space and time and so are l i k e l y to be located by herbivores. In such plants herbivore pressure should s e l e c t for "quantitative" defenses e f f e c t i v e against both adapted s p e c i a l i s t and unadapted generalist herbivores. Quantitative defenses (eg. tannins) were thought to a f f e c t feeding rates (via leaf toughness) and nutrient a v a i l a b i l i t y to the insect (via complexing with protein), and so would be impossible for insects to circumvent; however, they were believed to be metabolically expensive to produce i n the large quantities necessary f o r adequate protection. Unapparent plants, unpredictable i n space and time and mostly s h o r t - l i v e d early successional species, were believed to require defenses against generalist herbivores. In such plants " q u a l i t a t i v e " defenses (toxic secondary metabolites) would provide adequate defense. Although herbivores could evolve immunity to such defenses t h i s loss of e f f i c a c y would be balanced against t h e i r low cost of production. Among various d i f f i c u l t i e s with the plant apparency hypothesis (Fox, 1981; Bernays, 1978; Berenbaum, 1983), i t predicts that a l l plants should s u f f e r about equal rates of 47 herbivory, which i s not observed to be the case (Coley, 1983), and i t f a i l s to account for the observed differences i n types and extent of defense amongst perennial apparent plants. More recently these differences i n defense a l l o c a t i o n have been ascribed to the growth rate of plants, which i s c l o s e l y related to the a v a i l a b i l i t y of resources including water, l i g h t and nutrients (Coley et a l . , 1985). Fast-growing species, adapted to resource-rich habitats, replace leaves r e l a t i v e l y r a p i d l y and so are able to t o l e r a t e higher rates of herbivory. In resource-limited habitats, slow-growing species tend to replace leaves slowly, and so must l i m i t the rate of loss to herbivores. A given rate of herbivory w i l l remove a higher proportion of the primary productivity from slow-growing plants than from fa s t e r growing species. The model: dC/dt = G*C*(l-kD a) - (H-mD6) [where dC/dt i s the r e a l i z e d growth rate, G (g g" 1 d - 1 ) i s the maximum inherent growth rate without herbivores, C (g) i s the plant biomass at time 0, D (g g - 1 ) i s the investment i n defense, k (g d - 1 ) and a are constants r e l a t i n g investment i n defense to reduction i n growth, H (g d" 1) i s the p o t e n t i a l herbivore pressure i n a habitat, and m and 8 are constants r e l a t i n g the reduction i n herbivory to the investment i n defense] predicts that i n t r i n s i c a l l y slow-growing plants should invest more of t h e i r primary productivity i n defense than should fast-growing species. 48 Metabolically mobile secondary metabolite based defenses are thought to turn over r e l a t i v e l y r a p i d l y ; to maintain a given l e v e l of defense these defense compounds would have to be synthesized continuously, so the cost of these defenses would increase a r i t h m e t i c a l l y over the l i f e t i m e of the l e a f . However, because these defenses are metabolically mobile they may often be recovered from senescing leaves. On the other hand, quantitative defenses, e s p e c i a l l y l e a f toughness fac t o r s , are emplaced at leaf expansion; although i n i t i a l l y c o s t l y , such defenses are not metabolically mobile and cost l i t t l e to maintain once i n place, but they are l o s t at leaf senescence. Comparison of these defense types suggests that chemically based defenses should be selected for i n species with short leaf l i f e t i m e s (fast-growing plants), and immobile defenses should be favored i n species with long leaf l i f e t i m e s (slow-growing plants) (Figure 2-1). The model has been supported by studies of lowland r a i n f o r e s t species i n Panama (Coley, 1983, 1988), but measurement of investment i n secondary metabolite defenses was confined to assays of polyphenol content, which did not correlate with herbivory, leaf l i f e t i m e , or other leaf a t t r i b u t e s (Coley, 1983). To date, the hypothesis has been tested with plant species co-occurring i n the same habitat; i t i s unclear whether the model can also account f o r evolutionary patterns within c l o s e l y related groups of plants. The Meliaceae o f f e r s some p a r t i c u l a r advantages f o r a t e s t of t h i s hypothesis. Members of the family occur i n 49 habitats ranging from r a i n f o r e s t to mangrove swamp to semidesert (White, 1975). Few families embrace such a wide range of f l o r a l c h a r a c t e r i s t i c s , but the family has been considered to be monophyletic i n a l l taxonomic treatments to date (Pennington and Styles, 1975). Considerable time has been available for the evolution of the d i v e r s i t y observable today, as species c l e a r l y assignable to the modern genus Cedrela are c h a r a c t e r i s t i c of western North American Paleocene f l o r a s (Brown, 1965) and the family probably appeared i n the Cretaceous, at l e a s t 70 m i l l i o n years ago. The family lacks trichomes, thorns, and spines, and so physical defenses are confined to leaf toughness factors and pubescence. As well, despite the formidable chemistry of such species as Azadirachta indica (Siddiqui e_t a i . , 1988), a l l antifeedant or i n s e c t i c i d a l phytochemicals i d e n t i f i e d to date from t h i s family are limonoids; indeed the s c a r c i t y of other classes of secondary metabolites i s remarkable. The evolution of chemical defenses i n t h i s family may therefore be examined by a consideration of the r e l a t i o n s h i p between i n s e c t i c i d a l a c t i v i t y and evolutionary position of members of a single class of phytochemicals. Measurement of investment i n secondary metabolite based defenses has presented considerable d i f f i c u l t y ; previous attempts have la r g e l y centered on colorimetric assays f o r t o t a l phenolics (eg. Coley, 1983, 1988). This approach i s unsatisfactory, as most known i n s e c t i c i d a l or antifeedant compounds would not be detected i n such an assay, and the 50 assumption that quantity of phenolics i s d i r e c t l y r e l a t e d to the cost and e f f i c a c y of chemical defenses i s questionable. As the r o l e of secondary metabolite based defenses has been construed to be deterrence of unadapted generalist herbivores, i n t h i s study I have estimated r e l a t i v e investment i n phytochemical based defenses by the r e l a t i v e response of a highly polyphagous herbivore (Peridroma  saucia) to the ent i r e s u i t e of phytochemicals produced i n a plant, as obtained i n the methanolic extracts of mature f o l i a g e . As E . saucia lacks an evolutionary association with the Meliaceae or with plants containing limonoids, any observed response to the extracts may be construed as representing the response of a naive, unadapted herbivore encountering a potential meliaceous foodplant. This approach avoids the question of cost of production of chemical defenses, and rather focuses attention on the e f f i c a c y of those defenses. Relative leaf toughness was measured d i r e c t l y by determining the amount of force required to punch a 0.5 cm diameter f l a t - t i p p e d rod through a l e a f . Leaf l i f e t i m e s were not measured d i r e c t l y , but as an approximation species have been separated into deciduous and evergreen species. As presently understood, the resource a v a i l a b i l t y hypothesis would predict that species with short leaf l i f e t i m e s (deciduous) should produce leaves that are more t o x i c and 1 l e s s tough than the evergreen leaves of slow growing species. As well, a negative c o r r e l a t i o n between leaf 51 toughness and leaf t o x i c i t y may be expected. Overall, the perennial species may be expected to have a higher commitment to t o t a l defenses against herbivores. 52 Figure 2-1. Graphical depiction of the assumed r e l a t i v e cost of maintaining a chemical or physically-based defense against herbivores. Costs of the chemical defense increase a r i t h m e t i c a l l y over the l i f e of the l e a f , due to turnover. Physical defenses are i n i t i a l l y c o s t l y , during emplacement following leaf expansion; however as they are metabolically inactive (immobile), there i s l i t t l e or no cost associated with t h e i r maintenance once i n place. Consequently, plants with s h o r t - l i v e d leaves should be selected f o r the production of chemically-based defenses, and physical defenses should be favored i n species with long-lived leaves. 30 oH • 1 • 1 • 1 • 1 • 1 0 10 20 30 40 50 Leaf Lifetime 54 Materials and Methods F o l i a r samples of thirty-one species i n twenty genera of the Meliaceae were obtained for t h i s study. Most were c o l l e c t e d from the Gordon F a i r c h i l d Tropical Gardens and the USDA Plant Quarantine Center i n Miami, F l o r i d a , from the P a c i f i c T ropical Garden, Hawaii, or from the Kunming I n s t i t u t e , Kunming, China. Other species were f i e l d c o l l e c t e d i n Kenya, Thailand, Mauritius, and New Zealand. A few species are represented by c o l l e c t i o n s from more than one s i t e . A l l samples were of mature ( f u l l y expanded and greened) leaves c o l l e c t e d approximately i n the middle of the growing season. Sources f o r each species and c o l l e c t i o n dates are given i n Table 2-1. Samples of most species were received already powdered; where possible voucher specimens have been deposited i n the UBC Herbarium. Samples were a i r dried, ground to a fi n e powder i n a Wiley m i l l , weighed, and extracted i n three changes of MeOH, 24 h/change, 11/100 g dry weight (dwt). MeOH extracts were pooled and concentrated under vacuum; f i n a l concentrations were adjusted to 2 mis MeOH/g dwt l e a f . Aliquots of the MeOH extracts were dried and weighed to determine the extraction y i e l d and allow c a l c u l a t i o n of dose-response rela t i o n s h i p s i n terms of mg extract/g d i e t fresh weight (fwt). For bioassay, d i e t s were prepared according to the procedure of Isman and Rodriguez (1983). Aliquots of the 55 Table 2-1. Sources, collectors, and collection dates of plant material used in this study. Plants collected from botanical gardens are listed according to their accession numbers. Numbers beginning with PTBG are from the Pacific Tropical Garden, Maui, those beginning with PI are from the USDA Plant Introduction Quarantine center in Miami, Fla, and FG refers to the Gordon Fairchild Tropical Garden in Miami. Species Source Collector Date Family Meliaceae Subfamily Melioideae Tribe 1. Turreeae Turreae h o l s t i i Gurke Turreae mauritiana Tribe 2. Melieae EslXA azedirach L. (Fla) Melia azedirach L. (Ind) M£iia azedirach L. (Chi) Mfilia tPPSenden Azadirachta indica A.Juss. Azadirachta indica A.Juss. Azadirachta indica A.Juss. Tribe 4. Trichilieae T r j p h i l i a h i r t a L. T r i c h i l i a roka LgpidP t r i c h j i i a volKensii Mt Eldon, Mauritius Kenya (Fla) (Haw) PI073248 India Kunming, China Kunming, China PI137950 PTBG790480001 (Chi) Kunming, China FG PI (Gurke)Leroy Mt.Eldon, Kitale, Kenya PI105699 Trang, Maui Trang, Trang, Kunming R.N.Parker Manila PI Thai. Thai. Thai. EKebergia capensis Sparrm. Cipadessa baccifera (Roth) Miq. Tribe 5. Aglaieae Aglaia odorata Lour. (Thai) Aglaia odorata Lour. (Haw) Aglaia odoratissima Blume Aalaia araentia Blume Aphanamixus arandifolia Blume Aphanamjxus polvstachya (Wall) Lansium domesticum Corr. Tribe 6. Guareeae Guarea glabra Vahl Dyspxylum spectabile Hook. Opua Rec. Forest,N.Z. Tribe 7. Sandoriceae Sandoricum koetiape (Burman f.)Merrill PI Subfamily Swietenioideae Tribe 1. Cedreleae Cedrela odorata L. PI097976 Toona serrata (Royle) Penn.fi Styles PTG Tppna C i l i a t a M.J. Roemer PI lapjia australis (F. von Mueller) PI SD JTA DEC KP SQ SQ DEC TF SQ DEC TF SD SD DEC GHNT TF GHNT GHNT GHNT DEC GBS DEC GBS DEC DEC TF DEC DEC 03-85 06-86 06- 87 07- 85 05-86 05- 86 06- 87 05- 86 06- 87 03-85 03-85 06- 87 05- 85 12-86 07- 85 07- 85 08- 85 06- 87 08-88 06-87 12-87 06-87 06-87 56 Tribe 2. Svietenieae Khaya senegalensis (Desr)A.Juss. PTBG770642003 Chuckrassia tabularis A.Juss. PI Entandrophracrma caudatum (Sprague) Sprague PI Swietenia humilis Zuccarini PI092371 Swietenia mahoqani (L.)Jacquin PI Swietenia macrophylla King PI Tribe 3. Xylocarpeae Carapa auianensis Aubl. EI Collectors: DEC, D.E. Champagne, UBC; GHNT, G.H.N. Towers, UBC; JTA, J.T. Arnason, University of Ottawa; SQ, Song Qui-Si, Kunming Institute; TF, T. Flynn, Pacific Tropical Botanical Garden; GBS, G.B. Straley, UBC; SD, S. Dossaji, National Museums, Kenya. TF DEC 06-87 DEC 06-87 DEC 06= =87 DEC 06-87 DEC 06-87 DEC 06-87 57 extracts were added to the d i e t dry components (Velvetbeen C a t e r p i l l a r Diet, no. 9682, Bioserv Inc., Frenchtown, N.J.), i n i t i a l l y i n amounts calculated to produce concentrations of 25, 50, 75, and 100% of natural leaf concentrations, on a dwt leaf/dwt d i e t basis. I f necessary other concentrations were subsequently evaluated to f a c i l i t a t e determination of the E C 5 0 (concentration required to reduce l a r v a l growth by 50% r e l a t i v e to the co n t r o l s ) . The MeOH c a r r i e r was evaporated i n a fume hood, usually overnight. Controls were s i m i l a r l y treated with MeOH alone. Three neonate Peridroma saucia larvae were placed on 1 g fresh weight (fwt) d i e t i n a 30 ml p l a s t i c Solo cup; 10 cups were used per treatment for a t o t a l n=30. Experiments were r e p l i c a t e d three times. Rearing cups were put i n cl e a r p l a s t i c boxes, floored with moistened paper towels to maintain high humidity, and placed i n a growth cabinet at 27+1° C, 16:8 LD. Survivorship and l i v e l a r v a l weights were determined a f t e r seven days of growth; larvae were not weighed or handled on intervening days to minimize a r t i f i c i a l l y induced growth disturbances (Reese and Schmidt, 1986). Weights were l o g 1 0 transformed to correct f o r heteroscedasticity p r i o r to analysis by least-squares regression using the SAS GLM procedure to determine the EC 5 0 . Mortality values were corrected using Abbott's formula and L C 5 0 values (the concentration required to reduce survivorship by 50% r e l a t i v e to the controls ) were determined using the SAS Probit s t a t i s t i c a l package. 58 F o l i a r extracts were also examined for antifeedant a c t i v i t y against the migratory grasshopper, Melanoplus  sanguinipes. Extracts, s u f f i c i e n t to achieve 100% of natural concentration on a dwt/dwt leaf d i s c basis (20 u l ) , were applied evenly, using a 25 u l Hamilton syringe, to both surfaces of 1.5 cm diameter cabbage (Brassica oleraca cv. S i l v e r Queen) leaf d i s c s . After the extracts had dried, the leaf discs were presented to f i f t h i n s t a r nymphs i n seven cm diameter unwaxed paper cups; the bioassay was no-choice with one leaf disc/nymph. After 24 h uneaten leaf material was dried to constant weight (24 h § 60° C) and weighed; s t a r t i n g leaf weight was determined by drying and weighing samples of i n t a c t leaf d i s c s . Ten r e p l i c a t e s were used for each treatment. Extracts of Azadirachta i n d i c a , Melia azedirach  Aglaia odorata. and Turreae h o l s t i i were subsequently assayed using 1.5 cm diameter glass f i b r e f i l t e r d i s c s , treated with s u f f i c i e n t extract to achieve concentrations 1, 2.5 and 5-fold n a t u r a l l y occurring l e v e l s . After drying the discs were saturated with a 10% aqueous sucrose sol u t i o n and presented to f i f t h i n s t a r nymphs as described above. Leaf toughness was measured d i r e c t l y on some species (those available i n the USDA and F a i r c h i l d c o l l e c t i o n s i n Miami, Fla.) using a leaf "punchmeter" modelled a f t e r the design of Feeny (1970). This device measures the force required to punch a 5mm diameter, flat-ended rod through the l e a f , which i s clamped into place i n the base of the device. 59 Force was applied by adding water from a buiret to a beaker atop the rod; a f t e r penetration the beaker and water were weighed. Weights were converted to Newtons/cm2 according to the formula: F(Newtons) = wgt (kg) x 9.8 N/crn^ = wgt (kg) x 49 0.2 cm 2 A l l measurements were made on fr e s h l y c o l l e c t e d leaves. Care was taken to avoid primary and secondary veins, although t h i s was d i f f i c u l t to ensure i n the case of Azadirachta indica and Melia azadirach due to the close spacing of the secondary veins. Five to ten measurements were made on separate l e a f l e t s for each species. Species were compared using Duncan's multiple range t e s t . The rel a t i o n s h i p between leaf toughness and t o x i c i t y of the extracts, measured as the EC 5 0, was examined using regression analysis. Leaf extracts were also examined fo r the possible presence of a n t i b i o t i c or phototoxic compounds. The l a t t e r p o s s i b i l i t y was examined because of the presence of a d i v e r s i t y of known photosensitizers i n the c l o s e l y related plant family Rutaceae. The method of Daniels (1965) was used: extracts (equivalent to 5 mg leaf tissue) were dried on to s t e r i l e f i l t e r paper di s c s , which were placed on duplicate plates streaked with a lawn of the yeast Saccharomyces cerevisiae. One plate was incubated i n the dark (-UV) at 37° C; the second plate was i r r a d i a t e d by near-UV (a bank of four Black-Light Blue tubes 10 cm above the plates) for 4 h, after which the plates were incubated in the dark as per the -UV treatment. 61 Results A. Growth i n h i b i t i o n studies with Peridroma saucia A l l but three of the extracts tested produced marked i n h i b i t i o n of the growth of E . saucia neonates at concentrations below those occurring n a t u r a l l y i n the leaves. Growth curves f o r larvae fed Azadirachta i n d i c a f  Melia azedirach. and Melia toosenden extracts are shown i n F i g . 2-2 and are t y p i c a l of the more active extracts. E C 5 0 and L C 5 0 values, i n terms of both mg extract/g d i e t dwt and % of natural concentration, are given i n Table 2-2. Extraction e f f i c e n c i e s ( i n mg extract/g dry leaf) f o r a l l species are given i n Table 2-3, togeather with leaf toughness values ( i n N/cm2), leaf pubescence, and l e a f "habit" (deciduous or evergreen). The species which produced the most active extracts, i n terms of both growth i n h i b i t i o n and mortality, were a l l i n the subfamily Melioideae. Within t h i s subfamily, members of the t r i b e Melieae (Azadirachta i n d i c a . Melia azedirach, and Melia toosenden) were a l l highly i n h i b i t o r y towards E* saucia neonates, with E C 5 0 concentrations below 2% of natural concentration (0.59-2.10 mg/g). These species were also t o x i c , with L C 5 0 concentrations of about 5% of natural l e a f concentration. Dead larvae were a l l small and s t i l l i n the f i r s t i n s t a r ; none appeared to have died while molting. Azadirachta indica f o l i a g e samples from Hawaii, India, and 62 Figure 2-2. Growth (as % of Control) of neonate Peridroma  saucia fed a r t i f i c i a l d i e t treated with a MeOH extract of f o l i a g e of Azadirachta indica (Florida and Hawaiian samples), Melia toosenden f or Melia azedirach at 1, 2, or 3% of natural concentration. Each point shows the mean of three r e p l i c a t e s with 30 cohorts/replicate; standard errors were < 6% and are omitted f o r c l a r i t y . Concentration (% of Natural Leaf) \j4 64 Table 2-2. Growth inhibitory activity and toxicity of meliaceous leaf extracts on neonate £. saucia. Values given are the concentration (as % of natural leaf concentration and mg/g diet dwt) of the total MeOH extract administered in a r t i f i c i a l diet required to reduce growth (EC 5 0) or survivorship (LC 5 0) by 50% relative to the control, over a seven day assay. Numbers in a column followed by the same letter are not significantly different, based on overlap of their 95% confidence limits. E C5Q U J lag/a) m LC 50 (ma/cn Family Meliaceae Subfamily Melioideae Tribe 1. Turreeae Turreae holstii 1 Turreae mauritlana 37 Tribe 2. Melieae M£lia azedirach (Florida) 1 Melia azedirach (India) 1 Melia azedirach (China) 0 Melia toosenden 1 Azadirachta indica (Florida) o Azadirachta indica (Hawaii) 0 Azadirachta indica (China) 0 Tribe 4. Trichilieae Trichilia hir£a 12, Trichilia roka 18, Lepidotrichilia volkensii 12. Ekepergia capensis 43. Cipadessa baccifera 44. Tribe 5. Aglaieae Aglaia odorata (Thailand) 1, AoTaia odorata (Hawaii) 2. Aalaia odoratissima 11, Aalaia araentia 27. Aphanamixus grandifolla >100 Aphanamjxus polvstachya 74. Lansium domesticum >100 Tribe 6. Guareeae Guarea glabra 62. Dysoxvlum spectabile >100 Tribe 7. Sandoriceae Sandoricum Koetjape 17. .8' • 3< .0a .2a > 5ab .7a .6a .8a .5C .8C .5° .0e .0e 45.40 n iJ 1.75 2.10 1.58 2.29 cd Cd be cd 0.69a 0.59a 0.89 ab ef 18.88 26.14^9° 102.94* 133.021 cd °d 0 d2.07 3.29<* 4.27d 11.88e >63.1 77. 7 k >153.6 53.323 57.0 >100 7.1 7.3 5.6 4.7 4.8 4.8 5.2 >100 >100 >100 >100 >100 17.5 24.4 >100 >100 >100 >100 >100 >100 >100 >84.0 4° 22.79ef >100 90.7 >121.7 12.5 12.8 9.8 7.2 4.7 4.7 5.8 >151.0 >209.1 >239.4 >302.3 21.3 29.7 >38.8 >44.0 >63.1 >105.1 >153.6 >86.0 >84.0 >131.0 65 Subfamily Swietenioideae Tribe 1. Cedreleae Cedrela odorata  Toona serrata  Toona c i l i a t a  Toona a u s t r a l i s Tribe 2. Swietenieae Khaya senegalensis  Chuckrassia t a b u l a r i s  Entandrophraama caudatum  Swietenia humilis  Swietenia mahogani  Swietenia cand o l l e i Tribe 3. Xylocarpeae Carapa guianensis 53.6 f 24.66^9 27.0 d 47.63*3 28.2 d 28.20 f9 n 12.0 C 22.93 e f9 58.3 f 67.05 k . 12.5° 31.139 n i 22.6 c d 29.839 h 23.0 c d 23.64ffg 17.0 C 47.09 1? . 20.0 c d 42.36 n i3 53.2 f 29.689 h >100 >46.0 >100 >176.4 >100 >100.0 >100 >191.1 >100 >115.1 >100 >249.0 >100 >132.0 >100 >100.0 >100 >277.0 >100 >211.8 >100 >55.8 66 F l o r i d a d i d not d i f f e r s i g n i f i c a n t l y i n t h e i r t o x i c i t y to P. saucia neonates. S i m i l a r l y , Melia azedirach samples from Hawaii, India and F l o r i d a did not d i f f e r s i g n i f i c a n t l y i n these assays. Other t r i b e s i n the Melioideae were more variable i n t h e i r e f f e c t s . Among the Turreeae, Turrea h o l s t i i f o l i a g e extracts were comparable to Melia leaf extracts, i n h i b i t i n g the growth and survivorship of P.. saucia larvae (EC5o=1.8%, 2.8 mg/g). Unlike the s i t u a t i o n with Azadirachta indica or the two Melia species, numerous larvae were seen to have died at a f a i l e d molt attempt at the end of the f i r s t or second i n s t a r (LC5o=57.0%, 90.7 mg/g). Extracts of T_. mauritiana were much less active, with an E C 5 0 of 37.3% natural concentration (45.4 mg/g), and no t o x i c i t y at natural concentration. Within the Aglaieae, extracts of Thai and Hawaiian samples of the t r a d i t i o n a l medicinal plant Aglaia odorata were highly t o x i c to £. saucia neonates, with an E C 5 0 of 1.7% and 2.7% (2.07 and 3.29 mg/g) respectively, and L C 5 0 / s of 17.5 and 24.4% natural concentration. Other species of Aglaia were somewhat less active: A., odoratissima and A.. argentia had EC 5 0's of 11 and 27% natural leaf concentration (4.27 and 11.88 mg/g) respectively, and were not t o x i c i n the seven-day assay. When differences i n the extraction e f f i c i e n c i e s (Table 2-3) are taken into account, A., odorata i s about twice as active as A., odoratissima and about f i v e times more active than A., argentia. Of the two species of 67 Aphanamixus examined, neither were t o x i c at natural leaf concentration, but A., polystachya did i n h i b i t l a r v a l growth (EC 5 0=74%, 77.7 mg/g). Lansium domesticum was inactive at natural concentration (153.6 mg/g). Within the T r i c h i l e a e , extracts from T r i c h i l i a h i r t a and L e p i d o t r i c h i l i a v o l k e n s i i were equally active, with E C 5 0 / s of 12.5 % (18.88 and 26.14 mg/g r e s p e c t i v e l y ) . Neither species caused s i g n i f i c a n t mortality at 100% natural concentration. Cipadessa baccifera and Ekebergia capensis were active at threefold higher concentrations, with E C S Q ' S of 44 and 43% (102.9 and 133.0 mg/g) respectively. The two species of the Guareeae examined were r e l a t i v e l y i n a c t i v e ; Guarea glabra extracts had an E C 5 0 of 62% (53.3 mg/g) and Dysoxylum spectabile did not reduce P. saucia growth at 100%. Neither species caused s i g n i f i c a n t mortality i n the 7-day t r i a l . The singl e species of the Sandoriceae studied, Sandoricum koetiapte. was highly active with an E C 5 o of 17% natural concentration (22.79 mg/g). Species of the subfamily Swietenioideae were mostly less active than the Melioideae. Within the Cedrelae, Toona  a u s t r a l i s was the most active with an E C 5 0 of 12%. Two other species of Toona, T. c i l i a t a and T. serrata, were equally active with E C S Q ' S of 28 and 27% respectively. £. odorata was s i g n i f i c a n t l y less active, with an E C 5 0 of 53.6% natural concentration. Considering extraction y i e l d s , however, Toona a u s t r a l i s . Cedrela odorata. and Toona c i l i a t a were equally active, with EC 5 o's of 22.93, 24.66, and 28.20 68 mg/g respectively. Toona serrata was least active from t h i s perspective, with an E C 5 0 of 47.63 mg/g. No species caused s i g n i f i c a n t mortality. In the Swietenieae, Chuckrassia t a b u l a r i s was the most i n h i b i t o r y , with an E C 5 0 of 12.5% natural concentration. Similar a c t i v i t y was noted i n Swietenia mahogani. S_. macrophylla, S_. humilis, and Entandrophragma caudatum, with EC 5o's of 17.0, 20.0, 23.0, and 22.6% natural concentration. Considering extract y i e l d s , S_. humilis was the most active, followed by £. caudatum, C. t a b u l a r i s f S. macrophylla, and S. mahogani (EC 5 0= 23.64, 29.83, 31.13, 42.36, and 47.09 mg/g r e s p e c t i v e l y ) . Khaya senegalensis was the l e a s t a c t i v e , with an E C 5 0 of 53.6% (67.05 mg/g). The only member of the Xylocarpeae available f o r study, Carapa guianensis. also had low a c t i v i t y , with an E C 5 0 of 53.2% (29.68 mg/g). None of these extracts caused an increase i n P_. saucia mortality during the seven-day assay. B Antifeedant studies with Melanoplus sanguinipes When f o l i a r extracts were presented on cabbage leaf discs to M. sanguinipes nymphs, a l l were completely consumed within 24 h. However, when extracts of some species (chosen f o r re-examination because of t h e i r pronounced a c t i v i t y against £. saucia) were presented on glass f i b r e f i l t e r discs treated with 10% sucrose, markedly d i f f e r e n t r e s u l t s were obtained (Figure 2-3). Extracts of A. indica and M-azedirach were not s i g n i f i c a n t l y i n h i b i t o r y at natural 69 Figure 2-3. Consumption of g l a s s - f i b r e d i s c s , treated with 10% aqueous sucrose and MeOH extracts of Azadirachta  in d i c a , Melia azedirach. Turreae h o l s t i i , and Aglaia  odorata at 1, 2.5, and 5 times natural concentration (on a wt/wt b a s i s ) , by f i f t h i n s t a r nymphs of Melanoplus sanguinipes. In every case controls consumed 100% of the sucrose-treated discs during the 24 h assay. 100 90 80 70 60 50 40 30 20 10 0 % Feeding Inhibition / 7) A i f f i ftp l i t Hit i l l M I Azadirachta indica Melia azediracht Aglaia odorata Turreae holstii Plant Species and Concentration o 71 concentration, but did reduce feeding at 2.5 and 5 times that concentration. Even at the highest l e v e l s , feeding was only i n h i b i t e d by 73% (A., indica) and 43% (M. azedirach ). In contrast, f o l i a r extracts from A., odorata and T_. h o l s t i i both s i g n i f i c a n t l y reduced feeding at naturally occurring concentrations, and almost completely i n h i b i t e d feeding at 2.5 and 5 times natural concentration. C Bioassays for a n t i b i o t i c and phototoxic a c t i v i t y None of the extracts i n h i b i t e d the growth of Saccharomyces cerevisiae, e i t h e r i n the dark or following near-UV i r r a d i a t i o n . D Leaf Toughness Fi f t e e n species of Meliaceae were available f o r study i n the c o l l e c t i o n s of the USDA Plant Quarenteen Center and the Gordon F a i r c h i l d T r o p i c a l Gardens i n Miami, F l o r i d a These species were assayed f o r leaf toughness using a Mpunchmeter M modelled a f t e r a design by Feeny (1970). Values f o r leaf toughness given i n Table 2-3 are the mean of 5-10 measurements per species. Nearly a seven-fold range was observed, from a low of 12.4 N/cm2 i n Entandrophragma  caudatum to a high of 83.0 N/cm2 i n the mangrove species Carapa guianensis. The toughness of Azadirachta indica and M. azedirach are somewhat overestimated as i t was not possible to avoid the c l o s e l y spaced secondary veins i n these species. 72 E. Defensive C h a r a c t e r i s t i c s of Deciduous and Evergreen  Meliaceae Leaf l i f e t i m e was not measured d i r e c t l y i n t h i s study; as a f i r s t approximation a l l species were classed as deciduous or evergreen, as indicated by species descriptions i n various f l o r a s or monographs (Table 2-3). Carapa guianensis i s usually evergreen, but can be deciduous i n areas with a pronounced dry season (Pennington and Styles, 1981); I have classed i t as an evergreen species i n t h i s study. F o l i a r t o x i c i t y , evaluated as the E C 5 0 i n terms of % of natural leaf concentration, was s i g n i f i c a n t l y higher f o r deciduous species than f o r evergreen species (ANOVA, F^,29) = 6 - 9 6 / p = .015). When the E C 5 0 was expressed as mg extract/g d i e t fwt the difference was les s marked (F(j /29) = 3 - 7 9 » p = .0645). Much v a r i a t i o n was present within each "leaf age" cl a s s , and the evergreen c l a s s , although on the average less a c t i v e , included one of the most i n s e c t i c i d a l species i n t h i s study, Aglaia odorata. Leaf toughness data was available f o r a smaller sample of only 15 species, divided rather unevenly between the two classes (11 deciduous, 4 evergreen). Evergreen leaves were, on average, almost twice as tough as leaves of deciduous species (50.4 vs 33.1 N/cm2 r e s p e c t i v e l y ) . However again each group included a wide range of values (deciduous species:12.4-67.3 N/cm2; evergreen species 29.9-83.0 N/cm2), and the ANOVA was not s i g n i f i c a n t (ANOVA, F ( 1 1 5 j =2.263, 73 P=0.15). Aside from Carapa guianensis. the deciduous Swietenia species had the toughest leaves. Correlation of l e a f toughness and t o x i c i t y suggested the p o s s i b i l i t y of a negative r e l a t i o n s h i p between these two leaf characters (Figure 2 - 4 ) . The regression had a s i g n i f i c a n t negative slope at a= 0.1, but was not s i g n i f i c a n t at cr= 0.05, i n d i c a t i n g that there may be an inverse r e l a t i o n s h i p between leaf toughness and the t o x i c i t y of the MeOH extracts. Once again, much v a r i a b i l i t y was present and the regression equation accounted f o r only 23% of the observed v a r i a t i o n i n the data. 74 Table 2-3. Extraction yield (mg MeOH extract/g leaf dwt), leaf toughness (N/cm2), leaf pubescence (lower surface only) (glab=glabrous, axil=hairs in axils of main veins, pub=* pubescent), and "leaf habit" (deciduous [D] or evergreen [E]) for species of Meliaceae included in this study. Leaf toughness values are the mean ± 1 SD of 5-10 measurements/species; these have been ranked according to Duncan's New Multiple-Range test. "ND" indicates "not determined". Extract Toughness Pubes- Leaf ; Yield (N/cro2-) cence Habit Faaily Meliaceae Subfamily Melioideae Tribe 1. Turreeae Turreae holstii 158.0 ND glab D(l,5) Turreae mauritiana 213.0 ND glab D(5) Tribe 2. Melieae Melia azedirach (Floridai 174.7 20.0±2 .4b glab D(l) Melia azedirach (India) 165.3 ND glab D(l) Melia azedirach (Chinai 172.3 ND glab D(l) Melia toosenden 152.6 ND glab D(4) Azadirachta indica (Florida) 98.6 26.8±5 .6 c d glab D(4) Azadirachta indica (Hawaii) 112.3 ND glab D(4) Azadirachta indica (china) 107.7 ND glab D(4) Tribe 4. Trichilieae Trichilia hirta 151.0 16.0±1 .3a glab D(2) T r i c h i l i a reKa ND glab D(2) Lepidotrichilia volkensii 209.1 ND a x i l 1 E(l) Ekebergia capensis 239.4 ND glab E(l) Cipadessa baccifera 18.2±4 .4 a b glab D Tribe 5. Aglaieae Aalaia odorata (Thailand) 121.5 ND glab E(4) Aalaia odorata (Hawaiii 122.2 ND glab E(4) Aalaia odoratissima 38.8 ND glab E(4) Aalaia araentia 44.0 ND glab E(4) Aphanamixus arandlfolia 63.1 ND glab E Aphanamixus polystachya 105.0 ND glab E Lansium domesticum 153.6 ND glab E(4) Tribe 6. Guareeae Guarea glabra 86.0 60.1+3 .8e axil E(2) 2 Dysoxvlum SDectabile 84.0 ND glab E(4) Tribe 7. Sandoriceae .4d Sandoricvun Kcetjape 131.0 32.6+2 pub E(2,4) ibfaaily Swietenioideae Tribe 1. Cedreleae > 4 c d Cedrela odorata 46.2 27.3±1 glab D(2) Toona serrata 176.4 ND glab D(2) Toona c i l i a t a 100.0 27.8±1 .0d glab D(2) Toona australis 191.1 ND glab D(2) 75 Tribe 2. Swietenieae Khaya senegalensis 115.0 46.5±0.9® pub E(l) ChuckrasS3.a tabularis 249.7 21.2±1.0D C glab D(3,4) Entandrophracnna caudatum 132.0 12.4±1.2j glab D(l) Swietenia humUis 102.8 65.6±5.7t glab D(2) Swietenia mahogani 277.0 61.4±3.7* glab D(2) Swietenia candollei 211.8 67.3±5.0R glab D(2) Tribe 3. Xylocarpeae Carapa auianensis 3- 56.3 83 tp+lp ,3G Sllah EL2JL Notes: 1: red and black glands on lower surface (1) 2: individual leaf lifetime up to 52 months (6) 3: extrafloral nectaries at leaf margins (2) References: 1) White and Styles, 1963 2) Pennington and Styles, 1981 3) Pennington and Styles, 1975 4) Brandis, 1906 5) Palgare, 1977 6) Coley, 1988 76 Figure 2-3. Relationship between leaf toughness ( i n N/crn^) and b i o a c t i v i t y of the MeOH extract of f o l i a g e , calculated as 100-EC 5 0. y = 85.612 - 0.349*toughness r=0.55, F=3.362, p=0.0939 EC50 (% Natural Concentration) 78 Table 2-4. Comparison of MeOH extract t o x i c i t y (as E C 5 o to Peridroma saucia [% of natural concentration]) and toughness (N/cm2) between deciduous and evergreen species of Meliaceae. Means i n a column followed by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t (ANOVA). Toughness fN/cm^) Deciduous 18.1±15.7 (16) a 33.1±21.1 ( l l ) a Evergreen 44.4+35.8 (14^B 50.4+15.1 (51 3-Discussion A. Crude Extract Screening V i r t u a l l y a l l of the species of Meliaceae examined i n t h i s study appear to be well defended chemically against attack by generalist non-adapted insect herbivores, assuming that Peridroma saucia i s a v a l i d model species . Members of the subfamily Melioideae were on average better defended chemically, and members of the t r i b e Melieae were consistently highly t o x i c . The Melieae include the only species known to produce C-seco limonoids such as azadirachtin (Dreyer, 1983; Connoly, 1983), the most i n s e c t i c i d a l c lass of limonoids (Chapter 4 of t h i s Thesis). Correlations between phytochemistry and the i n s e c t i c i d a l or insect growth regulating a c t i v i t y of the extracts are discussed i n more d e t a i l i n Chapter 4 (pg 216); i n general the growth i n h i b i t o r y a c t i v i t y of the crude MeOH extracts agrees well with the expected a c t i v i t y i n those species where the limonoid phytochemistry i s known. Two of the three species which were inactive i n t h i s study (Lansium  domesticum and Dysoxylum spectabile) have been reported to lack limonoids. However, some species including Sandoricum  koetjapte, Aglaia odorata, A. odoratissima f and A,, argentia are i n h i b i t o r y to Peridroma saucia growth despite a reported lack of limonoids; these species deserve further phytochemical inve s t i g a t i o n . 80 Seed extracts of several of the species examined here have previously been tested f o r growth i n h i b i t i o n , feeding i n h i b i t i o n , and t o x i c i t y against the f a l l armyworm, Spodoptera frugiperda, f o r feeding i n h i b i t i o n against the s t r i p e d cucumber beetle, Acalymma vittatum, and f o r c y t o t o x i c i t y against the brine shrimp Artemia s a l i n a (Mikolajczak et aJL., 1987, 1989). This provides an opportunity to compare b i o l o g i c a l a c t i v i t i e s of seed and f o l i a r extracts. Hexane and ethanolic seed extracts of Aglaia cordata Hiern. markedly decreased feeding, growth, and survivorship of the f a l l armyworm at the lowest dose tested, 16 ppm; i n contrast A., odoratissima extracts were inactive at 10,000 ppm. These r e s u l t s support my observation of a wide range of a c t i v i t y between d i f f e r e n t Aglaia species. However, I found methanolic A., odoratissima f o l i a g e extracts to be i n h i b i t o r y to E . saucia growth at 4,000 ppm. Ethanolic seed extracts of Chuckrassia t a b u l a r i s i n h i b i t e d f a l l armyworm growth by 95% and surviorship by 50% at 400 ppm; f o l i a g e extracts i n h i b i t e d growth at 31,000 ppm and did not increase mortality. Seed extracts of Dysoxylum  spectabile and Lansium domesticum were active at 400 ppm; i n contrast f o l i a r extracts of neither species i n h i b i t e d E-saucia growth by as much as 50% at natural concentration (about 150,000 ppm). Seed extracts of Sandoricum koetiape. Azadirachta i n d i c a , Melia azedirach, Swietenia mahogani, and T r i c h i l i a roka a l l follow the same pattern of being highly 81 active at concentrations at lea s t two orders of magnitude lower than those of f o l i a r extracts. None of the f o l i a r extracts i n h i b i t e d feeding by the migratory grasshopper, Melanoplus sanguinipes. when assayed on cabbage leaf d i s c s . However, extracts from Aglaia  odorata and Turreae h o l s t i i were i n h i b i t o r y when tested at natural concentration (on a dwt l«af/dwt d i s c basis) on g l a s s - f i b r e discs provided with 10% sucrose as a phagostimulant. A s i m i l a r discrepancy between r e s u l t s with leaf and glass f i b r e discs was noted by Ascher (1981). I t i s l i k e l y that t h i s r e s u l t s from the a r t i f i c i a l circumstance of presenting the insect with a fr e s h l y cut leaf edge, which allows contact with phagostimulants present i n the leaf sap. Ord i n a r i l y such contact would not occur u n t i l the l a t e r stages of host-plant acceptance, a f t e r the leaf surface has been examined by contact chemosensilla (Chapman and Bernays, 1989). The phagostimulant a c t i v i t y of the sap must exceed the a c t i v i t y of the feeding i n h i b i t o r s i n the extracts, which i n turn are less active than 10% sucrose. However, the Aglaia odorata and Turreae h o l s t i i extracts might provide protection, i n an a g r i c u l t u r a l context, against t h i s grasshopper i f applied to i n t a c t f o l i a g e . The a c t i v i t y of the phytochemicals i n these extracts responsible f o r i n h i b i t i n g l a r v a l growth, and i n some cases molting, may be s p e c i f i c f o r insects, as no extracts were a n t i b i o t i c or phototoxic to Saccharomyces cereviseae. As well, several species found to be highly i n h i b i t o r y to 82 insect growth (including Azadirachta indica , Melia  azedirach f and Aalaia odorata) were inactive when tested against brine shrimp Artemia s a l i n a (Wiriyachitra and Towers, 1988). Together these r e s u l t s r u l e out general c y t o t o x i c i t y as a mode of action f o r the most active extracts. The lack of any phototoxic e f f e c t i n the Saccharomyces assay also suggests that the reported v i r t u a l absence i n the Meliaceae of furanocoumarins and other classes of photosensitizers, so t y p i c a l of the Rutaceae, i s r e a l and does not simply r e f l e c t a lack of attention from phytochemists. Several species investigated here f o r the f i r s t time appear to be s u f f i c i e n t l y i n h i b i t o r y to £. saucia neonates to warrant further attention. In p a r t i c u l a r , Aglaia odorata and Turreae h o l s t i i may have pote n t i a l as sources of useful extracts or phytochemicals. In the l a t t e r case, the active component(s) appears to i n h i b i t molting at doses which do not deter feeding. The phytochemistry of Turreae species i s almost unknown, but Taylor has reported limonoids s i m i l a r to cedrelone ( P e t t i t et, a i . , 1983), which can i n h i b i t molting i n the milkweed bug Oncopeltus fasciatus (Chapter 4). Several phytochemicals have been reported from Aglaia  odorata (Shiengthong et a i . , 1965, 1979) and the r o l e of these compounds i n the observed a c t i v i t y of the odorata extracts i s examined i n Chapter 3 of t h i s Thesis. 83 B. Resource A v a i l a b i l i t y Hypothesis Overall, the patterns seen here with regard to leaf t o x i c i t y , toughness, and l i f e t i m e , tend to support the resource a v a i l a b i l i t y hypothesis of Coley e_t a l . (1985) (termed the growth rate hypothesis i n Coley, 1 988 ) . I have assumed that the r e l a t i v e investment i n chemical (mobile) defenses may be estimated by comparing the r e l a t i v e e f f i c a c y of those defenses against a naive, non-adapted insect herbivore, Peridroma saucia. Physical (immobile) defenses including leaf toughness and pubescence were measured d i r e c t l y . As explained previously, the resource a v a i l a b i l i t y hypothesis predicts that investment i n chemical defenses should characterize plants with short leaf l i f e t i m e s , and investment i n physical defenses should be selected f o r i n plants with long l e a f l i f e t i m e s . I found that extracts from deciduous leaves were s i g n i f i c a n t l y more i n h i b i t o r y to E. saucia than were extracts from evergreen species, which suggests that deciduous species (short leaf l i f e t i m e ) r e l y more on chemical b a r r i e r s to herbivory than do evergreen (long leaf l i f e t i m e ) species. Previous attempts to demonstrate greater investment i n chemical defense i n species with short leaf l i f e t i m e s have not been successful, as they r e l i e d on col o r i m e t r i c assays f o r phenolics only ( i . e . Coley, 1 983 ) , which led Coley ( 1983, 1988) to suggest that the importance of plant chemistry had been overemphasized. In practice, i t i s generally impractical to determine the contribution of 84 each secondary metabolite to insect resistance ( i n most cases the compounds are unknown), and i t may be impossible to measure the cost of production of each compound. Cer t a i n l y i t i s unsatisfactory to estimate the importance of secondary metabolite-based defenses from a colo r i m e t r i c assay f o r a single c l a s s of compound. Rather, measurement of the response of the putative target of these defenses (generalist herbivores according to Feeny, 1976; Rhoades and Cates, 1976; Rhoades, 1979) appears to o f f e r a p r a c t i c a l a l t e r n a t i v e to t h i s dilemna. On average, the leaves of deciduous species were only h a l f as tough as leaves of evergreen species, but the range of v a r i a t i o n within each group was large. As a r e s u l t , the two groups d i d not d i f f e r s i g n i f i c a n t l y i n a s t a t i s t i c a l sense. However, numerous studies have demonstrated a strong c o r r e l a t i o n between leaf toughness and resistance to herbivory (Tanton, 1962; Grime gt a l . , 1968; Feeny, 1970; Rhoades, 1977; Rausher and Feeny, 1980; Coley, 1983; McKey, 1984; Coley, 1988), and t h i s factor i s also c l e a r l y p o s i t i v e l y correlated with leaf l i f e t i m e (Coley, 1983, 1988; Coley gt a l . , 1985) . The lack of s t a t i s t i c a l s i g n i f i c a n c e i n my study can be attributed to the small sample s i z e (11 deciduous and 4 evergreen species) and the wide range of v a r i a t i o n between ind i v i d u a l s i n each c l a s s . Similar v a r i a t i o n characterizes e a r l i e r studies as well and can only be overcome with appropriately large sample s i z e s . I attach more importance to the marked difference i n the means, which 85 suggest ( i n agreement with a l l e a r l i e r studies c i t e d above) that evergreen species invest more i n leaf toughness (immobile or quantitative defenses) than do deciduous species. Species which invest i n leaf toughness factors may r e l y less on phytochemical defenses, as there i s evidence for a negative c o r r e l a t i o n between leaf toughness and i n h i b i t o r y a c t i v i t y of the extracts. However, t h i s hypothesis c l e a r l y needs further study with larger sample s i z e s , again to overcome d i f f i c u l t i e s with the large amount of i n t e r s p e c i f i c v a r i a t i o n . Previous studies have compared defensive a t t r i b u t e s of pioneer and persistent species which occur together i n the same habitat. Although the species studied here were drawn from diverse habitats, i n t e r e s t i n g c orrelations can s t i l l be drawn between defensive strategies and ecological a t t r i b u t e s . The most i n s e c t i c i d a l species seen i n t h i s study, Azadirachta indica and the two Melia species, are noted f o r t h e i r extremely rapid growth, and are often planted as shade trees or f o r firewood f o r t h i s reason (Jacobson, 1988). They occur i n semi-arid areas where nutrients and l i g h t are often not l i m i t i n g (although water i s ) , and cannot compete well with other species (Jacobson, 1989), suggesting that they are adapted to "high resource a v a i l a b i l i t y " habitats. In times of drought they often constitute the only green plants ava i l a b l e , which may have provided the s e l e c t i o n pressure f o r the evolution of the highly active C-seco limonoids including azadirachtin 86 (Blaney, 1980). On the other hand, Khaya senegalensis, Ekebergia capensis, and Guarea glabra are slow-growing persistent r a i n - f o r e s t species which form a prominent component of the emergent f l o r a (Brandis, 1906; White and Styles, 1963; Pennington and Styles, 1981); these tend to produce tough, evergreen leaves low i n i n s e c t i c i d a l phytochemicals. Exceptions to t h i s pattern do occur, however. Swietenia macrophylla i s r e l a t i v e l y fast-growing, e s p e c i a l l y compared to other Swietenia species (Pennington and Styles, 1981), but a l l three species tested here produce equally tough leaves and appear to be about equally well defended chemically; the pattern of defense i n these species i s more t y p i c a l of the slow-growing evergreen species. Carapa guianensis i s a very variable species, occurring i n mangrove swamps, on rocky h i l l s i d e s , and even as an understory species i n the r a i n forest (Pennington and Styles, 1981). I t ' s defenses include very tough leaves, and investment i n phytochemical defenses appears to be low; i n addition, t h i s species has e x t r a f l o r a l nectaries along the lea f margin, and some populations appear to be well defended by ants (Pennington and Styles, 1981). The plants used i n t h i s study were c o l l e c t e d from diverse l o c a l i t i e s around the world; t h e i r only d i r e c t r e l a t i o n s h i p i s through phylogeny. Each species has had to respond, i n evolutionary time, to a unique suite of natural enemies (herbivores) and competitors. As a l l species are from t r o p i c a l habitats I have i m p l i c i t l y assumed (for 87 s i m p l i c i t y ) that p o t e n t i a l herbivores are numerous and consequently s e l e c t i o n pressure to reduce herbivory i s strong i n a l l cases. However, variations i n s e l e c t i o n pressure between d i f f e r e n t habitats may account f o r much of the v a r i a b i l i t y i n defense strategies noted i n t h i s study. As well, leaf toughness has been i m p l i c i t l y considered as a defense against herbivory, but tough leaves are also known to correlate with other factors including resistance to desiccation (Daubenmire, 1974) and possibly fungal attack, which may be more important s e l e c t i v e pressures than herbivory i n some habitats. Further, I have categorized species as deciduous or evergreen, but leaf l i f e t i m e can vary tremendously within these classes; p a r t i c u l a r l y , some species may f l u s h new leaves before dropping the old ones, i n which case the tree could be evergreen and yet each leaf may have a short l i f e t i m e . Despite these sources of v a r i a t i o n , an o v e r a l l pattern of defense a l l o c a t i o n between chemically- and physically-based strategies can be discerned and i s i n general agreement with the resource a v a i l a b i l i t y hypothesis. This suggests that the hypothesis i s s u f f i c i e n t l y robust to explain not only differences between plants within a single habitat, but also evolutionary patterns between rel a t e d plants occupying d i f f e r e n t habitats. In a study of defensive c h a r a c t e r i s t i c s of pioneer and persistent species occurring i n l i g h t gaps i n the Panamanian r a i n forest, members of the same family were consistently found to c l u s t e r according to ecological 88 a t t r i b u t e s rather than along taxonomic l i n e s , suggesting that habitat and l i f e h i s t o r y place greater constraints on defenses than do phylogenetic relationships (Coley, 1983). This conclusion i s supported by my findings. Detailed studies including leaf tagging experiments to determine leaf l i f e t i m e , and measurements of plant growth rates are underway i n conjunction with collaborators (Dr. K.R. Downum and associates) at F l o r i d a International University. 89 Chapter 3. Phytochemical Investigation of Aglaia Species Introduction Species of the genus Aglaia figure prominently i n t r a d i t i o n a l pharmacopeias throughout south-east Asia. For example, Aglaia odorata (Lour), known as Shu-Lan i n Taiwan, has been used for the treatment of coughs and inflammation (Kan, 1979) as well as "traumatic i n j u r y " (Hayashi ejb a l . , 1982). In Thailand the same plant i s t r a d i t i o n a l l y prescribed as a heart stimulant and febrifuge (Shiengthong et a l . , 1965). Leaves and roots of Aglaia p i r i f e r a Hance induce vomiting and are used as an antidote f o r poisoning i n Thailand (Saifah et a_l., 1988). As a r e s u l t of t h e i r reputed pharmacological a c t i v i t i e s , to date three species of Aglaia have been examined phytochemically. A., odorata has yielded the dammarane triterpenes a g l a i o l , aglaiondiol (1), and a g l a i t r i o l (2) (Shiengthong gt a l . , 1965, 1974) and the b i s -amides of 2-aminopyrrolidine, (+)-odorine ( 8 ) , (+)-odorinol (9) (Purushothaman e£ a l . , 1979) and (-)-odorinol (10) (Hayashi gt a l . , 1988). (+)-Odorine and (+)-odorinol were also i s o l a t e d from A., roxburgiana (Shiengthong gt a l . , 1979). A., p i r i f e r a was found to contain p i r i f e r i n e , i d e n t i c a l to (+)-odorine except for the loss of a terminal methyl from the 2-methylbutanoyl moiety (Saifah gt a l . , 1988). The only compound which has, to date, been shown to 90 possess any b i o l o g i c a l a c t i v i t y i s (-)-odorinol, which i n h i b i t s P-388 lymphocytic leukemia growth i n BDF1 male mice (T/C > 136%) at a dose of 5.0 mg/kg (Hayashi et a i . , 1982). In Chapter 2 of t h i s t h e s i s , MeOH extracts of A., odorata, &. odoratissima, and A., argentia were reported to i n h i b i t growth of the variegated cutworm, Peridroma saucia. This chapter i s a report of attempts to i s o l a t e and i d e n t i f y the phytochemical(s) responsible f o r t h i s a c t i v i t y i n A., odorata, and to determine the basis of the growth i n h i b i t i o n (feeding deterrence or t o x i c i t y ) . In addition, methods fo r the rapid q u a l i t a t i v e and quantitative analysis of Aalaia f o l i a g e were developed, based on TLC and HPLC. These methods were applied to compare the phytochemistries of the three available Aglaia species, including A., odorata samples from Thailand, Hawaii, and Taiwan. Several new natural products Were i s o l a t e d and i d e n t i f i e d from A., odorata f o l i a g e . 91 Materials and Methods A) Sources of Plant Material Sources of a l l plant samples are given i n Table 2-1 of Chapter 2. Additional material of Aalaia odorata from the P a c i f i c T r o p i c a l Gardens, Hawaii, was provided by Mr. T. Flynn of that i n s t i t u t i o n . Most of the phytochemical work reported i n t h i s Chapter i s based on t h i s l a t t e r material. A l l plant samples were a i r - d r i e d before shipping to UBC. B) I s o l a t i o n and i d e n t i f i c a t i o n of secondary metabolites i n  Aglaia f o l i a g e I s o l a t i o n of the i n s e c t i c i d a l components from A., odorata f o l i a g e was guided by bioassay (Section D). However, i n view of the unique phytochemistry of t h i s species within the Meliaceae, an attempt was made to i d e n t i f y a l l of the major secondary metabolites, whether or not they showed a c t i v i t y against E- saucia. I s o l a t i o n involved a preliminary solvent p a r t i t i o n i n g , followed by chromatographic p u r i f i c a t i o n of the most active solvent f r a c t i o n . Bl) Solvent p a r t i t i o n i n g A i r - d r i e d A. odorata f o l i a g e was weighed (130 g dwt), then extracted into three 1.5 1 changes of MeOH (3 x 24-72 h) at room temperature. The MeOH extracts were f i l t e r e d , pooled and concentrated under vacuum to a f i n a l volume of 2 ml/g leaf dwt equivalent. This extract was used for i n i t i a l 92 bioassays as described i n Chapter 2. Other species of Aglaia were extracted i n an i d e n t i c a l manner. The MeOH extract was di l u t e d to 1 1, combined with an equal volume of water and par t i t i o n e d successively (3 x per solvent) against an equal volume of hexane, d i e t h y l ether ( E t 2 0 ) , and dichloromethane (CH 2C1 2). Solvents were purchased from BDH and r e d i s t i l l e d p r i o r to use. A l l solvent phases, including the aqueous phase, were dried, weighed, and bioassayed at 10% of natural concentration (0.1 g leaf equivalent/ g dwt d i e t ) . The dried E t 2 0 phase was only p a r t i a l l y soluble i n anhydrous E t 2 0 ; t h i s phase was accordingly divided into anhydrous Et 20-soluble and -insoluble phases and both were bioassayed at 250 and 2500 ng extract/g d i e t dwt. B 2 i . Normal-Phase Chromatography The E t 2 0 soluble f r a c t i o n , which was the most active against E . saucia neonates, was f l a s h chromatographed ( S t i l l et a l . , 1975) i n 1 g l o t s on a 2.5 x 25 cm s i l i c a (Si) gel (230-400 mesh) column, using N 2 gas as the pressure source. The mobile phase was 2 column volumes each of E t 2 0 , Et 20:EtOAc (3:1), Et 20:EtOAC (1:1), EtOAc, and f i n a l l y MeOH. Chloroform was avoided because the bis-amides are unstable i n t h i s solvent (Shiengthong et al., 1979). The flow rate was 49 ml/min (2.5 cm/min), and 6 ml fr a c t i o n s were co l l e c t e d . Fractions were monitored by a n a l y t i c a l TLC (Si plates, 0.2 mm, Merck) developed i n EtOAc:PE 3:1; spots were 93 v i s u a l i z e d with Ehrlich's reagent (1 g p-dimethylaminobenzaldehyde i n 100 ml 2% ethanolic H 2S0 4) (Dreyer, 1964) and s i m i l a r f r a c t i o n s were pooled f o r bioassay. Fractions which were i n h i b i t o r y to £. saucia. or which contained major natural products (as evidenced by the appearance Of major spots on TLC or c r y s t a l l i z a t i o n of compounds on drying), were p u r i f i e d further by preparative TLC. Fractions were banded onto 1 mm thick S i gel TLC plates (Merck) and developed i n EtOAc:PE (1:1) i n an s e q u i l i b r a t e d TLC tank. Fractions containing highly polar compounds ( i . e . odorines) were developed up to 30 times to achieve separation of the (+)- and (-)- diastereomers. Bands were v i s u a l i z e d with UV l i g h t or by spraying only the edge of the plate with Ehrlich's reagent. Bands were then scraped from the plate and eluted with MeOH. Compounds were f i n a l l y p u r i f i e d by r e c r y s t a l l i z a t i o n , p r i o r to bioassay. B3) HPLC The most active f r a c t i o n s were chromatographed using preparative HPLC a f t e r TLC proved unsatisfactory f o r the i s o l a t i o n of the i n s e c t i c i d a l p r i n c i p l e ( s ) . Fractions were prepared for chromatography by passing them through a 3 cm s i l i c a plug i n a pasteur pipette, eluted with MeOH, followed by passage through two Sep-Pacs (Waters) packed with RP-18. Separation was achieved with a Lichrosorb Hibar® RP-18 (7/im) 10 X 250 mm column on a Varian Model 5000 HPLC equipped with 94 a UV/Vis detector and a Spectra-Physics SP4100 integrator. The solvent system was MeOH:H20 (1:1) f o r 10 minutes, followed by a gradient to 100% MeOH over 20 minutes, then e l u t i o n with 100% MeOH for 30 minutes. Flow rate was 3 ml/min, with UV monitoring at 217 nm. Six-ml f r a c t i o n s were c o l l e c t e d with an automatic f r a c t i o n c o l l e c t o r . Subsequently these f r a c t i o n s were examined by a n a l y t i c a l HPLC using a Lichrosorb RP-18 (5 /im) 4 X 250 mm column, i s o c r a t i c MeOH:H20 (7:3), with a flow of 1 ml/min. One twentieth of each f r a c t i o n was used f o r bioassay. C) Q u a l i t a t i v e and quantitative analyses Subsequently, techniques developed f o r the i s o l a t i o n of the active component(s) were adapted to provide rapid methods of analysis of Aglaia f o l i a g e . The Rfs of a l l compounds is o l a t e d were determined i n two a n a l y t i c a l TLC systems: EtOAC:PE (3:1) on S i gel (System A), and CHCl3:MeOH (49:1) on S i gel (System B). The color reactions of a l l compounds were determined with two spray reagents, Eh r l i c h ' s reagent (Dreyer, 1964), and V a n i l l i n reagent (2% v a n i l l i n i n EtOH/ overspray with 3M H 2S0 4) (Pieman e£ a l . , 1980). These TLC systems were used f o r q u a l i t a t i v e comparisons of Aalaia extracts. Quantitative analysis was achieved by a n a l y t i c a l HPLC, using a Lichrosorb RP-18 4 X 250 mm column, detection at 217 nm, and the following solvent system: 0-10 min, MeOH:H20 (1:1); 10-30 min, gradient to 100% MeOH; 30-60 min 100% 95 MeOH; flow = 1 ml/min throughout. Retention times and c a l i b r a t i o n curves were determined f o r a l l i d e n t i f i e d compounds i s o l a t e d from A., odorata. This method could not resolve the diastereomers of odorine and odorinol; to quantify these compounds I used 1H-NMR, comparing r e l a t i v e heights of the methyl peaks at 0.70 and 0.90 ppm to calcu l a t e the proportion of S,R and S,S odorine. Relative heights of the s i n g l e t s at 6 1.34 and 1.22 were used to calcu l a t e the r e l a t i v e proportions of S,R and S,S odorinol. D) Bioassay A l l f r a c t i o n s and pure compounds were bioassayed against neonate E- saucia, i n seven-day chronic feeding assays, as described i n Chapter 2. Solvent phases were bioassayed at 10% of the na t u r a l l y occurring l e v e l s ; f o r f r a c t i o n s c o l l e c t e d from column chromatography or HPLC, 1/20 of each f r a c t i o n was used. The maximum concentration of pure compounds bioassayed, given i n Table 3-7, was equal to or s l i g h t l y greater than the concentrations of the compounds i n the plant, as given i n Table 3-6. Various combinations of the p u r i f i e d compounds were also bioassayed (Table 3-8), to check for additive or sy n e r g i s t i c i n t e r a c t i o n s . The most active compound i s o l a t e d was assayed f o r i n h i b i t i o n of l a r v a l growth and survivorship at 0.4, 0.6, 1, 2, 3, 5, 10, and 15 ug/g d i e t dwt. A simple choice feeding assay was also used to determine the r o l e of antifeedant 96 e f f e c t s i n the a c t i v i t y of t h i s compound at 1, 2, and 3 ug/g d i e t ; t h i s assay i s described i n d e t a i l i n Chapter 4 . Results A) MeOH Extract Screening I n h i b i t i o n of neonate E- saucia growth by extracts from A.. odorata (Thailand sample), A., odoratissima, and A., argentia i s shown i n Figure 3-1. The A., odorata extract was about twice as active as the A., odoratissima extract, and f i v e times as active as the A., argentia extract. B) Solvent P a r t i t i o n i n g Bioassay of the various solvent phases from A., odorata indicated that the E t 2 0 phase was most i n h i b i t o r y to E . saucia growth and s u r v i v a l ; the CH 2C1 2 phase also retained some a c t i v i t y (Figure 3-2). The E t 2 0 phase was dried and divided into phases which were soluble or insoluble i n anhydrous E t 2 0 . The E t 2 0 soluble phase was about ten times more active than the insoluble phase when tested at 250 /jg extract/g d i e t dwt. Neither the hexane phase nor the remaining aqueous phase had an i n h i b i t o r y e f f e c t . Bioassay of the leaf marc, the residue a f t e r extraction, indicated that the i n s e c t i c i d a l p r i n c i p l e ( s ) had been completely removed by the MeOH extraction. Figure 3-2 also includes the y i e l d (as % of the MeOH extract) f o r each solvent phase. C1 Chromatography The i s o l a t i o n of phytochemicals was confined to the E t 2 0 phase as t h i s contained the i n s e c t i c i d a l compound(s). 98 Figure 3-1. E f f e c t of f o l i a r MeOH extracts of Aglaia  odorata (o), A., odoratissima ( A ) , and A., argentia (•) on the growth of neonate Peridroma saucia. Growth i s shown as % of the c o n t r o l . Each point indicates the mean of at l e a s t three determinations per treatment; standard deviations were <7% f o r a l l treatments. Larval Growth (% of Control) 100 Figure 3-2. Growth and survivorship of neonate Peridroma  saucia fed a r t i f i c i a l d i e t containing solvent extracts of Aglaia odorata (Hawaiian sample). The y i e l d of each solvent phase, as % of the t o t a l MeOH extract, i s also shown. Larval Growth & Survival (% of Control) 1201 102 Typical r e s u l t s of f l a s h chromatography of t h i s phase are given i n Table 3-1, along with bioassay r e s u l t s f o r each f r a c t i o n . Several f r a c t i o n s yielded c r y s t a l l i n e material on drying; these were investigated further whether or not they possessed i n s e c t i c i d a l a c t i v i t y . TLC analysis of the column fr a c t i o n s which were i n h i b i t o r y to £. saucia growth indicated the presence of three major compounds. Preparative TLC was used to i s o l a t e these compounds i n amounts s u f f i c i e n t f o r i d e n t i f i c a t i o n . Two compounds, the i d e n t i f i c a t i o n of which i s described below i n d e t a i l , were the very unusual flavanone naringenin 5,7,4'-trimethyl ether, known previously from Dahlia (Kaufmann and Lam, 1967; Lam and Wrang, 1975), and the novel natural product 3-hydroxy-5,7,4'-trimethoxyflavanone. The t h i r d component was not i d e n t i f i e d . Surprisingly, none of these i s o l a t e d compounds exhibited any a c t i v i t y against P. saucia (Table 3-9). Subsequently I eluted a l l remaining compounds from the TLC plate, recovering a small amount of o i l y material which also proved to be i n a c t i v e . The active p r i n c i p l e therefore appeared to be unstable on s i l i c a , degrading or isomerizing to an inactive form. Subsequent attempts to p u r i f y the active compound u t i l i z e d semipreparative reverse-phase HPLC. Bioassay r e s u l t s indicated that a l l the a c t i v i t y was associated with a single apparently homogeneous peak (Fig 3-3). HPLC was then used 103 Table 3 - 1 . Properties of fractio n s obtained from f l a s h column chromatography (Si g e l , 240-400 mesh) of A., odorata (Et 20 soluble phase), and t h e i r e f f e c t on the growth of neonate Peridroma saucia, reared f o r seven days on d i e t containing each f r a c t i o n at approximately natural concentration. Values followed by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t (Tukey's Studentized (HSD) Range t e s t ) . Fraction Description Growth # ; ; r% of Control) A 1-5 Empty 102 a B 6-9 Chlorophylls only 115 a C 10-15 Chlorophyll, copious white c r y s t a l s , red 97 a with Ehrlichs D 16-19 S t i l l some "red" cmpd (Rf=.525), 2 blue 2 b fluorescing cmps, yellow with E h r l i c h s , Rf= .85 & .89 E 20-35 Same two blue fluorescing cmpds, lower 6 b more abundant F 36-41 Empty 107 a G 42-60 White c r y s t a l s , purple with E h r l i c h s , Rf=.425 87 a H 61-95 Cry s t a l s , odorine (Rf=.344) and odorinol 83 a  (Rf=,24D Figure 3 - 3 . Preparative HPLC chromatograra of a growth i n h i b i t o r y f r a c t i o n from A., odorata. Insect growth-i n h i b i t i n g a c t i v i t y was associated with the shaded peak. 105 106 to i s o l a t e about 11 mg of t h i s compound, which was concentrated and c r y s t a l l i z e d from MeOH. The compound proved again to be the inactive 3-hydroxy-5,7,4'-trimethoxyflavanone, i s o l a t e d previously by TLC. A l l the i n h i b i t o r y a c t i v i t y remained i n the mother li q u o r ; when dried t h i s yielded about 400 pg of a mixture of the active compound (described as Compound 6 below) and the dihydroflavonol (Compound 4). Lack of plant material has precluded obtaining more of the active constituent. D) Phytochemistry In a l l , ten natural products were i s o l a t e d from A., odorata f o l i a g e , nine of which were characterized completely. Of the i d e n t i f i e d compounds, four had not previously been i d e n t i f i e d i n any Aglaia species, and two were new natural products. Only the compound responsible f o r the marked growth i n h i b i t i o n against E» saucia could not be i d e n t i f i e d . 1H-NMR and mass spectra of a l l compounds are given i n Appendix 2. Compound 1. Transparent needles, m/z (FAB): 460 (C3QH50O3). •'•H-NMR indicated the presence of seven methyl groups. This information suggested that the compound was possibly a dammarane triterpene, as such compounds have previously been i s o l a t e d from A. odorata. This i d e n t i f i c a t i o n was supported by CH 3 s i n g l e t s at cS 0.77 and 0.91, c h a r a c t e r i s t i c of geminal methyls at C-4, at 6 0.97 (6H), i n d i c a t i v e of 10B-107 and 8B-Me groups, and at 6 0.89, assigned to the 14a-Me group (Shiengthong et. a l . , 1965). As well a D 20 exchangeable proton at S 3.29 and a multiplet at 6 3.33 (s i m p l i f i e d to a quartet a f t e r D 20 exchange), were assignable to a 3B-0H and a 3a-proton respectively. As i n previously i d e n t i f i e d Aglaia dammaranes, a methylene group i s present at C-20, indicated by a broad doublet at 6 4.75 (2H, J= 10 Hz). The two C-25 methyls appear at S 1.15 and 1.17; these broaden to an overlapping s i n g l e t on D 20 exchange. These spectral data are consistent with the known dammarane triterpene a g l a i o n d i o l , previously described from Aglaia odorata (Shiengthong et a l . , 1965,1974) (Figure 3-4). Compound 2. White, f i n e needles, m/z (FAB):459 (C3QH50O3). This compound was also i d e n t i f i e d as a dammarane triterpene, with 1H-NMR CH 3 s i n g l e t s at 6 0.78, 0.85, 0.88, and 0.985; multiplets (IH each) at 6 3.41 and 3.22 were assigned to a geminal hydroxyl and proton on C-3. A broad doublet at 6 4.765 (2H, J= 10 Hz) indicated the presence of a methylene group at C-20. The C-25 methyl groups appear at 6 1.18 and 1.225. These data are consistent with l i t e r a t u r e values f o r the dammarane triterpene a g l a i t r i o l (Shiengthong et- a l . , 1974) (Figure 3-4). Compound 3. This compound formed white plates, m/z (EI, FAB) 330 ( C 1 8 H 1 8 0 6 ) . The UV absorbtion spectrum showed 108 Figure 3 - 4 . Structures of dammarane triterpenes i s o l a t e d from Hawaiian samples of Aglaia odorata. A g l a i o l was not i s o l a t e d but was assumed to be present i n the i n a c t i v e hexane phase. Amoora A 6 was i s o l a t e d from Amoora (=Aalaia) stellato-squamosa. Aglaiol Aglaiondiol 110 Table 3-2. —H-NMR spectral data of compounds 3. 4, and 5 . 1 3 4 5 H-2 4.99 d 5.37 dd 5.39 dd H-3 4.45 dd 3.05, 2.76 dd 3.12, 2.91 dd H-62 6.13 d 6.10 d 6.08 d H-82 6.14 d 6.15 d 6.09 d H-2',6' 7.50 d 7.39 d 7.40 d H-3',5' 7.00 d 6.95 d 6.98 d OH-3 4.05 d - -OH-5 - - 12.02 S OMe-5 3.82 s 3.83 S -OMe-7 3.84 s 3.84 S 3.83 S OMe-4' 3.92 s 3.89 S 3.85 S (1) 400 MHz i n CDC13, TMS as in t e r n a l standard; (2) assignments may be reversed J(Hz) 3:2,3=12.8; 3,OH=0.5; 6,8=1.2; 2',3'=9.2; 5',6'=9.2 4:2,3A=3.2; 2,3B=12.4; 3A,3B=16.4; 6,8=2.0; 2/,3/=8.0; 5',6'=8.0; S:2,3A=3.2; 2,3B=12.4; 3 A B=17.6; 6,8=2.4; 2/,3/=9.0; 5',6'=9.0 ' I l l intense peaks at 283 and 216 nm. H^-NMR (Table 3-2) showed an AA'BB' system with doublets at 7.00 and 7.50 ppm (2H, J=9.2 Hz, collapsed to a s i n g l e t when decoupled at 7.00 ppm), t y p i c a l of a para-substituted benzene r i n g . An ABX system included a doublet at S 4.99 (IH, J=12.8 Hz), coupled to a doublet of doublet at 6 4.45 (IH, J A X=12.8 Hz, J A B=0.5 Hz), which was i n turn coupled to a D 20 exchangeable doublet at S 4.05 (IH, J=0.5 Hz). This was interpreted as i n d i c a t i n g two protons, H-2 and H-3 i n c i s orientation, with H-3 coupled to a geminal OH. Three methoxy groups (3H, s, 3.82, 3.84, 3.92 ppm) and two meta-coupled protons (IH doublets, 6.13 and 6.14 ppm, J=MHz) were also indicated. This evidence was consistent with the compound being a dihydroflavonol ( c f . Balza and Towers, 1984). One methoxy group must be present at C-4' to account f o r the para-substituted B r i n g ; C-5 and C-7 of the A r i n g were also methoxylated, accounting for the meta-coupling between the C-6 and C-8 protons. The compound was therefore i d e n t i f i e d as the new natural product 3-hydroxy-5,7,4 trimethoxyflavanone (dihydrokaempferol 5,7,4'-trimethyl ether) (Figure 3-5). Further evidence supporting t h i s structure was obtained from the mass spectrum (Figures 3-6, 3-7). The i d e n t i t y of a l l the major fragments could be derived by comparison with the fragmentation pattern of 3,5,4'-trihydroxy-7-methoxyflavanone (Balza and Towers, 1984) and dihydrokaempferol 4'-methyl ether (Mabry and Markham, 1975) 112 (Figure 3-7). Fragments t y p i c a l of dihydroflavonols include the B r i n g fragments B 3 + (m/z 150), B 4 + (m/z 121, base peak), B 3-CH 3 (m/z 135), and [B 3-43]+ (m/z 107), and the A r i n g fragments [A1+H]+ (m/z 181), A 1 + (m/z 180), and A^—CO (m/z 152). The prominent peak at [M-CO]+ (m/z 301, C 1 7 H 1 7 0 5 ) , i s t y p i c a l of flavones, flavanones, and dihydroflavonols (Mabry and Markham, 1975). The s i z e of the B-ring fragments confirm that i t c a r r i e s a methoxy sub s t i t u t i o n . A-ring fragments at m/z 181 and 180, r e s u l t i n g from a re t r o Diels-Alder cleavage (Mabry and Markham, 1975), confirm the presence of two methoxy substitutions. Compound 4. White c r y s t a l s , m/z (EI) 314, C 1 8 H 1 8 0 5 . The •^H-NMR spectrum of t h i s compound was s i m i l a r to that of Compound 4, with a para-substituted benzene r i n g (2H, doublets, 6.95 and 7.39 ppm, J=8.0 Hz), two meta-coupled protons (1H, doublets, 6.10 and 6.15 ppm, J=2.0 Hz), and three methoxy groups (3H, s i n g l e t s , 3.83, 3.84, and 3.89 ppm) (Table 3-2). However, no D 20 exchangeable protons were present, and the ABX system showed doublets of doublet at 6 5.37 (1H, J A B = 0 - 4 J A X = 1 3 ' 5 H z ) ' 6 3 ' 0 5 ( 1 H' J A B = 0 * 4 J A X = 1 6 - ° Hz), and 2.76 ppm (1H, J A B = 0 , 4 J A X = 1 6 , 0 H z ) « This indicated that a proton at C-2 was coupled to geminal protons at C-3; the compound was therefore i d e n t i f i e d as 5,7,4'-trimethoxyflavanone (naringenin-5,7,4'-triroethyl ether) (Figure 3-5). 113 Figure 3-5. Structures of flavanones i s o l a t e d from Hawaiian samples of A a l a i a odorata. The stereochemistry shown assumes an S c o n f i g u r a t i o n at C-2 . C^OCH 3 CH30 O C H 3 o Compound 3 3-Hydroxy-5,7,4'-methoxyfIavanone Compound 5 5-Hydroxy-7,4'-dimethoxyflavanone 115 Figure 3-6. Mass spectrum (EI) of 3-hydroxy-5,7,4'-trimethoxyflavanone (Compound 3). 116 117 Figure 3-7. Mass spectrum fragmentation pattern of 3-hydroxy-5,7,4'-trimethoxyflavanone (Compound 3). 118 m/z 107 [B3 - 43]* 119 As with the previous compound, the mass spectrum showed A-ring fragments at m/z 181, 180, and 152 confirming two methoxy substitutions, and B-ring fragments at m/z 134 ( B 3 + , base peak), 119, and 91 confirm the location of the t h i r d methoxy group. Compound 5. White plates, m/z (EI) 300, C 1 7 H 1 6 0 5 ) . The 1H-NMR spectrum of t h i s compound was almost i d e n t i c a l to that of Compound 5, but showed only two methoxy groups (3H, 3.83 and 3.85 ppm) and a D 20 exchangeable s i n g l e t (1H) at 12.02 ppm (Table 3-2). The mass spectrum showed A-ring fragments at m/z 167 and 168, consistent with the presence of one methoxy and one hydroxy substitution on t h i s r i n g . B-rihg fragments at m/z 121 demonstrated the presence of a methoxy sub s t i t u t i o n , which must be at C-4' to account for the para-sub s t i t u t i o n pattern. The hydroxyl was assigned to C-5 rather than C-7, as hydrogen bonding with the adjacent carbonyl causes the sign a l to appear at 12.02 ppm; i f present at C-7 the sign a l would have occurred at 9.5 ppm (Balza and Towers, 1984). Compound 5 was therefore i d e n t i f i e d as 5-hydroxy-7,4'-dimethoxyflavanone (Figure 3-5). Compound 6. m/z (FAB) 661, MH+. Possibly C 3 6 H 3 6 0 1 2 . This compound produces a brown color reaction with Ehrlich's reagent, a reaction t y p i c a l of B-substituted furans (Dreyer, 1964). The mass and possible presence of a B-substituted 120 furan r i n g suggest that compound 6 i s an oxidized limonoid. However, the very li m i t e d amount of material on hand precludes the extensive NMR studies required f o r structure elu c i d a t i o n . Compound 7. White needles, m/z (EI) 300 (M+), C 1 8H 2 4N 20 2. 1H-NMR (Table 3-3) showed a benzene r i n g (5H, S 7.37, multiplet) coupled to an AB system (IH, S 7.06, J= Hz and 7.57, m, trans-substituted double bond), an NH doublet at 6 6.13 (IH, J=16 Hz), and methylene multiplets at S 3.70 (2H) and 2.00-2.26 (4H). Methyl functions were seen at 6 0.7 (3H, t , 3=7.9 Hz) and 1.10 (3H, d, J=7.0 Hz), a methylene was represented by multiplets at 6 1.32 and 1.56, and a single proton showed as a multiplet at 6 1.92. This information established the i d e n t i t y of Compound 8 as the known bis-amide odorine (Shiengthong et a i . , 1979; Purushothaman gt a i * , 1979) (Figure 3-8). This i d e n t i f i c a t i o n was confirmed by the mass spectrum, which showed major fragments at m/z 215 and 85, (r e s u l t i n g from loss of the 2-methylbutanoyl fragment), at m/z 169 and 131 (from loss of the cinnamoyl moiety), and at m/z 199 (from loss of 2-methylbutanoic acid amide). Mass spectral and ^ H-NMR data were c l o s e l y comparable to l i t e r a t u r e values, and with spectroscopic data obtained from a sample of odorine synthesized from dihydrocinnamoyl-L-proline according to the procedure of Purushothaman et al.(1979) by Dr. H. Barrios 121 Figure 3 - 8 . Structures of bis-amides i s o l a t e d from Hawaiian samples of Aglaia odorata. 123 Lopez, while he was a v i s i t i n g s c i e n t i s t with Dr. G.H.N. Towers. Odorine has c h i r a l centers at C-2 and C-2'. The C-2 s i t e i s p a r t i c u l a r l y s e n s i t i v e to epimerization i n a c i d i c solvents including CHC13 (Purushothaman et. ai., 1979). As a re s u l t chlorinated solvents were avoided during the i s o l a t i o n of Aglaia compounds. Samples of R,S and R,R odorine were synthesized, and then (separately) epimerized at C-2 with HC1 to produce a racemic mixture which was separated by preparative TLC. Comparison of the 1H-NMR (CDCI3) spectra indicate that i n the isomers which are R at C-2' the methyls of the 2-methylbutanoyl moiety appear at 6 0.70 and 1.10 ppm, whereas for the S isomers the methyls are at S 0.90 and 0.98 ppm. The enantiomeres R,R- and S,S-odorine had a Rf of .324 (Table 3-3), compared to a Rf of .331 f o r R,S and S,R-odorine. Compound 7 was therefore i d e n t i f i e d as the known S,R-odorine [(+)-odorine] (Figure 3-8). Compound 8. Needle c r y s t a l s , m/z (EI) 300, C 1 8H24N 202. The mass spectrum of compound 8 was i d e n t i c a l to compound 7. The 1H-NMR was also i d e n t i c a l except f o r the methyl signals at S 0.90 (3H, t , J=8.0 HZ) and 0.98 (3H, d, J=7.0 HZ). Compound 8 was therefore i d e n t i f i e d as S,S-odorine (Figure 3-8). This i d e n t i f i c a t i o n was supported by co-chromatography with synthetic S,S-odorine. This isomer of odorine has not previously been i d e n t i f i e d as a natural 124 product. To check the p o s s i b i l i t y that R,S-odorine could isomerize at C-2 i f stored i n MeOH (the solvent used to extract A., odorata) f o r extended periods, a sample of o r i g i n a l l y pure R,S-odorine was examined a f t e r s i x months i n MeOH at room temperature i n the dark. No evidence of isomerization to the S,S form was seen. S,S-Odorine appears to be an authentic novel natural product. Compound 9 . Needle c r y s t a l s , m/z (EI) 316, C 1 8H 2 4N 20 3. The 1H-NMR spectrum of t h i s compound was s i m i l a r to compound 8, except that the methyl group at C-2' appeared as a s i n g l e t at <S 1.22, the multiplet due to the C-2' proton was absent, and a D 20 exchange experiment showed an exchangeable proton at 6 2.16 ppm. The UV spectrum showed a single absorbance at 283 nm. This spectral data i s consistent with the known Aglaia bis-amide odorinol (Figure 3-8) (Shiengthong et a l . , 1979, Purushothaman et a i . , 1979, Hayashi et A l . , 1982). This structure was further confirmed by the mass spectrum, which indicated a cinnamoyl moiety with peaks at m/z 131 and 103, and a 2-methyl-2-hydroxybutanoyl moiety with fragments at m/z 231 and 101. An S,S stereochemistry i s indicated by methyl signals at 6 0.88 (3H, t , J=7.2 Hz) and S 1.22 (3H, s ) . Compound 9 was therefore assigned the structure S,S-odorinol (Figure 3-8). Compound 10 . Needle c r y s t a l s , m/z (EI) 316, C 1 8H24N 20 3. The mass spectrum of t h i s compound was i d e n t i c a l to compound 125 10. The 1H-NMR spectrum d i f f e r e d only i n that the methyls of the 2-methyl-2-hydroxybutanoyl moiety appeared at 6 0.70 (3H, t , J=7.2 Hz) and 1.34 (3H, s ) . These data indicate a structure of S,R-odorinol (Figure 3-8). £) Qu a l i t a t i v e and quantitative comparison of Aalaia species Two methods suitable for rapid phytochemical analysis of Aalaia species were developed. A q u a l i t a t i v e method was based on TLC on s i l i c a gel and use of color reactions with Ehrl i c h ' s and v a n i l l i n spray reagents (Table 3-3). Rf values of several compounds d i f f e r i n the two solvent systems used (EtOAc:PE 1:1 and CHCl3:MeOH 49:1), so these are suitable f o r two-dimensional TLC. The color reactions with the two spray reagents are highly c h a r a c t e r i s t i c and allow confident i d e n t i f i c a t i o n of most of the compounds. This method was applied to the analysis of A., odorata specimens from Thailand, Hawaii, and Taiwan, and to A.. odoratissima and A., argentia specimens from Thailand (Table 3-4). The second method was based on a n a l y t i c a l HPLC; retention times of pure standards are given i n Table 3-5. Chromatographic p r o f i l e s of the various Aglaia samples are shown i n Figures 3-9 to 3-13. As the concentrations of secondary metabolites i n plants may d i f f e r greatly from the amounts eventually i s o l a t e d , due to losses during extraction and p u r i f i c a t i o n , t h i s method was applied to determine the 126 concentration of Aglaia compounds i n f r e s h l y prepared E t 2 0 extracts (Table 3-6). Each method o f f e r s c e r t a i n advantages and weaknesses. The major advantage of the HPLC method l i e s i n the a b i l i t y to quantify concentrations of several compounds. However two major disadvantages e x i s t : HPLC cannot be used to detect the dammarane trit e r p e n o i d s , due to t h e i r low UV absorption c o e f f i c i e n t s , and HPLC does not resolve the various isomers of odorine or odorinol. For the l a t t e r purposes H^-NMR was used to quantify the proportion of each isomer, according to the r e l a t i v e peak heights of the 2-methylbutanoyl methyls. Although q u a l i t a t i v e i n nature, the TLC based system can r e a d i l y detect the presence of dammaranes i n very low concentration, and i t can resolve the diastereomers of the odorines and odorinols. In the l a t t e r case a minimumn of 4 -5 developments were required for c l e a r resolution of the diasteriomeres. Analysis of an E t 2 0 extract requires 1-3 h regardless of the method chosen, but TLC requires less sample preparation than HPLC. Tables 3-4 and 3-6 indicate that marked differences i n phytochemistry occur between most of the Aglaia species and samples examined. A., odorata samples from Thailand and Hawaii were most s i m i l a r ; both produced aglaiondiol and a g l a i t r i o l , and the three dihydroflavanones, although minor differences i n concentration were noted. The Hawaiian sample contained odorine B and odorinol A as the major amides, with minor amounts of odorine A and odorinol B 127 Table 3-3. Aglaia odorata compounds: chromatographic behavior and colour reactions with Ehrlichs reagent and vanillin reagent. Solvent A is EtOAC:PE (3:1), solvent B is CHCl3:MeOH (49:1). Where two colors are given the f i r s t applies to the color immediately after heating and the second occurs after cooling. Compound Rf(solvent A^  (solvent B^  Ehrlichs Vanillin 5-OH-7,4'-MF .912 .925 orange red Aglaiondiol .770 .394 red green Aglaitriol 5,7,4'-MF .689 .244 purple blue-purple .730 .844 yellow orange 3-OH-5,7,4'-MF .676 .788 yellow orange-brown limonoid .788 brown Odorine A .331 .400 yellow/purp1e pink Odorine A' .324 .363 yellow/purple pink Odorine B .324 .363 ye11ow/purp1e pink Odorine B' .331 .400 yellow/purple pink Odorinol A .297 .313 yellow/purp1e pink Odorinol A' .291 .275 yellow/purple pink B-Sitosterol ,868 purple-black areen 5-OH-7,4'-MF: 5-Hydroxy-7,4'-methoxyflavanone; 5,7,4'-MF: 5,7,4'-Trimethoxyflavanone; 3-OH-5,7,4'-MF: 3-Hydroxy-5,7,4'-trimethoxyflavanone. 126 Table 3-4. Qualitative TLC analysis of Aglaia samples. Amounts of compounds were assessed as: (-) not detected; (+) trace observed; (+++) major component; (++) intermediate concentration. Analysis was based on 2-D TLC with EtOAc:PE (3:1) and CHCl3:MeOH (39:1) on Si gel, followed by spraying with Ehrlichs or vanillin reagents. Cmpd Aalaia sample h. odorata A. odorata A. odorata A. odoratissima A. argentia (Hawaii) (Thailand\ (Taiwan^ (Thailand) (Thailand) 1 +++ +++ + + • -2 ++ ++ - -3 +++ ++ ++ - -4 ++ ++ - + + 5 ++ ++ 7 + - ++ ++ 8 ++ +++ +++ ++ ++ 9 ++ +++ +++ 10 + = = = ; Compounds: (1) Aglaiondiol; (2) Aglaitriol; (3) 5-hydroxy-7,4'-methoxy dihydroflavanone; (4) 5,7,4'-trimethoxy dihydroflavanone; (5) 3-hydroxy-5,7,4'-trimethoxy dihydroflavanone; (7) Odorine A; (8) Odorine B; (9) Odorinol A; (10) Odorinol B. Table 3 -5 . HPLC retention times (min) of Aalaia odorata compounds. Compound Rt (min} 1 Aglaiondiol 33.6 2 A g l a i t r i o l 33.48 3 5-OH-7,4'-MF 28.56 4 5,7,4'-MF 26.12 5 3-OH-5,7,4'-MF 22.80 6 limonoid 22.61 7 Odorine A 20.61 7' Odorine A' 20.65 8 Odorine B 20.65 8' Odorine B' 20.66 9 Odorinol A 16.79 10 Odorinol B 16.79 5-OH-7,4/-MF: 5-Hydroxy-7,4'-methoxyflavanone; 5,7,4'-MF 5,7,4'-Trimethoxyflavanone; 3-OH-5,7,4'-MF: 3-Hydroxy-5,7,4'-trimethoxyflavanone. 130 Figure 3-9. HPLC trace of E t 2 0 soluble f r a c t i o n from Aglaia  odorata (Hawaiian sample). I d e n t i f i e d compounds include: (I) Odorinol (S,R- and S,S-diastereomers unresolved); (II) Odorine (S,R- and S,S- diastereomers unresolved); (III) 3-OH-5,7,4'-trimethoxy dihydrof lavanone; (IV) 5,7,4'-trimethoxy dihydroflavanone; (V) 5-hydroxy-7,4'-dimethoxyflavanone. 131 Figure 3-10. HPLC trace of E t 2 0 soluble f r a c t i o n from Aglaia odorata (Thailand sample). I d e n t i f i e d compounds include: (I) Odorinol (S,R- and S,S-diastereomers unresolved); (II) Odorine (S,R- and S,S- diastereomers unresolved); (III) 3-OH-5,7,4'-trimethoxy dihydroflavanone (IV) 5,7,4'-trimethoxy dihydrof lavanone; (V) 5-hydroxy-7,4 dimethoxyflavanone. 133 Figure 3-11. HPLC trace of E t 2 0 soluble f r a c t i o n from Aglaia odorata (Taiwan sample). I d e n t i f i e d compounds include: (I) Odorinol (S,R- and S,S-diastereomers unresolved); (II) Odorine (S,R- and S,S- diastereomers unresolved); (III) 3-OH-5,7,4'-trimethoxy dihydrof lavanone (IV) 5,7,4'-trimethoxy dihydroflavanone; (V) 5-hydroxy-7,4 dimethoxyflavanone. 135 136 Figure 3-12. HPLC trace of the E t 2 0 soluble f r a c t i o n from Aalaia odoratissima. I d e n t i f i e d compounds include: (I) Odorinol (S,R- and S,S-diastereomers unresolved); (II) Odorine (S,R- and S,S- diastereomers unresolved); (III) 3-OH-5,7,4'-trimethoxy dihydroflavanone; (IV) 5,7,4 ' -trimethoxy dihydroflavanone; (V) 5-hydroxy-7,4'-dimethoxyflavanone. 137 138 Figure 3-13. HPLC trace of the E t 2 0 soluble f r a c t i o n from Aglaia argentia. I d e n t i f i e d compounds include: (I) Odorinol (S,R- and S,S-diastereomers unresolved); (II) Odorine (S,R-and S,S- diastereomers unresolved); (III) 3-OH-5,7,4'-trimethoxy dihydroflavanone; (IV) 5,7,4'-trimethoxy dihydroflavanone; (V) 5-hydroxy-7,4'-dimethoxyflavanone. 139 Table 3 - 6 . Concentration (ug/g ieaf dwt) of flavanones and bl amides In Aglaia species, determined by analytical HPLC. Compound Aglaia sample &. odorata &. odorata L- odorata &• odoratlaalma A- arqentla (Haval1) ( T h a i l a n d ) (Taiwan) (Thai l a n d ) ( T h a i l a n d ) 3 165.9 205.3 183.6 8.3 0 4 513.2 470.2 49.7 107.6 115.9 5 154.9 318.6 48.7 24.3 0 7 113.0 0 0 276.0 201.0 8 295.0 770.1 1298.9 422.7 189.8 9 143.7 704.1 1911.5 61.2 0 10 80.8 o o - 0 Compounds: (3) 5-hydroxy-7,4'-methoxy dihydroflavanone; (4) 5,7,4 '-trimethoxy dihydrof lavanone; (5) S-hydroxy-S,?,^-trlmethoxy dihydroflavanone; (7) Odorine A; (8) Odorine B; (9) Odorinol A; (10) Odorinol B. 141 present. In contrast the Thailand A., odorata sample contained larger amounts of odorine B and odorinol A, with odorine A and odorinol B t o t a l l y absent. The Taiwan sample of A. odorata contained only a trace of a g l a i o n d i o l , no a g l a i t r i o l , moderate concentrations of 5-hydroxy-7,4'-methoxy dihydroflavanone, and only traces of the other dihydroflavanones (detected by HPLC only). The amide p r o f i l e of t h i s sample was i d e n t i c a l to the Thai sample, with large amounts of odorine B and odorinol A and no odorine A or odorinol B. Aglaia odoratissima was found to contain small amounts of aglaiondiol and 5,7,4'-trimethoxy dihydroflavanone, and moderate amounts of odorine A and B i n equal proportions. HPLC analysis further revealed very low concentrations of the other dihydroflavanones and odorinol (isomer not i d e n t i f i e d ) . Aalaia argentia appeared to lack aglaiondiol and a g l a i t r i o l e n t i r e l y , but d i f f e r e n t dammaranes may be present as indicated by major components which show s i m i l a r color reactions. Only a small amount of 5,7,4'-trimethoxy dihydroflavanone was detected; the other dihydroflavanones were absent. Odorine A and B were found i n almost a 1:1 r a t i o , and odorinol appears to be absent. E) Insect Bioassays Bioassay-guided f r a c t i o n a t i o n of A., odorata led to a singl e compound, compound 6, with s i g n i f i c a n t growth i n h i b i t o r y a c t i v i t y against £. saucia neonates (Table 3-7). Although i n s u f f i c i e n t material was obtained f o r structure 142 elucid a t i o n , due to the pronounced a c t i v i t y of t h i s compound the dose-response r e l a t i o n s h i p could be established (Figure 2-14). The compound was about four times less active than azadirachtin i n i t s a b i l i t y to i n h i b i t E- saucia growth, with an E C 5 0 of 1.4 pg/g d i e t fwt compared to 0.36 ug/g for azadirachtin. The L C 5 0 was 11.2 ug/g, compared to 4.0 ug/g with azadirachtin. As the active compound was s t i l l admixed with some of the dihydroflavonol, the a c t i v i t y of the pure compound may be s t i l l c l oser to that of azadirachtin. The i n h i b i t o r y a c t i v i t y of compound 6 appeared not to be due to antifeedant a c t i v i t y . In choice tests with neonate £• saucia compound 6 had no s i g n i f i c a n t e f f e c t on feeding at concentrations which i n h i b i t e d growth i n no-choice assays (Table 3-9). On the other hand, the a c t i v i t y of the t o t a l MeOH extract, assayed at 50% of natural concentration, appeared to include an antifeedant e f f e c t (Table 3-9). No E- saucia larvae showed evidence of having died during a f a i l e d molt, but treatment with the known IGR compound azadirachtin also f a i l e d to produce obvious molt i n h i b i t i o n i n t h i s insect (Chapter 4). None of the other compounds i s o l a t e d from A. odorata i n h i b i t e d E- saucia growth or survivorship, over a seven-day assay beginning with neonates, when fed at concentrations equal to or greater than those occurring in planta (Table 3-7). The i s o l a t e d y i e l d of compound 6 was about 3 ng/g leaf dwt. However, an estimation based on the a c t i v i t y of the 143 Table 3-7. Aalaia odorata compounds: concentration bioassayed, and resultant E. saucia growth and survivorship (as % of Control). Values i n a column followed by the same l e t t e r do not d i f f e r s i g n i f i c a n t l y (Tukey's Studentized (HSD1 Range Test. Compound Max. Cone. Bioassayed E. fiflucia growth (%) E« saucia survivorship 1 2 3 4 5 6 7 7' 8 8' 9 10 Aglaiondiol A g l a i t r i o l 5-OH-7,4'-MF 5,7,4'-MF 3-OH-5,7,4'-MF "active" Odorine A Odorine A' Odorine B Odorine B* Odorinol A Odorinol B Amoora A6  1000 ug/g 1000 500 500 500 15 1000 1000 1000 1000 1000 1000 50Q 137.9a 132.0a 110.8a 108.3a 107.l a 2.6 b 114.8a 116.7a 121.2a 89.4a 114.8a 124.7a 103.2a 100 100 100 100 100 31 100 95 100 100 93 100 _10£ 144 Figure 3-14. E f f e c t of Compound 6 on the growth (•) and survivorship (A) of neonate Peridroma saucia. For comparison, the e f f e c t of azadirachtin on the growth (a) and su r v i v a l (&) of neonate Peridroma saucia i s also shown. Error bars indicate + one standard deviation. Larval Growth and Survivorship (% of Control) S+7l crude MeOH extract and assuming a l l the a c t i v i t y was due to compound 6 indicates an expected f o l i a r concentration of 98 fig/g. The discrepancy suggested that s y n e r g i s t i c interactions might be occurring between phytochemical constituents i n A., odorata. Consequently the a c t i v i t y of various combinations of the i s o l a t e d compounds were also assayed (Table 3-8). No combination of these secondary metabolites showed evidence of s y n e r g i s t i c a c t i v i t y . The putative synergist remains u n i d e n t i f i e d . Table 3-8. E f f e c t of Aglaia odorata compounds, tested i n combination, on growth of neonate Peridroma saucia i n a seven-day assay. Growth i s shown as % of Control; values followed by the same l e t t e r do not d i f f e r s i g n i f i c a n t l y (Tukey's Studentized (HSD) Range t e s t , <x=0.05). Compounds are numbered as i n Table 3-7. Compounds Concentrations Bioassayed (ua/a) Mean Growth 1+2+3+4+5+7+8+9 1,2:1000 96. 5 a 3,4,5:200 7,8,9:200 79.3 a b 1+6 1000 + 1 2+6 1000 + 1 68.3 b 3+6 200 + 2 34.6 C 4+6 200 + 2 26.4 C 5+6 200 + 2 28. 8 C 6 2 23. 8 C 7+6 100 + 2 29. 9 C 8+6 100 + 2 36.4° 9+6 100 + 2 22.6°-148 Table 3 -9 . E f f e c t of Compound 6 on d i e t choice by neonate Peridroma saucia. Figures shown indicate the percentage of larvae found on Control (C) or Treated (T) d i e t a f t e r 24 h of feeding; each value i s the mean of twelve r e p l i c a t i o n s . Observations were compared to a 50:50 d i s t r i b u t i o n using a G-test with oc=0.05; values which d i f f e r e d s i g n i f i c a n t l y are indicated by an a s t e r i s k . Also shown i s the l a r v a l growth (as % of control) a f t e r seven days of feeding i n a no-choice bioassay. Concentration Diet Choice Growth (ug/g d i e t dwt^ C T (% of Control) 1 49 51 69.7 2 40 60 36.9 3 44 56 15.3 MeOH extract (50%^ 92 8 - 0.2 Discussion A Phytochemistry Phytochemical examination of Aglaia species corroborated previous reports and added several new natural products to the l i s t . Most of these phytochemicals are unique to Aglaia; only the flavanones naringenin 5,7,4'-methyl ether (Compound 4) and 5-hydroxy-7,4'-dimethyl dihydroflavanone (Compound 3) have been reported from other plants. The f i r s t has been found only once, i n the composite Dahlia  t e n u i c a u l i s (Kaufmann and Lam, 1967; Lam and Wrang, 1975). The second i s known from a va r i e t y of unrelated taxa including Betula spp (Wollenweber, 1975), Prunus s a r a e n t i i (Wollenweber and Dietz, 1981), the composites Dahlia  te n u i c a u l i s (Lam and Wrang, 1975), Eupatorium odoratum (Arene e£ a l . , 1978),and Hieracium intybaceum (Wollenweber, 1984), the c a c t i Rhodocactus gr a n d i f o l i u s and Mamillaria  elongata (Burret gt a l . , 1982), and the fern Pityrogramma spp (Wollenweber and Dietz, 1980). A l l of the dihydroflavanones are here reported from the Meliaceae for the f i r s t time. Flavonoids previously known from Aglaia include glycosides of quercetin and kaempferol (Harborne, 1983). Quite d i s s i m i l a r C-8 prenylated flavonones have previously been reported from Azadirachta indica (Garg and Bhakuni, 1984). O-Methylation at C-5 i s uncommon amongst vascular plants i n general, but i s c h a r a c t e r i s t i c of flavonoids from species of the Rutaceae (Wollenweber and 150 Dietz, 1981; Harborne, 1983); the presence of such compounds in Aglaia may r e f l e c t the close r e l a t i o n s h i p between the Rutaceae and Meliaceae. Wollenweber and Dietz (1981) have commented on the common co-occurrence of methoxylated flavonoids and l i p o p h i l i c terpenoids, a pattern supported by the presence of both methylated flavanones and dammarane triterpenes i n Aglaia. The following discussion of phytochemical differences between samples of Aglaia must be prefaced by a caveat: as only s i n g l e samples were available from each c o l l e c t i o n s i t e , i t cannot be determined at t h i s time whether the var i a t i o n s observed r e f l e c t v a r i a t i o n between i n d i v i d u a l s or populations. Further analysis of large samples from dis c r e t e populations i s required to resolve t h i s question. Dammarane triterpenes are common among the Meliaceae, and some species, such as Cabralea e i c h l e r i a n a . may elaborate a considerable d i v e r s i t y of these compounds (Rao et a l . , 1975). A l l of the Aglaia dammaranes i s o l a t e d to date are characterized by a methylene group at C-20, and oxidation at C-24 and C-25. Hawaiian and Thai samples of A.. odorata contained both aglaiondiol and a g l a i t r i o l i n large amounts; i n contrast, only aglaiondiol was found i n A.. odorata from Taiwan, and i n A., odoratissima. Neither dammarane was present i n A., argentia. but other major compounds which give s i m i l a r color reactions on TLC may represent other dammaranes. 151 Dammarane triterpenes may serve as precursors i n the biosynthesis of euphane and apo-euphane compounds, which are themselves precursors of the limonoids (Devon and Scott, 1972; Nes and McLean, 1977). The production of large amounts of dammaranes, and apparent absence of limonoids (except possibly Compound 6), contrasts with most other Meliaceae and i n p a r t i c u l a r with other members of the t r i b e Aglaieae, and suggests that the pathway to limonoid synthesis i s blocked i n Aglaia. The bis-amide odorine was present i n a l l Aalaia samples examined i n t h i s study, but differences i n the s p e c i f i c diastereomers produced were noted. In a l l A., odorata samples examined odorine B predominates; lesser amounts of odorine A are also present only i n the sample from Hawaii i In contrast A. odoratissima and A., argentia produce the A and B forms i n approximately equal amounts. Odorine was not observed to isomerize i n MeOH i n v i t r o . These r e s u l t s indicate that the production of each diastereomere i s l i k e l y regulated by a separate enzyme. Odorinol was found i n a l l three samples of A., odorata. was present i n very low amounts i n A., odoratissima f and was absent from A. argentia. Odorinol A was the only isomer observed i n the Thai and Taiwan samples of A. odorata; i n the Hawaiian sample odorine A predominated but some odorine B was also present. Again t h i s argues for separate enymatic control for each isomer. The prevalence of the odorines within the genus i s unclear: a l l species examined to date contain odorine, but i n general 152 surveys of plants f o r alkaloids many Aglaia species gave a negative t e s t (Farnsworth et a l . , 1954). Certainly the odorines appear to be unique to Aglaia, and d i f f e r markedly from a l k a l o i d s present i n other Meliaceae such as Dysoxylum species (Aladesanmi and Ilesanmi, 1987; Aladesanmi et a l . , 1988) . The biosynthetic o r i g i n of these compounds i s unclear but they could be derived from ornithine, phenylalanine, and isoleucine. B I n s e c t i c i d a l A c t i v i t y Extracts of Aalaia odorata f o l i a g e from Thailand were as e f f e c t i v e as neem or chinaberry leaf extracts at i n h i b i t i n g the growth of £. saucia neonates. The degree of phytochemical defense appeared to vary between populations (or individuals) of A., odorata, as samples from Taiwan and Hawaii were somewhat less i n h i b i t o r y . Extracts of two other Aa l a i a species, A,, odoratissima and A., argentia. were even less active, i n d i c a t i n g i n t e r s p e c i f i c v a r i a t i o n i n the production of defense compound(s). Similar i n t e r s p e c i f i c v a r i a t i o n was noted i n the a c t i v i t y of Aglaia seed extracts against Spodoptera frugiperda (Mikolajczak et a l . , 1987, 1989) . Several secondary metabolites were present i n A.. odorata f o l i a g e i n high concentrations; however none of them exhibited i n h i b i t o r y a c t i v i t y against E . saucia when tested as pure compounds at e c o l o g i c a l l y relevant concentrations. Flavones and flavonols, e s p e c i a l l y those with catechol 153 substitutions, are known to i n h i b i t the growth of some phytophagous insects (Chan et a l . , 1978; Waiss gt a i . , 1979; E l l i g e r et a i - / 1980, 1981; Isman and Duffey, 1982; Hedin and Waage, 1986) and 5-methoxy flavones have recently been shown to be more i n h i b i t o r y than 5-hydroxy flavonoids (Mahoney et a l . , 1989). However the 5-methoxy and 5-hydroxy flavanones i s o l a t e d from h. odorata were inactive against P. saucia at naturally occurring concentrations, when tested as pure compounds or as mixtures. (-)-Odorinol i s cytotoxic to P-388 leukemia c e l l s j j i v i t r o and i n vivo (Hayashi gt a i . , 1988), but none of the isomers of odorine or odorinol, whether natural or synthetic, had any e f f e c t on £. saucia growth. Dammarane aglycones have apparently not previously been tested for a c t i v i t y against insect herbivores, but dammarane glycosides such as the saponins do i n h i b i t growth of some insects when present i n d i e t at high concentrations (1-5% fwt) (Applebaum and Birk, 1979). Other glycosides, the ginsenocides, produce a v a r i e t y of physiological e f f e c t s i n vertebrates, apparently as a r e s u l t of d i r e c t action on the hypothalamus and p i t u i t a r y r e s u l t i n g i n stimulation of the adrenal glands (Shibata, 1986). Aglaiondiol and a g l a i t r i o l , tested i n d i v i d u a l l y and as a mixture at combinations up to 1,000 ppm/component, were not i n h i b i t o r y to E . saucia growth. I t appears that the growth i n h i b i t o r y a c t i v i t y of A.. odorata may be ascribed almost e n t i r e l y to a single compound 154 (Compound 6). This contrasts with most other plants, where insect resistance may be ascribed to several co-occurring compounds (McKey, 1979). Feeding choice t e s t s indicated that the growth-inhibiting a c t i v i t y of Compound 6 was not due to antifeedant e f f e c t s and must r e f l e c t t o x i c i t y . This was s i m i l a r to the a c t i v i t y of some limonoids including cedrelone and anthothecol (Chapter 4). Although no evidence of IGR e f f e c t s on molting were seen, the known IGR compound azadirachtin also f a i l s to produce obvious e f f e c t s on molting when fed to E . saucia (Chapter 4). The concentration of Compound 6 i n the E t 2 0 extract could not be determined accurately by HPLC, as i t co-chromatographed with the 3-hydroxy-5,7,4'-methoxyflavanone. However, the i s o l a t e d y i e l d (3 ng/g leaf dwt) appeared to be much less than the expected concentration (98 fig/g) estimated from the a c t i v i t y of the crude MeOH extract. The discrepancy suggested the p o s s i b i l i t y of s y n e r g i s t i c interactions between phytochemical constituents i n A.. odorata. Several studies have now indicated the occurrance of additive or s y n e r g i s t i c interactions between phytochemicals i n other plants (Berenbaum, 1985); fo r instance an array of phenolics i n sorghum s i g n i f i c a n t l y deterred feeding by Locusta migratoria. even though i n d i v i d u a l compounds were not deterrent (Adams and Bernays, 1971). Combinations of isobutylamides, mixed i n proportions s i m i l a r to those occurring i n the plant, were more t o x i c than would be expected from simple addition of the 155 a c t i v i t i e s of the i n d i v i d u a l compounds (Miyakado et a l . , 1989), and N-isobutylundecylenamide synergises the a c t i v i t y of natural pyrethroids (Metcalf, 1967; Matsui and Yamamoto, 1971). In some plants, methylenedioxyphenyl (MDP) compounds may synergise t o x i c i t y by i n h i b i t i n g mixed-function based oxidative metabolism of xenobiotics i n the insect. For example, the MDP compounds m y r i s t i c i n , s a f r o l e , i s o s a f r o l e , and fagaramide synergise the t o x i c i t y of co-occurring furanocoumarins i n parsnip (Berenbaum and Neal, 1985; Neal, 1989). However, when various combinations of the Aalaia compounds here i s o l a t e d were tested f o r s y n e r g i s t i c interactions, no combination was more active than would be expected from the simple addition of the a c t i v i t y of the components i n i s o l a t i o n . The putative synergist remains to be i d e n t i f i e d . Although the structure of the active compound was not determined, the pronounced growth i n h i b i t o r y a c t i v i t y of A.. odorata extracts suggests that they may have some appl i c a t i o n i n pest management, p a r t i c u l a r l y i n areas where the plant occurs nat u r a l l y or as a r e s u l t of c u l t i v a t i o n . The phytochemical basis of t h i s a c t i v i t y requires further attention; the work presented i n t h i s Chapter should f a c i l i t a t e future attempts to i s o l a t e and i d e n t i f y the active compound and elucidate the parti c i p a n t s i n the sy n e r g i s t i c i n t e r a c t i o n . 156 Chapter 4 : E f f e c t s of Limonoids from the Rutales on Peridroma saucia and Oncopeltus fasciatus Introduction Limonoids are the most c h a r a c t e r i s t i c secondary metabolites i n the Meliaceae, and are also prominent i n the Rutaceae (reviewed i n the Introduction to t h i s t h e s i s , pg 16-27). Over 300 limonoid structures have been elucidated to date (Taylor, 1987), but only seventy such compounds have been examined fo r b i o l o g i c a l a c t i v i t y against insects. Table 4-1 summarizes the reported e f f e c t s of limonoids on insect feeding and growth; structures for these compounds are given i n Figure 4-1. Examination of t h i s compilation reveals two impediments to developing a quantitative understanding of s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s amongst limonoids. F i r s t l y , very few of the published studies u t i l i z e the same bioassay species; as a r e s u l t i t i s d i f f i c u l t to separate e f f e c t s owing to s t r u c t u r a l differences between compounds from e f f e c t s due to i n t e r s p e c i f i c differences i n the response of the t e s t insects. Secondly, the majority of studies were designed to detect feeding deterrence, and do not indicate non-behavioral e f f e c t s including t o x i c i t y or insect growth regulating (IGR) e f f e c t s . This s i t u a t i o n r e f l e c t s the p r e v a i l i n g b e l i e f that limonoids function p r i m a r i l y as antifeedants. For example, Taylor (1987) commented that "the limonoids seem to be remarkably bereft 157 of physiological properties; we have examined many without fi n d i n g anything beyond the c h a r a c t e r i s t i c b i t t e r t a ste...". In the most comprehensive survey to date, Kubo and Klocke (1987) assayed seventeen limonoids against three lepidopteran species. However, as they reported only E C 5 0 concentrations f o r growth i n h i b i t i o n , the roles of chemosensory (antifeedant) and post-ingestive e f f e c t s i n producing the observed growth i n h i b i t i o n cannot be separated. Despite such l i m i t a t i o n s , Table 4-1 i s of use i n i n d i c a t i n g general q u a l i t a t i v e trends i n s t r u c t u r e - a c t i v i t y r e l a t i o n s . The biosynthesis and evolution of the limonoids has been described recently by Das et a i . (1984, 1987). Biosynthesis apparently proceeds along four major pathways, a l l dominated by trends of increasing oxidation and s k e l e t a l rearrangement (Figure 4-2). If the primary raison d'etre fo r the production of limonoids i s to gain protection against insect herbivory, one may expect to f i n d that evolutionary trends ( i . e . increasing oxidation and s k e l e t a l rearrangement) correspond with increasing a c t i v i t y against insects. In the present study I bioassayed ten limonoids, including representatives of a l l the major classes, f o r growth and feeding i n h i b i t i o n against neonates of the variegated cutworm, Peridroma saucia, and for i n h i b i t i o n of molting (IGR e f f e c t s ) and reproduction against the large milkweed bug, Oncopeltus fascia t u s . The r e s u l t s of these 158 assays, and a compilation of the l i t e r a t u r e to date, were applied to t e s t the hypothesis that there i s a c o r r e l a t i o n between evolutionary advancement of the compounds (as defined by Das e i a i . , 1984,1987) and t h e i r a c t i v i t y against phytophagous insects. 159 Table 4-1. E f f e c t s of limonoids on insect feeding and growth. Abbreviations are: EC 5 0, concentration producing 50% growth i n h i b i t i o n ; F l , feeding i n h i b i t i o n ; GI, growth i n h i b i t i o n ; MI, molt i n h i b i t i o n ; IGR, insect growth regulator; MI, molt i n h i b i t i o n ; IA, in a c t i v e . Limonoid Test Insect E f f e c t i v e cone. Ref. Group 1. Protolimonoids M e l i a n t r i o l Schistocerca gregaria FI 1 0 0=8 ug/cm2 1 Melianone Epilachna v a r i v e s t r i s F l §0.05% 2 Azedarachol Agrotis seietum F I 1 0 0 § 5 0 0 PP m 3 Group 2. Intact apoeuphol skeleton limonoids Azadiron Epilacna v a r i v e s t i s FI 5 0=0.66% 4 Azadiradione H e l i o t h i s zea EC 5 0=250 ppm 5 He l i o t h i s virescens EC 5 0=560 ppm 6 Spodoptera frugiperda EC 5 0=130 ppm 5 Pectinophora gossypiella EC 5 0=42 ppm 5 Epilacna v a r i v e s t i s FI 5 0=0.033% 4 14-Epoxyazadiradione Epilacna v a r i v e s t i s FI 5 0=0.14% 4 7-Deacetylazadiradione H e l i o t h i s zea EC 5 0=3500 ppm 5 Hel i o t h i s virescens EC 5 0=1600 ppm 6 Spodoptera frugiperda EC 5 0=5000 ppm 5 Pectinophora gossypiella EC 5 0=290 ppm 5 7-Deacetyl-17 -hydroxyazadiradione H e l i o t h i s virescens EC 5 0=240 ppm 6 160 7 7 7 7 Diacetyoxyvilasinine Epilachna v a r i v e s t r i s FI 8 Cedrelone O s t r i n i a n u b i l a l i s 50 ppm FI, GI 9 Spodoptera l i t u r a 0.1% FI 10 Spodoptera frugiperda EC5o=2ppm 5 Pectinophora gossypiella EC5Q=3ppm 5 He l i o t h i s zea EC50=8ppm 5 Anthothecol O s t r i n i a n u b i l a l i s 50 ppm FI, GI 9 Spodoptera frugiperda EC50=3ppm 5 Pectinophora gossypiella EC5o=8ppm 5 He l i o t h i s zea EC50=24ppm 5 Nimocinolide Aedes aegypti IGR, LC50=0.625ppm 11 Isonimocinolide Aedes aegypti IGR, LC50=0.74ppm 11 Sendanin He l i o t h i s zea EC 5 0=45 ppm 7 He l i o t h i s virescens EC 5 0=60 ppm 7 Spodoptera frugiperda EC 5 0=11 ppm 7 Pectinophora gossypiella EC 5 0= 9 ppm 7 Trichirokanin H e l i o t h i s zea EC 5 0=41 ppm 7 He l i o t h i s virescens EC5Q=50 ppm 7 Spodoptera frugiperda EC 5 0=11 ppm 7 Pectinophora gossypiella EC 5 0= 9 ppm 7 Toosendanin O s t r i n i a f u r n a c a l i s 20 ppm FI 12 T r i c h i l i n A Spodoptera eridania FI§ 300ppm 13 T r i c h i l i n B Spodoptera eridania FI§ 200ppm 13 T r i c h i l i n C Spodoptera eridania inactive 13 Sendanal H e l i o t h i s zea EC 5 0=400 ppm He l i o t h i s virescens EC 5 0=400 ppm Spodoptera frugiperda EC 5 0=70 ppm Pectinophora gossypiella EC 5 0= 200 pp T r i c h i l i n D Spodoptera eridania FI§ 400ppm 13 Meliatoxin A 2 Spodoptera l i t u r a F l , GI@ 300 ppm 14 Meliatoxin Spodoptera l i t u r a GI@ 400 ppm 14 Group 3. D-ring seco limonoids Gedunin O s t r i n i a n u b i l a l i s 50 ppm, DS 6 Epilachna v a r i v e s t i s 50%FI § 0.1% 4 Pectinophora gossypiella EC 5 0=32 ppm 5 Spodoptera frugiperda EC5o=47 ppm 5 He l i o t h i s zea EC 5 0=50 ppm 5 7-Deacetylgedunin Spodoptera frugiperda EC5o=60ppm 5 Pectinophora go s s y p i e l l a EC50=22ppm 5 He l i o t h i s zea EC50=165ppm 5 7-Ketogedunin Spodoptera frugiperda EC50=800ppm 5 Pectinophora gossypiella EC5o=51ppm 5 He l i o t h i s zea EC50=900ppm 5 Group 9. A.D-ring seco limonoids Limonin Spodoptera l i t u r a 0.5% F l 10 Spodoptera frugiperda E C 5 0 = 756ppm 5 Spodoptera frugiperda PC 9 5=6.12ug/disk 7 Hel i o t h i s zea EC50=900ppm 5 He l i o t h i s zea PC 9c=60.8ug/disc 7 Choristoneura fumiferana IA @ l,000ppm 15 Spodoptera exempta IA § lOOug/disc 16 Eldana saccharina 61%FI @l00ug/disc 16 Maruca t e s t u l a l i s 58%FI § lOud/disc 16 162 Nomilin Spodoptera frugiperda E C 5 0 = 7 2 P P i n 5 Spodoptera frugiperda PC 9 5=0.66ug/disk 7 Spodoptera frugiperda F l 17 He l i o t h i s zea EC50=95ppm 5 He l i o t h i s zea PC 9 5=6.6ug/disk 7 Trichoplusia n i IA 17 O s t r i n i a n u b i l a l i s F l § 50ppm 9 Earias insulana ED50=0.05% 18 Deacetylnomi1in Spodoptera frugiperda IA § 2000ppm 5 Hel i o t h i s zea IA § 2000ppm 5 Pectinophora gossypiella EC50=950ppni 5 Obacunone O s t r i n i a n u b i l a l i s 50 ppm F l , GI 9 Spodoptera exempta 50%FI§100ug/disc 16 Eldana saccharina 79%FI§1 ug/disc 16 Maruca t e s t u l a l i s 61%FI@1 ug/disc 16 Spodoptera frugiperda EC50=70ppm 5 Spodoptera frugiperda PC 5 0=0.60ug/disk 7 H e l i o t h i s zea EC50=97ppm 5 He l i o t h i s zea PC 9 5=6.5ug/disk 7 Rutaevin He l i o t h i s zea PC 9 5=125ug/disc 7 C i t r o l i n Eldana saccharina 55%FI§100ug/disc 16 Maruca t e s t u l a l i s 66%FI@100ug/disc 16 Chorisoneura fumiferana 500 ppm GI 15 Spodoptera exempta IA § lOOug/disc 16 Harrisonin Spodoptera exempta 32%FI§100ug/disc 16 Eldana saccharina 74%FI@ lug/disc 16 Maruca t e s t u l a l i s 69%FI§ lOug/disc 16 Spodoptera exempta 20 ppm F l 19 12 -Acetoxyharrisonin Spodoptera exempta ,500 ppm F l 19,20,7 Eldana saccharina <50%FI@100ug/disc 21 Maruca t e s t u l a l i s >75%FI§100ug/disc 21 Spodoptera exempta IA § lOOug/disc 21 Pedonin Eldana saccharina >75%FI@lug/disc 21 Maruca t e s t u l a l i s >75%FI@10ug/disc 21 Spodoptera exempta <50%FI§100ug/disc 21 163 Groups 4,5. B fD-ring seco limonoids Methylangolensate H e l i o t h i s zea ED 5 0=60 ppm 5 Spodoptera frugiperda ED 5 0=40 ppm 5 Pectinophora gossypiella ED 5 0=15 ppm 5 Entandrophragmin O s t r i n i a n u b i l a l i s FI§500 ppm,DS@50 ppm 9 Bussein O s t r i n i a n u b i l a l i s FI§500 ppm 9 Methyl 3 -isobutyryloxy-l-oxomeliac-8(30)-enate Spodoptera frugiperda FI 22 Group 6. A-ring seco limonoids Evodulone H e l i o t h i s zea EC 5 0=80 ppm 5 Spodoptera frugiperda EC5o=120 ppm 5 Pectinophora go s s y p i e l l a EC 5 0=96 ppm 5 Tecleanine Spodoptera frugiperda EC 5 0=320 ppm 5 Pectinophora gossypiella EC 5 0=210 ppm 5 7-Deacetylproceranone H e l i o t h i s zea EC 5 0=740 ppm 5 Spodoptera frugiperda EC 5 0=350 ppm 5 Pectinophora gossypiella EC 5 0=175 ppm 5 Group 7. A fB-ring seco limonoids P r i e u r i a n i n H e l i o t h i s zea 60-90%FI§6ug/cm 2 23 Spodoptera frugiperda IA §19.8ug/cm 2 23 Epilachna v a r i v e s t i s 60-90%FI§19.8ug/cm 2 23 Prie u r i a n i n acetate H e l i o t h i s zea 60-90%FI § 6ug/cm2 23 Spodoptera frugiperda 60-90%FI @19.8ug/cm2 23 Epilachna v a r i v e s t i s 60-90%FI @1.5ug/cm2 23 Rohitiukin H e l i o t h i s zea IA @19.8ug/cm2 23 Spodoptera frugiperda IA §19.8ug/cm 2 23 Epilachna v a r i v e s t i s IA §19.8ug/cm 2 23 164 Rohitiuka-7 H e l i o t h i s zea  Spodoptera frugiperda  Epilachna v a r i v e s t i s "Tr—A" Agrotis sejetum "Tr-B" Agrotis sejetum "Tr-C" Agrotis seietum 60-90%FI ei9.8ug/cm 2 23 IA §19.8ug/cm 2 23 IA §19.8ug/cm 2 23 FI § 200 ppm FI @ 200 ppm FI @ 200 ppm 24 24 24 Group 10. B-ring seco limonoids Toonacilin Epilachna v a r i v e s t i s 6-Acetyoxytoonaci1in Epilachna v a r i v e s t i s 21-(R,S)-hydroxytoonacilid Epilachna v a r i v e s t i s 2 3-(R,S)-hydroxytoonac i 1 i d Epilachna v a r i v e s t i s FI FI FI§ 25-50 ppm FI§ 2000 ppm 25 25 Group 8. C-ring seco limonoids Azadirachtin Epilachna v a r i v e s t i s FI 5 o=0.0014% 4 Epilachna v a r i v e s t i s MI5o=1.66 ppm 26 H e l i o t h i s zea EC 5 0=0.7 ppm 5 Spodoptera frugiperda EC 5 0=0.4 ppm 5 Pectinophora gossypiella EC 5 0=0.4 ppm 5 7-Acetylazadirachtin Rhodnius prolixus MI50=0.45ug/ml 27 Rhodnius prolixus FI 5 0=30.Oug/ml 27 22,23-dihydro-23B-methoxyazadirachtin Epilachna v a r i v e s t i s IGR 8 Mythimna separata FI,IGR@ 0.01%, 0.1/ig/larva 28 3-Tigloylazadirachtol (=Azadirachtin B) (=Deacetylazadirachtinol) Epilachna v a r i v e s t i s MI5Q=1.30 ppm 26 H e l i o t h i s virescens ED50=0.17 ppm 5 165 l-Cinnamoyl-3-feruloyl-ll-hydroxymeliacarpin (=Azadirachtin D) Epilachna v a r i v e s t i s IGR 8 Epilachna v a r i v e s t i s MI50=1.57 ppm 26 l-Cinnamoyl-3-feruloyl-ll-hydroxy-22,23-dihydro-23-methoxymeliacarpin Epilachna v a r i v e s t i s IGR 8 l-Tigloyl-ll-methoxy-20-acetylmeliacarpinin Epilachna v a r i v e s t i s IGR 8 l-Tigloyl-3-acetyl-ll-methoxyazadirachtinin Epilachna v a r i v e s t i s IGR 8 Azadirachtin C Epilachna v a r i v e s t i s MI 5 0=1 .57 ppm 26 Azadirachtin F Epilachna v a r i v e s t i s MI 5 0=1 .57 ppm 26 Azadirachtin G Epilachna v a r i v e s t i s MI 5 0=1 .57 ppm 26 Nimbinen Epilachna v a r i v e s t i s F I 5 0 = 0 .018% 4 6-Deacetylnimbinen Epilachna v a r i v e s t i s FI 5 0=0 .0082% 4 Nimbandiol Epilachna v a r i v e s t i s FI 5 0=0 .01% 4 6-Acetylnimbandiol Epilachna v a r i v e s t i s FI 5 0=0 .011% 4 Salannin Spodoptera fruqiperda Spodoptera l i t t o r a l i s Earias insulana Acalymma v i t t a t a Musca domestica Epilachna v a r i v e s t i s Epilachna v a r i v e s t i s 50%FI@ 13ug/cm2 0.01% F l 0.01% F l F l 100%FI@0.1% FI 5 0=0.0082% no MI§ 100 ppm 29 30 30 31 32 4 26 3-Deacetylsalannin Epilachna v a r i v e s t i s FI 5 0=0 .0027% 4 Salannol Epilachna v a r i v e s t i s F l 8 Salannolacetate Epilachna v a r i v e s t i s FI 5 0=0 .00085% 4 166 Salannolactame-(21) Epilachna v a r i v e s t i s FI 8 Salannolactame-(23) Epilachna v a r i v e s t i s FI 8 Ochinolide B He l i o t h i s virescens EC 5 0=1500 ppm 7 Spodoptera frugiperda EC 5 0=600 ppm 7 Pectinophora gossypiella EC 5 0= 700 ppm 7 Ochinal H e l i o t h i s zea IA § 1000 ppm 7 He l i o t h i s virescens IA @ 1000 ppm 7 Spodoptera frugiperda IA § 1000 ppm 7 Pectinophora gossypiella EC 5 0= 1800 ppm 7 Volkensin Spodoptera frugiperda 50%FI@3.5ug/cm2 29 Volkensin hydroxylactone Spodoptera frugiperda 50%FI@6ug/cm2 29 Unknown structure Nkolbisonin H e l i o t h i s zea EC 5 0=71 ppm 5 Spodoptera frugiperda EC 5 0=65 ppm 5 Pectinophora gossypiella EC 5 0=20 ppm 5 1) Lavie et a l . , 1967. 2) Kraus and Grimminger, 1980. 3) Nakatani gt a l . , 198 4) Schwinger gt a l . , 1984. 5) Kubo and Klocke, 1986. 6) Lee gt a l . , 1988. 7) Kubo and Klocke, 1981. 8) Kraus gt a l . , 1987. 9) Arnason gt a l . , 1987. 10) Koul, 1983. 11) Naqui, 1987. 12) Chiu and Zhang, 1984. 13) Nakatani et a l . , 1981. 14) Koul gt a l . , unpublished data (1989). 15) A l f o r d and Bentley, 1986. 16) Hassanali gt a l . , 1986. 17) A l t i e r i gt al . , 1984. 18) Weissenburg gt al., 1986. 19) Kubo e t a l - , 1976. 20) L i u gt a l . , 1981. 21) Hassanali and Bentley, 1987. 22) Mikolajczak gt a l . , 1988. 23) L i d e r t gt a l « , 1985. 167 24) Nakatani et a l . , 1984. 25) Kraus et a l . , 1978. 26) Rembold, 1988. 27) Garcia et a l . , 1984. 28) Sankaram et a i - , 1987. 29) Rajab et a i . , 1988. 30) Meisner et a l . , 1981. 31) Reed et a l . , 1981. 32) Warthen et a l . , 1978. 168 Group 1: Proiolimoaoidi Meliaaoae Croup 2: Apo-Esphol LiaoBokU H 7-DeaccryIaxadiradioM O H 7-Deac«yl-17- hydrexjrazadindioM OAc A z a d i r a d i o n e or >OJU 14-epoxyazadiradioaa I Orriunia "A F>Ac Srnriania R - C O C H C H J C H J Trichirokaaia 12 - O H O Trichilia A 12 - O H O Trichilia B Figure 4-1. Structures of limonoids included in Table 3 169 170 Tecleanin 7-DeacetyIproceranone Evodulone OAe CH2CH3 Tr-A" CH3 Tr-C" H Toonacilin OAc 6-AcetoxytoonaciIin "O o Rohiiuka-7 Ac Prierianin accute H Prieriania 171 oco Eniandrophragmin 172 R=Ac Salannin R=H 3-Deacetylsalannin OR R=Ac Nimbinen R=H 6-Deacetylnimbinen Salannolactame-(21) O JO Sallanolactame-(23) R=H Salannol R=Ac Salannolacetate R = OH Volkensin R = O Volkensin hydroxylactone ' —6 OTig Ochinolide B R , 0 Ri R2 R3 R4 Rs Ac Tig COOCH3 H H Ac Tig COOCH3 2H OCH3/H Tig H COOCH3 H H Fer Cin C H 3 H H Fer Cin CH3 2H OCH3/H R , 0 ^ > ^ > X " ' O H Azadirachtin 3-Tigloylazadirachtol l-Cinnamoyl-3-feruloy 1-11 -hydroxy meliacarpin 173 Figure 4-2. Major biosynthetic routes of limonoids i n the Meliaceae. 174 175 Materials and Methods A. Sources of Chemicals Cedrelone, anthothecol, gedunin, nomilin, entandrophragmin, and bussein were obtained from Dr. J.T. Arnason, University of Ottawa, Canada. Harrisonin, obacunone, and pedonin, extracted from Harrisonia abyssinica (Hassanali et a l . , 1986, 1987) were provided by Dr. A. Hassanali, ICIPE, Nairobi. The puri t y of a l l compounds was assessed by HPLC, and compound i d e n t i t y and puri t y were also confirmed by 1H-NMR (400 MHz). Structures of a l l compounds are shown i n Figure 4-3. In many of the experiments the azadirachtin used was iso l a t e d by Dr. J . Kaminski, University of Ottawa. However, i n i t i a l experiments were done with azadirachtin i s o l a t e d according to an adaptation of the methods of Uebel gt a l (1979) and Yamasaki gt a_l. (1986). Azadirachta indica o i l (100 ml) (also supplied by Dr. J.T. Arnason) was d i l u t e d to 250 ml i n MeOH, defatted with three p a r t i t i o n i n g s against equal volumes of hexane, and then extracted three times with equivalent volumes of E t 2 0 . The E t 2 0 phase was concentrated under vacuum, and chromatographed i n 0.5 g l o t s by spinning-plate preparative TLC (Chromatotron, Model 7924, Harrison Research, Palo Alto, C a l i f o r n i a ) using a 2mm plate (Si gel 60 P F 2 5 4 , Merck). The solvent system consisted of 200 mis each of Et 2 0 , Et 20:Acetone (95:5), Et 20:Acetone (3:1), Et 20:Acetone (1:1), and f i n a l l y 100% MeOH; flow was 4 mis/ 176 Figure 4 -3 . Structures of limonoids examined i n t h i s s tudy. 177 AZADIRACHTIN BUSSEIN OCOCHMe, 178 min., and 8 ml fr a c t i o n s were c o l l e c t e d . Fractions were monitored for the presence of azadirachtin by a n a l y t i c a l HPLC (Varian Model 5000), using a MCH-10 5 x 250 mm column, MeOH:H20 (1:1), flow = l ml/ min. Peak detection was monitored at 217 nm, using a Varian Series 634 detector and a Spectra-Physics SP4100 recording integrator. Under these conditions the retention time of pure azadirachtin was 6.5 min. Fractions containing azadirachtin were pooled and rechromatographed using a MCH-10 1.0 x 30 cm semipreparative column with i s o c r a t i c ACHN:H20 67:33, flow = 3 ml/ min; 6 ml frac t i o n s were c o l l e c t e d . The i d e n t i t y and puri t y of the azadirachtin so obtained was established by co-chromatography ( a n a l y t i c a l HPLC) and 1H-NMR spectroscopy. The y i e l d of azadirachtin from t h i s o i l sample was quite low, about 1 mg/ 100 ml. Solvents were purchased from BDH and a l l except MeOH were r e d i s t i l l e d p r o i r to use. HPLC grade solvents were degassed p r i o r to use. B. Insects Peridroma saucia (Lepidoptera: Noctuidae) was obtained from a laboratory colony maintained as described previously (Chapter 2). Oncopeltus fasciatus (Hemiptera: Lygaeidae) nymphs were obtained from a colony maintained at room temperature on dry seeds of Asclepias speciosa. Water was supplied from a moist cotton r o l l , and cotton was provided as an ovi p o s i t i o n s i t e . 179 C. Growth Studies For growth studies, compounds i n methanol were added to the dry components of the a r t i f i c i a l d i e t (Velvetbean c a t e r p i l l a r d i e t , Bioserve Inc., Frenchtown, N.J. # 9682), the solvent was evaporated i n a fume hood, and the d i e t was prepared i n the usual manner. A l l compounds except azadirachtin were assayed at 0.01, 0.05, and 0.5 nmol/ q d i e t fwt. Two grams of d i e t and 10 neonate £. saucia were placed i n each of three 30 ml p l a s t i c cups per treatment; t h i s design made the most e f f i c i e n t use of scarce supplies of most of the compounds, and preliminary experiments indicated that both l a r v a l growth and survivorship up to seven days were independent of l a r v a l density. The en t i r e bioassay was repeated three times except i n the case of nomilin, where li m i t e d amounts of compound allowed only two r e p l i c a t i o n s . Larvae were reared under 16L:8D and 26° C, i n a c l e a r p l a s t i c box l i n e d with moistened paper towels to maintain high humidity. After seven days surviving larvae were counted and weighed. Live l a r v a l weights were l o g 1 0 transformed to correct f o r heteroscedasticity before analysis by ANOVA and Tukey's Studentized (HSD) Range Test. The e f f e c t of azadirachtin on growth and s u r v i v a l of neonate E- saucia was assessed i n two experiments. In the f i r s t , neonate E- saucia were reared f o r seven days on a r t i f i c i a l d i e t containing 0.5, 1.5, 4.5, 15, or 45 nmol/g d i e t fwt (0.36, 1.08, 3.24, 10.8, and 32.4 fxq/q d i e t fwt). 180 Control d i e t was treated with solvent (MeOH) alone. Larvae were reared, three to a cup (10 cups/ treatment) i n 30 ml cups with 2 g fwt d i e t , as previously described. Surviving larvae were counted and weighed a f t e r seven days. The experiment was re p l i c a t e d three times; l a r v a l weights were recalculated as % of control and loglO transformed p r i o r to analysis by l i n e a r regression against loglO dose. In the second experiment, neonate larvae (30 per treatment) were reared on d i e t containing 0, 0.15, 0.5, or 1.5 nmol azadirachtin/g fwt d i e t (0, 0.11, 0.36, or 1.08 ng/g r e s p e c t i v e l y ) . Conditions were the same as i n the f i r s t experiment, except that larvae were reared i n d i v i d u a l l y a f t e r the f i r s t seven days, as they tend to become c a n n i b a l i s t i c i n l a t e r i n s t a r s . Larvae were checked every 2-3 days and fresh d i e t was provided ad l i b . S i x t h - i n s t a r larvae were transferred to p l a s t i c cups containing s t e r i l e moistened s o i l to f a c i l i t a t e pupation. At t h i s point larvae were checked f o r pupation every two days. Parameters measured included % of larvae pupating, time to pupation, pupal weights, and % adult emergence. D. Feeding Assays Antifeedant e f f e c t s were examined using a simple choice t e s t . P e t r i dishes (5 cm diameter) were marked into quadrants, and control and treated d i e t cubes were placed i n alternating quadrants. Ten neonate £• saucia were released i n the center of t h i s feeding arena; the dishes were then 181 put into an opaque box to eliminate phototactic e f f e c t s . The number of larvae on the d i e t and i n each quadrant was determined a f t e r 24 h. Feeding was confirmed by the presence of f r a s s . Azadirachtin was tested at 0.5, 1.5, and 4.5 nmol/g d i e t fwt; other compounds were tested at 0.5 /imol/ g d i e t , and the assay was r e p l i c a t e d s i x times. Responses were compared to a random d i s t r i b u t i o n of larvae, expected i n the absence of antifeedant e f f e c t s , using a G-t e s t . E. N u t r i t i o n a l Analyses Growth, consumption, and dietary use by t h i r d i n s t a r £. saucia larvae ( s t a r t i n g weight, 11.2 + 0.9 mg, n=20/ treatment) were determined. Larvae were allowed to feed ( i n d i v i d u a l l y ) on about 1 g fwt of d i e t containing 0, 0.15, 0.5, or 1.5 nmol azadirachtin/g d i e t fwt. A f t e r three days the larvae, f r a s s , and uneaten d i e t were separated and dried to constant weight at 60° C. I n i t i a l dry weights of the larvae and d i e t s were calculated from fwt/dwt r a t i o s derived by determining the fwt/dwt r a t i o of 10 larvae and 5 d i e t aliquots at the s t a r t of the experiment. Relative growth rate (RGRi) and r e l a t i v e consumption rate (RCRi) were calculated r e l a t i v e to the i n i t i a l rather than the mean weight of the larvae, as t h i s measure i s independent of the ECI (Farrar et a l . , 1989). The e f f i c i e n c y of conversion of ingested (ECI) and digested (ECD) d i e t , and the approximate 182 d i g e s t i b i l i t y (AD), were calculated according to Reese and Beck (1976). RGRi = (Final l a r v a l weight - I n i t i a l l a r v a l weight /D I n i t i a l l a r v a l weight RCRi = Weight d i e t consumed /D I n i t i a l l a r v a l weight ECI = (Final l a r v a l weight - i n i t i a l l a r v a l weight) X 100 Weight d i e t consumed ECD = (Final - i n i t i a l l a r v a l weight) X 100 (Weight d i e t consumed - frass) AD = (Weight d i e t consumed - frass) X 100 Weight d i e t consumed F. Molt I n h i b i t i o n Assays The e f f e c t of limonoids on molting was assessed by t o p i c a l l y applying compounds i n acetone to the dorsal abdominal tergae of staged (< 24 h) f i f t h i n s t a r Q_. fasciatus nymphs. Each compound was tested twice (10 nymphs per rep l i c a t e ) at 10, 25, and 50 pg/ nymph. Cedrelone was tested three times at 5, 10, 15, 25, and 50 ng/ nymph. Azadirachtin was assayed at 1, 2, 3, 5, 8, 10, 15, and 20 ng/nymph. After a p p l i c a t i o n of the t e s t compound, nymphs were maintained i n 10 cm diameter glass p e t r i dishes at 27° C, 16L:8D, and provided with dry seeds of Asclepias speciosa and a 183 moistened cotton r o l l . Molting was scored according the following scale: category I was a normal molt, category II was a molt to a deformed adult (crumpled wings or i n a b i l i t y to completely shed the exuvia), category III was mortality during a molt attempt, and category IV was death without i n i t i a t i n g a molt attempt. Responses were compared by probit analysis (SAS, 1988) with categories I and II pooled as survivors and categories III and IV pooled as m o r t a l i t i e s . Following the molt, adults were maintained as described for the nymphs, but with cotton as an o v i p o s i t i o n s i t e , f o r a further two weeks and the occurrence of mating behavior, eggs, and neonates was recorded. G. Correlation Between Evolutionary Advancement and A c t i v i t y  Against Insects E C 5 0 values ( i n /imol/g d i e t fwt) of a l l limonoids tested here were correlated with measurements of molecular oxidation ( O ) and rearrangement ( S ) given by Das et a l . (1984, 1987). The same analysis was also applied to E C 5 0 values f o r 17 limonoids and three lepidopteran species given by Kubo and Klocke (1987). To eliminate E C 5 0 differences due to differences i n the molecular weight of compounds, a l l E C 5 0 values were converted from ppm to /imol/g. 184 Results A. Growth and Feeding Studies: Limonoids Other than  Azadirachtin Of the compounds tested, other than azadirachtin, cedrelone and anthothecol were the most i n h i b i t o r y to neonate E-saucia growth. Both compounds reduced growth to about 10% of control growth when incorporated into d i e t at 0.5 /mol/g d i e t fwt (Table 4-2); neither compound had any e f f e c t at 0.05 /xmol/g. (Control larvae mean weights were 45-70 mg a f t e r seven days.) Survivorship was not affected at 0.5 rtmol/g. The growth i n h i b i t i o n was apparently not due to a chemosensory antifeedant e f f e c t as neither compound s i g n i f i c a n t l y influenced neonate d i e t choice i n a 24 h assay (Table 4-2). Gedunin, s i m i l a r to cedrelone except f o r an epoxylactone D r i n g , had no e f f e c t on growth, survivorship, or feeding at 0.5 /imol/g. The A,D-seco limonoids obacunone and nomilin also d i d not a f f e c t d i e t choice or growth of E-saucia. Harrisonin had an antifeedant e f f e c t against neonate larvae, but t h i s must have been temporary as the l a r v a l weights did not d i f f e r s i g n i f i c a n t l y from the controls at the end of seven days of growth. The related spirolactone compound pedonin was not antifeedant and stimulated growth, as the larvae weighed s i g n i f i c a n t l y more than the controls by day seven. 185 Table 4-2. E f f e c t of limonoids on growth and d i e t choice of neonate Peridroma saucia. A l l compounds were administered at 0.5 /xraol/g d i e t fwt; concentrations i n ppm are also given. Larval growth i s given as % of control growth; numbers followed by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t (Tukey's Studentized [HSD] Range Test). In the choice t e s t s , l a r v a l d i s t r i b u t i o n i s compared to a n u l l hypothesis d i s t r i b u t i o n of 50:50 using a G-t e s t ; d i s t r i b u t i o n s d i f f e r i n g from 50:50 are indicated by an asteris k . Compound Cone. Growth Percentage of larvae on (% of Control) Control (C) or Treated (T) d i e t . (ppm) (mean + S.D.) C T Cedrelone 211 10.8 + 3.2 d 57 43 Anthothecol 248 12.9 + 7.5 d 57 43 Harrisonin 259 89.2 + 22.6 b 80 20* Obacunone 227 110.8 + 11. 8 b 61 39 Nomilin 265 118.3 + 18.3 b 63 37 Gedunin 249 96.8 + 23.7 b 43 57 Pedonin 250 133.3 + 8.6a 63 37 Entandrophragmin 416 96.8 + 18.3 b 66 34 Bussein 420 64.5 + 19.5°- 67 33 186 Of the B,D-seco limonoids tested, entandrophragmin did not a f f e c t P_. saucia feeding or growth at 0.5 jzmol/g d i e t fwt. Bussein s i g n i f i c a n t l y reduced l a r v a l growth (to 64.5% of the c o n t r o l s ) , although t h i s e f f e c t was much les s marked than was the case with cedrelone or anthothecol. Bussein did not s i g n i f i c a n t l y deter feeding by neonates i n the choice t e s t . B. Growth Studies with Azadirachtin Of the limonoids examined i n t h i s study, azadirachtin produced the greatest i n h i b i t i o n of growth and survivorship; as a r e s u l t i t s e f f e c t on E . saucia growth was examined i n more d e t a i l than were other compounds. In the seven-day assay, both growth and survivorship were reduced i n a dose-dependent manner (Figure 4-7); the E C 5 0 was 0.4 nmol/g d i e t fwt (0.29 M9/g d i e t fwt) and the L C 5 0 was 5.2 nmol/g d i e t fwt (3.7 /ig/g). Although a few larvae were seen with slipped head capsules or a half-shed band of c u t i c l e , i n most cases mortality was not obviously due to molt f a i l u r e . These e f f e c t s continued throughout the l i f e cycle of the insect. Regression analysis indicated a s i g n i f i c a n t negative e f f e c t of azadirachtin (at 0.15, 0.5 and 1.5 nmol/g d i e t fwt) on the % of larvae pupating (r 2=0.94, p< 0.01), and on pupal weight (r 2=0.94, p< 0.01) (Table 4-3). At 0.5 nmol/g, only 13% of the larvae pupated; only one larva survived to pupation at 1.5 nmol/g. Pupal weight at the Figure 4 - 4 . E f f e c t of dietary azadirachtin on growth and survivorship of Peridroma saucia neonates. Growth and survivorship were determined a f t e r seven days of feeding on azadirachtin-treated d i e t ; each point represents the mean of three r e p l i c a t i o n s , normalized to % of c o n t r o l . Azadirachtin Cone (nmot/g diet fwt) 189 Table 4 - 3 . Effect of azadirachtin on Peridroma saucia pupation and adult emergence. Concentration % Time to Pupal % Adult (nmol/g diet) Pupation Pupation wgt (mg) Emergence Control 75 24.8 ± 1.9 319.9 +44.1 75 0.15 60 26.3 ± 3.2 300.8 T 39.9 40 0.5 13 30.2 ± 4.8 282.3 ± 42.1 0 1 ^ 3 22 25Q.7 Q 190 higher dose was 78% of the controls. The time to pupation was increased i n a dose-dependent manner (r 2=0.83, p< 0.05), with controls requiring 24.8 ± 1.9 days fo r development, and larvae fed d i e t with 0.5 nmol/g azadirachtin requiring 30.2 days. None of the larvae reared on d i e t containing 0.5 or 1.5 nmol azadirachtin/g completed development to the adult stage; adult emergence was only 40% at 0.15 nmol azadirachtin/g d i e t , compared to 69% emergence fo r the controls. The data indicate an E C 5 0 f o r pupation of 0.16 nmol/g. C. Feeding Choice Tests with Azadirachtin In choice t e s t s , azadirachtin i n h i b i t e d feeding by neonate P.. saucia (Table 4-4). After only one hour there were s i g n i f i c a n t l y more larvae on control d i e t than on d i e t treated with 1.5 or 4.5 nmol azadirachtin/g fwt. A f t e r 24 h neonates were s i g n i f i c a n t l y deterred by 0.5 nmol azadirachtin/g d i e t as w e l l . As the s e n s i t i v i t y of larvae to antifeedants i s known to decline with age i n some cases (Reese, 1977; Isman and Duffey, 1982; Isman et a l . , 1989), these choice t e s t s were repeated with 6 day old second i n s t a r P. saucia larvae, which had not previously been exposed to azadirachtin. A f t e r 24 h, larvae were deterred by azadirachtin only at the 4.5 nmol/g l e v e l ; at 1.5 nmol/g there was no preference f o r eit h e r treated or control d i e t . 191 Table 4-4. E f f e c t of azadirachtin on d i e t choice by neonate and s i x day old, t h i r d i n s t a r Peridroma saucia. Values which d i f f e r from a 50:50 d i s t r i b u t i o n (G-test) are indicated with an a s t e r i s k . Azadirachtin % of larvae on control [C] or concentration treated [T] d i e t a f t e r 24 h Neonates Third Instar fnmol/a d i e t fwt) _C T_ C T 0 50 50 53 47 0.15 36 64 59 41 0.5 70 30* 1.5 89 11* 47 53 4.5 86 14* 72 28* 192 D. Diet U t i l i z a t i o n Experiments In a d e t a i l e d study of growth, feeding, and dietary u t i l i z a t i o n , azadirachtin reduced the r e l a t i v e growth rate (RGRi) of t h i r d - i n s t a r p_. saucia i n a dose-dependent manner (r 2=0.90, p<0.01) (Table 4-5). This e f f e c t was l a r g e l y owing to a dose-dependent decrease i n the r e l a t i v e consumption rate (RCRi) (r 2=.85, p<0.05). E f f e c t s on e f f i c i e n c y of conversion of ingested and digested food (ECI and ECD respectively) were not s i g n i f i c a n t l y correlated with azadirachtin dose; subsequent ANOVA and means-comparison analysis (Tukey's Studentized Range Test) indicated that both indices were s i g n i f i c a n t l y lower than the controls only at the highest azadirachtin dose tested. The approximate d i g e s t i b i l i t y (AD) was also not correlated with the azadirachtin dose, but was s i g n i f i c a n t l y higher than the control at 1.5 nmol azadirachtin/g. Analysis of the r e l a t i o n s h i p between RCRi and RGRi showed that a l l treatment groups f i t a regression l i n e of RGRi = 3.244*RCRi + 4.450 r 2=0.85, df=2, p<.05 although there were between-treatment differences i n the maximum values f o r RCRi (Figure 4-8). These r e s u l t s indicate that, i n P. saucia, the observed growth i n h i b i t i o n i s due to reduction i n feeding and not to d i r e c t t o x i c i t y (cf. B l a u e t a l . , 1978). 193 Table 4 - 5 . E f f e c t of azadirachtin on t h i r d i n s t a r Peridroma  saucia growth and n u t r i t i o n . Means i n a column with the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t (Tukey's Studentized Range (HSD) Test, a=0.05). An asterisk following a column heading indicates that index has a s i g n i f i c a n t o v e r a l l negative r e l a t i o n s h i p with azadirachtin dose. Treat RGRi* RCRi* ECI ECD AD Control 2.90 10.53 27.65 a 59.16 a 46.74 a 0.15 2.42 10.20 23.74 a 56.65 a 41.91 a 0.5 1.44 5.77 24.89 a 60.77 a 40.96 a 1.5 0.45 3.76 12.08^ 20.13^ 60.00^ Treat=treatment (/ig/g insect fwt), RGRi=Relative Growth Rate based on i n i t i a l wgt, RCRi=Relative Consumption Rate based on i n i t i a l wgt, ECI=Efficiency of Conversion of Ingested Food, ECD=Efficiency of Conversion of Digested Food, and AD=Approximate D i g e s t i b i l i t y . Values were calculated according to Reese and Beck (1976) except for the RGRi and RCRi, which were calculated according to Farrar e_£ a l . (1989). 194 Figure 4-5. Plot of RGR against RCR f o r larvae of Peridroma  saucia fed d i e t containing various concentrations of azadirachtin. For c l a r i t y , only every second data point i s ploted f o r each treatment. A l l treatments do not d i f f e r s i g n i f i c a n t l y from the regression equation: RGRi = 3.244*RCRi + 4.450 r 2 = 0.85 p<0.05 20 15 -a Control • 0.5 nmol/g • 1.5 nmol/g • 4.5 nmol/.g tr o 10 r— 1 0 — i — 2 0 —I— 30 40 RCRI 196 E. Molt I n h i b i t i o n Assays At 50 ug/nymph, only azadirachtin and cedrelone had any e f f e c t on molting of f i f t h i n s t a r O. fascia t u s. Tests with cedrelone were repeated at 5, 10, 15, 25, and 50 ^g cedrelone/nymph; these indicated a dose-dependent range of e f f e c t s which were divided into response categories as described previously. Cedrelone doses as low as f i v e /ig/nymph had a marked e f f e c t on molting success (Figure 4-5); at t h i s dose most nymphs molted to adults with curled wings (category I I ) , but some died a f t e r i n i t i a t i n g ecdysis (category I I I ) . At 10 and 15 pg cedrelone/nymph, an increasing proportion of the treated nymphs showed category III and IV responses; at 25 /ig/nymph, a l l responses f e l l into these two categories (each accounting for 50% of the nymphs). At the highest dose tested, 50 Mg/nymph, 70% of the nymphs died without molting, and 30% died i n a f a i l e d molt attempt. I f a l l category I and II responses are pooled as survivors, and a l l category III and IV responses are considered as m o r t a l i t i e s , the MD 5 0 (dose producing molt i n h i b i t i o n i n 50% of the treated nymphs) of t o p i c a l l y applied cedrelone was 12.24 ng/nywph (95% f i d u c i a l l i m i t s 9.29-16.09 jig/nymph). Azadirachtin was about f i v e thousand times more active than cedrelone i n the milkweed bug assay (Figure 4-6), but the same spectrum of e f f e c t s was produced. Even at 1 ng/nymph, most larvae showed a category II response, and a 197 Figure 4-6. E f f e c t of cedrelone on molting success i n Oncopeltus fa s c i a t u s . Responses were scored as: Category I, normal molt to an undeformed adult; Category I I , sucessful molt to a deformed adult (curled wings or legs ) ; Category I I I , death during a f a i l e d molt attempt; Category IV, death without i n i t i a t i n g ecdysis. Category I HI Category Ii III Category III % of treated population in category Category IV Control teg jamwmw III 11 III III i f 11 11 s i t 1 ; M 25 cedrelone dose ug/nymph 50 199 Figure 4 -7 . E f f e c t of azadirachtin on molting success i n Oncopeltus f a s c i a t u s . Responses were scored as: Category I, normal molt to an undeformed adult; Category I I , sucessful molt to a deformed adult (curled wings or l e g s ) ; Category I I I , death during a f a i l e d molt attempt; Category IV, death without i n i t i a t i n g ecdysis. H H Category I HH Category II 1111 Category III flU Category IV % of treated population in category Control 1 2 3 5 10 20 30 100 500 Azadirachtin dose ng/ nymph N5 O O 201 few died with and without i n i t i a t i n g ecdysis. At 10 /xg/nymph, no normal molts were recorded, and most nymphs died without i n i t i a t i n g ecdysis. Again pooling; category I and II responses, the MD 5 0 of azadirachtin was 3.0 ng/nymph. To check against the p o s s i b i l i t y that the r e s u l t s of the assays with cedrelone could be due to contamination of the cedrelone sample with traces of azadirachtin, I examined the purity of the cedrelone sample by HPLC and NMR. Neither technique indicated the presence of azadirachtin or other contaminants i n the cedrelone sample. Adults obtained from the molt i n h i b i t i o n assays were maintained for two weeks to check for possible long-term e f f e c t s on fecundity and f e r t i l i t y . Adults which had been treated with cedrelone and azadirachtin died within 48 h of molting without mating. Adults from a l l other treatments began mating 3-4 days a f t e r molting, and by the end of the observation period a l l treatments had produced vi a b l e eggs, indicated by the presence of f i r s t - i n s t a r nymphs i n the rearing arenas. F. Relationship of Anti-insect A c t i v i t y to Oxidation and  Skeletal Rearrangement No r e l a t i o n s h i p was found between the degree of b i o l o g i c a l a c t i v i t y of the limonoids studied herein ( i n terms of growth i n h i b i t i o n against £. saucia) and the degree of oxidation or rearrangement of the o r i g i n a l apo-euphol skeleton, as defined by Das gt a l (1984). However, only four compounds 202 exhibited appreciable b i o a c t i v i t y , an inadequate sample for comparison. The analysis was therefore extended to the comparative E C 5 0 data f o r seventeen limonoids and three lepidopterans published by Kubo and Klocke (1986). A l l E C 5 0 values were recalculated i n terms of nmol/g d i e t before comparison by regression analysis to measurements of oxidation and s k e l e t a l rearrangement given by Das et a l (1984). Separate regressions were performed using E C 5 0 data from each lepidopteran species. None of these comparisons indicated a s i g n i f i c a n t r e l a t i o n s h i p between the growth i n h i b i t o r y a c t i v i t y of limonoids and the degree of molecular oxidation and rearrangement (Figure 4-9); indeed the most active limonoids were those with the most (azadirachtin) or the l e a s t (cedrelone) derivation from the o r i g i n a l apo-euphol skeleton. Compounds with intermediate degrees of rearrangement and oxidation appeared to be the l e a s t a c t i v e . 203 Figure 4-8. Comparison of insect growth i n h i b i t i n g a c t i v i t y of limonoids with measurements of oxidation and s k e l e t a l rearrangement. The values f o r S and O used are from Das et a l (1984); the values for insect growth i n h i b i t i o n are ED 5 0 data for Spodoptera frugiperda, given by Kubo and Klocke (1987), corrected to /imol/g d i e t and log transformed. Neither S nor O are s i g n i f i c a n t l y related to the i n h i b i t o r y a c t i v i t y of the limonoids. Log ED50 (S. frugiperda) Log ED50 (S. frugiperda) 205 Discussion For purposes of discussion, compounds are grouped according to s k e l e t a l c l a s s as given i n F i g . 4-2. A. Group 2 Limonoids Cedrelone and anthothecol are simple limonoids which r e t a i n an i n t a c t apoeuphol skeleton (Fig. 4-3). Cedrelone occurs i n a v a r i e t y of Meliaceae including species of Cedrela, Chuckrasia, Khaya, and Toona; anthothecol d i f f e r s only i n possessing an acetate su b s t i t u t i o n at C l l of the C r i n g , and i s found i n species of Khaya (Das et a l . , 1984). Neither compound affected £. saucia growth at 0.05 /imol/ g d i e t , but at 0.5 /imol/ g d i e t both cedrelone and anthothecol reduced growth by almost 90% compared to the controls. This e f f e c t was evidently not due to a chemosensory antifeedant e f f e c t as neither compound had any e f f e c t on d i e t choice by neonate E . saucia i n the antifeedant assays. Cedrelone and anthothecol were also highly e f f e c t i v e growth i n h i b i t o r s against the European corn borer, O s t r i n i a  n u b i l a l i s (Arnason et ajL. , 1987). This a c t i v i t y included feeding i n h i b i t i o n at high doses, 50 and 500 ppm. Both limonoids reduced growth by reducing the e f f i c i e n c y of conversion of ingested and digested d i e t at lower doses at which consumption was a c t u a l l y enhanced (10 and 30 ppm), a r e s u l t which p a r a l l e l s my observation of growth i n h i b i t i o n 206 without antifeedant e f f e c t s i n E- saucia. In another study, cedrelone and anthothecol were the most i n h i b i t o r y of 17 limonoids (other than azadirachtin), reducing the growth of three lepidopterans, Spodoptera frugiperda. H e l i o t h i s zea, and Pectinophora gossypiella (Kubo and Klocke, 1986). Cedrelone also had growth i n h i b i t o r y a c t i v i t y against Spodoptera l i t u r a at 0.1%; i n t h i s case the observed i n h i b i t i o n could be e n t i r e l y ascribed to reduced consumption, due e i t h e r to behavioral or physiological e f f e c t s (Koul, 1983). R. saucia appears to be r e l a t i v e l y i n s e n s i t i v e to cedrelone and anthothecol, as growth i n h i b i t i o n occurred only at doses 100 times the E C 5 0 l e v e l s reported by Kubo and Klocke (1986) and 10 times the i n h i b i t o r y concentrations for O s t r i n i a n u b i l a l i s (Arnason et ai. , 1987). A marked difference was noted i n the response of 0. fasciatus nymphs to the two compounds. Cedrelone affected molting at doses as low as 5 ug/nymph, and had an L D 5 0 of 16.4 ug/nymph. Nymphs which molted successfully at the lower doses a l l exhibited deformities of the wings and died within 48 h of molting, without mating. These e f f e c t s were s i m i l a r to those r e s u l t i n g from much lower doses of azadirachtin ( L D 5 0 = 3.0 ng/nymph). Although cedrelone was not observed to i n h i b i t the molting of eith e r P. saucia (t h i s study) or of O. n u b i l a l i s (Arnason et ai., 1987), Spodoptera frugiperda, or H e l i o t h i s zea (Kubo and Klocke, 1986), i t has been reported to i n h i b i t ecdysis i n the pink 207 bollworm Pectinophora gossypiella at 150 ppm i n d i e t (Kubo and Klocke, 1986). The acetate substitution at C l l of the C ri n g abolished the mo l t - i n h i b i t i n g a c t i v i t y , as nymphs treated t o p i c a l l y with anthothecol doses up to 50 ug/nymph molted to undeformed adults which susequently mated and produced viable eggs. The nature of substituents at C12 of the C r i n g are known to markedly a f f e c t the antifeedant a c t i v i t y of limonoids. For example, the antifeedant a c t i v i t y of v i l a s i n i n e derivatives i s reduced by substituents at C12 (Pohnl, 1985). S i m i l a r l y , acetylation at C12 reduces the antifeedant a c t i v i t y of harrisonin 25-fold (Kubo et a l . , 1976). T r i c h i l i n s require an OH substitution at C12; a l t e r a t i o n to a ketone or acetate markedly reduce the antifeedant a c t i v i t y (Nakatani gt a l . , 1981) The r e s u l t s reported here suggest that substitutions at the C l l p o s i t i o n may also be important i n determining IGR a c t i v i t y . These r e s u l t s also indicate that even simple limonoids can have marked physiological e f f e c t s , and cannot be considered "harmless deterrents" (sensu Bernays and Graham, 1988). B. D-seco Limonoids Other comparisons suggest the importance of the epoxide sub s t i t u t i o n on an i n t a c t D r i n g . Cedrelone and anthothecol, the most active of the limonoids here tested, have such a su b s t i t u t i o n . Gedunin, which d i f f e r s i n that the D r i n g i s oxidized to a ^-lactone, as well as having 208 d i f f e r e n t substitutions on a saturated B r i n g , lacks antifeedant or IGR a c t i v i t y against £. saucia and Q_. fas c i a t u s . Gedunin was also much less active than cedrelone or anthothecol against O. n u b i l a l i s (Arnason e_£ a l . , 1 9 8 7 ) , P. go s s y p i e l l a . S_. frugiperda, and H. zea (Kubo and Klocke, 1 9 8 7 ) . This reduced a c t i v i t y i s not due to saturation i n the B r i n g since nimocinol, comparable to cedrelone except for the B r i n g saturation, has IGR, a n t i f e r t i l i t y , and molt i n h i b i t i n g a c t i v i t y against Musca domestica (Siddiqui et a l . , 1 9 8 8 ) and Aedes aegypti (Naqui, 1 9 8 7 ) . B r i n g substituents can, however, a f f e c t antifeedant a c t i v i t y as 7-deacetylgedunin and 7-ketogedunin are both less active than gedunin (Kubo and Klocke, 1 9 8 7 ) . Overall i t seems c l e a r that oxidation of the D r i n g epoxide to an epoxylactone r e s u l t s i n loss of IGR a c t i v i t y and a marked decrease i n antifeedant a c t i v i t y . Nevertheless t h i s type of oxidation i s c h a r a c t e r i s t i c of a l l known rutaceous and simaroubaceous limonoids, and i s t y p i c a l of most meliaceous limonoids as well (Das et a l . , 1 9 8 4 ) . C. A fD-seco Limonoids In addition to t h e i r c h a r a c t e r i s t i c D r i n g structure, t y p i c a l l y limonoids from the Rutaceae and Simaroubaceae have undergone Bayer-Villager oxidation of the A r i n g (A seco-limonoids) (Dreyer, 1 9 8 3 ) . None of the A,D-seco limonoids tested here (obacunone, nomilin, harrisonin, and pedonin) produced IGR or a n t i f e r t i l i t y e f f e c t s i n O. fa s c i a t u s . 209 Harrisonin had antifeedant a c t i v i t y i n a choice t e s t against neonate P. saucia, but did not s i g n i f i c a n t l y a f f e c t l a r v a l growth i n a seven-day no-choice bioassay. The antifeedant a c t i v i t y of harrisonin i s known to vary widely between species. Hassanali et al (1986) found i t to be deterrent to Eldana saccharina and Maruca t e s t u l a l i s at (respectively) 1 and 10 ug/cm2 d i s c , but i t had only marginal feeding i n h i b i t i o n against Spodoptera exempta at 100 ug/disc. Obacunone and nomilin had no antifeedant or IGR a c t i v i t y against £. saucia at concentrations up to 0.5 /nmol/g fwt. Again, these compounds are feeding deterrents at low concentrations f o r some insects, including JSj . saccharina and M« t e s t u l a l i s (Hassanali et al-, 1986), have only moderate a c t i v i t y against others, including Q. n u b i l a l i s (Arnason et a l . , 1987), £5. frugiperda, and H. zea (Kubo and Klocke, 1986), and are i n a c t i v e against yet other species including Trichoplusia n i ( A l t i e r i et ai., 1984). Similar C i t r u s limonoids were also i n a c t i v e against the spruce budworm, Choristoneura fumiferana (Alford and Bentley, 1985). In the related compound pedonin, the D r i n g has been opened to a y-ketofuran, and the r e s u l t i n g carbonyl group methylated to a carbomethoxy group; as well the A r i n g has undergone 1,2 s k e l e t a l rearrangement to a spiro structure (Hassanali et ai., 1987). Pedonin was stimulatory to £. saucia at 0.5 /nmol/g fwt, and was inactive against Q. fas c i a t u s . This compound was also inactive against the polyphagous £3. exempta, but was a strong antifeedant to the 210 more oligophagous M.- t e s t u l a l i s and E_. saccharina (Hassanali and Bentley, 1987). D. B,D-seco Limonoids Within the Swieteniodeae, D-seco limonoids may be further oxidized i n the B r i n g ; t h i s pathway produces a greater va r i e t y of limonoids than any other (Das et a l . , 1985; Connolly, 1983). Bussein and entandrophragmin are t y p i c a l of t h i s group of B,D r i n g seco limonoids, and are c h a r a c t e r i s t i c p a r t i c u l a r l y of species of Khaya and Entandrophraoma (Taylor, 1988). Entandrophragmin had no e f f e c t on either E. saucia or O. fa s c i a t u s . Bussein reduced the growth of E« saucia neonates, but was much les s active than eith e r cedrelone or anthothecol. The growth i n h i b i t i o n could not be ascribed to an antifeedant e f f e c t , as bussein d i d not have a s i g n i f i c a n t e f f e c t on d i e t choice. No IGR ef f e c t s were observed i n the Q. fasciatus assays. In the only other study to examine the e f f e c t s of entandrophragmin and bussein, both reduced feeding and survivorship of Q. n u b i l a l i s at 500 ppm i n d i e t (Arnason gt a l . . 1987), s l i g h t l y higher than the maximum concentration used i n my assays. Several B-seco limonoids are known to have antifeedant a c t i v i t y (Table 3-1), although t h i s can vary widely with r e l a t i v e l y minor changes i n structure. Although p r i e u r i a n i n and i t s acetate had antifeedant a c t i v i t y against H.. zea f S_. frugiperda (acetate only), and E. v a r i v e s t i s . the rela t e d r o h i t i u k i n and rohituka-7, which 211 d i f f e r i n that the B r i n g carboxyl fragment i s c y c l i z e d with C29 to form a new lactone r i n g , were inactive (Lidert e t a l . , 1985). Three compounds related to p i e r i a n i n are known to have antifeedant a c t i v i t y against Agrotis seietum (Nakatani et a l . , 1984). Toonacilin and 6-acetoxytoonacilin, which are s i m i l a r to cedrelone except f o r an o x i d a t i v e l y cleaved B r i n g , deter feeding by E_. v a r i v e s t i s (Kraus e t a l . , 1978). E. C-seco Limonoids (Azadirachtin) Azadirachtin had the most marked e f f e c t on £. saucia growth and feeding of any of the limonoids tested i n t h i s study, and was the only compound to s i g n i f i c a n t l y reduce survivorship. Choice t e s t s with neonate larvae indicated that an antifeedant e f f e c t may be an important component of the observed growth i n h i b i t i o n , as these larvae avoid d i e t treated with azadirachtin concentrations as low as 0.5 nmol/g fwt. Although insects are able to learn aversion to t o x i c d i e t s (Dethier, 1980), and can s e l e c t n u t r i t i o n a l l y optimal d i e t s based on changes i n brain serotonin (Cohen et a l . , 1988) or catecholamine neurotransmitters (Wurtman, 1981), the r a p i d i t y of the response to azadirachtin ( s i g n i f i c a n t a f t e r only 1 h) suggests a chemosensory basis f o r the avoidance. A "deterrent receptor", s e n s i t i v e to azadirachtin, i s present i n a number of oligophagous and polyphagous lepidopterans (Simmonds and Blaney, 1984; Schoonhoven and Jermy, 1977). This receptor does not 212 i n t e r f e r e with the a c t i v i t y of other nutrient receptors, but rather a l t e r s the c entral nervous processing of chemosensory information (Simmonds and Blaney, 1984). S e n s i t i v i t y to the antifeedant a c t i v i t y of azadirachtin declined markedly by the t h i r d i n s t a r . O s t r i n i a n u b i l a l i s larvae also show a decline i n the deterrent a c t i v i t y of azadirachtin between the f i r s t and the t h i r d i n s t a r (Arnason et a l . , 1985). The r e l a t i v e l y greater s e n s i t i v i t y of neonate c a t e r p i l l a r s to secondary metabolites has been noted previously (Reese, 1973), and includes s e n s i t i v i t y to phenolics (Isman and Duffey, 1982), polyacetylenes and thiophenes (Champagne e t aJL., 1986) and sesquiterpene lactones (Isman e t a l . , 1989). The growth i n h i b i t i n g a c t i v i t y of azadirachtin continued throughout the l a r v a l development of E. saucia, despite the decrease i n antifeedant a c t i v i t y . While the e f f e c t of azadirachtin concentration on pupal weight was s i g n i f i c a n t , the extent of i n h i b i t i o n was not large: at 0.5 /nmol/g the few pupae which were formed weighed 88% of the control pupae. While even small pupal weight reductions may correlate with reduced adult fecundity, the most b i o l o g i c a l l y s i g n i f i c a n t long-term e f f e c t of azadirachtin i s a marked decrease i n pupation and adult emergence. Similar r e s u l t s have been noted with H e l i o t h i s zea (Barnby and Klocke, 1987), O s t r i n i a n u b i l a l i s (Arnason e t a l . , 1985), the lepidopteran r i c e pests Mythimna separata and 213 Cnaphalocrocis medinialis (Schmutterrer et a i . , 1983) and the face f l y Musca autumnalis (Gaaboub and Hayes, 1984). F N u t r i t i o n a l Indices Azadirachtin markedly reduced growth and consumption i n t h i r d - i n s t a r E . saucia at dietary concentrations which did not a f f e c t the ECI, ECD, or AD. The reduced consumption i s u n l i k e l y to r e f l e c t a chemosensory antifeedant e f f e c t as consumption was reduced even at low concentrations which did not a f f e c t d i e t choice by second-instar larvae. Rather, the reduced RCR may r e f l e c t a d i r e c t action of azadirachtin on the gut or on neural regulation of feeding. Mordue et a l . (1985) have shown that azadirachtin reduces the rate of gut p e r i s t a l s i s i n Locusta r and indeed i n h i b i t s a l l p r o c t o l i n -mediated muscular a c t i v i t i e s (Mordue and Plane, 1988). Such a mechanism could well account f o r the observed decrease i n the RCR; consistent with t h i s i s the observation that the AD tended to increase with increasing azadirachtin concentration. This e f f e c t could r e s u l t from a prolonged exposure qf the food bolus to digestive enzymes (Slansky and Scriber, 1985). The decrease i n the ECI and ECD found at the highest azadirachtin concentration here tested could r e f l e c t increased metabolic costs of d e t o x i f i c a t i o n . However, reduced consumption can also r e s u l t d i r e c t l y i n a reduction i n ECI and ECD, as the f i x e d costs of metabolic a c t i v i t y w i l l consume a higher proportion of energy intake i f that intake i s small rather than large. In p a r t i c u l a r , 214 consumption at the highest azadirachtin concentration may be just s u f f i c i e n t to meet the metabolic requirements of the larvae, with no excess to support growth. This i n t e r p r e t a t i o n i s supported by the observation that, when RGRi i s plotted against RCRi, a l l treatments f i t the same regression l i n e ( F i g . 4-7). This r e s u l t strongly suggests that the growth-inhibiting a c t i v i t y of azadirachtin i s relat e d to reduced consumption and not to metabolic t o x i c i t y (cf. Blau et a i . , 1978). I t i s also noteworthy that very few larvae died while molting; Arnason e £ a i . (1985) noted a s i m i l a r r e s u l t with 0. n u b i l a l i s . The mo l t - i n h i b i t i n g a c t i v i t y of azadirachtin appears to be most prominent i n species which f a i l to respond to azadirachtin as an antifeedant ( i . e . Garcia and Rembold, 1983; Chapter 5 of t h i s t h e s i s ) , or i n bioassays where insects are a r t i f i c i a l l y exposed (via t o p i c a l a p plication, i n j e c t i o n , or gut cannulation) to doses of azadirachtin which would o r d i n a r i l y be avoided. The r e s u l t s of the n u t r i t i o n a l index study are comparable to r e s u l t s obtained with Melanoplus sanguinipes (Chapter IV), H e l i o t h i s virescens (Barnby and Klocke, 1987), and Crocidolomia b i n o t a l i s (Fagoonee, 1984). In the l a t t e r study, ECI and ECD were found to increase with azadirachtin concentration. In contrast to these r e s u l t s , Arnason et a i (1985) found no reduction i n consumption when 0. n u b i l a l i s larvae were exposed to azadirachtin; rather, a decrease i n the RGR was rela t e d to decreases i n the ECI and ECD. Rao 215 and Subramantham (1985) found that azadirachtin reduced consumption, ECI, and ECD i n a dose-dependent manner i n Schistocerca gregaria. This r e s u l t may r e f l e c t the extreme s e n s i t i v i t y of t h i s insect to azadirachtin (Blaney, 1980). G. Limonoid Evolution and S t r u c t u r e - A c t i v i t y Relationships The i n s e c t i c i d a l a c t i v i t y of limonoids does not appear to correlate with the evolutionary trends of increasing oxidation and s k e l e t a l rearrangement discussed by Das et a l (1984). This conclusion i s supported not only by the analysis i n Figure 4-8, but also by a consideration of Table 4-1 and the r e s u l t s of the bioassays presented here. The dammarane precursors of the limonoids are apparently inactive against insects (Chapter 3), but both the protolimonoids (Group 1 i n F i g . 4-2) and the simple apo-euphol type limonoids (Group 2) are active; i n the l a t t e r case the a c t i v i t y may include both t o x i c and IGR e f f e c t s ( i . e . i n the case of cedrelone). However, the dominant evolutionary pathways within most Meliaceae and a l l Rutaceae, giv i n g r i s e to D-seco, A,D-seco, and B,D-seco limonoids, lead to compounds with reduced antifeedant and apparently no IGR a c t i v i t y . In C i t r u s , the reduced a c t i v i t y may be to some extent compensated for by the very high concentrations of A,D-seco limonoids ( e s p e c i a l l y limonin) produced (> 1,000 ppm) (Rouseff and Nagy, 1982). Curiously, these pathways are the only ones expressed i n members of the Swietenioideae (Taylor, 1981), considered to be the most 216 advanced subfamily of the Meliaceae (Pennington and Styles, 1975). The only pathway which appears to lead to compounds with increased a c t i v i t y against generalist insects i s that giv i n g r i s e to the C-seco limonoids; these compounds may be derived v i a a d i f f e r e n t pathway from the other limonoids, involving an extra epoxidation step (Fig. 1-2) (Siddiqui et a l . , 1988). This pathway i s expressed only i n the t r i b e Melieae, which includes the (morphologically primitive) genera Azadirachta and Melia. Despite the absence of an obvious r e l a t i o n s h i p between limonoid evolution and a c t i v i t y against phytophagous insects, the p o s s i b i l i t y of a r o l e f o r limonoids i n a coevolutionary r e l a t i o n s h i p between plants and insects remains. A l l of the bioassay species used to date are polyphagous or oligophagous species which do not u t i l i z e Meliaceae as host plants. The p o s s i b i l i t y remains that s p e c i a l i s t insects, adapted to t o l e r a t e exposure to one c l a s s of limonoids, may be deterred or intoxicated by exposure to another c l a s s . For example, p a p i l i o n i d larvae able to t o l e r a t e large doses of l i n e a r furanocoumarins are susceptable to angular furanocoumarins (Berenbaum, 1978, 1981), even though the l a t t e r are considered less t o x i c as they are only able to form monofunctional adducts with DNA. The r o l e of limonoids i n a putative coevolutionary r e l a t i o n s h i p between the Meliaceae and insects would have to be examined i n the context of the adapted insect fauna found feeding on meliaceous species. For instance, the shoot 217 borers Hypsipyla spp. attack a l l species of Cedrela and Swietenia , to the point of l i m i t i n g t h e i r usefulness as plantation crops within t h e i r natural area of d i s t r i b u t i o n (Grijpma, 1973). These insects must be adapted to cope with Group 2 compounds such as cedrelone, which occur i n the shoots and heartwood as well as fo l i a g e of Cedrela. and may also be exposed to limonoids of groups 1, 3 and 4, known from the seeds of Cedrela and Swietenia species (Taylor, 1981). Cedrela and Swietenia often co-occur with species of Guarea (Pennington and Styles,1981), which are not attacked; i s the resistance due to limonoids of classes 5 and 7, found i n Guarea but not i n the other genera? Azadirachta indica has a small entomofauna of twelve species, almost a l l of which are monophagous (Warthen, 1979). Has t h i s s p e c i a l i z e d fauna resulted from a coevolutionary association during the evolution of the C-seco type limonoids? C l e a r l y the si g n i f i c a n c e of limonoids i n mediating such interactions requires further i n v e s t i g a t i o n . H. Correlation of Phytochemistry and Crude Extract Bioassays The s t r u c t u r e / a c t i v i t y conclusions drawn here are supported by a c o r r e l a t i o n of the r e s u l t s of the crude extract bioassays reported i n Chapter 2 with the known d i s t r i b u t i o n of limonoids i n the Meliaceae. Table 4-6 l i s t s the classes of limonoids known to occur i n the various genera of Meliaceae (updated from Taylor, 1981). Genera with group 8 limonoids were predicted to be highly active against E« 218 Table 4 - 6 . Comparison of E C 5 0 values of crude extracts of Meliaceae with predictions of a c t i v i t y based on classes of limonoids reported to occur i n the genera examined. Extracts were predicted to have high a c t i v i t y i f group 8 limonoids had been reported, moderate a c t i v i t y i f group 2 or 10 limonoids were known, low a c t i v i t y i f other limonoids had been reported, and no a c t i v i t y i f limonoids had not been saucia, those with group 2 limonoids were predicted to be moderately active, those with other classes of limonoids found. Phytochemical data i s taken from Taylor (1981), updated as noted. Limonoids known from species other than the one(s) bioassayed are i n parentheses. Observed E C 5 0 s are from Table 2-2 and are mg/g; where more than one species i n a genus was bioassayed the range of r e s u l t s i s shown. Genus Limonoid Groups Predicted Observed Reported A c t i v i t y •^^ 50 Aglaia dammaranes inactive 2.U7-11.88 Azadirachta 1,2,3,8 high 0.69-0.89 Carapa 3,4,(5,6) low 29.68 Cedrela 1,2,3,41 moderate 24.66 Chuckrasia 5 low 31.13 Dysoxylum ( 9 ) 2 low in a c t i v e Ekeberaia 4 low 102.94 Entandrophragma (1,3,4),5 low 29.83 Guarea (3,4,7) low 53.32 Khaya (2),3,4 moderate 67.05 Lansium none inactive i n a c t i v e Melia 1,2,3,8 high 1.58-2.10 Sandoricum none inactive 22.79 Swietenia 4 low 23.64-47.36 Toona 2,6,103 moderate 22.93-47.63 T r i c h i l i a 2,(7) moderate 18.88 Turreae 2 moderate 2.80-45.40 References:l) El-Shamy gt a i . , 1988. 2) Jogia and Andersen, i n press. 3) Kraus and Grimminger, 1978,1980. 219 were predicted to have low a c t i v i t y , and the genera without limonids were predicted to be in a c t i v e . Generally the c o r r e l a t i o n i s good: a l l species predicted to have high a c t i v i t y do, and those species found to be inactive had been predicted to have l i t t l e or no a c t i v i t y . Most species predicted to have moderate l e v e l s of a c t i v i t y had EC50 values close to 20 mg/g, and most of those predicted to have low a c t i v i t y had E C 5 0 values at or above 30 mg/g. Noteworthy exceptions were seen, however: Aglaia and Turreae were much more active than was predicted based on t h e i r known phytochemistry, and Sandoricum had a moderate l e v e l of a c t i v i t y despite the reported absence of limonoids. These species should be investigated further. On the other hand, Khaya was less active than expected, but the species investigated, K. senegalensis. has not been reported to contain group 2 compounds, although other members of the genus do. I. Comparison of I n s e c t i c i d a l and Cytotoxic A c t i v i t y The i n s e c t i c i d a l a c t i v i t y of limonoids i s to some extent p a r a l l e l e d by the cytotoxic a c t i v i t y of these compounds against murine P-388 lymphocytic leukemia c e l l s . P e t t i t et a l (1983) found that a 14-15 epoxide on an i n t a c t D r i n g was required f o r c y t o t o x i c i t y ; they suggested that the epoxide may be required f o r a l k y l a t i o n of bioamines or t h i o l s , but I found that azadirachtin does not spontaneously form adducts with the sul f h y d r y l cysteine i n v i t r o (Chapter 220 5). Limonoids with a 19-28 l a c t o l group were most active, but those with a 3-oxo-l-ene A r i n g structure (as i n cedrelone and anthothecol) were also highly active; A r i n g saturation abolished the c y t o t o x i c i t y . Conversely, Kraus gt a l . (1987) concluded that A-ring saturation, with oxygen functions at C-2 and C-3, was required for antifeedant a c t i v i t y against Epilachna v a r i v e s t i s , based on a comparison of the a c t i v i t y of v i l a s i n i n e derivatives with azadiradione. Compounds with a D r i n g epoxylactone, and the seco-ring A,D c i t r u s limonoids, were mostly not cytotoxic ( P e t t i t gt a l . , 1983). An i n t e r e s t i n g observation from t h i s and previous studies (Kubo and Klocke, 1987; Siddiqui gt a l . , 1988; 1988; Naqui, 1987) i s the IGR a c t i v i t y of cedrelone and re l a t e d compounds with a 14-156-epoxide on an otherwise i n t a c t D ring,an unsubstituted C r i n g , and a 3-oxo-l-ene A r i n g . These r e l a t i v e l y simple structures may be amenable to synthesis or manipulation, and so could provide leads f o r the development of synthetic limonoid-based i n s e c t i c i d e s . 221 Chapter 5: E f f e c t s of azadirachtin on the n u t r i t i o n and development of the migratory grasshopper, Melanoplus  sanguinipes Fab. Introduction Among the few insects reported to be r e s i s t a n t to azadirachtin are the New World grasshoppers, including the migratory grasshopper, Melanoplus sanguinipes Fab. (Orthoptera: Acrididae) (Mulkern and Mongolkiti, 1975). In i n i t i a l experiments I confirmed the absence of a chemosensory-based antifeedant e f f e c t against t h i s insect, but observed marked subsequent disruption of molting. This allowed me to compare the t o x i c i t y of azadirachtin following application o r a l l y , t o p i c a l l y , and v i a i n j e c t i o n , and so evaluate the s i g n i f i c a n c e of the gut and integument as factors l i m i t i n g the b i o a v a i l a b i l i t y of t h i s compound to putative target s i t e s within the insect. Azadirachtin-induced i n h i b i t i o n of molting has been shown, i n several insects, to involve a delay i n the appearance of the ecdysteroid peaks which regulate apolysis (reviewed i n Chapter 1); however the mechanism by which t h i s occurs i s s t i l l unclear. Mordue and Evans (1987) have suggested that such e f f e c t s may r e s u l t from a d i r e c t action on the gut rather than a d i r e c t action on ecdysteroid synthesis or metabolism. 222 The toxicology of azadirachtin i s i n many respects p a r a l l e l e d by the azasterols, which block the conversion of B - s i t o s t e r o l and other phytosterols to cholesterol by i n h i b i t i n g 2 4 and 22,24 s t e r o l reductases of insects (Svoboda et a l . , 1972; Svoboda and Robbins, 1971; Walker and Svoboda, 1973). S i m i l a r i t i e s include the i n h i b i t i o n of growth, molting and oogenesis at dietary concentrations as low as 3 /ig/g. As azasterol t o x i c i t y can be reversed by dietary supplementation with c h o l e s t e r o l , I investigated the e f f e c t of cholesterol and other phytosterols on azadirachtin t o x i c i t y . I also investigated the p o s s i b i l i t y that azadirachtin may i n t e r f e r e with s t e r o l transport through the hemolymph, a process dependent on the c a r r i e r l i p o p r o t e i n lipophorin (Chino and G i l b e r t , 1971; Chino, 1985). F i n a l l y , as both the transducing proteins involved i n chemoreception and the neurosecretory material formed i n the pars i n t e r c e r e b r a l i s are unusually r i c h i n s u l f h y d r y l residues (Norris, 1986; F r i e d e l and Loughton, 1980), and both s i t e s have been postulated to be putative molecular targets f o r azadirachtin a c t i v i t y , I tested the a b i l i t y of azadirachtin to form adducts with the s u l f h y d r y l amino acid cysteine i n v i t r o . 223 Materials and Methods A. Experimental Insects F i f t h i n s t a r Melanoplus sanguinipes nymphs, non-diapause s t r a i n , were obtained from a laboratory colony reared on fr e s h l y cut seedling wheat, dry wheat bran, and chickweed, Cerastium s t e l l a t a , (Isman, 1985). Synchronized groups of f i f t h i n s t a r nymphs were obtained by c l e a r i n g cages of f i f t h i n s t a r i n d i v i d u a l s 24 h p r i o r to s t a r t i n g an experiment, then u t i l i z i n g nymphs which molted overnight. In some cases ind i v i d u a l s so obtained were stored at 4° C f o r 24 h p r i o r to s t a r t i n g a bioassay, to allow c o l l e c t i o n of s u f f i c i e n t numbers of nymphs f o r the bioassay. Preliminary experiments established that grasshoppers could be stored at 4° C f o r up to 72 h without influencing subsequent growth rate, duration of the i n s t a r , or molting success. B. Source of Chemicals Azadirachtin used i n t h i s study was i s o l a t e d from Azadirachta indica seeds by Dr. J . Kaminski, and was kindly made availa b l e Dr. J . T. Arnason, University of Ottawa. Cholesterol and B - s i t o s t e r o l were purchased from Sigma Chemical Company, St. Louis, Mo. and were used without further p u r i f i c a t i o n . 4 - 1 4 C - B - s i t o s t e r o l (56 mCi/mmol) was purchased from Amersham. 224 C. Antifeedant A c t i v i t y Assays In no-choice antifeedant assays, azadirachtin i n acetone (0.5 or 0.05 mg/ml) was applied to both surfaces of fr e s h l y punched and weighed 1 cm diameter cabbage (Brassica oleracea cv. Early Copenhagen) leaf discs to achieve concentrations of 5, 10, 15, 20, 25, 50, 100, 200, 300, and 500 ug/ g leaf fresh weight (fwt). Control leaf discs were treated with 10 / i l acetone, a volume equivalent to that applied at the highest azadirachtin concentration. One leaf d i s c was presented to each f i f t h i n s t a r grasshopper i n a 4 oz unwaxed paper cup capped with a p l a s t i c p e t r i dish l i d ; grasshoppers were starved for two hours p r i o r to the t e s t . Ten nymphs were used f o r each concentration, and the t e s t was r e p l i c a t e d three times. The number of ind i v i d u a l s i n each treatment group which had completely consumed the d i s c a f t e r 15, 30, and 60 minutes was recorded. D. Dietary U t i l i z a t i o n Experiments The e f f e c t s of azadirachtin on r e l a t i v e growth, consumption, and digestive performance of nymphs were determined. Staged f i f t h i n s t a r nymphs (20 per treatment) were fed a single dose of 10 or 15 /ig/g insect fwt of azadirachtin applied to a 1 cm diameter cabbage leaf d i s c ; these doses were chosen as they produce markedly d i f f e r e n t e f f e c t s on molting success i n U. sanguinipes. Subsequently the nymphs were fed d a i l y with weighed aliquots of f r e s h l y cut seedling wheat. 225 I n i t i a l dry weight of the wheat was estimated by drying samples of wheat and c a l c u l a t i n g a dwt/fwt r a t i o . After 48 h the nymphs were weighed, then nymphs, fr a s s , and remaining wheat were dried to constant weight at 70 C and weighed. The i n i t i a l dw of the nymphs was calculated based on the dw/fw r a t i o of a sample of 10 nymphs. Indices determined included the approximate d i g e s t a b i l i t y (AD), e f f i c i e n c y of coversion of ingested food (ECI), and e f f i c i e n c y of conversion of digested food (ECD), calculated according to Reese and Beck (1976). In addition growth arid consumption rates were calculated i n r e l a t i o n to the weight of the nymphs at the s t a r t of the experiment (RGRi and RCRi respectively) (Farrar et a l , 1989). Formulas f o r c a l c u l a t i n g these indices are given i n Chapter 4. E. Molt I n h i b i t i o n Assays To assess the growth regulating a c t i v i t y of azadirachtin following o r a l administration, staged (<24 h) f i f t h i n s t a r nymphs were weighed (114.3 ± 1 2 . 1 mg/nymph), then fed s u f f i c i e n t azadirachtin applied i n acetone to a leaf d i s c to achieve a dose of 3, 5, 8, 10, 13, 15, or 25 fig/ g insect fwt. Controls were fed leaf discs treated with acetone only. Afte r the singl e dose of azadirachtin was consumed, usually i n less than 1 h, grasshoppers were maintained i n 4 oz paper cups , i n a controlled environment chamber at 30 + 1° C, about 40 % RH (ambient a i r RH), a 16L:8D photoperiod, and fed untreated wheat, bran, and chickweed ad l i b , u n t i l 226 molting or death occurred. Duration of the i n s t a r , molting success, and weight at molting were recorded. Ten nymphs per dose were used i n each of four r e p l i c a t e s . MD 5 0 values (the dose which i n h i b i t e d molting i n 50% of the treated nymphs) were determined by probit analysis, with category 3 and 4 responses (defined i n Results section) combined as m o r t a l i t i e s . Azadirachtin i n acetone was also applied t o p i c a l l y to the dorsal abdominal tergae of staged f i f t h i n s t a r nymphs, to achieve doses of 2, 4, 6, 8, and 10 /ig/g insect fwt. Controls were treated with 5 / i l acetone, equivalent to the volume used at the highest azadirachtin dose. After the single treatment insects were maintained as described above. Ten nymphs were used for each concentration i n each of three r e p l i c a t e s . For i n j e c t i o n experiments, staged f i f t h i n s t a r nymphs were weighed, c h i l l e d to 4° C, then injected mid-l a t e r a l l y between abdominal terga 3 and 4 , using a Hamilton syringe f i t t e d with a 26-gauge needle attached to a Hamilton repeating dispensor. Azadirachtin i n acetone (0.5 mg/ml) was applied at 3, 5, 8, 10, and 15 fig/ g insect fwt; controls were injected with 3 / i l acetone alone. Mortality i n the controls was about 6% (2 of 30 nymphs) with t h i s solvent. Three r e p l i c a t e s of ten insects per dose were performed. F. Piperonyl Butoxide Synergism Assay 227 The possible r o l e of mixed-function oxidases (MFOs) i n the metabolism of azadirachtin by M.. sanguinipes was investigated by feeding staged f i f t h i n s t a r nymphs azadirachtin at 2, 4, 6, 8, and 10 ng/ g insect fwt, coadministered with 500 /xg piperonyl butoxide (PBO) d i l u t e d 1:1 with acetone, on a cabbage leaf d i s c . Control insects were fed leaf discs treated with 500 ng PBO only. Insects were subsequently maintained as described above, and duration of the i n s t a r and molt success were recorded. The experiment involved three r e p l i c a t e s of ten nymphs per dose. G. Fecundity Experiment Staged teneral adult female M.. sanguinipes (<24 h from molting) were weighed, then fed s u f f i c i e n t azadirachtin, applied to a cabbage leaf d i s c , to achieve an o r a l dose of 0, 5, 10, 15, 25, or 50 pg/g insect fwt. Thereafter, females were maintained i n d i v i d u a l l y i n 4 oz unwaxed paper cups at 30° C, 18L:6D, and fed seedling wheat, chickweed, and bran ad l i b . The cups were modified as o v i p o s i t i o n arenas: a 1.5 cm diameter hole, punched i n the bottom of the cup, gave access to a 2 oz p l a s t i c cup f i l l e d with sieved, s t e r i l i z e d s o i l . One male, not treated with azadirachtin, was added to each cup 24 h a f t e r the female. Five females were used f o r each azadirachtin concentration tested; the experiment was repeated twice. The cups of s o i l were removed, examined for egg masses, and replaced with fresh s o i l every three days. Egg masses were disassembled to 228 count i n d i v i d u a l eggs; t h i s precluded the p o s s i b i l i t y of determining egg f e r t i l i t y . The bioassay was continued f o r si x weeks. Data were analysed by l i n e a r regression. H. E f f e c t of Dietary Sterols To determine the e f f e c t of supplementing the d i e t with s t e r o l s on azadirachtin t o x i c i t y , I established s i x treatment groups: (1) solvent control, (2) azadirachtin alone, (3) cholesterol alone, (4) cholesterol plus azadirachtin, (5) 8 - s i t o s t e r o l alone, and (6) B - s i t o s t e r o l plus azadirachtin. Insects were i n i t i a l l y fed 15 /ig/g insect fwt azadirachtin (treatments 2, 4, and 6) or a solvent control (treatments 1, 3, and 5). For the duration of the i n s t a r they were fed wheat dipped i n CHC13 (1 and 2), or a 10 mg/ml CHC13 solution of cholesterol (3 and 4) or B-s i t o s t e r o l (5 and 6). Duration of the in s t a r and molting success were recorded. Ten nymphs per concentration were used i n each of two r e p l i c a t e s . I. S t erol Transport Experiment I tested the hypothesis that azadirachtin i n t e r f e r e s with the transport of s t e r o l s i n the hemolymph by feeding 1 4C-B-s i t o s t e r o l (10,000 dpm/nymph) to control and azadirachtin (15 /ig/g insect fwt)-treated f i f t h - i n s t a r nymphs. Insects (36/treatment) were fed a leaf di s c treated with azadirachtin or acetone 24 h before the single pulse of rad i o l a b e l l e d s t e r o l . At hourly i n t e r v a l s f o r 12 h, three 229 insects from each treatment were randomly selected, a cut was made l a t e r a l l y along the abdominal wall, and 10 fil hemolymph was c o l l e c t e d with a microcapillary tube. The samples were i n d i v i d u a l l y digested f o r 1 h i n 1 ml Protosol, then 4 ml Aquasol was added i n p l a s t i c s c i n t i l l a t i o n tubes, and the c o c k t a i l was allowed to e q u i l i b r i a t e for 24 h to quench chemiluminescense before counting. Following the experiment (24 h) the nymphs from each treatment were pooled, homogenized i n CHC13, f i l t e r e d , and the extract was concentrated to 4 ml. A 1 ml aliquot was dried and prepared f o r s c i n t i l l a t i o n counting as described above. The remaining 3 mis were dried, derivatized with 30% t r i f l u r o a c e t i c acid (TFA) i n a c e t o n i t r i l e (ACHN) at 80° C for 1 h, then chromatographed by reverse-phase TLC developed 3 times i n ac e t i c acid:ACHN. Spots were v i s u a l i z e d with 3.0 M H 2S0 4, scraped from the plate, eluted with MeOH:H20 (1:1), and prepared f o r s c i n t i l l a t i o n counting i n Aquasol. Standards of TFA derivatives of cholesterol and 6 - s i t o s t e r o l were also prepared and chromatographed alongside the hemolymph extracts. J . Jn vitro assay f o r the formation of adducts Azadirachtin (0.02 /imoles = 1.8 mg) and cysteine (free base) (0.02 /xmoles = 0.3 mg) were mixed i n 1 ml of pH 7.0 phosphate buffer at room temperature. Aliquots were removed at ten minute i n t e r v a l s for the f i r s t hour, and thereafter hourly f o r f i v e hours, spotted on c e l l u l o s e TLC plates, and developed i n the upper phase of n-butanol:acetic acid:H20 (4:1:5) (Pieman et a l . , 1979). Spots were v i s u a l i z e d by spraying the plate with ninhydrin reagent (Stahl, 1972) followed by 2.0 M H 2S0 4, with heating a f t e r each spray reagent. 231 Results A. Antifeedant assays Azadirachtin had no antifeedant e f f e c t against M.. sanguinipes nymphs at any of the concentrations tested. Leaf discs were usually consumed within 30 minutes i n a sin g l e feeding bout; on occasion two feeding bouts were required. B. Growth and Dietary U t i l i z a t i o n Treatment with both 10 and 15 /ig/g azadirachtin resulted i n a s i g n i f i c a n t decrease i n the r e l a t i v e growth rate (RGRi: 38 and 40% of controls respectively, p<.0001, Tukey's (HSD) •i test) (Table 5-1). This was almost e n t i r e l y owing to a decrease i n the r e l a t i v e consumption rate (RCRi: 34 and 41%, p<.0001). There were no differences between the e f f e c t s at 10 and 15 /ng/g azadirachtin. The e f f i c i e n c y with which ingested (ECI) and digested (ECD) food was converted to new insect biomass was not s i g n i f i c a n t l y decreased. The approximate d i g e s t a b i l i t y was s l i g h t l y increased following azadirachtin treatment at the higher dose only (p<.017, Tukey's (HSD) t e s t ) . 232 Table 5 - 1 . E f f e c t of azadirachtin on Melanoplus sanguinipes growth and n u t r i t i o n . Means i n a column with the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t (Tukey's Studentized Range (HSD) Test, oc =0.05). Treat RGRi RCRi ECI ECD AD Control 0.243a 0.843a 30.2 a 60.3 a 53. 2 a 10 0.150° 0.569° 30. 5 a 54.0 a° 59. 4 a 15 0.145^ 0.500^ 25.9s- 44. 6^ 59. B3-Treat=treatment (/ig/g insect fwt), RGRi=Relative Growth Rate based on i n i t i a l wgt, RCRi=Relative Consumption Rate based on i n i t i a l wgt, ECI=Efficiency of Conversion of Ingested Food, ECD=Efficiency of Conversion of Digested Food, and AD=Approximate D i g e s t a b i l i t y . Values were calculated according to Reese and Beck (1976) except for the RGRi and RCRi, which were calculated according to Farrar et a l . ( 1 9 8 9 ) . 233 C. Molt I n h i b i t i o n Studies Melanoplus sanguinipes nymphs that consumed azadirachtin subsequently showed a range of dose-dependent e f f e c t s which are here a r b i t r a r i l y divided into four categories of response. Results f o r males and females did not d i f f e r and were therefore combined. Controls and nymphs consuming azadirachtin at 3 jxg/g insect fwt molted normally to adults (=Category I ) . At 5, 8, and 10 \xq/q, increasing proportions of the treated nymphs molted to adults with deformed wings and, at higher doses, deformed legs (=Category I I ) . These ind i v i d u a l s took longer to complete the molt. The deformities may have resulted from the nymphs i n i t i a t i n g s c l e r o t i z a t i o n of the adult structures before ecdysis was complete. The duration of the in s t a r was also s i g n i f i c a n t l y increased at 8 and 10 nq/q azadirachtin (12.0 + 1.4 days c f . 8.0 + 1.0 days f o r the co n t r o l s ) . At 8 and 10 nq/q, a small proportion (<10%) of treated nymphs died during an incomplete molt attempt (=Category I I I ) . A notable t r a n s i t i o n was seen between 10 and 13 nq/q: at the lower dose most nymphs showed a category II response, but at 13 nq/q a l l nymphs died i n the molt. At 15 nq/q about 25% of the nymphs eventually died without i n i t i a t i n g any molt attempt (Category IV), and at 25 nq/q 80% o f the nymphs showed t h i s response. In some cases category IV insects were observed to l i v e f or over 60 days without i n i t i a t i n g the molt. At day 13 post-treatment the weight of category IV nymphs exceeded the weight of the controls at molting 234 Figure 5 - 1 . Morphogenic e f f e c t s of o r a l l y administered azadirachtin on f i f t h - i n s t a r nymphs of Melanoplus  sanguinipes. Response categories include: Category I: no morphological e f f e c t ; Category I I : molt to an adult with deformed wings or legs; Category I I I : death at a f a i l e d molt attempt; Category IV: death without i n i t i a t i n g a molt attempt. 236 Figure 5-2. E f f e c t of o r a l l y administered azadirachtin on molting success of f i f t h - i n s t a r nymphs of Melanoplus  sanguinipes. Responses are divided into four categories: I, no response except delay of molt; I I , molt to an adult with deformed wings or legs; I I I , death at a f a i l e d molt attempt; and IV, death without i n i t i a t i n g a molt attempt. Percent of treated population Control 3 5 8 10 13 15 Azadirachtin dose ug/g insect fwt 25 Category 1 Category 2 Category 3 Pill Category 4 238 (232.1 + 17.6 mg vs 207.6 ± 25.4 mg re s p e c t i v e l y ) . Grouping category 1 and 2 responses as survivors and category 3 and 4 as m o r t a l i t i e s , the o r a l MD 5 0 was 10.83 /ig/g insect fwt (95% f i d u c i a l l i m i t s 10.34-11.44 pg/g). Topical administration of azadirachtin produced the same range of e f f e c t s , but at lower doses. At 2 /ig/g only 55% of the nymphs molted to normal adults, and at 4 pg/g no nymphs molted normally. Category 3 responses dominated at 6 /ig/g, and at 8 and 10 /ig/g category 4 responses were most prevalent. The t o p i c a l MD 5 0 was 3.80 /ig/g insect fwt (95% f i d u c i a l l i m i t s 3.196-4.353 /ig/g). When azadirachtin was injected d i r e c t l y into the hemocoel, the same spectrum of e f f e c t s was produced (Figure 5-3). Even at 3 ng/g only 20% of the nymphs molted normally; category II and III responses were each 40%. At doses of 5, and p a r t i c u l a r l y 8 and 10 /ig/g, category III responses were most common. At 8 and 10 /ig/g, some nymphs f a i l e d to molt; t h i s respose accounts f o r 80% of the treatment group at 15 /ig/g. The MD 5 0 was 3.01 /ig/g (95% f i d u c i a l l i m i t s 2.49-3.50 /i/g). D. Synergism by piperonyl butoxide Coadministration of piperonyl butoxide (PBO) s i g n i f i c a n t l y increased the o r a l t o x i c i t y of azadirachtin i n M« sanguinipes nymphs. At 4 ng/g, 100% of the treated nymphs showed a category I or II response, but at 6 /ig/g only 57% Figure 5 - 3 . E f f e c t of injected azadirachtin on molting success of f i f t h i n s t a r nymphs of Melanoplus sanguinipes. Responses are divided into four categories: I, no response except delay of molt; I I , molt to an adult with deformed wings or legs; I I I , death at a f a i l e d molt attempt; and IV, death without i n i t i a t i n g a molt attempt. Percent of treated population Injected Control 3 5 8 10 15 Azadirachtin dose ug/g Insect fwt Category 1 i i i l Category 2 Category 3 Category 4 ro o 241 Figure 5-4. E f f e c t of t o p i c a l l y applied azadirachtin on molting success of f i f t h - i n s t a r nymphs of Melanoplus  sanguinipes. Responses are divided into four categories: I, no response except delay of molt; I I , molt to an adult with deformed wings or legs; I I I , death at a f a i l e d molt attempt; and IV, death without i n i t i a t i n g a molt attempt. Percent of treated population Control 2 4 6 8 Azadirachtin dose ug/g Insect fwt Category 1 i l l Category 2 Category 3 Category 4 - F 243 Figure 5 - 5 . E f f e c t of co-administered piperonyl butoxide (PBO) on the molt i n h i b i t o r y a c t i v i t y of o r a l l y administered azadirachtin. In t h i s figure category I and II responses are combined to give survivorship curves. The e f f e c t of injected and t o p i c a l l y applied azadirachtin i s also shown. D O M (ug/g Intact fwt) 245 molted successfully. At 8 /ig/g most nymphs showed a type III response and at 10 ng/g most showed a category IV response. The o r a l MD 5 0 of azadirachtin plus PBO was 6.5 /xg/g insect fwt. PBO alone had no e f f e c t on i n s t a r length or molt success. E. F e r t i l i t y Experiment Azadirachtin decreased the number of egg masses and eggs produced over the f i r s t s i x weeks of the adult stage, i n a dose-dependent manner (Figure 5-6). Overall there was a highly s i g n i f i c a n t dose-response, with an F I 5 0 (50% f e r t i l i t y i n h i b i t i o n ) of 35 /ig azadirachtin/g insect. This appeared to be due to an azadirachtin-induced dose-dependent delay i n the time to production and s i z e of the f i r s t egg mass. Survivorship to the end of the 6-week assay was 100% i n a l l treatment groups. F. Sterol supplementation assays Supplementing the d i e t with cholesterol or B - s i t o s t e r o l did not s i g n i f i c a n t l y influence the t o x i c i t y of o r a l l y -administered azadirachtin. The s t e r o l s alone had no e f f e c t on molt success or duration of the i n s t a r . Treatment with azadirachtin did not abolish the movement of r a d i o l a b e l l e d s t e r o l into the hemolymph from the gut (Figure 5-7). In both control and treated nymphs radio l a b e l f i r s t appeared i n the hemolymph 4 h a f t e r feeding with 1 4 C - B - s i t o s t e r o l . In controls, the amount of Figure 5 - 6 . E f f e c t of o r a l l y administered azadirachtin on adult female fecundity. Each point represents the mean of f i v e females; controls produced 4 2 + 1 1 eggs/female. Fedundity (% ofControl) = 110-1.7[Aazdirachtin] r 2=.96 cn Figure 5-7. Pharmacokinetics of r a d i o l a b e l l e d s t e r o l s i n the hemolymph of control and azadirachtin treated nymphs. Each point represents the mean of three i n d i v i d u a l nymphs. 250 r a d i o a c t i v i t y increased to a peak at 10 h post-feeding whereas, i n azadirachtin-treated nymphs, the increase was more gradual and d i d not peak during the course of the experiment. Chromatography of TFA derivatives of the s t e r o l f r a c t i o n from control and azadirachtin treated insects indicated that /3-sitosterol was primarily metabolized to choleste r o l i n M - sanguinipes. No differences i n the r e l a t i v e proportion of ch o l e s t e r o l , 6 - s i t o s t e r o l , and desmosterol were evident between the two treatment groups (Table 5-2). G. In v i t r o assay f o r adduct formation Chromatography of aliquots of an equimolar mixture of azadirachtin and cysteine i n pH 7.0 phosphate buffer did not indicate the formation of adducts between these compounds. Azadirachtin (Rf=0.98) and cysteine (Rf=0.37) could be detected i n aliquots taken at a l l time i n t e r v a l s ; a f t e r one hour cystine (Rf=0.31), the oxidation product of cysteine, was also detected. 251 Table 5-2. Radioladelled s t e r o l composition of control and azadirachtin-treated M. sanguinipes fed 4 - 1 4 C - B - s i t o s t e r o l . Treatment % Cholesterol % B-Sitosterol % Desmosterol Control 72.7 26.1 1.2 15 ug/g aza 68.7 29.9 1.4 Discussion A. Antifeedant and n u t r i t i o n a l e f f e c t s Azadirachtin had no chemosensory-based antifeedant a c t i v i t y against M. sanguinipes nymphs at concentrations up to 500 /ig/g, as treated leaf discs were usually consumed i n a single feeding bout. However, the RCRi was s i g n i f i c a n t l y reduced following azadirachtin ingestion, i n d i c a t i n g an action against the gut or on the neural regulation of feeding. This e f f e c t resulted i n a s i g n i f i c a n t decrease i n RGRi. Reduced consumption following azadirachtin administration, i n the absence of chemosensory i n h i b i t i o n , has been noted with other insects (Rembold et a l . , 1980; Redfern et a l . , 1981; Schmutterrer, 1985; Mordue et a l . , 1985; Simmonds and Blaney, 1984) and i n Locusta may be due to a decreased rate of gut p e r i s t a l s i s (Mordue et a l . , 1985). These r e s u l t s suggest that bioassays extending beyond a singl e feeding bout cannot d i s t i n g u i s h between a true chemosensory response (deterrency) and t o x i c i t y with t h i s compound; at l e a s t some of the many reports of the antifeedant e f f e c t of azadirachtin may be due to post-ingestive e f f e c t s . However, ele c t r o p h y s i o l o g i c a l studies and choice tests show unambiguously that azadirachtin does have a chemosensory-based antifeedant e f f e c t against some insects (Schoonhoven, 1982; Simmonds and Blaney, 1984). In Schistocerca gregaria azadirachtin may activate a s p e c i f i c " l a b e l l e d l i n e " deterrent receptor (Blaney, 1980). Neither 253 the ECI nor the ECD were reduced s i g n i f i c a n t l y ; the AD was increased only at the higher concentration. Similar r e s u l t s have been noted i n studies with the lepidopterans H e l i o t h i s  virescens (Barnby and Klocke, 1987) and Crocidolomia  b i n o t a l i s (Fagoonee, 1984); i n both cases consumption was decreased by azadirachtin without concomitant reductions i n e f f i c i e n c y of dietary u t i l i z a t i o n , and i n f a c t f o r the l a t t e r insect both the ECI and ECD increased to compensate fora reduction i n food volume. In contrast, Arnason et a l . (1985) found that dose-dependent reductions i n growth of O s t r i n i a n u b i l a l i s were due to a reduction i n ECI and ECD; consumption rate was unaffected by azadirachtin concentrations i n a r t i f i c i a l d i e t up to 30 ppm. Their r e s u l t s could be owing to the obstruction of the s e n s i l l a r pores on the mouthparts by the agar-based d i e t (Arnason, pers. comm. 1988). The limonoids cedrelone and anthothecol also reduce the ECI and ECD at concentrations which a c t u a l l y increase the consumption rate i n O s t r i n i a n u b i l a l i s (Arnason et a l . , 1987). Rao and Subramanyam (1986) found that azadirachtin lowers the RCR, ECI, and ECD i n f i f t h - i n s t a r Schistocerca gregaria. In a l l studies to date the AD i s unaffected or increases with increasing azadirachtin concentration. Such a phenomena would r e s u l t from a decreased rate of gut p e r i s t a l s i s (Mordue §t a l . , 1985), increasing the amount of time during which the food bolus i s exposed to digestive enzymes (Slansky and Scriber, 1985). 254 Most notable i s the absence of any difference i n growth, consumption, or n u t r i t i o n a l u t i l i z a t i o n indices between 10 and 15 nq/q insect, as these doses produce remarkably d i f f e r e n t e f f e c t s on molting. At the lower dose, most nymphs molted successfully, a l b i e t to malformed adults, whereas at the higher dose a l l nymphs died i n the molt. The lack of c o r r e l a t i o n between n u t r i t i o n a l and presumably endocrine-mediated e f f e c t s suggests that separate physiological targets may be involved. B. Oral, t o p i c a l , and i n j e c t i o n experiments M. sanguinipes nymphs which consumed azadirachtin at the beginning of the i n s t a r subsequently displayed a dose-dependent range of e f f e c t s . Low doses (<10 nq/q insect) resulted i n molt delay and deformity of adult appendages, intermediate doses (13-15 nq/q) resulted i n death at a f a i l e d molt attempt, and high doses (>25 nq/q) produced permanent f i f t h i n s t a r nymphs i n which the molting response was abolished. These e f f e c t s p a r a l l e l those seen with Locusta (Mordue et a l . , 1985, 1986), Oncopeltus (Redfern et a l - , 1982), and other insects (Gaaboub and Hayes, 1984; Koul, 1984; Ladd et a l . , 1984). Molt f a i l u r e has been attributed to disruption of the normal ecdysteroid t i t r e s i n several species (Koul et a l . . 1987; Rembold and Sieber, 1981; Rembold gt a l . , 1984; Schluter et a l . , 1985; Mordue and Evans, 1987; Min-Li and Shin-Foon, 1987). However, the physiological basis of t h i s 255 disruption i s not known. Mordue et aJL.(1986) suggested that f a i l u r e to a t t a i n c r i t i c a l weight was responsible for f a i l e d ecdysis i n Locusta migratoria, but the lowest dose at which they measured growth (7.5 /ig) was almost three times the dose required to i n h i b i t molting. In t h i s study f a i l u r e to molt was not due to a f a i l u r e to reach c r i t i c a l body mass as the weight of Category IV nymphs at d 13 post-treatment (232.1 + 17.6 mg) exceeded the weight of the controls at molting (207.6 ± 25.4 mg). This implies a more d i r e c t action on the endocrine system, rather than an i n d i r e c t e f f e c t operating through i n t e r n a l measures of growth. Application of azadirachtin t o p i c a l l y or by intrahemocoelic i n j e c t i o n also reulted i n dose-dependent i n h i b i t i o n of molting. The MD 5 0 f o r injected azadirachtin i n M. sanguinipes. 3.01 /ig/g, was higher than that reported for Locusta migratoria , 2.0 /ig/g (Mordue et a l . , 1985) or Schistocerca gregaria, 1.66 /ig/g (Rao and Subrahmanyam, 1986). Azadirachtin i s also acutely t o x i c to some insect s when injected at high doses: the 24 h L D 5 0 for Locusta  migratoria i s 80 /ig/g (Mordue ej£ a l . , 1985? Cottee et a l . . 1988), and f o r Schistocerca gregaria the L D 5 0 i s 330 /ig/g (Cottee et a l . , 1988). Acute t o x i c i t y to M. sanguinipes was not observed at the highest dose tested i n t h i s study, 25 The MD 5 0 v i a o r a l administration (10.83 /ig/g) was over three times that v i a i n j e c t i o n (3.01 /ig/g), i n d i c a t i n g that the gut poses a physical or physiological b a r r i e r to 256 azadirachtin b i o a v a i l a b i l i t y . The gut presents a s i m i l a r b a r r i e r to several other natural products i n Locusta  migratoria and Schistocerca gregaria, indicated by s i m i l a r r a t i o s of o r a l to injected acute t o x i c i t y (Cottee ej: a l . , 1988). The observation that the o r a l t o x i c i t y of azadirachtin can be synergised i n M- sanguinipes by coadministration of the mixed-function oxidase i n h i b i t o r piperonyl butoxide suggests that the b a r r i e r i s la r g e l y owing to MFO based oxidative metabolism. The slope of the dose-response curve was the same i n the o r a l and i n j e c t i o n assays. The t o x i c i t y curve f o r the o r a l dose-response i s s h i f t e d by 10 /ig/g r e l a t i v e to the injected dose-response suggesting that the gut MFO's are able to metabolise up to 10 ng azadirachtin/g insect, allowing azadirachtin i n excess of t h i s dose to penetrate to target s i t e s within the insect. In contrast, azadirachtin was equitoxic when applied t o p i c a l l y (MD50= 3.80 /ig/g) or v i a i n j e c t i o n (95% f i d u c i a l l i m i t s overlap), i n d i c a t i n g that i n t h i s insect the integument does not s i g n i f i c a n t l y l i m i t penetration and therefore b i o a v a i l a b i l i t y of azadirachtin to putative target s i t e s within the insect. In each case azadirachtin must have considerable s t a b i l i t y once inside the insect, as i t s e f f e c t s on molting are expressed 8 to 14 days a f t e r exposure to a singl e dose. Rembold et a l (1984) found the h a l f - l i f e of injected 3H-dihydroazadirachtin to be greater than 1 week in Locusta migratoria. 257 The response of M.. sanguinipes to azadirachtin contrasts with i t s response to sesquiterpene lactones, b i t t e r p r i n c i p l e s from Asteraceous plants (Isman, 1985). Parthenin, the most active of s i x compounds tested, had a L D 5 0 of 1.5 /imol/g insect fwt when injected into the hemocoel, compared to a L D 5 0 of 4.4 nmol/g (= 3.2 /ig/g) for azadirachtin. However, adult males were able to t o l e r a t e exposure to doses up to 12 /imol/g, applied t o p i c a l l y or o r a l l y , without t o x i c symptoms. The gut and integument therefore provide e f f e c t i v e b a r r i e r s to b i o a v a i l a b i l i t y of sesquiterpene lactones, unlike the s i t u a t i o n with azadirachtin. The factors allowing penetration of some compounds and exclusion of others are not well understood i n t h i s insect: although l i p o p h i l i c i t y may play a r o l e i t i s u n l i k e l y to be a dominant factor as both hydrophilic ( i . e . azadirachtin) and strongly hydrophobic compounds (eg a-t e r t h i e n y l , a thiophene) (Smirle, Champagne, Isman, unpublished data) are absorbed e f f i c i e n t l y . C. Fecundity Experiment Azadirachtin, fed to adult females, reduced egg production during the f i r s t s i x weeks of the adult stage. This e f f e c t i s s i m i l a r to the reported chemosterilization of female Locusta migratoria (Rembold and Sieber, 1981), Oncopeltus  fasciatus (Dorn et a l . , 1986), Dysdercus koe n i g i i (Koul, 1984), and Rhodnius prolixus (Feder et a l . , 1989). In Locusta t h i s e f f e c t has been ascribed to diminished JH and 258 ecdysteroid t i t r e s , and i n Rhodnius azadirachtin lowered hemolymph ecdysteroid and v i t e l l o g e n i n t i t r e s , and impared ecdysteroid synthesis i n the ovaries. As adult female Melanoplus sanguinipes survive i n the f i e l d f o r only 25-33 days on average, the e f f e c t s reported here could r e s u l t i n a s i g n i f i c a n t decline i n f i e l d populations. D. Sterol studies Molting i s the end r e s u l t of a complex series of physiological events dependent on proper functioning of several organ systems including the gut (Nijhout, 1981). As suggested by Mordue et a l . (1985, 1986; Mordue and Evans, 1987), the observed endocrine e f f e c t s of azadirachtin could be due to the disruption of some aspect of gut function necessary f o r the i n i t i a t i o n of the molting process. Azasterols i n h i b i t the conversion of phytosterols such as 6-s i t o s t e r o l to cholesterol by i n h i b i t i n g the A 2 2 and A22,24 s t e r o l reductases, r e s u l t i n g i n the accumulation of the intermediate desmosterol (Svoboda and Robbins, 1971; Svoboda et a l . , 1972; Walker and Svoboda, 1973; Svoboda and Thompson, 1985). As cholesterol i s required for the assembly of normal membranes and as a precursor f o r ecdysteriod synthesis, azasterol t o x i c i t y i s characterized by i n h i b i t i o n of growth and molting, and by chemosterilization of adult females. These e f f e c t s may be a l l e v i a t e d by providing a source of cholesterol i n the d i e t (Walker and Svoboda, 1973). The molt i n h i b i t i n g e f f e c t s of 259 azadirachtin were not a l l e v i a t e d by supplementation with dietary c h o l e s t e r o l , i n d i c a t i n g that the target s i t e involved i s probably not the A 2 2 and ^22,24 s t e r o l reductases. As insects are unable to synthesize s t e r o l s de novo (Clarke and Bloch, 1959), transport of s t e r o l from the s i t e of absorbtion to target tissues i s of c r i t i c a l importance. This transport i s accomplished v i a a hemolymph l i p o p r o t e i n termed lipophorin (Chino, 1985), which loads cholesterol d i r e c t l y from the midgut (Chino and G i l b e r t , 1971). Lipophorin i s also involved i n the transport of f a t t y acids, mainly i n the form of d i a c y l g l y c e r o l , and i s able to bind l i p o p h i l i c pesticides and allelochemicals (Haunerland and Bowers, 1986). In M. sanguinipes nymphs, r a d i o l a b e l l e d B-s i t o s t e r o l was observed to cross the gut and appear i n the hemolymph, r u l i n g out i n h i b i t i o n of s t e r o l transport as a mechanism of action f o r azadirachtin i n t h i s insect. The three-hour time lag between consumption and the f i r s t appearance of r a d i o l a b e l l e d s t e r o l i n the hemolymph suggests that s t e r o l adsorbtion probably occurrs from the midgut i n M. sanguinipes. In general, the s i t e of s t e r o l absorbtion i n insects appears to be related to the type of food used: carnivorous and omnivorous insects adsorb from the crop, whereas phytophagous insects absorb s t e r o l s from the midgut (Clayton et a l . , 1964; Joshi and Agarwal, 1977; Kuthiala and R i t t e r , 1988). In p a r t i c u l a r , i n the few phytophagous orthopterans studied to date, including Schistocerca 260 gregaria, s t e r o l absorbtion occurs from the g a s t r i c caceae (Joshi and Agarwal, 1977). The decreased rate of appearance of r a d i o l a b e l i n the hemolymph of azadirachtin-treated nymphs i s consistant with a decreased rate of gut p e r i s t a l s i s . The decrease i n rad i o l a b e l content of control hemolymph at 11 and 12 h i s probably due to s t e r o l unloading at the f a t body and other target t i s s u e s ; i n H e l i o t h i s zea a s i m i l a r decrease i n hemolymph s t e r o l was associated with an increase i n the amount of rad i o l a b e l associated with the f a t body (Kuthiala and R i t t e r , 1988). Unfortunately t h i s experiment did not extend f o r long enough to observe a s i m i l a r decrease i n radiolabel content of hemolymph from azadirachtin-treated nymphs, so the e f f e c t of azadirachtin on s t e r o l unloading remains unknown. I t i s not known i f cholesterol unloading i s passive or an active, receptor-mediated process i n insects (Chino, 1985). The i d e n t i t y of r a d i o l a b e l l e d s t e r o l s i n the insect 24 h a f t e r feeding with 1 4 C - B - s i t o s t e r o l i s given i n Table 5-2. The s t e r o l composition of control and azadirachtin-treated insects does not d i f f e r , corroborating the r e s u l t s of the s t e r o l supplementation experiment and r u l i n g out s t e r o l reductase i n h i b i t i o n as a mechanism of action. The s t e r o l composition of M. sanguinipes i s dominated by cho l e s t e r o l , with a lesser amount of B - s i t o s t e r o l and < 2% of the intermediate desmosterol. This pattern i s common to most phytophagous insects, which are able to dealkylate C 2 Q and C 2g phytosterols to cholesterol (Svoboda and Thompson, 261 1985). Although some insects accumulate large amounts of cholesterol esters (Svoboda and Thompson, 1985), these were not observed i n the M.. sanguinipes extracts; however d e r i v i t i z a t i o n with t r i f l u o r o a c e t i c acid may remove the ester function. E . In vitro formation of adducts The t o x i c action of a number of plant products involves the formation of covalent Michael adducts between the allelochemical and s u l f h y d r y l residues of proteins; examples include the sesquiterpene lactones (Pieman et a l . , 1979) and warburganal (Ma, 1977). Such a mechanism of action has been proposed to account f o r the c y t o t o x i c i t y of several limonoids to murine P-388 lymphocytic leukemia c e l l s ( P e t t i t et a l . , 1983). Two tissues possibly involved i n azadirachtin t o x i c i t y , mouthpart chemoreceptors and the pars i n t e r c e r e b r a l i s , contain protein unusually r i c h i n s u l f h y d r y l residues (Norris, 1988; F r i e d e l and Loughton, 1980). I found that azadirachtin does not spontaneously form adducts with cysteine, at l e a s t at neutral pH, suggesting that non-specific binding to s u l f h y d r y l - r i c h protein i s u n l i k e l y to play an important r o l e i n azadirachtin t o x i c i t y . This i s consistant with the observation that unbound azadirachtin may be r e a d i l y extracted from Locusta brains as much as one week a f t e r i n j e c t i o n of the compound (H. Rembold, pers comm 1988). As well, azadirachtin treated s e n s i l l a e return to normal 262 funtioning within 2-5 min (Simmonds and Blaney, 1984), i n d i c a t i n g that the receptors have not suffered irreversable damage such as i s produced by sulf h y d r y l reagents (Ma. 1977). The p o s s i b i l i t y remains that other targets i n the gut can be affected by azadirachtin, f o r example the release of factors involved i n stimulating elevated rates of protein synthesis i n the f a t body, recently demonstrated i n Locusta  migratoria (Laughton et a l . , 1987). As the r e l a t i o n s h i p between feeding and endocrine events has been p a r t i c u l a r l y well studied i n Jf- sanguinipes (Dogra and G i l l o t t , 1971; E l l i o t t and G i l l o t t , 1977a,b), t h i s insect may provide a convenient model system f o r further studies on the mode of action of azadirachtin. F. A g r i c u l t u r a l implications Grasshoppers p e r i o d i c a l l y i n f l i c t severe damage on cereal crops and rangeland forage (Bierne, 1971; Hewitt and Onsager, 1983). Melanoplus sanguinipes i s always a major contributor to grasshopper outbreaks, and o v e r a l l i s considered the fourth most damaging pest insect to Canadian agricult u r e (Bierne, 1971). Currently, control r e l i e s on a e r i a l spraying of the pyrethroids deltamethrin (Johnson et a l . , 1986), cypermethrin, and carbaryl (Mukerji and Ewen, 1984); dimethoate, malathion, and methamidophos are also used (Harris, 1985). As t h i s method of application may have severe impact on non-target insects, e s p e c i a l l y p o l l i n a t o r s , 263 recent work has focussed on use of b a i t , p a r t i c u l a r l y wheat bran, impregnated with i n s e c t i c i d e (Onsager et a i . , 1980a,b; Mukerji et a l . , 1981; Mukerji and Ewen, 1984; Johnson and Henry, 1986) or the pathogen Nosema locustae (Henry, 1972; Johnson and Pavlikova, 1986; Johnson and Henry, 1986). At sublethal doses N. locustae reduces feeding and reproduction. The application of bran b a i t i s f a c i l i t a t e d by the d i s t r i b u t i o n of grasshoppers, which tend to concentrate within 10-15 m of roadsides (Bierne, 1971; Johnson and Henry, 1986). As azadirachtin does not produce an antifeedant response i n M. sanguinipes. i t would appear to have pot e n t i a l as an i n s e c t i c i d e applied to b a i t s . Application would have to be timed to coincide with the presence of early i n s t a r s , before the nymphs are capable of economic l e v e l s of damage, as mortality would occur only at molting, some days a f t e r a p p l i c a t i o n . Application l a t e r i n the l i f e c ycle could produce permanent nymphs. Advantages to the use of azadirachtin could include minimal non-target impact, low residue l e v e l s due to the rapid photodegradation of azadirachtin i n sunlight (Yamasaki et a l . , 1988), and possibly compatability with b i o l o g i c a l control measures including N. locustae. Even at sublethal doses azadirachtin based treatments could be expected to reduce feeding and reproduction. A l l the currently available i n s e c t i c i d e s share a common mode of action, neurotoxicity; azadirachtin could provide an 264 alt e r n a t i v e with a r a d i c a l l y d i f f e r e n t mode of action, minimizing the chances of cross-resistance. In insects Where azadirachtin has antifeedant a c t i v i t y ( i e P.. saucia), the development of resistance would involve overcoming both the chemosensory and the physiological e f f e c t s . For example two s t r a i n s of P l u t e l l a x y l o s t e l l a showed no evidence of resistance i n feeding and fecundity t e s t s a f t e r 35 generations of exposure to neem seed extract; the same two st r a i n s developed resistance factors of 20-35 to deltamethrin i n the same time period (Vollinger, 1986). Populations which became r e s i s t a n t to deltamethrin d i d not show cross-resistance to neem ssed extract. However, M -sanguinipes already posseses s i g n i f i c a n t MFO a c t i v i t y against azadirachtin and lacks an antifeedant response, and so could be expected to develop resistance f a i r l y r a p i d l y i n the presence of strong s e l e c t i o n pressure. A sucessful pest management strategy w i l l have to r e l y on a vari e t y of control measures, employing a vari e t y of modes of action, j u d i c i o u s l y applied to minimize the chance of developing resistance. 265 Chapter 6: General Summary The work described i n the previous four chapters examined several aspects of the putative defenses against herbivorous insects found i n members of the plant family Meliaceae. The studies began with a preliminary examination of the r e l a t i o n s h i p between defense strategies and plant l i f e -h i s t o r y c h a r a c t e r i s t i c s , moved to an attempt to i d e n t i f y the phytochemicals involved, and concluded with d e t a i l e d investigations of the e f f e c t s and mode of action of the major group of phytochemicals involved, the limonoids, i n three model insect species. In the f i r s t study, r e l a t i v e investment i n phytochemical-based defenses i n t h i r t y species of Meliaceae was estimated, by investigating the response of an unadapted, generalist herbivore, Peridroma saucia f to the enti r e s u i t e of phytochemicals produced, as included i n the methanolic extract of mature f o l i a g e . Physical defenses i n the Meliaceae are l a r g e l y confined to leaf toughness facto r s ; consequently leaf toughness was measured on sixteen species. Leaf l i f e t i m e was not measured d i r e c t l y , but a l l species were classed as deciduous or evergreen, with the assumption that leaf l i f e t i m e s would be longer for the evergreen species. Large differences were found between species i n the r e l a t i v e investment i n phytochemical defenses. Extracts of some species, p a r t i c u l a r l y i n the t r i b e Melieae, were i n h i b i t o r y to E . saucia growth at 266 concentrations only 1% of those occurring naturally i n the f o l i a g e . The most active extracts were a l l from members of the subfamily Melioideae, whereas extracts from the subfamily Swietenioideae were on average less a c t i v e . Only three species appeared to lack phytochemical defenses against generalist herbivores, assuming £. saucia i s a v a l i d model species. The plant apparency hypothesis of Feeny (1976) and Rhoades and Cates (1976) predicts s i m i l a r defenses i n a l l the species studied here, as they are a l l "apparent", perennial tree species. This prediction was not supported by my data, as large differences between species i n defensive at t r i b u t e s were found. The resource a v a i l a b i l i t y hypothesis of Coley e_t a l . (1985), on the other hand, predicts that species with short leaf l i f e t i m e s ( i . e . deciduous species i n t h i s study) should be selected f o r phytochemically-based defenses, and species with long l e a f l i f e t i m e s ( i . e . evergreen species) should elaborate physical defenses including leaf toughness. In t h i s study, extracts from deciduous species were found to be s i g n i f i c a n t l y more i n h i b i t o r y to E- saucia growth. Previous attempts to quantify investment i n chemical defenses have r e l i e d on colorimetric assays for phenolics only, and these did not correlate with leaf l i f e t i m e or show an inverse r e l a t i o n s h i p with herbivory (Coley, 1983, 1988), leading Coley (1988) to suggest that the importance of plant chemistry as a defense had been 267 overestimated. As generalist or unadapted herbivores ( i . e . P.saucia) have been postulated to be the target of t o x i c or antifeedant natural products (Feeny,1976; Rhoades and Cates, 1976; Coley et a l . , 1985), bioassays with such species "may represent the most relevant method fo r assessing r e l a t i v e investment i n phytochemical-based defenses. Therefore, my r e s u l t s indicate that Meliaceae with short leaf l i f e t i m e s do invest more i n secondary-metabolite based defenses than do the evergreen species. Leaves of evergreen species were almost twice as tough as leaves of deciduous species. However, the r e l a t i o n s h i p of increasing leaf toughness with increasing leaf l i f e t i m e i s well established from previous studies (Coley, 1983, 1985), as i s the r e l a t i o n s h i p between leaf toughness and reduced rates of herbivory. As well, there i s evidence i n my data to suggest an inverse r e l a t i o n s h i p between leaf t o x i c i t y and leaf toughness, suggesting that species with tough leaves require lower l e v e l s of production of phytochemical defenses. However, t h i s hypothesis needs further examination. Species not previously known fo r the production of insect i n h i b i t o r y natural products and i d e n t i f i e d here for the f i r s t time included Aglaia odorata and Turreae h o l s t i i . The three available Aglaia species showed a range of b i o a c t i v i t y against P. saucia, and so t h i s genus was chosen for an inves t i g a t i o n into the phytochemical basis of resistance to herbivory. The natural product chemistry of A., odorata proved complex; compounds i s o l a t e d and i d e n t i f i e d 268 using spectroscopic methods included the known dammaranes a g l a i t r i o l and a g l a i o n d i o l , and the bis-amides (S,S)-odorine, (S,R)-odorine (a new natural product), (S,S)-odorinol, and (S,R)-odorinol. As well a s e r i e s of methylated flavanones, previously unknown i n the Meliaceae, were i d e n t i f i e d , including 3-hydroxy-5,7,4'-trimethoxyflavanone (also a new natural product), 5,7,4'-trimethoxyflavanone, and 5-hydroxy-7,4'-dimethoxyflavanone. These compounds, however, a l l proved to be inactive against P. saucia, when tested s i n g l y or i n combination. Rather, the active constituent appeared to be a single compound, t e n t a t i v e l y i d e n t i f i e d as a limonoid. This compound i n h i b i t s P. saucia growth, with an E C 5 0 o f I - 4 /ig/g d i e t fwt and an L C 5 0 of 11.2 /ig/g, concentrations which do not a f f e c t feeding behaviour. The a c t i v i t y therefore appears to be due to post-ingestive t o x i c e f f e c t s . The i s o l a t e d y i e l d of the compound, 3 /ig/g leaf dwt, was less than the expected concentration, 98 /ig/g, based on the a c t i v i t y of the methanolic extract; the discrepancy suggests the p o s s i b i l i t y of a s y n e r g i s t i c i n t e r a c t i o n , but combinations of the active compound and the other phytochemicals i s o l a t e d from A. odorata produced only additive e f f e c t s . The t h i r d study began with a review of the current l i t e r a t u r e on the e f f e c t s of limonoids on phytophagous insects. Most studies have focussed on assays f o r feeding i n h i b i t i o n , r e f l e c t i n g the p r e v a i l i n g b e l i e f that limonoids function mostly to deter insect feeding, but pre-ingestive 269 chemosensory-based e f f e c t s are not c l e a r l y separated from post-ingestive t o x i c e f f e c t s , including growth regulating e f f e c t s . As well, the a c t i v i t y of these compounds has not been evaluated i n r e l a t i o n to t h e i r proposed evolution. Consequently, I investigated ten limonoids, representing a l l the major biosynthetic classes, f o r i n h i b i t i o n of growth and feeding against £. saucia, and for e f f e c t s on molting and reproduction against Oncopeltus  f a s c i a t u s . The simple apo-euphol type limonoids cedrelone and anthothecol, with an i n t a c t s t e r o i d skeleton, were highly i n h i b i t o r y to E . saucia growth at 0.5 umol/g d i e t fwt, a concentration which did not a f f e c t feeding i n a choice t e s t . As well, cedrelone i n h i b i t e d Q. fasciatus molting, with an L D 5 0 of 12.2 /ig/nymph, indicating'that even simple limonoids may have IGR a c t i v i t y . Anthothecol, which d i f f e r s from cedrelone i n having an acetoxy s u b s t i t u t i o n at C - l l , was inactive i n the Oncopeltus assay, i n d i c a t i n g the importance of C-ring substitutions i n determining b i o l o g i c a l a c t i v i t y , and suggesting that growth and molt i n h i b i t i o n involve separate physiological targets. The dammarane precursors of these simple limonoids were inactive against E- saucia, so to t h i s point biosynthetic evolution coincides with an increase i n i n s e c t i c i d a l a c t i v i t y . However, oxidative opening ofthe D r i n g , as i n gedunin, leads to a pronounced drop i n a c t i v i t y . Paradoxically, t h i s i s a major step i n the biosynthetic evolution of limonoids, and characterizes a l l limonoids found i n the Rutaceae and 270 Simaroubaceae, and most limonoids found i n the Swietenioideae. Further oxidation to produce the A,D-seco limonoids leads to compounds, including obacunone, nomilin, harrisonin, and pedonin, which were also inactive against p.. saucia and O. f a s c i a t u s . Of two B,D-seco limonids tested, entandrophragmin was i n a c t i v e and bussein weakly i n h i b i t e d P. saucia growth; such limonoids are c h a r a c t e r i s t i c of many Swietenioideae including Khaya and Entandrophragma (Taylor, 1981), considered to be advanced genera on morphological grounds (Pennington and Styles, 1975). The main l i n e s of limonoid evolution i n most Meliaceae, therefore, do not appear to correlate with i n s e c t i c i d a l a c t i v i t y against ge n e r a l i s t herbivores. This conclusion was supported by an attempt to correlate measures of s k e l e t a l oxidation and rearrangement, i d e n t i f i e d by Das e t a l . (1984, 1987) as the dominant themes i n limonoid evolution, with i n h i b i t o r y a c t i v i t y against four species of polyphagous lepidopterans (data from t h i s study and Kubo and Klocke, 1986). No c o r r e l a t i o n was found. The C-seco limonoids, including azadirachtin, may represent a possible exception to the above conclusion. These limonoids may be derived from euphol or t i r u c a l l o l precursors independently of the other limonoids (Siddiqui et a l . , 1988), or they may be formed from an i n t a c t apo-euphol type limonoid with oxidation at C-12 (Jones et a l . , 1988). This class of limonoid i s the most active, and the most 271 advanced C-seco limonoid, azadirachtin, i s known to be active against more than two hundred species of a g r i c u l t u r a l l y important pest insects (Warthen, 1979, 1989; Saxena, 1989). Azadirachtin was highly i n h i b i t o r y against P.. saucia, with an E C 5 0 of 0.4 nmol/g d i e t fwt and an L C 5 0 of 5.2 nmol/g. Chemosensory-based antifeedant e f f e c t s were most pronounced against neonate c a t e r p i l l a r s , but became much reduced by the early t h i r d i n s t a r . However, pronounced growth i n h i b i t i o n and mortality continued throughout the l i f e c y c l e . Analysis of dietary use and e f f i c i e n c y demonstrated that azadirachtin led to decreased consumption at concentrations which did not a f f e c t measures of dietary e f f i c i e n c y or feeding i n a choice t e s t , suggesting post-ingestive e f f e c t s , perhaps involving the gut d i r e c t l y or neural regulation of feeding. As well, the d i g e s t a b i l i t y was increased at the highest concentration tested. Together these r e s u l t s concur with the mechanism of action proposed by Mordue et a l . (1985), who suggested that i n h i b i t i o n of gut p e r i s t a l s i s could l i m i t feeding and growth by l i m i t i n g the rate at which a food bolus could move through the gut. Azadirachtin was also highly i n h i b i t o r y to O. f a s c i a t u s . disrupting molting (MD 5 0 = 3.8 ng/nymph) and decreasing adult s u r v i v a l , i n agreement with e a r l i e r studies (Dorn, 1983, 1987). The f i n a l study focussed on the migratory grasshopper, Melanoplus sanguinipes. as t h i s insect had been reported to 272 be r e s i s t a n t to azadirachtin (Mulkern and Mongolkitti, 1975). I n i t i a l experiments confirmed a complete lack of an antifeedant response when nymphs were fed leaf disks treated with up to 500 ppm azadirachtin. However, nymphs fed azadirachtin were subsequently unable to molt. A dose-response experiment indicated a dose-dependant range of ef f e c t s , from delay of molt at doses below 5 /ig/g insect fwt, to deformation of adult structures at doses up to 10 /ig/g, to death during an incomplete molt attempt at 13 and 15 /ig/g, culminating i n complete blockage of the molt at doses of 15 /ig/g and above. These e f f e c t s were s i m i l a r to those reported e a r l i e r i n Locusta (Rembold and Seiber, 1981, Mordue et a l . , 1985; Mordue, 1988), but are noteworthy as they followed o r a l application, whereas i n e a r l i e r studies the azadirachtin was applied by i n j e c t i o n , bypassing the normal chemosensory mechanisms and gut defenses. The consumption of p h y s i o l o g i c a l l y active doses of azadirachtin by M.- sanguinipes nymphs allowed me to evaluate the importance of the gut and integument to b i o a v a i l a b i l i t y of azadirachtin, by comparing o r a l and t o p i c a l a c t i v i t y with the a c t i v i t y of injected azadirachtin. There was no s i g n i f i c a n t difference between t o p i c a l and injected azadirachtin (MD 5 0 3.8 and 3.01 /ig/g r e s p e c t i v e l y ) , i n d i c a t i n g the absence of ba r r i e r s to azadirachtin b i o a v a i l a b i l i t y i n the integument. However, the o r a l MD50, 10.8 /ig/g, was s i g n i f i c a n t l y higher than the injected MD50. The b a r r i e r associated with the gut l i k e l y involves the MFO 273 system and oxidative metabolism, as the a c t i v i t y of o r a l l y administered azadirachtinis s i g n i f i c a n t l y increased by co-app l i c a t i o n of the MFO i n h i b i t o r piperonyl butoxide. Analysis of growth, food consumption, and measures of dietary e f f i c i e n c y following consumption of azadirachtin showed that the consumption rate was s i g n i f i c a n t l y decreased, despite the lack of an antifeedant response, again i n d i c a t i n g an e f f e c t on the gut or on the neural regulation of feeding. However, there was no difference i n n u t r i t i o n a l performance between 10 and 15 /ig/g, although these doses produced markedly d i f f e r e n t e f f e c t s on molt success, suggesting that e f f e c t s on endocrine events leading to molt i n h i b i t i o n are not d i r e c t l y r e l a t e d to the e f f e c t s on gut p e r i s t a l s i s . Two hypotheses f o r the mechanism of action of azadirachtin were tested. The symptoms of azadirachtin treatment c l o s e l y p a r a l l e l the symptoms produced by treatment with azasterols, compounds known to i n h i b i t the conversion of phytosterols to cholesterol (Svoboda and Thompson, 1985). However, azadirachtin t o x i c i t y could not be rescued by supplementing the d i e t with cho l e s t e r o l , and azadirachtin treatment did not a f f e c t the a b i l i t y of M. sanguinipes nymphs to metabolize B - s i t o s t e r o l to desmosterol and c h o l e s t e r o l . As well, the a b i l i t y of hemolymph lipophbrin to transport s t e r o l s was not reduced, although the time course of s t e r o l pharmacokinetics was s l i g h t l y altered, presumably due to a slower rate of gut p e r i s t a l s i s . 274 Secondly, P e t t i t et a l . (1983) suggested that the c y t o t o x i c i t y of some limonoids might be due to the a b i l i t y to form Michael adducts with su l f h y d r y l residues, and the two most conspicuous targets f o r azadirachtin, neurosecretory material and transducing protein i n chemosensillae, are unusually r i c h i n cysteine residues (Norris, 1988; F r i e d e l and Laughton,1980). However, I found that azadirachtin did not spontaneously form adducts with cysteine FB, suggesting that non-specific binding to s u l f h y d r y l - r i c h protein i s u n l i k e l y to be involved i n the mechanism of action. 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Stone and M.V. Darlington 1986. I s o l a t i o n and p u r i f i c a t i o n of azadirachtin from neem (Azadirachta indica) seeds using f l a s h chromatography and high-performance l i q u i d chromatography. J . Chromatog. 356:220-226. Yu, S.J. 1983. Induction of detoxifying enzymes by allelochemicals and host plants i n the f a l l armyworm. Pest. Biochem. Physiol. 19:330-336. Yu, S.J., 1986. Consequences of induction of foreign-compound metabolizing enzymes i n insects. In "Molecular Aspects of Insect-Plant Associations", L.B. Brattsten and S. Ahmad, eds., Plenum Press, New York. Zanno, P.R., I. Miura, K. Nakanishi and D.L. Elder 1975. Structure ofthe insect phagorepellant azadirachtin: Application of PRFT/CWD carbon-13 nuclear magnetic resonance. J . Am. Chem. Soc. 97:1975. Zebitz, C.P.W., 1986. E f f e c t s of three d i f f e r e n t neem seed kernel extracts and azadirachtin on larvae of d i f f e r e n t mosquito species. J . Appl. Ent. 102:455-463. Zittear, T.A. 1984. E f f e c t s of c i t i z e n chemophobia on plant pathology. Plant Dis. 68:655. Appendix 1: Host plants of Peridroma saucia (after T i e t z , 1972; Bierne, 1972). Abies balsamea (Linn) seedlings Abies grandis (Dougl.) Acer sp. Acer negundo Linn. Acer saccharinum Linn. Allium cepa Linn. Alnus rubra Bong. Althaea rosea Cav. Ambrosia a r t e m i s i i f o l i a Linn. Amelanchier f l o r i d a L i n d l . Anthemis cotula Linn. Asparagus medeoloides Thunb. Asparagus o f f i c i n a l i s Linn. Asparagus plumosa Baker Beta vu l g a r i s Linn. Brassica nigra (Linn.) Brassica h i r t a Moench Brassica oleracea Linn. Brassica rapa Linn. Capselja bursa-pastoris (Linn.) Capsicum annum Linn.(peppers) Chamecyparis thyoides (Linn.) seedlings Chrysanthemum sp. Cirsium sp. Cirsium vulaare (Savi) Citrus limon (Linn.) lemon Cit r u s sinensis (Linn.) orange Conyza canadensis (Linn.) Cucumus sativus Linn. Datura stramonium Linn. Daucus carota v. sativus Hoff. Dianthus caryophyllus Linn. Epilobium anaustifolium (Linn.) Eupatorium sp. Fragaria c h i l o e n s i s (Linn.) Geranium sp. G l e d i t s i a triacanthos Linn. Gossypium herbaceum Linn. Helianthus annuus Linn. Humulus lupulus Linn. Lactuca sativa Linn. Lathyrus odoratus Linn. Lycopersicon esculentum (Linn.) Maclura pomifera (Raf.) Maius pumila M i l l . Medicago sativa Linn. Melilotus alba Desr. Morus sp. Nasturtium sp. Nicotiana tabacum Linn. Parthenium araentatum Gray 314 Persea americana M i l l . Phieuro pratense Linn. Plantago lanceolata Linn. Plantago sp. Polygonum aviculare Linn. Portulaca oleracea Linn. Prunus americana Marsh. Prunus cerasus Linn. Prunus armeniaca Linn. Prunus domestica Linn. Prunus domestica v. galatensis ex Hook Prunus emarginata Dougl. Prunus persica (Linn.) Pteridium latiusculum (Desv.) Ouercus sp. Ouercus alba Linn. Raphanus sativus Linn. Rheum rhaponticum Linn. Rhus sp. Rhus c o p a l l i n a Linn. Ribes lacustre (Pers.) Ribes sanouineum Pursh. Ribes sativum Syme Rosa sp. Rubus allegheniensis Porter Rubus oc c i d e n t a l i s Linn. Rubus idaeus v. strioosus (Michx.) Rumex crispus Linn. Sal i x sp. S a l i x hookeriana Barr. S a l i x xcouleriana Barr. S a l v i a o f f i c i n a l i s Linn. Smilax r o t u n d i f o l i a Linn. Solanum tuberosum Linn. Solidago leavenworthii T.&G. S t e l l a r i a media (Linn.) T r i f o l i u m sp. Triticum aestivum Linn. Tropaelum majus Linn. Tsuga canadensis (Linn.) seedlings U r t i c a sp. Vaccinium angustifolium A i t . Vaccinium corymbosum Linn. Vaccinium m y r t i l l o i d e s Michx. V i o l a sp. V i o l a t r i c o l o r Linn. V i t i s sp. V i t i s v i n i f e r a Linn. Xanthium strumarium Linn. Zea mays Linn. Ferns, general feeder on f i e l d crops, forest trees, f r u i t trees, grass, low plants, most anything, shrubs, vegetables. 315 Appendix 2: 1H-NMR and mass spectra of compounds i s o l a t e d from Aalaia odorata. Compound 3: 3-hydroxy-5,7,4'-trimethoxyflavanone 00 lea.e - i 30.0-M'E 100.0• se.e -N H S S S P E C T R U M ej-21 -as i J ienee • 1140 S M M P L E I HD-2 •98 TO I I M SUMMED - «3 TO «10 XI.80 134 OATAi octei t33 BASE H'Ei 134 RICi 33213288. 91 63 41 33 3d jjj] 7 ? iiiJllpiL 96 JL 189 tJl,J 1111^,4, Jpiln tee 132 90 ,J,iJkl,L, 29? LL.I,ji>llly I l 130 tu\% ,T, , T . r 230 3330710. 10. r 3330710. 10. 3 4 286 rvE 29? Compound 4 : S^^'-trimethoxyflavanone I 3:>» 341 3 ^ 363 • * I - I • • i ' i • * i 388 I 338 .13? jae .lag . , I ' I ' 1 430 I • I ' I t—1 U3 IOP.O -, 50.9-1 •^ •i TO «6? SUMMED - «I0 TO »I3 XI.«e 134 DATAi PC 102 »39 BASE M'El 134 PICi 3345Sieg. 91 63 5.1 41 -U 77 103 M-E lee.e 58 l u l l p 2617349. ie. 166 156 193 181 243 —f 2se 29? 272 r 2617340. ie. 300 50.PH Compound 5: 5-hydroxy-7,4/-di.Heth.oxyflavanone 1*1 to to Compound 7: (S,R)-odorine iae.0 - i 90.0 H MtiSS S P E C T R U M u-t i ; 83 9 I 4 7 I 6 6 * 1102 S H U P L E I FLrtU •S3 T O « 6 3 SUMMED - #10 T O 113 M l . 8 6 134 DATAI 0C1B2 639 BASE M^El 134 RICi 33436160. 1?» 9.1 63 91 41 M'E 166.6 -Ik-Li 36 I 77 163 -I 129 166 In.*.']!  >., Jin i  130 193 101 I* |U> 2 2 297 272 1T,l..2f .^ ,9 -gt3 ...if. 200 2617340. 10. 290 3 6 . 6 H 366 M E^ Compound 7 : (S,R)-odorine 366 3>9 , , 3j6, , ^  3 7 7 ; jB? ,186, t 442, 336 I • I ' I • I 430 ' s i . ' ' ' ' 2617340. 10. ro Compound 8 : (S,S)-odorine iee.e-i 30.0-100.0-se.e -M - E MMiS S P E C T R U M 6412/89 I 8 H 4 I 0 0 • 1sse S H M P I E I A M I O E • 1 1 8 T O « 1 1 4 S U M M E D - « 7 T O « 1 4 X I . 0 0 1 3 1 DATAI DC183 1112 BASE M ' E I 1 3 1 RIC i 1 7 8 3 6 9 8 0 . 103 73 39 49 91 1 Jj.lu 63 77 83 Ililly.lt.J.. 3 0 T 9 ^ 4 201 109 4 3 I 3 T 244 'I J 190 I- , ™ t 1966710. 10. 290 I- 19C6710. 10 . Compound 9: (S,S)-odorinol .1. 388 36? , ,383, ,399, 1U , .128 • ,11? , 338 I • I • I 408 490 Compound 10 : (S,R)-odorinol 

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