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

in

THE FACULTY OF GRADUATE STUDIES

(BIOLOGY)

We accept this 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 sauciaf and for feeding inhibition against the migratory grasshopper, Melanoplus sanguinipes. All 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 holstii. Deciduous species produced extracts which were significantly more active than evergreen species, indicating a greater reliance on phytochemical-based defenses. Evidence is also presented to suggest that the leaves of evergreen species are tougher than deciduous species, and that there is a negative correlation between leaf toughness factors (physical defenses) and phytochemical-based defenses. These results are in agreement with predictions of the resource availability hypothesis.

The phytochemistry of Aglaia odorata, A. odoratissima. and A. argentia was examined in detail. Compounds iii

identified included the dammaranes, aglaiondiol and aglaitriol, and the bis-amides (S,S)-odorine, (S,R)-odorine

(a new natural product), (S,S)-odorinol, and (S,R)-odorincT.

Three dihydroflavanones were identified from the Meliaceae for the first time: 3-hydroxy-5,7,4'-trimethoxyflavanone (a new natural product), 5,7,4'-trimethoxyflavanone, and 5- hydroxy-7,4'-dimethoxyflavanone. All compounds were inactive against E« saucia. The inhibitory activity of A.. odorata appeared to be due to a compound, tentatively identified as a limonoid, which may be acting in conjunction with a synergist. This compound inhibits E- saucia larval growth in the absence of antifeedant activity.

The toxicology of limonoids, representing the major biosynthetic classes, was examined against E> saucia and the large milkweed bug, Oncopeltus fasciatus. Cedrelone and anthothecol inhibited E« saucia growth by 90%, but not feeding, when applied in diet at 0.5 /imol/g fwt. Cedrelone

also inhibited O. fasciatus molting, with an MD50 of 12.2

^g/nymph. In contrast, anthothecol, with an acetoxy function at C-ll, was inactive against O. fasciatus. The D- seco compound gedunin, and the A,D-seco limonoids obacunone, nomilin, and pedonin were inactive in these assays; harrisonin initially inhibited feeding by neonate P. saucia but produced no long-term effects on growth rate. Bussein inhibited growth by 35% but entandrophragmin had no effect.

Azadirachtin was the most toxic compound examined in

this study. Peridroma saucia growth (EC50 =0.4 nmol/g diet iv

fwt), survivorship (LC50 =5.2 nmol/g), pupation, pupal weight, and adult emergence were decreased in a dose- dependent manner. Chemosensory antifeedant activity was

implicated in neonates but was much less marked with third

instar larvae. Azadirachtin decreased relative growth and

consumption rates at doses lower than those affecting

nutritional efficiency, or feeding in the choice tests.

This suggests an action directly on the gut or on the neural regulation of feeding. Bioactivity of other limonoids did not correlate with measures of skeletal oxidation or rearrangement, although these are dominant themes in 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 orally, topically, or by injection, allowed determination of the role of the gut and integument in

limiting the bioavailability of this compound to putative

target site(s) within the insect. The oral MD50, 10.8 ng/g

insect fwt, was significantly higher than the injected MD50,

3.01 pg/g, indicating a barrier to bioavailability in the gut. The oral activity of azadirachtin was synergised by coadministration of piperonyl butoxide, indicating that the barrier is due largely to oxidative metabolism. There was no significant difference between topical (3.8 ng/g) and

injected activity, indicating that the integument does not pose a barrier to bioavailability. Azadirachtin decreased V

growth and consumption at doses which did not affect nutritional efficiency, again indicating an effect on the gut or neural regulation of feeding. No difference was seen in nutritional indices of nymphs treated with azadirachtin at 10 and 15 pq/q, although these doses produced markedly different effects on molting. This observation suggested that effects on endocrine events are not directly related to nutritional effects. The effects of azadirachtin treatment were not alleviated by dietary supplementation with cholesterol, and azadirachtin did not affect the hemolymph transport or metabolism of 14C-B-sitosterol, indicating that sterol metabolism is not the target for azadirachtin activity. Azadirachtin also did not form adducts with cysteine, suggesting that non-specific binding to sulfhydryl-rich protein is also unlikely as a mechanism of action. vi

Table of Contents Page

Abstract ii Table of Contents vi

List of Tables xi List of Figures xiii List 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 vii

C) Bioassays for antibiotic and phototoxic

activity . 71

D Leaf Toughness 71

E- Defensive Characteristics of Deciduous and

Evergreen Meliaceae 72

Discussion 79

A) Crude Extract Screening .. 79 B) Resource Availability Hypothesis 83

Chapter 3: Phytochemical Investigation of Aglaia Species

Introduction 89

Materials and Methods 91

A) Sources of Plant Material 91

B) Isolation and identification of secondary

metabolites in Aglaia foliage 91

Bl) Solvent partitioning. 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 Partitioning 97

C) Chromatography 97

D) Phy tochemi stry 106 E) Qualitative and quantitative comparison of Aglaia species 125

F) Insect Bioassays 141

Discussion 149 viii

A) Phytochemistry .149

B) Insecticidal Activity 152

Chapter 4: Effects 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) Nutritional Analyses 179

F) Molt Inhibition Assays 182

G) Correlation Between Evolutionary Advancement

and Activity 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 Utilization Experiments 192

E) Molt Inhibition Assays 196

F) Relationship of Anti-insect Activity to

Oxidation and Skeletal Rearrangement 201

Discussion 205

A) Group 2 Limonoids 205

B) D-seco Limonoids 207 ix

C) A,D-seco Limonoids 208

D) B,D-seco Limonoids 210

E) C-seco Limonoids (Azadirachtin). 211

F) Nutritional Indices 213

G) Limonoid Evolution and Structure-Activity Relationships 215

H) Correlation of Phytochemistry with Methanolic Extract Screening 217

I) Comparison of Insecticidal and Cytotoxic Activity 219

Chapter 5; Effects of azadirachtin on the nutrition 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 Activity Assays 224

D) Dietary Utilization Experiments 224

E) Molt Inhibition Assays 225

F) Piperonyl Butoxide Synergism Assay 226

G) Fecundity Experiment 227

H) Effect of Dietary Sterols 228

I) Sterol Transport Experiment 228

J) In vitro assay for the formation of adducts....229

Results 231

A) Antifeedant assays 231 X

B) Growth and Dietary Utilization 231

C) Molt Inhibition Studies... 233

D) Synergism by piperonyl butoxide 238

E) Fecundity Experiment 245

F) Sterol supplementation assays 245

G) In vitro assay for adduct formation 250

Discussion 252

A) Antifeedant and nutritional effects. 252

B) Oral, topical, and injection experiments 254

C) Fecundity Experiment 257

D) Sterol studies... ..258

E) In vitro formation of adducts 261

F) Agricultural implications 262

Chapter 6: General Summary 265

References: 275

Appendix 1: 3/3

Appendix 2: 3/5 xi

List of Tables

Table 2-1. Sources, collectors, and collection dates of plant material used in this study.... 55

Table 2-2. Growth inhibitory activity of meliaceous leaf extracts on neonate P. saucia 64

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 74

Table 2-4. Comparison of MeOH extract toxicity (as EC50 to Peridroma saucia [% of natural concentration]) and toughness (N/cm2) between deciduous and evergreen species of Meliaceae 78

Table 3-1. Typical results of flash column chromatography

(Si gel, 240-400 mesh) of A., odorata (Et20 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 Ehrlichs reagent and vanillin 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 in Aglaia species, determined by analytical HPLC 140

Table 3-7. Aglaia odorata compounds: concentration bioassayed, and resultant p_. saucia growth and survivorship (as % of Control) 143 Table 3-8. Effect of combinations of phytochemicals from Aglaia odorata on the growth of neonate Peridroma saucia (as % of Control) 147

Table 3-9. Effect of Compound 6 on diet choice by neonate Peridroma saucia 148

Table 4-1. Effects of limonoids on insect feeding and growth 159 xii

Table 4-2. Effect of limonoids on growth and diet choice of neonate Peridroma saucia ....185

Table 4-3. Effect of azadirachtin on Peridroma saucia pupation and adult emergence 189

Table 4-4. Effect of azadirachtin on diet choice by neonate and third instar Peridroma saucia 191

Table 4-5. Effect of azadirachtin on third instar Peridroma saucia growth and nutrition 193

Table 4-6. Comparison of EC50 values of crude extracts of Meliaceae with predictions of activity based on classes of limonoids reported to occur in the genera examined .218

Table 5-1. Effect of azadirachtin on Melanoplus sanguinipes growth and nutrition 232

Table 5-2. Radioladelled sterol composition of control and azadirachtin-treated Melanoplus sanguinipes fed 4-14C-8-sitosterol 251 xiii

List of Figures

Figure 1-1. Biosynthetic pathway leading to the formation of an apo-euphol type limonoid (modified from Siddiqui e_t al., 1988) 19

Figure 1-2, Biosynthetic pathway leading to the formation of a C-seco limonoid (modified from Siddiqui et al., 1988) 21

Figure 1-3. Biosynthetic pathway leading to the formation of a C-seco limonoid, according to Jones et al. (1988) 23

Figure 1-4, Relationship of neurosecretory and neurohemal organs involved in the endocrine regulation of molting in insects 53

Figure 2-1, Graphical depiction of the assumed relative cost of maintaining a chemical or physically -based defense against herbivores. 52

Figure 2-2, Growth (as % of control) of neonate Peridroma saucia fed artificial diet treated with a MeOH extract of foliage of Azadirachta indica, Melia toosenden, or Melia azedirach at 1, 2, or 3% of natural concentration 62

Figure 2-3, Consumption of glass-fibre discs, treated with 10% aqueous sucrose and MeOH extracts of Azadirachta indica, Melia azedirach, Turreae holstii, and Aglaia odorata at 1, 2.5, and 5 times natural concentration (on a wt/wt basis), by fifth instar nymphs of Melanoplus sanguinipes 69

Figure 2-4, Relationship between leaf toughness (in N/cm2) and bioactivity of the MeOH extract of foliage,

calculated as 100-EC50 • 76

Figure 3-1. Effect of foliar MeOH extracts of Aglaia odorata, A., odoratissima, and A., argentia on the growth of neonate Peridroma saucia 98

Figure 3-2. Growth and survivorship of neonate Peridroma saucia fed artificial diet containing solvent extracts of Aglaia odorata (Hawaiian sample)...100

Figure 3-3. Preparative HPLC chromatogram of a growth inhibitory fraction from Aglaia odorata 104

Figure 3-4, Structures of dammarane triterpenes isolated from Hawaiian samples of Aglaia odorata 108 xiv

Figure 3-5. Structures of flavanones isolated 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 isolated from Hawaiian samples of Aglaia odorata 121

Figure 3-9. HPLC trace of Et20 soluble fraction from Aglaia odorata (Hawaiian sample) 130

Figure 3-10 HPLC trace of EtoO soluble fraction from Aglaia odorata (Thailand sample) 132

Figure 3-11. HPLC trace of Et20 soluble fraction from Aglaia odorata (Taiwan sample) 134

Figure 3-12. HPLC trace of the Et20 soluble fraction from Aglaia odoratissima 136

Figure 3-13. HPLC trace of the Et20 soluble fraction from Aglaia argentia 138

Figure 3-14. Effect of Compound 6 on the growth and

survivorship of neonate Peridroma saucia 144

Figure 4-1. Structures of limonoids included in Table 4-1..168

Figure 4-2. Major biosynthetic routes of limonoids in the

Meliaceae 173

Figure 4-3. Structures of limonoids examined in this study.176

Figure 4-4. Effect 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 diet containing various concentrations of azadirachtin 194 Figure 4-6. Effect of cedrelone on molting success in Oncopeltus fasciatus 196

Figure 4-7. Effect of azadirachtin on molting success in Oncopeltus fasciatus 199

Figure 4-8. Comparison of insect growth inhibiting activity of limonoids with measurements of oxidation and skeletal rearrangement 203 XV

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 fifth instar nymphs of Melanoplus sanguinipes 239 Figure 5-4. Effect of topically applied azadirachtin on molting success of fifth-instar 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 List of Abbreviations

ACHN: Acetonitrile.

AD: Approximate Digestability.

ANOVA: Analysis of variance.

CH2C12: Dichloromethane.

CHCI3: Chloroform.

EC50: Effective concentration for 50% effect (ie growth inhibition).

ECD: Efficiency of conversion of digested food.

ECI: Efficiency of conversion of ingested food.

ED50: Effective dose for 50% effect (ie growth inhibition).

Et20: Ethyl ether.

EtOAc: Ethyl acetate.

EtOH: Ethanol (ethyl alcohol).

HPLC: High pressure liquid chromatography.

IGR: Insect growth regulator.

JH: Juvenile hormone.

LC50: Lethal concentration for 50% of treated population.

LD50: Lethal dose for 50% of treated population.

MC50: Concentration which inhibits molting in 50% of the treated insects.

MD50: Dose which inhibits molting in 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 xviii

Acknowledgements

Firstly I thank my wife, Christy, for all her love, support, and patience. This thesis, and all that I do, is dedicated to her.

I thank my supervisor, Dr. G.H.N. Towers, for his support, encouragement, and enthusiasm. Dr. M.B. Isman has also been a de facto supervisor of this project, and gave generously of his time and knowledge, not to mention support and lab space. Without his assistance this thesis would not have been possible. I would also like to thank my committee members, Drs. I.E.P. Taylor and M. Shaw, for all their advice.

Numerous people have contributed materially to the research reported here, by providing plant material or pure compounds for bioassay. In particular I would like to thank

Dr. Kelsey Downum and Lee Swain, of Florida International

University, Miami, for their generous assistance and hospitality. Plant material was provided by: Tim Flynn,

Pacific Tropical Gardens, Maui; Dr. S. Dossagi, Kenyan

National Museum, Nairobi; Dr. Zhun Jun, Kunming Institute 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 in the field in 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 identification of the plant consituents. Felipe Balza provided invaluble assistance with the identification of the flavanones. Dr. B. Bhom generously allowed me access to his computerized library of the flavonoid literature.

Finally, I wish to acknowledge the contribution of my fellow graduate students, particularly Paul Spencer, Murray

Webb, Cris Guppy, Greg Salloom, Sue Dreier, Shona Ellis,

Cathy McDougall, and many others, for much pleasant conversation, coffee, and particularly for helping me keep a sense of perspective and humour about the last few years. 1

Chapter 1: General Introduction

Interactions between phytophagous insects and their host plants constitute perhaps the largest class of interspecific interactions in the terrestrial biosphere. Study of the factors which serve to regulate these interactions has become one of the most dynamic fields in ecology. Many of the interactions are of direct relevance to Man, especially when the plants involved are of economic importance.

Chemical ecology (the study of biochemically mediated interactions between species) therefore has two facets: advances in theory may yield novel pest management strategies (Raffa, 1986), and the study of applied agricultural problems may increase our understanding of problems ranging from the structure of communities to the nature of evolutionary processes.

This thesis reports a series of investigations of the effect of phytochemicals from members of the tropical plant family Meliaceae on herbivorous insects. This family was chosen for investigation because, although some species such as the neem tree, Azadirachta indica, are known to produce insecticidal phytochemicals, most members of the family have not yet been examined for this property. The phytochemistry of the family is fairly well known, which provides a convenient basis for such studies. Finally, the toxicology and mode of action of several known insecticidal compounds requires clairification. 2

Literature Review

The potential impact of insect herbivory on plant populations is well illustrated by examples from the biological control of weeds (Crawley, 1989; Krischik and

Denno, 1983). For instance, klamath weed (Hypericum perforatum) (Hypericaceae) populations in western North

America were reduced by over 95% following the introduction of the beetle Chrysolina quadrigemina (Chrysomelidae)

(Holloway, 1964). At present klamath weed is largely confined to shaded habitats where C_. quadrigemina prefers not to oviposit. Opuntia stricta (Cactaceae) was introduced into Australia in 1839. By 1920 this cactus had spread to cover 24 X 106 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 in

1925, and by 1930 most areas of Opuntia had been killed.

Opuntia is presently restricted to small isolated populations.

Insect exclusion studies, using insecticides or exclusion cages, provide the strongest evidence that insect herbivory can affect the structure of plant communities

(Crawley, 1989). Despite potential methodological problems, including phytotoxic or stimulatory effects of insecticides and herbivore rebound in the absence of natural enemies, over half of the studies indicate changes in species composition when herbivorous insects are excluded from plant communities (Crawley, 1989). In some cases, even a single 3

herbivore species can exclude a plant from potential habitats. When caged and uncaged Machaeranthera canescans

(Asteraceae) were transplanted to areas populated by the grasshopper Hesperotettix viridis, the uncaged plants were completely defoliated after an average of 7.4 days, but 77% of the caged plants survived to flowering (Parker and Root,

1981).

Despite this potential impact on plants, the terrestrial environment remains green. Indeed, the effects of herbivory are rarely evident in the field, and plant population dynamics have generally been thought to be regulated by resource limitations (i.e. Slobodkin et, al.,

1967) or competition (Hairston et al., 1960). According to this view insect populations are regulated at low densities by natural enemies, particularly predators, parasites, and diseases, and so cannot have a major impact on plant populations (Hassell and Anderson, 1984; Strong et, al. ,

1984; Bernays and Graham, 1988).

However, it also has been realized that most plants possess resistance mechanisms that limit both the range of herbivore species which can inflict damage and the rate at which the damage can occur. Consequently, each plant species is susceptible to herbivory by only a small percentage of the phytophagous insects to which it is exposed. For example, in a Costa Rican dry forest each plant species supports an average of four to eight lepidopteran species; the greatest herbivore load supported 4

by any one plant is 17 out of the 3140 species of caterpillars known from this habitat (Janzen, 1988). Over

50% of the caterpillars are monophagous, and virtually all of the rest feed on less than five related plant species; fewer than 10% of the lepidopteran species can be considered polyphagous. Tabulations from temperate localities indicate a similar pattern (Strong et al., 1984; Thorsteinson, 1960;

Scott, 1988).

Even acceptable host plants may provide less than optimal substrates for growth. Black cutworms, Aarotis

ipsilon. fed corn (Zea mays)r a preferred host plant, grew at only 15% the rate of siblings reared on an artificial diet (Reese and Field, 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 in water (Scriber, 1979; Scriber and Slansky, 1981) and nitrogen content (Mattson, 1980;

Scriber, 1984) may affect insects, most plants appear to contain sufficient nutrients to support insect growth

(Fraenkel, 1959). Resistance to herbivory is to a large extent expressed at the stage of host plant selection and results from chemical and tactile stimuli received by the insect. Plant architecture, profile, colour, and odor are perceived by mobile phytophagous insects and often have a role in locating potential host plants (Miller and

Strickler, 1984). However, the primary factor governing 5

host plant acceptance or rejection appears to be the profile of "secondary metabolites" present in 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 first used by the

German chemist A. Kossel in 1891; in 1896 he defined this term (cited in Mothes, 1980; Schneider, 1988):

"The search and description of those atomic complexes, which are the essence of life are the foundation for the investigation of the life processes. I propose to call the essential components of the cell primary and those that are not found in all the cells that have the capacity to develop, secondary. The decision whether a substance is a primary or a secondary one is in some cases difficult."

The diversity of the secondary metabolites is astonishing: Swain (1977) estimated that 100,000-400,000 such natural products may exist. Each plant contains from a few to hundreds of these compounds; typically they vary qualitatively and quantitatively between organs of the same individual, between tissues of different ages, and between individuals and populations within a species (McKey, 1979).

Although roles in "primary" metabolism have been identified or postulated for a few secondary metabolites (Seigler and

Price, 1976), for the most part they seem to function as signals mediating interspecific interactions (Swain, 1977).

Specific terms have been developed to describe the signalling role of secondary metabolites (Shorey, 1977;

Nordlund, 1981). Semiochemicals, chemicals which mediate 6

interactions between organisms, can be divided into pheromones, which mediate intraspecific interactions, and allelochemics, which mediate interspecific 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 in mutualistic interactions.

Allomones are deleterious to the receiving organism, and include repellants and toxins used for defense.

As implied from the above definitions, secondary metabolites may affect both the behavior and the physiology of phytophagous insects. The range of toxic (physiological) effects of plant allelochemicals is impressive. Non-protein amino acids may be incorporated into proteins, resulting in non-functional enzymes or structural protein (Rosenthal and

Bell, 1979). Many alkaloids are neurotoxic to vertebrate and insect herbivores (Robinson, 1979), and one, nicotine, found use as an insecticide (Schmeltz, 1971; Jacobson and

Crosby, 1971). Other potent neurotoxins include the pyrethrins (Matsui and Yamamoto, 1971; Mabry and Gill,

1979), isobutylamides (Jacobson, 1971; Miyakado et al.,

1989), some aliphatic acetylenes including cicutitoxin

(Towers and Wat, 1978; Robinson, 1980), and some mono- and diterpenes (Ryan and Byrne, 1988). Sesquiterpene lactones

(Mabry and Gill, 1979; Pieman et al., 1979), drimane sesquiterpenes (Ma, 1975; D'Ischia et al., 1982; Asakawa et al., 1988) and some hydroxamic acids (Niemeyer et al., 1982) 7

react with amines or sulfhydryl amino acids to alkylate proteins. Saponins (Applebaum and Birk, 1979) and the steroidal alkaloid tomatine (Duffey and Bloem, 1987; Bloem et al., 1989) complex with dietary sterols and reduce their availability to the insect. Some compounds require near-UV light for toxicity: these include the linear and angular furanocoumarins, B-carboline alkaloids, and isoquinoline alkaloids, which photobind to DNA (Berenbaum, 1978, 1983;

Berenbaum and Feeny, 1981; Towers and Champagne, 1987), and many aliphatic and phenyl acetylenes and thiophenes, which disrupt membranes (Downum et al., 1984; Champagne et al.,

1986).

Some natural products disrupt hormonal regulation of physiological processes in insects. Juvenile hormone titres are reduced by the allaticidal precocenes (Bowers, 1983); other compounds are juvenile hormone analogues and produce non-reproductive supernumerary instars in sensitive insects

(Slama, 1979; Bowers, 1983). The protective role of the phytoecdysones is controversial (Jones, 1983): while these molting hormone analogues are highly toxic when injected into the hemocoel, they may be much less active following oral administration, perhaps due to rapid metabolism and excretion (Feyereisen et. al. , 1976). As will be discussed in detail later, the limonoid azadirachtin disrupts both juvenile hormone and ecdysone titres, resulting in molt failure or chemosterilization of adults. 8

Many secondary metabolites deter feeding or oviposition by insects. Detection of such compounds is accomplished via chemoreceptors located on the maxillary palps, labrum, in the epipharyngeal cavity, and frequently on the tarsi and ovipositors (Chapman and Blaney, 1979; Chapman, 1982;

Hanson, 1983, 1987). Sampling of potential host plants is usually accomplished by stereotypic behaviors which maximize contact of the chemoreceptors with the plant surface

(Frazier, 1986; Chapman and Bernays, 1989). Butterflies sample potential oviposition sites by rapidly drumming their tarsi against or scratching on the leaf surface (Rothschild,

1987; Renwick, 1989). Locusts initially touch a leaf with their antennae, then bring the labrum into contact, and rapidly touch the tips of the maxillary palpi to the leaf

(Williams, 1954). Finally, a test bite may be taken, initially expressing leaf sap without removing tissue. In grasshoppers a similar sequence is seen but the antennae play a lesser role (Mulkern, 1969). Further examples are given by Chapman and Bernays (1989).

Contact chemoreceptor morphology and physiology have recently been reviewed (Stadler 1984; Frazier, 1986; Hanson,

1987). In brief, chemoreceptors (sensory hairs or sensilla styloconica) are characterized by a single apical pore which admits to a cavity filled with a "sensillum liquor" bathing dendrites. Compounds are detected as they bind to specific receptors or "transducing proteins" (Norris, 1988), eliciting an action potential. Classically four types of 9

chemosensory cells have been recognized, based on the substances to which they show the maximum response. These are the sugar, salt, water, and anion (or second salt) cells; however, each cell also has other functions which include the detection of feeding deterrents. Alternatively to stimulating a deterrent cell, some secondary metabolites inhibit the activity of the sugar receptor cell (Ma, 1977;

Mitchell and Sutcliff, 1984). In a few monophagous insects one receptor has become specialized for the detection of specific secondary metabolites ("sign stimuli") characteristic of their host plant. Examples include the glucosinolate receptor in Pieris brassicae (Nielsen et al.,

1979) and the hypericin receptor in Chrysolina brunsvicensis

(Rees, 1969).

Only in a few cases do secondary metabolites mediate an

"all-or-none" response by activating a specific neural pathway, termed a "labelled line". Diabroticite beetles are stimulated to feed on any substrate containing cucurbitacins, triterpenes characteristic of their normal host plant (Metcalf and Lampman, 1989). The desert locust,

S. gregaria, will starve to death rather than consume food treated with azadirachtin at concentrations as low as 4 ng/cm2 (Blaney, 1980). However, in most cases the insect response is more labile, and may be affected by the combination of deterrents and stimulants present and the physiological state of the insect (Dethier, 1982; Miller and

Strickler, 1984; Reese and Schmidt, 1986). A plant which is 10

unacceptable to a satiated insect may well be consumed, albeit in small meals, by a hungry one. Processing of sensory information appears to occur at the level of the central nervous system rather than at the peripheral sensilla, probably via a cross-fibre patterning (Dethier,

1982). Schoonhoven and Blom (1988) suggested a simple model for neural processing in Pieris brassicae in which phagostimulant and deterrent signals are simply summed, with each deterrent action potential equivalent to 2.5 stimulant action potentials.

The Insect Response

Exposure to allelochemicals in host plant tissues can select for resistance mechanisms in phytophagous insects; this selection may involve behavioral, physiological, or biochemical resistance. Ingested phytochemicals may be excreted intact, presumably due to non-absorption; examples include cocaine in Etoria noyesi feeding on Erythroxylum coca (Blum et al., 1981), cardenolides in Schistocerca gregaria (Scudder and Meredeth, 1982), and thiophenes in

Melanoplus sanguinipes (Smirle, Champagne, and Isman, unpublished data). Once a compound penetrates the mucosal membrane of the gut it may be confronted by a variety of physiological or biochemical resistance mechanisms

(Brattsten, 1988, 1986, 1979; Ahmad, 1986; Ahmad et al..

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 affinities, whose net effect is to oxidize lipophilic compounds to more water-soluble forms and thereby facilitate excretion. Other enzyme systems involved in detoxification and excretion include the glutathione S transferases (GSTs), which conjugate compounds with an electrophilic 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 in the excretion of insecticides, their importance in plant-insect interactions has not been extensively studied (Brattsten, 1988).

These enzyme systems are present in high concentrations particularly in the midgut and fat body but may also occur in other tissues including the Malpighian tubules. The MFOs in particular may be rapidly induced by exposure to certain secondary metabolites (Terriere, 1984; Yu, 1983, 1986).

Resistance may also involve target-site insensitivity

(Berenbaum, 1986; Brattsten, 1988). Examples include the insensitivity of Na+/K+ ATPases from milkweed bugs and monarch butterflies to inactivation by cardenolides (Vaughn and Jungreis, 1977; Moore and Scudder, 1985). Studies with synthetic insecticides suggest that target-site insensitivity appears after metabolism-based resistance 12

(Brattsten, 1986; Berenbaum, 1986). It may occur when selection pressure comes from extremely toxic allelochemicals, or when an allelochemical is sequestered for defensive uses.

Coevolution

The tendency for particular herbivores to associate with particular host plants has long been noted (Brues, 1924) and was attributed to coevolution by Ehrlich and Raven (1964).

According to their model, the appearance, by mutation or recombination, of a novel allelochemical in a plant will result in a decrease in herbivore pressure, leading to evolutionary radiation. The availability of competitor-free space will then select for resistant populations among the herbivore fauna. Once able to exploit this resource, the herbivores will also undergo evolutionary radiation.

Eventually the resistant herbivores will again select for further novel allelochemicals, which will continue the selection for resistance.

This pattern of reciprocal evolution may lead to progressively tighter associations between host plants and their adapted insect fauna, in part at least due to the cost of maintaining metabolic defenses against the entire range of allelochemicals to which a generalist is potentially exposed. However, Neal (1985) was unable to show any metabolic cost associated with the induction of nine-fold higher MFO titres in the generalist caterpillar Heliothis 13

zea, suggesting that other factors are involved in the evolution of a limited host-plant range.

Although there is no doubt that evolution of plant- insect associations has occurred, few examples are known where there is evidence of the reciprocal selection pressures essential to coevolutionary theory (Janzen, 1980;

Thompson, 1982). Possibly the most convincing case is the association of a specialized insect fauna, particularly papilionid butterflies, with plants which contain linear and angular furanocoumarins (Berenbaum, 1978, 1981, 1983;

Berenbaum and Feeny, 1981). In this case the plant elaborates not only the toxic furanocoumarins but also a series of synergists, the methylenedioxyphenyl compounds myristicin, safrole, and isosafrole (Berenbaum and Neal,

1987; Neal, 1989). However, the insect mixed-function oxidases are not only resistant to the synergists (Neal and

Berenbaum, 1989) but are specifically induced by the furanocoumarins (Cohen et ai., 1989).

More commonly herbivore selection pressure results from a diverse fauna, which may also interact as competitors; coevolution in such a case is said to be "diffuse" (Fox,

1982, 1988; Fox and Morrow, 1981). In such a situation the evolution of tight associations between host plants and their insect fauna is less likely; rather, any change in selection pressure due to a change in any of the participants will affect all members of the association. 14

Evolution of Deterrent Responses

According to the prevailing view, the presence of allelochemicals in a plant can select for physiological resistance or the ability to detect and avoid those allelochemicals (feeding and oviposition deterrence)

(Scriber, 1984; Berenbaum, 1986). It is generally thought that, due to the adaptive nature of insect behavior, antifeedants which are not accompanied by toxicity will be quickly overcome in evolutionary time. Most or all antifeedants, therefore, should be toxic if ingested, or invariably be associated with toxins. Recently Bernays and

Chapman (1987) (also Bernays and Graham, 1988) have challenged this view; they point to several studies in which non-host plants were shown to support normal larval growth, to studies which have failed to find toxicity associated with antifeedant activity, and to studies of oviposition behavior of butterflies in which plants which were rejected could sustain larval development. They suggest that factors other than physiological specialization and the avoidance of toxicity may mediate host plant specialization; in particular that generalist insects are subject to higher rates of predation from generalist predators, and that specialist herbivores enjoy a reduced predation rate. In support of this concept, Bernays (1988) has shown that generalist herbivores are more likely to be found and eaten by the generalist wasp Mischocyttarus flavitarsus than are specialists when both are exposed to the predator on the 15

same host plant. Polyphagous caterpillars are also more palatable than aposematic or cryptic specialists to the generalist predator Iridomyrmex humilis (Bernays and

Cornelius, 1989).

Crucial to the rejection of plant chemistry as an important primary factor driving the evolution of host-plant specificity is the conclusion that most deterrents are

"harmless", that is, that they are not in themselves toxic , nor are they usually associated with toxins (Bernays and

Graham, 1988). However, as noted by Schultz (1988), this is argued largely from "absence of evidence" rather than

"evidence of absence". The limonoids are a case in point: due to their bitter taste to vertebrates, they have been considered to be primarily feeding deterrents. As a result, the majority of published assays of these compounds have been designed to detect only antifeedant activity (reviewed in Chapter 4), creating the impression that this is the only biological activity these compounds possess (Taylor, 1987).

Limonoids, in common with most other known antifeedants, need re-evaluation with bioassays capable of detecting biological activity other than feeding inhibition; this is one focus of Chapter 4. 16

Phybochemistry of the Meliaceae

Phytochemically, the family Meliaceae is characterized by the oxidized triterpenoids known variously as limonoids, meliacins, or tetranortriterpenoids (Taylor, 1981).

Limonoids, named for limonin, the first such structure to be

elucidated (Arigoni et al., 1960), possess a C22 4,4,8- trimethyl steroid nucleus with a furan ring 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 in a reasonable biosynthetic sequence which also derives support from in vitro synthetic studies (Buchanan and Halsal, 1970; Lavie and Levy, 1971;

Siddiqui et al., 1988). To form the protolimonoids, euphanol (20BH) or tirucallol (20crH) derivatives give rise to a A7 derivative such as butyrospermol, which is subsequently epoxidized at C7-8 (Figure 1-1). Opening of the 7B, 8B-epoxide induces a euphol-apo-euphol (or tirucallol-apo-tirucallol) rearrangement, with formation of

a C-7 OH, migration of the C-14 CH3 to C-8, and formation of a double bond at C-14,15, resulting in the characteristic

limonoid nucleus. The existence of protolimonoids with a C8 aliphatic side chain, including meliantriol, suggests that this rearrangement precedes the cyclization of the side

chain. The furan ring is formed by cyclization of the C8 chain to a cyclic hemiacetal, followed by the loss of four carbons (hence tetranortriterpenoid). 17

Subsequent oxidations and rearrangements give rise to a diversity of structures; more than 300 have been described to date (Taylor, 1987). The major biosynthetic pathways have been described by Das et al. (1984, 1987). The most structurally diverse pathway begins with oxidation of the D ring to an epoxylactone (see Figure 4-2). The parent apoeuphol, with a double bond at C-14,15, undergoes allylic oxidation to a A14,1 6-ketone, which is oxidized to a 14,15- epoxy-16-keto derivative. Baeyer-Villiger oxidation then yields the epoxylactone. This sequence is exemplified by the series azadirone-azadiradione-epoxyazadiradione-gedunin, isolated from Azadirachta indica (Kraus, 1981; Schwinger et al., 1983) (see Figure 4-1). Subsequent Baeyer-Villiger reactions may lead to oxidation of either the A ring (ring-

A,D seco limonoids) or the B ring (ring-B,D seco limonoids).

This pathway is particularly characteristic of the subfamily

Swietenioideae (Taylor, 1981; Das ejfe ai., 1984).

In the subfamily Meliodeae, two additional pathways are also expressed. The A ring may be oxidized to a lactone; rarely this may be followed by oxidative opening of the B ring. Finally, the intact precursor may undergo ring-C opening and rearrangement (Figure 1-2). Radiotracer experiments have established that, contrary to the generally accepted scheme for limonoid biosynthesis, the isomers of

3H-euphol, tirucallol, and butyrospermol were more efficiently used than were the A7-isomers in the biosynthesis of the ring-C-seco limonoid nimbolide (Ekong et 18

al., 1971; Ekong and Ibiyemi, 1985). This suggested that a

7 9 7 8ott9 tx—epoxide gives rise to a A ' (H)-diene; the A function leads to the 7B-0H, and the A9^11^ function would activate C-12, leading to oxidative fission of the C ring.

Also (perhaps simultaneously), the 7

Melieae, consisting of the two genera Azadirachta and Melia

(Das et ai., 1984).

The Meliaceae appear to be remarkable for the paucity of phytochemical constituents other than limonoids; in this respect they contrast strongly with their 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 siderin from Toona ciliata (Gray, 1983). The only chromone known is 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. al.. 1988). 20 21

Figure 1-2. Biosynthetic pathway leading to the formation

of a C-seco limonoid (modified from Siddiqui et aj,.,

1988). 22 23

Figure 1-3. Biosynthetic pathway leading to the formation

of a C-seco limonoid, according to Jones gt ai. (1988). 2k 25

methylpiperid-4-yl) substituent, isolated from Amoora rohituka (Harmon et al., 1979). Amoora is now considered a

junior synonym of Aglaia (Pennington and Styles, 1975). The only furanocoumarin described to date is bergapten from

Toona ciliata seeds (Chatterjee et al., 1971).

The diversity of alkaloids is equally limited.

Xylocarpus granatum produces the angular pyranoquinoline N- methylflindersine and a benzo[c]phenanthride (Chou et al.,

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 al.f

1979; Purushothaman gt aJL., 1979). The closely related compound piriferine occurs in foliage of A., pirifera (Saifah et al., 1988). An unidentified Aglaia species contained tiglamide (Johns and Lamberton, 1969). Recently a series of cardioactive 1-phenylethylisoquinoline alkaloids were isolated from the foliage of the traditional medicinal plant

Dysoxylum lenticellare (Aladesanmi and Ilesanmi, 1987); they co-occur with the molluscicidal alkaloid lenticellarine

(Aladesanmi et al., 1988).

Few flavonoids have been identified in 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 results of a preliminary survey of 12 species (W. Crins and D. Champagne, unpublished data), the diversity of flavonoids in the Meliaceae is probably under-represented in the literature.

Diterpenes include sugiol, nimbiol, nimosone, nimbosone, methyl nimbiol, methyl nimbionone, nimbionone, and nimbionol from A., indica bark (Hegenauer, 1969; Ara et al., 1988; Siddiqui et al., 1988) and eperu-13-en-8B,15-diol from Aphanamixus polystachya (Chandrasekharan and

Chakrabortty, 1968). Dysoxylum lenticellare has recently yielded two diterpenes, phyllocladene and fi- hydroxysandaracopimarene (Aladesanmi et al., 1986).

Sesquiterpenes, known from Lansium anamalayanum, include (-

)-K-gurjunene, (-)-

(Krishnappa and Dev, 1973). Pentacyclic triterpenes include betulin, betulinic acid, katonic and indicic acids, walsurenol, onoceradienone, and lansic acid (Hegnauer,

1983). Aphanamixus polystachya seeds contain a saponin, stigmastienol diglycoside (Bhatt gt ai., 1981).

Uncharacterized monoterpenes occur in the leaf glands, and a few species produce 2,6-dimethoxy-benzoquinone (Hegnauer,

1983).

Antifeedant and Insecticidal Activity of Azadirachtin

Leaves and fruits of the neem tree, Azadirachta indica A.

Juss., have long been used in traditional medicine and 27

agriculture in India; Sanskrit writings from 2,000 B.C. describe the preparation of water extracts for use against locust plagues (Pradhan gt al., 1962). Pradhan and coworkers (1962) confirmed the locust repellent activity of neem extracts. As well, neem leaves are still used, mixed with grain or placed in woolen clothing, to protect against insect damage (Saxena, 1989).

Work in Israel resulted in the description of the protolimonoid meliantriol as a locust feeding deterrent from foliage of A. indica and the closely related chinaberry,

Melia azedirach L. (Lavie et al., 1967). A year later

Butterworth and Morgan (1968) isolated a microcrystalline compound, which they named azadirachtin, as a potent locust antifeedant from neem seed. The structure of azadirachtin

(see Figure 3-3) proved elusive: a partial structure

(Butterworth and Morgan, 1971; Butterworth gt al., 1972), later completed by Zanno gt al. (1975), was subsequently revised based on 13C-NMR (Kraus et al., 1985) and X-ray diffraction studies of detigloyl azadirachtin (Bilton gt al., 1985; Broughton gt ai., 1986).

The phytochemistry of the neem tree is complex. Jones et al. (1988) list 53 limonoids and two protolimonoids, most of which have been isolated from the seed oil. Some of these are very similar 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 itself (reviewed in

Chapter 4).

Azadirachtin completely inhibits 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 is remarkably sensitive, as it does not feed on cabbage treated with a 0.001% aqueous solution of neem kernel extract (Pradhan gt al., 1962). A ten-fold higher concentration was needed to deter feeding by

L. migratoria. Subsequently either pure azadirachtin or neem oil preparations have been shown to inhibit feeding or growth in 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 cell responds to azadirachtin; in some insects interaction occurs between these cells at the peripheral level, reducing the frequency of action potentials, but in Mamestra brassicae and Spodoptera exempta no interaction occurs and the conflict between signals from the deterrent and sugar receptors are resolved in the central nervous system (Simmonds and Blaney, 1983).

Habituation to azadirachtin can occur, as larvae exposed to the compound for two days showed a markedly reduced neurophysiological response (Simmonds and Blaney, 1983). 29

Soon after the discovery of the antifeedant activity of azadirachtin, it was noted that this compound also delayed or inhibited the molting of a variety of insects, resulting in death or the malformation of adults (Ruscoe, 1972;

Steets, 1975; Meisner et al., 1976; Ladd et al., 1978).

(Readers unfamiliar with the endocrine regulation of molting in insects are referred to the review of this topic given on pp 32- 3

Rembold, 1983) and Galleria mellonella (Malczewska et al.,

1988). The appearance of ecdysone peaks was delayed in a dose-dependent manner in Locusta migratoria (Sieber and

Rembold, 1983; Mordue et al., 1986; Mordue and Evans, 1987),

Oncopeltus fasciatus (Redfern et al., 1982), Manduca sexta

(Schluter et al., 1985; Pener g£ al., 1988), Ostrinia furnacalis (Min-Li and Shin-Foon, 1987), Calliphora vicina

(Koolman et al., 1988), and Galleria mellonella (Malczewska et al., 1988). In most of these cases ecdysone titres were also decreased, but in some (i.e. Schluter et al., 1985;

Malczewska et al., 1988) the delayed ecdysone peak was actually higher than the controls, and appeared to fall more gradually.

Azadirachtin also inhibits oogenesis in Locusta migratoria (Rembold and Sieber, 1981), Oncopeltus fasciatus

(Dorn et al., 1986), and Dysdercus koenigii (Koul, 1984).

Similar effects have been produced by exposure to neem 30

extracts in Epilachna varivestis (Steets and Schmutterer,

1975) and Leptinotarsa decemlineata (Steets, 1976;

Schmutterer, 1986). Again this effect is related to inhibition of JH production and ovarian ecdysteroid titres

(Rembold and Sieber, 1981). Curiously, last-instar nymphs treated with sufficient azadirachtin to completely inhibit molting can show development of the ovaries (Dorn et ai.,

1986a,b; Shalom et al., 1988). Vitellogenin concentrations in over-age L- migratoria nymphs were 6-7 times the levels in control adult females (Shalom g£ ai., 1988).

The mechanism by which azadirachtin interferes with molting and the production of neurohormones remains unclear.

The prothoracic glands are not directly affected, as these glands remain able to synthesize ecdysone and respond to prothoracicotrophic hormone (PTTH) in vitro (Koul et al.,

1987; Pener et ai-, 1988), although Koolman gt ai. (1988) found inhibition of release but not synthesis of ecdysone in the brain-ring gland complex of Calliphora vicina. Indeed prothoracic glands of azadirachtin-treated insects remained intact and able to secrete ecdysone long after the glands degenerated in control insects (Pener et al., 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 35S-labelled cysteine in neurosecretory material. This evidence points 31

to a delay in, and in some cases complete inhibition of, the release of PTTH from the corpora cardiaca.

That azadirachtin toxicity involves more than simple inhibition of the release of ecdysone or JH is indicated by the failure of subsequently applied hormones to reverse toxicity in Manduca sexta (Schluter gt ai., 1985). Chilling induces the production of supernumerary instars in Galleria mellonella larvae; this effect was abolished by azadirachtin

(due to marked inhibition of JH synthesis) and the inhibition was not reversed by application of the JH analogue ZR512 (Malczewska et al., 1988). However, azadirachtin-induced inhibition of molting was reversed in

Rhodnius prolixus by oral application of ecdysone or topical application of a JH analogue (Garcia and Rembold, 1984).

Azadirachtin does not inhibit binding of ecdysteroids to their receptors (Koolman gt ai-, 1988), apparently because of a different conformation of the A and B rings (Rembold,

1988).

In addition to inhibition of molting and reproduction, azadirachtin inhibits gut peristalsis in vitro (Mordue gt ai., 1985; Mordue and Evans, 1987). This effect is related to a generalized inhibition of proctolin-induced muscle contraction (Mordue and Evans, 1987; Mordue gt al., 1989).

Mordue has suggested that the observed endocrine effects of azadirachtin could be due to disruption of normal gut functioning, which is involved in feedback loops regulating the timing of some endocrine events (Nijhout, 1981). To 32

that end she has shown that azadirachtin can inhibit molting even if applied after the ecdysteroid peak and at a time when JH titres should be low. In this case azadirachtin inhibits the air-swallowing behavior necessary to split the old cuticle and may inhibit the release of bursicon (Mordue et al., 1985). The only other physiological effect reported to date which can be attributed to azadirachtin is a marked decrease in the rate of synthesis of RNA and subsequently

DNA in a suspension culture of the protist Tetrahymena thermophila (Fritzsche and CTeffman, 1987).

In spite of the confusion surrounding the molecular- mode of action of azadirachtin, this compound and neem preparations have been applied to a wide variety of agricultural problems (Schmutterer, 1988). Such applications rely on neem oil, cake, or semipurified extracts due to the prohibitive expense of pure azadirachtin

(Schmutterer and Hellpap, 1988). Promising activity has been found against pests of vegetables and fruit trees

(Schmutterer and Hellpap, 1988), rice (Saxena, 1989), stored grains (Saxena et al., 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 al., 1989). A neem oil preparation, Marigosan 0, has now been registered for use on ornamental crops in the

United States (Larson, 1988).

Toxicological testing has indicated that azadirachtin itself is not toxic to mammals, birds, or fish (Schmutterer, 33

1988; Jacobson, 1986;, Okpanyi and Ezeukwu, 1981). However, neem oil has a large range of pharmacological effects including antipyretic and diuretic activity, promoting smooth muscle contraction, and inhibition of fungal skin parasites, scabies, and eczema (Jacobson, 1988). Neem oil also inactivated the potato-X virus (Singh, 1971), and has spermicidal and antifertility activity in various mammals

(Jacobson, 1988). However, the principles responsible for these activities were nimbin and nimbidin and not azadirachtin.

A further favorable aspect of neem is its systemic activity in a variety of plants including corn and rice

(Saxena et al., 1983). Azadirachtin apparently translocates into and protects new foliage or fruits which appear after the application of the neem extract.

Regulation of Molting

To facilitate discussion of the mode of action of azadirachtin, a review of the endocrine regulation of molting is given here. Figure 1-4 illustrates the relationship of the various secretory and neurohaemal organs in the insect. Molting is a two-step process: apolysis, the secretion of new cuticle and partial resorption of the endocuticle, is followed by ecdysis, the process of actually shedding the old cuticle.

Apolysis is regulated by the prohormone B-ecdysone

(ecdysterone), produced in the prothoracic glands in 34

response to the neuropeptide prothoracicotrophic hormone

(PTTH). In Manduca sexta, PTTH is synthesized in the pars intercerebralis, each hemisphere having a single prothoracicotropic cell located in the lateral protocerebrum

(Gilbert gt al (1981). Although the corpus cardiacum (CC) has generally been considered the site of PTTH release

(Gilbert and King, 1973; Gillott, 1982), in Manduca the prothoracicotropes are connected via axons to the corpus allatum (CA) and this is the site 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 gt al., 1984). Corpora Cardiacum

Figure 1-4. Neuroendocrine structures discussed in the text. Modified from Gillott, 1980. 36

The factors which trigger PTTH release are poorly understood but are related to internal measurements of factors correlated with feeding, size, and photoperiod

(Nijhout, 1981). In Rhodnius prolixus molting follows feeding after a characteristic period of time. Nutritional 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 stretch 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- nutritive saline meal could initiate molting. A similar

system initiates molting in Oncopeltus fasciatusr as molting may be triggered by inflating the abdomen with saline or air

(Nijhout, 1979). The required degree of stretching is associated with a sharply defined critical weight, which is usually attained within the first 24 h of the instar

(Nijhout, 1979; Blakley and Goodner, 1978). The critical weight depends on some factor or structure whose dimensions are determined at the previous molt, as there are positive linear correlations between the critical weight and dimensions of sclerotized structures such as femur length.

In Manduca sexta the release of PTTH is regulated by both a critical weight (5 g in the final instar) (Nijhout and

Williams, 1974) and a photoperiod-controlled "gating"

(Truman, 1972; Truman and Riddiford, 1974; Riddiford and 37

Curtis, 1978). In the 5th instar the gate opens shortly after lights-off and closes at the beginning of the next light phase. Larvae must attain critical weight at least 24 h before a particular gate closes; otherwise that gate is bypassed and PTTH is not secreted until the subsequent gate

(Nijhout, 1981). A physiological event associated with the attainment of critical weight is the cessation of juvenile hormone (JH) synthesis: the 24 h latent period represents the time necessary to clear JH from the hemolymph. In the last instar the presence of JH is sufficient to inhibit PTTH secretion, but this mechanism does not operate during earlier larval-larval molts. In the saturniid moth Samia cynthia, photoperiodic control of molting is regulated by an endogenous circadian clock located in the prothoracic glands

(Mizoguchi and Ishizaki, 1982).

In the prothoracic glands the PTTH is thought to bind to cell-surface receptors associated with a Ca2+ channel, leading to an influx of extracellular Ca2+ (Smith and

Gilbert, 1986). Subsequent stimulation of a Ca2+ sensitive adenylate cyclase results in an increase in intracellular cAMP; calmodulin may mediate this stimulation as

Ca2+/calmodulin sensitive adenylate cyclases have been identified in other insect tissues (Combest et al., 1985).

Additionally, some receptors are associated directly with the adenylate cyclase. The cAMP may then activate a cAMP- dependent protein kinase responsible for phosphorylating a rate-limiting enzyme(s) involved in ecdysone synthesis. 38

Although PTTH has been considered the principal hormone regulating ecdysone production, recent evidence suggests that other factors may modulate prothoracic gland activity.

In Manduca sexta JH was shown to have a prothoracicotropic action on ecdysone synthesis (Gruetzmacher gt a_l., 1984a).

Subsequently, this was shown to be an indirect effect: JH stimulates the production of a 30 kD hemolymph factor from the fat body (Gruetzmacher et al., 1984b; Watson et al.,

1985, 1988). Stimulation of ecdysone synthesis occurred even in the presence of saturating titres of PTTH. The factor may be a hemolymph carrier protein which transports a sterol substrate used by the glands in the production of ecdysone. In Bombyx mori this function may be fulfilled by high molecular weight lipoproteins (>200 kD) which transport cholesterol to the prothoracic glands (Chino et al., 1974).

In Heliothis zea, diapause is terminated by an increase in ecdysone synthesis, mediated by a temperature-dependent humoral factor (Meola and Gray, 1984).

In the Lepidoptera, ecdysis is triggered by the release of eclosion hormone (EC) in response to falling ecdysteroid titers (Truman, 1981; Reynolds and Truman, 1983). EC is apparently released from the ventral ganglion at larval- larval and larval-pupal molts, and from the CC at adult eclosion. Injection of exogenous ecdysterone inhibits both the release of EC and sensitivity to exogenously applied EC, and so results in a dose-dependent delay of eclosion. In larvae, high doses of ecdysterone can permanently inhibit 39

ecdysis. In adults, where ecdysis is gated by photoperiod, eclosion may be delayed until a gate several days subsequent to the ecdysterone dose. EC release triggers a series of stereotyped behaviors which serve to release the insect from the old cuticle, including swallowing air to expand the body and rhythmic peristaltic muscular contractions. These actions apparently result from EC binding to receptors located on each of the abdominal ganglia.

The presence of EC in 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 in Locusta (Rembold, 198 ). Truman et al. (1981) found that extracts from the nervous system of insects from five orders (other than Lepidoptera) initiated ecdysis when applied to Manduca pupae, suggesting the presence of an EC-like hormone.

Selection of Test Insects

Factors to be considered in the selection of an appropriate insect species for bioassay have been discussed by Berenbaum

(1986). If the goal of a study is the development of a control strategy for a particular test, the pest naturally becomes the bioassay target. If questions of an evolutionary or ecological nature are addressed, the choice becomes more restrictive. Oligophagous and monophagous insects usually require specific "sign stimuli", secondary 40

metabolites characteristic of their host plants, before feeding will be initiated (Dethier, 1941). Such insects are also usually highly sensitive to the presence of secondary metabolites foreign to their normal food plants, and so tend to show an exaggerated antifeedant response. Further, these insects appear to elaborate a limited range of MFO's, and are possibly more susceptible to intoxication by allelochemicals found in non-host plants (Kreiger et al.,

1971). A more conservative bioassay is provided by polyphagous insects: host-plant choice in these insects is 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 ai., 1971), they are less likely to show an exaggerated response to the intrinsic toxicity of a given allelochemical.

Most of the bioassays reported in this thesis involve the variegated cutworm, Peridroma saucia (Hubner). This is 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 toxic to insects (Bierne, 1971).

Both herbs and deciduous and coniferous trees are included;

P. saucia generally attacks the foliage but may also consume fruits (Bierne, 1971). The only species on its host plant list known to contain limonoids are some species of Citrus, but as these are recent introductions to North America it seems unlikely that exposure to these compounds could have 41

influenced the evolution of deterrent or physiological responses in E- saucia. As E. saucia is a constant background pest which occasionally outbreaks to major pest status (Bierne, 1971), its use is justified to develop novel pest-control strategies relevant to Canadian agriculture.

Although E- saucia does not ordinarily encounter limonoids, its use as an evolutionary or ecological model may also be justified, as its response to these compounds may be taken as representative of unadapted, polyphagous herbivores encountering limonoid-containing plants for the first time.

Such unadapted herbivores are generally assumed to be the primary target of allelochemical-based defenses (Feeny,

1976; Rhoades and Cates, 1976; Coley et al., 1985).

Similar arguments may be made for the use of the migratory grasshopper, Melanoplus sanguinipes. This polyphagous insect is considered the fourth most serious pest in Canadian agriculture (Bierne, 1971). Further advantages to the use of this insect are discussed in

Chapter 5. In brief, the lack of an antifeedant response to azadirachtin makes this a good model insect in which to study the physiological effects of that compound.

In some assays, I used the milkweed bug Oncopeltus fasciatus. This species was used as a model to compare the relative molt-inhibiting activity of several limonoids, as it is known to be sensitive to topically applied IGR compounds, including azadirachtin (Dorn, 1983). 42

Objectives of the Thesis

The objectives of the work reported in this thesis were to examine aspects of the efficacy and mode of action of phytochemicals from species of the plant family Meliaceae for the control of phytophagous insects. In the first investigation, foliar extracts from thirty species of

Meliaceae were screened for growth-inhibiting activity and toxicity against the variegated cutworm, Peridroma saucia.

In addition, results of the screening assays and measurements of leaf toughness from fifteen species were used to assess aspects of the resource availability hypothesis of Coley e£ al (1985). As extracts of Aglaia odorata were highly active in the crude extract screening, the phytochemistry of three species of the genus Aglaia were examined, and are reported in Chapter 3.

In Chapter 4, I describe the comparison of ten limonoids, representing the major pathways of limonoid biosynthesis, for inhibition of feeding and growth in p_.

sauciaf and for inhibition of molting and reproduction in the milkweed bug, Oncopeltus fasciatus. The toxicology of one of these compounds, azadirachtin, was examined in detail in these species. Results are discussed in terms of mechanism of action, structure/function relationships, and the significance of insecticidal activity in the evolution of limonoids. In Chapter 5 I reexamined the reported resistance of the migratory grasshopper, Melanoplus sanguinipes. to azadirachtin. I found that this compound 43 lacked antifeedant activity against M.. sanguinipes. but, once ingested, it produced a variety of physiological effects. The toxicology of azadirachtin and factors involved in its bioavailability in this insect were examined in detail. Finally, I proposed and tested two hypotheses as to the mode of action of azadirachtin. 44

Chapter 2: Insecticidal and Growth-Reducing Activity of

Foliar Extracts from the Meliaceae

Introduction

Prior to the advent of synthetic insecticides, pest-control strategies relied largely on plant-derived extracts and preparations (Jacobson and Crosby, 1971). In certain regions, members of the Meliaceae figured prominently in this respect. In particular, the neem tree, Azadirachta indica A. Juss., has long been noted for its effectiveness in protecting crops, clothing, and stored grains from attack by insects. Sanskrit writings over 2,000 years old detail proceedures for preparing water extracts of the foliage to protect grain fields from locusts (Radwanski, 1977). A similar ancient history of use pertains to Melia azedirach in the Middle East (Lavie gt al., 1967). Subsequent work has confirmed the potent antifeedant and insecticidal activity of these preparations and has led to the isolation of a variety of triterpenoid constituents, exclusively of the limonoid or protolimonoid class, as the active principles (reviewed in Chapter 4 of this Thesis).

The use of plant extracts declined as inexpensive synthetic insecticides became available forty years ago

(Jacobson and Crosby, 1971). However, intensive use of synthetic compounds has resulted in numerous environmental problems including impact on non-target organisms (including 45

humans), contamination of water and soil with persistant residues (Zitter, 1985), and the appearance of resistance in over 400 species of economically important pest insect species (Luck et al., 1977). As well, the cost of synthetic insecticides has escalated (Kinoshita, 1985) and such compounds are now less economically attractive, particularly in the Third World. As a result, interest in botanical insecticides has been rekindled in recent years (Balandrin et al., 1985; Hedin, 1982). Plants may provide useful compounds or extracts directly, or insecticidal and antifeedant phytochemicals may provide leads for the synthesis of new compounds.

Given the remarkable insecticidal activity 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 identifying further species of potential interest. To this end a collection of foliage samples of thirty-one species, out of about 500 in the family (Pennington and Styles, 1975), in twenty-two genera

(out of 51) was assembled and bioassayed against the variegated cutworm, Peridroma saucia. Extracts which showed the strongest activity against E. saucia were also assayed for feeding inhibition against the migratory grasshopper,

Melanoplus sanguinipes.

A primary goal in the study of plant-herbivore interactions is to explain why plants differ in their commitment to defenses and consequently in their 46

susceptability to herbivores (Coley et al., 1985). In the first attempt at such a synthesis, Feeny (1976) and Rhoades and Cates (1976) independently proposed that the probability of discovery by herbivores, termed plant apparency by Feeny, governs the evolution of defensive strategy. According to the hypothesis, apparent plants, which are generally late- successional perennials, are distributed predictably in space and time and so are likely to be located by herbivores. In such plants herbivore pressure should select for "quantitative" defenses effective against both adapted specialist and unadapted generalist herbivores.

Quantitative defenses (eg. tannins) were thought to affect feeding rates (via leaf toughness) and nutrient availability 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 in the large quantities necessary for adequate protection.

Unapparent plants, unpredictable in space and time and mostly short-lived early successional species, were believed to require defenses against generalist herbivores. In such plants "qualitative" defenses (toxic secondary metabolites) would provide adequate defense. Although herbivores could evolve immunity to such defenses this loss of efficacy would be balanced against their low cost of production. Among various difficulties with the plant apparency hypothesis

(Fox, 1981; Bernays, 1978; Berenbaum, 1983), it predicts that all plants should suffer about equal rates of 47

herbivory, which is not observed to be the case (Coley,

1983), and it fails to account for the observed differences in types and extent of defense amongst perennial apparent plants.

More recently these differences in defense allocation have been ascribed to the growth rate of plants, which is closely related to the availability of resources including water, light and nutrients (Coley et al., 1985). Fast- growing species, adapted to resource-rich habitats, replace leaves relatively rapidly and so are able to tolerate higher rates of herbivory. In resource-limited habitats, slow- growing species tend to replace leaves slowly, and so must limit the rate of loss to herbivores. A given rate of herbivory will remove a higher proportion of the primary productivity from slow-growing plants than from faster growing species. The model:

dC/dt = G*C*(l-kDa) - (H-mD6)

[where dC/dt is the realized growth rate, G (g g"1 d-1) is the maximum inherent growth rate without herbivores, C (g) is the plant biomass at time 0, D (g g-1) is the investment in defense, k (g d-1) and a are constants relating investment in defense to reduction in growth, H (g d"1) is the potential herbivore pressure in a habitat, and m and 8 are constants relating the reduction in herbivory to the investment in defense] predicts that intrinsically slow- growing plants should invest more of their primary productivity in defense than should fast-growing species. 48

Metabolically mobile secondary metabolite based defenses are thought to turn over relatively rapidly; to maintain a given level of defense these defense compounds would have to be synthesized continuously, so the cost of these defenses would increase arithmetically over the lifetime of the leaf.

However, because these defenses are metabolically mobile they may often be recovered from senescing leaves. On the other hand, quantitative defenses, especially leaf toughness factors, are emplaced at leaf expansion; although initially costly, such defenses are not metabolically mobile and cost little to maintain once in place, but they are lost at leaf senescence. Comparison of these defense types suggests that chemically based defenses should be selected for in species with short leaf lifetimes (fast-growing plants), and immobile defenses should be favored in species with long leaf lifetimes (slow-growing plants) (Figure 2-1). The model has been supported by studies of lowland rainforest species in Panama (Coley, 1983, 1988), but measurement of investment in secondary metabolite defenses was confined to assays of polyphenol content, which did not correlate with herbivory, leaf lifetime, or other leaf attributes (Coley,

1983). To date, the hypothesis has been tested with plant species co-occurring in the same habitat; it is unclear whether the model can also account for evolutionary patterns within closely related groups of plants.

The Meliaceae offers some particular advantages for a test of this hypothesis. Members of the family occur in 49

habitats ranging from rainforest to mangrove swamp to semidesert (White, 1975). Few families embrace such a wide range of floral characteristics, but the family has been considered to be monophyletic in all taxonomic treatments to date (Pennington and Styles, 1975). Considerable time has been available for the evolution of the diversity observable today, as species clearly assignable to the modern genus

Cedrela are characteristic of western North American

Paleocene floras (Brown, 1965) and the family probably appeared in the Cretaceous, at least 70 million 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 ai., 1988), all antifeedant or insecticidal phytochemicals identified to date from this family are limonoids; indeed the scarcity of other classes of secondary metabolites is remarkable. The evolution of chemical defenses in this family may therefore be examined by a consideration of the relationship between insecticidal activity and evolutionary position of members of a single class of phytochemicals.

Measurement of investment in secondary metabolite based defenses has presented considerable difficulty; previous attempts have largely centered on colorimetric assays for total phenolics (eg. Coley, 1983, 1988). This approach is unsatisfactory, as most known insecticidal or antifeedant compounds would not be detected in such an assay, and the 50

assumption that quantity of phenolics is directly related to the cost and efficacy of chemical defenses is questionable.

As the role of secondary metabolite based defenses has been construed to be deterrence of unadapted generalist herbivores, in this study I have estimated relative investment in phytochemical based defenses by the relative response of a highly polyphagous herbivore (Peridroma saucia) to the entire suite of phytochemicals produced in a plant, as obtained in the methanolic extracts of mature foliage. 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 efficacy of those defenses.

Relative leaf toughness was measured directly by determining the amount of force required to punch a 0.5 cm diameter flat-tipped rod through a leaf. Leaf lifetimes were not measured directly, but as an approximation species have been separated into deciduous and evergreen species.

As presently understood, the resource availabilty hypothesis would predict that species with short leaf lifetimes

(deciduous) should produce leaves that are more toxic and1 less tough than the evergreen leaves of slow growing species. As well, a negative correlation between leaf 51

toughness and leaf toxicity may be expected. Overall, the perennial species may be expected to have a higher commitment to total defenses against herbivores. 52

Figure 2-1. Graphical depiction of the assumed relative

cost of maintaining a chemical or physically-based

defense against herbivores. Costs of the chemical

defense increase arithmetically over the life of the

leaf, due to turnover. Physical defenses are initially

costly, during emplacement following leaf expansion;

however as they are metabolically inactive (immobile),

there is little or no cost associated with their

maintenance once in place. Consequently, plants with

short-lived leaves should be selected for the

production of chemically-based defenses, and physical

defenses should be favored in species with long-lived

leaves. 30

oH • 1 • 1 • 1 • 1 • 1 0 10 20 30 40 50

Leaf Lifetime 54

Materials and Methods

Foliar samples of thirty-one species in twenty genera of the

Meliaceae were obtained for this study. Most were collected from the Gordon Fairchild Tropical Gardens and the USDA

Plant Quarantine Center in Miami, Florida, from the Pacific

Tropical Garden, Hawaii, or from the Kunming Institute,

Kunming, China. Other species were field collected in

Kenya, Thailand, Mauritius, and New Zealand. A few species are represented by collections from more than one site. All samples were of mature (fully expanded and greened) leaves collected approximately in the middle of the growing season.

Sources for each species and collection dates are given in

Table 2-1. Samples of most species were received already powdered; where possible voucher specimens have been deposited in the UBC Herbarium.

Samples were air dried, ground to a fine powder in a

Wiley mill, weighed, and extracted in three changes of MeOH,

24 h/change, 11/100 g dry weight (dwt). MeOH extracts were pooled and concentrated under vacuum; final concentrations were adjusted to 2 mis MeOH/g dwt leaf. Aliquots of the

MeOH extracts were dried and weighed to determine the extraction yield and allow calculation of dose-response relationships in terms of mg extract/g diet fresh weight

(fwt).

For bioassay, diets 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 holstii Gurke Mt Eldon, Kenya SD 03-85 Turreae mauritiana Mauritius JTA 06-86 Tribe 2. Melieae EslXA azedirach L. (Fla) PI073248 DEC 06- 87 Melia azedirach L. (Ind) India KP 07- 85 M£iia azedirach L. (Chi) Kunming, China SQ 05-86 Mfilia tPPSenden Kunming, China SQ 05- 86 Azadirachta indica A.Juss. (Fla) PI137950 DEC 06- 87 Azadirachta indica A.Juss. (Haw) PTBG790480001 TF Azadirachta indica A.Juss. (Chi) Kunming, China SQ 05- 86 Tribe 4. Trichilieae Trjphilia hirta L. FG DEC 06- 87 Trichilia roka PI TF LgpidPtrichjiia volKensii (Gurke)Leroy Mt.Eldon, SD 03-85 EKebergia capensis Sparrm. Kitale, Kenya SD 03-85 Cipadessa baccifera (Roth) Miq. PI105699 DEC 06- 87 Tribe 5. Aglaieae Aglaia odorata Lour. (Thai) Trang, Thai. GHNT 05- 85 Aglaia odorata Lour. (Haw) Maui TF 12-86 Aglaia odoratissima Blume Trang, Thai. GHNT 07- 85 Aalaia araentia Blume Trang, Thai. GHNT 07- 85 Aphanamixus arandifolia Blume Kunming GHNT 08- 85 Aphanamjxus polvstachya (Wall) R.N.Parker DEC 06- 87 Lansium domesticum Corr. Manila GBS 08-88 Tribe 6. Guareeae Guarea glabra Vahl PI DEC 06-87 Dyspxylum spectabile Hook. Opua Rec. Forest,N.Z. GBS 12-87 Tribe 7. Sandoriceae Sandoricum koetiape (Burman f.)Merrill PI DEC 06-87 Subfamily Swietenioideae Tribe 1. Cedreleae Cedrela odorata L. PI097976 DEC 06-87 Toona serrata (Royle) Penn.fi Styles PTG TF Tppna Ciliata M.J. Roemer PI DEC lapjia australis (F. von Mueller) PI DEC 56

Tribe 2. Svietenieae Khaya senegalensis (Desr)A.Juss. PTBG770642003 TF Chuckrassia tabularis A.Juss. PI DEC 06-87 Entandrophracrma caudatum (Sprague) Sprague PI DEC 06-87 Swietenia humilis Zuccarini PI092371 DEC 06==8 7 Swietenia mahoqani (L.)Jacquin PI DEC 06-87 Swietenia macrophylla King PI DEC 06-87 Tribe 3. Xylocarpeae Carapa auianensis Aubl. EI DEC 06-87 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. 57

extracts were added to the diet dry components (Velvetbeen

Caterpillar Diet, no. 9682, Bioserv Inc., Frenchtown, N.J.), initially in amounts calculated to produce concentrations of

25, 50, 75, and 100% of natural leaf concentrations, on a dwt leaf/dwt diet basis. If necessary other concentrations were subsequently evaluated to facilitate determination of

the EC50 (concentration required to reduce larval growth by

50% relative to the controls). The MeOH carrier was evaporated in a fume hood, usually overnight. Controls were similarly treated with MeOH alone.

Three neonate Peridroma saucia larvae were placed on 1 g fresh weight (fwt) diet in a 30 ml plastic Solo cup; 10 cups were used per treatment for a total n=30. Experiments were replicated three times. Rearing cups were put in clear plastic boxes, floored with moistened paper towels to maintain high humidity, and placed in a growth cabinet at

27+1° C, 16:8 LD. Survivorship and live larval weights were determined after seven days of growth; larvae were not weighed or handled on intervening days to minimize artificially induced growth disturbances (Reese and Schmidt,

1986). Weights were log10 transformed to correct for heteroscedasticity prior to analysis by least-squares regression using the SAS GLM procedure to determine the

EC50. Mortality values were corrected using Abbott's

formula and LC50 values (the concentration required to reduce survivorship by 50% relative to the controls ) were determined using the SAS Probit statistical package. 58

Foliar extracts were also examined for antifeedant activity against the migratory grasshopper, Melanoplus sanguinipes. Extracts, sufficient to achieve 100% of natural concentration on a dwt/dwt leaf disc basis (20 ul), were applied evenly, using a 25 ul Hamilton syringe, to both surfaces of 1.5 cm diameter cabbage (Brassica oleraca cv.

Silver Queen) leaf discs. After the extracts had dried, the leaf discs were presented to fifth instar nymphs in 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; starting leaf weight was determined by drying and weighing samples of intact leaf discs. Ten replicates were used for each treatment.

Extracts of Azadirachta indica, Melia azedirach

Aglaia odorata. and Turreae holstii were subsequently assayed using 1.5 cm diameter glass fibre filter discs, treated with sufficient extract to achieve concentrations 1,

2.5 and 5-fold naturally occurring levels. After drying the discs were saturated with a 10% aqueous sucrose solution and presented to fifth instar nymphs as described above.

Leaf toughness was measured directly on some species

(those available in the USDA and Fairchild collections in

Miami, Fla.) using a leaf "punchmeter" modelled after the design of Feeny (1970). This device measures the force required to punch a 5mm diameter, flat-ended rod through the leaf, which is clamped into place in the base of the device. 59

Force was applied by adding water from a buiret to a beaker atop the rod; after 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 cm2

All measurements were made on freshly collected leaves.

Care was taken to avoid primary and secondary veins, although this was difficult to ensure in 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 leaflets for each species. Species were compared using Duncan's multiple range test. The relationship between leaf toughness and toxicity of the

extracts, measured as the EC50, was examined using regression analysis.

Leaf extracts were also examined for the possible presence of antibiotic or phototoxic compounds. The latter possibility was examined because of the presence of a diversity of known photosensitizers in the closely related plant family Rutaceae. The method of Daniels (1965) was used: extracts (equivalent to 5 mg leaf tissue) were dried on to sterile filter paper discs, which were placed on duplicate plates streaked with a lawn of the yeast

Saccharomyces cerevisiae. One plate was incubated in the dark (-UV) at 37° C; the second plate was irradiated 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 inhibition studies with Peridroma saucia

All but three of the extracts tested produced marked inhibition of the growth of E. saucia neonates at concentrations below those occurring naturally in the

leaves. Growth curves for larvae fed Azadirachta indicaf

Melia azedirach. and Melia toosenden extracts are shown in

Fig. 2-2 and are typical of the more active extracts. EC50

and LC50 values, in terms of both mg extract/g diet dwt and

% of natural concentration, are given in Table 2-2.

Extraction efficencies (in mg extract/g dry leaf) for all species are given in Table 2-3, togeather with leaf toughness values (in N/cm2), leaf pubescence, and leaf

"habit" (deciduous or evergreen).

The species which produced the most active extracts, in terms of both growth inhibition and mortality, were all in the subfamily Melioideae. Within this subfamily, members of the tribe Melieae (Azadirachta indica. Melia azedirach, and

Melia toosenden) were all highly inhibitory towards E*

saucia neonates, with EC50 concentrations below 2% of natural concentration (0.59-2.10 mg/g). These species were

also toxic, with LC50 concentrations of about 5% of natural leaf concentration. Dead larvae were all small and still in the first instar; none appeared to have died while molting.

Azadirachta indica foliage samples from Hawaii, India, and 62

Figure 2-2. Growth (as % of Control) of neonate Peridroma

saucia fed artificial diet treated with a MeOH extract

of foliage of Azadirachta indica (Florida and Hawaiian

samples), Melia toosendenf or Melia azedirach at 1, 2,

or 3% of natural concentration. Each point shows the

mean of three replicates with 30 cohorts/replicate;

standard errors were < 6% and are omitted for clarity. 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 artificial diet

required to reduce growth (EC50) or survivorship (LC50) 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.

EC5Q LC 50 UJ lag/a) m (ma/cn Family Meliaceae Subfamily Melioideae Tribe 1. Turreeae Turreae holstii 1 .8' 57.0 90.7 Turreae mauritlana 37 • 3< 45.40niJ >100 >121.7 Tribe 2. Melieae M£lia azedirach (Florida) 1 .0a 1.75c d 7.1 12.5 Melia azedirach (India) 1 .2a 2.10C d 7.3 12.8 Melia azedirach (China) 0 1.58b e 5.6 9.8 Melia toosenden 1 >5ab 2.29 cd 4.7 7.2 Azadirachta indica (Florida) o .7a 0.69a 4.8 4.7 Azadirachta indica (Hawaii) 0 .6a 0.59a 4.8 4.7 Azadirachta indica (China) 0 .8a 0.89a b 5.2 5.8 Tribe 4. Trichilieae Trichilia hir£a 12,.5 C 18.88 ef >100 >151.0 Trichilia roka 18,.8 C >100 Lepidotrichilia volkensii 12..5 ° 26.14^9° >100 >209.1 .0e 102.94* >100 >239.4 Ekepergia capensis 43. e 1 >100 Cipadessa baccifera 44..0 133.02 >302.3 Tribe 5. Aglaieae 2.07c d 17.5 21.3 Aglaia odorata (Thailand) 1, 3.29<* 24.4 29.7 AoTaia odorata (Hawaii) 2. 4.27d >100 >38.8 Aalaia odoratissima 11, 0°dd 11.88e >100 >44.0 Aalaia araentia 27. >63.1 >100 >63.1 Aphanamixus grandifolla >100 k Aphanamjxus polvstachya 74. 77. 7 >100 >105.1 Lansium domesticum >100 >153.6 >100 >153.6

Tribe 6. Guareeae 3 Guarea glabra 62. 53.32 >100 >86.0 Dysoxvlum spectabile >100 >84.0 >100 >84.0

Tribe 7. Sandoriceae ef Sandoricum Koetjape 17. 4° 22.79 >100 >131.0 65

Subfamily Swietenioideae Tribe 1. Cedreleae Cedrela odorata 53.6f 24.66^9 >100 >46.0 Toona serrata 27.0d 47.63*3 >100 >176.4 Toona ciliata 28.2d 28.20f9n >100 >100.0 Toona australis 12.0C 22.93ef9 >100 >191.1

Tribe 2. Swietenieae Khaya senegalensis 58.3f 67.05k . >100 >115.1 Chuckrassia tabularis 12.5° 31.139ni >100 >249.0 Entandrophraama caudatum 22.6cd 29.839h >100 >132.0 Swietenia humilis 23.0cd 23.64ffg >100 >100.0 Swietenia mahogani 17.0C 47.091? . >100 >277.0 Swietenia candollei 20.0cd 42.36ni3 >100 >211.8 Tribe 3. Xylocarpeae Carapa guianensis 53.2f 29.689h >100 >55.8 66

Florida did not differ significantly in their toxicity to P. saucia neonates. Similarly, Melia azedirach samples from

Hawaii, India and Florida did not differ significantly in these assays.

Other tribes in the Melioideae were more variable in their effects. Among the Turreeae, Turrea holstii foliage extracts were comparable to Melia leaf extracts, inhibiting the growth and survivorship of P.. saucia larvae (EC5o=1.8%,

2.8 mg/g). Unlike the situation with Azadirachta indica or the two Melia species, numerous larvae were seen to have died at a failed molt attempt at the end of the first or

second instar (LC5o=57.0%, 90.7 mg/g). Extracts of T_.

mauritiana were much less active, with an EC50 of 37.3% natural concentration (45.4 mg/g), and no toxicity at natural concentration.

Within the Aglaieae, extracts of Thai and Hawaiian samples of the traditional medicinal plant Aglaia odorata

were highly toxic to £. saucia neonates, with an EC50 of

/ 1.7% and 2.7% (2.07 and 3.29 mg/g) respectively, and LC50 s of 17.5 and 24.4% natural concentration. Other species of

Aglaia were somewhat less active: A., odoratissima and A..

argentia had EC50's of 11 and 27% natural leaf concentration

(4.27 and 11.88 mg/g) respectively, and were not toxic in the seven-day assay. When differences in the extraction efficiencies (Table 2-3) are taken into account, A., odorata is about twice as active as A., odoratissima and about five times more active than A., argentia. Of the two species of 67

Aphanamixus examined, neither were toxic at natural leaf concentration, but A., polystachya did inhibit larval growth

(EC50=74%, 77.7 mg/g). Lansium domesticum was inactive at natural concentration (153.6 mg/g).

Within the Trichileae, extracts from Trichilia hirta and Lepidotrichilia volkensii were equally active, with

/ EC50 s of 12.5 % (18.88 and 26.14 mg/g respectively).

Neither species caused significant mortality at 100% natural concentration. Cipadessa baccifera and Ekebergia capensis were active at threefold higher concentrations, with ECSQ'S of 44 and 43% (102.9 and 133.0 mg/g) respectively.

The two species of the Guareeae examined were

relatively inactive; Guarea glabra extracts had an EC50 of

62% (53.3 mg/g) and Dysoxylum spectabile did not reduce P. saucia growth at 100%. Neither species caused significant mortality in the 7-day trial. The single species of the

Sandoriceae studied, Sandoricum koetiapte. was highly active

with an EC5o of 17% natural concentration (22.79 mg/g).

Species of the subfamily Swietenioideae were mostly less active than the Melioideae. Within the Cedrelae, Toona

australis was the most active with an EC50 of 12%. Two other species of Toona, T. ciliata and T. serrata, were equally active with ECSQ'S of 28 and 27% respectively. £.

odorata was significantly less active, with an EC50 of 53.6% natural concentration. Considering extraction yields, however, Toona australis. Cedrela odorata. and Toona ciliata

were equally active, with EC5o's of 22.93, 24.66, and 28.20 68

mg/g respectively. Toona serrata was least active from this

perspective, with an EC50 of 47.63 mg/g. No species caused significant mortality.

In the Swietenieae, Chuckrassia tabularis was the most

inhibitory, with an EC50 of 12.5% natural concentration.

Similar activity was noted in Swietenia mahogani. S_. macrophylla, S_. humilis, and Entandrophragma caudatum, with

EC5o's of 17.0, 20.0, 23.0, and 22.6% natural concentration.

Considering extract yields, S_. humilis was the most active,

followed by £. caudatum, C. tabularisf S. macrophylla, and

S. mahogani (EC50= 23.64, 29.83, 31.13, 42.36, and 47.09 mg/g respectively). Khaya senegalensis was the least

active, with an EC50 of 53.6% (67.05 mg/g). The only member of the Xylocarpeae available for study, Carapa guianensis.

also had low activity, with an EC50 of 53.2% (29.68 mg/g).

None of these extracts caused an increase in P_. saucia mortality during the seven-day assay.

B Antifeedant studies with Melanoplus sanguinipes

When foliar extracts were presented on cabbage leaf discs to

M. sanguinipes nymphs, all were completely consumed within

24 h. However, when extracts of some species (chosen for re-examination because of their pronounced activity against

£. saucia) were presented on glass fibre filter discs treated with 10% sucrose, markedly different results were obtained (Figure 2-3). Extracts of A. indica and M- azedirach were not significantly inhibitory at natural 69

Figure 2-3. Consumption of glass-fibre discs, treated with

10% aqueous sucrose and MeOH extracts of Azadirachta

indica, Melia azedirach. Turreae holstii, and Aglaia

odorata at 1, 2.5, and 5 times natural concentration

(on a wt/wt basis), by fifth instar nymphs of

Melanoplus sanguinipes. In every case controls

consumed 100% of the sucrose-treated discs during the

24 h assay. % Feeding Inhibition 100 90 iffi 80 / 7) 70 60 ftp lit 50 40 A 30 20 Hit 10 ill 0 MI 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 levels, feeding was only inhibited by 73% (A., indica) and 43% (M. azedirach ).

In contrast, foliar extracts from A., odorata and T_. holstii both significantly reduced feeding at naturally occurring concentrations, and almost completely inhibited feeding at

2.5 and 5 times natural concentration.

C Bioassays for antibiotic and phototoxic activity

None of the extracts inhibited the growth of Saccharomyces cerevisiae, either in the dark or following near-UV irradiation.

D Leaf Toughness

Fifteen species of Meliaceae were available for study in the collections of the USDA Plant Quarenteen Center and the

Gordon Fairchild Tropical Gardens in Miami, Florida These species were assayed for leaf toughness using a MpunchmeterM modelled after a design by Feeny (1970). Values for leaf toughness given in 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 in Entandrophragma caudatum to a high of 83.0 N/cm2 in the mangrove species

Carapa guianensis. The toughness of Azadirachta indica and

M. azedirach are somewhat overestimated as it was not possible to avoid the closely spaced secondary veins in these species. 72

E. Defensive Characteristics of Deciduous and Evergreen

Meliaceae

Leaf lifetime was not measured directly in this study; as a first approximation all species were classed as deciduous or evergreen, as indicated by species descriptions in various floras or monographs (Table 2-3). Carapa guianensis is usually evergreen, but can be deciduous in areas with a pronounced dry season (Pennington and Styles, 1981); I have classed it as an evergreen species in this study. Foliar

toxicity, evaluated as the EC50 in terms of % of natural leaf concentration, was significantly higher for deciduous species than for evergreen species (ANOVA, F^,29) = 6-96/ p

= .015). When the EC50 was expressed as mg extract/g diet

= 3 79 p = fwt the difference was less marked (F(j/29) - »

.0645). Much variation was present within each "leaf age" class, and the evergreen class, although on the average less active, included one of the most insecticidal species in this study, Aglaia odorata.

Leaf toughness data was available for 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 respectively). 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 significant (ANOVA, F(115j =2.263, 73

P=0.15). Aside from Carapa guianensis. the deciduous

Swietenia species had the toughest leaves.

Correlation of leaf toughness and toxicity suggested the possibility of a negative relationship between these two leaf characters (Figure 2-4). The regression had a significant negative slope at a= 0.1, but was not significant at cr= 0.05, indicating that there may be an inverse relationship between leaf toughness and the toxicity of the MeOH extracts. Once again, much variability was present and the regression equation accounted for only 23% of the observed variation in 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 .6cd 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) Trichilia reKa ND glab D(2) Lepidotrichilia volkensii 209.1 ND axil1 E(l) Ekebergia capensis 239.4 ND glab E(l) Cipadessa baccifera 18.2±4 .4ab 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 Sandoricvun Kcetjape 131.0 32.6+2 .4d pub E(2,4)

ibfaaily Swietenioideae Tribe 1. Cedreleae Cedrela odorata 46.2 27.3±1 >4cd glab D(2) Toona serrata 176.4 ND glab D(2) Toona ciliata 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±1.0DC 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 3 G Carapa auianensis - 56.3 83tp+lp,3 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 (in N/crn^)

and bioactivity of the MeOH extract of foliage,

calculated as 100-EC50.

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 toxicity (as EC5o to

Peridroma saucia [% of natural concentration]) and toughness

(N/cm2) between deciduous and evergreen species of

Meliaceae. Means in a column followed by the same letter are not significantly different (ANOVA).

Toughness fN/cm^)

Deciduous 18.1±15.7 (16)a 33.1±21.1 (ll)a

Evergreen 44.4+35.8 (14^B 50.4+15.1 (513- Discussion

A. Crude Extract Screening

Virtually all of the species of Meliaceae examined in this study appear to be well defended chemically against attack by generalist non-adapted insect herbivores, assuming that

Peridroma saucia is a valid model species . Members of the subfamily Melioideae were on average better defended chemically, and members of the tribe Melieae were consistently highly toxic. The Melieae include the only species known to produce C-seco limonoids such as azadirachtin (Dreyer, 1983; Connoly, 1983), the most insecticidal class of limonoids (Chapter 4 of this Thesis).

Correlations between phytochemistry and the insecticidal or insect growth regulating activity of the extracts are discussed in more detail in Chapter 4 (pg 216); in general the growth inhibitory activity of the crude MeOH extracts agrees well with the expected activity in those species where the limonoid phytochemistry is known. Two of the three species which were inactive in this study (Lansium domesticum and Dysoxylum spectabile) have been reported to lack limonoids. However, some species including Sandoricum

koetjapte, Aglaia odorata, A. odoratissimaf and A,, argentia are inhibitory to Peridroma saucia growth despite a reported lack of limonoids; these species deserve further phytochemical investigation. 80

Seed extracts of several of the species examined here have previously been tested for growth inhibition, feeding inhibition, and toxicity against the fall armyworm,

Spodoptera frugiperda, for feeding inhibition against the striped cucumber beetle, Acalymma vittatum, and for cytotoxicity against the brine shrimp Artemia salina

(Mikolajczak et aJL., 1987, 1989). This provides an opportunity to compare biological activities of seed and foliar extracts. Hexane and ethanolic seed extracts of

Aglaia cordata Hiern. markedly decreased feeding, growth, and survivorship of the fall armyworm at the lowest dose tested, 16 ppm; in contrast A., odoratissima extracts were inactive at 10,000 ppm. These results support my observation of a wide range of activity between different

Aglaia species. However, I found methanolic A., odoratissima foliage extracts to be inhibitory to E. saucia growth at

4,000 ppm. Ethanolic seed extracts of Chuckrassia tabularis inhibited fall armyworm growth by 95% and surviorship by 50% at 400 ppm; foliage extracts inhibited growth at 31,000 ppm and did not increase mortality. Seed extracts of Dysoxylum spectabile and Lansium domesticum were active at 400 ppm; in contrast foliar extracts of neither species inhibited E- saucia growth by as much as 50% at natural concentration

(about 150,000 ppm). Seed extracts of Sandoricum koetiape.

Azadirachta indica, Melia azedirach, Swietenia mahogani, and

Trichilia roka all follow the same pattern of being highly 81

active at concentrations at least two orders of magnitude lower than those of foliar extracts.

None of the foliar extracts inhibited feeding by the migratory grasshopper, Melanoplus sanguinipes. when assayed on cabbage leaf discs. However, extracts from Aglaia odorata and Turreae holstii were inhibitory when tested at natural concentration (on a dwt l«af/dwt disc basis) on glass-fibre discs provided with 10% sucrose as a phagostimulant. A similar discrepancy between results with leaf and glass fibre discs was noted by Ascher (1981). It is likely that this results from the artificial circumstance of presenting the insect with a freshly cut leaf edge, which allows contact with phagostimulants present in the leaf sap.

Ordinarily such contact would not occur until the later stages of host-plant acceptance, after the leaf surface has been examined by contact chemosensilla (Chapman and Bernays,

1989). The phagostimulant activity of the sap must exceed the activity of the feeding inhibitors in the extracts, which in turn are less active than 10% sucrose. However, the Aglaia odorata and Turreae holstii extracts might provide protection, in an agricultural context, against this grasshopper if applied to intact foliage.

The activity of the phytochemicals in these extracts responsible for inhibiting larval growth, and in some cases molting, may be specific for insects, as no extracts were antibiotic or phototoxic to Saccharomyces cereviseae. As well, several species found to be highly inhibitory to 82

insect growth (including Azadirachta indica, Melia

azedirachf and Aalaia odorata) were inactive when tested against brine shrimp Artemia salina (Wiriyachitra and

Towers, 1988). Together these results rule out general cytotoxicity as a mode of action for the most active extracts. The lack of any phototoxic effect in the

Saccharomyces assay also suggests that the reported virtual absence in the Meliaceae of furanocoumarins and other classes of photosensitizers, so typical of the Rutaceae, is real and does not simply reflect a lack of attention from phytochemists.

Several species investigated here for the first time appear to be sufficiently inhibitory to £. saucia neonates to warrant further attention. In particular, Aglaia odorata and Turreae holstii may have potential as sources of useful extracts or phytochemicals. In the latter case, the active component(s) appears to inhibit molting at doses which do not deter feeding. The phytochemistry of Turreae species is almost unknown, but Taylor has reported limonoids similar to cedrelone (Pettit et, ai., 1983), which can inhibit molting in the milkweed bug Oncopeltus fasciatus (Chapter 4).

Several phytochemicals have been reported from Aglaia odorata (Shiengthong et ai., 1965, 1979) and the role of these compounds in the observed activity of the odorata extracts is examined in Chapter 3 of this Thesis. 83

B. Resource Availability Hypothesis

Overall, the patterns seen here with regard to leaf toxicity, toughness, and lifetime, tend to support the resource availability hypothesis of Coley e_t al. (1985)

(termed the growth rate hypothesis in Coley, 1988). I have assumed that the relative investment in chemical (mobile) defenses may be estimated by comparing the relative efficacy of those defenses against a naive, non-adapted insect herbivore, Peridroma saucia. Physical (immobile) defenses including leaf toughness and pubescence were measured directly. As explained previously, the resource availability hypothesis predicts that investment in chemical defenses should characterize plants with short leaf lifetimes, and investment in physical defenses should be selected for in plants with long leaf lifetimes.

I found that extracts from deciduous leaves were significantly more inhibitory to E. saucia than were extracts from evergreen species, which suggests that deciduous species (short leaf lifetime) rely more on chemical barriers to herbivory than do evergreen (long leaf lifetime) species. Previous attempts to demonstrate greater investment in chemical defense in species with short leaf lifetimes have not been successful, as they relied on colorimetric assays for phenolics only (i.e. Coley, 1983), which led Coley (1983, 1988) to suggest that the importance of plant chemistry had been overemphasized. In practice, it is generally impractical to determine the contribution of 84 each secondary metabolite to insect resistance (in most cases the compounds are unknown), and it may be impossible to measure the cost of production of each compound.

Certainly it is unsatisfactory to estimate the importance of secondary metabolite-based defenses from a colorimetric assay for a single class 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 offer a practical alternative to this dilemna.

On average, the leaves of deciduous species were only half as tough as leaves of evergreen species, but the range of variation within each group was large. As a result, the two groups did not differ significantly in a statistical sense. However, numerous studies have demonstrated a strong correlation between leaf toughness and resistance to herbivory (Tanton, 1962; Grime gt al., 1968; Feeny, 1970;

Rhoades, 1977; Rausher and Feeny, 1980; Coley, 1983; McKey,

1984; Coley, 1988), and this factor is also clearly positively correlated with leaf lifetime (Coley, 1983, 1988;

Coley gt al., 1985). The lack of statistical significance in my study can be attributed to the small sample size (11 deciduous and 4 evergreen species) and the wide range of variation between individuals in each class. Similar variation characterizes earlier studies as well and can only be overcome with appropriately large sample sizes. I attach more importance to the marked difference in the means, which 85

suggest (in agreement with all earlier studies cited above) that evergreen species invest more in leaf toughness

(immobile or quantitative defenses) than do deciduous species. Species which invest in leaf toughness factors may rely less on phytochemical defenses, as there is evidence for a negative correlation between leaf toughness and inhibitory activity of the extracts. However, this hypothesis clearly needs further study with larger sample sizes, again to overcome difficulties with the large amount of interspecific variation.

Previous studies have compared defensive attributes of pioneer and persistent species which occur together in the same habitat. Although the species studied here were drawn from diverse habitats, interesting correlations can still be drawn between defensive strategies and ecological attributes. The most insecticidal species seen in this study, Azadirachta indica and the two Melia species, are noted for their extremely rapid growth, and are often planted as shade trees or for firewood for this reason

(Jacobson, 1988). They occur in semi-arid areas where nutrients and light are often not limiting (although water is), and cannot compete well with other species (Jacobson,

1989), suggesting that they are adapted to "high resource availability" habitats. In times of drought they often constitute the only green plants available, which may have provided the selection pressure for 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 rain-forest species which form a prominent component of the emergent flora (Brandis, 1906; White and

Styles, 1963; Pennington and Styles, 1981); these tend to produce tough, evergreen leaves low in insecticidal phytochemicals. Exceptions to this pattern do occur, however. Swietenia macrophylla is relatively fast-growing, especially compared to other Swietenia species (Pennington and Styles, 1981), but all three species tested here produce equally tough leaves and appear to be about equally well defended chemically; the pattern of defense in these species is more typical of the slow-growing evergreen species.

Carapa guianensis is a very variable species, occurring in mangrove swamps, on rocky hillsides, and even as an understory species in the rain forest (Pennington and

Styles, 1981). It's defenses include very tough leaves, and investment in phytochemical defenses appears to be low; in addition, this species has extrafloral nectaries along the leaf margin, and some populations appear to be well defended by ants (Pennington and Styles, 1981).

The plants used in this study were collected from diverse localities around the world; their only direct relationship is through phylogeny. Each species has had to respond, in evolutionary time, to a unique suite of natural enemies (herbivores) and competitors. As all species are from tropical habitats I have implicitly assumed (for 87

simplicity) that potential herbivores are numerous and consequently selection pressure to reduce herbivory is strong in all cases. However, variations in selection pressure between different habitats may account for much of the variability in defense strategies noted in this study.

As well, leaf toughness has been implicitly 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 selective pressures than herbivory in some habitats. Further, I have categorized species as deciduous or evergreen, but leaf lifetime can vary tremendously within these classes; particularly, some species may flush new leaves before dropping the old ones, in which case the tree could be evergreen and yet each leaf may have a short lifetime. Despite these sources of variation, an overall pattern of defense allocation between chemically- and physically-based strategies can be discerned and is in general agreement with the resource availability hypothesis. This suggests that the hypothesis is sufficiently robust to explain not only differences between plants within a single habitat, but also evolutionary patterns between related plants occupying different habitats. In a study of defensive characteristics of pioneer and persistent species occurring in light gaps in the Panamanian rain forest, members of the same family were consistently found to cluster according to ecological 88

attributes rather than along taxonomic lines, suggesting that habitat and life history place greater constraints on defenses than do phylogenetic relationships (Coley, 1983).

This conclusion is supported by my findings.

Detailed studies including leaf tagging experiments to determine leaf lifetime, and measurements of plant growth rates are underway in conjunction with collaborators (Dr.

K.R. Downum and associates) at Florida International

University. 89

Chapter 3. Phytochemical Investigation of Aglaia Species

Introduction

Species of the genus Aglaia figure prominently in traditional pharmacopeias throughout south-east Asia. For example, Aglaia odorata (Lour), known as Shu-Lan in Taiwan, has been used for the treatment of coughs and inflammation

(Kan, 1979) as well as "traumatic injury" (Hayashi ejb al.,

1982). In Thailand the same plant is traditionally prescribed as a heart stimulant and febrifuge (Shiengthong et al., 1965). Leaves and roots of Aglaia pirifera Hance induce vomiting and are used as an antidote for poisoning in

Thailand (Saifah et a_l., 1988).

As a result of their reputed pharmacological activities, to date three species of Aglaia have been examined phytochemically. A., odorata has yielded the dammarane triterpenes aglaiol, aglaiondiol (1), and aglaitriol (2) (Shiengthong gt al., 1965, 1974) and the bis- amides of 2-aminopyrrolidine, (+)-odorine (8), (+)-odorinol

(9) (Purushothaman e£ al., 1979) and (-)-odorinol (10)

(Hayashi gt al., 1988). (+)-Odorine and (+)-odorinol were also isolated from A., roxburgiana (Shiengthong gt al.,

1979). A., pirifera was found to contain piriferine, identical to (+)-odorine except for the loss of a terminal methyl from the 2-methylbutanoyl moiety (Saifah gt al.,

1988). The only compound which has, to date, been shown to 90

possess any biological activity is (-)-odorinol, which inhibits P-388 lymphocytic leukemia growth in BDF1 male mice

(T/C > 136%) at a dose of 5.0 mg/kg (Hayashi et ai., 1982).

In Chapter 2 of this thesis, MeOH extracts of A., odorata, &. odoratissima, and A., argentia were reported to inhibit growth of the variegated cutworm, Peridroma saucia.

This chapter is a report of attempts to isolate and identify the phytochemical(s) responsible for this activity in A., odorata, and to determine the basis of the growth inhibition

(feeding deterrence or toxicity). In addition, methods for the rapid qualitative and quantitative analysis of Aalaia foliage 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 isolated and identified from A., odorata foliage. 91

Materials and Methods

A) Sources of Plant Material

Sources of all plant samples are given in Table 2-1 of

Chapter 2. Additional material of Aalaia odorata from the

Pacific Tropical Gardens, Hawaii, was provided by Mr. T.

Flynn of that institution. Most of the phytochemical work reported in this Chapter is based on this latter material.

All plant samples were air-dried before shipping to UBC.

B) Isolation and identification of secondary metabolites in

Aglaia foliage

Isolation of the insecticidal components from A., odorata foliage was guided by bioassay (Section D). However, in view of the unique phytochemistry of this species within the

Meliaceae, an attempt was made to identify all of the major secondary metabolites, whether or not they showed activity against E- saucia. Isolation involved a preliminary solvent partitioning, followed by chromatographic purification of the most active solvent fraction.

Bl) Solvent partitioning

Air-dried A. odorata foliage 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 filtered, pooled and concentrated under vacuum to a final volume of 2 ml/g leaf dwt equivalent. This extract was used for initial 92

bioassays as described in Chapter 2. Other species of

Aglaia were extracted in an identical manner.

The MeOH extract was diluted to 1 1, combined with an equal volume of water and partitioned successively (3 x per solvent) against an equal volume of hexane, diethyl ether

(Et20), and dichloromethane (CH2C12). Solvents were purchased from BDH and redistilled prior to use. All solvent phases, including the aqueous phase, were dried, weighed, and bioassayed at 10% of natural concentration (0.1

g leaf equivalent/ g dwt diet). The dried Et20 phase was

only partially soluble in anhydrous Et20; this phase was

accordingly divided into anhydrous Et20-soluble and

-insoluble phases and both were bioassayed at 250 and 2500 ng extract/g diet dwt.

B2i. Normal-Phase Chromatography

The Et20 soluble fraction, which was the most active against

E. saucia neonates, was flash chromatographed (Still et al.,

1975) in 1 g lots on a 2.5 x 25 cm silica (Si) gel (230-400

mesh) column, using N2 gas as the pressure source. The

mobile phase was 2 column volumes each of Et20, Et20:EtOAc

(3:1), Et20:EtOAC (1:1), EtOAc, and finally MeOH.

Chloroform was avoided because the bis-amides are unstable in this solvent (Shiengthong et al., 1979). The flow rate was 49 ml/min (2.5 cm/min), and 6 ml fractions were collected. Fractions were monitored by analytical TLC (Si plates, 0.2 mm, Merck) developed in EtOAc:PE 3:1; spots were 93

visualized with Ehrlich's reagent (1 g p-

dimethylaminobenzaldehyde in 100 ml 2% ethanolic H2S04)

(Dreyer, 1964) and similar fractions were pooled for bioassay.

Fractions which were inhibitory to £. saucia. or which contained major natural products (as evidenced by the appearance Of major spots on TLC or crystallization of compounds on drying), were purified further by preparative

TLC. Fractions were banded onto 1 mm thick Si gel TLC plates (Merck) and developed in EtOAc:PE (1:1) in ans equilibrated 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 visualized with UV light 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 finally purified by recrystallization, prior to bioassay.

B3) HPLC

The most active fractions were chromatographed using preparative HPLC after TLC proved unsatisfactory for the isolation of the insecticidal principle(s). Fractions were prepared for chromatography by passing them through a 3 cm silica plug in 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) for 10 minutes,

followed by a gradient to 100% MeOH over 20 minutes, then

elution with 100% MeOH for 30 minutes. Flow rate was 3

ml/min, with UV monitoring at 217 nm. Six-ml fractions were

collected with an automatic fraction collector.

Subsequently these fractions were examined by analytical

HPLC using a Lichrosorb RP-18 (5 /im) 4 X 250 mm column,

isocratic MeOH:H20 (7:3), with a flow of 1 ml/min. One

twentieth of each fraction was used for bioassay.

C) Qualitative and quantitative analyses

Subsequently, techniques developed for the isolation of the

active component(s) were adapted to provide rapid methods of

analysis of Aglaia foliage. The Rfs of all compounds

isolated were determined in two analytical TLC systems:

EtOAC:PE (3:1) on Si gel (System A), and CHCl3:MeOH (49:1)

on Si gel (System B). The color reactions of all compounds were determined with two spray reagents, Ehrlich's reagent

(Dreyer, 1964), and Vanillin reagent (2% vanillin in EtOH/

overspray with 3M H2S04) (Pieman e£ al., 1980). These TLC

systems were used for qualitative comparisons of Aalaia

extracts.

Quantitative analysis was achieved by analytical 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 calibration curves were determined for all identified compounds isolated from A., odorata. This method could not resolve the diastereomers of odorine and odorinol; to quantify these compounds I used 1H-NMR, comparing relative heights of the methyl peaks at 0.70 and 0.90 ppm to calculate the proportion of S,R and S,S odorine. Relative heights of the singlets at 6 1.34 and 1.22 were used to calculate the relative proportions of S,R and S,S odorinol.

D) Bioassay

All fractions and pure compounds were bioassayed against neonate E- saucia, in seven-day chronic feeding assays, as described in Chapter 2. Solvent phases were bioassayed at

10% of the naturally occurring levels; for fractions collected from column chromatography or HPLC, 1/20 of each fraction was used. The maximum concentration of pure compounds bioassayed, given in Table 3-7, was equal to or slightly greater than the concentrations of the compounds in the plant, as given in Table 3-6. Various combinations of the purified compounds were also bioassayed (Table 3-8), to check for additive or synergistic interactions.

The most active compound isolated was assayed for inhibition of larval growth and survivorship at 0.4, 0.6, 1,

2, 3, 5, 10, and 15 ug/g diet dwt. A simple choice feeding assay was also used to determine the role of antifeedant 96

effects in the activity of this compound at 1, 2, and 3 ug/g diet; this assay is described in detail in Chapter 4. Results

A) MeOH Extract Screening

Inhibition of neonate E- saucia growth by extracts from A.. odorata (Thailand sample), A., odoratissima, and A., argentia is shown in Figure 3-1. The A., odorata extract was about twice as active as the A., odoratissima extract, and five times as active as the A., argentia extract.

B) Solvent Partitioning

Bioassay of the various solvent phases from A., odorata

indicated that the Et20 phase was most inhibitory to E.

saucia growth and survival; the CH2C12 phase also retained

some activity (Figure 3-2). The Et20 phase was dried and divided into phases which were soluble or insoluble in

anhydrous Et20. The Et20 soluble phase was about ten times more active than the insoluble phase when tested at 250 /jg extract/g diet dwt. Neither the hexane phase nor the remaining aqueous phase had an inhibitory effect. Bioassay of the leaf marc, the residue after extraction, indicated that the insecticidal principle(s) had been completely removed by the MeOH extraction. Figure 3-2 also includes the yield (as % of the MeOH extract) for each solvent phase.

C1 Chromatography

The isolation of phytochemicals was confined to the Et20 phase as this contained the insecticidal compound(s). 98

Figure 3-1. Effect of foliar MeOH extracts of Aglaia odorata (o), A., odoratissima (A), and A., argentia (•) on the growth of neonate Peridroma saucia. Growth is shown as % of the control. Each point indicates the mean of at least three determinations per treatment; standard deviations were

<7% for all treatments. Larval Growth (% of Control) 100

Figure 3-2. Growth and survivorship of neonate Peridroma

saucia fed artificial diet containing solvent extracts of

Aglaia odorata (Hawaiian sample). The yield of each solvent

phase, as % of the total MeOH extract, is also shown. Larval Growth & Survival (% of Control) 1201 102

Typical results of flash chromatography of this phase are given in Table 3-1, along with bioassay results for each fraction. Several fractions yielded crystalline material on drying; these were investigated further whether or not they possessed insecticidal activity.

TLC analysis of the column fractions which were inhibitory to £. saucia growth indicated the presence of three major compounds. Preparative TLC was used to isolate these compounds in amounts sufficient for identification.

Two compounds, the identification of which is described below in detail, 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 third component was not identified. Surprisingly, none of these isolated compounds exhibited any activity against P. saucia (Table 3-9). Subsequently I eluted all remaining compounds from the TLC plate, recovering a small amount of oily material which also proved to be inactive. The active principle therefore appeared to be unstable on silica, degrading or isomerizing to an inactive form.

Subsequent attempts to purify the active compound utilized semipreparative reverse-phase HPLC. Bioassay results indicated that all the activity was associated with a single apparently homogeneous peak (Fig 3-3). HPLC was then used 103

Table 3-1. Properties of fractions obtained from flash column

chromatography (Si gel, 240-400 mesh) of A., odorata (Et20 soluble phase), and their effect on the growth of neonate Peridroma saucia, reared for seven days on diet containing each fraction at approximately natural concentration. Values followed by the same letter are not significantly different (Tukey's Studentized (HSD) Range test).

Fraction Description Growth

# ; ; r% of Control) A 1-5 Empty 102a B 6-9 Chlorophylls only 115a C 10-15 Chlorophyll, copious white crystals, red 97a with Ehrlichs D 16-19 Still some "red" cmpd (Rf=.525), 2 blue 2b fluorescing cmps, yellow with Ehrlichs, Rf= .85 & .89 E 20-35 Same two blue fluorescing cmpds, lower 6b more abundant F 36-41 Empty 107a G 42-60 White crystals, purple with Ehrlichs, Rf=.425 87a H 61-95 Crystals, odorine (Rf=.344) and odorinol 83a (Rf=,24D Figure 3-3. Preparative HPLC chromatograra of a growth inhibitory fraction from A., odorata. Insect growth- inhibiting activity was associated with the shaded peak. 105 106

to isolate about 11 mg of this compound, which was concentrated and crystallized from MeOH. The compound proved again to be the inactive 3-hydroxy-5,7,4'- trimethoxyflavanone, isolated previously by TLC. All the inhibitory activity remained in the mother liquor; when dried this 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 all, ten natural products were isolated from A., odorata foliage, nine of which were characterized completely. Of the identified compounds, four had not previously been identified in any Aglaia species, and two were new natural products. Only the compound responsible for the marked growth inhibition against E» saucia could not be identified.

1H-NMR and mass spectra of all compounds are given in

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 isolated from A. odorata. This identification was supported

by CH3 singlets at cS 0.77 and 0.91, characteristic of geminal methyls at C-4, at 6 0.97 (6H), indicative of 10B- 107

and 8B-Me groups, and at 6 0.89, assigned to the 14a-Me

group (Shiengthong et. al., 1965). As well a D20 exchangeable proton at S 3.29 and a multiplet at 6 3.33

(simplified to a quartet after D20 exchange), were assignable to a 3B-0H and a 3a-proton respectively.

As in previously identified Aglaia dammaranes, a methylene group is 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

singlet on D20 exchange. These spectral data are consistent with the known dammarane triterpene aglaiondiol, previously described from Aglaia odorata (Shiengthong et al.,

1965,1974) (Figure 3-4).

Compound 2. White, fine needles, m/z (FAB):459 (C3QH50O3).

This compound was also identified as a dammarane triterpene,

1 with H-NMR CH3 singlets 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 literature values for the dammarane triterpene aglaitriol (Shiengthong et- al. ,

1974) (Figure 3-4).

Compound 3. This compound formed white plates, m/z (EI,

FAB) 330 (C18H1806). The UV absorbtion spectrum showed 108

Figure 3-4. Structures of dammarane triterpenes isolated from Hawaiian samples of Aglaia odorata. Aglaiol was not isolated but was assumed to be present in the inactive

hexane phase. Amoora A6 was isolated 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 3.8-2 s 3.83 S OMe-7 3.84 s 3.84 S 3.8-3 S OMe-4' 3.92 s 3.89 S 3.85 S

(1) 400 MHz in CDC13, TMS as internal 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; 3A B=17.6; 6,8=2.4; 2/,3/=9.0; 5',6'=9.0 ' Ill

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 singlet when decoupled at 7.00 ppm), typical of a para-substituted benzene ring. 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, JAX=12.8 Hz, JAB=0.5

Hz), which was in turn coupled to a D20 exchangeable doublet at S 4.05 (IH, J=0.5 Hz). This was interpreted as indicating two protons, H-2 and H-3 in cis 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 (cf. Balza and Towers, 1984). One methoxy group must be present at C-4' to account for the para- substituted B ring; C-5 and C-7 of the A ring were also methoxylated, accounting for the meta-coupling between the

C-6 and C-8 protons. The compound was therefore identified as the new natural product 3-hydroxy-5,7,4 trimethoxyflavanone (dihydrokaempferol 5,7,4'-trimethyl ether) (Figure 3-5).

Further evidence supporting this structure was obtained from the mass spectrum (Figures 3-6, 3-7). The identity of all 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 typical of dihydroflavonols include

the B ring fragments B3+ (m/z 150), B4+ (m/z 121, base

3 3 peak), B -CH3 (m/z 135), and [B -43]+ (m/z 107), and the A

ring fragments [A1+H]+ (m/z 181), A1+ (m/z 180), and A^—CO

(m/z 152). The prominent peak at [M-CO]+ (m/z 301,

C17H1705), is typical of flavones, flavanones, and

dihydroflavonols (Mabry and Markham, 1975). The size of the

B-ring fragments confirm that it carries a methoxy

substitution. A-ring fragments at m/z 181 and 180,

resulting from a retro Diels-Alder cleavage (Mabry and

Markham, 1975), confirm the presence of two methoxy

substitutions.

Compound 4. White crystals, m/z (EI) 314, C18H1805. The

^•H-NMR spectrum of this compound was similar to that of

Compound 4, with a para-substituted benzene ring (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, singlets, 3.83, 3.84, and 3.89

ppm) (Table 3-2). However, no D20 exchangeable protons were

present, and the ABX system showed doublets of doublet at 6

5.37 (1H, JAB=0-4 JAX=13'5 Hz)' 6 3'05 (1H' JAB=0*4 JAX=16-°

Hz), and 2.76 ppm (1H, JAB=0,4 JAX=16,0 Hz)« This indicated

that a proton at C-2 was coupled to geminal protons at C-3;

the compound was therefore identified as 5,7,4'-

trimethoxyflavanone (naringenin-5,7,4'-triroethyl ether)

(Figure 3-5). 113

Figure 3-5. Structures of flavanones isolated from Hawaiian samples of Aalaia odorata. The stereochemistry shown assumes an S configuration at C-2. ^OCH3 CH30 C

OCH3 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 (B3+,

base peak), 119, and 91 confirm the location of the third methoxy group.

1 Compound 5. White plates, m/z (EI) 300, C17H1605). The H-

NMR spectrum of this compound was almost identical to that of Compound 5, but showed only two methoxy groups (3H, 3.83

and 3.85 ppm) and a D20 exchangeable singlet (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 this ring. B-rihg

fragments at m/z 121 demonstrated the presence of a methoxy

substitution, which must be at C-4' to account for the para-

substitution pattern. The hydroxyl was assigned to C-5 rather than C-7, as hydrogen bonding with the adjacent

carbonyl causes the signal to appear at 12.02 ppm; if present at C-7 the signal would have occurred at 9.5 ppm

(Balza and Towers, 1984). Compound 5 was therefore

identified as 5-hydroxy-7,4'-dimethoxyflavanone (Figure 3-

5).

Compound 6. m/z (FAB) 661, MH+. Possibly C36H36012. This

compound produces a brown color reaction with Ehrlich's

reagent, a reaction typical of B-substituted furans (Dreyer,

1964). The mass and possible presence of a B-substituted 120

furan ring suggest that compound 6 is an oxidized limonoid.

However, the very limited amount of material on hand precludes the extensive NMR studies required for structure elucidation.

Compound 7. White needles, m/z (EI) 300 (M+), C18H24N202.

1H-NMR (Table 3-3) showed a benzene ring (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 identity of Compound 8 as the known bis-amide odorine (Shiengthong et ai., 1979;

Purushothaman gt ai*, 1979) (Figure 3-8). This identification was confirmed by the mass spectrum, which showed major fragments at m/z 215 and 85, (resulting 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 closely comparable to literature 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 isolated from Hawaiian samples of Aglaia odorata.

123

Lopez, while he was a visiting scientist with Dr. G.H.N.

Towers.

Odorine has chiral centers at C-2 and C-2'. The C-2

site is particularly sensitive to epimerization in acidic

solvents including CHC13 (Purushothaman et. ai., 1979). As a

result chlorinated solvents were avoided during the

isolation 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 the1 H-NMR

(CDCI3) spectra indicate that in 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 for R,S and S,R-odorine. Compound 7 was therefore

identified as the known S,R-odorine [(+)-odorine] (Figure 3-

8).

Compound 8. Needle crystals, m/z (EI) 300, C18H24N202. The mass spectrum of compound 8 was identical to compound 7.

The 1H-NMR was also identical except for 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 identified as S,S-odorine (Figure

3-8). This identification was supported by co-

chromatography with synthetic S,S-odorine. This isomer of odorine has not previously been identified as a natural 124

product. To check the possibility that R,S-odorine could

isomerize at C-2 if stored in MeOH (the solvent used to extract A., odorata) for extended periods, a sample of originally pure R,S-odorine was examined after six months in

MeOH at room temperature in 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 crystals, m/z (EI) 316, C18H24N203. The

1H-NMR spectrum of this compound was similar to compound 8, except that the methyl group at C-2' appeared as a singlet at

and a D20 exchange experiment showed an exchangeable proton at 6 2.16 ppm. The UV spectrum showed a single absorbance at 283 nm. This spectral data is consistent with the known

Aglaia bis-amide odorinol (Figure 3-8) (Shiengthong et al.,

1979, Purushothaman et ai., 1979, Hayashi et Al., 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 is 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 crystals, m/z (EI) 316, C18H24N203.

The mass spectrum of this compound was identical to compound 125

10. The 1H-NMR spectrum differed only in 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).

£) Qualitative and quantitative comparison of Aalaia species

Two methods suitable for rapid phytochemical analysis of Aalaia species were developed. A qualitative method was based on TLC on silica gel and use of color reactions with

Ehrlich's and vanillin spray reagents (Table 3-3). Rf values of several compounds differ in the two solvent

systems used (EtOAc:PE 1:1 and CHCl3:MeOH 49:1), so these are suitable for two-dimensional TLC. The color reactions with the two spray reagents are highly characteristic and allow confident identification 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 analytical HPLC; retention times of pure standards are given in Table 3-5.

Chromatographic profiles of the various Aglaia samples are shown in Figures 3-9 to 3-13. As the concentrations of secondary metabolites in plants may differ greatly from the amounts eventually isolated, due to losses during extraction and purification, this method was applied to determine the 126

concentration of Aglaia compounds in freshly prepared Et20 extracts (Table 3-6).

Each method offers certain advantages and weaknesses.

The major advantage of the HPLC method lies in the ability to quantify concentrations of several compounds. However two major disadvantages exist: HPLC cannot be used to detect the dammarane triterpenoids, due to their low UV absorption coefficients, and HPLC does not resolve the various isomers of odorine or odorinol. For the latter purposes ^H-NMR was used to quantify the proportion of each isomer, according to the relative peak heights of the 2-methylbutanoyl methyls.

Although qualitative in nature, the TLC based system can readily detect the presence of dammaranes in very low concentration, and it can resolve the diastereomers of the odorines and odorinols. In the latter case a minimumn of 4-

5 developments were required for clear resolution of the

diasteriomeres. Analysis of an Et20 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 in phytochemistry occur between most of the Aglaia species and samples examined. A., odorata samples from Thailand and

Hawaii were most similar; both produced aglaiondiol and aglaitriol, and the three dihydroflavanones, although minor differences in 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 first 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 .689 .244 purple blue-purple 5,7,4'-MF .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 Aglaitriol 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 Et20 soluble fraction from Aglaia odorata (Hawaiian sample). Identified 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 Et20 soluble fraction from

Aglaia odorata (Thailand sample). Identified 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 Et20 soluble fraction from

Aglaia odorata (Taiwan sample). Identified 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 Et20 soluble fraction from

Aalaia odoratissima. Identified 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 Et20 soluble fraction from

Aglaia argentia. Identified 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) (Thailand) (Taiwan) (Thai land) (Thailand) 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 totally absent. The Taiwan sample

of A. odorata contained only a trace of aglaiondiol, no

aglaitriol, moderate concentrations of 5-hydroxy-7,4'- methoxy dihydroflavanone, and only traces of the other dihydroflavanones (detected by HPLC only). The amide profile of this sample was identical 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 in equal proportions. HPLC analysis further revealed very low concentrations of the other dihydroflavanones and odorinol

(isomer not identified). Aalaia argentia appeared to lack aglaiondiol and aglaitriol entirely, but different dammaranes may be present as indicated by major components which show similar 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

in almost a 1:1 ratio, and odorinol appears to be absent.

E) Insect Bioassays

Bioassay-guided fractionation of A., odorata led to a

single compound, compound 6, with significant growth

inhibitory activity against £. saucia neonates (Table 3-7).

Although insufficient material was obtained for structure 142

elucidation, due to the pronounced activity of this compound the dose-response relationship could be established (Figure

2-14). The compound was about four times less active than azadirachtin in its ability to inhibit E- saucia growth,

with an EC50 of 1.4 pg/g diet fwt compared to 0.36 ug/g for

azadirachtin. The LC50 was 11.2 ug/g, compared to 4.0 ug/g with azadirachtin. As the active compound was still admixed with some of the dihydroflavonol, the activity of the pure compound may be still closer to that of azadirachtin.

The inhibitory activity of compound 6 appeared not to be due to antifeedant activity. In choice tests with neonate £• saucia compound 6 had no significant effect on feeding at concentrations which inhibited growth in no- choice assays (Table 3-9). On the other hand, the activity of the total MeOH extract, assayed at 50% of natural concentration, appeared to include an antifeedant effect

(Table 3-9). No E- saucia larvae showed evidence of having died during a failed molt, but treatment with the known IGR compound azadirachtin also failed to produce obvious molt inhibition in this insect (Chapter 4).

None of the other compounds isolated from A. odorata

inhibited 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 isolated yield of compound 6 was about 3 ng/g leaf dwt. However, an estimation based on the activity of the 143

Table 3-7. Aalaia odorata compounds: concentration bioassayed, and resultant E. saucia growth and survivorship (as % of Control). Values in a column followed by the same letter do not differ significantly (Tukey's Studentized (HSD1 Range Test.

Compound Max. Cone. E. fiflucia E« saucia Bioassayed growth (%) survivorship 1 Aglaiondiol 1000 ug/g 137.9a 100 2 Aglaitriol 1000 132.0a 100 3 5-OH-7,4'-MF 500 110.8a 100 4 5,7,4'-MF 500 108.3a 100 5 3-OH-5,7,4'-MF 500 107.la 100 6 "active" 15 2.6b 31 7 Odorine A 1000 114.8a 100 7' Odorine A' 1000 116.7a 95 8 Odorine B 1000 121.2a 100 8' Odorine B* 1000 89.4a 100 9 Odorinol A 1000 114.8a 93 10 Odorinol B 1000 124.7a 100 Amoora A6 50Q 103.2a _10£ 144

Figure 3-14. Effect of Compound 6 on the growth (•) and

survivorship (A) of neonate Peridroma saucia. For comparison, the effect of azadirachtin on the growth (a) and

survival (&) of neonate Peridroma saucia is also shown.

Error bars indicate + one standard deviation. Larval Growth and Survivorship (% of Control)

S+7l crude MeOH extract and assuming all the activity was due to compound 6 indicates an expected foliar concentration of 98 fig/g. The discrepancy suggested that synergistic interactions might be occurring between phytochemical constituents in A., odorata. Consequently the activity of various combinations of the isolated compounds were also assayed (Table 3-8). No combination of these secondary metabolites showed evidence of synergistic activity. The putative synergist remains unidentified. Table 3-8. Effect of Aglaia odorata compounds, tested in combination, on growth of neonate Peridroma saucia in a seven-day assay. Growth is shown as % of Control; values followed by the same letter do not differ significantly (Tukey's Studentized (HSD) Range test,

Compounds Concentrations Mean Bioassayed (ua/a) Growth 1+2+3+4+5+7+8+9 1,2:1000 96. 5a 3,4,5:200 7,8,9:200 1+6 1000 +1 79.3ab 2+6 1000 + 1 68.3b 3+6 200 + 2 34.6C 4+6 200 + 2 26.4C 5+6 200 + 2 28. 8C 6 2 23. 8C 7+6 100 + 2 29. 9C 8+6 100 + 2 36.4° 9+6 100 + 2 22.6°- 148

Table 3-9. Effect of Compound 6 on diet choice by neonate Peridroma saucia. Figures shown indicate the percentage of larvae found on Control (C) or Treated (T) diet after 24 h of feeding; each value is the mean of twelve replications. Observations were compared to a 50:50 distribution using a G-test with oc=0.05; values which differed significantly are indicated by an asterisk. Also shown is the larval growth (as % of control) after seven days of feeding in a no-choice bioassay.

Concentration Diet Choice Growth (ug/g diet 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 list. 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 first has been found only once, in the composite Dahlia tenuicaulis (Kaufmann and Lam, 1967; Lam and Wrang, 1975).

The second is known from a variety of unrelated taxa including Betula spp (Wollenweber, 1975), Prunus saraentii

(Wollenweber and Dietz, 1981), the composites Dahlia tenuicaulis (Lam and Wrang, 1975), Eupatorium odoratum

(Arene e£ al., 1978),and Hieracium intybaceum (Wollenweber,

1984), the cacti Rhodocactus grandifolius and Mamillaria elongata (Burret gt al., 1982), and the fern Pityrogramma spp (Wollenweber and Dietz, 1980). All of the dihydroflavanones are here reported from the Meliaceae for the first time. Flavonoids previously known from Aglaia include glycosides of quercetin and kaempferol (Harborne,

1983). Quite dissimilar C-8 prenylated flavonones have previously been reported from Azadirachta indica (Garg and

Bhakuni, 1984). O-Methylation at C-5 is uncommon amongst vascular plants in general, but is characteristic of flavonoids from species of the Rutaceae (Wollenweber and 150

Dietz, 1981; Harborne, 1983); the presence of such compounds

in Aglaia may reflect the close relationship between the

Rutaceae and Meliaceae. Wollenweber and Dietz (1981) have

commented on the common co-occurrence of methoxylated

flavonoids and lipophilic terpenoids, a pattern supported by the presence of both methylated flavanones and dammarane triterpenes in Aglaia.

The following discussion of phytochemical differences between samples of Aglaia must be prefaced by a caveat: as only single samples were available from each collection site, it cannot be determined at this time whether the variations observed reflect variation between individuals or populations. Further analysis of large samples from discrete populations is required to resolve this question.

Dammarane triterpenes are common among the Meliaceae, and some species, such as Cabralea eichleriana. may elaborate a considerable diversity of these compounds (Rao et al., 1975). All of the Aglaia dammaranes isolated 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 aglaitriol in large

amounts; in contrast, only aglaiondiol was found in A.. odorata from Taiwan, and in A., odoratissima. Neither dammarane was present in A., argentia. but other major compounds which give similar color reactions on TLC may represent other dammaranes. 151

Dammarane triterpenes may serve as precursors in 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 in particular with other members of the tribe

Aglaieae, and suggests that the pathway to limonoid synthesis is blocked in Aglaia.

The bis-amide odorine was present in all Aalaia samples examined in this study, but differences in the specific diastereomers produced were noted. In all A., odorata samples examined odorine B predominates; lesser amounts of odorine A are also present only in the sample from Hawaiii

In contrast A. odoratissima and A., argentia produce the A and B forms in approximately equal amounts. Odorine was not observed to isomerize in MeOH in vitro. These results indicate that the production of each diastereomere is likely regulated by a separate enzyme. Odorinol was found in all three samples of A., odorata. was present in very low amounts

in A., odoratissimaf and was absent from A. argentia.

Odorinol A was the only isomer observed in the Thai and

Taiwan samples of A. odorata; in the Hawaiian sample odorine

A predominated but some odorine B was also present. Again this argues for separate enymatic control for each isomer.

The prevalence of the odorines within the genus is unclear: all species examined to date contain odorine, but in general 152

surveys of plants for alkaloids many Aglaia species gave a

negative test (Farnsworth et al., 1954). Certainly the

odorines appear to be unique to Aglaia, and differ markedly

from alkaloids present in other Meliaceae such as Dysoxylum

species (Aladesanmi and Ilesanmi, 1987; Aladesanmi et al.,

1988) . The biosynthetic origin of these compounds is

unclear but they could be derived from ornithine, phenylalanine, and isoleucine.

B Insecticidal Activity

Extracts of Aalaia odorata foliage from Thailand were as

effective as neem or chinaberry leaf extracts at inhibiting 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 inhibitory. Extracts of two other

Aalaia species, A,, odoratissima and A., argentia. were even

less active, indicating interspecific variation in the

production of defense compound(s). Similar interspecific variation was noted in the activity of Aglaia seed extracts

against Spodoptera frugiperda (Mikolajczak et al., 1987, 1989) .

Several secondary metabolites were present in A..

odorata foliage in high concentrations; however none of them

exhibited inhibitory activity against E. saucia when tested

as pure compounds at ecologically relevant concentrations.

Flavones and flavonols, especially those with catechol 153

substitutions, are known to inhibit the growth of some phytophagous insects (Chan et al., 1978; Waiss gt ai., 1979;

Elliger et ai-/ 1980, 1981; Isman and Duffey, 1982; Hedin

and Waage, 1986) and 5-methoxy flavones have recently been

shown to be more inhibitory than 5-hydroxy flavonoids

(Mahoney et al., 1989). However the 5-methoxy and 5-hydroxy

flavanones isolated from h. odorata were inactive against P. saucia at naturally occurring concentrations, when tested as pure compounds or as mixtures. (-)-Odorinol is cytotoxic to

P-388 leukemia cells jji vitro and in vivo (Hayashi gt ai.,

1988), but none of the isomers of odorine or odorinol, whether natural or synthetic, had any effect on £. saucia growth.

Dammarane aglycones have apparently not previously been tested for activity against insect herbivores, but dammarane glycosides such as the saponins do inhibit growth of some

insects when present in diet at high concentrations (1-5% fwt) (Applebaum and Birk, 1979). Other glycosides, the ginsenocides, produce a variety of physiological effects in vertebrates, apparently as a result of direct action on the hypothalamus and pituitary resulting in stimulation of the adrenal glands (Shibata, 1986). Aglaiondiol and aglaitriol, tested individually and as a mixture at combinations up to

1,000 ppm/component, were not inhibitory to E. saucia growth.

It appears that the growth inhibitory activity of A.. odorata may be ascribed almost entirely 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 tests indicated that the growth-inhibiting activity of Compound 6 was not due to antifeedant effects and must reflect toxicity. This was similar to the activity of some limonoids including cedrelone and anthothecol (Chapter 4). Although no evidence of IGR effects on molting were seen, the known IGR compound azadirachtin also fails to produce obvious effects on molting when fed to E. saucia (Chapter 4).

The concentration of Compound 6 in the Et20 extract could not be determined accurately by HPLC, as it co- chromatographed with the 3-hydroxy-5,7,4'-methoxyflavanone.

However, the isolated yield (3 ng/g leaf dwt) appeared to be much less than the expected concentration (98 fig/g) estimated from the activity of the crude MeOH extract. The discrepancy suggested the possibility of synergistic interactions between phytochemical constituents in A.. odorata. Several studies have now indicated the occurrance of additive or synergistic interactions between phytochemicals in other plants (Berenbaum, 1985); for instance an array of phenolics in sorghum significantly deterred feeding by Locusta migratoria. even though individual compounds were not deterrent (Adams and Bernays,

1971). Combinations of isobutylamides, mixed in proportions similar to those occurring in the plant, were more toxic than would be expected from simple addition of the 155

activities of the individual compounds (Miyakado et al.,

1989), and N-isobutylundecylenamide synergises the activity of natural pyrethroids (Metcalf, 1967; Matsui and Yamamoto,

1971). In some plants, methylenedioxyphenyl (MDP) compounds may synergise toxicity by inhibiting mixed-function based oxidative metabolism of xenobiotics in the insect. For example, the MDP compounds myristicin, safrole, isosafrole, and fagaramide synergise the toxicity of co-occurring furanocoumarins in parsnip (Berenbaum and Neal, 1985; Neal,

1989). However, when various combinations of the Aalaia compounds here isolated were tested for synergistic interactions, no combination was more active than would be expected from the simple addition of the activity of the components in isolation. The putative synergist remains to be identified.

Although the structure of the active compound was not determined, the pronounced growth inhibitory activity of A.. odorata extracts suggests that they may have some application in pest management, particularly in areas where the plant occurs naturally or as a result of cultivation.

The phytochemical basis of this activity requires further attention; the work presented in this Chapter should facilitate future attempts to isolate and identify the active compound and elucidate the participants in the synergistic interaction. 156

Chapter 4: Effects of Limonoids from the Rutales on

Peridroma saucia and Oncopeltus fasciatus

Introduction

Limonoids are the most characteristic secondary metabolites in the Meliaceae, and are also prominent in the Rutaceae

(reviewed in the Introduction to this thesis, pg 16-27).

Over 300 limonoid structures have been elucidated to date

(Taylor, 1987), but only seventy such compounds have been examined for biological activity against insects. Table 4-1 summarizes the reported effects of limonoids on insect feeding and growth; structures for these compounds are given in Figure 4-1. Examination of this compilation reveals two impediments to developing a quantitative understanding of structure-activity relationships amongst limonoids.

Firstly, very few of the published studies utilize the same bioassay species; as a result it is difficult to separate effects owing to structural differences between compounds from effects due to interspecific differences in the response of the test insects. Secondly, the majority of studies were designed to detect feeding deterrence, and do not indicate non-behavioral effects including toxicity or insect growth regulating (IGR) effects. This situation reflects the prevailing belief that limonoids function primarily as antifeedants. For example, Taylor (1987) commented that "the limonoids seem to be remarkably bereft 157

of physiological properties; we have examined many without finding anything beyond the characteristic bitter taste...".

In the most comprehensive survey to date, Kubo and Klocke

(1987) assayed seventeen limonoids against three

lepidopteran species. However, as they reported only EC50 concentrations for growth inhibition, the roles of chemosensory (antifeedant) and post-ingestive effects in producing the observed growth inhibition cannot be separated. Despite such limitations, Table 4-1 is of use in indicating general qualitative trends in structure-activity relations.

The biosynthesis and evolution of the limonoids has been described recently by Das et ai. (1984, 1987).

Biosynthesis apparently proceeds along four major pathways, all dominated by trends of increasing oxidation and skeletal rearrangement (Figure 4-2). If the primary raison d'etre for the production of limonoids is to gain protection against insect herbivory, one may expect to find that evolutionary trends (i.e. increasing oxidation and skeletal rearrangement) correspond with increasing activity against insects.

In the present study I bioassayed ten limonoids, including representatives of all the major classes, for growth and feeding inhibition against neonates of the variegated cutworm, Peridroma saucia, and for inhibition of molting (IGR effects) and reproduction against the large milkweed bug, Oncopeltus fasciatus. The results of these 158

assays, and a compilation of the literature to date, were applied to test the hypothesis that there is a correlation between evolutionary advancement of the compounds (as defined by Das ei ai., 1984,1987) and their activity against phytophagous insects. 159

Table 4-1. Effects of limonoids on insect feeding and growth.

Abbreviations are: EC50, concentration producing 50% growth inhibition; Fl, feeding inhibition; GI, growth inhibition; MI, molt inhibition; IGR, insect growth regulator; MI, molt inhibition; IA, inactive.

Limonoid Test Insect Effective cone. Ref.

Group 1. Protolimonoids

Meliantriol

2 Schistocerca gregaria FI100=8 ug/cm 1

Melianone

Epilachna varivestris Fl §0.05% 2

Azedarachol m 3 Agrotis seietum FI100§500 PP Group 2. Intact apoeuphol skeleton limonoids

Azadiron

Epilacna varivestis FI50=0.66% 4

Azadiradione

Heliothis zea EC50=250 ppm 5

Heliothis virescens EC50=560 ppm 6

Spodoptera frugiperda EC50=130 ppm 5

Pectinophora gossypiella EC50=42 ppm 5

Epilacna varivestis FI50=0.033% 4

14-Epoxyazadiradione

Epilacna varivestis FI50=0.14% 4

7-Deacetylazadiradione

Heliothis zea EC50=3500 ppm 5

Heliothis virescens EC50=1600 ppm 6

Spodoptera frugiperda EC50=5000 ppm 5

Pectinophora gossypiella EC50=290 ppm 5

7-Deacetyl-17 -hydroxyazadiradione

Heliothis virescens EC50=240 ppm 6 160

Sendanal

Heliothis zea EC50=400 ppm 7

Heliothis virescens EC50=400 ppm 7

Spodoptera frugiperda EC50=70 ppm 7

Pectinophora gossypiella EC50= 200 pp 7

Diacetyoxyvilasinine Epilachna varivestris FI 8

Cedrelone Ostrinia nubilalis 50 ppm FI, GI 9 Spodoptera litura 0.1% FI 10

Spodoptera frugiperda EC5o=2ppm 5

Pectinophora gossypiella EC5Q=3ppm 5

Heliothis zea EC50=8ppm 5

Anthothecol Ostrinia nubilalis 50 ppm FI, GI 9

Spodoptera frugiperda EC50=3ppm 5

Pectinophora gossypiella EC5o=8ppm 5

Heliothis zea EC50=24ppm 5

Nimocinolide

Aedes aegypti IGR, LC50=0.625ppm 11

Isonimocinolide

Aedes aegypti IGR, LC50=0.74ppm 11

Sendanin

Heliothis zea EC50=45 ppm 7

Heliothis virescens EC50=60 ppm 7

Spodoptera frugiperda EC50=11 ppm 7

Pectinophora gossypiella EC50= 9 ppm 7

Trichirokanin

Heliothis zea EC50=41 ppm 7

Heliothis virescens EC5Q=50 ppm 7

Spodoptera frugiperda EC50=11 ppm 7

Pectinophora gossypiella EC50= 9 ppm 7

Toosendanin Ostrinia furnacalis 20 ppm FI 12

Trichilin A

Spodoptera eridania FI§ 300ppm 13

Trichilin B

Spodoptera eridania FI§ 200ppm 13

Trichilin C Spodoptera eridania inactive 13 Trichilin D Spodoptera eridania FI§ 400ppm 13

Meliatoxin A2

Spodoptera litura Fl, GI@ 300 ppm 14

Meliatoxin

Spodoptera litura GI@ 400 ppm 14

Group 3. D-ring seco limonoids

Gedunin Ostrinia nubilalis 50 ppm, DS 6 Epilachna varivestis 50%FI § 0.1% 4

Pectinophora gossypiella EC50=32 ppm 5

Spodoptera frugiperda EC5o=47 ppm 5

Heliothis zea EC50=50 ppm 5 7-Deacetylgedunin

Spodoptera frugiperda EC5o=60ppm 5

Pectinophora gossypiella EC50=22ppm 5

Heliothis zea EC50=165ppm 5

7-Ketogedunin

Spodoptera frugiperda EC50=800ppm 5

Pectinophora gossypiella EC5o=51ppm 5

Heliothis zea EC50=900ppm 5

Group 9. A.D-ring seco limonoids

Limonin Spodoptera litura 0.5% Fl 10 Spodoptera frugiperda EC50 = 756ppm 5

Spodoptera frugiperda PC95=6.12ug/disk 7

Heliothis zea EC50=900ppm 5

Heliothis zea PC9c=60.8ug/disc 7 Choristoneura fumiferana IA @ l,000ppm 15 Spodoptera exempta IA § lOOug/disc 16 Eldana saccharina 61%FI @l00ug/disc 16 Maruca testulalis 58%FI § lOud/disc 16 162

Nomilin Spodoptera frugiperda EC50 =72PPin 5

Spodoptera frugiperda PC95=0.66ug/disk 7 Spodoptera frugiperda Fl 17

Heliothis zea EC50=95ppm 5

Heliothis zea PC95=6.6ug/disk 7 Trichoplusia ni IA 17 Ostrinia nubilalis Fl § 50ppm 9

Earias insulana ED50=0.05% 18

Deacetylnomi1in Spodoptera frugiperda IA § 2000ppm 5 Heliothis zea IA § 2000ppm 5

Pectinophora gossypiella EC50=950ppni 5

Obacunone Ostrinia nubilalis 50 ppm Fl, GI 9 Spodoptera exempta 50%FI§100ug/disc 16 Eldana saccharina 79%FI§1 ug/disc 16 Maruca testulalis 61%FI@1 ug/disc 16

Spodoptera frugiperda EC50=70ppm 5

Spodoptera frugiperda PC50=0.60ug/disk 7

Heliothis zea EC50=97ppm 5

Heliothis zea PC95=6.5ug/disk 7

Rutaevin

Heliothis zea PC95=125ug/disc 7

Citrolin Eldana saccharina 55%FI§100ug/disc 16 Maruca testulalis 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 testulalis 69%FI§ lOug/disc 16 Spodoptera exempta 20 ppm Fl 19

12 -Acetoxyharrisonin Spodoptera exempta ,500 ppm Fl 19,20,7 Eldana saccharina <50%FI@100ug/disc 21 Maruca testulalis >75%FI§100ug/disc 21 Spodoptera exempta IA § lOOug/disc 21

Pedonin Eldana saccharina >75%FI@lug/disc 21 Maruca testulalis >75%FI@10ug/disc 21 Spodoptera exempta <50%FI§100ug/disc 21 163

Groups 4,5. BfD-ring seco limonoids

Methylangolensate

Heliothis zea ED50=60 ppm 5

Spodoptera frugiperda ED50=40 ppm 5

Pectinophora gossypiella ED50=15 ppm 5

Entandrophragmin Ostrinia nubilalis FI§500 ppm,DS@50 ppm 9

Bussein Ostrinia nubilalis FI§500 ppm 9

Methyl 3 -isobutyryloxy-l-oxomeliac-8(30)-enate Spodoptera frugiperda FI 22

Group 6. A-ring seco limonoids

Evodulone

Heliothis zea EC50=80 ppm 5

Spodoptera frugiperda EC5o=120 ppm 5

Pectinophora gossypiella EC50=96 ppm 5 Tecleanine

Spodoptera frugiperda EC50=320 ppm 5

Pectinophora gossypiella EC50=210 ppm 5

7-Deacetylproceranone

Heliothis zea EC50=740 ppm 5

Spodoptera frugiperda EC50=350 ppm 5

Pectinophora gossypiella EC50=175 ppm 5

Group 7. AfB-ring seco limonoids

Prieurianin Heliothis zea 60-90%FI§6ug/cm2 23 Spodoptera frugiperda IA §19.8ug/cm2 23 Epilachna varivestis 60-90%FI§19.8ug/cm2 23 Prieurianin acetate Heliothis zea 60-90%FI § 6ug/cm2 23 Spodoptera frugiperda 60-90%FI @19.8ug/cm2 23 Epilachna varivestis 60-90%FI @1.5ug/cm2 23

Rohitiukin Heliothis zea IA @19.8ug/cm2 23 Spodoptera frugiperda IA §19.8ug/cm2 23 Epilachna varivestis IA §19.8ug/cm2 23 164

Rohitiuka-7 Heliothis zea 60-90%FI ei9.8ug/cm2 23 Spodoptera frugiperda IA §19.8ug/cm2 23 Epilachna varivestis IA §19.8ug/cm2 23 "Tr—A" Agrotis sejetum FI § 200 ppm 24

"Tr-B" Agrotis sejetum FI @ 200 ppm 24

"Tr-C" Agrotis seietum FI @ 200 ppm 24

Group 10. B-ring seco limonoids

Toonacilin

Epilachna varivestis FI 25

6-Acetyoxytoonaci1in Epilachna varivestis FI 25

21-(R,S)-hydroxytoonacilid Epilachna varivestis FI§ 25-50 ppm

2 3-(R,S)-hydroxytoonac i1id Epilachna varivestis FI§ 2000 ppm

Group 8. C-ring seco limonoids

Azadirachtin

Epilachna varivestis FI5o=0.0014% 4

Epilachna varivestis MI5o=1.66 ppm 26

Heliothis zea EC50=0.7 ppm 5

Spodoptera frugiperda EC50=0.4 ppm 5

Pectinophora gossypiella EC50=0.4 ppm 5

7-Acetylazadirachtin

Rhodnius prolixus MI50=0.45ug/ml 27

Rhodnius prolixus FI50=30.Oug/ml 27

22,23-dihydro-23B-methoxyazadirachtin Epilachna varivestis IGR 8 Mythimna separata FI,IGR@ 0.01%, 0.1/ig/larva 28

3-Tigloylazadirachtol (=Azadirachtin B) (=Deacetylazadirachtinol)

Epilachna varivestis MI5Q=1.30 ppm 26

Heliothis virescens ED50=0.17 ppm 5 165

l-Cinnamoyl-3-feruloyl-ll-hydroxymeliacarpin (=Azadirachtin D) Epilachna varivestis IGR 8

Epilachna varivestis MI50=1.57 ppm 26 l-Cinnamoyl-3-feruloyl-ll-hydroxy-22,23-dihydro-23- methoxymeliacarpin Epilachna varivestis IGR 8 l-Tigloyl-ll-methoxy-20-acetylmeliacarpinin Epilachna varivestis IGR 8 l-Tigloyl-3-acetyl-ll-methoxyazadirachtinin Epilachna varivestis IGR 8

Azadirachtin C

Epilachna varivestis MI50=1 .57 ppm 26

Azadirachtin F

Epilachna varivestis MI50=1 .57 ppm 26

Azadirachtin G

Epilachna varivestis MI50=1 .57 ppm 26

Nimbinen 0 Epilachna varivestis FI50= .018% 4

6-Deacetylnimbinen

Epilachna varivestis FI50=0 .0082% 4

Nimbandiol

Epilachna varivestis FI50=0 .01% 4

6-Acetylnimbandiol

Epilachna varivestis FI50=0 .011% 4

Salannin Spodoptera fruqiperda 50%FI@ 13ug/cm2 29 Spodoptera littoralis 0.01% Fl 30 Earias insulana 0.01% Fl 30 Acalymma vittata Fl 31 Musca domestica 100%[email protected]% 32

Epilachna varivestis FI50=0.0082% 4 Epilachna varivestis no MI§ 100 ppm 26

3-Deacetylsalannin

Epilachna varivestis FI50=0 .0027% 4

Salannol Epilachna varivestis Fl 8

Salannolacetate

Epilachna varivestis FI50=0 .00085% 4 166

Salannolactame-(21) Epilachna varivestis FI 8

Salannolactame-(23) Epilachna varivestis FI 8

Ochinolide B

Heliothis virescens EC50=1500 ppm 7

Spodoptera frugiperda EC50=600 ppm 7

Pectinophora gossypiella EC50= 700 ppm 7

Ochinal Heliothis zea IA § 1000 ppm 7 Heliothis virescens IA @ 1000 ppm 7 Spodoptera frugiperda IA § 1000 ppm 7

Pectinophora gossypiella EC50= 1800 ppm 7

Volkensin Spodoptera frugiperda 50%[email protected]/cm2 29

Volkensin hydroxylactone Spodoptera frugiperda 50%FI@6ug/cm2 29

Unknown structure

Nkolbisonin

Heliothis zea EC50=71 ppm 5

Spodoptera frugiperda EC50=65 ppm 5

Pectinophora gossypiella EC50=20 ppm 5

1) Lavie et al., 1967. 2) Kraus and Grimminger, 1980. 3) Nakatani gt al., 198 4) Schwinger gt al. , 1984. 5) Kubo and Klocke, 1986. 6) Lee gt al., 1988. 7) Kubo and Klocke, 1981. 8) Kraus gt al., 1987. 9) Arnason gt al., 1987. 10) Koul, 1983. 11) Naqui, 1987. 12) Chiu and Zhang, 1984. 13) Nakatani et al., 1981. 14) Koul gt al., unpublished data (1989). 15) Alford and Bentley, 1986. 16) Hassanali gt al., 1986. 17) Altieri gt al., 1984. 18) Weissenburg gt al., 1986. 19) Kubo etal-, 1976. 20) Liu gt al., 1981. 21) Hassanali and Bentley, 1987. 22) Mikolajczak gt al., 1988. 23) Lidert gt al«, 1985. 167

24) Nakatani et al., 1984. 25) Kraus et al., 1978. 26) Rembold, 1988. 27) Garcia et al., 1984. 28) Sankaram et ai-, 1987. 29) Rajab et ai., 1988. 30) Meisner et al. , 1981. 31) Reed et al., 1981. 32) Warthen et al., 1978. 168

Group 1: Proiolimoaoidi

Meliaaoae

Croup 2: Apo-Esphol LiaoBokU

H 7-DeaccryIaxadiradioM OH 7-Deac«yl-17- hydrexjrazadindioM

OAc Azadiradione

or >OJU 14-epoxyazadiradioaa I Orriunia

"A 12 -OH O Trichilia A F>Ac Srnriania 12 -OH O Trichilia B R-COCHCHJCHJ Trichirokaaia

Figure 4-1. Structures of limonoids included in Table 3 169 170

Tecleanin 7-DeacetyIproceranone Evodulone

OAe

CH2CH3 Tr-A" CH3 Tr-C"

"O o Rohiiuka-7 H Toonacilin OAc 6-AcetoxytoonaciIin

Ac Prierianin accute H Prieriania 171

oco

Eniandrophragmin 172

OR R=Ac Nimbinen R=H 6-Deacetylnimbinen

R=Ac Salannin Salannolactame-(21) R=H 3-Deacetylsalannin O JO

Sallanolactame-(23)

R=H Salannol R=Ac Salannolacetate

OTig R = OH Volkensin '—6 Ochinolide B R = O Volkensin hydroxylactone

R,0

R,0^>^>X"'OH

Ri R2 R3 R4 Rs Ac Tig COOCH3 H H Azadirachtin Ac Tig COOCH3 2H OCH3/H Tig H COOCH3 H H 3-Tigloylazadirachtol

Fer Cin CH3 H H l-Cinnamoyl-3-feruloy 1-11 -hydroxy meliacarpin Fer Cin CH3 2H OCH3/H 173

Figure 4-2. Major biosynthetic routes of limonoids in 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 al.,

1986, 1987) were provided by Dr. A. Hassanali, ICIPE,

Nairobi. The purity of all compounds was assessed by HPLC, and compound identity and purity were also confirmed by 1H-

NMR (400 MHz). Structures of all compounds are shown in

Figure 4-3.

In many of the experiments the azadirachtin used was isolated by Dr. J. Kaminski, University of Ottawa. However, initial experiments were done with azadirachtin isolated according to an adaptation of the methods of Uebel gt al

(1979) and Yamasaki gt a_l. (1986). Azadirachta indica oil

(100 ml) (also supplied by Dr. J.T. Arnason) was diluted to

250 ml in MeOH, defatted with three partitionings against equal volumes of hexane, and then extracted three times with

equivalent volumes of Et20. The Et20 phase was concentrated under vacuum, and chromatographed in 0.5 g lots by spinning- plate preparative TLC (Chromatotron, Model 7924, Harrison

Research, Palo Alto, California) using a 2mm plate (Si gel

60 PF254, Merck). The solvent system consisted of 200 mis

each of Et20, Et20:Acetone (95:5), Et20:Acetone (3:1),

Et20:Acetone (1:1), and finally 100% MeOH; flow was 4 mis/ 176

Figure 4-3. Structures of limonoids examined in this study. 177

BUSSEIN OCOCHMe, AZADIRACHTIN 178

min., and 8 ml fractions were collected. Fractions were monitored for the presence of azadirachtin by analytical

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 isocratic ACHN:H20 67:33, flow = 3 ml/ min; 6 ml fractions were collected. The identity and purity of the azadirachtin so obtained was established by co- chromatography (analytical HPLC) and1 H-NMR spectroscopy.

The yield of azadirachtin from this oil sample was quite low, about 1 mg/ 100 ml.

Solvents were purchased from BDH and all except MeOH were redistilled proir to use. HPLC grade solvents were degassed prior 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 roll, and cotton was provided as an oviposition site. 179

C. Growth Studies

For growth studies, compounds in methanol were added to the dry components of the artificial diet (Velvetbean caterpillar diet, Bioserve Inc., Frenchtown, N.J. # 9682), the solvent was evaporated in a fume hood, and the diet was prepared in the usual manner. All compounds except azadirachtin were assayed at 0.01, 0.05, and 0.5 nmol/ q diet fwt. Two grams of diet and 10 neonate £. saucia were placed in each of three 30 ml plastic cups per treatment; this design made the most efficient use of scarce supplies of most of the compounds, and preliminary experiments indicated that both larval growth and survivorship up to seven days were independent of larval density. The entire bioassay was repeated three times except in the case of nomilin, where limited amounts of compound allowed only two replications. Larvae were reared under 16L:8D and 26° C, in a clear plastic box lined with moistened paper towels to maintain high humidity. After seven days surviving larvae

were counted and weighed. Live larval weights were log10 transformed to correct for heteroscedasticity before analysis by ANOVA and Tukey's Studentized (HSD) Range Test.

The effect of azadirachtin on growth and survival of neonate E- saucia was assessed in two experiments. In the first, neonate E- saucia were reared for seven days on artificial diet containing 0.5, 1.5, 4.5, 15, or 45 nmol/g diet fwt (0.36, 1.08, 3.24, 10.8, and 32.4 fxq/q diet fwt). 180

Control diet was treated with solvent (MeOH) alone. Larvae were reared, three to a cup (10 cups/ treatment) in 30 ml cups with 2 g fwt diet, as previously described. Surviving

larvae were counted and weighed after seven days. The experiment was replicated three times; larval weights were recalculated as % of control and loglO transformed prior to analysis by linear regression against loglO dose. In the second experiment, neonate larvae (30 per treatment) were reared on diet containing 0, 0.15, 0.5, or 1.5 nmol azadirachtin/g fwt diet (0, 0.11, 0.36, or 1.08 ng/g respectively). Conditions were the same as in the first experiment, except that larvae were reared individually after the first seven days, as they tend to become cannibalistic in later instars. Larvae were checked every

2-3 days and fresh diet was provided ad lib. Sixth-instar larvae were transferred to plastic cups containing sterile moistened soil to facilitate pupation. At this point larvae were checked for pupation every two days. Parameters measured included % of larvae pupating, time to pupation, pupal weights, and % adult emergence.

D. Feeding Assays

Antifeedant effects were examined using a simple choice test. Petri dishes (5 cm diameter) were marked into quadrants, and control and treated diet cubes were placed in alternating quadrants. Ten neonate £• saucia were released in the center of this feeding arena; the dishes were then 181

put into an opaque box to eliminate phototactic effects.

The number of larvae on the diet and in each quadrant was determined after 24 h. Feeding was confirmed by the presence of frass. Azadirachtin was tested at 0.5, 1.5, and

4.5 nmol/g diet fwt; other compounds were tested at 0.5

/imol/ g diet, and the assay was replicated six times.

Responses were compared to a random distribution of larvae, expected in the absence of antifeedant effects, using a G- test.

E. Nutritional Analyses

Growth, consumption, and dietary use by third instar £. saucia larvae (starting weight, 11.2 + 0.9 mg, n=20/ treatment) were determined. Larvae were allowed to feed

(individually) on about 1 g fwt of diet containing 0, 0.15,

0.5, or 1.5 nmol azadirachtin/g diet fwt. After three days the larvae, frass, and uneaten diet were separated and dried to constant weight at 60° C. Initial dry weights of the larvae and diets were calculated from fwt/dwt ratios derived by determining the fwt/dwt ratio of 10 larvae and 5 diet aliquots at the start of the experiment. Relative growth rate (RGRi) and relative consumption rate (RCRi) were calculated relative to the initial rather than the mean weight of the larvae, as this measure is independent of the

ECI (Farrar et al., 1989). The efficiency of conversion of ingested (ECI) and digested (ECD) diet, and the approximate 182

digestibility (AD), were calculated according to Reese and

Beck (1976).

RGRi = (Final larval weight - Initial larval weight /D Initial larval weight

RCRi = Weight diet consumed /D Initial larval weight

ECI = (Final larval weight - initial larval weight) X 100 Weight diet consumed

ECD = (Final - initial larval weight) X 100 (Weight diet consumed - frass)

AD = (Weight diet consumed - frass) X 100 Weight diet consumed

F. Molt Inhibition Assays

The effect of limonoids on molting was assessed by topically applying compounds in acetone to the dorsal abdominal tergae of staged (< 24 h) fifth instar Q_. fasciatus nymphs. Each compound was tested twice (10 nymphs per replicate) 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 application of the test compound, nymphs were maintained in

10 cm diameter glass petri dishes at 27° C, 16L:8D, and provided with dry seeds of Asclepias speciosa and a 183

moistened cotton roll. 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 inability to completely shed the exuvia), category III was mortality during a molt attempt, and category IV was death without initiating 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 mortalities. Following the molt, adults were maintained as described for the nymphs, but with cotton as an oviposition site, for a further two weeks and the occurrence of mating behavior, eggs, and neonates was recorded.

G. Correlation Between Evolutionary Advancement and Activity

Against Insects

EC50 values (in /imol/g diet fwt) of all limonoids tested here were correlated with measurements of molecular oxidation (O) and rearrangement (S) given by Das et al.

(1984, 1987). The same analysis was also applied to EC50 values for 17 limonoids and three lepidopteran species given

by Kubo and Klocke (1987). To eliminate EC50 differences due to differences in the molecular weight of compounds, all

EC50 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 inhibitory to neonate E- saucia growth. Both compounds reduced growth to about 10% of control growth when incorporated into diet at 0.5 /mol/g diet fwt (Table 4-2); neither compound had any effect at

0.05 /xmol/g. (Control larvae mean weights were 45-70 mg after seven days.) Survivorship was not affected at 0.5 rtmol/g. The growth inhibition was apparently not due to a chemosensory antifeedant effect as neither compound significantly influenced neonate diet choice in a 24 h assay

(Table 4-2).

Gedunin, similar to cedrelone except for an epoxylactone D ring, had no effect on growth, survivorship, or feeding at 0.5 /imol/g. The A,D-seco limonoids obacunone and nomilin also did not affect diet choice or growth of E- saucia. Harrisonin had an antifeedant effect against neonate larvae, but this must have been temporary as the larval weights did not differ significantly 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 significantly more than the controls by day seven. 185

Table 4-2. Effect of limonoids on growth and diet choice of neonate Peridroma saucia. All compounds were administered at 0.5 /xraol/g diet fwt; concentrations in ppm are also given. Larval growth is given as % of control growth; numbers followed by the same letter are not significantly different (Tukey's Studentized [HSD] Range Test). In the choice tests, larval distribution is compared to a null hypothesis distribution of 50:50 using a G- test; distributions differing from 50:50 are indicated by an asterisk.

Compound Cone. Growth Percentage of larvae on (% of Control) Control (C) or Treated (T) diet. (ppm) (mean + S.D.) C T

Cedrelone 211 10.8 + 3.2d 57 43

Anthothecol 248 12.9 + 7.5d 57 43

Harrisonin 259 89.2 + 22.6b 80 20*

Obacunone 227 110.8 + 11. 8b 61 39

Nomilin 265 118.3 + 18.3b 63 37

Gedunin 249 96.8 + 23.7b 43 57

Pedonin 250 133.3 + 8.6a 63 37

Entandrophragmin 416 96.8 + 18.3b 66 34

Bussein 420 64.5 + 19.5°- 67 33 186

Of the B,D-seco limonoids tested, entandrophragmin did not affect P_. saucia feeding or growth at 0.5 jzmol/g diet fwt. Bussein significantly reduced larval growth (to 64.5% of the controls), although this effect was much less marked than was the case with cedrelone or anthothecol. Bussein did not significantly deter feeding by neonates in the choice test.

B. Growth Studies with Azadirachtin

Of the limonoids examined in this study, azadirachtin produced the greatest inhibition of growth and survivorship; as a result its effect on E. saucia growth was examined in more detail than were other compounds. In the seven-day assay, both growth and survivorship were reduced in a dose-

dependent manner (Figure 4-7); the EC50 was 0.4 nmol/g diet

fwt (0.29 M9/g diet fwt) and the LC50 was 5.2 nmol/g diet fwt (3.7 /ig/g). Although a few larvae were seen with slipped head capsules or a half-shed band of cuticle, in most cases mortality was not obviously due to molt failure.

These effects continued throughout the life cycle of the insect. Regression analysis indicated a significant negative effect of azadirachtin (at 0.15, 0.5 and 1.5 nmol/g diet fwt) on the % of larvae pupating (r2=0.94, p< 0.01), and on pupal weight (r2=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. Effect of dietary azadirachtin on growth and survivorship of Peridroma saucia neonates. Growth and survivorship were determined after seven days of feeding on azadirachtin-treated diet; each point represents the mean of three replications, normalized to % of control. 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 in a dose-dependent manner (r2=0.83, p< 0.05), with controls requiring 24.8 ± 1.9 days for development, and larvae fed diet with 0.5 nmol/g azadirachtin requiring 30.2 days. None of the larvae reared on diet 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 diet, compared to 69% emergence for the

controls. The data indicate an EC50 for pupation of 0.16 nmol/g.

C. Feeding Choice Tests with Azadirachtin

In choice tests, azadirachtin inhibited feeding by neonate P.. saucia (Table 4-4). After only one hour there were significantly more larvae on control diet than on diet treated with 1.5 or 4.5 nmol azadirachtin/g fwt. After 24 h neonates were significantly deterred by 0.5 nmol azadirachtin/g diet as well.

As the sensitivity of larvae to antifeedants is known to decline with age in some cases (Reese, 1977; Isman and

Duffey, 1982; Isman et al., 1989), these choice tests were repeated with 6 day old second instar P. saucia larvae, which had not previously been exposed to azadirachtin.

After 24 h, larvae were deterred by azadirachtin only at the

4.5 nmol/g level; at 1.5 nmol/g there was no preference for either treated or control diet. 191

Table 4-4. Effect of azadirachtin on diet choice by neonate and six day old, third instar Peridroma saucia. Values which differ from a 50:50 distribution (G-test) are indicated with an asterisk.

Azadirachtin % of larvae on control [C] or concentration treated [T] diet after 24 h Neonates Third Instar fnmol/a diet 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 Utilization Experiments

In a detailed study of growth, feeding, and dietary utilization, azadirachtin reduced the relative growth rate

(RGRi) of third-instar p_. saucia in a dose-dependent manner

(r2=0.90, p<0.01) (Table 4-5). This effect was largely owing to a dose-dependent decrease in the relative consumption rate (RCRi) (r2=.85, p<0.05). Effects on efficiency of conversion of ingested and digested food (ECI and ECD respectively) were not significantly correlated with azadirachtin dose; subsequent ANOVA and means-comparison analysis (Tukey's Studentized Range Test) indicated that both indices were significantly lower than the controls only at the highest azadirachtin dose tested. The approximate digestibility (AD) was also not correlated with the azadirachtin dose, but was significantly higher than the control at 1.5 nmol azadirachtin/g.

Analysis of the relationship between RCRi and RGRi showed that all treatment groups fit a regression line of

RGRi = 3.244*RCRi + 4.450 r2=0.85, df=2, p<.05 although there were between-treatment differences in the maximum values for RCRi (Figure 4-8). These results indicate that, in P. saucia, the observed growth inhibition is due to reduction in feeding and not to direct toxicity

(cf. Blauetal., 1978). 193

Table 4-5. Effect of azadirachtin on third instar Peridroma saucia growth and nutrition. Means in a column with the same letter are not significantly different (Tukey's Studentized Range (HSD) Test, a=0.05). An asterisk following a column heading indicates that index has a significant overall negative relationship with azadirachtin dose.

Treat RGRi* RCRi* ECI ECD AD Control 2.90 10.53 27.65a 59.16a 46.74a 0.15 2.42 10.20 23.74a 56.65a 41.91a 0.5 1.44 5.77 24.89a 60.77a 40.96a 1.5 0.45 3.76 12.08^ 20.13^ 60.00^ Treat=treatment (/ig/g insect fwt), RGRi=Relative Growth Rate based on initial wgt, RCRi=Relative Consumption Rate based on initial wgt, ECI=Efficiency of Conversion of Ingested Food, ECD=Efficiency of Conversion of Digested Food, and AD=Approximate Digestibility. Values were calculated according to Reese and Beck (1976) except for the RGRi and RCRi, which were calculated according to Farrar e_£ al. (1989). 194

Figure 4-5. Plot of RGR against RCR for larvae of Peridroma saucia fed diet containing various concentrations of azadirachtin. For clarity, only every second data point is ploted for each treatment. All treatments do not differ significantly from the regression equation:

RGRi = 3.244*RCRi + 4.450 r2 = 0.85 p<0.05 20

a Control • 0.5 nmol/g 15 - • 1.5 nmol/g • 4.5 nmol/.g tr 10 o

r— —i— —I— 1 0 2 0 30 40 RCRI 196

E. Molt Inhibition Assays

At 50 ug/nymph, only azadirachtin and cedrelone had any effect on molting of fifth instar O. fasciatus. Tests with cedrelone were repeated at 5, 10, 15, 25, and 50 ^g cedrelone/nymph; these indicated a dose-dependent range of effects which were divided into response categories as described previously. Cedrelone doses as low as five

/ig/nymph had a marked effect on molting success (Figure 4-

5); at this dose most nymphs molted to adults with curled wings (category II), but some died after initiating ecdysis

(category III). At 10 and 15 pg cedrelone/nymph, an increasing proportion of the treated nymphs showed category

III and IV responses; at 25 /ig/nymph, all responses fell 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 in a failed molt attempt. If all category I and II responses are pooled as survivors, and all category III and IV responses are

considered as mortalities, the MD50 (dose producing molt inhibition in 50% of the treated nymphs) of topically applied cedrelone was 12.24 ng/nywph (95% fiducial limits

9.29-16.09 jig/nymph).

Azadirachtin was about five thousand times more active than cedrelone in the milkweed bug assay (Figure 4-6), but the same spectrum of effects was produced. Even at 1 ng/nymph, most larvae showed a category II response, and a 197

Figure 4-6. Effect of cedrelone on molting success in Oncopeltus fasciatus. Responses were scored as: Category I, normal molt to an undeformed adult; Category II, sucessful molt to a deformed adult (curled wings or legs); Category III, death during a failed molt attempt; Category IV, death without initiating ecdysis. Category I HI Category Ii III Category III Category IV % of treated population in category

tegjamwmw III 1II1I II11I if 11 sit 1 ;M Control 25 50 cedrelone dose ug/nymph 199

Figure 4-7. Effect of azadirachtin on molting success in Oncopeltus fasciatus. Responses were scored as: Category I, normal molt to an undeformed adult; Category II, sucessful molt to a deformed adult (curled wings or legs); Category III, death during a failed molt attempt; Category IV, death without initiating ecdysis. HH 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

N5 O Azadirachtin dose ng/ nymph O 201

few died with and without initiating ecdysis. At 10

/xg/nymph, no normal molts were recorded, and most nymphs died without initiating ecdysis. Again pooling; category I

and II responses, the MD50 of azadirachtin was 3.0 ng/nymph.

To check against the possibility that the results 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 in the cedrelone sample.

Adults obtained from the molt inhibition assays were maintained for two weeks to check for possible long-term effects on fecundity and fertility. Adults which had been treated with cedrelone and azadirachtin died within 48 h of molting without mating. Adults from all other treatments began mating 3-4 days after molting, and by the end of the observation period all treatments had produced viable eggs, indicated by the presence of first-instar nymphs in the rearing arenas.

F. Relationship of Anti-insect Activity to Oxidation and

Skeletal Rearrangement

No relationship was found between the degree of biological activity of the limonoids studied herein (in terms of growth inhibition against £. saucia) and the degree of oxidation or rearrangement of the original apo-euphol skeleton, as defined by Das gt al (1984). However, only four compounds 202

exhibited appreciable bioactivity, an inadequate sample for comparison. The analysis was therefore extended to the

comparative EC50 data for seventeen limonoids and three

lepidopterans published by Kubo and Klocke (1986). All EC50 values were recalculated in terms of nmol/g diet before comparison by regression analysis to measurements of oxidation and skeletal rearrangement given by Das et al

(1984). Separate regressions were performed using EC50 data from each lepidopteran species. None of these comparisons indicated a significant relationship between the growth inhibitory activity 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 least (cedrelone) derivation from the original apo- euphol skeleton. Compounds with intermediate degrees of rearrangement and oxidation appeared to be the least active. 203

Figure 4-8. Comparison of insect growth inhibiting activity of limonoids with measurements of oxidation and skeletal rearrangement. The values for S and O used are from Das et al (1984); the values for insect growth

inhibition are ED50 data for Spodoptera frugiperda, given by Kubo and Klocke (1987), corrected to /imol/g diet and log transformed. Neither S nor O are significantly related to the inhibitory activity of the limonoids. Log ED50 (S. frugiperda) Log ED50 (S. frugiperda) 205

Discussion

For purposes of discussion, compounds are grouped according to skeletal class as given in Fig. 4-2.

A. Group 2 Limonoids

Cedrelone and anthothecol are simple limonoids which retain an intact apoeuphol skeleton (Fig. 4-3). Cedrelone occurs in a variety of Meliaceae including species of

Cedrela, Chuckrasia, Khaya, and Toona; anthothecol differs only in possessing an acetate substitution at Cll of the C ring, and is found in species of Khaya (Das et al., 1984).

Neither compound affected £. saucia growth at 0.05 /imol/ g diet, but at 0.5 /imol/ g diet both cedrelone and anthothecol reduced growth by almost 90% compared to the controls. This effect was evidently not due to a chemosensory antifeedant effect as neither compound had any effect on diet choice by neonate E. saucia in the antifeedant assays.

Cedrelone and anthothecol were also highly effective growth inhibitors against the European corn borer, Ostrinia nubilalis (Arnason et ajL. , 1987). This activity included feeding inhibition at high doses, 50 and 500 ppm. Both limonoids reduced growth by reducing the efficiency of conversion of ingested and digested diet at lower doses at which consumption was actually enhanced (10 and 30 ppm), a result which parallels my observation of growth inhibition 206

without antifeedant effects in E- saucia. In another study, cedrelone and anthothecol were the most inhibitory of 17 limonoids (other than azadirachtin), reducing the growth of three lepidopterans, Spodoptera frugiperda. Heliothis zea, and Pectinophora gossypiella (Kubo and Klocke, 1986).

Cedrelone also had growth inhibitory activity against

Spodoptera litura at 0.1%; in this case the observed inhibition could be entirely ascribed to reduced consumption, due either to behavioral or physiological effects (Koul, 1983). R. saucia appears to be relatively insensitive to cedrelone and anthothecol, as growth

inhibition occurred only at doses 100 times the EC50 levels reported by Kubo and Klocke (1986) and 10 times the inhibitory concentrations for Ostrinia nubilalis (Arnason et ai. , 1987).

A marked difference was noted in the response of 0. fasciatus nymphs to the two compounds. Cedrelone affected

molting at doses as low as 5 ug/nymph, and had an LD50 of

16.4 ug/nymph. Nymphs which molted successfully at the lower doses all exhibited deformities of the wings and died within 48 h of molting, without mating. These effects were similar to those resulting from much lower doses of

azadirachtin (LD50 = 3.0 ng/nymph). Although cedrelone was not observed to inhibit the molting of either P. saucia

(this study) or of O. nubilalis (Arnason et ai., 1987),

Spodoptera frugiperda, or Heliothis zea (Kubo and Klocke,

1986), it has been reported to inhibit ecdysis in the pink 207

bollworm Pectinophora gossypiella at 150 ppm in diet (Kubo and Klocke, 1986). The acetate substitution at Cll of the C ring abolished the molt-inhibiting activity, as nymphs treated topically 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 ring are known to markedly affect the antifeedant activity of limonoids. For example, the antifeedant activity of vilasinine derivatives is reduced by substituents at C12

(Pohnl, 1985). Similarly, acetylation at C12 reduces the antifeedant activity of harrisonin 25-fold (Kubo et al.,

1976). Trichilins require an OH substitution at C12; alteration to a ketone or acetate markedly reduce the antifeedant activity (Nakatani gt al., 1981) The results reported here suggest that substitutions at the Cll position may also be important in determining IGR activity. These results also indicate that even simple limonoids can have marked physiological effects, and cannot be considered

"harmless deterrents" (sensu Bernays and Graham, 1988).

B. D-seco Limonoids

Other comparisons suggest the importance of the epoxide substitution on an intact D ring. Cedrelone and anthothecol, the most active of the limonoids here tested, have such a substitution. Gedunin, which differs in that the D ring is oxidized to a ^-lactone, as well as having 208

different substitutions on a saturated B ring, lacks antifeedant or IGR activity against £. saucia and Q_. fasciatus. Gedunin was also much less active than cedrelone or anthothecol against O. nubilalis (Arnason e_£ al., 1987),

P. gossypiella. S_. frugiperda, and H. zea (Kubo and Klocke,

1987). This reduced activity is not due to saturation in the B ring since nimocinol, comparable to cedrelone except for the B ring saturation, has IGR, antifertility, and molt inhibiting activity against Musca domestica (Siddiqui et al. , 1988) and Aedes aegypti (Naqui, 1987). B ring substituents can, however, affect antifeedant activity as 7- deacetylgedunin and 7-ketogedunin are both less active than gedunin (Kubo and Klocke, 1987). Overall it seems clear that oxidation of the D ring epoxide to an epoxylactone results in loss of IGR activity and a marked decrease in antifeedant activity. Nevertheless this type of oxidation is characteristic of all known rutaceous and simaroubaceous limonoids, and is typical of most meliaceous limonoids as well (Das et al., 1984).

C. AfD-seco Limonoids

In addition to their characteristic D ring structure, typically limonoids from the Rutaceae and Simaroubaceae have undergone Bayer-Villager oxidation of the A ring (A seco- limonoids) (Dreyer, 1983). None of the A,D-seco limonoids tested here (obacunone, nomilin, harrisonin, and pedonin) produced IGR or antifertility effects in O. fasciatus. 209

Harrisonin had antifeedant activity in a choice test against neonate P. saucia, but did not significantly affect larval growth in a seven-day no-choice bioassay. The antifeedant activity of harrisonin is known to vary widely between species. Hassanali et al (1986) found it to be deterrent to

Eldana saccharina and Maruca testulalis at (respectively) 1 and 10 ug/cm2 disc, but it had only marginal feeding inhibition against Spodoptera exempta at 100 ug/disc.

Obacunone and nomilin had no antifeedant or IGR activity against £. saucia at concentrations up to 0.5 /nmol/g fwt.

Again, these compounds are feeding deterrents at low concentrations for some insects, including JSj. saccharina and

M« testulalis (Hassanali et al-, 1986), have only moderate activity against others, including Q. nubilalis (Arnason et al. , 1987), £5. frugiperda, and H. zea (Kubo and Klocke,

1986), and are inactive against yet other species including

Trichoplusia ni (Altieri et ai., 1984). Similar Citrus limonoids were also inactive against the spruce budworm,

Choristoneura fumiferana (Alford and Bentley, 1985).

In the related compound pedonin, the D ring has been opened to a y-ketofuran, and the resulting carbonyl group methylated to a carbomethoxy group; as well the A ring has undergone 1,2 skeletal 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. fasciatus. This compound was also inactive against the polyphagous £3. exempta, but was a strong antifeedant to the 210

more oligophagous M.- testulalis and E_. saccharina (Hassanali and Bentley, 1987).

D. B,D-seco Limonoids

Within the Swieteniodeae, D-seco limonoids may be further oxidized in the B ring; this pathway produces a greater variety of limonoids than any other (Das et al., 1985;

Connolly, 1983). Bussein and entandrophragmin are typical of this group of B,D ring seco limonoids, and are characteristic particularly of species of Khaya and

Entandrophraoma (Taylor, 1988). Entandrophragmin had no effect on either E. saucia or O. fasciatus. Bussein reduced the growth of E« saucia neonates, but was much less active than either cedrelone or anthothecol. The growth inhibition could not be ascribed to an antifeedant effect, as bussein did not have a significant effect on diet choice. No IGR effects were observed in the Q. fasciatus assays.

In the only other study to examine the effects of entandrophragmin and bussein, both reduced feeding and survivorship of Q. nubilalis at 500 ppm in diet (Arnason gt al.. 1987), slightly higher than the maximum concentration used in my assays. Several B-seco limonoids are known to have antifeedant activity (Table 3-1), although this can vary widely with relatively minor changes in structure.

Although prieurianin and its acetate had antifeedant

activity against H.. zeaf S_. frugiperda (acetate only), and

E. varivestis. the related rohitiukin and rohituka-7, which 211

differ in that the B ring carboxyl fragment is cyclized with

C29 to form a new lactone ring, were inactive (Lidert et al., 1985). Three compounds related to pierianin are known to have antifeedant activity against Agrotis seietum

(Nakatani et al., 1984). Toonacilin and 6- acetoxytoonacilin, which are similar to cedrelone except for an oxidatively cleaved B ring, deter feeding by E_. varivestis (Kraus et al., 1978).

E. C-seco Limonoids (Azadirachtin)

Azadirachtin had the most marked effect on £. saucia growth and feeding of any of the limonoids tested in this study, and was the only compound to significantly reduce survivorship. Choice tests with neonate larvae indicated that an antifeedant effect may be an important component of the observed growth inhibition, as these larvae avoid diet treated with azadirachtin concentrations as low as 0.5 nmol/g fwt. Although insects are able to learn aversion to toxic diets (Dethier, 1980), and can select nutritionally optimal diets based on changes in brain serotonin (Cohen et al., 1988) or catecholamine neurotransmitters (Wurtman,

1981), the rapidity of the response to azadirachtin

(significant after only 1 h) suggests a chemosensory basis for the avoidance. A "deterrent receptor", sensitive to azadirachtin, is present in a number of oligophagous and polyphagous lepidopterans (Simmonds and Blaney, 1984;

Schoonhoven and Jermy, 1977). This receptor does not 212

interfere with the activity of other nutrient receptors, but rather alters the central nervous processing of chemosensory information (Simmonds and Blaney, 1984).

Sensitivity to the antifeedant activity of azadirachtin declined markedly by the third instar. Ostrinia nubilalis larvae also show a decline in the deterrent activity of azadirachtin between the first and the third instar (Arnason et al., 1985). The relatively greater sensitivity of neonate caterpillars to secondary metabolites has been noted previously (Reese, 1973), and includes sensitivity to phenolics (Isman and Duffey, 1982), polyacetylenes and thiophenes (Champagne et aJL., 1986) and sesquiterpene lactones (Isman et al., 1989).

The growth inhibiting activity of azadirachtin continued throughout the larval development of E. saucia, despite the decrease in antifeedant activity. While the effect of azadirachtin concentration on pupal weight was significant, the extent of inhibition 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 biologically significant long-term effect of azadirachtin is a marked decrease in pupation and adult emergence. Similar results have been noted with Heliothis zea (Barnby and

Klocke, 1987), Ostrinia nubilalis (Arnason et al., 1985), the lepidopteran rice pests Mythimna separata and 213

Cnaphalocrocis medinialis (Schmutterrer et ai., 1983) and the face fly Musca autumnalis (Gaaboub and Hayes, 1984).

F Nutritional Indices

Azadirachtin markedly reduced growth and consumption in third-instar E. saucia at dietary concentrations which did not affect the ECI, ECD, or AD. The reduced consumption is unlikely to reflect a chemosensory antifeedant effect as consumption was reduced even at low concentrations which did not affect diet choice by second-instar larvae. Rather, the reduced RCR may reflect a direct action of azadirachtin on the gut or on neural regulation of feeding. Mordue et al.

(1985) have shown that azadirachtin reduces the rate of gut

peristalsis in Locusta r and indeed inhibits all proctolin- mediated muscular activities (Mordue and Plane, 1988). Such a mechanism could well account for the observed decrease in the RCR; consistent with this is the observation that the AD tended to increase with increasing azadirachtin concentration. This effect could result from a prolonged exposure qf the food bolus to digestive enzymes (Slansky and

Scriber, 1985). The decrease in the ECI and ECD found at the highest azadirachtin concentration here tested could reflect increased metabolic costs of detoxification.

However, reduced consumption can also result directly in a reduction in ECI and ECD, as the fixed costs of metabolic activity will consume a higher proportion of energy intake if that intake is small rather than large. In particular, 214

consumption at the highest azadirachtin concentration may be just sufficient to meet the metabolic requirements of the larvae, with no excess to support growth. This interpretation is supported by the observation that, when

RGRi is plotted against RCRi, all treatments fit the same regression line (Fig. 4-7). This result strongly suggests that the growth-inhibiting activity of azadirachtin is related to reduced consumption and not to metabolic toxicity

(cf. Blau et ai., 1978). It is also noteworthy that very few larvae died while molting; Arnason e£ai. (1985) noted a similar result with 0. nubilalis. The molt-inhibiting activity of azadirachtin appears to be most prominent in species which fail to respond to azadirachtin as an antifeedant (i.e. Garcia and Rembold, 1983; Chapter 5 of this thesis), or in bioassays where insects are artificially exposed (via topical application, injection, or gut cannulation) to doses of azadirachtin which would ordinarily be avoided.

The results of the nutritional index study are comparable to results obtained with Melanoplus sanguinipes

(Chapter IV), Heliothis virescens (Barnby and Klocke, 1987), and Crocidolomia binotalis (Fagoonee, 1984). In the latter study, ECI and ECD were found to increase with azadirachtin concentration. In contrast to these results, Arnason et ai

(1985) found no reduction in consumption when 0. nubilalis larvae were exposed to azadirachtin; rather, a decrease in the RGR was related to decreases in the ECI and ECD. Rao 215

and Subramantham (1985) found that azadirachtin reduced consumption, ECI, and ECD in a dose-dependent manner in

Schistocerca gregaria. This result may reflect the extreme sensitivity of this insect to azadirachtin (Blaney, 1980).

G. Limonoid Evolution and Structure-Activity Relationships

The insecticidal activity of limonoids does not appear to correlate with the evolutionary trends of increasing oxidation and skeletal rearrangement discussed by Das et al

(1984). This conclusion is supported not only by the analysis in Figure 4-8, but also by a consideration of Table

4-1 and the results of the bioassays presented here. The dammarane precursors of the limonoids are apparently inactive against insects (Chapter 3), but both the protolimonoids (Group 1 in Fig. 4-2) and the simple apo- euphol type limonoids (Group 2) are active; in the latter case the activity may include both toxic and IGR effects

(i.e. in the case of cedrelone). However, the dominant evolutionary pathways within most Meliaceae and all

Rutaceae, giving rise to D-seco, A,D-seco, and B,D-seco limonoids, lead to compounds with reduced antifeedant and apparently no IGR activity. In Citrus, the reduced activity may be to some extent compensated for by the very high concentrations of A,D-seco limonoids (especially limonin) produced (> 1,000 ppm) (Rouseff and Nagy, 1982). Curiously, these pathways are the only ones expressed in 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 activity against generalist insects is that giving rise to the C-seco limonoids; these compounds may be derived via a different pathway from the other limonoids, involving an extra epoxidation step (Fig. 1-2) (Siddiqui et al., 1988). This pathway is expressed only in the tribe

Melieae, which includes the (morphologically primitive) genera Azadirachta and Melia.

Despite the absence of an obvious relationship between limonoid evolution and activity against phytophagous insects, the possibility of a role for limonoids in a coevolutionary relationship between plants and insects remains. All of the bioassay species used to date are polyphagous or oligophagous species which do not utilize

Meliaceae as host plants. The possibility remains that specialist insects, adapted to tolerate exposure to one class of limonoids, may be deterred or intoxicated by exposure to another class. For example, papilionid larvae able to tolerate large doses of linear furanocoumarins are susceptable to angular furanocoumarins (Berenbaum, 1978,

1981), even though the latter are considered less toxic as they are only able to form monofunctional adducts with DNA.

The role of limonoids in a putative coevolutionary relationship between the Meliaceae and insects would have to be examined in the context of the adapted insect fauna found feeding on meliaceous species. For instance, the shoot 217

borers Hypsipyla spp. attack all species of Cedrela and

Swietenia , to the point of limiting their usefulness as plantation crops within their natural area of distribution

(Grijpma, 1973). These insects must be adapted to cope with

Group 2 compounds such as cedrelone, which occur in the shoots and heartwood as well as foliage 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; is the resistance due to limonoids of classes 5 and 7, found in Guarea but not in the other genera? Azadirachta indica has a small entomofauna of twelve species, almost all of which are monophagous (Warthen, 1979). Has this specialized fauna resulted from a coevolutionary association during the evolution of the C-seco type limonoids? Clearly the significance of limonoids in mediating such interactions requires further investigation.

H. Correlation of Phytochemistry and Crude Extract Bioassays

The structure/activity conclusions drawn here are supported by a correlation of the results of the crude extract bioassays reported in Chapter 2 with the known distribution of limonoids in the Meliaceae. Table 4-6 lists the classes of limonoids known to occur in 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 EC50 values of crude extracts of Meliaceae with predictions of activity based on classes of limonoids reported to occur in the genera examined. Extracts were predicted to have high activity if group 8 limonoids had been reported, moderate activity if group 2 or 10 limonoids were known, low activity if other limonoids had been reported, and no activity if 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 is taken from Taylor (1981), updated as noted. Limonoids known from species other than the one(s) bioassayed are in parentheses. Observed EC50s are from Table 2-2 and are mg/g; where more than one species in a genus was bioassayed the range of results is shown.

Genus Limonoid Groups Predicted Observed Reported Activity ^•^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 inactive 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 inactive 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 Trichilia 2,(7) moderate 18.88 Turreae 2 moderate 2.80-45.40 References:l) El-Shamy gt ai., 1988. 2) Jogia and Andersen, in press. 3) Kraus and Grimminger, 1978,1980. 219

were predicted to have low activity, and the genera without limonids were predicted to be inactive. Generally the correlation is good: all species predicted to have high activity do, and those species found to be inactive had been predicted to have little or no activity. Most species predicted to have moderate levels of activity had EC50 values close to 20 mg/g, and most of those predicted to have

low activity had EC50 values at or above 30 mg/g.

Noteworthy exceptions were seen, however: Aglaia and Turreae were much more active than was predicted based on their known phytochemistry, and Sandoricum had a moderate level of activity 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 Insecticidal and Cytotoxic Activity

The insecticidal activity of limonoids is to some extent paralleled by the cytotoxic activity of these compounds against murine P-388 lymphocytic leukemia cells. Pettit et al (1983) found that a 14-15 epoxide on an intact D ring was required for cytotoxicity; they suggested that the epoxide may be required for alkylation of bioamines or thiols, but I found that azadirachtin does not spontaneously form adducts with the sulfhydryl cysteine in vitro (Chapter 220

5). Limonoids with a 19-28 lactol group were most active, but those with a 3-oxo-l-ene A ring structure (as in cedrelone and anthothecol) were also highly active; A ring saturation abolished the cytotoxicity. Conversely, Kraus gt al. (1987) concluded that A-ring saturation, with oxygen functions at C-2 and C-3, was required for antifeedant activity against Epilachna varivestis, based on a comparison of the activity of vilasinine derivatives with azadiradione.

Compounds with a D ring epoxylactone, and the seco-ring A,D citrus limonoids, were mostly not cytotoxic (Pettit gt al.,

1983).

An interesting observation from this and previous studies (Kubo and Klocke, 1987; Siddiqui gt al., 1988; 1988;

Naqui, 1987) is the IGR activity of cedrelone and related compounds with a 14-156-epoxide on an otherwise intact D ring,an unsubstituted C ring, and a 3-oxo-l-ene A ring.

These relatively simple structures may be amenable to synthesis or manipulation, and so could provide leads for the development of synthetic limonoid-based insecticides. 221

Chapter 5: Effects of azadirachtin on the nutrition and development of the migratory grasshopper, Melanoplus sanguinipes Fab.

Introduction

Among the few insects reported to be resistant to azadirachtin are the New World grasshoppers, including the migratory grasshopper, Melanoplus sanguinipes Fab.

(Orthoptera: Acrididae) (Mulkern and Mongolkiti, 1975). In

initial experiments I confirmed the absence of a chemosensory-based antifeedant effect against this insect, but observed marked subsequent disruption of molting. This

allowed me to compare the toxicity of azadirachtin following

application orally, topically, and via injection, and so evaluate the significance of the gut and integument as

factors limiting the bioavailability of this compound to putative target sites within the insect.

Azadirachtin-induced inhibition of molting has been

shown, in several insects, to involve a delay in the appearance of the ecdysteroid peaks which regulate apolysis

(reviewed in Chapter 1); however the mechanism by which this occurs is still unclear. Mordue and Evans (1987) have

suggested that such effects may result from a direct action

on the gut rather than a direct action on ecdysteroid

synthesis or metabolism. 222

The toxicology of azadirachtin is in many respects paralleled by the azasterols, which block the conversion of

B-sitosterol and other phytosterols to cholesterol by

inhibiting 24 and 22,24 sterol reductases of insects

(Svoboda et al., 1972; Svoboda and Robbins, 1971; Walker and

Svoboda, 1973). Similarities include the inhibition of growth, molting and oogenesis at dietary concentrations as

low as 3 /ig/g. As azasterol toxicity can be reversed by dietary supplementation with cholesterol, I investigated the effect of cholesterol and other phytosterols on azadirachtin toxicity. I also investigated the possibility that azadirachtin may interfere with sterol transport through the hemolymph, a process dependent on the carrier lipoprotein

lipophorin (Chino and Gilbert, 1971; Chino, 1985).

Finally, as both the transducing proteins involved in chemoreception and the neurosecretory material formed in the pars intercerebralis are unusually rich in sulfhydryl residues (Norris, 1986; Friedel and Loughton, 1980), and both sites have been postulated to be putative molecular targets for azadirachtin activity, I tested the ability of azadirachtin to form adducts with the sulfhydryl amino acid cysteine in vitro. 223

Materials and Methods

A. Experimental Insects

Fifth instar Melanoplus sanguinipes nymphs, non-diapause

strain, were obtained from a laboratory colony reared on

freshly cut seedling wheat, dry wheat bran, and chickweed,

Cerastium stellata, (Isman, 1985). Synchronized groups of

fifth instar nymphs were obtained by clearing cages of fifth

instar individuals 24 h prior to starting an experiment, then utilizing nymphs which molted overnight. In some cases

individuals so obtained were stored at 4° C for 24 h prior to starting a bioassay, to allow collection of sufficient numbers of nymphs for the bioassay. Preliminary experiments established that grasshoppers could be stored at 4° C for up to 72 h without influencing subsequent growth rate, duration of the instar, or molting success.

B. Source of Chemicals

Azadirachtin used in this study was isolated from

Azadirachta indica seeds by Dr. J. Kaminski, and was kindly made available Dr. J. T. Arnason, University of Ottawa.

Cholesterol and B-sitosterol were purchased from Sigma

Chemical Company, St. Louis, Mo. and were used without

further purification. 4-14C-B-sitosterol (56 mCi/mmol) was purchased from Amersham. 224

C. Antifeedant Activity Assays

In no-choice antifeedant assays, azadirachtin in acetone

(0.5 or 0.05 mg/ml) was applied to both surfaces of freshly 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

/il acetone, a volume equivalent to that applied at the highest azadirachtin concentration. One leaf disc was presented to each fifth instar grasshopper in a 4 oz unwaxed paper cup capped with a plastic petri dish lid; grasshoppers were starved for two hours prior to the test. Ten nymphs were used for each concentration, and the test was replicated three times. The number of individuals in each treatment group which had completely consumed the disc after

15, 30, and 60 minutes was recorded.

D. Dietary Utilization Experiments

The effects of azadirachtin on relative growth, consumption, and digestive performance of nymphs were determined. Staged fifth instar 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 disc; these doses were chosen as they produce markedly different effects on molting success in U. sanguinipes. Subsequently the nymphs were fed daily with weighed aliquots of freshly cut seedling wheat. 225

Initial dry weight of the wheat was estimated by drying samples of wheat and calculating a dwt/fwt ratio. After 48 h the nymphs were weighed, then nymphs, frass, and remaining wheat were dried to constant weight at 70 C and weighed.

The initial dw of the nymphs was calculated based on the dw/fw ratio of a sample of 10 nymphs. Indices determined included the approximate digestability (AD), efficiency of coversion of ingested food (ECI), and efficiency of conversion of digested food (ECD), calculated according to

Reese and Beck (1976). In addition growth arid consumption rates were calculated in relation to the weight of the nymphs at the start of the experiment (RGRi and RCRi respectively) (Farrar et al, 1989). Formulas for calculating these indices are given in Chapter 4.

E. Molt Inhibition Assays

To assess the growth regulating activity of azadirachtin following oral administration, staged (<24 h) fifth instar nymphs were weighed (114.3 ±12.1 mg/nymph), then fed sufficient azadirachtin applied in acetone to a leaf disc 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. After the single dose of azadirachtin was consumed, usually in less than 1 h, grasshoppers were maintained in 4 oz paper cups , in a controlled environment chamber at 30 +

1° C, about 40 % RH (ambient air RH), a 16L:8D photoperiod, and fed untreated wheat, bran, and chickweed ad lib, until 226

molting or death occurred. Duration of the instar, molting success, and weight at molting were recorded. Ten nymphs

per dose were used in each of four replicates. MD50 values

(the dose which inhibited molting in 50% of the treated nymphs) were determined by probit analysis, with category 3 and 4 responses (defined in Results section) combined as mortalities.

Azadirachtin in acetone was also applied topically to the dorsal abdominal tergae of staged fifth instar nymphs, to achieve doses of 2, 4, 6, 8, and 10 /ig/g insect fwt.

Controls were treated with 5 /il 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 in each of three replicates. For injection experiments, staged fifth instar nymphs were weighed, chilled to 4° C, then injected mid- laterally between abdominal terga 3 and 4 , using a Hamilton syringe fitted with a 26-gauge needle attached to a Hamilton repeating dispensor. Azadirachtin in acetone (0.5 mg/ml) was applied at 3, 5, 8, 10, and 15 fig/ g insect fwt; controls were injected with 3 /il acetone alone. Mortality in the controls was about 6% (2 of 30 nymphs) with this solvent. Three replicates of ten insects per dose were performed.

F. Piperonyl Butoxide Synergism Assay 227

The possible role of mixed-function oxidases (MFOs) in the metabolism of azadirachtin by M.. sanguinipes was

investigated by feeding staged fifth instar nymphs azadirachtin at 2, 4, 6, 8, and 10 ng/ g insect fwt, coadministered with 500 /xg piperonyl butoxide (PBO) diluted

1:1 with acetone, on a cabbage leaf disc. Control insects were fed leaf discs treated with 500 ng PBO only. Insects were subsequently maintained as described above, and duration of the instar and molt success were recorded. The experiment involved three replicates of ten nymphs per dose.

G. Fecundity Experiment

Staged teneral adult female M.. sanguinipes (<24 h from molting) were weighed, then fed sufficient azadirachtin, applied to a cabbage leaf disc, to achieve an oral dose of

0, 5, 10, 15, 25, or 50 pg/g insect fwt. Thereafter, females were maintained individually in 4 oz unwaxed paper cups at 30° C, 18L:6D, and fed seedling wheat, chickweed, and bran ad lib. The cups were modified as oviposition arenas: a 1.5 cm diameter hole, punched in the bottom of the cup, gave access to a 2 oz plastic cup filled with sieved, sterilized soil. One male, not treated with azadirachtin, was added to each cup 24 h after the female. Five females were used for each azadirachtin concentration tested; the experiment was repeated twice. The cups of soil were removed, examined for egg masses, and replaced with fresh soil every three days. Egg masses were disassembled to 228

count individual eggs; this precluded the possibility of determining egg fertility. The bioassay was continued for six weeks. Data were analysed by linear regression.

H. Effect of Dietary Sterols

To determine the effect of supplementing the diet with sterols on azadirachtin toxicity, I established six treatment groups: (1) solvent control, (2) azadirachtin alone, (3) cholesterol alone, (4) cholesterol plus azadirachtin, (5) 8-sitosterol alone, and (6) B-sitosterol plus azadirachtin. Insects were initially 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 instar they were fed wheat dipped in CHC13 (1 and 2),

or a 10 mg/ml CHC13 solution of cholesterol (3 and 4) or B- sitosterol (5 and 6). Duration of the instar and molting success were recorded. Ten nymphs per concentration were used in each of two replicates.

I. Sterol Transport Experiment

I tested the hypothesis that azadirachtin interferes with the transport of sterols in the hemolymph by feeding 14C-B- sitosterol (10,000 dpm/nymph) to control and azadirachtin

(15 /ig/g insect fwt)-treated fifth-instar nymphs. Insects

(36/treatment) were fed a leaf disc treated with azadirachtin or acetone 24 h before the single pulse of radiolabelled sterol. At hourly intervals for 12 h, three 229

insects from each treatment were randomly selected, a cut was made laterally along the abdominal wall, and 10 fil hemolymph was collected with a microcapillary tube. The samples were individually digested for 1 h in 1 ml Protosol, then 4 ml Aquasol was added in plastic scintillation tubes,

and the cocktail was allowed to equilibriate for 24 h to quench chemiluminescense before counting. Following the experiment (24 h) the nymphs from each treatment were

pooled, homogenized in CHC13, filtered, and the extract was concentrated to 4 ml. A 1 ml aliquot was dried and prepared

for scintillation counting as described above. The remaining 3 mis were dried, derivatized with 30% trifluroacetic acid (TFA) in acetonitrile (ACHN) at 80° C for 1 h, then chromatographed by reverse-phase TLC developed

3 times in acetic acid:ACHN. Spots were visualized with 3.0

M H2S04, scraped from the plate, eluted with MeOH:H20 (1:1), and prepared for scintillation counting in Aquasol.

Standards of TFA derivatives of cholesterol and 6-sitosterol were also prepared and chromatographed alongside the hemolymph extracts.

J. Jn vitro assay for the formation of adducts

Azadirachtin (0.02 /imoles = 1.8 mg) and cysteine (free base)

(0.02 /xmoles = 0.3 mg) were mixed in 1 ml of pH 7.0 phosphate buffer at room temperature. Aliquots were removed at ten minute intervals for the first hour, and thereafter hourly for five hours, spotted on cellulose TLC plates, and developed in the upper phase of n-butanol:acetic acid:H20

(4:1:5) (Pieman et al., 1979). Spots were visualized by spraying the plate with ninhydrin reagent (Stahl, 1972)

followed by 2.0 M H2S04, with heating after each spray reagent. 231

Results

A. Antifeedant assays

Azadirachtin had no antifeedant effect against M..

sanguinipes nymphs at any of the concentrations tested.

Leaf discs were usually consumed within 30 minutes in a

single feeding bout; on occasion two feeding bouts were

required.

B. Growth and Dietary Utilization

Treatment with both 10 and 15 /ig/g azadirachtin resulted in

a significant decrease in the relative growth rate (RGRi: 38

and 40% of controls respectively, p<.0001, Tukey's (HSD)

•i test) (Table 5-1). This was almost entirely owing to a

decrease in the relative consumption rate (RCRi: 34 and 41%,

p<.0001). There were no differences between the effects at

10 and 15 /ng/g azadirachtin. The efficiency with which

ingested (ECI) and digested (ECD) food was converted to new

insect biomass was not significantly decreased. The

approximate digestability was slightly increased following

azadirachtin treatment at the higher dose only (p<.017,

Tukey's (HSD) test). 232

Table 5-1. Effect of azadirachtin on Melanoplus sanguinipes growth and nutrition. Means in a column with the same

letter are not significantly different (Tukey's Studentized

Range (HSD) Test, oc =0.05).

Treat RGRi RCRi ECI ECD AD

Control 0.243a 0.843a 30.2a 60.3a 53. 2a

10 0.150° 0.569° 30. 5a 54.0a° 59. 4a

15 0.145^ 0.500^ 25.9s- 44. 6^ 59. B3- Treat=treatment (/ig/g insect fwt), RGRi=Relative Growth Rate based on initial wgt, RCRi=Relative Consumption Rate based on initial wgt, ECI=Efficiency of Conversion of Ingested Food, ECD=Efficiency of Conversion of Digested Food, and AD=Approximate Digestability. Values were calculated according to Reese and Beck (1976) except for the RGRi and RCRi, which were calculated according to Farrar et al.(1989). 233

C. Molt Inhibition Studies

Melanoplus sanguinipes nymphs that consumed azadirachtin

subsequently showed a range of dose-dependent effects which

are here arbitrarily divided into four categories of

response. Results for males and females did not differ 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 II). These

individuals took longer to complete the molt. The deformities may have resulted from the nymphs initiating

sclerotization of the adult structures before ecdysis was complete. The duration of the instar was also significantly

increased at 8 and 10 nq/q azadirachtin (12.0 + 1.4 days cf.

8.0 + 1.0 days for the controls). At 8 and 10 nq/q, a small proportion (<10%) of treated nymphs died during an

incomplete molt attempt (=Category III). A notable transition was seen between 10 and 13 nq/q: at the lower dose most nymphs showed a category II response, but at 13 nq/q all nymphs died in the molt. At 15 nq/q about 25% of the nymphs eventually died without initiating any molt attempt (Category IV), and at 25 nq/q 80% of the nymphs showed this response. In some cases category IV insects were observed to live for over 60 days without initiating 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 effects of orally administered azadirachtin on fifth-instar nymphs of Melanoplus sanguinipes. Response categories include: Category I: no morphological effect; Category II: molt to an adult with deformed wings or legs; Category III: death at a failed molt

attempt; Category IV: death without initiating a molt

attempt.

236

Figure 5-2. Effect of orally administered azadirachtin on molting success of fifth-instar nymphs of Melanoplus sanguinipes. Responses are divided into four categories: I, no response except delay of molt; II, molt to an adult with deformed wings or legs; III, death at a failed molt attempt; and IV, death without initiating a molt attempt. Percent of treated population

Control 3 5 8 10 13 15 25 Azadirachtin dose ug/g insect fwt

Category 1 Category 2 Category 3 Pill Category 4 238

(232.1 + 17.6 mg vs 207.6 ± 25.4 mg respectively). Grouping category 1 and 2 responses as survivors and category 3 and 4

as mortalities, the oral MD50 was 10.83 /ig/g insect fwt (95% fiducial limits 10.34-11.44 pg/g).

Topical administration of azadirachtin produced the same range of effects, 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 topical MD50 was 3.80 /ig/g insect fwt (95% fiducial limits 3.196-4.353 /ig/g).

When azadirachtin was injected directly into the hemocoel, the same spectrum of effects 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 particularly 8 and 10 /ig/g, category III responses were most common. At 8 and 10 /ig/g, some nymphs failed to molt; this respose accounts for 80% of the

treatment group at 15 /ig/g. The MD50 was 3.01 /ig/g (95% fiducial limits 2.49-3.50 /i/g).

D. Synergism by piperonyl butoxide

Coadministration of piperonyl butoxide (PBO) significantly

increased the oral toxicity of azadirachtin in 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. Effect of injected azadirachtin on molting success of fifth instar nymphs of Melanoplus sanguinipes.

Responses are divided into four categories: I, no response except delay of molt; II, molt to an adult with deformed wings or legs; III, death at a failed molt attempt; and IV, death without initiating a molt attempt. Percent of treated population Injected

Control 3 5 8 10 15 Azadirachtin dose ug/g Insect fwt

Category 1 iiil Category 2 Category 3 Category 4 ro o 241

Figure 5-4. Effect of topically applied azadirachtin on molting success of fifth-instar nymphs of Melanoplus sanguinipes. Responses are divided into four categories: I, no response except delay of molt; II, molt to an adult with deformed wings or legs; III, death at a failed molt attempt; and IV, death without initiating a molt attempt. Percent of treated population

Control 2 4 6 8 Azadirachtin dose ug/g Insect fwt

Category 1 ill Category 2 Category 3 Category 4

-F 243

Figure 5-5. Effect of co-administered piperonyl butoxide

(PBO) on the molt inhibitory activity of orally administered azadirachtin. In this figure category I and II responses are combined to give survivorship curves. The effect of injected and topically applied azadirachtin is also shown. DOM (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 oral MD50 of azadirachtin plus PBO was 6.5

/xg/g insect fwt. PBO alone had no effect on instar length or molt success.

E. Fertility Experiment

Azadirachtin decreased the number of egg masses and eggs produced over the first six weeks of the adult stage, in a dose-dependent manner (Figure 5-6). Overall there was a

highly significant dose-response, with an FI50 (50% fertility inhibition) of 35 /ig azadirachtin/g insect. This appeared to be due to an azadirachtin-induced dose-dependent delay in the time to production and size of the first egg mass. Survivorship to the end of the 6-week assay was 100% in all treatment groups.

F. Sterol supplementation assays

Supplementing the diet with cholesterol or B-sitosterol did not significantly influence the toxicity of orally- administered azadirachtin. The sterols alone had no effect on molt success or duration of the instar.

Treatment with azadirachtin did not abolish the movement of radiolabelled sterol into the hemolymph from the gut (Figure 5-7). In both control and treated nymphs radiolabel first appeared in the hemolymph 4 h after feeding with 14C-B-sitosterol. In controls, the amount of Figure 5-6. Effect of orally administered azadirachtin on adult female fecundity. Each point represents the mean of five females; controls produced 42+11 eggs/female.

Fedundity (% ofControl) = 110-1.7[Aazdirachtin] r2=.96 cn Figure 5-7. Pharmacokinetics of radiolabelled sterols in the hemolymph of control and azadirachtin treated nymphs.

Each point represents the mean of three individual nymphs.

250

radioactivity increased to a peak at 10 h post-feeding whereas, in azadirachtin-treated nymphs, the increase was more gradual and did not peak during the course of the experiment.

Chromatography of TFA derivatives of the sterol fraction from control and azadirachtin treated insects indicated that /3-sitosterol was primarily metabolized to cholesterol in M- sanguinipes. No differences in the relative proportion of cholesterol, 6-sitosterol, and desmosterol were evident between the two treatment groups

(Table 5-2).

G. In vitro assay for adduct formation

Chromatography of aliquots of an equimolar mixture of azadirachtin and cysteine in 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 in aliquots taken at all time intervals; after one hour cystine (Rf=0.31), the oxidation product of cysteine, was also detected. 251

Table 5-2. Radioladelled sterol composition of control and azadirachtin-treated M. sanguinipes fed 4-14C-B-sitosterol.

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 nutritional effects

Azadirachtin had no chemosensory-based antifeedant activity against M. sanguinipes nymphs at concentrations up to 500

/ig/g, as treated leaf discs were usually consumed in a single feeding bout. However, the RCRi was significantly reduced following azadirachtin ingestion, indicating an action against the gut or on the neural regulation of feeding. This effect resulted in a significant decrease in

RGRi. Reduced consumption following azadirachtin administration, in the absence of chemosensory inhibition, has been noted with other insects (Rembold et al., 1980;

Redfern et al., 1981; Schmutterrer, 1985; Mordue et al.,

1985; Simmonds and Blaney, 1984) and in Locusta may be due to a decreased rate of gut peristalsis (Mordue et al.,

1985). These results suggest that bioassays extending beyond a single feeding bout cannot distinguish between a true chemosensory response (deterrency) and toxicity with this compound; at least some of the many reports of the antifeedant effect of azadirachtin may be due to post- ingestive effects. However, electrophysiological studies and choice tests show unambiguously that azadirachtin does have a chemosensory-based antifeedant effect against some insects (Schoonhoven, 1982; Simmonds and Blaney, 1984). In

Schistocerca gregaria azadirachtin may activate a specific

"labelled line" deterrent receptor (Blaney, 1980). Neither 253

the ECI nor the ECD were reduced significantly; the AD was

increased only at the higher concentration. Similar results have been noted in studies with the lepidopterans Heliothis virescens (Barnby and Klocke, 1987) and Crocidolomia binotalis (Fagoonee, 1984); in both cases consumption was decreased by azadirachtin without concomitant reductions in efficiency of dietary utilization, and in fact for the

latter insect both the ECI and ECD increased to compensate

fora reduction in food volume. In contrast, Arnason et al.

(1985) found that dose-dependent reductions in growth of

Ostrinia nubilalis were due to a reduction in ECI and ECD; consumption rate was unaffected by azadirachtin concentrations in artificial diet up to 30 ppm. Their results could be owing to the obstruction of the sensillar pores on the mouthparts by the agar-based diet (Arnason, pers. comm. 1988). The limonoids cedrelone and anthothecol also reduce the ECI and ECD at concentrations which actually increase the consumption rate in Ostrinia nubilalis (Arnason et al., 1987). Rao and Subramanyam (1986) found that azadirachtin lowers the RCR, ECI, and ECD in fifth-instar

Schistocerca gregaria. In all studies to date the AD is unaffected or increases with increasing azadirachtin concentration. Such a phenomena would result from a decreased rate of gut peristalsis (Mordue §t al., 1985), increasing the amount of time during which the food bolus is exposed to digestive enzymes (Slansky and Scriber, 1985). 254

Most notable is the absence of any difference in growth, consumption, or nutritional utilization indices between 10 and 15 nq/q insect, as these doses produce remarkably different effects on molting. At the lower dose, most nymphs molted successfully, albiet to malformed adults, whereas at the higher dose all nymphs died in the molt. The lack of correlation between nutritional and presumably endocrine-mediated effects suggests that separate physiological targets may be involved.

B. Oral, topical, and injection experiments

M. sanguinipes nymphs which consumed azadirachtin at the beginning of the instar subsequently displayed a dose- dependent range of effects. Low doses (<10 nq/q insect) resulted in molt delay and deformity of adult appendages, intermediate doses (13-15 nq/q) resulted in death at a failed molt attempt, and high doses (>25 nq/q) produced permanent fifth instar nymphs in which the molting response was abolished. These effects parallel those seen with

Locusta (Mordue et al., 1985, 1986), Oncopeltus (Redfern et al-, 1982), and other insects (Gaaboub and Hayes, 1984;

Koul, 1984; Ladd et al., 1984).

Molt failure has been attributed to disruption of the normal ecdysteroid titres in several species (Koul et al..

1987; Rembold and Sieber, 1981; Rembold gt al., 1984;

Schluter et al., 1985; Mordue and Evans, 1987; Min-Li and

Shin-Foon, 1987). However, the physiological basis of this 255

disruption is not known. Mordue et aJL.(1986) suggested that

failure to attain critical weight was responsible for failed ecdysis in Locusta migratoria, but the lowest dose at which they measured growth (7.5 /ig) was almost three times the dose required to inhibit molting. In this study failure to molt was not due to a failure to reach critical 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 direct action on the endocrine system, rather than an indirect effect operating through internal measures of growth.

Application of azadirachtin topically or by

intrahemocoelic injection also reulted in dose-dependent

inhibition of molting. The MD50 for injected azadirachtin

in M. sanguinipes. 3.01 /ig/g, was higher than that reported

for Locusta migratoria, 2.0 /ig/g (Mordue et al., 1985) or

Schistocerca gregaria, 1.66 /ig/g (Rao and Subrahmanyam,

1986). Azadirachtin is also acutely toxic to some insects

when injected at high doses: the 24 h LD50 for Locusta migratoria is 80 /ig/g (Mordue ej£ al., 1985? Cottee et al. .

1988), and for Schistocerca gregaria the LD50 is 330 /ig/g

(Cottee et al., 1988). Acute toxicity to M. sanguinipes was not observed at the highest dose tested in this study, 25

The MD50 via oral administration (10.83 /ig/g) was over three times that via injection (3.01 /ig/g), indicating that the gut poses a physical or physiological barrier to 256

azadirachtin bioavailability. The gut presents a similar barrier to several other natural products in Locusta migratoria and Schistocerca gregaria, indicated by similar ratios of oral to injected acute toxicity (Cottee ej: al.,

1988). The observation that the oral toxicity of azadirachtin can be synergised in M- sanguinipes by coadministration of the mixed-function oxidase inhibitor piperonyl butoxide suggests that the barrier is largely owing to MFO based oxidative metabolism. The slope of the dose-response curve was the same in the oral and injection assays. The toxicity curve for the oral dose-response is shifted by 10 /ig/g relative to the injected dose-response suggesting that the gut MFO's are able to metabolise up to

10 ng azadirachtin/g insect, allowing azadirachtin in excess of this dose to penetrate to target sites within the insect.

In contrast, azadirachtin was equitoxic when applied

topically (MD50= 3.80 /ig/g) or via injection (95% fiducial limits overlap), indicating that in this insect the integument does not significantly limit penetration and therefore bioavailability of azadirachtin to putative target sites within the insect. In each case azadirachtin must have considerable stability once inside the insect, as its effects on molting are expressed 8 to 14 days after exposure to a single dose. Rembold et al (1984) found the half-life of injected 3H-dihydroazadirachtin to be greater than 1 week in Locusta migratoria. 257

The response of M.. sanguinipes to azadirachtin contrasts with its response to sesquiterpene lactones, bitter principles from Asteraceous plants (Isman, 1985).

Parthenin, the most active of six compounds tested, had a

LD50 of 1.5 /imol/g insect fwt when injected into the

hemocoel, compared to a LD50 of 4.4 nmol/g (= 3.2 /ig/g) for azadirachtin. However, adult males were able to tolerate exposure to doses up to 12 /imol/g, applied topically or orally, without toxic symptoms. The gut and integument therefore provide effective barriers to bioavailability of sesquiterpene lactones, unlike the situation with azadirachtin. The factors allowing penetration of some compounds and exclusion of others are not well understood in this insect: although lipophilicity may play a role it is unlikely to be a dominant factor as both hydrophilic (i.e. azadirachtin) and strongly hydrophobic compounds (eg a- terthienyl, a thiophene) (Smirle, Champagne, Isman, unpublished data) are absorbed efficiently.

C. Fecundity Experiment

Azadirachtin, fed to adult females, reduced egg production during the first six weeks of the adult stage. This effect is similar to the reported chemosterilization of female

Locusta migratoria (Rembold and Sieber, 1981), Oncopeltus fasciatus (Dorn et al., 1986), Dysdercus koenigii (Koul,

1984), and Rhodnius prolixus (Feder et al., 1989). In

Locusta this effect has been ascribed to diminished JH and 258

ecdysteroid titres, and in Rhodnius azadirachtin lowered hemolymph ecdysteroid and vitellogenin titres, and impared ecdysteroid synthesis in the ovaries. As adult female

Melanoplus sanguinipes survive in the field for only 25-33 days on average, the effects reported here could result in a significant decline in field populations.

D. Sterol studies

Molting is the end result 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 al. (1985, 1986; Mordue and Evans,

1987), the observed endocrine effects of azadirachtin could be due to the disruption of some aspect of gut function necessary for the initiation of the molting process.

Azasterols inhibit the conversion of phytosterols such as 6- sitosterol to cholesterol by inhibiting the A22 and

A22,24 sterol reductases, resulting in the accumulation of the intermediate desmosterol (Svoboda and Robbins, 1971; Svoboda et al., 1972; Walker and Svoboda, 1973; Svoboda and

Thompson, 1985). As cholesterol is required for the assembly of normal membranes and as a precursor for ecdysteriod synthesis, azasterol toxicity is characterized by inhibition of growth and molting, and by chemosterilization of adult females. These effects may be alleviated by providing a source of cholesterol in the diet

(Walker and Svoboda, 1973). The molt inhibiting effects of 259

azadirachtin were not alleviated by supplementation with

dietary cholesterol, indicating that the target site

involved is probably not the A22 and ^22,24 sterol

reductases.

As insects are unable to synthesize sterols de novo

(Clarke and Bloch, 1959), transport of sterol from the site

of absorbtion to target tissues is of critical importance.

This transport is accomplished via a hemolymph lipoprotein

termed lipophorin (Chino, 1985), which loads cholesterol

directly from the midgut (Chino and Gilbert, 1971).

Lipophorin is also involved in the transport of fatty acids,

mainly in the form of diacylglycerol, and is able to bind

lipophilic pesticides and allelochemicals (Haunerland and

Bowers, 1986). In M. sanguinipes nymphs, radiolabelled B-

sitosterol was observed to cross the gut and appear in the

hemolymph, ruling out inhibition of sterol transport as a mechanism of action for azadirachtin in this insect. The

three-hour time lag between consumption and the first

appearance of radiolabelled sterol in the hemolymph suggests

that sterol adsorbtion probably occurrs from the midgut in

M. sanguinipes. In general, the site of sterol absorbtion

in insects appears to be related to the type of food used:

carnivorous and omnivorous insects adsorb from the crop,

whereas phytophagous insects absorb sterols from the midgut

(Clayton et al., 1964; Joshi and Agarwal, 1977; Kuthiala and

Ritter, 1988). In particular, in the few phytophagous

orthopterans studied to date, including Schistocerca 260

gregaria, sterol absorbtion occurs from the gastric caceae

(Joshi and Agarwal, 1977). The decreased rate of appearance of radiolabel in the hemolymph of azadirachtin-treated nymphs is consistant with a decreased rate of gut peristalsis. The decrease in radiolabel content of control hemolymph at 11 and 12 h is probably due to sterol unloading at the fat body and other target tissues; in Heliothis zea a similar decrease in hemolymph sterol was associated with an

increase in the amount of radiolabel associated with the fat body (Kuthiala and Ritter, 1988). Unfortunately this experiment did not extend for long enough to observe a similar decrease in radiolabel content of hemolymph from azadirachtin-treated nymphs, so the effect of azadirachtin on sterol unloading remains unknown. It is not known if cholesterol unloading is passive or an active, receptor- mediated process in insects (Chino, 1985).

The identity of radiolabelled sterols in the insect 24 h after feeding with 14C-B-sitosterol is given in Table 5-2.

The sterol composition of control and azadirachtin-treated insects does not differ, corroborating the results of the sterol supplementation experiment and ruling out sterol reductase inhibition as a mechanism of action. The sterol composition of M. sanguinipes is dominated by cholesterol, with a lesser amount of B-sitosterol and < 2% of the intermediate desmosterol. This pattern is common to most phytophagous insects, which are able to dealkylate C2Q and

C2g 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 in the M.. sanguinipes extracts; however derivitization with trifluoroacetic acid may remove the ester function.

E. In vitro formation of adducts

The toxic action of a number of plant products involves the formation of covalent Michael adducts between the allelochemical and sulfhydryl residues of proteins; examples

include the sesquiterpene lactones (Pieman et al., 1979) and warburganal (Ma, 1977). Such a mechanism of action has been proposed to account for the cytotoxicity of several

limonoids to murine P-388 lymphocytic leukemia cells (Pettit et al., 1983). Two tissues possibly involved in azadirachtin toxicity, mouthpart chemoreceptors and the pars intercerebralis, contain protein unusually rich in sulfhydryl residues (Norris, 1988; Friedel and Loughton,

1980). I found that azadirachtin does not spontaneously form adducts with cysteine, at least at neutral pH, suggesting that non-specific binding to sulfhydryl-rich protein is unlikely to play an important role in azadirachtin toxicity. This is consistant with the observation that unbound azadirachtin may be readily extracted from Locusta brains as much as one week after injection of the compound (H. Rembold, pers comm 1988). As well, azadirachtin treated sensillae return to normal 262

funtioning within 2-5 min (Simmonds and Blaney, 1984), indicating that the receptors have not suffered irreversable damage such as is produced by sulfhydryl reagents (Ma.

1977).

The possibility remains that other targets in the gut can be affected by azadirachtin, for example the release of factors involved in stimulating elevated rates of protein synthesis in the fat body, recently demonstrated in Locusta migratoria (Laughton et al., 1987). As the relationship between feeding and endocrine events has been particularly well studied in Jf- sanguinipes (Dogra and Gillott, 1971;

Elliott and Gillott, 1977a,b), this insect may provide a convenient model system for further studies on the mode of action of azadirachtin.

F. Agricultural implications

Grasshoppers periodically inflict severe damage on cereal crops and rangeland forage (Bierne, 1971; Hewitt and

Onsager, 1983). Melanoplus sanguinipes is always a major contributor to grasshopper outbreaks, and overall is considered the fourth most damaging pest insect to Canadian agriculture (Bierne, 1971). Currently, control relies on aerial spraying of the pyrethroids deltamethrin (Johnson et al., 1986), cypermethrin, and carbaryl (Mukerji and Ewen,

1984); dimethoate, malathion, and methamidophos are also used (Harris, 1985). As this method of application may have severe impact on non-target insects, especially pollinators, 263

recent work has focussed on use of bait, particularly wheat bran, impregnated with insecticide (Onsager et ai., 1980a,b;

Mukerji et al., 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 bait is facilitated by the distribution 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 in M. sanguinipes. it would appear to have potential as an insecticide applied to baits. Application would have to be timed to coincide with the presence of early instars, before the nymphs are capable of economic levels of damage, as mortality would occur only at molting, some days after application. Application later in the life cycle could produce permanent nymphs. Advantages to the use of azadirachtin could include minimal non-target impact, low residue levels due to the rapid photodegradation of azadirachtin in sunlight (Yamasaki et al., 1988), and possibly compatability with biological control measures including N. locustae. Even at sublethal doses azadirachtin based treatments could be expected to reduce feeding and reproduction.

All the currently available insecticides share a common mode of action, neurotoxicity; azadirachtin could provide an 264

alternative with a radically different mode of action, minimizing the chances of cross-resistance. In insects

Where azadirachtin has antifeedant activity (ie P.. saucia), the development of resistance would involve overcoming both the chemosensory and the physiological effects. For example two strains of Plutella xylostella showed no evidence of resistance in feeding and fecundity tests after 35 generations of exposure to neem seed extract; the same two strains developed resistance factors of 20-35 to deltamethrin in the same time period (Vollinger, 1986).

Populations which became resistant to deltamethrin did not show cross-resistance to neem ssed extract. However, M- sanguinipes already posseses significant MFO activity against azadirachtin and lacks an antifeedant response, and so could be expected to develop resistance fairly rapidly in the presence of strong selection pressure. A sucessful pest management strategy will have to rely on a variety of control measures, employing a variety of modes of action, judiciously applied to minimize the chance of developing resistance. 265

Chapter 6: General Summary

The work described in the previous four chapters examined

several aspects of the putative defenses against herbivorous

insects found in members of the plant family Meliaceae. The

studies began with a preliminary examination of the

relationship between defense strategies and plant life-

history characteristics, moved to an attempt to identify the

phytochemicals involved, and concluded with detailed

investigations of the effects and mode of action of the major group of phytochemicals involved, the limonoids, in

three model insect species.

In the first study, relative investment in

phytochemical-based defenses in thirty species of Meliaceae was estimated, by investigating the response of an

unadapted, generalist herbivore, Peridroma sauciaf to the

entire suite of phytochemicals produced, as included in the methanolic extract of mature foliage. Physical defenses in the Meliaceae are largely confined to leaf toughness

factors; consequently leaf toughness was measured on sixteen

species. Leaf lifetime was not measured directly, but all

species were classed as deciduous or evergreen, with the

assumption that leaf lifetimes would be longer for the

evergreen species. Large differences were found between

species in the relative investment in phytochemical

defenses. Extracts of some species, particularly in the

tribe Melieae, were inhibitory to E. saucia growth at 266

concentrations only 1% of those occurring naturally in the foliage. The most active extracts were all from members of the subfamily Melioideae, whereas extracts from the subfamily Swietenioideae were on average less active. Only three species appeared to lack phytochemical defenses against generalist herbivores, assuming £. saucia is a valid model species.

The plant apparency hypothesis of Feeny (1976) and

Rhoades and Cates (1976) predicts similar defenses in all the species studied here, as they are all "apparent", perennial tree species. This prediction was not supported by my data, as large differences between species in defensive attributes were found. The resource availability hypothesis of Coley e_t al. (1985), on the other hand, predicts that species with short leaf lifetimes (i.e. deciduous species in this study) should be selected for phytochemically-based defenses, and species with long leaf lifetimes (i.e. evergreen species) should elaborate physical defenses including leaf toughness.

In this study, extracts from deciduous species were found to be significantly more inhibitory to E- saucia growth. Previous attempts to quantify investment in chemical defenses have relied on colorimetric assays for phenolics only, and these did not correlate with leaf lifetime or show an inverse relationship 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 toxic or

antifeedant natural products (Feeny,1976; Rhoades and Cates,

1976; Coley et al., 1985), bioassays with such species "may

represent the most relevant method for assessing relative

investment in phytochemical-based defenses. Therefore, my

results indicate that Meliaceae with short leaf lifetimes do

invest more in 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 relationship of increasing leaf toughness with

increasing leaf lifetime is well established from previous

studies (Coley, 1983, 1985), as is the relationship between

leaf toughness and reduced rates of herbivory. As well, there is evidence in my data to suggest an inverse relationship between leaf toxicity and leaf toughness,

suggesting that species with tough leaves require lower

levels of production of phytochemical defenses. However, this hypothesis needs further examination.

Species not previously known for the production of

insect inhibitory natural products and identified here for the first time included Aglaia odorata and Turreae holstii.

The three available Aglaia species showed a range of bioactivity against P. saucia, and so this genus was chosen

for an investigation into the phytochemical basis of resistance to herbivory. The natural product chemistry of

A., odorata proved complex; compounds isolated and identified 268

using spectroscopic methods included the known dammaranes aglaitriol and aglaiondiol, and the bis-amides (S,S)- odorine, (S,R)-odorine (a new natural product), (S,S)- odorinol, and (S,R)-odorinol. As well a series of methylated flavanones, previously unknown in the Meliaceae, were identified, 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, all proved to be inactive against

P. saucia, when tested singly or in combination. Rather, the active constituent appeared to be a single compound, tentatively identified as a limonoid. This compound

of 4 inhibits P. saucia growth, with an EC50 I- /ig/g diet fwt

and an LC50 of 11.2 /ig/g, concentrations which do not affect feeding behaviour. The activity therefore appears to be due to post-ingestive toxic effects. The isolated yield of the compound, 3 /ig/g leaf dwt, was less than the expected concentration, 98 /ig/g, based on the activity of the methanolic extract; the discrepancy suggests the possibility of a synergistic interaction, but combinations of the active compound and the other phytochemicals isolated from A. odorata produced only additive effects.

The third study began with a review of the current literature on the effects of limonoids on phytophagous insects. Most studies have focussed on assays for feeding inhibition, reflecting the prevailing belief that limonoids function mostly to deter insect feeding, but pre-ingestive 269

chemosensory-based effects are not clearly separated from

post-ingestive toxic effects, including growth regulating

effects. As well, the activity of these compounds has not been evaluated in relation to their proposed evolution.

Consequently, I investigated ten limonoids,

representing all the major biosynthetic classes, for

inhibition of growth and feeding against £. saucia, and for

effects on molting and reproduction against Oncopeltus

fasciatus. The simple apo-euphol type limonoids cedrelone

and anthothecol, with an intact steroid skeleton, were highly inhibitory to E. saucia growth at 0.5 umol/g diet

fwt, a concentration which did not affect feeding in a choice test. As well, cedrelone inhibited Q. fasciatus

molting, with an LD50 of 12.2 /ig/nymph, indicating'that even

simple limonoids may have IGR activity. Anthothecol, which differs from cedrelone in having an acetoxy substitution at

C-ll, was inactive in the Oncopeltus assay, indicating the

importance of C-ring substitutions in determining biological activity, and suggesting that growth and molt inhibition

involve separate physiological targets. The dammarane precursors of these simple limonoids were inactive against

E- saucia, so to this point biosynthetic evolution coincides with an increase in insecticidal activity. However, oxidative opening ofthe D ring, as in gedunin, leads to a pronounced drop in activity. Paradoxically, this is a major

step in the biosynthetic evolution of limonoids, and

characterizes all limonoids found in the Rutaceae and 270

Simaroubaceae, and most limonoids found in 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. fasciatus. Of two B,D-seco limonids tested,

entandrophragmin was inactive and bussein weakly inhibited

P. saucia growth; such limonoids are characteristic of many

Swietenioideae including Khaya and Entandrophragma (Taylor,

1981), considered to be advanced genera on morphological grounds (Pennington and Styles, 1975). The main lines of

limonoid evolution in most Meliaceae, therefore, do not

appear to correlate with insecticidal activity against generalist herbivores. This conclusion was supported by an attempt to correlate measures of skeletal oxidation and

rearrangement, identified by Das et al. (1984, 1987) as the dominant themes in limonoid evolution, with inhibitory activity against four species of polyphagous lepidopterans

(data from this study and Kubo and Klocke, 1986). No correlation 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 tirucallol precursors independently of the other limonoids (Siddiqui et al., 1988), or they may be formed from an intact apo-euphol type limonoid with oxidation at C-12 (Jones et al., 1988).

This class of limonoid is the most active, and the most 271

advanced C-seco limonoid, azadirachtin, is known to be

active against more than two hundred species of

agriculturally important pest insects (Warthen, 1979, 1989;

Saxena, 1989).

Azadirachtin was highly inhibitory against P.. saucia,

with an EC50 of 0.4 nmol/g diet fwt and an LC50 of 5.2 nmol/g. Chemosensory-based antifeedant effects were most pronounced against neonate caterpillars, but became much reduced by the early third instar. However, pronounced growth inhibition and mortality continued throughout the

life cycle. Analysis of dietary use and efficiency demonstrated that azadirachtin led to decreased consumption at concentrations which did not affect measures of dietary efficiency or feeding in a choice test, suggesting post-

ingestive effects, perhaps involving the gut directly or neural regulation of feeding. As well, the digestability was increased at the highest concentration tested. Together these results concur with the mechanism of action proposed by Mordue et al. (1985), who suggested that inhibition of gut peristalsis could limit feeding and growth by limiting the rate at which a food bolus could move through the gut.

Azadirachtin was also highly inhibitory to O. fasciatus.

disrupting molting (MD50 = 3.8 ng/nymph) and decreasing adult survival, in agreement with earlier studies (Dorn,

1983, 1987).

The final study focussed on the migratory grasshopper,

Melanoplus sanguinipes. as this insect had been reported to 272

be resistant to azadirachtin (Mulkern and Mongolkitti,

1975). Initial 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

effects, 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 in complete blockage of the molt at doses of 15 /ig/g and above. These effects were similar to those reported earlier in Locusta (Rembold and Seiber, 1981,

Mordue et al., 1985; Mordue, 1988), but are noteworthy as they followed oral application, whereas in earlier studies the azadirachtin was applied by injection, bypassing the normal chemosensory mechanisms and gut defenses.

The consumption of physiologically active doses of

azadirachtin by M.- sanguinipes nymphs allowed me to evaluate the importance of the gut and integument to bioavailability of azadirachtin, by comparing oral and topical activity with the activity of injected azadirachtin. There was no

significant difference between topical and injected

azadirachtin (MD50 3.8 and 3.01 /ig/g respectively),

indicating the absence of barriers to azadirachtin

bioavailability in the integument. However, the oral MD50,

10.8 /ig/g, was significantly higher than the injected MD50.

The barrier associated with the gut likely involves the MFO 273

system and oxidative metabolism, as the activity of orally administered azadirachtinis significantly increased by co- application of the MFO inhibitor piperonyl butoxide.

Analysis of growth, food consumption, and measures of dietary efficiency following consumption of azadirachtin showed that the consumption rate was significantly decreased, despite the lack of an antifeedant response, again indicating an effect on the gut or on the neural regulation of feeding. However, there was no difference in nutritional performance between 10 and 15 /ig/g, although these doses produced markedly different effects on molt success, suggesting that effects on endocrine events leading to molt inhibition are not directly related to the effects on gut peristalsis.

Two hypotheses for the mechanism of action of azadirachtin were tested. The symptoms of azadirachtin treatment closely parallel the symptoms produced by treatment with azasterols, compounds known to inhibit the conversion of phytosterols to cholesterol (Svoboda and

Thompson, 1985). However, azadirachtin toxicity could not be rescued by supplementing the diet with cholesterol, and azadirachtin treatment did not affect the ability of M. sanguinipes nymphs to metabolize B-sitosterol to desmosterol and cholesterol. As well, the ability of hemolymph lipophbrin to transport sterols was not reduced, although the time course of sterol pharmacokinetics was slightly altered, presumably due to a slower rate of gut peristalsis. 274

Secondly, Pettit et al. (1983) suggested that the

cytotoxicity of some limonoids might be due to the ability

to form Michael adducts with sulfhydryl residues, and the

two most conspicuous targets for azadirachtin,

neurosecretory material and transducing protein in

chemosensillae, are unusually rich in cysteine residues

(Norris, 1988; Friedel and Laughton,1980). However, I found

that azadirachtin did not spontaneously form adducts with

cysteine FB, suggesting that non-specific binding to

sulfhydryl-rich protein is unlikely to be involved in the mechanism of action.

These studies suggest that azadirachtin does have a

pronounced effect on gut physiology, as has been claimed by

Mordue (1988) (Mordue et al., 1985), but this effect is

probably not directly related to the disruption of endocrine

events leading to the molt. The results are consistant with

a mechanism involving specific inhibition of the release of

neurosecretory material from neurohaemal organs, as has been

suggested by Rembold (Subrahmanyam et al-, 1989). The mechanism by which this occurs is not yet known. 275

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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 artemisiifolia Linn. Amelanchier florida Lindl. Anthemis cotula Linn. Asparagus medeoloides Thunb. Asparagus officinalis Linn. Asparagus plumosa Baker Beta vulgaris Linn. Brassica nigra (Linn.) Brassica hirta 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 Citrus 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 chiloensis (Linn.) Geranium sp. Gleditsia 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 Mill. Medicago sativa Linn. Melilotus alba Desr. Morus sp. Nasturtium sp. Nicotiana tabacum Linn. Parthenium araentatum Gray 314

Persea americana Mill. 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 copallina Linn. Ribes lacustre (Pers.) Ribes sanouineum Pursh. Ribes sativum Syme Rosa sp. Rubus allegheniensis Porter Rubus occidentalis Linn. Rubus idaeus v. strioosus (Michx.) Rumex crispus Linn. Salix sp. Salix hookeriana Barr. Salix xcouleriana Barr. Salvia officinalis Linn. Smilax rotundifolia Linn. Solanum tuberosum Linn. Solidago leavenworthii T.&G. Stellaria media (Linn.) Trifolium sp. Triticum aestivum Linn. Tropaelum majus Linn. Tsuga canadensis (Linn.) seedlings Urtica sp. Vaccinium angustifolium Ait. Vaccinium corymbosum Linn. Vaccinium myrtilloides Michx. Viola sp. Viola tricolor Linn. Vitis sp. Vitis vinifera Linn. Xanthium strumarium Linn. Zea mays Linn. Ferns, general feeder on field crops, forest trees, fruit trees, grass, low plants, most anything, shrubs, vegetables. 315

Appendix 2: 1H-NMR and mass spectra of compounds isolated from Aalaia odorata.

Compound 3: 3-hydroxy-5,7,4'-trimethoxyflavanone 00 NHSS SPECTRUM OATAi octei t33 BASE H'Ei 134 ej-21 -as iJienee • 1140 RICi 33213288. SMMPLEI HD-2 •98 TO IIM SUMMED - «3 TO «10 XI.80 lea.e -i 134 3330710. 10.

tee

132 91 30.0-

63 29? 7? 189 33 41 3d 96

jjj] tJl,J 1111^,4, Jpiln LL.I,ji>llly I l iiiJllpiL JL l,J,iJkl,L, tu\% r,T , ,T. M'E 90 130 230 100.0• r 3330710. 10.

3 4

se.e - Compound 4: S^^'-trimethoxyflavanone

t—1 286 29? U3

I 3:>» 341 3^ 363 • * I - I • • I i ' i • * i .13? jae .lag . , I ' I ' 1 I • I ' I rvE 388 338 430

BASE M'El 134 DATAi PC 102 »39 PICi 3345Sieg. •^•i TO «6? SUMMED - «I0 TO »I3 XI.«e IOP.O -, 134 p 2617349. ie.

91 50.9-1 166

193 63 77 5.1

181 103 29? 272 41 -U lull 243 M-E 58 156 —f 2se lee.e r 2617340. ie.

300

50.PH Compound 5: 5-hydroxy-7,4/-di.Heth.oxyflavanone 1*1 to to Compound 7: (S,R)-odorine MtiSS SPECTRUM DATAI 0C1B2 639 BASE M^El 134 u-t i; 83 9I47I66 * 1102 RICi 33436160. SHUPLEI FLrtU •S3 TO «63 SUMMED - #10 TO 113 Ml.86 134 iae.0 -i 2617340. 10.

1?»

9.1 90.0 H 166

193 63 77 91

163 129 101 2 2 297 272 2 9 3 41 -Li -I -Ik In.*.']!! >., JiI* n|U i>i 1T,l.. f .^, -gt ...if. M'E I 36 130 200 290 166.6 2617340. 10.

366

Compound 7: (S,R)-odorine 36.6 H

ro

3>9 , , 3j6, , ^ 377; jB? ,186, t 442, I • I ' I • I 366 M^E 336 430 'si.' ' ' ' Compound 8: (S,S)-odorine

MMiS SPECTRUM DATAI DC183 1112 BASE M'E I 131 6412/89 I8H4I00 • 1sse RICi 17836980. SHMPIEI AMIOE •118 TO «114 SUMMED - «7 TO «14 XI.00 131 iee.e-i 1966710. 10.

103

73 201

30.0- 77 109 39

83 49

9 244 91

43 63 T ^4 I 3 T 1 Jj.lu Ililly.lt.J.. 'I J I- ,™ t 30 190 290 100.0- I- 19C6710. 10.

se.e - Compound 9: (S,S)-odorinol

.1. 36? , ,383, ,399, 1U , .128 • ,11? , I • I • I M-E 388 338 408 490 Compound 10: (S,R)-odorinol