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

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INSECT ANTIFEEDANT AND GROWTH REGULATING ACTIVITY OF PHYTOCHEMICALS AND EXTRACTS FROM THE PLANT FAMILY MELIACEAE By DONALD EDMOND CHAMPAGNE B.Sc., The U n i v e r s i t y o f Ottawa, 1981 M . S c , The Ottawa-Carleton Center f o r 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 a c c e p t t h i s t h e s i s 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  DE-6 (2/88)  OCT- /a.  (1%*)  Abstract  This thesis represents studies on aspects of the defenses against insect herbivores i n species of the plant family Meliaceae, p a r t i c u l a r l y with regard to phytochemicals.  Methanolic extracts of foliage from t h i r t y  species i n twenty-two genera were bioassayed f o r t o x i c i t y and growth i n h i b i t o r y a c t i v i t y against the variegated cutworm, Peridroma s a u c i a and f o r feeding i n h i b i t i o n f  against the migratory grasshopper, Melanoplus sanguinipes. A l l but three species were i n h i b i t o r y to £. saucia, members of the t r i b e Melieae being most i n h i b i t o r y .  Members of the  subfamily Melioideae were more active than members of the Swieteniodeae.  Newly i d e n t i f i e d species with a c t i v i t y  comparable to neem (Azadirachta indica) foliage extracts included Aglaia odorata and Turreae h o l s t i i .  Deciduous  species produced extracts which were s i g n i f i c a n t l y more active than evergreen species, i n d i c a t i n g a greater r e l i a n c e on phytochemical-based defenses.  Evidence i s also presented  to suggest that the leaves of evergreen species are tougher than deciduous species, and that there i s a negative c o r r e l a t i o n between leaf toughness factors (physical defenses) and phytochemical-based  defenses.  These r e s u l t s  are i n agreement with predictions of the resource a v a i l a b i l i t y hypothesis. The phytochemistry of Aglaia odorata, A. odoratissima. and A. argentia was examined i n d e t a i l .  Compounds  iii  i d e n t i f i e d i n c l u d e d t h e dammaranes, a g l a i o n d i o l and a g l a i t r i o l , and t h e bis-amides (S,S)-odorine,  (S,R)-odorine  (a new n a t u r a l p r o d u c t ) ,  (S,R)-odorincT.  Three d i h y d r o f l a v a n o n e s for  ( S , S ) - o d o r i n o l , and  were i d e n t i f i e d from t h e M e l i a c e a e  t h e f i r s t time: 3-hydroxy-5,7,4'-trimethoxyflavanone (a  new n a t u r a l p r o d u c t ) ,  5,7,4'-trimethoxyflavanone, and 5-  hydroxy-7,4'-dimethoxyflavanone. i n a c t i v e a g a i n s t E« s a u c i a . odorata  A l l compounds were  The i n h i b i t o r y a c t i v i t y o f A..  appeared t o be due t o a compound, t e n t a t i v e l y  i d e n t i f i e d as a l i m o n o i d , which may be a c t i n g i n c o n j u n c t i o n with a s y n e r g i s t .  T h i s compound i n h i b i t s E - s a u c i a  growth i n t h e absence o f a n t i f e e d a n t The  toxicology of limonoids,  larval  activity.  r e p r e s e n t i n g the major  b i o s y n t h e t i c c l a s s e s , was examined a g a i n s t E> s a u c i a and t h e l a r g e milkweed bug, Oncopeltus f a s c i a t u s . anthothecol  Cedrelone and  i n h i b i t e d E« s a u c i a growth by 90%, but not  f e e d i n g , when a p p l i e d i n d i e t a t 0.5 /imol/g fwt.  Cedrelone  a l s o i n h i b i t e d O. f a s c i a t u s m o l t i n g , w i t h an M D  o f 12.2  ^g/nymph.  In c o n t r a s t , a n t h o t h e c o l ,  50  w i t h an acetoxy  f u n c t i o n a t C - l l , was i n a c t i v e a g a i n s t O. f a s c i a t u s . seco compound gedunin, and t h e A,D-seco limonoids n o m i l i n , and pedonin were i n a c t i v e i n these harrisonin i n i t i a l l y  The D-  obacunone,  assays;  i n h i b i t e d f e e d i n g by neonate P. s a u c i a  but produced no long-term e f f e c t s on growth r a t e .  Bussein  i n h i b i t e d growth by 35% but entandrophragmin had no e f f e c t . A z a d i r a c h t i n was t h e most t o x i c compound examined i n t h i s study.  Peridroma s a u c i a growth ( E C  5 0  =0.4 nmol/g d i e t  iv  fwt), survivorship ( L C  5 0  =5.2 nmol/g), pupation,  pupal  weight, and a d u l t emergence were decreased i n a dosedependent manner.  Chemosensory a n t i f e e d a n t a c t i v i t y was  i m p l i c a t e d i n neonates but was much l e s s marked with instar larvae.  third  A z a d i r a c h t i n decreased r e l a t i v e growth  and  consumption r a t e s a t doses lower than those a f f e c t i n g n u t r i t i o n a l e f f i c i e n c y , o r f e e d i n g i n the c h o i c e  tests.  T h i s suggests an a c t i o n d i r e c t l y on the gut o r on the regulation of feeding.  neural  B i o a c t i v i t y o f other limonoids  did  not c o r r e l a t e w i t h measures o f s k e l e t a l o x i d a t i o n o r rearrangement, although these are dominant themes i n the e v o l u t i o n o f the  limonoids.  Melanoplus s a n a u i n i p e s  l a c k e d an a n t i f e e d a n t  t o a z a d i r a c h t i n , up t o c o n c e n t r a t i o n s  o f 500 ppm.  subsequent m o l t i n g was markedly e f f e c t e d .  However,  Application of  a z a d i r a c h t i n o r a l l y , t o p i c a l l y , o r by i n j e c t i o n , determination  response  allowed  o f the r o l e o f the gut and integument i n  l i m i t i n g the b i o a v a i l a b i l i t y o f t h i s compound t o p u t a t i v e t a r g e t s i t e ( s ) w i t h i n the i n s e c t .  The o r a l MD , 10.8 50  ng/g  i n s e c t fwt, was s i g n i f i c a n t l y h i g h e r than the i n j e c t e d MD , 50  3.01 gut.  pg/g,  i n d i c a t i n g a b a r r i e r t o b i o a v a i l a b i l i t y i n the  The o r a l a c t i v i t y o f a z a d i r a c h t i n was s y n e r g i s e d by  coadministration  o f p i p e r o n y l butoxide,  indicating that  b a r r i e r i s due l a r g e l y t o o x i d a t i v e metabolism. no s i g n i f i c a n t d i f f e r e n c e between t o p i c a l  (3.8  the  There was ng/g) and  i n j e c t e d a c t i v i t y , i n d i c a t i n g t h a t the integument does not pose a b a r r i e r t o b i o a v a i l a b i l i t y .  A z a d i r a c h t i n decreased  V  growth and consumption a t doses which d i d not a f f e c t n u t r i t i o n a l e f f i c i e n c y , again i n d i c a t i n g an e f f e c t on t h e gut o r n e u r a l r e g u l a t i o n o f f e e d i n g .  No d i f f e r e n c e was seen  i n n u t r i t i o n a l i n d i c e s o f nymphs t r e a t e d w i t h a t 10 and 15 pq/q,  azadirachtin  although these doses produced markedly  d i f f e r e n t e f f e c t s on m o l t i n g .  This observation  suggested  t h a t e f f e c t s on endocrine events a r e not d i r e c t l y r e l a t e d t o nutritional effects.  The e f f e c t s o f a z a d i r a c h t i n  treatment  were not a l l e v i a t e d by d i e t a r y supplementation w i t h c h o l e s t e r o l , and a z a d i r a c h t i n d i d not a f f e c t t h e hemolymph t r a n s p o r t o r metabolism o f  1 4  C - B - s i t o s t e r o l , indicating that  s t e r o l metabolism i s not t h e t a r g e t f o r a z a d i r a c h t i n activity.  Azadirachtin  a l s o d i d not form adducts w i t h  cysteine, suggesting that non-specific binding t o s u l f h y d r y l - r i c h p r o t e i n i s a l s o u n l i k e l y as a mechanism o f action.  vi  Table of Contents Page Abstract  i i  Table of Contents  vi  L i s t of Tables  xi  L i s t of Figures  xiii  L i s t of Abbreviations  xiv  Acknowledgements  xv  Chapter 1: General Introduction Literature Review  1 2  The Insect Response  10  Coevolution.  12  Evolution of Deterrent Responses  14  Phytochemistry  16  of the Meliaceae  Antifeedant and I n s e c t i c i d a l A c t i v i t y of Azadirachtin..  26  Regulation of Molting  33  Selection of Test Insects  39  Objectives of the Thesis  42  Chapter 2: I n s e c t i c i d a l and Growth-Reducing A c t i v i t y of F o l i a r Extracts from the Meliaceae Introduction  .  44  Materials and Methods  54  Results  61  A) Growth i n h i b i t i o n studies with Peridroma saucia  61  B) Antifeedant studies with Melanoplus sanauinipes  68  vii  C) Bioassays f o r a n t i b i o t i c and activity  phototoxic .  71  D Leaf Toughness  71  E- D e f e n s i v e C h a r a c t e r i s t i c s of Deciduous  and  Evergreen Meliaceae  72  Discussion  79  A) Crude E x t r a c t Screening B) Resource A v a i l a b i l i t y Hypothesis Chapter 3:  Phytochemical I n v e s t i g a t i o n of Aglaia  ..  Species  Introduction M a t e r i a l s and  79 83  89 Methods  91  A)  Sources of P l a n t M a t e r i a l  B)  I s o l a t i o n and  i d e n t i f i c a t i o n of secondary  metabolites i n Aglaia f o l i a g e Bl) Solvent  91  partitioning.  91 91  B2)  Normal-Phase Chromatography  92  B3)  HPLC  93  C)  Q u a l i t a t i v e and  D)  Bioassay  q u a n t i t a t i v e analyses  94 95  Results  97  A)  MeOH E x t r a c t S c r e e n i n g  97  B)  Solvent  97  C)  Chromatography  97  D) E)  Phy tochemi s t r y Q u a l i t a t i v e and Aglaia species  106  F)  Insect Bioassays  Discussion  Partitioning  q u a n t i t a t i v e comparison of  125 141 149  viii  A) Phytochemistry  .149  B) I n s e c t i c i d a l A c t i v i t y  Chapter 4 : E f f e c t s of Limonoids Peridroma  saucia  152  from the R u t a l e s on  and Oncopeltus  fasciatus  Introduction  156  M a t e r i a l s and Methods  175  A) Sources of Chemicals  175  B) I n s e c t s  .178  C) Growth S t u d i e s  179  D) Feeding Assays  180  E) N u t r i t i o n a l A n a l y s e s  179  F) Molt I n h i b i t i o n Assays  182  G) C o r r e l a t i o n Between E v o l u t i o n a r y Advancement and A c t i v i t y A g a i n s t I n s e c t s  183  Results  184  A) Growth and Feeding S t u d i e s : Limonoids  Other  than A z a d i r a c h t i n  184  B) Growth S t u d i e s w i t h A z a d i r a c h t i n  186  C) Feeding Choice T e s t s w i t h A z a d i r a c h t i n  190  D) D i e t U t i l i z a t i o n Experiments  192  E) Molt I n h i b i t i o n Assays  196  F) R e l a t i o n s h i p o f A n t i - i n s e c t A c t i v i t y t o O x i d a t i o n and S k e l e t a l Rearrangement Discussion  201 205  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  211  (Azadirachtin).  F) N u t r i t i o n a l I n d i c e s G) Limonoid E v o l u t i o n and Relationships  Chapter 5;  213 Structure-Activity  215  H) C o r r e l a t i o n of Phytochemistry w i t h M e t h a n o l i c Extract Screening  217  I) Comparison of I n s e c t i c i d a l and C y t o t o x i c Activity  219  E f f e c t s o f a z a d i r a c h t i n on the n u t r i t i o n development of the m i g r a t o r y grasshopper, sanguinipes  and Melanoplus  Fab.  Introduction  221  M a t e r i a l s and Methods  223  A) Experimental I n s e c t s  223  B) Source o f Chemicals.  223  C) A n t i f e e d a n t A c t i v i t y Assays  224  D) D i e t a r y U t i l i z a t i o n Experiments  224  E) Molt I n h i b i t i o n Assays  225  F) P i p e r o n y l Butoxide Synergism Assay  226  G) F e c u n d i t y Experiment  227  H) E f f e c t o f D i e t a r y S t e r o l s  228  I) S t e r o l T r a n s p o r t Experiment  228  J ) In v i t r o assay f o r the f o r m a t i o n of Results A) A n t i f e e d a n t assays  adducts....229 231 231  X  B) Growth and D i e t a r y U t i l i z a t i o n  231  C) Molt I n h i b i t i o n S t u d i e s . . .  233  D) Synergism by p i p e r o n y l butoxide  238  E) F e c u n d i t y Experiment  245  F) S t e r o l supplementation assays  245  G) In v i t r o assay f o r adduct f o r m a t i o n  250  Discussion  252  A) A n t i f e e d a n t and n u t r i t i o n a l e f f e c t s .  252  B) O r a l , t o p i c a l , and i n j e c t i o n experiments  254  C) F e c u n d i t y Experiment  257  D) S t e r o l s t u d i e s . . .  ..258  E) In v i t r o f o r m a t i o n of adducts  261  F) A g r i c u l t u r a l i m p l i c a t i o n s  262  Chapter 6 :  General Summary  265  References:  275  Appendix 1:  3/3  Appendix 2:  3/5  xi L i s t o f Tables T a b l e 2-1.  Sources, c o l l e c t o r s , and c o l l e c t i o n dates o f p l a n t m a t e r i a l used i n t h i s study....  T a b l e 2-2. Growth i n h i b i t o r y a c t i v i t y o f meliaceous e x t r a c t s on neonate P. s a u c i a  55  leaf 64  T a b l e 2-3. E x t r a c t i o n y i e l d (mg MeOH e x t r a c t / g l e a f dwt), l e a f toughness (N/cm ), l e a f pubescence (lower s u r f a c e o n l y ) (glab=glabrous, a x i l = h a i r s i n a x i l s o f main v e i n s , pub= pubescent), and " l e a f h a b i t " (deciduous [D] o r evergreen [E]) f o r s p e c i e s o f Meliaceae i n c l u d e d i n t h i s study 74 2  T a b l e 2-4. Comparison o f MeOH e x t r a c t t o x i c i t y (as E C t o Peridroma s a u c i a [% o f n a t u r a l c o n c e n t r a t i o n ] ) and toughness (N/cm ) between deciduous and evergreen s p e c i e s o f Meliaceae 78 5 0  2  T a b l e 3-1.  T y p i c a l r e s u l t s o f f l a s h column chromatography ( S i g e l , 240-400 mesh) o f A., odorata ( E t 0 s o l u b l e phase) 2  103  T a b l e 3-2. H-NMR s p e c t r a l data o f compounds 3, 4, and 5...110 1  T a b l e 3-3. A g l a i a odorata compounds: chromatographic b e h a v i o r and c o l o u r r e a c t i o n s w i t h E h r l i c h s reagent and v a n i l l i n reagent  127  T a b l e 3-4. Q u a l i t a t i v e TLC a n a l y s i s o f A g l a i a samples  128  T a b l e 3-5. HPLC r e t e n t i o n times compounds  129  (min) o f A g l a i a odorata  T a b l e 3-6. C o n c e n t r a t i o n (nq/q l e a f dwt) o f flavanones and bis-amides i n A g l a i a s p e c i e s , determined by a n a l y t i c a l HPLC 140 T a b l e 3-7. A g l a i a odorata compounds: c o n c e n t r a t i o n b i o a s s a y e d , and r e s u l t a n t p_. s a u c i a growth and s u r v i v o r s h i p (as % o f C o n t r o l )  143  T a b l e 3-8. E f f e c t o f combinations o f phytochemicals A g l a i a odorata on t h e growth o f neonate Peridroma s a u c i a (as % o f C o n t r o l )  147  from  T a b l e 3-9. E f f e c t o f Compound 6 on d i e t c h o i c e by neonate Peridroma s a u c i a  148  T a b l e 4-1. E f f e c t s o f limonoids on i n s e c t f e e d i n g and growth  159  xii Table 4 - 2 .  E f f e c t of limonoids on growth and d i e t c h o i c e of neonate Peridroma s a u c i a ....185  Table 4 - 3 .  E f f e c t of a z a d i r a c h t i n on Peridroma pupation and a d u l t emergence  saucia 189  T a b l e 4 - 4 . E f f e c t of a z a d i r a c h t i n on d i e t c h o i c e by neonate and t h i r d i n s t a r Peridroma s a u c i a 191 Table 4 - 5 .  E f f e c t of a z a d i r a c h t i n on t h i r d i n s t a r s a u c i a growth and n u t r i t i o n  Peridroma 193  Table 4 - 6 .  Comparison of E C v a l u e s of crude e x t r a c t s of Meliaceae w i t h p r e d i c t i o n s of a c t i v i t y based on c l a s s e s of limonoids r e p o r t e d t o occur i n the genera examined .218  T a b l e 5-1.  E f f e c t of a z a d i r a c h t i n on Melanoplus growth and n u t r i t i o n  T a b l e 5-2.  5 0  sanguinipes  R a d i o l a d e l l e d s t e r o l composition of c o n t r o l and a z a d i r a c h t i n - t r e a t e d Melanoplus s a n g u i n i p e s f e d 4- C-8-sitosterol 1 4  232  251  xiii  L i s t of Figures Figure 1-1.  Figure 1-2,  B i o s y n t h e t i c pathway l e a d i n g t o t h e formation of an apo-euphol type limonoid ( m o d i f i e d from S i d d i q u i e_t al., 1988)  19  B i o s y n t h e t i c pathway l e a d i n g t o t h e formation of a C-seco l i m o n o i d (modified from S i d d i q u i et a l . , 1988)  21  Figure 1-3.  B i o s y n t h e t i c pathway l e a d i n g t o t h e formation of a C-seco l i m o n o i d , a c c o r d i n g t o Jones e t a l . (1988) 23  Figure 1-4,  R e l a t i o n s h i p o f n e u r o s e c r e t o r y and neurohemal organs i n v o l v e d i n t h e endocrine r e g u l a t i o n o f molting i n i n s e c t s 53  Figure 2-1,  G r a p h i c a l d e p i c t i o n o f t h e assumed r e l a t i v e c o s t o f m a i n t a i n i n g a chemical o r p h y s i c a l l y -based defense a g a i n s t h e r b i v o r e s .  52  Figure 2-2,  Growth (as % o f c o n t r o l ) o f neonate Peridroma s a u c i a f e d a r t i f i c i a l d i e t t r e a t e d w i t h a MeOH extract of f o l i a g e of Azadirachta i n d i c a , M e l i a toosenden, o r M e l i a a z e d i r a c h a t 1, 2, or 3% o f n a t u r a l c o n c e n t r a t i o n 62  Figure 2-3,  Consumption o f g l a s s - f i b r e d i s c s , t r e a t e d w i t h 10% aqueous sucrose and MeOH e x t r a c t s o f A z a d i r a c h t a i n d i c a , M e l i a a z e d i r a c h , Turreae h o l s t i i , and A g l a i a odorata a t 1, 2.5, and 5 times n a t u r a l c o n c e n t r a t i o n (on a wt/wt b a s i s ) , by f i f t h i n s t a r nymphs o f Melanoplus sanguinipes 69  Figure 2-4,  R e l a t i o n s h i p between l e a f toughness ( i n N/cm ) and b i o a c t i v i t y o f t h e MeOH e x t r a c t o f f o l i a g e , c a l c u l a t e d as 1 0 0 - E C • 76 2  50  Figure 3-1.  E f f e c t o f f o l i a r MeOH e x t r a c t s o f A g l a i a odorata, A., o d o r a t i s s i m a , and A., a r g e n t i a on the growth o f neonate Peridroma s a u c i a  98  Figure 3-2.  Growth and s u r v i v o r s h i p o f neonate Peridroma saucia fed a r t i f i c i a l d i e t containing solvent e x t r a c t s o f A g l a i a odorata (Hawaiian sample)...100  Figure 3-3.  P r e p a r a t i v e HPLC chromatogram o f a growth i n h i b i t o r y f r a c t i o n from A g l a i a odorata  104  Figure 3-4,  S t r u c t u r e s o f dammarane t r i t e r p e n e s i s o l a t e d from Hawaiian samples o f A g l a i a odorata  108  xiv F i g u r e 3-5.  S t r u c t u r e s o f flavanones i s o l a t e d from Hawaiian samples o f A g l a i a odorata 113  Figure 3 - 6 .  Mass spectrum o f 3-hydroxy-5,7,4' -trimethoxyf lavanone  115  F i g u r e 3-7.  Mass spectrum fragments from 3'-hydroxy-5,7,4'trimethoxy d i h y d r o f l a v a n o n e 117  Figure 3 - 8 .  S t r u c t u r e s o f bis-amides i s o l a t e d from Hawaiian samples o f A g l a i a odorata 121  Figure 3 - 9 .  HPLC t r a c e o f E t 0 s o l u b l e f r a c t i o n from A g l a i a odorata (Hawaiian sample) 130  F i g u r e 3-10  HPLC t r a c e o f E t o O s o l u b l e f r a c t i o n from A g l a i a odorata ( T h a i l a n d sample) 132  2  F i g u r e 3-11. HPLC t r a c e o f E t 0 s o l u b l e f r a c t i o n from A g l a i a odorata (Taiwan sample) 134 2  F i g u r e 3-12. HPLC t r a c e o f t h e E t 0 s o l u b l e f r a c t i o n from Aglaia odoratissima  136  F i g u r e 3-13. HPLC t r a c e o f t h e E t 0 s o l u b l e f r a c t i o n from Aglaia argentia  138  2  2  F i g u r e 3-14. E f f e c t o f Compound 6 on t h e growth and s u r v i v o r s h i p o f neonate Peridroma s a u c i a F i g u r e 4-1.  S t r u c t u r e s o f limonoids  F i g u r e 4-2.  Major b i o s y n t h e t i c r o u t e s o f limonoids  144  i n c l u d e d i n T a b l e 4-1..168 i n the  Meliaceae  173  Figure 4 - 3 .  S t r u c t u r e s o f limonoids  Figure 4 - 4 .  E f f e c t o f d i e t a r y a z a d i r a c h t i n on growth and s u r v i v o r s h i p o f Peridroma s a u c i a neonates 187 P l o t o f RGR a g a i n s t RCR f o r l a r v a e o f Peridroma saucia fed d i e t containing various concentrations of azadirachtin 194 E f f e c t o f c e d r e l o n e on m o l t i n g success i n Oncopeltus f a s c i a t u s 196  F i g u r e 4-5. Figure 4 - 6 . F i g u r e 4-7. Figure 4 - 8 .  examined i n t h i s study.176  E f f e c t o f a z a d i r a c h t i n on m o l t i n g Oncopeltus f a s c i a t u s  success i n 199  Comparison o f i n s e c t growth i n h i b i t i n g a c t i v i t y of limonoids w i t h measurements o f o x i d a t i o n and s k e l e t a l rearrangement 203  XV  Figure 5-1.  Figure 5-2.  Figure 5-3.  Figure 5-4.  Morphogenic e f f e c t s of o r a l l y administered azadirachtin on f i f t h - i n s t a r nymphs of Melanoplus sanguinipes  234  E f f e c t of o r a l l y administered azadirachtin on molting success of f i f t h - i n s t a r nymphs of Melanoplus sanguinipes  236  E f f e c t of injected azadirachtin on molting success of f i f t h instar nymphs of Melanoplus sanguinipes  239  E f f e c t of t o p i c a l l y applied azadirachtin on molting success of f i f t h - i n s t a r nymphs of Melanoplus sanguinipes  241  Figure 5-5.  E f f e c t of co-administered piperonyl butoxide (PBO) on the molt i n h i b i t o r y a c t i v i t y of o r a l l y administered azadirachtin 243  Figure 5-6.  E f f e c t of o r a l l y administered azadirachtin on Melanoplus sanguinipes fecundity 246  Figure 5-7.  Pharmacokinetics of r a d i o l a b e l l e d s t e r o l s i n the hemolymph of control and azadirachtin treated nymphs  248  L i s t of Abbreviations  ACHN:  Acetonitrile.  AD:  Approximate D i g e s t a b i l i t y .  ANOVA:  Analysis of variance.  C H C 1 : Dichloromethane. 2  2  CHCI3: EC  :  5 0  Chloroform. E f f e c t i v e c o n c e n t r a t i o n f o r 50% e f f e c t inhibition).  ( i e growth  ECD:  E f f i c i e n c y of conversion of digested  food.  ECI:  E f f i c i e n c y o f conversion  food.  ED :  E f f e c t i v e dose f o r 50% e f f e c t inhibition).  Et 0:  Ethyl ether.  EtOAc:  Ethyl acetate.  EtOH:  Ethanol  HPLC:  High p r e s s u r e  IGR:  I n s e c t growth r e g u l a t o r .  JH:  J u v e n i l e hormone.  5 0  2  LC  5 0  :  of ingested  ( i e growth  (ethyl alcohol). liquid  chromatography.  L e t h a l c o n c e n t r a t i o n f o r 50% o f t r e a t e d p o p u l a t i o n .  LD :  L e t h a l dose f o r 50% o f t r e a t e d p o p u l a t i o n .  MC :  C o n c e n t r a t i o n which i n h i b i t s m o l t i n g i n 50% o f the t r e a t e d i n s e c t s .  MD :  Dose which i n h i b i t s m o l t i n g i n 50% o f t h e t r e a t e d insects.  MeOH:  Methanol (methyl a l c o h o l ) .  5 0  50  50  H-NMR: Proton n u c l e a r magnetic resonance  1  P.E.:  Petroleum e t h e r , b.p. .  PTTH:  P r o t h o r a c i c o t r o p h i c hormone.  spectroscopy.  R e l a t i v e consumption r a t e R e l a t i v e growth r a t e . T h i n l a y e r chromatography  xviii  Acknowledgements  F i r s t l y I thank my w i f e , C h r i s t y , f o r a l l h e r l o v e , support,  and p a t i e n c e .  dedicated  T h i s t h e s i s , and a l l t h a t I do, i s  t o her.  I thank my s u p e r v i s o r , Dr. G.H.N. Towers, f o r h i s support,  encouragement, and enthusiasm.  a l s o been a de facto generously and  Dr. M.B. Isman has  s u p e r v i s o r o f t h i s p r o j e c t , and gave  of h i s time and knowledge, not t o mention  l a b space.  support  Without h i s a s s i s t a n c e t h i s t h e s i s would not  have been p o s s i b l e .  I would a l s o l i k e t o thank my committee  members, Drs. I.E.P. T a y l o r and M. Shaw, f o r a l l t h e i r advice. Numerous people have c o n t r i b u t e d m a t e r i a l l y t o t h e r e s e a r c h r e p o r t e d here, by p r o v i d i n g p l a n t m a t e r i a l o r pure compounds f o r b i o a s s a y .  In p a r t i c u l a r I would l i k e t o thank  Dr. Kelsey Downum and Lee Swain, o f F l o r i d a I n t e r n a t i o n a l U n i v e r s i t y , Miami, f o r t h e i r generous a s s i s t a n c e and hospitality.  P l a n t m a t e r i a l was p r o v i d e d  by: Tim F l y n n ,  P a c i f i c T r o p i c a l Gardens, Maui; Dr. S. Dossagi,  Kenyan  N a t i o n a l Museum, N a i r o b i ; Dr. Zhun Jun, Kunming I n s t i t u t e o f Botany, China; Dr. G.B.S. S t r a l e y , U.B.C.; Dr. J.T. Arnason, U n i v e r s i t y o f Ottawa; and K a n t i P a t e l , U.B.C.  Dr. J.C.  Maxwell a s s i s t e d Dr. Towers i n t h e f i e l d i n T h a i l a n d . Samples o f limonoids  were p r o v i d e d  by: Dr. A. H a s s a n a l i ,  I.C.I.P.E., N a i r o b i ; Dr. I . Kubo, Berkley;  Dr. J.T. Arnason,  U n i v e r s i t y o f Ottawa; and Dr. J . Kaminski, U n i v e r s i t y o f  xix  Ottawa.  Dr, Hector B a r r i s o s - L o p e z s y n t h e s i z e d t h e odorine  isomers which allowed c o n f i d e n t i d e n t i f i c a t i o n o f t h e p l a n t consituents.  F e l i p e Balza provided invaluble assistance  w i t h t h e i d e n t i f i c a t i o n o f the f l a v a n o n e s .  Dr. B. Bhom  generously allowed me access t o h i s computerized the f l a v o n o i d Finally,  l i b r a r y of  literature. I wish t o acknowledge t h e c o n t r i b u t i o n o f my  f e l l o w graduate  students, p a r t i c u l a r l y  Webb, C r i s Guppy, Greg Salloom, Cathy McDougall,  Paul Spencer,  Murray  Sue D r e i e r , Shona E l l i s ,  and many o t h e r s , f o r much p l e a s a n t  c o n v e r s a t i o n , c o f f e e , and p a r t i c u l a r l y  f o r h e l p i n g me keep a  sense o f p e r s p e c t i v e and humour about t h e l a s t few y e a r s .  1  Chapter 1 : General  Introduction  I n t e r a c t i o n s between phytophagous i n s e c t s and t h e i r  host  p l a n t s c o n s t i t u t e perhaps the l a r g e s t c l a s s o f i n t e r s p e c i f i c i n t e r a c t i o n s i n t h e t e r r e s t r i a l biosphere.  Study o f t h e  f a c t o r s which serve t o r e g u l a t e these i n t e r a c t i o n s has become one o f the most dynamic f i e l d s i n e c o l o g y . the i n t e r a c t i o n s a r e o f d i r e c t r e l e v a n c e  Many o f  t o Man, e s p e c i a l l y  when t h e p l a n t s i n v o l v e d a r e o f economic importance. Chemical ecology ( t h e study o f b i o c h e m i c a l l y  mediated  i n t e r a c t i o n s between s p e c i e s ) t h e r e f o r e has two f a c e t s : advances i n theory may y i e l d novel p e s t management s t r a t e g i e s ( R a f f a , 1986), and t h e study o f a p p l i e d a g r i c u l t u r a l problems may i n c r e a s e our understanding o f problems ranging  from t h e s t r u c t u r e o f communities t o t h e  nature o f e v o l u t i o n a r y  processes.  This t h e s i s reports a s e r i e s of i n v e s t i g a t i o n s of the e f f e c t o f phytochemicals from members o f the t r o p i c a l f a m i l y M e l i a c e a e on h e r b i v o r o u s i n s e c t s .  plant  T h i s f a m i l y was  chosen f o r i n v e s t i g a t i o n because, although some s p e c i e s as t h e neem t r e e , A z a d i r a c h t a  such  i n d i c a , a r e known t o produce  i n s e c t i c i d a l phytochemicals, most members o f t h e f a m i l y have not y e t been examined f o r t h i s p r o p e r t y .  The phytochemistry  of t h e f a m i l y i s f a i r l y w e l l known, which p r o v i d e s a convenient b a s i s f o r such s t u d i e s .  F i n a l l y , the toxicology  and mode o f a c t i o n o f s e v e r a l known i n s e c t i c i d a l compounds requires  clairification.  2  L i t e r a t u r e Review The p o t e n t i a l impact of i n s e c t h e r b i v o r y on p l a n t p o p u l a t i o n s i s w e l l i l l u s t r a t e d by examples from the b i o l o g i c a l c o n t r o l o f weeds (Crawley, 1989; K r i s c h i k and Denno, 1983).  For i n s t a n c e , klamath weed  (Hypericum  perforatum) (Hypericaceae) p o p u l a t i o n s i n western North America were reduced by over 95% f o l l o w i n g the i n t r o d u c t i o n of the b e e t l e C h r y s o l i n a quadrigemina (Holloway, 1964).  (Chrysomelidae)  At p r e s e n t klamath weed i s l a r g e l y  c o n f i n e d t o shaded h a b i t a t s where C_. quadrigemina  prefers  not t o o v i p o s i t .  introduced  Opuntia s t r i c t a  i n t o A u s t r a l i a i n 1839. cover 24 X 1 0  6  (Cactaceae) was  By 1920 t h i s c a c t u s had spread t o  ha, about h a l f of which was  i n f e s t e d by  stands so dense as t o be impenetrable (Holloway, 1964). O p u n t i a - f e e d i n a moth C a c t o b l a s t i s cactorum was 1925,  The  introduced i n  and by 1930 most areas o f Opuntia had been k i l l e d .  Opuntia i s p r e s e n t l y r e s t r i c t e d t o s m a l l  isolated  populations. Insect exclusion studies, using i n s e c t i c i d e s or e x c l u s i o n cages, p r o v i d e the s t r o n g e s t evidence t h a t h e r b i v o r y can a f f e c t the s t r u c t u r e of p l a n t (Crawley, 1989).  insect  communities  D e s p i t e p o t e n t i a l m e t h o d o l o g i c a l problems,  i n c l u d i n g p h y t o t o x i c o r s t i m u l a t o r y e f f e c t s of i n s e c t i c i d e s and h e r b i v o r e rebound i n the absence o f n a t u r a l over h a l f of the s t u d i e s i n d i c a t e changes  enemies,  i n species  composition when h e r b i v o r o u s i n s e c t s are excluded from p l a n t communities  (Crawley, 1989).  In some c a s e s , even a s i n g l e  3  herbivore  s p e c i e s can exclude a p l a n t from p o t e n t i a l  habitats.  When caged and uncaged Machaeranthera canescans  (Asteraceae) were t r a n s p l a n t e d grasshopper H e s p e r o t e t t i x  t o areas populated by  the  v i r i d i s , the uncaged p l a n t s were  completely d e f o l i a t e d a f t e r an average o f 7.4 of the caged p l a n t s s u r v i v e d t o f l o w e r i n g  days, but  (Parker  and  77%  Root,  1981). Despite terrestrial  t h i s p o t e n t i a l impact on p l a n t s , environment remains green.  of h e r b i v o r y population  are r a r e l y e v i d e n t  Indeed, the e f f e c t s  i n the f i e l d , and  or competition  l i m i t a t i o n s ( i . e . Slobodkin ( H a i r s t o n e t a l . , 1960).  t h i s view i n s e c t p o p u l a t i o n s  and  populations 1984;  et, a l . , to  densities  parasites,  so cannot have a major impact on  ( H a s s e l l and Anderson, 1984;  be  According  are r e g u l a t e d a t low  by n a t u r a l enemies, p a r t i c u l a r l y p r e d a t o r s , diseases,  plant  dynamics have g e n e r a l l y been thought t o  r e g u l a t e d by r e s o u r c e 1967)  the  and  plant  Strong et, a l . ,  Bernays and Graham, 1988). However, i t a l s o has  been r e a l i z e d t h a t most p l a n t s  possess r e s i s t a n c e mechanisms t h a t l i m i t both the range of herbivore  s p e c i e s which can i n f l i c t damage and  which the damage can occur.  the r a t e a t  Consequently, each p l a n t  species i s susceptible to herbivory  by o n l y a s m a l l  percentage of the phytophagous i n s e c t s t o which i t i s exposed.  For example, i n a Costa R i c a n dry f o r e s t each  p l a n t s p e c i e s supports an average of f o u r t o e i g h t lepidopteran  s p e c i e s ; the g r e a t e s t h e r b i v o r e  l o a d supported  4  by any  one  p l a n t i s 17 out of the  3140  c a t e r p i l l a r s known from t h i s h a b i t a t 50%  species  of  (Janzen, 1988).  of the c a t e r p i l l a r s are monophagous, and  virtually a l l  of the r e s t feed on l e s s than f i v e r e l a t e d p l a n t fewer than 10% polyphagous.  of the l e p i d o p t e r a n Tabulations  a s i m i l a r pattern Scott,  (Strong  indicate  Thorsteinson,  1960;  1988).  optimal substrates  f o r growth.  i p s i l o n . f e d corn (Zea mays)  diet  considered  from temperate l o c a l i t i e s  Even a c c e p t a b l e h o s t p l a n t s may  at only  species;  s p e c i e s can be  e t a l . , 1984;  Over  r  p r o v i d e l e s s than  B l a c k cutworms, A a r o t i s a p r e f e r r e d h o s t p l a n t , grew  15% the r a t e of s i b l i n g s r e a r e d on an  (Reese and  F i e l d , 1986).  artificial  Low-quality food may  i n c r e a s e the e f f e c t i v e n e s s of n a t u r a l enemies and herbivore  populations  also  maintain  a t low d e n s i t i e s (Lawton and  McNeil,  Although d i f f e r e n c e s i n water ( S c r i b e r , 1979;  Scriber  1979).  and  Slansky, 1981)  S c r i b e r , 1984) contain  may  and  nitrogen  content (Mattson,  1980;  a f f e c t i n s e c t s , most p l a n t s appear t o  s u f f i c i e n t n u t r i e n t s t o support i n s e c t growth  (Fraenkel,  1959).  Resistance to herbivory  i s to a  large  e x t e n t expressed a t the stage of host p l a n t s e l e c t i o n  and  r e s u l t s from chemical and  the  insect. perceived  t a c t i l e s t i m u l i r e c e i v e d by  P l a n t a r c h i t e c t u r e , p r o f i l e , c o l o u r , and by mobile phytophagous i n s e c t s and  r o l e i n l o c a t i n g p o t e n t i a l host p l a n t s S t r i c k l e r , 1984).  odor  are  o f t e n have a  (Miller  and  However, the primary f a c t o r governing  5  h o s t p l a n t acceptance o r r e j e c t i o n appears t o be the p r o f i l e of  "secondary m e t a b o l i t e s " p r e s e n t i n the p l a n t (Whittaker  and Feeny, 1971;  Feeny, 1976; Rhoades and Cates,  Bernays and Chapman, 1977,  1976;  1978; Rosenthal and Janzen,  1979). The term "secondary m e t a b o l i t e " was German chemist A. K o s s e l i n 1891; term ( c i t e d i n Mothes, 1980;  first  used by the  i n 1896 he d e f i n e d  this  Schneider, 1988):  "The s e a r c h and d e s c r i p t i o n of those atomic complexes, which are the essence o f l i f e are the f o u n d a t i o n f o r the i n v e s t i g a t i o n o f the l i f e p r o c e s s e s . I propose t o c a l l the e s s e n t i a l components of the c e l l primary and those t h a t are not found i n a l l the c e l l s t h a t have the c a p a c i t y t o develop, secondary. The d e c i s i o n whether a substance i s a primary o r a secondary one i s i n some cases d i f f i c u l t . " The d i v e r s i t y of the secondary m e t a b o l i t e s i s a s t o n i s h i n g : Swain (1977) e s t i m a t e d t h a t 100,000-400,000 such n a t u r a l p r o d u c t s may  exist.  Each p l a n t c o n t a i n s from a  few t o hundreds o f t h e s e compounds; t y p i c a l l y they v a r y q u a l i t a t i v e l y and q u a n t i t a t i v e l y between organs o f the same i n d i v i d u a l , between t i s s u e s o f d i f f e r e n t ages, and between i n d i v i d u a l s and p o p u l a t i o n s w i t h i n a s p e c i e s (McKey, 1979). Although r o l e s i n "primary" metabolism have been i d e n t i f i e d or  p o s t u l a t e d f o r a few secondary m e t a b o l i t e s ( S e i g l e r and  P r i c e , 1976), f o r the most p a r t they seem t o f u n c t i o n as s i g n a l s m e d i a t i n g i n t e r s p e c i f i c i n t e r a c t i o n s (Swain,  1977).  S p e c i f i c terms have been developed t o d e s c r i b e the s i g n a l l i n g r o l e o f secondary m e t a b o l i t e s (Shorey,  1977;  Nordlund, 1981).  mediate  Semiochemicals, chemicals which  6  i n t e r a c t i o n s between organisms, can be d i v i d e d  into  pheromones, which mediate i n t r a s p e c i f i c i n t e r a c t i o n s , and a l l e l o c h e m i c s , which mediate i n t e r s p e c i f i c (Nordlund, 1981).  interactions  Kairomones a r e compounds which, when  r e l e a s e d from t h e e m i t t i n g organism, b e n e f i t t h e r e c e i v i n g organism.  Synomones b e n e f i t both t h e e m i t t e r and t h e  r e c e i v e r , and f u n c t i o n i n m u t u a l i s t i c  interactions.  Allomones a r e d e l e t e r i o u s t o t h e r e c e i v i n g organism, and i n c l u d e r e p e l l a n t s and t o x i n s used f o r defense. As i m p l i e d from t h e above d e f i n i t i o n s , secondary m e t a b o l i t e s may a f f e c t both t h e b e h a v i o r and t h e p h y s i o l o g y of  phytophagous  insects.  The range o f t o x i c  (physiological)  e f f e c t s of plant a l l e l o c h e m i c a l s i s impressive.  Non-protein  amino a c i d s may be i n c o r p o r a t e d i n t o p r o t e i n s , r e s u l t i n g i n n o n - f u n c t i o n a l enzymes o r s t r u c t u r a l p r o t e i n (Rosenthal and B e l l , 1979).  Many a l k a l o i d s a r e n e u r o t o x i c t o v e r t e b r a t e  and i n s e c t h e r b i v o r e s (Robinson, 1979), and one, n i c o t i n e , found use as an i n s e c t i c i d e (Schmeltz, 1971; Jacobson and Crosby, 1971).  Other p o t e n t n e u r o t o x i n s i n c l u d e t h e  p y r e t h r i n s (Matsui and Yamamoto, 1971; Mabry and G i l l , 1979), i s o b u t y l a m i d e s (Jacobson, 1971; Miyakado e t a l . , 1989), some a l i p h a t i c a c e t y l e n e s i n c l u d i n g  cicutitoxin  (Towers and Wat, 1978; Robinson, 1980), and some mono- and d i t e r p e n e s (Ryan and Byrne, 1988). (Mabry and G i l l ,  Sesquiterpene lactones  1979; Pieman e t a l . ,  1979), drimane  s e s q u i t e r p e n e s (Ma, 1975; D ' I s c h i a e t a l . , al.,  1982; Asakawa e t  1988) and some hydroxamic a c i d s (Niemeyer e t a l . ,  1982)  7  r e a c t w i t h amines o r s u l f h y d r y l amino a c i d s t o a l k y l a t e proteins.  Saponins (Applebaum and B i r k , 1979) and t h e  s t e r o i d a l a l k a l o i d tomatine (Duffey  and Bloem, 1987; Bloem  e t a l . , 1989) complex w i t h d i e t a r y s t e r o l s and reduce t h e i r a v a i l a b i l i t y t o the i n s e c t .  Some compounds r e q u i r e  near-UV  l i g h t f o r t o x i c i t y : these i n c l u d e t h e l i n e a r and angular furanocoumarins, B - c a r b o l i n e  a l k a l o i d s , and i s o q u i n o l i n e  a l k a l o i d s , which photobind t o DNA  (Berenbaum, 1978, 1983;  Berenbaum and Feeny, 1981; Towers and Champagne, 1987), and many a l i p h a t i c and phenyl a c e t y l e n e s d i s r u p t membranes  and thiophenes, which  (Downum e t a l . , 1984; Champagne e t a l . ,  1986). Some n a t u r a l p r o d u c t s d i s r u p t hormonal r e g u l a t i o n o f p h y s i o l o g i c a l processes i n i n s e c t s .  J u v e n i l e hormone t i t r e s  are reduced by t h e a l l a t i c i d a l precocenes (Bowers, 1983); o t h e r compounds a r e j u v e n i l e hormone analogues and produce n o n - r e p r o d u c t i v e supernumerary i n s t a r s i n s e n s i t i v e i n s e c t s (Slama, 1979; Bowers, 1983).  The p r o t e c t i v e r o l e o f t h e  phytoecdysones i s c o n t r o v e r s i a l (Jones, 1983): w h i l e these molting  hormone analogues a r e h i g h l y t o x i c when i n j e c t e d  i n t o t h e hemocoel, they may be much l e s s a c t i v e oral administration, excretion  (Feyereisen  following  perhaps due t o r a p i d metabolism and et. a l . , 1976).  i n d e t a i l l a t e r , the l i m o n o i d  As w i l l be  discussed  a z a d i r a c h t i n d i s r u p t s both  j u v e n i l e hormone and ecdysone t i t r e s , r e s u l t i n g i n molt f a i l u r e or chemosterilization of adults.  8  Many secondary m e t a b o l i t e s d e t e r f e e d i n g o r o v i p o s i t i o n by  insects.  D e t e c t i o n o f such compounds i s accomplished v i a  chemoreceptors l o c a t e d the  on t h e m a x i l l a r y  epipharyngeal c a v i t y , and f r e q u e n t l y  ovipositors  on t h e t a r s i and  (Chapman and Blaney, 1979; Chapman, 1982;  Hanson, 1983, 1987). usually  p a l p s , labrum, i n  Sampling o f p o t e n t i a l h o s t p l a n t s i s  accomplished by s t e r e o t y p i c  b e h a v i o r s which maximize  c o n t a c t o f t h e chemoreceptors w i t h t h e p l a n t  surface  ( F r a z i e r , 1986; Chapman and Bernays, 1989).  Butterflies  sample p o t e n t i a l o v i p o s i t i o n s i t e s by r a p i d l y drumming t h e i r t a r s i against or scratching 1987;  Renwick, 1989).  on t h e l e a f s u r f a c e  Locusts i n i t i a l l y  (Rothschild,  touch a l e a f w i t h  t h e i r antennae, then b r i n g t h e labrum i n t o c o n t a c t , and r a p i d l y touch t h e t i p s o f t h e m a x i l l a r y ( W i l l i a m s , 1954). initially  p a l p i t o the leaf  F i n a l l y , a t e s t b i t e may be taken,  e x p r e s s i n g l e a f sap without removing t i s s u e .  In  grasshoppers a s i m i l a r sequence i s seen b u t t h e antennae p l a y a l e s s e r r o l e (Mulkern, 1969).  F u r t h e r examples a r e  g i v e n by Chapman and Bernays (1989). Contact chemoreceptor morphology and p h y s i o l o g y have recently 1987).  been reviewed ( S t a d l e r  1984; F r a z i e r , 1986; Hanson,  In b r i e f , chemoreceptors (sensory h a i r s o r s e n s i l l a  styloconica)  are characterized  by a s i n g l e a p i c a l pore which  admits t o a c a v i t y f i l l e d w i t h a " s e n s i l l u m dendrites.  l i q u o r " bathing  Compounds a r e d e t e c t e d as they b i n d t o s p e c i f i c  receptors or "transducing proteins" e l i c i t i n g an a c t i o n p o t e n t i a l .  (Norris,  1988),  C l a s s i c a l l y f o u r types o f  9  chemosensory c e l l s have been r e c o g n i z e d ,  based on  the  substances t o which they show the maximum response. are the sugar, s a l t , water, and anion  These  (or second s a l t )  c e l l s ; however, each c e l l a l s o has o t h e r f u n c t i o n s which i n c l u d e the d e t e c t i o n of f e e d i n g d e t e r r e n t s . to stimulating a deterrent c e l l ,  Alternatively  some secondary  metabolites  i n h i b i t the a c t i v i t y of the sugar r e c e p t o r c e l l M i t c h e l l and S u t c l i f f , one  1984).  (Ma,  1977;  In a few monophagous i n s e c t s  r e c e p t o r has become s p e c i a l i z e d f o r the d e t e c t i o n of  s p e c i f i c secondary m e t a b o l i t e s  ("sign s t i m u l i " )  c h a r a c t e r i s t i c of t h e i r host p l a n t .  Examples i n c l u d e  glucosinolate receptor i n P i e r i s brassicae 1979)  the  (Nielsen et a l . ,  and the h y p e r i c i n r e c e p t o r i n C h r y s o l i n a  brunsvicensis  (Rees, 1969). Only i n a few  cases do secondary m e t a b o l i t e s  " a l l - o r - n o n e " response by a c t i v a t i n g a s p e c i f i c  mediate an  neural  pathway, termed a " l a b e l l e d l i n e " .  D i a b r o t i c i t e beetles  s t i m u l a t e d t o feed on any  containing  substrate  are  c u c u r b i t a c i n s , t r i t e r p e n e s c h a r a c t e r i s t i c of t h e i r normal host p l a n t ( M e t c a l f and  Lampman, 1989).  The  desert locust,  S. g r e g a r i a , w i l l s t a r v e t o death r a t h e r than consume food t r e a t e d with a z a d i r a c h t i n at concentrations ng/cm  2  (Blaney,  1980).  as low as 4  However, i n most cases the i n s e c t  response i s more l a b i l e , and may combination of d e t e r r e n t s and  be a f f e c t e d by  stimulants present  p h y s i o l o g i c a l s t a t e of the i n s e c t ( D e t h i e r , 1982; S t r i c k l e r , 1984;  Reese and  Schmidt, 1986).  the and  the  Miller  and  A p l a n t which i s  10  unacceptable t o a s a t i a t e d i n s e c t may  w e l l be consumed,  a l b e i t i n s m a l l meals, by a hungry one. sensory i n f o r m a t i o n appears  P r o c e s s i n g of  t o occur a t the l e v e l of the  c e n t r a l nervous system r a t h e r than a t the p e r i p h e r a l s e n s i l l a , probably v i a a c r o s s - f i b r e p a t t e r n i n g ( D e t h i e r , 1982).  Schoonhoven and Blom (1988) suggested  a simple model  f o r n e u r a l p r o c e s s i n g i n P i e r i s b r a s s i c a e i n which phagostimulant  and d e t e r r e n t s i g n a l s are simply summed, with  each d e t e r r e n t a c t i o n p o t e n t i a l e q u i v a l e n t t o 2.5  stimulant  action potentials.  The  I n s e c t Response  Exposure t o a l l e l o c h e m i c a l s i n host p l a n t t i s s u e s can f o r r e s i s t a n c e mechanisms i n phytophagous i n s e c t s ; s e l e c t i o n may  Ingested phytochemicals  may  e x c r e t e d i n t a c t , presumably due t o non-absorption; i n c l u d e c o c a i n e i n E t o r i a n o y e s i f e e d i n g on (Blum e t a l . ,  gregaria  this  i n v o l v e b e h a v i o r a l , p h y s i o l o g i c a l , or  biochemical resistance.  coca  select  be  examples  Erythroxylum  1981), c a r d e n o l i d e s i n S c h i s t o c e r c a  (Scudder and Meredeth, 1982), and thiophenes i n  Melanoplus  sanguinipes  unpublished d a t a ) .  ( S m i r l e , Champagne, and Isman,  Once a compound p e n e t r a t e s the mucosal  membrane of the gut i t may  be c o n f r o n t e d by a v a r i e t y of  p h y s i o l o g i c a l or b i o c h e m i c a l r e s i s t a n c e mechanisms ( B r a t t s t e n , 1988, 1986).  1986,  1979;  Ahmad, 1986;  Ahmad e t a l . .  The major enzyme systems i n v o l v e d are the cytochrome  P450-based microsomal m i x e d - f u n c t i o n o x i d a s e s  (MFOs),  11  r e c e n t l y renamed p o l y s u b s t r a t e  monooxygenases (PSMOs)  ( B r a t t s t e n , 1988), a s s o c i a t e d with the smooth endoplasmic reticulum.  The  overlapping,  MFOs are a f a m i l y of monooxygenases w i t h  broad s u b s t r a t e a f f i n i t i e s , whose net e f f e c t i s  t o o x i d i z e l i p o p h i l i c compounds t o more w a t e r - s o l u b l e and thereby f a c i l i t a t e e x c r e t i o n . i n v o l v e d i n d e t o x i f i c a t i o n and glutathione  S transferases  Other enzyme systems  excretion include  MFOs, the GSTs are not membrane bound and  Unlike  Hexose t r a n s f e r a s e s  conjugate Although they  i n v o l v e d i n the e x c r e t i o n of i n s e c t i c i d e s ,  t h e i r importance i n p l a n t - i n s e c t i n t e r a c t i o n s has extensively studied  not been  ( B r a t t s t e n , 1988).  These enzyme systems are present p a r t i c u l a r l y i n the midgut and  i n high  concentrations  f a t body but may  a l s o occur  i n other t i s s u e s i n c l u d i n g the M a l p i g h i a n t u b u l e s . i n p a r t i c u l a r may  may  (Berenbaum, 1986;  ( T e r r i e r e , 1984;  Yu,  1983,  1986).  also involve t a r g e t - s i t e i n s e n s i t i v i t y  B r a t t s t e n , 1988).  i n s e n s i t i v i t y of N a / +  K +  Examples i n c l u d e  ATPases from milkweed bugs  monarch b u t t e r f l i e s t o i n a c t i v a t i o n by c a r d e n o l i d e s and J u n g r e i s ,  The MFOs  be r a p i d l y induced by exposure t o c e r t a i n  secondary m e t a b o l i t e s Resistance  the  can a t t a c k water-  a l l e l o c h e m i c a l s t o g l u c o s e or other sugars. are known t o be  the  (GSTs), which conjugate compounds  w i t h an e l e c t r o p h i l i c c e n t e r t o g l u t a t h i o n e .  soluble allelochemicals.  forms  1977;  Moore and  Scudder, 1985).  the  and (Vaughn  Studies  s y n t h e t i c i n s e c t i c i d e s suggest t h a t t a r g e t - s i t e i n s e n s i t i v i t y appears a f t e r metabolism-based r e s i s t a n c e  with  12  ( B r a t t s t e n , 1986;  Berenbaum, 1986).  I t may  s e l e c t i o n p r e s s u r e comes from extremely  occur when  toxic  a l l e l o c h e m i c a l s , or when an a l l e l o c h e m i c a l i s sequestered for defensive  uses.  Coevolution The tendency f o r p a r t i c u l a r h e r b i v o r e s t o a s s o c i a t e w i t h p a r t i c u l a r host p l a n t s has l o n g been noted was  (Brues, 1924)  and  a t t r i b u t e d t o c o e v o l u t i o n by E h r l i c h and Raven (1964).  A c c o r d i n g t o t h e i r model, the appearance, by mutation or recombination,  of a novel a l l e l o c h e m i c a l i n a p l a n t w i l l  r e s u l t i n a decrease  i n herbivore pressure, leading to  evolutionary radiation.  The  a v a i l a b i l i t y of  competitor-free  space w i l l then s e l e c t f o r r e s i s t a n t p o p u l a t i o n s among the h e r b i v o r e fauna.  Once a b l e t o e x p l o i t t h i s r e s o u r c e ,  the  h e r b i v o r e s w i l l a l s o undergo e v o l u t i o n a r y r a d i a t i o n . E v e n t u a l l y the r e s i s t a n t h e r b i v o r e s w i l l again s e l e c t f o r f u r t h e r n o v e l a l l e l o c h e m i c a l s , which w i l l continue  the  selection for resistance. T h i s p a t t e r n of r e c i p r o c a l e v o l u t i o n may  lead to  p r o g r e s s i v e l y t i g h t e r a s s o c i a t i o n s between host p l a n t s and t h e i r adapted i n s e c t fauna,  i n p a r t a t l e a s t due t o the c o s t  of m a i n t a i n i n g m e t a b o l i c defenses of  a g a i n s t the e n t i r e range  a l l e l o c h e m i c a l s t o which a g e n e r a l i s t i s p o t e n t i a l l y  exposed.  However, Neal  (1985) was  unable t o show any  m e t a b o l i c c o s t a s s o c i a t e d with the i n d u c t i o n of n i n e - f o l d h i g h e r MFO  titres  i n the g e n e r a l i s t c a t e r p i l l a r  Heliothis  13  zea,  suggesting that other f a c t o r s are involved  evolution  i n the  of a l i m i t e d h o s t - p l a n t range.  Although t h e r e i s no doubt t h a t e v o l u t i o n insect associations  of plant-  has o c c u r r e d , few examples a r e known  where t h e r e i s evidence o f the r e c i p r o c a l  selection  p r e s s u r e s e s s e n t i a l t o c o e v o l u t i o n a r y t h e o r y (Janzen, 1 9 8 0 ; Thompson, 1 9 8 2 ) . association  Possibly  the most c o n v i n c i n g case i s the  o f a s p e c i a l i z e d i n s e c t fauna, p a r t i c u l a r l y  p a p i l i o n i d b u t t e r f l i e s , with plants  which c o n t a i n l i n e a r and  angular furanocoumarins (Berenbaum, 1 9 7 8 , 1 9 8 1 , Berenbaum and Feeny, 1 9 8 1 ) .  1983;  In t h i s case t h e p l a n t  e l a b o r a t e s not o n l y the t o x i c furanocoumarins but a l s o a s e r i e s of synergists,  t h e methylenedioxyphenyl compounds  m y r i s t i c i n , s a f r o l e , and i s o s a f r o l e (Berenbaum and Neal, 1 9 8 7 ; Neal, 1 9 8 9 ) .  However, the i n s e c t  mixed-function  o x i d a s e s a r e not o n l y r e s i s t a n t t o the s y n e r g i s t s Berenbaum,  (Neal and  1989) but a r e s p e c i f i c a l l y induced by t h e  furanocoumarins (Cohen e t a i . , 1 9 8 9 ) . More commonly h e r b i v o r e s e l e c t i o n p r e s s u r e r e s u l t s from a diverse  fauna, which may a l s o i n t e r a c t as c o m p e t i t o r s ;  c o e v o l u t i o n i n such a case i s s a i d t o be " d i f f u s e " (Fox, 1 9 8 2 , 1 9 8 8 ; Fox and Morrow, 1 9 8 1 ) . evolution  of t i g h t a s s o c i a t i o n s  In such a s i t u a t i o n the  between h o s t p l a n t s  t h e i r i n s e c t fauna i s l e s s l i k e l y ; r a t h e r ,  and  any change i n  s e l e c t i o n p r e s s u r e due t o a change i n any of the participants  w i l l a f f e c t a l l members o f the  association.  14  E v o l u t i o n of D e t e r r e n t  Responses  A c c o r d i n g t o the p r e v a i l i n g view, the presence of a l l e l o c h e m i c a l s i n a p l a n t can s e l e c t f o r p h y s i o l o g i c a l r e s i s t a n c e o r the a b i l i t y t o d e t e c t and allelochemicals  ( f e e d i n g and  ( S c r i b e r , 1984;  Berenbaum, 1986).  t h a t , due  those  o v i p o s i t i o n deterrence) I t i s generally  thought  t o the a d a p t i v e nature of i n s e c t b e h a v i o r ,  antifeedants  which are not accompanied by t o x i c i t y w i l l  q u i c k l y overcome i n e v o l u t i o n a r y antifeedants,  therefore,  time.  Chapman (1987) ( a l s o Bernays and  be  Most or a l l  should be t o x i c i f i n g e s t e d ,  i n v a r i a b l y be a s s o c i a t e d w i t h t o x i n s .  challenged  avoid  or  R e c e n t l y Bernays  Graham, 1988)  and  have  t h i s view; they p o i n t t o s e v e r a l s t u d i e s i n which  non-host p l a n t s were shown t o support normal l a r v a l growth, t o s t u d i e s which have f a i l e d t o f i n d t o x i c i t y with antifeedant  a c t i v i t y , and  associated  t o s t u d i e s of o v i p o s i t i o n  b e h a v i o r of b u t t e r f l i e s i n which p l a n t s which were r e j e c t e d c o u l d s u s t a i n l a r v a l development.  They suggest t h a t  o t h e r than p h y s i o l o g i c a l s p e c i a l i z a t i o n and t o x i c i t y may  factors  the avoidance of  mediate h o s t p l a n t s p e c i a l i z a t i o n ; i n  p a r t i c u l a r t h a t g e n e r a l i s t i n s e c t s are s u b j e c t t o h i g h e r r a t e s of p r e d a t i o n  from g e n e r a l i s t p r e d a t o r s ,  s p e c i a l i s t herbivores  enjoy a reduced p r e d a t i o n  support of t h i s concept, Bernays (1988) has g e n e r a l i s t herbivores  and  that  rate.  In  shown t h a t  are more l i k e l y t o be found and  eaten  by the g e n e r a l i s t wasp M i s c h o c y t t a r u s f l a v i t a r s u s than s p e c i a l i s t s when both are exposed t o the p r e d a t o r on  are  the  15  same host p l a n t .  Polyphagous c a t e r p i l l a r s are a l s o more  p a l a t a b l e than aposematic or c r y p t i c s p e c i a l i s t s t o g e n e r a l i s t predator  Iridomyrmex h u m i l i s  (Bernays  the  and  C o r n e l i u s , 1989). C r u c i a l t o the r e j e c t i o n of p l a n t chemistry  as  important primary f a c t o r d r i v i n g the e v o l u t i o n of s p e c i f i c i t y i s the c o n c l u s i o n t h a t most d e t e r r e n t s  an  host-plant are  "harmless", t h a t i s , t h a t they are not i n themselves t o x i c , nor are they u s u a l l y a s s o c i a t e d w i t h t o x i n s (Bernays Graham, 1988).  However, as noted by S c h u l t z  argued l a r g e l y from "absence of evidence" "evidence of absence". due  The  limonoids  and  (1988), t h i s i s  r a t h e r than  are a case i n p o i n t :  t o t h e i r b i t t e r t a s t e t o v e r t e b r a t e s , they have been  considered  t o be p r i m a r i l y f e e d i n g d e t e r r e n t s .  As a r e s u l t ,  the m a j o r i t y of p u b l i s h e d assays of these compounds have been designed t o d e t e c t o n l y a n t i f e e d a n t a c t i v i t y i n Chapter 4 ) , c r e a t i n g the impression  (reviewed  t h a t t h i s i s the  only  b i o l o g i c a l a c t i v i t y these compounds possess ( T a y l o r , 1987). Limonoids, i n common w i t h most o t h e r known a n t i f e e d a n t s , need r e - e v a l u a t i o n w i t h b i o a s s a y s  capable of d e t e c t i n g  b i o l o g i c a l a c t i v i t y other than f e e d i n g i n h i b i t i o n ; t h i s i s one  focus of Chapter  4.  16 Phybochemistry o f t h e M e l i a c e a e Phytochemically,  t h e f a m i l y Meliaceae i s c h a r a c t e r i z e d by  the o x i d i z e d t r i t e r p e n o i d s known v a r i o u s l y as  limonoids,  m e l i a c i n s , o r t e t r a n o r t r i t e r p e n o i d s ( T a y l o r , 1981). Limonoids, named f o r l i m o n i n , t h e f i r s t such s t r u c t u r e t o be elucidated  ( A r i g o n i e t a l . , 1960), possess a C  2 2  4,4,8-  t r i m e t h y l s t e r o i d nucleus w i t h a f u r a n r i n g attached 17,  and may be e x t e n s i v e l y o x i d i z e d and r e a r r a n g e d  1981,  1983; C o n n o l l y ,  1983).  a t C(Taylor,  F o r t h e most p a r t ,  b i o s y n t h e t i c s t u d i e s w i t h r a d i o t r a c e r s a r e l a c k i n g , but known compounds may be arranged i n a reasonable b i o s y n t h e t i c sequence which a l s o d e r i v e s support from i n v i t r o studies  (Buchanan and H a l s a l , 1970; L a v i e and Levy, 1971;  S i d d i q u i e t a l . , 1988).  To form t h e  euphanol (20BH) o r t i r u c a l l o l to a A  synthetic  protolimonoids,  (20crH) d e r i v a t i v e s g i v e  rise  d e r i v a t i v e such as butyrospermol, which i s  7  subsequently e p o x i d i z e d  a t C7-8 ( F i g u r e 1-1).  Opening o f  the 7B, 8B-epoxide induces a euphol-apo-euphol ( o r t i r u c a l l o l - a p o - t i r u c a l l o l ) rearrangement, w i t h formation o f a C-7  OH, m i g r a t i o n  o f the C-14  CH  3  t o C-8, and  formation  of a double bond a t C-14,15, r e s u l t i n g i n t h e c h a r a c t e r i s t i c limonoid  nucleus.  The e x i s t e n c e  a l i p h a t i c side chain,  of protolimonoids  with a C  i n c l u d i n g m e l i a n t r i o l , suggests t h a t  t h i s rearrangement precedes t h e c y c l i z a t i o n o f t h e s i d e chain.  The f u r a n r i n g i s formed by c y c l i z a t i o n o f t h e C  chain t o a c y c l i c hemiacetal,  8  f o l l o w e d by t h e l o s s o f f o u r  carbons (hence t e t r a n o r t r i t e r p e n o i d ) .  8  17  Subsequent o x i d a t i o n s and rearrangements g i v e r i s e t o a d i v e r s i t y of s t r u c t u r e s ; more than 300 t o date ( T a y l o r , 1987).  have been d e s c r i b e d  The major b i o s y n t h e t i c pathways  have been d e s c r i b e d by Das  e t a l . (1984, 1987).  The  most  s t r u c t u r a l l y d i v e r s e pathway begins with o x i d a t i o n of the r i n g t o an epoxylactone (see F i g u r e 4-2).  The  D  parent  apoeuphol, w i t h a double bond a t C-14,15, undergoes a l l y l i c oxidation to a A  1 4  , 1 6-ketone, which i s o x i d i z e d t o a 14,15-  epoxy-16-keto d e r i v a t i v e . y i e l d s the epoxylactone. the s e r i e s isolated al.,  indica  (see F i g u r e 4-1).  r e a c t i o n s may A,D  T h i s sequence i s e x e m p l i f i e d  (Kraus,  1981;  Subsequent B a e y e r - V i l l i g e r  o r the B r i n g  (ring-B,D seco  T h i s pathway i s p a r t i c u l a r l y c h a r a c t e r i s t i c Swietenioideae  ( T a y l o r , 1981;  In the s u b f a m i l y a l s o expressed. r a r e l y t h i s may  The  Das  ejfe a i . ,  Meliodeae, two A r i n g may  limonoids).  o f the  3  subfamily  1984).  be o x i d i z e d t o a l a c t o n e ;  be f o l l o w e d by o x i d a t i v e opening of the B  rearrangement ( F i g u r e 1-2).  undergo r i n g - C Radiotracer  experiments have e s t a b l i s h e d t h a t , c o n t r a r y t o the accepted  (ring-  a d d i t i o n a l pathways are  F i n a l l y , the i n t a c t p r e c u r s o r may  opening and  Schwinger e t  l e a d t o o x i d a t i o n of e i t h e r the A r i n g  seco limonoids)  ring.  by  azadirone-azadiradione-epoxyazadiradione-gedunin,  from A z a d i r a c h t a  1983)  B a e y e r - V i l l i g e r o x i d a t i o n then  scheme f o r l i m o n o i d b i o s y n t h e s i s , the  generally isomers of  H-euphol, t i r u c a l l o l , and butyrospermol were more  e f f i c i e n t l y used than were the A - i s o m e r s i n the 7  b i o s y n t h e s i s of the r i n g - C - s e c o  l i m o n o i d nimbolide  (Ekong e t  18  al.,  1971;  Ekong and  Ibiyemi, 1985).  T h i s suggested  8ot 9 tx—epoxide g i v e s r i s e t o a A ' ( H ) - d i e n e ; 7  9  t  f u n c t i o n leads t o the 7B-0H, and the A ^ 9  a c t i v a t e C-12,  1 1  ^  the  A  that a 7  f u n c t i o n would  l e a d i n g t o o x i d a t i v e f i s s i o n of the C r i n g .  A l s o (perhaps s i m u l t a n e o u s l y ) , the 7<x-0H c o u l d b i n d t o of the D r i n g t o form a new  C-15  furan r i n g connecting r i n g s B  and D ( S i d d i q u i e t a l . , 1988).  F u r t h e r a d d i t i o n of  s u b s t i t u e n t s and oxygen b r i d g e s g i v e s r i s e t o a d i v e r s i t y of h i g h l y o x i d i z e d compounds i n c l u d i n g a z a d i r a c h t i n . a l t e r n a t i v e pathway, based on i n v i t r o  An  chemical  t r a n s f o r m a t i o n s , has the C-seco limonoids a r i s i n g from an intact  A  apo-euphol type l i m o n o i d w i t h a c a r b o n y l  1 4  f u n c t i o n a t C-12  ( F i g u r e 1-3)  (Jones e t a i . , 1988).  pathway t o r i n g - C seco limonoids i s c o n f i n e d t o the M e l i e a e , c o n s i s t i n g of the two (Das e t a i . ,  The tribe  genera A z a d i r a c h t a and  Melia  1984).  The Meliaceae of phytochemical  appear t o be remarkable f o r the p a u c i t y  c o n s t i t u e n t s o t h e r than l i m o n o i d s ; i n t h i s  r e s p e c t they c o n t r a s t s t r o n g l y w i t h t h e i r s i s t e r - g r o u p , the Rutaceae.  Known coumarins i n c l u d e o n l y the 6,7-oxygenated  compounds a e s c u l e t i n , s c o p o l e t i n , i s o s c o p o l e t i n , and scoparon,  common t o many t a x a , and the more  e k e r s e n i n from Ekebergia ciliata  (Gray,  1983).  unusual  s e n e a a l e n s i s and s i d e r i n from Toona  The o n l y chromone known i s  r o h i t u k i n e , which has a v e r y unusual  8-(3-hydroxy-l-  F i g u r e 1-1.  B i o s y n t h e t i c pathway l e a d i n g t o the  of an apo-euphol type l i m o n o i d al..  1988).  formation  ( m o d i f i e d from S i d d i q u i et.  20  21  F i g u r e 1-2.  B i o s y n t h e t i c pathway l e a d i n g t o the  of a C-seco l i m o n o i d 1988).  formation  (modified from S i d d i q u i e t aj,.,  22  23  Figure 1-3.  B i o s y n t h e t i c pathway l e a d i n g t o the  of a C-seco l i m o n o i d ,  formation  a c c o r d i n g t o Jones g t a i .  (1988).  2k  25  methylpiperid-4-yl) rohituka  s u b s t i t u e n t , i s o l a t e d from Amoora  (Harmon e t a l . , 1979).  Amoora i s now  considered  j u n i o r synonym of A g l a i a (Pennington and S t y l e s , 1975).  a The  o n l y furanocoumarin d e s c r i b e d t o date i s bergapten from Toona c i l i a t a seeds ( C h a t t e r j e e e t a l . , 1971). The  d i v e r s i t y of a l k a l o i d s i s e q u a l l y l i m i t e d .  Xylocarpus  granatum produces the angular p y r a n o q u i n o l i n e  N-  m e t h y l f l i n d e r s i n e and a benzo[c]phenanthride (Chou e t a l . , 1977), and Ekeberaia hydroxylpyridine  c y l i n d r i c u m produces the simple  (Menster, 1983).  A g l a i a odorata  roxburghiana l e a v e s c o n t a i n bis-amides o f a r o i n o p y r r o l i d i n e , odorine 1979;  Purushothaman gt aJL.,  and A..  2-  and o d o r i n o l (Shiengthong e t a l . 1979).  The  f  closely related  compound p i r i f e r i n e occurs i n f o l i a g e of A., p i r i f e r a e t a l . , 1988).  3-  An u n i d e n t i f i e d A g l a i a s p e c i e s  t i g l a m i d e (Johns and Lamberton, 1969).  (Saifah  contained  Recently  a s e r i e s of  c a r d i o a c t i v e 1 - p h e n y l e t h y l i s o q u i n o l i n e a l k a l o i d s were i s o l a t e d from the f o l i a g e of the t r a d i t i o n a l m e d i c i n a l p l a n t Dysoxylum l e n t i c e l l a r e co-occur  Ilesanmi,  w i t h the m o l l u s c i c i d a l a l k a l o i d  (Aladesanmi e t a l . , Few  (Aladesanmi and  1987); they  lenticellarine  1988).  f l a v o n o i d s have been i d e n t i f i e d i n the  most common are g l y c o s i d e s of q u e r c e t i n and  Meliaceae;  kaempferol,  i n c l u d i n g 3 - g a l a c t o s i d e s , a r a b i n o s i d e s , rhamnosides, r u t i n o s i d e s , and g l u c o s i d e s (Harborne, 1983). i n d i c a produces, as w e l l , the r a r e  Azadirachta  myricetin-3'-arabinoside.  Aglycones i n c l u d e o n l y nimbaflavone (5,7-hydroxy-4'-methoxy-  26  8,3'-di-C-prenyl flavanone) from A., i n d i c a (Garg and Bhakuni, 1984).  Based on the r e s u l t s o f a p r e l i m i n a r y  survey o f 12 s p e c i e s (W. C r i n s and D. Champagne, u n p u b l i s h e d data), the d i v e r s i t y of flavonoids i n the Meliaceae i s probably under-represented i n t h e l i t e r a t u r e . Diterpenes include s u g i o l , nimbiol,  nimosone,  nimbosone, methyl n i m b i o l , methyl nimbionone,  nimbionone,  and nimbionol from A., i n d i c a bark (Hegenauer,  1969; A r a e t  al.,  1988; S i d d i q u i e t a l . ,  1988) and eperu-13-en-8B,15-diol  from Aphanamixus p o l y s t a c h y a (Chandrasekharan and Chakrabortty, 1968).  Dysoxylum  l e n t i c e l l a r e has r e c e n t l y  y i e l d e d two d i t e r p e n e s , p h y l l o c l a d e n e and fihydroxysandaracopimarene  (Aladesanmi e t a l . ,  1986).  S e s q u i t e r p e n e s , known from Lansium anamalayanum, i n c l u d e ()-K-gurjunene,  (-)-<x-trans-bergamotene,  (Krishnappa and Dev, 1973).  and (-)-<x-bisabolene  Pentacyclic triterpenes include  b e t u l i n , b e t u l i n i c a c i d , k a t o n i c and i n d i c i c w a l s u r e n o l , onoceradienone, and l a n s i c a c i d 1983).  acids, (Hegnauer,  Aphanamixus p o l y s t a c h y a seeds c o n t a i n a saponin,  s t i g m a s t i e n o l d i g l y c o s i d e (Bhatt g t a i . , 1981). U n c h a r a c t e r i z e d monoterpenes  o c c u r i n t h e l e a f g l a n d s , and a  few s p e c i e s produce 2,6-dimethoxy-benzoquinone  (Hegnauer,  1983).  A n t i f e e d a n t and I n s e c t i c i d a l A c t i v i t y o f A z a d i r a c h t i n Leaves and f r u i t s o f t h e neem t r e e , A z a d i r a c h t a i n d i c a A. J u s s . , have long been used i n t r a d i t i o n a l medicine and  27  agriculture i n India; describe  Sanskrit  writings  from 2,000 B.C.  t h e p r e p a r a t i o n o f water e x t r a c t s  l o c u s t plagues (Pradhan g t a l . , 1962).  f o r use a g a i n s t  Pradhan and  coworkers (1962) confirmed t h e l o c u s t r e p e l l e n t a c t i v i t y o f neem e x t r a c t s .  As w e l l , neem l e a v e s a r e s t i l l used, mixed  w i t h g r a i n o r p l a c e d i n woolen c l o t h i n g , t o p r o t e c t  against  i n s e c t damage (Saxena, 1989). Work i n I s r a e l r e s u l t e d i n t h e d e s c r i p t i o n o f t h e p r o t o l i m o n o i d m e l i a n t r i o l as a l o c u s t f e e d i n g  deterrent  f o l i a g e o f A. i n d i c a and t h e c l o s e l y r e l a t e d  chinaberry,  Melia  a z e d i r a c h L. ( L a v i e e t a l . , 1967).  from  A year l a t e r  Butterworth and Morgan (1968) i s o l a t e d a m i c r o c r y s t a l l i n e compound, which they named a z a d i r a c h t i n , antifeedant  from neem seed.  as a potent  The s t r u c t u r e  (see F i g u r e 3-3) proved e l u s i v e : a p a r t i a l  locust  of azadirachtin structure  (Butterworth and Morgan, 1971; Butterworth g t a l . ,  1972),  l a t e r completed by Zanno g t a l . (1975), was subsequently r e v i s e d based on C-NMR (Kraus e t a l . , 1985) and X-ray 13  d i f f r a c t i o n studies al.,  of d e t i g l o y l azadirachtin  1985; Broughton g t a i . , The  (Bilton gt  1986).  phytochemistry o f t h e neem t r e e i s complex.  e t a l . (1988) l i s t  Jones  53 limonoids and two p r o t o l i m o n o i d s , most  of which have been i s o l a t e d from t h e seed o i l . these a r e v e r y s i m i l a r t o a z a d i r a c h t i n , designated "azadirachtins  Some o f  and indeed have been  A-G" by Rembold (1987, 1988).  S e v e r a l neem l i m o n o i d s a r e a c t i v e a g a i n s t  insects,  although  28  none are as a c t i v e as a z a d i r a c h t i n i t s e l f  (reviewed  in  Chapter 4 ) . A z a d i r a c h t i n completely  i n h i b i t s f e e d i n g by the  locust, Schistocerca gregaria, at concentrations 70 ng/1 1968,  (= 4 ng/cm  1971).  2  l e a f d i s c ) (Butterworth  desert  as low  as  and Morgan,  S. g r e g a r i a i s remarkably s e n s i t i v e , as i t  does not feed on cabbage t r e a t e d w i t h a 0.001% aqueous s o l u t i o n of neem k e r n e l e x t r a c t (Pradhan gt a l . , 1962). t e n - f o l d h i g h e r c o n c e n t r a t i o n was L. m i g r a t o r i a .  needed t o d e t e r f e e d i n g  by  Subsequently e i t h e r pure a z a d i r a c h t i n o r  neem o i l p r e p a r a t i o n s growth i n n e a r l y two (Warthen, 1979;  A  have been shown t o i n h i b i t f e e d i n g  hundred s p e c i e s of phytophagous i n s e c t s  Jacobson, 1986;  Saxena, 1989).  The  n e u r o p h y s i o l o g y u n d e r l y i n g the a n t i f e e d a n t response has examined by s e v e r a l authors (Blaney, and Jenny, 1977;  or  1980,  1981;  Simmonds and Blaney, 1983).  A  been  Schoonhoven receptor  o t h e r than the sugar c e l l responds t o a z a d i r a c h t i n ; i n some i n s e c t s i n t e r a c t i o n occurs between these c e l l s a t  the  p e r i p h e r a l l e v e l , r e d u c i n g the frequency of a c t i o n p o t e n t i a l s , but i n Mamestra b r a s s i c a e and no i n t e r a c t i o n occurs the d e t e r r e n t and  Spodoptera exempta  and the c o n f l i c t between s i g n a l s from  sugar r e c e p t o r s are r e s o l v e d i n the  c e n t r a l nervous system (Simmonds and Blaney, 1983). H a b i t u a t i o n t o a z a d i r a c h t i n can occur, the compound f o r two  as l a r v a e exposed t o  days showed a markedly reduced  n e u r o p h y s i o l o g i c a l response (Simmonds and  Blaney, 1983).  29  Soon a f t e r the d i s c o v e r y of the a n t i f e e d a n t a c t i v i t y o f a z a d i r a c h t i n , i t was noted t h a t t h i s compound a l s o d e l a y e d o r i n h i b i t e d the m o l t i n g o f a v a r i e t y o f i n s e c t s , i n death o r the malformation of a d u l t s (Ruscoe, S t e e t s , 1975; Meisner e t a l . ,  1976; Ladd e t a l . ,  resulting  1972; 1978).  (Readers u n f a m i l i a r w i t h the e n d o c r i n e r e g u l a t i o n of m o l t i n g i n i n s e c t s a r e r e f e r r e d t o t h e review o f t h i s t o p i c g i v e n on pp 32- 3<r o f t h i s I n t r o d u c t i o n ) .  Injection of azadirachtin  was f o l l o w e d by an immediate r a p i d d e c l i n e i n j u v e n i l e hormone (JH) t i t r e s i n L o c u s t a m i a r a t o r i a  ( S i e b e r and  Rembold, 1983) and G a l l e r i a m e l l o n e l l a (Malczewska e t a l . , 1988).  The appearance o f ecdysone peaks was d e l a y e d i n a  dose-dependent manner i n L o c u s t a m i g r a t o r i a ( S i e b e r and Rembold, 1983; Mordue e t a l . ,  1986; Mordue and Evans, 1987),  Oncopeltus f a s c i a t u s (Redfern e t a l . , (Schluter et a l . ,  1985; Pener g£ a l . ,  1982), Manduca s e x t a 1988), O s t r i n i a  f u r n a c a l i s ( M i n - L i and Shin-Foon, 1987), C a l l i p h o r a v i c i n a (Koolman e t a l . , 1988), and G a l l e r i a m e l l o n e l l a e t a l . , 1988).  (Malczewska  In most o f t h e s e cases ecdysone t i t r e s were  a l s o decreased, but i n some ( i . e . S c h l u t e r e t a l . , 1985; Malczewska e t a l . , 1988) the d e l a y e d ecdysone peak was a c t u a l l y h i g h e r than the c o n t r o l s , and appeared t o f a l l more gradually. A z a d i r a c h t i n a l s o i n h i b i t s oogenesis i n L o c u s t a m i g r a t o r i a (Rembold and S i e b e r , 1981), Oncopeltus f a s c i a t u s (Dorn e t a l . , 1986), and Dysdercus k o e n i g i i  (Koul, 1984).  S i m i l a r e f f e c t s have been produced by exposure t o neem  30  extracts i n Epilachna v a r i v e s t i s 1975)  and  ( S t e e t s and  L e p t i n o t a r s a decemlineata ( S t e e t s ,  Schmutterer, 1986).  Sieber,  1976;  Again t h i s e f f e c t i s r e l a t e d t o  i n h i b i t i o n of JH p r o d u c t i o n (Rembold and  Schmutterer,  and  1981).  ovarian ecdysteroid  C u r i o u s l y , l a s t - i n s t a r nymphs  t r e a t e d w i t h s u f f i c i e n t a z a d i r a c h t i n t o completely molting  can show development of the o v a r i e s  1986a,b; Shalom e t a l . , 1988). i n over-age L-  m i g r a t o r i a nymphs were 6-7  molting The  inhibit  (Dorn e t a i . ,  Vitellogenin  i n c o n t r o l a d u l t females (Shalom g£ a i . , The  titres  concentrations  times the  levels  1988).  mechanism by which a z a d i r a c h t i n i n t e r f e r e s w i t h and the p r o d u c t i o n  of neurohormones remains  unclear.  p r o t h o r a c i c glands are not d i r e c t l y a f f e c t e d , as these  glands remain a b l e t o s y n t h e s i z e prothoracicotrophic 1987;  Pener e t a i - ,  ecdysone and respond t o  hormone (PTTH) i n v i t r o  (Koul e t a l . ,  1988), although Koolman g t a i .  (1988)  found i n h i b i t i o n of r e l e a s e but not s y n t h e s i s o f ecdysone i n the b r a i n - r i n g gland complex of C a l l i p h o r a v i c i n a .  Indeed  p r o t h o r a c i c glands of a z a d i r a c h t i n - t r e a t e d i n s e c t s remained i n t a c t and  a b l e t o s e c r e t e ecdysone long a f t e r the  glands  degenerated i n c o n t r o l i n s e c t s (Pener e t a l . , 1988). of a z a d i r a c h t i n - t r e a t e d Manduca sexta c o n t a i n e d  Brains  as much PTTH  as d i d c o n t r o l animals (Pener e t a l . , 1988), but Subrahmanyam e t al-  (1989) found t h a t a z a d i r a c h t i n reduced  the r a t e of i n c o r p o r a t i o n and cysteine i n neurosecretory  turnover  material.  of  3 5  S-labelled  T h i s evidence p o i n t s  31  to a delay  i n , and  i n some cases complete i n h i b i t i o n o f ,  r e l e a s e of PTTH from the c o r p o r a  the  cardiaca.  That a z a d i r a c h t i n t o x i c i t y i n v o l v e s more than simple i n h i b i t i o n of the r e l e a s e of ecdysone or JH i s i n d i c a t e d the f a i l u r e of subsequently a p p l i e d hormones t o t o x i c i t y i n Manduca sexta induces the p r o d u c t i o n  (Schluter gt a i . ,  (due  not r e v e r s e d  1988).  Chilling  in Galleria  by  azadirachtin  and  the  by a p p l i c a t i o n of the  analogue ZR512 (Malczewska e t a l . , azadirachtin-induced  1985).  abolished  t o marked i n h i b i t i o n of JH s y n t h e s i s )  i n h i b i t i o n was  reverse  of supernumerary i n s t a r s  m e l l o n e l l a l a r v a e ; t h i s e f f e c t was  JH  However,  i n h i b i t i o n of m o l t i n g  was  reversed  Rhodnius p r o l i x u s by o r a l a p p l i c a t i o n of ecdysone o r a p p l i c a t i o n of a JH analogue ( G a r c i a and A z a d i r a c h t i n does not i n h i b i t b i n d i n g (Koolman g t a i - ,  t h e i r receptors of a d i f f e r e n t  in  topical  Rembold, 1984).  of e c d y s t e r o i d s  1988), a p p a r e n t l y  conformation o f the A and  by  B rings  to  because (Rembold,  1988). In a d d i t i o n t o i n h i b i t i o n of m o l t i n g  and  a z a d i r a c h t i n i n h i b i t s gut p e r i s t a l s i s i n v i t r o ai.,  1985;  Mordue and  to a generalized contraction Mordue has  Evans, 1987).  reproduction, (Mordue g t  This e f f e c t  i n h i b i t i o n of p r o c t o l i n - i n d u c e d  (Mordue and  Evans, 1987;  i s related muscle  Mordue g t a l . ,  1989).  suggested t h a t the observed endocrine e f f e c t s  a z a d i r a c h t i n c o u l d be due  t o d i s r u p t i o n of normal  of  gut  f u n c t i o n i n g , which i s i n v o l v e d i n feedback loops r e g u l a t i n g the t i m i n g of some endocrine events ( N i j h o u t ,  1981).  To  32  t h a t end she has shown t h a t a z a d i r a c h t i n can i n h i b i t  molting  even i f a p p l i e d a f t e r t h e e c d y s t e r o i d peak and a t a time when JH t i t r e s should  be low.  i n h i b i t s the air-swallowing  In t h i s case a z a d i r a c h t i n  b e h a v i o r necessary t o s p l i t t h e  o l d c u t i c l e and may i n h i b i t t h e r e l e a s e o f b u r s i c o n e t a l . , 1985).  (Mordue  The o n l y other p h y s i o l o g i c a l e f f e c t r e p o r t e d  t o date which can be a t t r i b u t e d t o a z a d i r a c h t i n i s a marked decrease i n t h e r a t e o f s y n t h e s i s o f RNA and subsequently DNA i n a suspension c u l t u r e o f t h e p r o t i s t Tetrahymena thermophila ( F r i t z s c h e and CTeffman, 1987). In s p i t e o f t h e c o n f u s i o n  s u r r o u n d i n g t h e molecular-  mode o f a c t i o n o f a z a d i r a c h t i n , t h i s compound and neem preparations  have been a p p l i e d t o a wide v a r i e t y o f  a g r i c u l t u r a l problems (Schmutterer, 1988). a p p l i c a t i o n s r e l y on neem o i l ,  Such  cake, o r s e m i p u r i f i e d  e x t r a c t s due t o t h e p r o h i b i t i v e expense o f pure a z a d i r a c h t i n (Schmutterer and H e l l p a p ,  1988).  P r o m i s i n g a c t i v i t y has  been found a g a i n s t p e s t s o f v e g e t a b l e s and f r u i t (Schmutterer and H e l l p a p , grains 1988).  1988), r i c e  (Saxena, 1989), s t o r e d  (Saxena e t a l . , 1988), and ornamental crops (Larew, Various  household p e s t s and d i s e a s e v e c t o r s may a l s o  be c o n t r o l l e d by neem e x t r a c t s Rembold e t a l . , 1989). has  trees  (Ascher and Meisner, 1988;  A neem o i l p r e p a r a t i o n ,  Marigosan 0,  now been r e g i s t e r e d f o r use on ornamental crops i n t h e  United  States  (Larson,  1988).  T o x i c o l o g i c a l t e s t i n g has i n d i c a t e d t h a t a z a d i r a c h t i n i t s e l f i s n o t t o x i c t o mammals, b i r d s , o r f i s h  (Schmutterer,  33  1988;  Jacobson, 1986;, Okpanyi and Ezeukwu, 1981).  However,  neem o i l has a l a r g e range o f p h a r m a c o l o g i c a l e f f e c t s i n c l u d i n g a n t i p y r e t i c and d i u r e t i c a c t i v i t y , promoting smooth muscle c o n t r a c t i o n , and i n h i b i t i o n o f f u n g a l p a r a s i t e s , s c a b i e s , and eczema (Jacobson, 1988). a l s o i n a c t i v a t e d t h e potato-X v i r u s (Singh,  skin  Neem o i l  1971), and has  s p e r m i c i d a l and a n t i f e r t i l i t y a c t i v i t y i n v a r i o u s mammals (Jacobson, 1988).  However, t h e p r i n c i p l e s r e s p o n s i b l e  these a c t i v i t i e s were nimbin and nimbidin  for  and not  azadirachtin. A f u r t h e r f a v o r a b l e aspect  o f neem i s i t s systemic  a c t i v i t y i n a v a r i e t y o f p l a n t s i n c l u d i n g corn and r i c e (Saxena e t a l . , 1983).  Azadirachtin apparently  translocates  i n t o and p r o t e c t s new f o l i a g e o r f r u i t s which appear a f t e r the a p p l i c a t i o n o f t h e neem e x t r a c t .  Regulation of Molting To f a c i l i t a t e d i s c u s s i o n o f t h e mode o f a c t i o n o f a z a d i r a c h t i n , a review o f t h e endocrine r e g u l a t i o n o f molting  i s g i v e n here.  F i g u r e 1-4 i l l u s t r a t e s t h e  r e l a t i o n s h i p o f t h e v a r i o u s s e c r e t o r y and neurohaemal organs i n the i n s e c t .  Molting  i s a two-step p r o c e s s :  apolysis, the  s e c r e t i o n o f new c u t i c l e and p a r t i a l r e s o r p t i o n o f t h e endocuticle,  i s f o l l o w e d by e c d y s i s , t h e p r o c e s s o f a c t u a l l y  shedding t h e o l d c u t i c l e . A p o l y s i s i s r e g u l a t e d by t h e prohormone B-ecdysone (ecdysterone),  produced i n t h e p r o t h o r a c i c glands i n  34  response t o t h e neuropeptide p r o t h o r a c i c o t r o p h i c hormone (PTTH).  In Manduca s e x t a , PTTH i s s y n t h e s i z e d i n t h e pars  i n t e r c e r e b r a l i s , each hemisphere  having a s i n g l e  prothoracicotropic c e l l  l o c a t e d i n t h e l a t e r a l protocerebrum  ( G i l b e r t g t a l (1981).  Although t h e corpus cardiacum (CC)  has g e n e r a l l y been c o n s i d e r e d t h e s i t e o f PTTH r e l e a s e ( G i l b e r t and K i n g , 1973; G i l l o t t ,  1982), i n Manduca t h e  p r o t h o r a c i c o t r o p e s a r e connected v i a axons t o the corpus a l l a t u m (CA) and t h i s i s t h e s i t e o f PTTH r e l e a s e t o t h e hemolymph.  Two forms o f PTTH, termed  " b i g " and " s m a l l " ,  w i t h m o l e c u l a r weights o f about 22,000 and 7,000 r e s p e c t i v e l y , are present (Bollenbacher g t a l . ,  1984).  Corpora Cardiacum  Figure 1-4.  Neuroendocrine structures discussed i n the text. from G i l l o t t ,  1980.  Modified  36  The  f a c t o r s which t r i g g e r PTTH r e l e a s e are  poorly  understood but are r e l a t e d t o i n t e r n a l measurements of f a c t o r s c o r r e l a t e d w i t h f e e d i n g , s i z e , and ( N i j h o u t , 1981).  photoperiod  In Rhodnius p r o l i x u s m o l t i n g  follows  f e e d i n g a f t e r a c h a r a c t e r i s t i c p e r i o d of time.  Nutritional  f a c t o r s are not i n v o l v e d as a s e r i e s of s m a l l blood meals do not provoke m o l t i n g but a s i n g l e l a r g e meal (>100 (Wigglesworth, 1934).  mg)  does  Wigglesworth suggested t h a t nervous  impulses from abdominal s t r e t c h r e c e p t o r s were r e q u i r e d ,  and  showed t h a t s e v e r i n g the v e n t r a l nerve c o r d between the head and the thorax prevented the response t o a blood meal. Beckel  and  F r i e n d (1964) subsequently showed t h a t a  n u t r i t i v e s a l i n e meal c o u l d i n i t i a t e m o l t i n g . system i n i t i a t e s m o l t i n g may  non-  A similar  i n Oncopeltus f a s c i a t u s as  molting  r  be t r i g g e r e d by i n f l a t i n g the abdomen w i t h s a l i n e o r a i r  ( N i j h o u t , 1979).  The  r e q u i r e d degree of s t r e t c h i n g i s  a s s o c i a t e d w i t h a s h a r p l y d e f i n e d c r i t i c a l weight, which i s u s u a l l y a t t a i n e d w i t h i n the f i r s t 24 h of the ( N i j h o u t , 1979;  B l a k l e y and Goodner, 1978).  instar The  critical  weight depends on some f a c t o r or s t r u c t u r e whose dimensions are determined a t the p r e v i o u s molt, as t h e r e are p o s i t i v e l i n e a r c o r r e l a t i o n s between the c r i t i c a l weight  and  dimensions of s c l e r o t i z e d s t r u c t u r e s such as femur l e n g t h . In Manduca sexta the r e l e a s e of PTTH i s r e g u l a t e d by both a c r i t i c a l weight (5 g i n the f i n a l i n s t a r ) ( N i j h o u t Williams,  1974)  (Truman, 1972;  and a p h o t o p e r i o d - c o n t r o l l e d Truman and R i d d i f o r d , 1974;  and  "gating"  R i d d i f o r d and  37  C u r t i s , 1978).  In the 5th i n s t a r the gate opens s h o r t l y  a f t e r l i g h t s - o f f and l i g h t phase. h before  c l o s e s a t the b e g i n n i n g of the next  Larvae must a t t a i n c r i t i c a l weight a t l e a s t 24  a p a r t i c u l a r gate c l o s e s ; otherwise t h a t gate i s  bypassed and  PTTH i s not s e c r e t e d u n t i l the subsequent gate  ( N i j h o u t , 1981).  A p h y s i o l o g i c a l event a s s o c i a t e d w i t h  the  attainment of c r i t i c a l weight i s the c e s s a t i o n of j u v e n i l e hormone (JH) s y n t h e s i s : the 24 h l a t e n t p e r i o d  represents  the time necessary t o c l e a r JH from the hemolymph.  In  the  l a s t i n s t a r the presence of JH i s s u f f i c i e n t t o i n h i b i t PTTH s e c r e t i o n , but t h i s mechanism does not operate e a r l i e r l a r v a l - l a r v a l molts. cynthia, photoperiodic  during  In the s a t u r n i i d moth Samia  c o n t r o l of m o l t i n g  i s r e g u l a t e d by  endogenous c i r c a d i a n c l o c k l o c a t e d i n the p r o t h o r a c i c (Mizoguchi and  an  glands  I s h i z a k i , 1982).  In the p r o t h o r a c i c glands the PTTH i s thought t o to c e l l - s u r f a c e receptors associated with a C a l e a d i n g t o an i n f l u x of e x t r a c e l l u l a r C a G i l b e r t , 1986).  2 +  2 +  (Smith  Subsequent s t i m u l a t i o n of a C a  2 +  bind  channel, and sensitive  adenylate c y c l a s e r e s u l t s i n an i n c r e a s e i n i n t r a c e l l u l a r cAMP; c a l m o d u l i n  may  mediate t h i s s t i m u l a t i o n  as  C a / c a l m o d u l i n s e n s i t i v e adenylate c y c l a s e s have been 2 +  i d e n t i f i e d i n other  i n s e c t t i s s u e s (Combest e t a l . , 1985).  A d d i t i o n a l l y , some r e c e p t o r s the adenylate c y c l a s e . dependent p r o t e i n k i n a s e  The  are a s s o c i a t e d d i r e c t l y w i t h cAMP may  then a c t i v a t e a cAMP-  responsible f o r phosphorylating  r a t e - l i m i t i n g enzyme(s) i n v o l v e d i n ecdysone s y n t h e s i s .  a  38  Although PTTH has been c o n s i d e r e d t h e p r i n c i p a l hormone r e g u l a t i n g ecdysone p r o d u c t i o n , r e c e n t evidence suggests t h a t o t h e r f a c t o r s may modulate p r o t h o r a c i c g l a n d a c t i v i t y . In Manduca sexta JH was shown t o have a p r o t h o r a c i c o t r o p i c a c t i o n on ecdysone s y n t h e s i s (Gruetzmacher g t a_l.,  1984a).  Subsequently, t h i s was shown t o be an i n d i r e c t e f f e c t : JH s t i m u l a t e s t h e p r o d u c t i o n o f a 30 kD hemolymph f a c t o r from the f a t body (Gruetzmacher e t a l . , 1985,  1988).  1984b; Watson e t a l . ,  S t i m u l a t i o n o f ecdysone s y n t h e s i s o c c u r r e d  even i n t h e presence o f s a t u r a t i n g t i t r e s o f PTTH.  The  f a c t o r may be a hemolymph c a r r i e r p r o t e i n which t r a n s p o r t s a s t e r o l s u b s t r a t e used by t h e glands i n t h e p r o d u c t i o n o f ecdysone.  In Bombyx mori t h i s f u n c t i o n may be f u l f i l l e d by  h i g h m o l e c u l a r weight l i p o p r o t e i n s (>200 kD) which t r a n s p o r t c h o l e s t e r o l t o t h e p r o t h o r a c i c g l a n d s (Chino e t a l . ,  1974).  In H e l i o t h i s zea, diapause i s t e r m i n a t e d by an i n c r e a s e i n ecdysone s y n t h e s i s , mediated by a temperature-dependent humoral f a c t o r (Meola and Gray,  1984).  In t h e L e p i d o p t e r a , e c d y s i s i s t r i g g e r e d by t h e r e l e a s e of e c l o s i o n hormone (EC) i n response t o f a l l i n g titers  ecdysteroid  (Truman, 1981; Reynolds and Truman, 1983).  EC i s  a p p a r e n t l y r e l e a s e d from t h e v e n t r a l g a n g l i o n a t l a r v a l l a r v a l and l a r v a l - p u p a l m o l t s , and from t h e CC a t a d u l t eclosion.  I n j e c t i o n o f exogenous ecdysterone i n h i b i t s both  the r e l e a s e o f EC and s e n s i t i v i t y t o exogenously a p p l i e d EC, and so r e s u l t s i n a dose-dependent  delay of e c l o s i o n .  l a r v a e , h i g h doses o f ecdysterone can permanently  In  inhibit  39  ecdysis.  In a d u l t s , where e c d y s i s i s gated by  e c l o s i o n may  photoperiod,  be delayed u n t i l a gate s e v e r a l days subsequent  t o the ecdysterone dose.  EC r e l e a s e t r i g g e r s a s e r i e s of  s t e r e o t y p e d b e h a v i o r s which serve t o r e l e a s e the i n s e c t the o l d c u t i c l e , i n c l u d i n g swallowing  from  a i r t o expand the body  and rhythmic p e r i s t a l t i c muscular c o n t r a c t i o n s .  These  a c t i o n s a p p a r e n t l y r e s u l t from EC b i n d i n g t o r e c e p t o r s l o c a t e d on each of the abdominal g a n g l i a . The presence of EC i n n o n - l e p i d o p t e r a n i n s e c t s has been u n e q u i v o c a l l y demonstrated.  However, l o c u s t s and  not other  i n s e c t s a l s o have s t e r e o t y p e d b e h a v i o r s a s s o c i a t e d w i t h e c d y s i s , and the onset of these b e h a v i o r s can be delayed exogenous ecdysterone i n Locusta (Rembold, 198 al.  ).  by  Truman e t  (1981) found t h a t e x t r a c t s from the nervous system of  i n s e c t s from f i v e o r d e r s (other than L e p i d o p t e r a )  initiated  e c d y s i s when a p p l i e d t o Manduca pupae, s u g g e s t i n g the presence of an E C - l i k e hormone.  S e l e c t i o n of Test Insects F a c t o r s t o be c o n s i d e r e d i n the s e l e c t i o n of an a p p r o p r i a t e i n s e c t s p e c i e s f o r b i o a s s a y have been d i s c u s s e d by Berenbaum (1986).  I f the g o a l of a study i s the development of a  c o n t r o l s t r a t e g y f o r a p a r t i c u l a r t e s t , the p e s t n a t u r a l l y becomes the b i o a s s a y t a r g e t .  I f q u e s t i o n s of an  e v o l u t i o n a r y o r e c o l o g i c a l nature are addressed, the c h o i c e becomes more r e s t r i c t i v e .  Oligophagous  and monophagous  insects usually require s p e c i f i c "sign s t i m u l i " ,  secondary  40  metabolites  c h a r a c t e r i s t i c o f t h e i r host p l a n t s ,  f e e d i n g w i l l be i n i t i a t e d  ( D e t h i e r , 1941).  before  Such i n s e c t s a r e  a l s o u s u a l l y h i g h l y s e n s i t i v e t o t h e presence o f secondary metabolites  f o r e i g n t o t h e i r normal food p l a n t s , and so tend  t o show an exaggerated a n t i f e e d a n t i n s e c t s appear t o e l a b o r a t e  response.  Further,  these  a l i m i t e d range o f MFO's, and  are p o s s i b l y more s u s c e p t i b l e t o i n t o x i c a t i o n by a l l e l o c h e m i c a l s found i n non-host p l a n t s 1971).  A more c o n s e r v a t i v e  bioassay  polyphagous i n s e c t s : h o s t - p l a n t constrained  (Kreiger e t a l . ,  i s provided  choice  by  i n these i n s e c t s i s  by t h e presence o f a n t i f e e d a n t s  r a t h e r than t h e  absence o f phagostimulants (Bernays, 1983) and, as they elaborate  a wider range o f MFOs ( K r e i g e r e t a i . , 1971), they  are l e s s l i k e l y t o show an exaggerated response t o t h e intrinsic  t o x i c i t y of a given a l l e l o c h e m i c a l .  Most o f t h e b i o a s s a y s the v a r i e g a t e d  reported  i n t h i s thesis involve  cutworm, Peridroma s a u c i a  (Hubner).  a h i g h l y polyphagous s p e c i e s whose known h o s t - p l a n t  This i s range  i n c l u d e s s p e c i e s from over twenty p l a n t f a m i l i e s (Appendix 1); some o f t h e s p e c i e s a t t a c k e d , generally considered  i n c l u d i n g w i l d onion, a r e  q u i t e t o x i c t o i n s e c t s (Bierne,  1971).  Both herbs and deciduous and c o n i f e r o u s t r e e s are i n c l u d e d ; P. s a u c i a g e n e r a l l y a t t a c k s the f o l i a g e but may a l s o consume fruits  ( B i e r n e , 1971).  The o n l y s p e c i e s on i t s host  l i s t known t o c o n t a i n limonoids but as these a r e r e c e n t  plant  a r e some s p e c i e s o f C i t r u s ,  i n t r o d u c t i o n s t o North America i t  seems u n l i k e l y t h a t exposure t o these compounds c o u l d have  41  i n f l u e n c e d the e v o l u t i o n of d e t e r r e n t o r p h y s i o l o g i c a l responses i n E-  saucia.  As E .  saucia i s a  constant  background p e s t which o c c a s i o n a l l y outbreaks t o major p e s t s t a t u s ( B i e r n e , 1971), i t s use  i s j u s t i f i e d t o develop novel  p e s t - c o n t r o l s t r a t e g i e s r e l e v a n t t o Canadian a g r i c u l t u r e . Although E-  s a u c i a does not o r d i n a r i l y encounter  limonoids,  i t s use as an e v o l u t i o n a r y o r e c o l o g i c a l model may j u s t i f i e d , as i t s response t o these compounds may as r e p r e s e n t a t i v e of unadapted, polyphagous encountering  limonoid-containing  Such unadapted h e r b i v o r e s  migratory  herbivores  are g e n e r a l l y assumed t o be  Rhoades and Cates, 1976; S i m i l a r arguments may  be taken  p l a n t s f o r the f i r s t  primary t a r g e t of a l l e l o c h e m i c a l - b a s e d 1976;  a l s o be  time. the  defenses (Feeny,  Coley e t a l . , 1985).  be made f o r the use of  grasshopper, Melanoplus s a n g u i n i p e s .  polyphagous i n s e c t i s c o n s i d e r e d  the  This  the f o u r t h most s e r i o u s  p e s t i n Canadian a g r i c u l t u r e ( B i e r n e , 1971).  Further  advantages t o the use of t h i s i n s e c t are d i s c u s s e d i n Chapter 5.  In b r i e f , the l a c k of an a n t i f e e d a n t response t o  a z a d i r a c h t i n makes t h i s a good model i n s e c t i n which t o study the p h y s i o l o g i c a l e f f e c t s of t h a t compound. In some assays, fasciatus.  I used the milkweed bug  T h i s s p e c i e s was  Oncopeltus  used as a model t o compare the  r e l a t i v e m o l t - i n h i b i t i n g a c t i v i t y of s e v e r a l limonoids, it  i s known t o be s e n s i t i v e t o t o p i c a l l y a p p l i e d  compounds, i n c l u d i n g a z a d i r a c h t i n (Dorn, 1983).  IGR  as  42  Objectives The  of the Thesis  o b j e c t i v e s o f t h e work r e p o r t e d  i n t h i s t h e s i s were t o  examine a s p e c t s o f t h e e f f i c a c y and mode o f a c t i o n o f phytochemicals from s p e c i e s o f t h e p l a n t f a m i l y Meliaceae f o r t h e c o n t r o l o f phytophagous i n s e c t s .  In t h e f i r s t  i n v e s t i g a t i o n , f o l i a r e x t r a c t s from t h i r t y s p e c i e s o f M e l i a c e a e were screened f o r g r o w t h - i n h i b i t i n g a c t i v i t y and t o x i c i t y against the variegated  cutworm, Peridroma s a u c i a .  In a d d i t i o n , r e s u l t s o f t h e s c r e e n i n g  assays and  measurements o f l e a f toughness from f i f t e e n s p e c i e s were used t o a s s e s s a s p e c t s o f t h e r e s o u r c e h y p o t h e s i s o f Coley e£ a l (1985).  availability  As e x t r a c t s o f A g l a i a  odorata were h i g h l y a c t i v e i n t h e crude e x t r a c t  screening,  the phytochemistry o f t h r e e s p e c i e s o f t h e genus A g l a i a were examined, and a r e r e p o r t e d  i n Chapter 3.  In Chapter 4, I d e s c r i b e t h e comparison o f t e n limonoids,  representing  t h e major pathways o f limonoid  b i o s y n t h e s i s , f o r i n h i b i t i o n o f f e e d i n g and growth i n p_. saucia  f  and f o r i n h i b i t i o n o f m o l t i n g  and r e p r o d u c t i o n i n  the milkweed bug, Oncopeltus f a s c i a t u s . one  The t o x i c o l o g y o f  o f these compounds, a z a d i r a c h t i n , was examined i n d e t a i l  i n these s p e c i e s .  Results are discussed  i n terms o f  mechanism o f a c t i o n , s t r u c t u r e / f u n c t i o n r e l a t i o n s h i p s , and the s i g n i f i c a n c e o f i n s e c t i c i d a l a c t i v i t y i n t h e e v o l u t i o n of l i m o n o i d s .  In Chapter 5 I reexamined t h e r e p o r t e d  r e s i s t a n c e o f t h e m i g r a t o r y grasshopper, Melanoplus sanguinipes.  to azadirachtin.  I found t h a t t h i s compound  43  lacked antifeedant once i n g e s t e d , effects. involved  a c t i v i t y a g a i n s t M.. s a n g u i n i p e s . but,  i t produced a v a r i e t y o f p h y s i o l o g i c a l  The t o x i c o l o g y o f a z a d i r a c h t i n and f a c t o r s in i t s bioavailability  in detail.  Finally,  i n t h i s i n s e c t were examined  I proposed and t e s t e d two hypotheses as  t o the mode o f a c t i o n o f a z a d i r a c h t i n .  44  Chapter 2: I n s e c t i c i d a l and Growth-Reducing A c t i v i t y o f F o l i a r E x t r a c t s from t h e M e l i a c e a e  Introduction  P r i o r t o t h e advent o f s y n t h e t i c i n s e c t i c i d e s , p e s t - c o n t r o l s t r a t e g i e s r e l i e d l a r g e l y on p l a n t - d e r i v e d e x t r a c t s and preparations  (Jacobson and Crosby, 1 9 7 1 ) .  In c e r t a i n  r e g i o n s , members o f t h e Meliaceae f i g u r e d prominently i n t h i s respect.  In p a r t i c u l a r , t h e neem t r e e ,  Azadirachta  i n d i c a A. J u s s . , has long been noted f o r i t s e f f e c t i v e n e s s i n p r o t e c t i n g crops, by i n s e c t s .  c l o t h i n g , and s t o r e d g r a i n s from  S a n s k r i t w r i t i n g s over 2,000 y e a r s o l d d e t a i l  proceedures f o r p r e p a r i n g  water e x t r a c t s o f t h e f o l i a g e t o  p r o t e c t g r a i n f i e l d s from l o c u s t s (Radwanski, 1 9 7 7 ) . s i m i l a r a n c i e n t h i s t o r y o f use p e r t a i n s t o M e l i a i n t h e Middle East has  attack  (Lavie g t a l . , 1967).  confirmed t h e potent a n t i f e e d a n t  a c t i v i t y o f these p r e p a r a t i o n s  A  azedirach  Subsequent work  and i n s e c t i c i d a l  and has l e d t o t h e i s o l a t i o n  of a v a r i e t y o f t r i t e r p e n o i d c o n s t i t u e n t s , e x c l u s i v e l y o f the l i m o n o i d principles The  or protolimonoid  c l a s s , as t h e a c t i v e  (reviewed i n Chapter 4 o f t h i s T h e s i s ) .  use o f p l a n t e x t r a c t s d e c l i n e d as  inexpensive  s y n t h e t i c i n s e c t i c i d e s became a v a i l a b l e f o r t y y e a r s ago (Jacobson and Crosby, 1 9 7 1 ) .  However, i n t e n s i v e use o f  s y n t h e t i c compounds has r e s u l t e d i n numerous environmental problems i n c l u d i n g impact on non-target organisms ( i n c l u d i n g  45  humans), contamination o f water and s o i l w i t h p e r s i s t a n t residues  ( Z i t t e r , 1985), and t h e appearance o f r e s i s t a n c e i n  over 400 s p e c i e s o f e c o n o m i c a l l y species  (Luck e t a l . , 1977).  i n s e c t i c i d e s has e s c a l a t e d  important p e s t i n s e c t  As w e l l , t h e c o s t o f s y n t h e t i c  ( K i n o s h i t a , 1985) and such  compounds a r e now l e s s economically i n t h e T h i r d World.  attractive, particularly  As a r e s u l t , i n t e r e s t i n b o t a n i c a l  i n s e c t i c i d e s has been r e k i n d l e d i n r e c e n t y e a r s  (Balandrin  e t a l . , 1985; Hedin, 1982).  useful  P l a n t s may p r o v i d e  compounds o r e x t r a c t s d i r e c t l y , o r i n s e c t i c i d a l and antifeedant  phytochemicals may p r o v i d e  leads f o r the  s y n t h e s i s o f new compounds. Given t h e remarkable i n s e c t i c i d a l a c t i v i t y o f e x t r a c t s o f neem and c h i n a b e r r y ,  I decided  t o bioassay  methanolic  e x t r a c t s o f a d i v e r s e assemblage o f s p e c i e s o f t h e M e l i a c e a e , w i t h t h e aim of i d e n t i f y i n g f u r t h e r s p e c i e s o f potential interest.  To t h i s end a c o l l e c t i o n o f f o l i a g e  samples o f t h i r t y - o n e s p e c i e s , out o f about 500 i n t h e family  (Pennington and S t y l e s , 1975), i n twenty-two genera  (out o f 51) was assembled and b i o a s s a y e d a g a i n s t the variegated  cutworm, Peridroma s a u c i a .  the s t r o n g e s t  E x t r a c t s which showed  a c t i v i t y a g a i n s t E . s a u c i a were a l s o assayed  f o r f e e d i n g i n h i b i t i o n a g a i n s t t h e m i g r a t o r y grasshopper, Melanoplus  sanguinipes.  A primary g o a l i n t h e study o f p l a n t - h e r b i v o r e i n t e r a c t i o n s i s t o e x p l a i n why p l a n t s d i f f e r i n t h e i r commitment t o defenses and consequently i n t h e i r  46  s u s c e p t a b i l i t y to herbivores  (Coley e t a l . , 1985).  In  f i r s t attempt a t such a s y n t h e s i s , Feeny (1976) and and  the  Rhoades  Cates (1976) independently proposed t h a t the p r o b a b i l i t y  of d i s c o v e r y by h e r b i v o r e s ,  termed p l a n t apparency by Feeny,  governs the e v o l u t i o n of d e f e n s i v e the h y p o t h e s i s ,  strategy.  According  apparent p l a n t s , which are g e n e r a l l y  to  late-  s u c c e s s i o n a l p e r e n n i a l s , are d i s t r i b u t e d p r e d i c t a b l y i n space and  time and  herbivores.  so are l i k e l y t o be l o c a t e d  In such p l a n t s h e r b i v o r e  pressure  by should  select  f o r " q u a n t i t a t i v e " defenses e f f e c t i v e a g a i n s t both adapted s p e c i a l i s t and unadapted g e n e r a l i s t Q u a n t i t a t i v e defenses (eg. t a n n i n s ) feeding rates  herbivores. were thought t o a f f e c t  ( v i a l e a f toughness) and  nutrient a v a i l a b i l i t y  t o the i n s e c t ( v i a complexing w i t h p r o t e i n ) , and impossible  so would  be  f o r i n s e c t s t o circumvent; however, they were  b e l i e v e d t o be m e t a b o l i c a l l y expensive t o produce i n the l a r g e q u a n t i t i e s necessary f o r adequate p r o t e c t i o n . Unapparent p l a n t s , u n p r e d i c t a b l e  i n space and time  and  mostly s h o r t - l i v e d e a r l y s u c c e s s i o n a l s p e c i e s , were b e l i e v e d t o r e q u i r e defenses a g a i n s t g e n e r a l i s t h e r b i v o r e s . p l a n t s " q u a l i t a t i v e " defenses ( t o x i c secondary would p r o v i d e evolve  adequate defense.  In such  metabolites)  Although h e r b i v o r e s  could  immunity t o such defenses t h i s l o s s of e f f i c a c y would  be balanced a g a i n s t t h e i r low c o s t of p r o d u c t i o n .  Among  v a r i o u s d i f f i c u l t i e s w i t h the p l a n t apparency h y p o t h e s i s (Fox,  1981;  Bernays, 1978;  t h a t a l l p l a n t s should  Berenbaum, 1983), i t p r e d i c t s  s u f f e r about equal r a t e s of  47  h e r b i v o r y , which i s not observed t o be the case 1983), and  (Coley,  i t f a i l s t o account f o r the observed d i f f e r e n c e s  i n types and e x t e n t of defense amongst p e r e n n i a l apparent plants. More r e c e n t l y these d i f f e r e n c e s i n defense a l l o c a t i o n have been a s c r i b e d t o the growth r a t e of p l a n t s , which i s c l o s e l y r e l a t e d t o the a v a i l a b i l i t y  of r e s o u r c e s i n c l u d i n g  water, l i g h t and n u t r i e n t s (Coley e t a l . , 1985).  Fast-  growing s p e c i e s , adapted t o r e s o u r c e - r i c h h a b i t a t s , r e p l a c e l e a v e s r e l a t i v e l y r a p i d l y and so are a b l e t o t o l e r a t e h i g h e r r a t e s of h e r b i v o r y .  In r e s o u r c e - l i m i t e d h a b i t a t s , slow-  growing s p e c i e s tend t o r e p l a c e l e a v e s s l o w l y , and so must l i m i t the r a t e of l o s s t o h e r b i v o r e s .  A g i v e n r a t e of  h e r b i v o r y w i l l remove a h i g h e r p r o p o r t i o n of the primary p r o d u c t i v i t y from slow-growing p l a n t s than from f a s t e r growing s p e c i e s .  The  model:  dC/dt = G*C*(l-kD ) - (H-mD ) a  6  [where dC/dt i s the r e a l i z e d growth r a t e , G (g g " the maximum i n h e r e n t growth r a t e without  d )  is  herbivores, C  (g)  1  i s the p l a n t biomass a t time 0, D (g g )  i s the  i n defense,  relating  - 1  k (g d ) - 1  and  a are c o n s t a n t s  - 1  investment  investment i n defense t o r e d u c t i o n i n growth, H (g d" ) 1  the p o t e n t i a l h e r b i v o r e p r e s s u r e are c o n s t a n t s  is  i n a h a b i t a t , and m and  r e l a t i n g the r e d u c t i o n i n h e r b i v o r y t o the  investment i n defense] p r e d i c t s t h a t i n t r i n s i c a l l y growing p l a n t s should  slow-  i n v e s t more of t h e i r primary  p r o d u c t i v i t y i n defense than should fast-growing  species.  8  48  M e t a b o l i c a l l y mobile secondary m e t a b o l i t e based defenses a r e thought t o t u r n over r e l a t i v e l y r a p i d l y ; t o m a i n t a i n a g i v e n l e v e l o f defense these defense compounds would have t o be s y n t h e s i z e d c o n t i n u o u s l y , so t h e c o s t o f these defenses would i n c r e a s e a r i t h m e t i c a l l y over t h e l i f e t i m e o f t h e l e a f . However, because these defenses a r e m e t a b o l i c a l l y mobile they may o f t e n be r e c o v e r e d from s e n e s c i n g l e a v e s .  On t h e  o t h e r hand, q u a n t i t a t i v e defenses, e s p e c i a l l y l e a f f a c t o r s , a r e emplaced a t l e a f expansion; although  toughness initially  c o s t l y , such defenses a r e n o t m e t a b o l i c a l l y mobile and c o s t l i t t l e t o m a i n t a i n once i n p l a c e , but they a r e l o s t a t l e a f senescence.  Comparison o f these defense types suggests t h a t  c h e m i c a l l y based defenses should be s e l e c t e d f o r i n s p e c i e s w i t h s h o r t l e a f l i f e t i m e s ( f a s t - g r o w i n g p l a n t s ) , and immobile  defenses s h o u l d be f a v o r e d i n s p e c i e s w i t h l o n g  l e a f l i f e t i m e s (slow-growing p l a n t s ) ( F i g u r e 2-1).  The  model has been supported by s t u d i e s o f lowland r a i n f o r e s t s p e c i e s i n Panama (Coley, 1983, 1988), b u t measurement o f investment i n secondary m e t a b o l i t e defenses was c o n f i n e d t o assays o f polyphenol c o n t e n t , which d i d n o t c o r r e l a t e w i t h h e r b i v o r y , l e a f l i f e t i m e , o r o t h e r l e a f a t t r i b u t e s (Coley, 1983).  To date, t h e h y p o t h e s i s has been t e s t e d w i t h p l a n t  s p e c i e s c o - o c c u r r i n g i n t h e same h a b i t a t ; i t i s u n c l e a r whether t h e model can a l s o account f o r e v o l u t i o n a r y p a t t e r n s w i t h i n c l o s e l y r e l a t e d groups o f p l a n t s . The Meliaceae o f f e r s some p a r t i c u l a r advantages t e s t of t h i s hypothesis.  for a  Members o f t h e f a m i l y occur i n  49  h a b i t a t s r a n g i n g from r a i n f o r e s t t o mangrove swamp t o semidesert (White, 1975).  Few  f a m i l i e s embrace such a wide  range of f l o r a l c h a r a c t e r i s t i c s , but the f a m i l y has been c o n s i d e r e d t o be monophyletic i n a l l taxonomic treatments t o date (Pennington and S t y l e s , 1975).  C o n s i d e r a b l e time has  been a v a i l a b l e f o r the e v o l u t i o n o f the d i v e r s i t y o b s e r v a b l e today, as s p e c i e s c l e a r l y a s s i g n a b l e t o the modern genus C e d r e l a a r e c h a r a c t e r i s t i c o f western North American Paleocene f l o r a s (Brown, 1965)  and the f a m i l y p r o b a b l y  appeared i n the Cretaceous, a t l e a s t 70 m i l l i o n y e a r s ago. The f a m i l y l a c k s trichomes, t h o r n s , and s p i n e s , and so p h y s i c a l defenses are c o n f i n e d t o l e a f toughness f a c t o r s and pubescence.  As w e l l , d e s p i t e the f o r m i d a b l e c h e m i s t r y of  such s p e c i e s as A z a d i r a c h t a i n d i c a all  ( S i d d i q u i e_t a i . ,  1988),  a n t i f e e d a n t o r i n s e c t i c i d a l phytochemicals i d e n t i f i e d t o  date from t h i s f a m i l y are l i m o n o i d s ; indeed the s c a r c i t y o f o t h e r c l a s s e s o f secondary m e t a b o l i t e s i s remarkable. e v o l u t i o n of chemical defenses i n t h i s f a m i l y may  The  therefore  be examined by a c o n s i d e r a t i o n of the r e l a t i o n s h i p between i n s e c t i c i d a l a c t i v i t y and e v o l u t i o n a r y p o s i t i o n o f members of a s i n g l e c l a s s o f phytochemicals. Measurement of investment i n secondary m e t a b o l i t e based defenses has p r e s e n t e d c o n s i d e r a b l e d i f f i c u l t y ; p r e v i o u s attempts have l a r g e l y c e n t e r e d on c o l o r i m e t r i c assays f o r t o t a l p h e n o l i c s (eg. C o l e y , 1983,  1988).  T h i s approach i s  u n s a t i s f a c t o r y , as most known i n s e c t i c i d a l o r a n t i f e e d a n t compounds would not be d e t e c t e d i n such an assay, and the  50  assumption  t h a t q u a n t i t y of p h e n o l i c s i s d i r e c t l y r e l a t e d t o  the c o s t and e f f i c a c y of chemical defenses i s q u e s t i o n a b l e . As the r o l e of secondary m e t a b o l i t e based defenses has been construed t o be d e t e r r e n c e of unadapted g e n e r a l i s t h e r b i v o r e s , i n t h i s study I have estimated investment  i n phytochemical  relative  based defenses by the r e l a t i v e  response of a h i g h l y polyphagous h e r b i v o r e  (Peridroma  s a u c i a ) t o the e n t i r e s u i t e of phytochemicals  produced  in a  p l a n t , as o b t a i n e d i n the methanolic e x t r a c t s of mature foliage.  As E .  s a u c i a l a c k s an e v o l u t i o n a r y a s s o c i a t i o n  w i t h the Meliaceae o r w i t h p l a n t s c o n t a i n i n g l i m o n o i d s , any observed response t o the e x t r a c t s may  be c o n s t r u e d as  r e p r e s e n t i n g the response of a n a i v e , unadapted h e r b i v o r e e n c o u n t e r i n g a p o t e n t i a l meliaceous  foodplant.  This  approach avoids the q u e s t i o n of c o s t of p r o d u c t i o n of chemical defenses, and r a t h e r f o c u s e s a t t e n t i o n on t h e e f f i c a c y o f those  defenses.  R e l a t i v e l e a f toughness was  measured d i r e c t l y  by  d e t e r m i n i n g the amount of f o r c e r e q u i r e d t o punch a 0.5 diameter  f l a t - t i p p e d rod through a l e a f .  Leaf  lifetimes  were not measured d i r e c t l y , but as an approximation have been separated i n t o deciduous As p r e s e n t l y understood,  species  and evergreen s p e c i e s .  the r e s o u r c e a v a i l a b i l t y  would p r e d i c t t h a t s p e c i e s w i t h s h o r t l e a f  hypothesis  lifetimes  (deciduous) s h o u l d produce l e a v e s t h a t are more t o x i c l e s s tough than the evergreen l e a v e s of slow growing species.  cm  As w e l l , a n e g a t i v e c o r r e l a t i o n between l e a f  and  1  51  toughness and  l e a f t o x i c i t y may  p e r e n n i a l s p e c i e s may  be expected.  Overall,  be expected t o have a h i g h e r  commitment t o t o t a l defenses a g a i n s t h e r b i v o r e s .  the  52  F i g u r e 2-1.  G r a p h i c a l d e p i c t i o n of the assumed r e l a t i v e  c o s t o f m a i n t a i n i n g a chemical o r p h y s i c a l l y - b a s e d defense a g a i n s t h e r b i v o r e s .  Costs of t h e chemical  defense i n c r e a s e a r i t h m e t i c a l l y over the l i f e l e a f , due t o t u r n o v e r .  P h y s i c a l defenses  are i n i t i a l l y  c o s t l y , d u r i n g emplacement f o l l o w i n g l e a f  expansion;  however as they a r e m e t a b o l i c a l l y i n a c t i v e there i s l i t t l e  o r no c o s t a s s o c i a t e d w i t h  maintenance once i n p l a c e .  o f the  (immobile), their  Consequently, p l a n t s w i t h  s h o r t - l i v e d l e a v e s should be s e l e c t e d f o r the production of chemically-based defenses leaves.  defenses,  and p h y s i c a l  should be f a v o r e d i n s p e c i e s with l o n g - l i v e d  30  oH 0  •  1  10  •  1  20  •  1  30  Leaf Lifetime  •  1  40  •  1 50  54  M a t e r i a l s and Methods  F o l i a r samples o f t h i r t y - o n e s p e c i e s i n twenty genera o f t h e M e l i a c e a e were o b t a i n e d f o r t h i s study.  Most were c o l l e c t e d  from the Gordon F a i r c h i l d T r o p i c a l Gardens  and the USDA  P l a n t Quarantine Center i n Miami, F l o r i d a , from the P a c i f i c T r o p i c a l Garden, Hawaii, o r from the Kunming I n s t i t u t e , Kunming, China.  Other s p e c i e s were f i e l d c o l l e c t e d i n  Kenya, T h a i l a n d , M a u r i t i u s , and New Zealand.  A few s p e c i e s  are r e p r e s e n t e d by c o l l e c t i o n s from more than one s i t e . A l l samples were o f mature ( f u l l y expanded and greened) l e a v e s c o l l e c t e d approximately i n t h e middle o f t h e growing season. Sources f o r each s p e c i e s and c o l l e c t i o n dates are g i v e n i n T a b l e 2-1. Samples o f most s p e c i e s were r e c e i v e d a l r e a d y powdered; where p o s s i b l e voucher specimens have been d e p o s i t e d i n the UBC Herbarium. Samples were a i r d r i e d , ground t o a f i n e powder i n a Wiley m i l l , weighed,  and e x t r a c t e d i n t h r e e changes o f MeOH,  24 h/change, 11/100 g dry weight (dwt).  MeOH e x t r a c t s were  pooled and c o n c e n t r a t e d under vacuum; f i n a l c o n c e n t r a t i o n s were a d j u s t e d t o 2 mis MeOH/g dwt l e a f .  Aliquots of the  MeOH e x t r a c t s were d r i e d and weighed t o determine t h e e x t r a c t i o n y i e l d and a l l o w c a l c u l a t i o n o f dose-response r e l a t i o n s h i p s i n terms o f mg e x t r a c t / g d i e t f r e s h weight (fwt). For b i o a s s a y , d i e t s were prepared a c c o r d i n g t o t h e procedure o f 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 l i s t e d 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 i n Miami, F l a , and FG refers to the Gordon Fairchild Tropical Garden in Miami. Species Family Meliaceae Subfamily Melioideae Tribe 1. Turreeae  Turreae h o l s t i i Gurke  Turreae mauritiana Tribe 2. Melieae EslXA azedirach L. (Fla) Melia azedirach L. (Ind) M£iia azedirach L. (Chi) Mfilia tPPSenden Azadirachta indica A.Juss. (Fla) Azadirachta indica A.Juss. (Haw) Azadirachta indica A.Juss. (Chi) Tribe 4. T r i c h i l i e a e  Source  Collector Date  Mt Eldon, Kenya Mauritius  SD JTA  03-85 06-86  PI073248 India Kunming, China Kunming, China PI137950 PTBG790480001 Kunming, China  DEC KP SQ SQ DEC TF SQ  06- 87 07- 85 05-86 05- 86 06- 87  DEC TF SD SD DEC  06- 87 03-85 03-85 06- 87  GHNT TF GHNT GHNT GHNT DEC GBS  05- 85 12-86 07- 85 07- 85 08- 85 06- 87 08-88  DEC GBS  06-87 12-87  DEC  06-87  DEC TF DEC DEC  06-87  FG T r j p h i l i a h i r t a L. PI T r i c h i l i a roka L g p i d P t r i c h j i i a v o l K e n s i i (Gurke)Leroy  Mt.Eldon, Kitale, Kenya EKebergia capensis Sparrm. Cipadessa baccifera (Roth) Miq. PI105699 Tribe 5. Aglaieae Trang, Thai. A g l a i a odorata Lour. (Thai) Maui Aglaia odorata Lour. (Haw) Trang, Thai. A g l a i a odoratissima Blume Trang, Thai. Aalaia araentia Blume Kunming Aphanamixus arandifolia Blume Aphanamjxus polvstachya (Wall) R.N.Parker Manila Lansium domesticum Corr. Tribe 6. Guareeae PI Guarea glabra Vahl Dyspxylum spectabile Hook. Opua Rec. Forest,N.Z. Tribe 7. Sandoriceae Sandoricum koetiape (Burman f . ) M e r r i l l PI Subfamily Swietenioideae Tribe 1. Cedreleae Cedrela odorata L. PI097976 Toona serrata (Royle) Penn.fi Styles PTG Tppna C i l i a t a M.J. Roemer PI lapjia australis (F. von Mueller) PI  05- 86  56 Tribe 2. Svietenieae Khaya senegalensis (Desr)A.Juss. PTBG770642003 TF Chuckrassia tabularis A.Juss. PI DEC Entandrophracrma caudatum (Sprague) Sprague PI DEC Swietenia humilis Zuccarini PI092371 DEC Swietenia mahoqani (L.)Jacquin PI DEC Swietenia macrophylla King PI DEC Tribe 3. Xylocarpeae DEC Carapa auianensis Aubl. EI Collectors: DEC, D.E. Champagne, UBC; GHNT, G.H.N. Towers, UBC; JTA, J.T. Arnason, University of Ottawa; SQ, Song Qui-Si, Kunming Institute; TF, T. Flynn, P a c i f i c Tropical Botanical Garden; GBS, G.B. Straley, UBC; SD, S. Dossaji, National Museums, Kenya.  06-87 06-87 06==87 06-87 06-87 06- 87  57  e x t r a c t s were added t o t h e d i e t d r y components  (Velvetbeen  C a t e r p i l l a r D i e t , no. 9682, B i o s e r v I n c . , Frenchtown, N.J.), initially  i n amounts c a l c u l a t e d t o produce c o n c e n t r a t i o n s o f  25, 50, 75, and 100% o f n a t u r a l l e a f c o n c e n t r a t i o n s , on a dwt leaf/dwt d i e t b a s i s . were subsequently the E C  5 0  I f necessary  other  concentrations  evaluated t o f a c i l i t a t e determination of  ( c o n c e n t r a t i o n r e q u i r e d t o reduce l a r v a l growth by  50% r e l a t i v e t o t h e c o n t r o l s ) . evaporated  The MeOH c a r r i e r was  i n a fume hood, u s u a l l y o v e r n i g h t .  C o n t r o l s were  s i m i l a r l y t r e a t e d w i t h MeOH a l o n e . Three neonate Peridroma s a u c i a l a r v a e were p l a c e d on 1 g f r e s h weight (fwt) d i e t i n a 30 ml p l a s t i c Solo cup; 10 cups were used p e r treatment were r e p l i c a t e d t h r e e times.  f o r a t o t a l n=30.  Experiments  Rearing cups were p u t i n c l e a r  p l a s t i c boxes, f l o o r e d w i t h moistened paper towels t o m a i n t a i n h i g h humidity, 27+1° C, 16:8 LD.  and p l a c e d i n a growth c a b i n e t a t  S u r v i v o r s h i p and l i v e l a r v a l weights were  determined a f t e r seven days o f growth; l a r v a e were n o t weighed o r handled  on i n t e r v e n i n g days t o minimize  a r t i f i c i a l l y induced growth d i s t u r b a n c e s 1986).  Weights were l o g  1 0  transformed  (Reese and Schmidt,  to correct f o r  h e t e r o s c e d a s t i c i t y p r i o r t o a n a l y s i s by l e a s t - s q u a r e s r e g r e s s i o n u s i n g t h e SAS GLM procedure t o determine t h e EC  5 0  .  formula  M o r t a l i t y v a l u e s were c o r r e c t e d u s i n g and L C  5 0  values  Abbott's  (the c o n c e n t r a t i o n r e q u i r e d t o  reduce s u r v i v o r s h i p by 50% r e l a t i v e t o t h e c o n t r o l s ) were determined u s i n g t h e SAS P r o b i t s t a t i s t i c a l package.  58  F o l i a r e x t r a c t s were a l s o examined f o r a n t i f e e d a n t a c t i v i t y a g a i n s t the migratory sanguinipes.  grasshopper,  Melanoplus  E x t r a c t s , s u f f i c i e n t t o a c h i e v e 100%  of  n a t u r a l c o n c e n t r a t i o n on a dwt/dwt l e a f d i s c b a s i s (20 u l ) , were a p p l i e d evenly, u s i n g a 25 u l Hamilton s y r i n g e , t o both s u r f a c e s of 1.5  cm diameter cabbage ( B r a s s i c a o l e r a c a cv.  S i l v e r Queen) l e a f d i s c s . l e a f d i s c s were presented  A f t e r the e x t r a c t s had d r i e d , the to f i f t h  i n s t a r nymphs i n seven cm  diameter unwaxed paper cups; the b i o a s s a y was one  l e a f disc/nymph.  no-choice  A f t e r 24 h uneaten l e a f m a t e r i a l  with was  d r i e d t o c o n s t a n t weight (24 h § 60° C) and weighed; s t a r t i n g l e a f weight was  determined by d r y i n g and  samples o f i n t a c t l e a f d i s c s . each  weighing  Ten r e p l i c a t e s were used f o r  treatment. E x t r a c t s of A z a d i r a c h t a i n d i c a , M e l i a a z e d i r a c h  A g l a i a o d o r a t a . and Turreae h o l s t i i were assayed  u s i n g 1.5  subsequently  cm diameter g l a s s f i b r e f i l t e r  discs,  t r e a t e d w i t h s u f f i c i e n t e x t r a c t t o achieve c o n c e n t r a t i o n s 2.5  and  5-fold naturally occurring levels.  A f t e r d r y i n g the  d i s c s were s a t u r a t e d with a 10% aqueous sucrose s o l u t i o n presented  to f i f t h  1,  and  i n s t a r nymphs as d e s c r i b e d above.  Leaf toughness was  measured d i r e c t l y on some s p e c i e s  (those a v a i l a b l e i n the USDA and F a i r c h i l d c o l l e c t i o n s i n Miami, F l a . ) u s i n g a l e a f "punchmeter" modelled a f t e r the d e s i g n of Feeny (1970). r e q u i r e d t o punch a 5mm  T h i s d e v i c e measures the f o r c e diameter,  f l a t - e n d e d rod through the  l e a f , which i s clamped i n t o p l a c e i n the base of the d e v i c e .  59  Force was a p p l i e d by adding water from a b u i r e t t o a beaker atop t h e rod; a f t e r p e n e t r a t i o n weighed. the  t h e beaker and water were  Weights were converted t o Newtons/cm  2  according t o  formula:  F(Newtons) = wgt (kg) x 9.8 N/crn^ 0.2 cm  =  wgt (kg) x 49  2  A l l measurements were made on f r e s h l y c o l l e c t e d l e a v e s . Care was taken t o a v o i d primary and secondary v e i n s , although t h i s was d i f f i c u l t t o ensure i n t h e case o f Azadirachta spacing  i n d i c a and M e l i a a z a d i r a c h  o f t h e secondary v e i n s .  due t o t h e c l o s e  F i v e t o t e n measurements  were made on s e p a r a t e l e a f l e t s f o r each s p e c i e s .  Species  were compared u s i n g Duncan's m u l t i p l e range t e s t .  The  r e l a t i o n s h i p between l e a f toughness and t o x i c i t y o f t h e e x t r a c t s , measured as t h e E C , was examined u s i n g 5 0  regression analysis. Leaf e x t r a c t s were a l s o examined f o r t h e p o s s i b l e presence o f a n t i b i o t i c o r p h o t o t o x i c  compounds.  The l a t t e r  p o s s i b i l i t y was examined because o f t h e presence o f a d i v e r s i t y o f known p h o t o s e n s i t i z e r s i n t h e c l o s e l y r e l a t e d p l a n t f a m i l y Rutaceae.  The method o f D a n i e l s  (1965) was  used: e x t r a c t s ( e q u i v a l e n t t o 5 mg l e a f t i s s u e ) were d r i e d on t o s t e r i l e f i l t e r paper d i s c s , which were p l a c e d on d u p l i c a t e p l a t e s s t r e a k e d w i t h a lawn o f t h e y e a s t Saccharomyces c e r e v i s i a e .  One p l a t e was incubated  i n the  dark (-UV) a t 37° C; t h e second p l a t e was i r r a d i a t e d by near-UV (a bank o f f o u r B l a c k - L i g h t Blue tubes 10 cm above  the plates) for 4 h, a f t e r which the plates were incubated i n the dark as per the -UV  treatment.  61  Results  A. Growth i n h i b i t i o n s t u d i e s w i t h Peridroma s a u c i a A l l but t h r e e of the e x t r a c t s t e s t e d produced marked i n h i b i t i o n of the growth of E .  s a u c i a neonates a t  c o n c e n t r a t i o n s below those o c c u r r i n g n a t u r a l l y i n the leaves.  Growth curves f o r l a r v a e f e d A z a d i r a c h t a  indica  f  M e l i a a z e d i r a c h . and M e l i a toosenden e x t r a c t s are shown i n F i g . 2-2  and are t y p i c a l of the more a c t i v e e x t r a c t s .  and L C  v a l u e s , i n terms of both mg  5 0  e x t r a c t / g d i e t dwt  % of n a t u r a l c o n c e n t r a t i o n , are g i v e n i n Table Extraction efficencies  ( i n mg  The  t o g e a t h e r with  leaf  2  or  and  2-2.  ( i n N/cm ), l e a f pubescence, and  " h a b i t " (deciduous  5 0  e x t r a c t / g dry l e a f ) f o r a l l  s p e c i e s are g i v e n i n Table 2-3, toughness v a l u e s  EC  leaf  evergreen).  s p e c i e s which produced the most a c t i v e e x t r a c t s , i n  terms of both growth i n h i b i t i o n and m o r t a l i t y , were a l l i n the s u b f a m i l y M e l i o i d e a e . the t r i b e Melieae  W i t h i n t h i s s u b f a m i l y , members of  (Azadirachta i n d i c a . Melia azedirach,  and  M e l i a toosenden) were a l l h i g h l y i n h i b i t o r y towards E* s a u c i a neonates, w i t h E C  5 0  c o n c e n t r a t i o n s below 2% of  n a t u r a l c o n c e n t r a t i o n (0.59-2.10 mg/g). a l s o t o x i c , with L C leaf concentration.  5 0  These s p e c i e s were  c o n c e n t r a t i o n s of about 5% of n a t u r a l Dead l a r v a e were a l l s m a l l and s t i l l  the f i r s t i n s t a r ; none appeared t o have d i e d w h i l e A z a d i r a c h t a i n d i c a f o l i a g e samples from Hawaii,  in  molting.  I n d i a , and  62  F i g u r e 2-2.  Growth (as % of C o n t r o l ) o f neonate Peridroma  s a u c i a f e d a r t i f i c i a l d i e t t r e a t e d w i t h a MeOH e x t r a c t of f o l i a g e o f A z a d i r a c h t a i n d i c a samples), M e l i a toosenden  f  ( F l o r i d a and Hawaiian  o r M e l i a a z e d i r a c h a t 1,  o r 3% o f n a t u r a l c o n c e n t r a t i o n .  2,  Each p o i n t shows t h e  mean o f t h r e e r e p l i c a t e s with 30 c o h o r t s / r e p l i c a t e ; standard e r r o r s were < 6% and a r e omitted f o r c l a r i t y .  Concentration (% of Natural Leaf) \j4  64 Table 2-2. Growth inhibitory a c t i v i t y and t o x i c i t y of meliaceous leaf extracts on neonate £. saucia. Values given are the concentration (as % of natural leaf concentration and mg/g diet dwt) of the t o t a l MeOH extract administered i n a r t i f i c i a l diet required to reduce growth (EC ) or survivorship ( L C ) by 50% relative to the control, over a seven day assay. Numbers i n a column followed by the same l e t t e r are not significantly different, based on overlap of their 95% confidence l i m i t s . 50  50  EC  UJ  5Q  lag/a)  Family Meliaceae Subfamily Melioideae Tribe 1. Turreeae Turreae h o l s t i i 1 .8' Turreae mauritlana 37 • 3< 45.40 J Tribe 2. Melieae M£lia azedirach (Florida) 1 .0 1.75cd Melia azedirach (India) 1 .2 2.10Cd Melia azedirach (China) 0 1.58be a b Melia toosenden 1 2.29 cd 0.69 Azadirachta indica (Florida) o .7 0.59 Azadirachta indica (Hawaii) 0 .6 0.89ab Azadirachta indica (China) 0 .8 Tribe 4. T r i c h i l i e a e 18.88 ef T r i c h i l i a hir£a 12,.5 T r i c h i l i a roka 18,.8 Lepidotrichilia volkensii 12..5° 26.14^9° Ekepergia capensis 43..0 102.94* Cipadessa baccifera 44..0 133.02 Tribe 5. Aglaieae 2.07cd Aglaia odorata (Thailand) 1, 3.29<* AoTaia odorata (Hawaii) 2. 4.27 Aalaia odoratissima 11, °d 11.88 Aalaia araentia 27. 0 >63.1 Aphanamixus grandifolla >100 77. 7 Aphanamjxus polvstachya 74. >153.6 Lansium domesticum >100 Tribe 6. Guareeae 53.32 Guarea glabra 62. >84.0 Dysoxvlum spectabile >100 Tribe 7. Sandoriceae Sandoricum Koetjape 17. 4° 22.79 ni  a  a  >5  a  a  a  a  a  C  C  e  1  e  d e  d  k  3  ef  m  LC 50  57.0 >100 7.1 7.3 5.6 4.7 4.8 4.8 5.2  (ma/cn  90.7 >121.7 12.5 12.8 9.8 7.2 4.7 4.7 5.8  >100 >100 >100 >100 >100  >151.0  17.5 24.4 >100 >100 >100 >100 >100  21.3 29.7 >38.8 >44.0 >63.1 >105.1 >153.6  >209.1 >239.4 >302.3  >100 >100  >86.0 >84.0  >100  >131.0  65  Subfamily S w i e t e n i o i d e a e T r i b e 1. Cedreleae C e d r e l a odorata Toona s e r r a t a Toona c i l i a t a Toona a u s t r a l i s T r i b e 2. Swietenieae Khaya s e n e g a l e n s i s Chuckrassia t a b u l a r i s Entandrophraama caudatum Swietenia h u m i l i s Swietenia mahogani Swietenia c a n d o l l e i T r i b e 3. Xylocarpeae Carapa g u i a n e n s i s  53.6 27.0 28.2 12.0  f d d  67.05 . 31.139 29.839 23.64ffg 47.09 ? . 42.36 3  >100 >100 >100 >100 >100 >100  >115.1 >249.0 >132.0 >100.0 >277.0 >211.8  29.689  >100  >55.8  n  k  ni  c d c d  C  f  >46.0 >176.4 >100.0 >191.1  ef  f  53.2  >100 >100 >100 >100  f  C  58.3 12.5° 22.6 23.0 17.0 20.0  24.66^9 47.63*3 28.20 9 22.93 9  h  1  c d  ni  h  66  F l o r i d a d i d not d i f f e r s i g n i f i c a n t l y i n t h e i r t o x i c i t y t o P. s a u c i a neonates.  S i m i l a r l y , Melia azedirach  samples from  Hawaii, I n d i a and F l o r i d a d i d not d i f f e r s i g n i f i c a n t l y i n these assays. Other t r i b e s i n t h e M e l i o i d e a e their effects.  were more v a r i a b l e i n  Among t h e Turreeae, Turrea  holstii foliage  e x t r a c t s were comparable t o M e l i a l e a f e x t r a c t s ,  (EC5o=1.8%,  the growth and s u r v i v o r s h i p o f P.. s a u c i a l a r v a e 2.8 mg/g).  inhibiting  Unlike the s i t u a t i o n with Azadirachta  indica or  the two M e l i a s p e c i e s , numerous l a r v a e were seen t o have d i e d a t a f a i l e d molt attempt a t t h e end o f t h e f i r s t o r second i n s t a r (LC o=57.0%, 90.7 mg/g). 5  mauritiana  were much l e s s a c t i v e , w i t h an E C  natural concentration natural  E x t r a c t s o f T_. 5 0  of 37.3%  (45.4 mg/g), and no t o x i c i t y a t  concentration.  Within  the Aglaieae,  e x t r a c t s o f T h a i and Hawaiian  samples o f t h e t r a d i t i o n a l m e d i c i n a l  p l a n t A g l a i a odorata  were h i g h l y t o x i c t o £. s a u c i a neonates, w i t h an E C 1.7% and 2.7% (2.07 and 3.29 mg/g)  of  r e s p e c t i v e l y , and L C  of 17.5 and 24.4% n a t u r a l c o n c e n t r a t i o n .  / 5 0  s  Other s p e c i e s o f  A g l a i a were somewhat l e s s a c t i v e : A., o d o r a t i s s i m a a r g e n t i a had E C ' s o f 11 and 27% n a t u r a l l e a f 5 0  (4.27  5 0  and A..  concentration  and 11.88 mg/g) r e s p e c t i v e l y , and were not t o x i c i n  the seven-day assay. efficiencies  When d i f f e r e n c e s i n t h e e x t r a c t i o n  (Table 2-3) a r e taken i n t o account, A., odorata  i s about t w i c e as a c t i v e as A., o d o r a t i s s i m a times more a c t i v e than A., a r g e n t i a .  and about f i v e  Of t h e two s p e c i e s of  67  Aphanamixus examined, n e i t h e r were t o x i c a t n a t u r a l l e a f c o n c e n t r a t i o n , but A., p o l y s t a c h y a d i d i n h i b i t l a r v a l growth (EC =74%, 50  77.7 mg/g).  Lansium domesticum was i n a c t i v e a t  n a t u r a l c o n c e n t r a t i o n (153.6 mg/g). W i t h i n the T r i c h i l e a e , e x t r a c t s from T r i c h i l i a and L e p i d o t r i c h i l i a v o l k e n s i i were e q u a l l y a c t i v e , EC  / 5 0  s  o f 12.5 % (18.88 and 26.14 mg/g  hirta with  respectively).  N e i t h e r s p e c i e s caused s i g n i f i c a n t m o r t a l i t y a t 100% n a t u r a l concentration.  Cipadessa b a c c i f e r a and Ekebergia  capensis  were a c t i v e a t t h r e e f o l d h i g h e r c o n c e n t r a t i o n s , w i t h of  44 and 43% (102.9 and 133.0 mg/g)  ECSQ'S  respectively.  The two s p e c i e s o f the Guareeae examined were r e l a t i v e l y i n a c t i v e ; Guarea g l a b r a e x t r a c t s had an E C 62% (53.3 mg/g)  N e i t h e r s p e c i e s caused  m o r t a l i t y i n the 7-day t r i a l .  w i t h an E C  of  and Dysoxylum s p e c t a b i l e d i d n o t reduce P.  s a u c i a growth a t 100%.  Sandoriceae  5 0  significant  The s i n g l e s p e c i e s o f t h e  s t u d i e d , Sandoricum k o e t i a p t e . was h i g h l y a c t i v e 5 o  of 17% n a t u r a l c o n c e n t r a t i o n (22.79 mg/g).  S p e c i e s o f t h e s u b f a m i l y S w i e t e n i o i d e a e were l e s s a c t i v e than t h e M e l i o i d e a e .  mostly  W i t h i n the C e d r e l a e , Toona  a u s t r a l i s was the most a c t i v e w i t h an E C  5 0  o f 12%.  Two  o t h e r s p e c i e s of Toona, T. c i l i a t a and T. s e r r a t a , were equally a c t i v e with  ECSQ'S  o f 28 and 27% r e s p e c t i v e l y .  odorata was s i g n i f i c a n t l y l e s s a c t i v e , w i t h an E C natural concentration.  5 0  £.  of 53.6%  Considering e x t r a c t i o n y i e l d s ,  however, Toona a u s t r a l i s . C e d r e l a o d o r a t a . and Toona c i l i a t a were e q u a l l y a c t i v e , w i t h E C o ' s of 22.93, 24.66, and 28.20 5  68  mg/g  respectively.  Toona s e r r a t a was l e a s t a c t i v e from t h i s  p e r s p e c t i v e , with an E C  5 0  of 47.63 mg/g.  No s p e c i e s caused  significant mortality. In t h e Swietenieae, C h u c k r a s s i a t a b u l a r i s was the most i n h i b i t o r y , w i t h an E C  5 0  o f 12.5% n a t u r a l c o n c e n t r a t i o n .  S i m i l a r a c t i v i t y was noted i n S w i e t e n i a mahogani. S_. macrophylla, S_. h u m i l i s , and Entandrophragma caudatum, w i t h E C o ' s o f 17.0, 20.0, 23.0, and 22.6% n a t u r a l c o n c e n t r a t i o n . 5  C o n s i d e r i n g e x t r a c t y i e l d s , S_. h u m i l i s was the most a c t i v e , f o l l o w e d by £. caudatum, C. t a b u l a r i s  f  S. macrophylla, and  S. mahogani ( E C = 23.64, 29.83, 31.13, 42.36, and 47.09 5 0  mg/g  respectively).  a c t i v e , w i t h an E C  5 0  Khaya s e n e g a l e n s i s was the l e a s t o f 53.6% (67.05 mg/g).  The o n l y member  of t h e Xylocarpeae a v a i l a b l e f o r study, Carapa g u i a n e n s i s . a l s o had low a c t i v i t y , w i t h an E C  5 0  o f 53.2% (29.68 mg/g).  None o f these e x t r a c t s caused an i n c r e a s e i n P_. s a u c i a m o r t a l i t y d u r i n g the seven-day assay.  B A n t i f e e d a n t s t u d i e s w i t h Melanoplus s a n g u i n i p e s When f o l i a r e x t r a c t s were presented on cabbage l e a f d i s c s t o M. s a n g u i n i p e s nymphs, a l l were completely consumed w i t h i n 24 h.  However, when e x t r a c t s o f some s p e c i e s (chosen f o r  re-examination because of t h e i r pronounced a c t i v i t y a g a i n s t £. s a u c i a ) were presented on g l a s s f i b r e f i l t e r  discs  t r e a t e d w i t h 10% s u c r o s e , markedly d i f f e r e n t r e s u l t s were o b t a i n e d ( F i g u r e 2-3). azedirach  E x t r a c t s o f A. i n d i c a and M-  were not s i g n i f i c a n t l y i n h i b i t o r y a t n a t u r a l  69  F i g u r e 2-3.  Consumption o f g l a s s - f i b r e d i s c s , t r e a t e d with  10% aqueous sucrose and MeOH e x t r a c t s o f A z a d i r a c h t a i n d i c a , M e l i a a z e d i r a c h . Turreae h o l s t i i , and A g l a i a odorata  a t 1, 2.5, and 5 times n a t u r a l c o n c e n t r a t i o n  (on a wt/wt b a s i s ) , by f i f t h i n s t a r nymphs o f Melanoplus s a n g u i n i p e s .  In every case c o n t r o l s  consumed 100% o f t h e s u c r o s e - t r e a t e d d i s c s d u r i n g t h e 24 h assay.  100  % Feeding Inhibition  90 iffi  80 70  /  7)  ftp lit  60 50 40  A  30  Hit ill  20 10 0  MI  Azadirachta indica  Melia azediracht  Aglaia odorata  Turreae holstii  Plant Species and Concentration o  71  c o n c e n t r a t i o n , but d i d reduce f e e d i n g a t 2.5 and 5 times that concentration.  Even a t t h e h i g h e s t l e v e l s , f e e d i n g was  o n l y i n h i b i t e d by 73% (A., i n d i c a ) and 43% ( M . a z e d i r a c h ). In c o n t r a s t , f o l i a r e x t r a c t s from A., odorata  and T_. h o l s t i i  both s i g n i f i c a n t l y reduced f e e d i n g a t n a t u r a l l y c o n c e n t r a t i o n s , and almost completely 2.5  occurring  i n h i b i t e d feeding a t  and 5 times n a t u r a l c o n c e n t r a t i o n .  C Bioassays  f o r a n t i b i o t i c and p h o t o t o x i c a c t i v i t y  None o f t h e e x t r a c t s i n h i b i t e d t h e growth o f Saccharomyces cerevisiae,  either  i n t h e dark o r f o l l o w i n g near-UV  irradiation.  D Leaf Toughness F i f t e e n s p e c i e s o f Meliaceae  were a v a i l a b l e  f o r study  i n the  c o l l e c t i o n s o f t h e USDA P l a n t Quarenteen Center and t h e Gordon F a i r c h i l d T r o p i c a l  Gardens i n Miami, F l o r i d a  These  s p e c i e s were assayed f o r l e a f toughness u s i n g a punchmeter M  modelled a f t e r a d e s i g n by Feeny (1970). toughness g i v e n i n Table measurements p e r s p e c i e s .  2-3 a r e t h e mean o f 5-10 Nearly a s e v e n - f o l d range was  observed, from a low o f 12.4 N/cm caudatum t o a h i g h o f 83.0 N/cm Carapa g u i a n e n s i s . M. a z e d i r a c h  Values f o r l e a f  2  2  i n Entandrophragma  i n t h e mangrove s p e c i e s  The toughness o f A z a d i r a c h t a  a r e somewhat overestimated  i n d i c a and  as i t was not  p o s s i b l e t o a v o i d t h e c l o s e l y spaced secondary v e i n s i n these  species.  M  72  E. D e f e n s i v e C h a r a c t e r i s t i c s o f Deciduous and Evergreen Meliaceae Leaf l i f e t i m e was n o t measured d i r e c t l y i n t h i s study; as a f i r s t approximation a l l s p e c i e s were c l a s s e d as deciduous o r evergreen, as i n d i c a t e d by s p e c i e s d e s c r i p t i o n s i n v a r i o u s f l o r a s o r monographs (Table 2-3).  Carapa g u i a n e n s i s i s  u s u a l l y evergreen, b u t can be deciduous pronounced d r y season  i n areas w i t h a  (Pennington and S t y l e s , 1981); I have  c l a s s e d i t as an evergreen s p e c i e s i n t h i s study. t o x i c i t y , e v a l u a t e d as t h e E C  5 0  Foliar  i n terms o f % o f n a t u r a l  l e a f c o n c e n t r a t i o n , was s i g n i f i c a n t l y h i g h e r f o r deciduous s p e c i e s than f o r evergreen s p e c i e s (ANOVA, F ^ , 2 9 ) = .015).  When t h e E C  5 0  6  -  9 6  /  p  was expressed as mg e x t r a c t / g d i e t  fwt t h e d i f f e r e n c e was l e s s marked ( F ( j 2 9 )  =  3  /  .0645).  =  -  7 9  »  p  =  Much v a r i a t i o n was p r e s e n t w i t h i n each " l e a f age"  c l a s s , and t h e evergreen c l a s s , although on t h e average  less  a c t i v e , i n c l u d e d one o f t h e most i n s e c t i c i d a l s p e c i e s i n t h i s study, A g l a i a o d o r a t a . Leaf toughness  data was a v a i l a b l e f o r a s m a l l e r sample  of o n l y 15 s p e c i e s , d i v i d e d r a t h e r unevenly between t h e two c l a s s e s (11 deciduous, 4 e v e r g r e e n ) .  Evergreen l e a v e s were,  on average, almost t w i c e as tough as l e a v e s o f deciduous s p e c i e s (50.4 v s 33.1 N/cm  2  respectively).  each group i n c l u d e d a wide range o f v a l u e s species:12.4-67.3  However a g a i n (deciduous  N/cm ; evergreen s p e c i e s 29.9-83.0 N/cm ), 2  and t h e ANOVA was n o t s i g n i f i c a n t  2  (ANOVA, F (  1  1  5  j  =2.263,  73  P=0.15).  A s i d e from Carapa g u i a n e n s i s . the deciduous  S w i e t e n i a s p e c i e s had the toughest  leaves.  C o r r e l a t i o n o f l e a f toughness and t o x i c i t y  suggested  the p o s s i b i l i t y o f a n e g a t i v e r e l a t i o n s h i p between these two leaf characters (Figure 2 - 4 ) .  The r e g r e s s i o n had a  s i g n i f i c a n t n e g a t i v e s l o p e a t a= 0.1, but was not s i g n i f i c a n t a t cr= 0.05, i n d i c a t i n g t h a t t h e r e may be an i n v e r s e r e l a t i o n s h i p between l e a f toughness and the t o x i c i t y of the MeOH e x t r a c t s .  Once a g a i n , much v a r i a b i l i t y  was  p r e s e n t and t h e r e g r e s s i o n equation accounted f o r o n l y 23% of t h e observed  v a r i a t i o n i n the d a t a .  74 Table 2-3. Extraction y i e l d (mg MeOH extract/g leaf dwt), leaf toughness (N/cm ), leaf pubescence (lower surface only) (glab=glabrous, axil=hairs i n axils of main veins, pub=* pubescent), and "leaf habit" (deciduous [D] or evergreen [E]) f o r species of Meliaceae included i n 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". 2  ; Faaily Meliaceae Subfamily Melioideae Tribe 1. Turreeae Turreae h o l s t i i Turreae mauritiana Tribe 2. Melieae Melia azedirach (Floridai Melia azedirach (India) Melia azedirach (Chinai  Extract Yield 158.0 213.0  174.7 165.3 172.3 152.6 Melia toosenden Azadirachta i n d i c a (Florida) 98.6 Azadirachta i n d i c a (Hawaii) 112.3 107.7 Azadirachta i n d i c a (china) Tribe 4. T r i c h i l i e a e Trichilia hirta 151.0  T r i c h i l i a reKa  Lepidotrichilia volkensii Ekebergia capensis Cipadessa baccifera Tribe 5. Aglaieae Aalaia odorata (Thailand) Aalaia odorata (Hawaiii Aalaia odoratissima Aalaia araentia Aphanamixus arandlfolia Aphanamixus polystachya Lansium domesticum Tribe 6. Guareeae Guarea glabra Dysoxvlum SDectabile Tribe 7. Sandoriceae  Sandoricvun Kcetjape  ibfaaily Swietenioideae Tribe 1. Cedreleae Cedrela odorata  Toona serrata Toona c i l i a t a  Toona a u s t r a l i s  209.1 239.4 121.5 122.2 38.8 44.0 63.1 105.0 153.6  Toughness (N/cro -) 2  ND ND 20.0±2 .4 ND ND ND 26.8±5 . 6 ND ND  b  16.0±1 .3 ND ND ND 18.2±4 . 4  cd  a  ab  ND ND ND ND ND ND ND  Pubescence  Leaf Habit  glab glab  D(l,5) D(5)  glab glab glab glab glab glab glab  D(l) D(l) D(l) D(4) D(4) D(4) D(4)  glab glab axil glab glab  D(2) D(2) E(l) E(l) D  1  glab glab glab glab glab glab glab  E(4) E(4) E(4) E(4) E E E(4)  86.0 84.0  60.1+3 .8 ND  e  axil glab  E(2) E(4)  131.0  32.6+2 .4  d  pub  E(2,4)  46.2 176.4 100.0 191.1  27.3±1 c d ND 27.8±1 .0 ND  glab glab glab glab  D(2) D(2) D(2) D(2)  > 4  d  2  75 Tribe 2.  Swietenieae  Khaya senegalensis  ChuckrasS3.a tabularis Entandrophracnna caudatum Swietenia humUis Swietenia mahogani Swietenia candollei Tribe 3. Xylocarpeae  115.0  249.7 132.0 102.8 277.0 211.8  46.5±0.9®  21.2±1.0 12.4±1.2j 65.6±5.7t 61.4±3.7* 67.3±5.0  DC  R  pub  glab glab glab glab glab  56.3 83 p+lp,3 Sllah 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 Carapa auianensis 3  G  t  E(l)  D(3,4) D(l) D(2) D(2) D(2) EL2JL  76  F i g u r e 2-3.  R e l a t i o n s h i p between l e a f toughness ( i n N/crn^)  and b i o a c t i v i t y o f the MeOH e x t r a c t o f f o l i a g e , calculated  as  100-EC . 50  y = 85.612 - 0.349*toughness  r=0.55, F=3.362, p=0.0939  EC50 (% Natural Concentration)  78  T a b l e 2-4.  Comparison o f MeOH e x t r a c t t o x i c i t y  (as E C  5 o  to  Peridroma s a u c i a [% o f n a t u r a l c o n c e n t r a t i o n ] ) and toughness (N/cm ) between deciduous and evergreen s p e c i e s o f 2  Meliaceae.  Means i n a column f o l l o w e d by t h e same l e t t e r  are not s i g n i f i c a n t l y d i f f e r e n t  (ANOVA). Toughness  fN/cm^)  Deciduous  18.1±15.7 (16)a  33.1±21.1 ( l l )  Evergreen  44.4+35.8 (14^B  50.4+15.1 (51 3  a  Discussion  A. Crude E x t r a c t  Screening  V i r t u a l l y a l l of the s p e c i e s of Meliaceae  examined i n t h i s  study appear t o be w e l l defended c h e m i c a l l y a g a i n s t  attack  by g e n e r a l i s t non-adapted i n s e c t h e r b i v o r e s , assuming t h a t Peridroma s a u c i a i s a v a l i d model s p e c i e s  .  Members of the  s u b f a m i l y M e l i o i d e a e were on average b e t t e r defended c h e m i c a l l y , and members of the t r i b e Melieae consistently highly toxic.  The Melieae  were  i n c l u d e the  only  s p e c i e s known t o produce C-seco limonoids such as a z a d i r a c h t i n (Dreyer,  1983;  Connoly, 1983), the most  i n s e c t i c i d a l c l a s s of limonoids  (Chapter  C o r r e l a t i o n s between phytochemistry  4 of t h i s T h e s i s ) .  and the i n s e c t i c i d a l  or  i n s e c t growth r e g u l a t i n g a c t i v i t y o f the e x t r a c t s are d i s c u s s e d i n more d e t a i l i n Chapter 4 (pg 216);  i n general  the growth i n h i b i t o r y a c t i v i t y of the crude MeOH e x t r a c t s agrees w e l l with the expected a c t i v i t y i n those where the l i m o n o i d phytochemistry  i s known.  Two  t h r e e s p e c i e s which were i n a c t i v e i n t h i s study  species of  the  (Lansium  domesticum and Dysoxylum s p e c t a b i l e ) have been r e p o r t e d t o lack limonoids.  However, some s p e c i e s i n c l u d i n g Sandoricum  k o e t j a p t e , A g l a i a odorata,  A. o d o r a t i s s i m a  f  and A,, a r g e n t i a  are i n h i b i t o r y t o Peridroma s a u c i a growth d e s p i t e a r e p o r t e d l a c k of l i m o n o i d s ; these s p e c i e s deserve f u r t h e r phytochemical i n v e s t i g a t i o n .  80  Seed e x t r a c t s of s e v e r a l of the s p e c i e s examined here have p r e v i o u s l y been t e s t e d f o r growth i n h i b i t i o n , i n h i b i t i o n , and t o x i c i t y a g a i n s t the f a l l  feeding  armyworm,  Spodoptera f r u g i p e r d a , f o r f e e d i n g i n h i b i t i o n a g a i n s t s t r i p e d cucumber b e e t l e , Acalymma v i t t a t u m ,  and  the  for  c y t o t o x i c i t y a g a i n s t the b r i n e shrimp Artemia s a l i n a (Mikolajczak opportunity  e t aJL., 1987,  A g l a i a cordata  Hexane and  t e s t e d , 16 ppm;  armyworm a t the lowest dose  my  However, I found methanolic A.,  odoratissima  s a u c i a growth a t  E t h a n o l i c seed e x t r a c t s of C h u c k r a s s i a  inhibited f a l l  and  e x t r a c t s were  These r e s u l t s support  f o l i a g e e x t r a c t s t o be i n h i b i t o r y t o E .  400  growth,  of a wide range of a c t i v i t y between d i f f e r e n t  Aglaia species.  at  ppm;  and  e t h a n o l i c seed e x t r a c t s of  i n c o n t r a s t A., o d o r a t i s s i m a  i n a c t i v e a t 10,000 ppm.  4,000 ppm.  an  H i e r n . markedly decreased f e e d i n g ,  s u r v i v o r s h i p of the f a l l  observation  This provides  t o compare b i o l o g i c a l a c t i v i t i e s of seed  f o l i a r extracts.  and  1989).  armyworm growth by 95%  and  tabularis  s u r v i o r s h i p by  f o l i a g e e x t r a c t s i n h i b i t e d growth a t 31,000  d i d not i n c r e a s e m o r t a l i t y .  ppm;  c o n t r a s t f o l i a r e x t r a c t s of n e i t h e r s p e c i e s i n h i b i t e d  (about 150,000 ppm). Azadirachta Trichilia  ppm  Seed e x t r a c t s of Dysoxylum  s p e c t a b i l e and Lansium domesticum were a c t i v e a t 400  s a u c i a growth by as much as 50%  50%  at natural  E-  concentration  Seed e x t r a c t s of Sandoricum  i n d i c a , Melia azedirach,  in  Swietenia  koetiape.  mahogani,  roka a l l f o l l o w the same p a t t e r n of being  and  highly  81  a c t i v e a t c o n c e n t r a t i o n s a t l e a s t two o r d e r s o f magnitude lower than those o f f o l i a r  extracts.  None o f t h e f o l i a r e x t r a c t s i n h i b i t e d f e e d i n g by t h e m i g r a t o r y grasshopper, Melanoplus on cabbage l e a f d i s c s .  s a n g u i n i p e s . when assayed  However, e x t r a c t s from A g l a i a  odorata and Turreae h o l s t i i were i n h i b i t o r y when t e s t e d a t n a t u r a l c o n c e n t r a t i o n (on a dwt l«af/dwt d i s c b a s i s ) on g l a s s - f i b r e d i s c s p r o v i d e d w i t h 10% s u c r o s e as a phagostimulant.  A s i m i l a r d i s c r e p a n c y between r e s u l t s w i t h  l e a f and g l a s s f i b r e d i s c s was noted by Ascher (1981). i s l i k e l y t h a t t h i s r e s u l t s from t h e a r t i f i c i a l  It  circumstance  of p r e s e n t i n g t h e i n s e c t w i t h a f r e s h l y c u t l e a f edge, which a l l o w s c o n t a c t w i t h phagostimulants p r e s e n t i n t h e l e a f sap. O r d i n a r i l y such c o n t a c t would n o t occur u n t i l t h e l a t e r stages o f h o s t - p l a n t acceptance, a f t e r t h e l e a f s u r f a c e has been examined by c o n t a c t c h e m o s e n s i l l a (Chapman and Bernays, 1989).  The phagostimulant a c t i v i t y o f t h e sap must exceed  the a c t i v i t y o f t h e f e e d i n g i n h i b i t o r s i n t h e e x t r a c t s , which i n t u r n a r e l e s s a c t i v e than 10% s u c r o s e .  However,  the A g l a i a odorata and Turreae h o l s t i i e x t r a c t s might p r o v i d e p r o t e c t i o n , i n an a g r i c u l t u r a l c o n t e x t , a g a i n s t t h i s grasshopper i f a p p l i e d t o i n t a c t  foliage.  The a c t i v i t y o f t h e phytochemicals i n these e x t r a c t s r e s p o n s i b l e f o r i n h i b i t i n g l a r v a l growth, and i n some cases m o l t i n g , may be s p e c i f i c f o r i n s e c t s , as no e x t r a c t s were a n t i b i o t i c o r p h o t o t o x i c t o Saccharomyces c e r e v i s e a e . w e l l , s e v e r a l s p e c i e s found t o be h i g h l y i n h i b i t o r y t o  As  82  i n s e c t growth ( i n c l u d i n g A z a d i r a c h t a i n d i c a , M e l i a azedirach  f  and A a l a i a odorata) were i n a c t i v e when t e s t e d  a g a i n s t b r i n e shrimp Artemia s a l i n a Towers, 1 9 8 8 ) .  (Wiriyachitra  and  Together these r e s u l t s r u l e out g e n e r a l  c y t o t o x i c i t y as a mode of a c t i o n f o r the most a c t i v e extracts.  The l a c k of any p h o t o t o x i c e f f e c t i n the  Saccharomyces assay a l s o suggests t h a t the r e p o r t e d v i r t u a l absence  i n the M e l i a c e a e of furanocoumarins  and o t h e r  c l a s s e s of p h o t o s e n s i t i z e r s , so t y p i c a l of the Rutaceae, i s r e a l and does not simply r e f l e c t a l a c k of a t t e n t i o n  from  phytochemists. S e v e r a l s p e c i e s i n v e s t i g a t e d here f o r the f i r s t appear t o be s u f f i c i e n t l y i n h i b i t o r y t o £. s a u c i a t o warrant f u r t h e r a t t e n t i o n . and Turreae h o l s t i i may  time  neonates  In p a r t i c u l a r , A g l a i a odorata  have p o t e n t i a l as sources of u s e f u l  e x t r a c t s or phytochemicals.  In the l a t t e r case, the a c t i v e  component(s) appears t o i n h i b i t m o l t i n g a t doses which do not d e t e r f e e d i n g .  The phytochemistry of Turreae s p e c i e s i s  almost unknown, but T a y l o r has r e p o r t e d l i m o n o i d s s i m i l a r t o c e d r e l o n e ( P e t t i t et, a i . ,  1 9 8 3 ) , which can i n h i b i t m o l t i n g  i n the milkweed bug Oncopeltus f a s c i a t u s (Chapter 4 ) . S e v e r a l phytochemicals have been r e p o r t e d from A g l a i a odorata (Shiengthong e t a i . ,  1965,  1979)  and the r o l e of  these compounds i n the observed a c t i v i t y of the e x t r a c t s i s examined i n Chapter 3 of t h i s T h e s i s .  odorata  83  B. Resource A v a i l a b i l i t y  Hypothesis  O v e r a l l , the p a t t e r n s seen here w i t h r e g a r d t o l e a f t o x i c i t y , toughness, and l i f e t i m e , tend t o support the r e s o u r c e a v a i l a b i l i t y h y p o t h e s i s of Coley e_t a l . ( 1 9 8 5 ) (termed the growth r a t e h y p o t h e s i s i n Coley, 1 9 8 8 ) . assumed t h a t the r e l a t i v e investment i n chemical defenses may  be e s t i m a t e d by comparing  I have  (mobile)  the r e l a t i v e  efficacy  of those defenses a g a i n s t a n a i v e , non-adapted i n s e c t h e r b i v o r e , Peridroma s a u c i a . i n c l u d i n g l e a f toughness directly.  Physical  (immobile)  defenses  and pubescence were measured  As e x p l a i n e d p r e v i o u s l y , the r e s o u r c e  a v a i l a b i l i t y h y p o t h e s i s p r e d i c t s t h a t investment i n chemical defenses should c h a r a c t e r i z e p l a n t s w i t h s h o r t l e a f l i f e t i m e s , and investment i n p h y s i c a l defenses should be s e l e c t e d f o r i n p l a n t s with l o n g l e a f  lifetimes.  I found t h a t e x t r a c t s from deciduous l e a v e s were s i g n i f i c a n t l y more i n h i b i t o r y t o E.  s a u c i a than were  e x t r a c t s from evergreen s p e c i e s , which suggests t h a t deciduous s p e c i e s ( s h o r t l e a f l i f e t i m e ) r e l y more on chemical b a r r i e r s t o h e r b i v o r y than do evergreen ( l o n g l e a f lifetime) species.  P r e v i o u s attempts t o demonstrate g r e a t e r  investment i n chemical defense i n s p e c i e s w i t h s h o r t l e a f l i f e t i m e s have not been s u c c e s s f u l , as they r e l i e d  on  c o l o r i m e t r i c assays f o r p h e n o l i c s o n l y ( i . e . Coley, 1 9 8 3 ) , which l e d Coley ( 1 9 8 3 , 1988)  t o suggest t h a t the  of p l a n t chemistry had been overemphasized.  importance  In p r a c t i c e , i t  i s g e n e r a l l y i m p r a c t i c a l t o determine the c o n t r i b u t i o n of  84  each secondary m e t a b o l i t e t o i n s e c t r e s i s t a n c e ( i n most cases t h e compounds a r e unknown), and i t may be i m p o s s i b l e t o measure t h e c o s t o f p r o d u c t i o n o f each compound. C e r t a i n l y i t i s u n s a t i s f a c t o r y t o estimate t h e importance o f secondary m e t a b o l i t e - b a s e d defenses from a c o l o r i m e t r i c assay f o r a s i n g l e c l a s s o f compound.  Rather, measurement  of t h e response o f t h e p u t a t i v e t a r g e t o f these defenses ( g e n e r a l i s t h e r b i v o r e s a c c o r d i n g t o Feeny, 1976; Rhoades and Cates, 1976; Rhoades, 1979) appears t o o f f e r a p r a c t i c a l a l t e r n a t i v e t o t h i s dilemna. On average, t h e l e a v e s o f deciduous s p e c i e s were o n l y h a l f as tough as l e a v e s o f evergreen s p e c i e s , but t h e range of v a r i a t i o n w i t h i n each group was l a r g e .  As a r e s u l t , t h e  two groups d i d n o t d i f f e r s i g n i f i c a n t l y i n a s t a t i s t i c a l sense.  However, numerous s t u d i e s have demonstrated  a strong  c o r r e l a t i o n between l e a f toughness and r e s i s t a n c e t o h e r b i v o r y (Tanton, 1962; Grime g t a l . , 1968; Feeny, 1970; Rhoades, 1977; Rausher 1984; Coley, 1 9 8 8 ) ,  and Feeny, 1980; C o l e y , 1983; McKey,  and t h i s f a c t o r i s a l s o c l e a r l y  p o s i t i v e l y c o r r e l a t e d w i t h l e a f l i f e t i m e ( C o l e y , 1983, Coley g t a l . , 1 9 8 5 ) .  The l a c k o f s t a t i s t i c a l  1988;  significance  i n my study can be a t t r i b u t e d t o t h e s m a l l sample s i z e  (11  deciduous and 4 evergreen s p e c i e s ) and t h e wide range o f v a r i a t i o n between i n d i v i d u a l s i n each c l a s s .  Similar  v a r i a t i o n c h a r a c t e r i z e s e a r l i e r s t u d i e s as w e l l and can o n l y be overcome w i t h a p p r o p r i a t e l y l a r g e sample s i z e s .  I attach  more importance t o t h e marked d i f f e r e n c e i n t h e means, which  85  suggest  ( i n agreement w i t h a l l e a r l i e r s t u d i e s c i t e d above)  t h a t evergreen s p e c i e s i n v e s t more i n l e a f toughness (immobile  or q u a n t i t a t i v e defenses) than do  species.  Species which i n v e s t i n l e a f toughness f a c t o r s  r e l y l e s s on phytochemical  deciduous may  defenses, as t h e r e i s evidence  f o r a n e g a t i v e c o r r e l a t i o n between l e a f toughness and i n h i b i t o r y a c t i v i t y o f the e x t r a c t s .  However, t h i s  h y p o t h e s i s c l e a r l y needs f u r t h e r study w i t h l a r g e r sample s i z e s , a g a i n t o overcome d i f f i c u l t i e s w i t h the l a r g e amount of i n t e r s p e c i f i c  variation.  P r e v i o u s s t u d i e s have compared d e f e n s i v e a t t r i b u t e s o f p i o n e e r and p e r s i s t e n t s p e c i e s which occur t o g e t h e r i n the same h a b i t a t .  Although the s p e c i e s s t u d i e d here were drawn  from d i v e r s e h a b i t a t s , i n t e r e s t i n g c o r r e l a t i o n s can s t i l l drawn between d e f e n s i v e s t r a t e g i e s and attributes.  ecological  The most i n s e c t i c i d a l s p e c i e s seen i n t h i s  study, A z a d i r a c h t a i n d i c a and the two M e l i a s p e c i e s , are noted f o r t h e i r extremely r a p i d growth, and are o f t e n p l a n t e d as shade t r e e s or f o r firewood f o r t h i s (Jacobson, 1988).  reason  They occur i n s e m i - a r i d areas where  n u t r i e n t s and l i g h t are o f t e n not l i m i t i n g  (although water  i s ) , and cannot compete w e l l w i t h o t h e r s p e c i e s 1989), s u g g e s t i n g t h a t they are adapted a v a i l a b i l i t y " habitats.  (Jacobson,  t o "high r e s o u r c e  In times of drought they o f t e n  c o n s t i t u t e the o n l y green p l a n t s a v a i l a b l e , which may  have  p r o v i d e d the s e l e c t i o n p r e s s u r e f o r the e v o l u t i o n of the h i g h l y a c t i v e C-seco l i m o n o i d s i n c l u d i n g a z a d i r a c h t i n  be  86  (Blaney, 1980). Ekebergia  On t h e other hand, Khaya s e n e g a l e n s i s ,  c a p e n s i s , and Guarea g l a b r a a r e slow-growing  p e r s i s t e n t r a i n - f o r e s t s p e c i e s which form a prominent component o f t h e emergent f l o r a  ( B r a n d i s , 1906; White and  S t y l e s , 1963; Pennington and S t y l e s , 1981); these tend t o produce tough, evergreen phytochemicals. however.  l e a v e s low i n i n s e c t i c i d a l  E x c e p t i o n s t o t h i s p a t t e r n do occur,  Swietenia macrophylla  i s relatively  e s p e c i a l l y compared t o o t h e r Swietenia s p e c i e s  fast-growing, (Pennington  and S t y l e s , 1981), b u t a l l t h r e e s p e c i e s t e s t e d here produce e q u a l l y tough l e a v e s and appear t o be about e q u a l l y w e l l defended c h e m i c a l l y ; t h e p a t t e r n o f defense i n these i s more t y p i c a l o f t h e slow-growing evergreen  species  species.  Carapa g u i a n e n s i s i s a v e r y v a r i a b l e s p e c i e s , o c c u r r i n g i n mangrove swamps, on rocky h i l l s i d e s , and even as an understory  species i n the r a i n forest  S t y l e s , 1981). investment  I t ' s defenses  i n phytochemical  (Pennington and  i n c l u d e v e r y tough l e a v e s , and  defenses  appears t o be low; i n  a d d i t i o n , t h i s s p e c i e s has e x t r a f l o r a l n e c t a r i e s a l o n g the l e a f margin, and some p o p u l a t i o n s appear t o be w e l l defended by ants  (Pennington  and S t y l e s , 1981).  The p l a n t s used i n t h i s study were c o l l e c t e d from d i v e r s e l o c a l i t i e s around the world; t h e i r o n l y d i r e c t r e l a t i o n s h i p i s through phylogeny.  Each s p e c i e s has had t o  respond, i n e v o l u t i o n a r y time, t o a unique s u i t e o f n a t u r a l enemies ( h e r b i v o r e s ) and c o m p e t i t o r s .  As a l l s p e c i e s a r e  from t r o p i c a l h a b i t a t s I have i m p l i c i t l y assumed ( f o r  87  s i m p l i c i t y ) t h a t p o t e n t i a l h e r b i v o r e s a r e numerous and consequently  s e l e c t i o n p r e s s u r e t o reduce h e r b i v o r y i s  s t r o n g i n a l l cases.  However, v a r i a t i o n s i n s e l e c t i o n  p r e s s u r e between d i f f e r e n t h a b i t a t s may account f o r much o f the v a r i a b i l i t y i n defense s t r a t e g i e s noted i n t h i s  study.  As w e l l , l e a f toughness has been i m p l i c i t l y c o n s i d e r e d as a defense a g a i n s t h e r b i v o r y , but tough l e a v e s a r e a l s o known t o c o r r e l a t e with o t h e r f a c t o r s i n c l u d i n g r e s i s t a n c e t o d e s i c c a t i o n (Daubenmire, 1974) and p o s s i b l y f u n g a l a t t a c k , which may be more important h e r b i v o r y i n some h a b i t a t s .  s e l e c t i v e pressures  than  F u r t h e r , I have c a t e g o r i z e d  s p e c i e s as deciduous o r evergreen,  b u t l e a f l i f e t i m e can  v a r y tremendously w i t h i n these c l a s s e s ; p a r t i c u l a r l y , some s p e c i e s may f l u s h new l e a v e s b e f o r e dropping  t h e o l d ones,  i n which case the t r e e c o u l d be evergreen and y e t each l e a f may have a s h o r t l i f e t i m e .  D e s p i t e these sources o f  v a r i a t i o n , an o v e r a l l p a t t e r n o f defense a l l o c a t i o n between c h e m i c a l l y - and p h y s i c a l l y - b a s e d s t r a t e g i e s can be d i s c e r n e d and  i s i n g e n e r a l agreement with t h e r e s o u r c e  hypothesis.  availability  T h i s suggests t h a t t h e h y p o t h e s i s i s  s u f f i c i e n t l y r o b u s t t o e x p l a i n n o t o n l y d i f f e r e n c e s between p l a n t s w i t h i n a s i n g l e h a b i t a t , but a l s o e v o l u t i o n a r y p a t t e r n s between r e l a t e d p l a n t s occupying habitats.  different  In a study o f d e f e n s i v e c h a r a c t e r i s t i c s o f  p i o n e e r and p e r s i s t e n t s p e c i e s o c c u r r i n g i n l i g h t gaps i n the Panamanian r a i n f o r e s t , members o f t h e same f a m i l y were c o n s i s t e n t l y found t o c l u s t e r a c c o r d i n g t o e c o l o g i c a l  88  a t t r i b u t e s r a t h e r than along taxonomic l i n e s , t h a t h a b i t a t and  l i f e h i s t o r y place greater c o n s t r a i n t s  defenses than do p h y l o g e n e t i c This conclusion  suggesting  r e l a t i o n s h i p s (Coley,  i s supported by my  on  1983).  findings.  Detailed studies i n c l u d i n g l e a f tagging  experiments t o  determine l e a f l i f e t i m e , and measurements of p l a n t growth r a t e s are underway i n c o n j u n c t i o n K.R.  Downum and  University.  associates)  with c o l l a b o r a t o r s  at F l o r i d a International  (Dr.  89  Chapter 3.  Phytochemical  Investigation of A g l a i a Species  Introduction  S p e c i e s of the genus A g l a i a f i g u r e prominently i n t r a d i t i o n a l pharmacopeias throughout s o u t h - e a s t A s i a .  For  example, A g l a i a odorata ( L o u r ) , known as Shu-Lan i n Taiwan, has been used f o r the treatment of coughs and (Kan, 1979) 1982).  inflammation  as w e l l as " t r a u m a t i c i n j u r y " (Hayashi ejb a l . ,  In T h a i l a n d the same p l a n t i s t r a d i t i o n a l l y  p r e s c r i b e d as a h e a r t s t i m u l a n t and f e b r i f u g e et a l . ,  1965).  (Shiengthong  Leaves and r o o t s of A g l a i a p i r i f e r a Hance  induce v o m i t i n g and are used as an a n t i d o t e f o r p o i s o n i n g i n Thailand  ( S a i f a h e t a_l., 1988).  As a r e s u l t of t h e i r reputed p h a r m a c o l o g i c a l a c t i v i t i e s , t o date t h r e e s p e c i e s of A g l a i a have been examined p h y t o c h e m i c a l l y .  A., odorata has y i e l d e d the  dammarane t r i t e r p e n e s a g l a i o l , a g l a i o n d i o l aglaitriol  (2) (Shiengthong g t a l . , 1965,  amides of 2 - a m i n o p y r r o l i d i n e , (+)-odorine (9) (Purushothaman e£ a l . , 1979)  and  (Hayashi g t a l . , 1988). (+)-Odorine  ( 1 ) , and 1974) (8),  and the b i s (+)-odorinol  ( - ) - o d o r i n o l (10) and  ( + ) - o d o r i n o l were  a l s o i s o l a t e d from A., roxburgiana (Shiengthong g t a l . , 1979).  A., p i r i f e r a was  found t o c o n t a i n p i r i f e r i n e ,  i d e n t i c a l t o (+)-odorine except f o r the l o s s of a t e r m i n a l methyl 1988).  from the 2-methylbutanoyl  moiety  (Saifah gt a l . ,  The o n l y compound which has, t o date, been shown t o  90  possess any  b i o l o g i c a l a c t i v i t y i s (-)-odorinol,  which  i n h i b i t s P-388 lymphocytic leukemia growth i n BDF1 (T/C  > 136%)  a t a dose of 5.0  male mice  mg/kg (Hayashi et a i . , 1982).  In Chapter 2 of t h i s t h e s i s , MeOH e x t r a c t s of A., odorata, &.  odoratissima,  and A., a r g e n t i a were r e p o r t e d  i n h i b i t growth of the v a r i e g a t e d  to  cutworm, Peridroma s a u c i a .  T h i s c h a p t e r i s a r e p o r t of attempts t o i s o l a t e and  identify  the p h y t o c h e m i c a l ( s ) r e s p o n s i b l e f o r t h i s a c t i v i t y i n A., o d o r a t a , and  t o determine the b a s i s of the growth i n h i b i t i o n  (feeding deterrence  or t o x i c i t y ) .  In a d d i t i o n , methods f o r  the r a p i d q u a l i t a t i v e and q u a n t i t a t i v e a n a l y s i s of A a l a i a f o l i a g e were developed, based on TLC  and  HPLC.  These  methods were a p p l i e d t o compare the p h y t o c h e m i s t r i e s  of  the  t h r e e a v a i l a b l e A g l a i a s p e c i e s , i n c l u d i n g A., odorata samples from T h a i l a n d , Hawaii, and  Taiwan.  products Were i s o l a t e d and  i d e n t i f i e d from A., odorata  foliage.  S e v e r a l new  natural  91  M a t e r i a l s and Methods  A)  Sources o f P l a n t M a t e r i a l  Sources o f a l l p l a n t samples a r e g i v e n i n Table Chapter 2.  A d d i t i o n a l m a t e r i a l o f A a l a i a odorata  Pacific Tropical  Gardens, Hawaii, was p r o v i d e d  Flynn of that i n s t i t u t i o n . reported  2-1 o f from t h e  by Mr. T.  Most o f t h e phytochemical work  i n t h i s Chapter i s based on t h i s l a t t e r m a t e r i a l .  A l l p l a n t samples were a i r - d r i e d b e f o r e s h i p p i n g t o UBC.  B)  Isolation  Aglaia  and i d e n t i f i c a t i o n o f secondary m e t a b o l i t e s i n  foliage  Isolation  o f t h e i n s e c t i c i d a l components from A.,  f o l i a g e was guided by b i o a s s a y  ( S e c t i o n D).  odorata  However, i n  view o f the unique phytochemistry o f t h i s s p e c i e s w i t h i n t h e Meliaceae,  an attempt was made t o i d e n t i f y a l l o f the major  secondary m e t a b o l i t e s , against E- saucia. partitioning,  whether o r n o t they showed a c t i v i t y  Isolation  Solvent  Air-dried  solvent  f o l l o w e d by chromatographic p u r i f i c a t i o n o f  the most a c t i v e s o l v e n t  Bl)  involved a preliminary  fraction.  partitioning  A. odorata  f o l i a g e was weighed (130  g dwt),  then  e x t r a c t e d i n t o t h r e e 1.5 1 changes o f MeOH (3 x 24-72 h) a t room temperature. and c o n c e n t r a t e d  The MeOH e x t r a c t s were f i l t e r e d ,  pooled  under vacuum t o a f i n a l volume o f 2 ml/g  l e a f dwt e q u i v a l e n t .  T h i s e x t r a c t was used f o r  initial  92  bioassays  as d e s c r i b e d i n Chapter 2.  Other s p e c i e s o f  A g l a i a were e x t r a c t e d i n an i d e n t i c a l manner. The MeOH e x t r a c t was d i l u t e d t o 1 1, combined w i t h an equal volume o f water and p a r t i t i o n e d s u c c e s s i v e l y (3 x p e r s o l v e n t ) a g a i n s t an equal volume o f hexane, d i e t h y l ( E t 0 ) , and dichloromethane ( C H C 1 ) . 2  2  ether  S o l v e n t s were  2  purchased from BDH and r e d i s t i l l e d p r i o r t o use. A l l s o l v e n t phases, i n c l u d i n g the aqueous phase, were d r i e d , weighed, and bioassayed  a t 10% o f n a t u r a l c o n c e n t r a t i o n (0.1  g l e a f e q u i v a l e n t / g dwt d i e t ) .  The d r i e d E t 0 phase was 2  o n l y p a r t i a l l y s o l u b l e i n anhydrous E t 0 ; t h i s phase was 2  a c c o r d i n g l y d i v i d e d i n t o anhydrous E t 0 - s o l u b l e and 2  - i n s o l u b l e phases and both were b i o a s s a y e d  a t 250 and 2500  ng e x t r a c t / g d i e t dwt.  B2i. The E.  Normal-Phase Chromatography E t 0 s o l u b l e f r a c t i o n , which was the most a c t i v e a g a i n s t 2  s a u c i a neonates, was f l a s h chromatographed ( S t i l l e t a l . ,  1975)  i n 1 g l o t s on a 2.5 x 25 cm s i l i c a  mesh) column, u s i n g N  2  ( S i ) g e l (230-400  gas as t h e p r e s s u r e source.  mobile phase was 2 column volumes each o f E t 0 , 2  The  Et 0:EtOAc 2  (3:1), Et 0:EtOAC (1:1), EtOAc, and f i n a l l y MeOH. 2  Chloroform  was avoided  because t h e bis-amides are  i n t h i s s o l v e n t (Shiengthong e t al., 1979). was  unstable  The flow r a t e  49 ml/min (2.5 cm/min), and 6 ml f r a c t i o n s were  collected.  F r a c t i o n s were monitored by a n a l y t i c a l TLC ( S i  p l a t e s , 0.2 mm, Merck) developed i n EtOAc:PE 3:1; spots were  93  v i s u a l i z e d w i t h E h r l i c h ' s reagent  (1 g p-  dimethylaminobenzaldehyde i n 100 ml 2% e t h a n o l i c H S 0 ) 2  4  (Dreyer, 1964) and s i m i l a r f r a c t i o n s were pooled f o r bioassay. F r a c t i o n s which were i n h i b i t o r y t o £ . s a u c i a . o r which c o n t a i n e d major n a t u r a l products  (as evidenced by t h e  appearance Of major s p o t s on TLC o r c r y s t a l l i z a t i o n o f compounds on d r y i n g ) , were p u r i f i e d f u r t h e r by p r e p a r a t i v e TLC.  F r a c t i o n s were banded onto 1 mm t h i c k S i g e l TLC  p l a t e s (Merck) and developed e q u i l i b r a t e d TLC tank.  i n EtOAc:PE (1:1) i n a n  s  Fractions containing highly polar  compounds ( i . e . o d o r i n e s ) were developed up t o 30 times t o achieve s e p a r a t i o n o f t h e (+)- and (-)- d i a s t e r e o m e r s . Bands were v i s u a l i z e d w i t h UV l i g h t o r by s p r a y i n g o n l y t h e edge o f t h e p l a t e w i t h E h r l i c h ' s reagent.  Bands were then  scraped from t h e p l a t e and e l u t e d w i t h MeOH.  Compounds were  f i n a l l y p u r i f i e d by r e c r y s t a l l i z a t i o n , p r i o r t o b i o a s s a y .  B3) HPLC The most a c t i v e f r a c t i o n s were chromatographed u s i n g p r e p a r a t i v e HPLC a f t e r TLC proved u n s a t i s f a c t o r y f o r t h e i s o l a t i o n of the i n s e c t i c i d a l p r i n c i p l e ( s ) .  F r a c t i o n s were  prepared f o r chromatography by p a s s i n g them through a 3 cm s i l i c a p l u g i n a p a s t e u r p i p e t t e , e l u t e d w i t h MeOH, f o l l o w e d by passage through two Sep-Pacs (Waters) packed w i t h RP-18. S e p a r a t i o n was achieved w i t h a L i c h r o s o r b Hibar® RP-18 10 X 250 mm column on a V a r i a n Model 5000 HPLC equipped  (7/im) with  94  a UV/Vis d e t e c t o r and a S p e c t r a - P h y s i c s SP4100 i n t e g r a t o r . The s o l v e n t system was MeOH:H 0 (1:1) f o r 10 minutes, 2  f o l l o w e d by a g r a d i e n t t o 100% MeOH over 20 minutes, e l u t i o n w i t h 100% MeOH f o r 30 minutes. ml/min, w i t h UV m o n i t o r i n g a t 217 nm. c o l l e c t e d w i t h an automatic Subsequently  fraction  then  Flow r a t e was 3 Six-ml f r a c t i o n s were  collector.  these f r a c t i o n s were examined by a n a l y t i c a l  HPLC u s i n g a L i c h r o s o r b RP-18 (5 /im) 4 X 250 mm column, i s o c r a t i c MeOH:H 0 (7:3), w i t h a flow o f 1 ml/min. 2  One  t w e n t i e t h o f each f r a c t i o n was used f o r b i o a s s a y .  C)  Q u a l i t a t i v e and q u a n t i t a t i v e a n a l y s e s  Subsequently,  t e c h n i q u e s developed  f o r the i s o l a t i o n o f t h e  a c t i v e component(s) were adapted t o p r o v i d e r a p i d methods o f analysis of Aglaia foliage. i s o l a t e d were determined  The Rfs o f a l l compounds  i n two a n a l y t i c a l TLC systems:  EtOAC:PE (3:1) on S i g e l (System A ) , and CHCl :MeOH (49:1) 3  on S i g e l (System were determined  B).  The c o l o r r e a c t i o n s o f a l l compounds  w i t h two spray reagents, E h r l i c h ' s  (Dreyer, 1964), and V a n i l l i n reagent  (2% v a n i l l i n i n EtOH/  o v e r s p r a y w i t h 3M H S 0 ) (Pieman e £ a l . , 2  4  reagent  1980).  These TLC  systems were used f o r q u a l i t a t i v e comparisons o f A a l a i a extracts. Q u a n t i t a t i v e a n a l y s i s was achieved by a n a l y t i c a l HPLC, u s i n g a L i c h r o s o r b RP-18 4 X 250 mm column, d e t e c t i o n a t 217 nm, and the f o l l o w i n g s o l v e n t system: 0-10 min, (1:1); 10-30 min,  MeOH:H 0 2  g r a d i e n t t o 100% MeOH; 30-60 min 100%  95  MeOH; flow = 1 ml/min throughout.  R e t e n t i o n times and  c a l i b r a t i o n curves were determined f o r a l l i d e n t i f i e d compounds i s o l a t e d from A., odorata. r e s o l v e t h e diastereomers  T h i s method c o u l d not  o f odorine and o d o r i n o l ; t o  q u a n t i f y these compounds I used H-NMR, comparing r e l a t i v e 1  h e i g h t s of t h e methyl peaks a t  0.70 and 0.90 ppm t o  c a l c u l a t e t h e p r o p o r t i o n o f S,R and S,S o d o r i n e .  Relative  h e i g h t s o f t h e s i n g l e t s a t 6 1.34 and 1.22 were used t o c a l c u l a t e t h e r e l a t i v e p r o p o r t i o n s o f S,R and S,S o d o r i n o l .  D)  Bioassay  All  f r a c t i o n s and pure compounds were b i o a s s a y e d  against  neonate E- s a u c i a , i n seven-day c h r o n i c f e e d i n g assays, as d e s c r i b e d i n Chapter 2.  S o l v e n t phases were b i o a s s a y e d a t  10% o f t h e n a t u r a l l y o c c u r r i n g l e v e l s ; f o r f r a c t i o n s c o l l e c t e d from column chromatography o r HPLC, 1/20 of each f r a c t i o n was used.  The maximum c o n c e n t r a t i o n o f pure  compounds bioassayed,  g i v e n i n Table 3-7, was equal t o o r  s l i g h t l y g r e a t e r than t h e c o n c e n t r a t i o n s o f t h e compounds i n the p l a n t , as g i v e n i n Table 3-6.  V a r i o u s combinations o f  the p u r i f i e d compounds were a l s o b i o a s s a y e d check f o r a d d i t i v e o r s y n e r g i s t i c  (Table 3-8), t o  interactions.  The most a c t i v e compound i s o l a t e d was assayed f o r i n h i b i t i o n of l a r v a l growth and s u r v i v o r s h i p a t 0.4, 0.6, 1, 2, 3, 5, 10, and 15 ug/g d i e t dwt.  A simple c h o i c e f e e d i n g  assay was a l s o used t o determine t h e r o l e of a n t i f e e d a n t  96  e f f e c t s i n the a c t i v i t y of t h i s compound a t 1, 2, and 3 ug/g d i e t ; t h i s assay i s d e s c r i b e d i n d e t a i l i n Chapter  4.  Results  A)  MeOH E x t r a c t  Screening  I n h i b i t i o n o f neonate E - s a u c i a growth by e x t r a c t s from A.. odorata ( T h a i l a n d sample), A., o d o r a t i s s i m a , i s shown i n F i g u r e  argentia  3-1. The A., odorata e x t r a c t was about  t w i c e as a c t i v e as t h e A., o d o r a t i s s i m a times as a c t i v e as the A., a r g e n t i a  B)  and A.,  e x t r a c t , and f i v e  extract.  Solvent P a r t i t i o n i n g  B i o a s s a y o f the v a r i o u s s o l v e n t phases from A., odorata i n d i c a t e d t h a t the E t 0 phase was most i n h i b i t o r y t o E . 2  s a u c i a growth and s u r v i v a l ; t h e C H C 1 2  some a c t i v i t y  ( F i g u r e 3-2).  2  phase a l s o r e t a i n e d  The E t 0 phase was d r i e d and 2  d i v i d e d i n t o phases which were s o l u b l e o r i n s o l u b l e i n anhydrous E t 0 . 2  The E t 0 s o l u b l e phase was about ten times 2  more a c t i v e than the i n s o l u b l e phase when t e s t e d a t 250 /jg e x t r a c t / g d i e t dwt.  N e i t h e r the hexane phase nor t h e  remaining aqueous phase had an i n h i b i t o r y e f f e c t .  Bioassay  of t h e l e a f marc, t h e r e s i d u e a f t e r e x t r a c t i o n , i n d i c a t e d t h a t t h e i n s e c t i c i d a l p r i n c i p l e ( s ) had been completely removed by the MeOH e x t r a c t i o n . the y i e l d  Figure  3-2 a l s o  includes  (as % o f the MeOH e x t r a c t ) f o r each s o l v e n t phase.  C1  Chromatography  The  i s o l a t i o n o f phytochemicals was c o n f i n e d  phase as t h i s c o n t a i n e d  t o the E t 0  the i n s e c t i c i d a l compound(s).  2  98  F i g u r e 3-1.  E f f e c t o f f o l i a r MeOH e x t r a c t s of A g l a i a  odorata ( o ) ,  A., o d o r a t i s s i m a  ( A ) , and A., a r g e n t i a  growth o f neonate Peridroma s a u c i a . the c o n t r o l .  Growth i s shown as % of  Each p o i n t i n d i c a t e s t h e mean o f a t l e a s t  t h r e e d e t e r m i n a t i o n s per treatment; standard <7%  (•) on t h e  f o r a l l treatments.  d e v i a t i o n s were  Larval Growth (% of Control)  100  F i g u r e 3-2. Growth and s u r v i v o r s h i p o f neonate  Peridroma  s a u c i a f e d a r t i f i c i a l d i e t c o n t a i n i n g s o l v e n t e x t r a c t s of A g l a i a o d o r a t a (Hawaiian sample).  The y i e l d of each  phase, as % o f the t o t a l MeOH e x t r a c t , i s a l s o shown.  solvent  Larval Growth & Survival (% of Control)  1201  102  T y p i c a l r e s u l t s o f f l a s h chromatography o f t h i s phase a r e g i v e n i n Table 3-1, along with b i o a s s a y r e s u l t s f o r each fraction.  S e v e r a l f r a c t i o n s y i e l d e d c r y s t a l l i n e m a t e r i a l on  d r y i n g ; these were i n v e s t i g a t e d f u r t h e r whether o r not they possessed TLC  insecticidal  activity.  a n a l y s i s o f t h e column f r a c t i o n s which were  i n h i b i t o r y t o £. s a u c i a growth i n d i c a t e d t h e presence o f t h r e e major compounds.  P r e p a r a t i v e TLC was used t o i s o l a t e  these compounds i n amounts s u f f i c i e n t f o r i d e n t i f i c a t i o n . Two compounds, t h e i d e n t i f i c a t i o n o f which i s d e s c r i b e d below i n d e t a i l , were t h e v e r y unusual 5,7,4'-trimethyl  flavanone  naringenin  e t h e r , known p r e v i o u s l y from D a h l i a  (Kaufmann and Lam, 1967; Lam and Wrang, 1975), and t h e n o v e l n a t u r a l product  3-hydroxy-5,7,4'-trimethoxyflavanone.  t h i r d component was not i d e n t i f i e d .  The  S u r p r i s i n g l y , none o f  these i s o l a t e d compounds e x h i b i t e d any a c t i v i t y a g a i n s t P. s a u c i a (Table 3-9).  Subsequently I e l u t e d a l l remaining  compounds from t h e TLC p l a t e , r e c o v e r i n g a s m a l l amount o f o i l y m a t e r i a l which a l s o proved t o be i n a c t i v e .  The a c t i v e  p r i n c i p l e t h e r e f o r e appeared t o be u n s t a b l e on s i l i c a , degrading  o r i s o m e r i z i n g t o an i n a c t i v e form.  Subsequent attempts t o p u r i f y t h e a c t i v e compound semipreparative  reverse-phase  HPLC.  Bioassay r e s u l t s i n d i c a t e d  t h a t a l l t h e a c t i v i t y was a s s o c i a t e d w i t h a s i n g l e homogeneous peak ( F i g 3-3).  utilized  HPLC was then used  apparently  103  T a b l e 3 - 1 . P r o p e r t i e s o f f r a c t i o n s o b t a i n e d from f l a s h column chromatography ( S i g e l , 240-400 mesh) o f A., odorata ( E t 0 s o l u b l e phase), and t h e i r e f f e c t on the growth o f neonate Peridroma s a u c i a , r e a r e d f o r seven days on d i e t c o n t a i n i n g each f r a c t i o n a t approximately n a t u r a l c o n c e n t r a t i o n . Values f o l l o w e d by t h e same l e t t e r a r e not s i g n i f i c a n t l y d i f f e r e n t (Tukey's S t u d e n t i z e d (HSD) Range t e s t ) . 2  Fraction # A 1-5 B 6-9 C 10-15 D 16-19 E 20-35 F 36-41 G 42-60 H 61-95  Description  Growth r% o f C o n t r o l ) Empty 102 Chlorophylls only 115 C h l o r o p h y l l , copious white c r y s t a l s , r e d 97 with E h r l i c h s S t i l l some " r e d " cmpd (Rf=.525), 2 b l u e 2 f l u o r e s c i n g cmps, y e l l o w w i t h E h r l i c h s , Rf= .85 & .89 Same two b l u e f l u o r e s c i n g cmpds, lower 6 more abundant Empty 107 White c r y s t a l s , p u r p l e w i t h E h r l i c h s , Rf=.425 8 7 C r y s t a l s , o d o r i n e (Rf=.344) and o d o r i n o l 83 (Rf=,24D ;  ;  a  a  a  b  b  a  a  a  Figure 3 - 3 .  P r e p a r a t i v e HPLC chromatograra of a growth  i n h i b i t o r y f r a c t i o n from A., odorata. i n h i b i t i n g a c t i v i t y was  I n s e c t growth-  a s s o c i a t e d w i t h the shaded peak.  105  106  t o i s o l a t e about 11 mg o f t h i s compound, which was concentrated  and c r y s t a l l i z e d from MeOH.  The compound  proved again t o be the i n a c t i v e 3-hydroxy-5,7,4't r i m e t h o x y f l a v a n o n e , i s o l a t e d p r e v i o u s l y by TLC.  A l l the  i n h i b i t o r y a c t i v i t y remained i n the mother l i q u o r ; when d r i e d t h i s y i e l d e d about 400 pg o f a mixture o f t h e a c t i v e compound ( d e s c r i b e d as Compound 6 below) and the dihydroflavonol precluded  D)  (Compound 4 ) .  Lack o f p l a n t m a t e r i a l has  o b t a i n i n g more o f t h e a c t i v e c o n s t i t u e n t .  Phytochemistry  In a l l ,  t e n n a t u r a l products were i s o l a t e d from A., odorata  f o l i a g e , nine o f which were c h a r a c t e r i z e d c o m p l e t e l y .  Of  the i d e n t i f i e d compounds, f o u r had not p r e v i o u s l y been i d e n t i f i e d i n any A g l a i a s p e c i e s , and two were new n a t u r a l products.  Only t h e compound r e s p o n s i b l e f o r t h e marked  growth i n h i b i t i o n a g a i n s t E» s a u c i a c o u l d not be i d e n t i f i e d . H-NMR and mass s p e c t r a o f a l l compounds a r e g i v e n i n  1  Appendix 2.  Compound 1.  Transparent needles, m/z (FAB): 460 (C3QH50O3).  •'•H-NMR i n d i c a t e d t h e presence o f seven methyl groups. information  This  suggested t h a t the compound was p o s s i b l y a  dammarane t r i t e r p e n e , as such compounds have p r e v i o u s l y been i s o l a t e d from A. o d o r a t a .  T h i s i d e n t i f i c a t i o n was supported  by C H s i n g l e t s a t cS 0.77 and 0.91, c h a r a c t e r i s t i c o f 3  geminal methyls a t C-4, a t 6 0.97 (6H), i n d i c a t i v e o f 10B-  107  and 8B-Me groups, and a t 6 0.89, a s s i g n e d t o t h e 14a-Me group (Shiengthong et. a l . , 1965).  As w e l l a D 0 2  exchangeable p r o t o n a t S 3.29 and a m u l t i p l e t a t 6 3.33 ( s i m p l i f i e d t o a q u a r t e t a f t e r D 0 exchange), were 2  a s s i g n a b l e t o a 3B-0H and a 3a-proton r e s p e c t i v e l y . As i n p r e v i o u s l y i d e n t i f i e d A g l a i a dammaranes, a methylene group i s p r e s e n t a t C-20, i n d i c a t e d by a broad d o u b l e t a t 6 4.75 (2H, J= 10 H z ) .  The two C-25 methyls  appear a t S 1.15 and 1.17; these broaden t o an o v e r l a p p i n g s i n g l e t on D 0 exchange. 2  These s p e c t r a l d a t a a r e c o n s i s t e n t  w i t h t h e known dammarane t r i t e r p e n e a g l a i o n d i o l ,  previously  d e s c r i b e d from A g l a i a odorata (Shiengthong e t a l . , 1965,1974) ( F i g u r e 3-4).  Compound 2.  White, f i n e n e e d l e s , m/z  (FAB):459  T h i s compound was a l s o i d e n t i f i e d as a dammarane w i t h H-NMR CH  (C3QH50O3). triterpene,  s i n g l e t s a t 6 0.78, 0.85, 0.88, and 0.985;  1  3  m u l t i p l e t s (IH each) a t 6 3.41 and 3.22 were a s s i g n e d t o a geminal h y d r o x y l and p r o t o n on C-3.  A broad d o u b l e t a t 6  4.765 (2H, J= 10 Hz) i n d i c a t e d t h e presence o f a methylene The C-25 methyl groups appear a t 6 1.18 and  group a t C-20. 1.225.  These data a r e c o n s i s t e n t w i t h l i t e r a t u r e v a l u e s f o r  the dammarane t r i t e r p e n e a g l a i t r i o l 1974)  (Shiengthong et- a l . ,  ( F i g u r e 3-4).  Compound 3 . FAB) 330 ( C  T h i s compound formed white p l a t e s , m/z ( E I , 1 8  H  1 8  0 ). 6  The UV a b s o r b t i o n spectrum showed  108  Figure 3 - 4 .  S t r u c t u r e s of dammarane t r i t e r p e n e s i s o l a t e d  from Hawaiian samples of A g l a i a o d o r a t a . i s o l a t e d but was hexane phase.  A g l a i o l was  assumed t o be p r e s e n t i n the  Amoora A  stellato-squamosa.  6  was  not  inactive  i s o l a t e d from Amoora  (=Aalaia)  Aglaiol  Aglaiondiol  110 T a b l e 3-2. —H-NMR s p e c t r a l data o f compounds 3. 4, and 5 . 4 3 5 5.39 dd 5.37 dd 4.99 d H-2 3.12, 2.91 dd 4.45 dd 3.05, 2.76 dd H-3 6.08 d 6.13 d 6.10 d H-6 6.09 d 6.14 d 6.15 d H-8 7.40 d 7.50 d 7.39 d H-2',6' 6.98 d 7.00 d 6.95 d H-3',5' 4.05 d OH-3 12.02 S OH-5 3.82 s 3.83 S OMe-5 3.83 S 3.84 s 3.84 S OMe-7 3.89 S 3.85 S 3.92 s OMe-4' (1) 400 MHz i n CDC1 , TMS as i n t e r n a l standard; (2) assignments may be r e v e r s e d J(Hz) 3:2,3=12.8; 3,OH=0.5; 6,8=1.2; 2',3'=9.2; 5',6'=9.2 4:2,3 =3.2; 2,3 =12.4; 3 ,3 =16.4; 6,8=2.0; 2 ,3 =8.0; 5',6'=8.0; S:2,3 =3.2; 2,3 =12.4; 3 =17.6; 6,8=2.4; 2 ,3 =9.0; 5',6'=9.0 ' 2  2  -  -  -  -  3  A  /  B  A  B  /  A  /  /  B  A  B  1  Ill  i n t e n s e peaks a t 283 and 216 nm.  ^H-NMR (Table 3-2)  an AA'BB' system w i t h d o u b l e t s a t 7.00  and 7.50  J=9.2 Hz, c o l l a p s e d t o a s i n g l e t when decoupled ppm),  t o a d o u b l e t of d o u b l e t a t 6 4.45 Hz), which was a t S 4.05  and H-3  coupled t o a geminal OH. 3.84,  3.92  d o u b l e t s , 6.13  ppm)  dihydroflavonol  ppm,  as the new  coupled J =0.5 AB  i n t e r p r e t e d as i n c i s o r i e n t a t i o n , with  Three methoxy groups (3H,  J=MHz) were a l s o  ( c f . B a l z a and Towers, 1984).  s u b s t i t u t e d B r i n g ; C-5  and C-8  ABX  s,  (IH  indicated.  c o n s i s t e n t w i t h the compound b e i n g a  group must be p r e s e n t a t C-4'  C-6  An  exchangeable d o u b l e t  and two meta-coupled protons  and 6.14  T h i s evidence was  methoxylated,  AX  2  i n d i c a t i n g two p r o t o n s , H-2  3.82,  (IH, J = 1 2 . 8 Hz,  T h i s was  7.00  (IH, J=12.8 Hz),  i n t u r n coupled t o a D 0  (IH, J=0.5 Hz).  (2H,  at  t y p i c a l of a p a r a - s u b s t i t u t e d benzene r i n g .  system i n c l u d e d a d o u b l e t a t S 4.99  H-3  ppm  showed  methoxy  t o account f o r the p a r a -  and C-7  of the A r i n g were a l s o  a c c o u n t i n g f o r the meta-coupling  protons.  One  The compound was  between the  therefore identified  n a t u r a l product 3-hydroxy-5,7,4  trimethoxyflavanone  (dihydrokaempferol  5,7,4'-trimethyl  e t h e r ) ( F i g u r e 3-5). F u r t h e r evidence s u p p o r t i n g t h i s s t r u c t u r e was from the mass spectrum  ( F i g u r e s 3-6,  3-7).  obtained  The i d e n t i t y of  a l l the major fragments c o u l d be d e r i v e d by comparison w i t h the fragmentation p a t t e r n of  3,5,4'-trihydroxy-7-  methoxyflavanone ( B a l z a and Towers, 1984) dihydrokaempferol  and  4'-methyl e t h e r (Mabry and Markham,  1975)  112  ( F i g u r e 3-7).  Fragments t y p i c a l of d i h y d r o f l a v o n o l s  the B r i n g fragments B peak), B - C H  (m/z  3  3  (m/z  3 +  135),  and  r i n g fragments [A +H]+ (m/z  C  1 7  H  152). 1 7  The  0 ),  B  (m/z  4 +  [B -43]+ (m/z 3  181),  1  (m/z  150),  A  (m/z  1 +  121,  and  180),  and A^—CO  prominent peak a t [M-CO]+ (m/z  dihydroflavonols  (Mabry and  B - r i n g fragments c o n f i r m substitution.  base  107),  Markham, 1975).  the  A  301,  i s t y p i c a l of f l a v o n e s , f l a v a n o n e s ,  5  include  and  The  s i z e of  the  t h a t i t c a r r i e s a methoxy  A - r i n g fragments a t m/z  181  and  180,  r e s u l t i n g from a r e t r o D i e l s - A l d e r c l e a v a g e (Mabry Markham, 1975), c o n f i r m the presence of two  and  methoxy  substitutions.  Compound 4.  White c r y s t a l s , m/z  (EI) 314,  ^•H-NMR spectrum of t h i s compound was Compound 4, w i t h a p a r a - s u b s t i t u t e d doublets,  6.95  protons (1H,  and  7.39  doublets,  ppm, 6.10  t h r e e methoxy groups (3H, ppm)  (Table  present, 5.37  (1H,  Hz),  and  3-2).  and JAB 2.76  = 0  -  4  ppm  J  AX  the compound was  = 1 3  '  5  H  JAB  was  z  ) '  =  0 , 4  6  3  J  coupled  '  1 8  3-5).  The  5  (2H,  meta-coupled  3.83,  3.84,  and  and 3.89  exchangeable protons were  ( '  0 5  AX  =  1 H  1  6  ,  0  H z  J  of d o u b l e t  AB  )«  = 0  *  4  J  AX  = 1 6  at 6 -°  This indicated  t o geminal protons a t  t h e r e f o r e i d e n t i f i e d as 5,7,4'-  trimethoxyflavanone (naringenin-5,7,4'-triroethyl (Figure  0 .  J=2.0 Hz),  system showed d o u b l e t s  (1H,  t h a t a p r o t o n a t C-2  two  ppm,  singlets, 2  the ABX  H  benzene r i n g  6.15  However, no D 0  1 8  s i m i l a r t o t h a t of  J=8.0 Hz), and  C  ether)  C-3;  113  F i g u r e 3-5.  S t r u c t u r e s of flavanones  samples of A a l a i a o d o r a t a .  i s o l a t e d from Hawaiian  The s t e r e o c h e m i s t r y shown  assumes an S c o n f i g u r a t i o n at  C-2.  C  ^OCH  CH 0 3  3  OCH3 o  Compound 3 3-Hydroxy-5,7,4'-methoxyfIavanone  Compound 5 5-Hydroxy-7,4'-dimethoxyflavanone  115  F i g u r e 3-6.  Mass spectrum (EI) o f 3-hydroxy-5,7,4'-  t r i m e t h o x y f l a v a n o n e (Compound 3 ) .  116  117  F i g u r e 3-7.  Mass spectrum fragmentation p a t t e r n o f 3-  hydroxy-5,7,4'-trimethoxyflavanone (Compound 3 ) .  118  m/z 107 [B - 43]* 3  119  As w i t h t h e p r e v i o u s compound, t h e mass spectrum showed A - r i n g fragments a t m/z  181, 180, and 152 c o n f i r m i n g two  methoxy s u b s t i t u t i o n s , and B - r i n g fragments a t m/z  134 ( B  3 +  ,  base peak), 119, and 91 c o n f i r m t h e l o c a t i o n o f t h e t h i r d methoxy group.  Compound 5. NMR  White p l a t e s , m/z  (EI) 300, C  H  1 7  1 6  0 ).  The H1  5  spectrum o f t h i s compound was almost i d e n t i c a l t o t h a t  of Compound 5, but showed o n l y two methoxy groups (3H, 3.83 and 3.85 ppm) and a D 0 exchangeable s i n g l e t 2  ppm  (Table 3-2).  a t m/z  (1H) a t 12.02  The mass spectrum showed A - r i n g  fragments  167 and 168, c o n s i s t e n t w i t h t h e presence o f one  methoxy and one hydroxy s u b s t i t u t i o n on t h i s r i n g . fragments a t m/z  B-rihg  121 demonstrated t h e presence of a methoxy  s u b s t i t u t i o n , which must be a t C-4' t o account f o r t h e p a r a substitution pattern.  The h y d r o x y l was a s s i g n e d t o C-5  r a t h e r than C-7, as hydrogen bonding w i t h t h e a d j a c e n t c a r b o n y l causes t h e s i g n a l t o appear a t 12.02 ppm; i f p r e s e n t a t C-7 t h e s i g n a l would have o c c u r r e d a t 9.5 ( B a l z a and Towers, 1984).  ppm  Compound 5 was t h e r e f o r e  i d e n t i f i e d as 5-hydroxy-7,4'-dimethoxyflavanone  ( F i g u r e 3-  5).  Compound 6.  m/z  (FAB) 661, MH+.  Possibly C  3 6  H  3 6  0  1 2  .  This  compound produces a brown c o l o r r e a c t i o n w i t h E h r l i c h ' s reagent, a r e a c t i o n t y p i c a l o f B - s u b s t i t u t e d f u r a n s (Dreyer, 1964).  The mass and p o s s i b l e presence o f a B - s u b s t i t u t e d  120  f u r a n r i n g suggest t h a t compound 6 i s an o x i d i z e d l i m o n o i d . However, t h e v e r y l i m i t e d amount o f m a t e r i a l on hand p r e c l u d e s t h e e x t e n s i v e NMR s t u d i e s r e q u i r e d f o r s t r u c t u r e elucidation.  Compound 7.  White needles, m/z (EI) 300 (M+), C  H-NMR (Table 3-3) showed a benzene r i n g  1  1 8  H  2 4  N 0 . 2  2  (5H, S 7.37,  m u l t i p l e t ) coupled t o an AB system (IH, S 7.06, J=  Hz and  7.57, m, t r a n s - s u b s t i t u t e d double bond), an NH d o u b l e t a t 6 6.13  (IH, J=16 Hz), and methylene m u l t i p l e t s a t S 3.70 (2H) Methyl f u n c t i o n s were seen a t 6 0.7  and 2.00-2.26  (4H).  (3H, t , 3=7.9  Hz) and 1.10 (3H, d, J=7.0 Hz), a methylene  was r e p r e s e n t e d by m u l t i p l e t s a t 6 1.32 and 1.56, and a s i n g l e proton showed as a m u l t i p l e t a t 6 1.92.  This  i n f o r m a t i o n e s t a b l i s h e d t h e i d e n t i t y o f Compound 8 as t h e known bis-amide o d o r i n e (Shiengthong  e t a i . , 1979;  Purushothaman gt a i * , 1979) ( F i g u r e 3-8).  This  i d e n t i f i c a t i o n was confirmed by t h e mass spectrum, which showed major fragments a t m/z 215 and 85, ( r e s u l t i n g l o s s o f t h e 2-methylbutanoyl (from l o s s o f t h e cinnamoyl l o s s o f 2-methylbutanoic  fragment),  from  a t m/z 169 and 131  m o i e t y ) , and a t m/z 199 (from  a c i d amide).  Mass s p e c t r a l and ^H-  NMR data were c l o s e l y comparable t o l i t e r a t u r e v a l u e s , and w i t h s p e c t r o s c o p i c data o b t a i n e d from a sample o f o d o r i n e s y n t h e s i z e d from d i h y d r o c i n n a m o y l - L - p r o l i n e a c c o r d i n g t o t h e procedure o f Purushothaman e t al.(1979) by Dr. H. B a r r i o s  121  Figure 3-8.  S t r u c t u r e s of bis-amides i s o l a t e d from Hawaiian  samples of A g l a i a o d o r a t a .  123  Lopez, w h i l e he was a v i s i t i n g s c i e n t i s t w i t h Dr. G.H.N. Towers. Odorine has c h i r a l c e n t e r s a t C-2 and C-2'. site i s particularly  sensitive t o epimerization i n acidic  s o l v e n t s i n c l u d i n g CHC1 result  The C-2  3  (Purushothaman  et. a i . , 1979).  As a  c h l o r i n a t e d s o l v e n t s were avoided d u r i n g t h e  i s o l a t i o n o f A g l a i a compounds.  Samples o f R,S and R,R  o d o r i n e were s y n t h e s i z e d , and then ( s e p a r a t e l y ) e p i m e r i z e d a t C-2 w i t h HC1 t o produce a racemic mixture which was s e p a r a t e d by p r e p a r a t i v e TLC.  Comparison o f t h e H-NMR 1  (CDCI3) s p e c t r a i n d i c a t e t h a t i n t h e isomers which a r e R a t C-2' t h e methyls o f t h e 2-methylbutanoyl moiety appear a t 6 0.70 and 1.10 ppm, whereas f o r t h e S isomers t h e methyls a r e a t S 0.90 and 0.98 ppm. The enantiomeres R,R- and S,So d o r i n e had a Rf o f .324 (Table 3-3), compared t o a Rf o f .331 f o r R,S and S,R-odorine.  Compound 7 was t h e r e f o r e  i d e n t i f i e d as t h e known S,R-odorine  [ ( + ) - o d o r i n e ] ( F i g u r e 3-  8).  Compound 8.  Needle c r y s t a l s ,  m/z (EI) 300, C H24N 02. 18  2  The  mass spectrum o f compound 8 was i d e n t i c a l t o compound 7. The H-NMR was a l s o i d e n t i c a l 1  except f o r t h e methyl s i g n a l s  a t S 0.90 (3H, t , J=8.0 HZ) and 0.98 (3H, d, J=7.0 HZ). Compound 8 was t h e r e f o r e i d e n t i f i e d as S,S-odorine 3-8).  (Figure  T h i s i d e n t i f i c a t i o n was supported by c o -  chromatography  w i t h s y n t h e t i c S,S-odorine.  T h i s isomer o f  o d o r i n e has not p r e v i o u s l y been i d e n t i f i e d as a n a t u r a l  124  product.  To check t h e p o s s i b i l i t y t h a t R,S-odorine c o u l d  i s o m e r i z e a t C-2 i f s t o r e d i n MeOH (the s o l v e n t used t o e x t r a c t A., odorata) f o r extended p e r i o d s , a sample o f o r i g i n a l l y pure R,S-odorine was examined a f t e r s i x months i n MeOH a t room temperature i n t h e dark.  No evidence o f  i s o m e r i z a t i o n t o t h e S,S form was seen.  S,S-Odorine  appears  t o be an a u t h e n t i c n o v e l n a t u r a l p r o d u c t .  Compound 9 .  Needle c r y s t a l s , m/z (EI) 316, C  1 8  H  2 4  N 0 . 2  3  The  H-NMR spectrum o f t h i s compound was s i m i l a r t o compound 8,  1  except t h a t t h e methyl group a t C-2' appeared as a s i n g l e t a t <S 1.22, t h e m u l t i p l e t due t o t h e C-2' proton was absent, and a D 0 exchange experiment showed an exchangeable p r o t o n 2  a t 6 2.16 ppm. a t 283 nm.  The UV spectrum showed a s i n g l e absorbance  T h i s s p e c t r a l d a t a i s c o n s i s t e n t w i t h t h e known  A g l a i a bis-amide o d o r i n o l  ( F i g u r e 3-8) (Shiengthong e t a l . ,  1979, Purushothaman e t a i . , 1979, Hayashi e t A l . ,  1982).  T h i s s t r u c t u r e was f u r t h e r confirmed by t h e mass spectrum, which i n d i c a t e d a cinnamoyl moiety w i t h peaks a t m/z 131 and 103, and a 2-methyl-2-hydroxybutanoyl moiety w i t h fragments a t m/z 231 and 101.  An S,S s t e r e o c h e m i s t r y i s i n d i c a t e d by  methyl s i g n a l s a t 6 0.88 (3H, t , J=7.2 Hz) and S 1.22 (3H, s).  Compound 9 was t h e r e f o r e a s s i g n e d t h e s t r u c t u r e S,S-  o d o r i n o l ( F i g u r e 3-8).  Compound 1 0 .  Needle c r y s t a l s , m/z (EI) 316, C H 2 4 N 0 . 18  2  3  The mass spectrum o f t h i s compound was i d e n t i c a l t o compound  125  10.  The H-NMR spectrum d i f f e r e d o n l y i n t h a t t h e methyls 1  of t h e 2-methyl-2-hydroxybutanoyl moiety appeared a t 6 0.70 (3H, t , J=7.2 Hz) and  1.34  (3H, s ) . These data i n d i c a t e a  s t r u c t u r e o f S,R-odorinol ( F i g u r e 3-8).  £) Q u a l i t a t i v e and q u a n t i t a t i v e comparison of A a l a i a s p e c i e s Two methods s u i t a b l e f o r r a p i d phytochemical a n a l y s i s of A a l a i a s p e c i e s were developed.  A q u a l i t a t i v e method was  based on TLC on s i l i c a g e l and use o f c o l o r r e a c t i o n s w i t h E h r l i c h ' s and v a n i l l i n spray reagents (Table 3-3).  Rf  v a l u e s o f s e v e r a l compounds d i f f e r i n t h e two s o l v e n t systems used (EtOAc:PE 1:1 and CHCl :MeOH 49:1), so t h e s e 3  are s u i t a b l e f o r two-dimensional TLC.  The c o l o r r e a c t i o n s  w i t h t h e two spray reagents a r e h i g h l y c h a r a c t e r i s t i c and a l l o w c o n f i d e n t i d e n t i f i c a t i o n of most o f t h e compounds. T h i s method was a p p l i e d t o t h e a n a l y s i s o f A., o d o r a t a specimens from T h a i l a n d , Hawaii, and Taiwan, and t o A.. o d o r a t i s s i m a and A., a r g e n t i a specimens from T h a i l a n d  (Table  3-4). The second method was based on a n a l y t i c a l  HPLC;  r e t e n t i o n times of pure standards a r e g i v e n i n T a b l e 3-5. Chromatographic p r o f i l e s o f t h e v a r i o u s A g l a i a samples a r e shown i n F i g u r e s 3-9 t o 3-13.  As t h e c o n c e n t r a t i o n s o f  secondary m e t a b o l i t e s i n p l a n t s may d i f f e r g r e a t l y from t h e amounts e v e n t u a l l y i s o l a t e d , due t o l o s s e s d u r i n g e x t r a c t i o n and p u r i f i c a t i o n , t h i s method was a p p l i e d t o determine t h e  126  c o n c e n t r a t i o n of A g l a i a compounds i n f r e s h l y prepared e x t r a c t s (Table  Et 0 2  3-6).  Each method o f f e r s c e r t a i n advantages and weaknesses. The major advantage of the HPLC method l i e s i n the to q u a n t i f y c o n c e n t r a t i o n s of s e v e r a l compounds. two  ability However  major disadvantages e x i s t : HPLC cannot be used t o d e t e c t  the dammarane t r i t e r p e n o i d s , due  t o t h e i r low UV  absorption  c o e f f i c i e n t s , and HPLC does not r e s o l v e the v a r i o u s isomers of odorine or o d o r i n o l .  For the l a t t e r purposes ^H-NMR  was  used t o q u a n t i f y the p r o p o r t i o n of each isomer, a c c o r d i n g  to  the r e l a t i v e peak h e i g h t s of the 2-methylbutanoyl methyls. Although q u a l i t a t i v e i n nature, the TLC based system can r e a d i l y d e t e c t the presence of dammaranes i n very c o n c e n t r a t i o n , and odorines  low  i t can r e s o l v e the diastereomers  and o d o r i n o l s .  o f the  In the l a t t e r case a minimumn of  4-  5 developments were r e q u i r e d f o r c l e a r r e s o l u t i o n of the diasteriomeres.  A n a l y s i s of an E t 0 e x t r a c t r e q u i r e s 1-3 2  r e g a r d l e s s o f the method chosen, but TLC  h  requires less  sample p r e p a r a t i o n than HPLC. Tables  3-4  phytochemistry  and  3-6  i n d i c a t e t h a t marked d i f f e r e n c e s i n  occur between most of the A g l a i a s p e c i e s  samples examined.  A., odorata  samples from T h a i l a n d  and  and  Hawaii were most s i m i l a r ; both produced a g l a i o n d i o l and aglaitriol,  and the t h r e e d i h y d r o f l a v a n o n e s ,  d i f f e r e n c e s i n c o n c e n t r a t i o n were noted.  The  although Hawaiian  sample c o n t a i n e d odorine B and o d o r i n o l A as the major amides, w i t h minor amounts of odorine A and o d o r i n o l B  minor  127 Table 3-3. Aglaia odorata compounds: chromatographic behavior and colour reactions with Ehrlichs reagent and v a n i l l i n reagent. Solvent A i s EtOAC:PE (3:1), solvent B i s CHCl :MeOH (49:1). Where two colors are given the f i r s t applies to the color immediately after heating and the second occurs after cooling. 3  Rf(solvent A^ (solvent B^ Ehrlichs Vanillin Compound red orange 5-OH-7,4'-MF .912 .925 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-Hydroxy5,7,4'-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 CHCl :MeOH (39:1) on Si gel, followed by spraying with Ehrlichs or v a n i l l i n reagents. 3  Cmpd  h. odorata (Hawaii)  A a l a i a sample A. odorata A . odorata (Thailand\ (Taiwan^  A. odoratissima  (Thailand)  A. argentia  (Thailand)  1 +++ +++ + + • 2 ++ ++ 3 +++ ++ ++ 4 ++ ++ + + 5 ++ ++ 7 + ++ ++ 8 ++ +++ +++ ++ ++ 9 ++ +++ +++ 10 + = = = ; Compounds: (1) Aglaiondiol; (2) A g l a i t r i o l ; (3) 5-hydroxy7,4'-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.  T a b l e 3 - 5 . HPLC r e t e n t i o n times (min) o f A a l a i a o d o r a t a compounds. Rt (min} Compound 33.6 1 Aglaiondiol 33.48 2 Aglaitriol 28.56 3 5-OH-7,4'-MF 4 5,7,4'-MF 26.12 22.80 5 3-OH-5,7,4'-MF 6 limonoid 22.61 20.61 7 Odorine A 20.65 7' Odorine A' 20.65 8 Odorine B 20.66 8' Odorine B' 16.79 9 Odorinol A 16.79 10 Odorinol B 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-Hydroxy5,7,4'-trimethoxyflavanone. /  130  F i g u r e 3-9.  HPLC t r a c e o f E t 0 s o l u b l e f r a c t i o n from A g l a i a  o d o r a t a (Hawaiian sample).  2  I d e n t i f i e d compounds i n c l u d e :  (I) O d o r i n o l (S,R- and S,S-diastereomers u n r e s o l v e d ) ; ( I I ) Odorine  (S,R- and S,S- diastereomers u n r e s o l v e d ) ; ( I I I ) 3-  OH-5,7,4'-trimethoxy d i h y d r o f lavanone; (IV) 5,7,4'trimethoxy d i h y d r o f l a v a n o n e ; (V) 5-hydroxy-7,4'dimethoxyflavanone.  131  F i g u r e 3-10.  HPLC t r a c e o f E t 0 s o l u b l e f r a c t i o n 2  A g l a i a odorata ( T h a i l a n d sample). include:  (I) Odorinol  from  I d e n t i f i e d compounds  (S,R- and S,S-diastereomers  unresolved);  ( I I ) Odorine (S,R- and S,S- diastereomers  unresolved);  ( I I I ) 3-OH-5,7,4'-trimethoxy d i h y d r o f l a v a n o n e  (IV) 5,7,4'-trimethoxy dimethoxyflavanone.  d i h y d r o f lavanone; (V) 5-hydroxy-7,4  133  F i g u r e 3-11.  HPLC t r a c e o f E t 0 s o l u b l e f r a c t i o n 2  A g l a i a o d o r a t a (Taiwan sample).  from  I d e n t i f i e d compounds  i n c l u d e : ( I ) O d o r i n o l (S,R- and S,S-diastereomers u n r e s o l v e d ) ; ( I I ) Odorine  (S,R- and S,S- d i a s t e r e o m e r s  u n r e s o l v e d ) ; ( I I I ) 3-OH-5,7,4'-trimethoxy d i h y d r o f lavanone (IV) 5,7,4'-trimethoxy dimethoxyflavanone.  d i h y d r o f l a v a n o n e ; (V) 5-hydroxy-7,4  135  136  F i g u r e 3-12.  HPLC t r a c e o f the E t 0 s o l u b l e f r a c t i o n from  Aalaia odoratissima.  2  I d e n t i f i e d compounds i n c l u d e : ( I )  O d o r i n o l (S,R- and S,S-diastereomers u n r e s o l v e d ) ; ( I I ) Odorine (S,R- and S,S- diastereomers  u n r e s o l v e d ) ; ( I I I ) 3-  OH-5,7,4'-trimethoxy d i h y d r o f l a v a n o n e ; trimethoxy d i h y d r o f l a v a n o n e ; dimethoxyflavanone.  (IV) 5 , 7 , 4 ' -  (V) 5-hydroxy-7,4'-  137  138  F i g u r e 3-13.  HPLC t r a c e o f t h e E t 0 s o l u b l e f r a c t i o n from  Aglaia argentia. (S,R-  2  I d e n t i f i e d compounds i n c l u d e : ( I ) O d o r i n o l  and S,S-diastereomers  and S,S- diastereomers  unresolved);  trimethoxy d i h y d r o f l a v a n o n e ; dihydroflavanone;  u n r e s o l v e d ) ; ( I I ) Odorine (S,R( I I I ) 3-OH-5,7,4'-  (IV) 5,7,4'-trimethoxy  (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 a n a l y t i c a l HPLC. Compound &. odorata (Haval1)  &. odorata (Thailand)  A g l a i a sample L- odorata &• odoratlaalma (Taiwan)  (Thai land)  165.9 205.3 183.6 8.3 3 4 513.2 470.2 49.7 107.6 5 154.9 318.6 48.7 24.3 7 113.0 0 0 276.0 8 295.0 770.1 1298.9 422.7 9 143.7 704.1 1911.5 61.2 80.8 o o 10 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.  A- arqentla (Thailand)  0 115.9 0 201.0 189.8 0 0  141  present.  In c o n t r a s t the T h a i l a n d A., odorata sample  c o n t a i n e d l a r g e r amounts of odorine B and o d o r i n o l A, o d o r i n e A and o d o r i n o l B t o t a l l y absent.  with  The Taiwan sample  of A. odorata c o n t a i n e d o n l y a t r a c e of a g l a i o n d i o l ,  no  a g l a i t r i o l , moderate c o n c e n t r a t i o n s of 5-hydroxy-7,4'methoxy d i h y d r o f l a v a n o n e , and o n l y t r a c e s of the o t h e r dihydroflavanones  ( d e t e c t e d by HPLC o n l y ) .  p r o f i l e of t h i s sample was  The  amide  i d e n t i c a l t o the T h a i sample,  w i t h l a r g e amounts o f odorine B and o d o r i n o l A and o d o r i n e A or o d o r i n o l B.  A g l a i a o d o r a t i s s i m a was  c o n t a i n s m a l l amounts of a g l a i o n d i o l and  no found t o  5,7,4'-trimethoxy  d i h y d r o f l a v a n o n e , and moderate amounts of odorine A and B i n equal p r o p o r t i o n s .  HPLC a n a l y s i s f u r t h e r r e v e a l e d v e r y  low  c o n c e n t r a t i o n s of the o t h e r d i h y d r o f l a v a n o n e s and o d o r i n o l (isomer not i d e n t i f i e d ) .  A a l a i a a r g e n t i a appeared t o l a c k  a g l a i o n d i o l and a g l a i t r i o l e n t i r e l y , but d i f f e r e n t dammaranes may  be p r e s e n t as i n d i c a t e d by major components  which show s i m i l a r c o l o r r e a c t i o n s .  Only a s m a l l amount of  5,7,4'-trimethoxy d i h y d r o f l a v a n o n e was d i h y d r o f l a v a n o n e s were absent. i n almost a 1:1  E)  d e t e c t e d ; the o t h e r  Odorine A and B were found  r a t i o , and o d o r i n o l appears t o be  absent.  Insect Bioassays Bioassay-guided f r a c t i o n a t i o n of A., odorata l e d t o a  s i n g l e compound, compound 6, w i t h s i g n i f i c a n t growth i n h i b i t o r y a c t i v i t y a g a i n s t £. s a u c i a neonates (Table Although  i n s u f f i c i e n t m a t e r i a l was  3-7).  obtained f o r s t r u c t u r e  142  e l u c i d a t i o n , due t o t h e pronounced a c t i v i t y o f t h i s compound the dose-response r e l a t i o n s h i p c o u l d be e s t a b l i s h e d 2-14).  (Figure  The compound was about f o u r times l e s s a c t i v e than  a z a d i r a c h t i n i n i t s a b i l i t y t o i n h i b i t E- s a u c i a growth, w i t h an E C  5 0  o f 1.4 pg/g d i e t fwt compared t o 0.36 ug/g f o r  azadirachtin.  The L C  with a z a d i r a c h t i n .  5 0  was 11.2 ug/g, compared t o 4.0 ug/g  As t h e a c t i v e compound was s t i l l  admixed  w i t h some o f t h e d i h y d r o f l a v o n o l , t h e a c t i v i t y o f t h e pure compound may be s t i l l The  c l o s e r to that of azadirachtin.  i n h i b i t o r y a c t i v i t y o f compound 6 appeared not t o  be due t o a n t i f e e d a n t a c t i v i t y .  In c h o i c e t e s t s w i t h  neonate £• s a u c i a compound 6 had no s i g n i f i c a n t e f f e c t on feeding at concentrations  which i n h i b i t e d growth i n no-  c h o i c e assays (Table 3-9).  On t h e o t h e r hand, t h e a c t i v i t y  of t h e t o t a l MeOH e x t r a c t , assayed a t 50% o f n a t u r a l concentration, (Table 3-9).  appeared t o i n c l u d e an a n t i f e e d a n t  effect  No E - s a u c i a l a r v a e showed evidence o f having  d i e d d u r i n g a f a i l e d molt, but treatment with the known IGR compound a z a d i r a c h t i n a l s o f a i l e d t o produce obvious molt i n h i b i t i o n i n t h i s i n s e c t (Chapter 4 ) . None o f t h e o t h e r compounds i s o l a t e d from A.  odorata  i n h i b i t e d E - s a u c i a growth o r s u r v i v o r s h i p , over a seven-day assay beginning  w i t h neonates, when f e d a t c o n c e n t r a t i o n s  equal t o o r g r e a t e r than those o c c u r r i n g in  p l a n t a (Table 3-  7). The dwt.  i s o l a t e d y i e l d o f compound 6 was about 3 ng/g  leaf  However, an e s t i m a t i o n based on t h e a c t i v i t y o f t h e  143 Table 3-7. A a l a i a odorata compounds: concentration bioassayed, and r e s u l t a n t E. saucia growth and s u r v i v o r s h i p (as % of C o n t r o l ) . Values i n a column followed by the same l e t t e r do not d i f f e r s i g n i f i c a n t l y (Tukey's Studentized (HSD1 Range T e s t . Compound 1 2 3 4 5 6  7 7'  8 8' 9 10  Aglaiondiol Aglaitriol 5-OH-7,4'-MF 5,7,4'-MF 3-OH-5,7,4'-MF "active" Odorine A Odorine A' Odorine B Odorine B* Odorinol A Odorinol B Amoora A6  Max. Cone. Bioassayed 1000 ug/g 1000 500 500 500 15 1000 1000 1000 1000 1000 1000 50Q  E. fiflucia growth (%) 137.9 132.0 110.8 108.3 107.l 2.6 114.8 116.7 121.2 89.4 114.8 124.7 103.2  a  a a a  a  b a a  a a a a a  E« saucia  survivorship 100 100 100 100 100 31 100 95 100 100 93 100 _10£  144  Figure 3-14. survivorship  E f f e c t o f Compound 6 on t h e growth ( A ) o f neonate Peridroma s a u c i a .  (•) and  For  comparison, t h e e f f e c t o f a z a d i r a c h t i n on t h e growth survival  (&) o f neonate Peridroma s a u c i a  (a) and  i s a l s o shown.  E r r o r bars i n d i c a t e + one s t a n d a r d d e v i a t i o n .  Larval Growth and Survivorship (%  S+7l  of Control)  crude MeOH e x t r a c t and assuming a l l t h e a c t i v i t y was due t o compound 6 i n d i c a t e s an expected f o l i a r c o n c e n t r a t i o n of 98 fig/g.  The d i s c r e p a n c y suggested t h a t  i n t e r a c t i o n s might be o c c u r r i n g constituents  i n A., o d o r a t a .  synergistic  between phytochemical  Consequently t h e a c t i v i t y o f  v a r i o u s combinations o f t h e i s o l a t e d compounds were a l s o assayed (Table 3-8).  No combination of these secondary  m e t a b o l i t e s showed evidence o f s y n e r g i s t i c a c t i v i t y . putative  synergist  remains  unidentified.  The  T a b l e 3-8. E f f e c t o f A g l a i a odorata compounds, t e s t e d i n combination, on growth o f neonate Peridroma s a u c i a i n a seven-day assay. Growth i s shown as % o f C o n t r o l ; v a l u e s f o l l o w e d by t h e same l e t t e r do not d i f f e r s i g n i f i c a n t l y (Tukey's S t u d e n t i z e d (HSD) Range t e s t , <x=0.05). Compounds are numbered as i n T a b l e 3-7. Compounds 1+2+3+4+5+7+8+9 1+6 2+6 3+6 4+6 5+6 6 7+6 8+6 9+6  Concentrations Bioassayed (ua/a) 1,2:1000 3,4,5:200 7,8,9:200 1000 + 1 1000 + 1 200 + 2 200 + 2 200 + 2 2 100 + 2 100 + 2 100 + 2  Mean Growth 96. 5  a  79.3 68.3 34.6 26.4 28. 8 23. 8 29. 9 36.4° 22.6°-  a b  b  C  C  C C  C  148  Table 3-9. E f f e c t of Compound 6 on d i e t c h o i c e by neonate Peridroma s a u c i a . F i g u r e s shown i n d i c a t e the percentage of l a r v a e found on C o n t r o l (C) or T r e a t e d (T) d i e t a f t e r 24 h of f e e d i n g ; each v a l u e i s the mean of twelve r e p l i c a t i o n s . O b s e r v a t i o n s were compared t o a 50:50 d i s t r i b u t i o n u s i n g a G - t e s t w i t h oc=0.05; v a l u e s which d i f f e r e d s i g n i f i c a n t l y a r e i n d i c a t e d by an a s t e r i s k . A l s o shown i s the l a r v a l growth (as % of c o n t r o l ) a f t e r seven days of f e e d i n g i n a no-choice bioassay. Concentration (ug/g d i e t dwt^  D i e t Choice C T  MeOH e x t r a c t (50%^  92  1 2 3  49 40 44  51 60 56  8-  Growth (% of C o n t r o l ) 69.7 36.9 15.3  0.2  Discussion  A Phytochemistry Phytochemical examination o f A g l a i a s p e c i e s c o r r o b o r a t e d p r e v i o u s r e p o r t s and added s e v e r a l new n a t u r a l products t o the l i s t .  Most o f these phytochemicals a r e unique t o  A g l a i a ; o n l y t h e f l a v a n o n e s n a r i n g e n i n 5,7,4'-methyl e t h e r (Compound 4) and 5-hydroxy-7,4'-dimethyl  dihydroflavanone  (Compound 3) have been r e p o r t e d from o t h e r p l a n t s .  The  f i r s t has been found o n l y once, i n t h e composite D a h l i a t e n u i c a u l i s (Kaufmann and Lam, 1967; Lam and Wrang, 1975). The second i s known from a v a r i e t y o f u n r e l a t e d t a x a i n c l u d i n g B e t u l a spp (Wollenweber,  1975), Prunus s a r a e n t i i  (Wollenweber and D i e t z , 1981), t h e composites D a h l i a tenuicaulis  (Lam and Wrang, 1975), Eupatorium  (Arene e £ a l . , 1978),and Hieracium intybaceum  odoratum (Wollenweber,  1984), t h e c a c t i Rhodocactus g r a n d i f o l i u s and M a m i l l a r i a e l o n g a t a ( B u r r e t gt a l . , 1982), and t h e f e r n Pityrogramma spp (Wollenweber and D i e t z , 1980).  A l l of the  d i h y d r o f l a v a n o n e s a r e here r e p o r t e d from t h e Meliaceae f o r the f i r s t time.  F l a v o n o i d s p r e v i o u s l y known from A g l a i a  i n c l u d e g l y c o s i d e s o f q u e r c e t i n and kaempferol 1983).  (Harborne,  Q u i t e d i s s i m i l a r C-8 p r e n y l a t e d flavonones have  p r e v i o u s l y been r e p o r t e d from A z a d i r a c h t a i n d i c a Bhakuni,  1984).  (Garg and  O-Methylation a t C-5 i s uncommon amongst  v a s c u l a r p l a n t s i n g e n e r a l , but i s c h a r a c t e r i s t i c o f f l a v o n o i d s from s p e c i e s o f t h e Rutaceae  (Wollenweber and  150  D i e t z , 1981; Harborne, 1983); t h e presence o f such compounds i n A g l a i a may r e f l e c t t h e c l o s e r e l a t i o n s h i p between t h e Rutaceae and M e l i a c e a e .  Wollenweber and D i e t z  (1981) have  commented on the common co-occurrence o f methoxylated f l a v o n o i d s and l i p o p h i l i c t e r p e n o i d s ,  a p a t t e r n supported by  the presence o f both methylated f l a v a n o n e s and dammarane triterpenes i n Aglaia. The  f o l l o w i n g d i s c u s s i o n o f phytochemical d i f f e r e n c e s  between samples o f A g l a i a must be p r e f a c e d  by a c a v e a t : as  o n l y s i n g l e samples were a v a i l a b l e from each c o l l e c t i o n site,  i t cannot be determined a t t h i s time whether t h e  v a r i a t i o n s observed r e f l e c t v a r i a t i o n between i n d i v i d u a l s o r populations.  Further  d i s c r e t e populations  a n a l y s i s o f l a r g e samples from i s required to resolve t h i s  question.  Dammarane t r i t e r p e n e s a r e common among t h e M e l i a c e a e , and  some s p e c i e s , such as C a b r a l e a e i c h l e r i a n a . may  elaborate  a considerable  e t a l . , 1975).  d i v e r s i t y o f these compounds (Rao  A l l o f t h e A g l a i a dammaranes i s o l a t e d t o  date a r e c h a r a c t e r i z e d by a methylene group a t C-20, and o x i d a t i o n a t C-24 and C-25. odorata contained  Hawaiian and Thai samples o f A..  both a g l a i o n d i o l and a g l a i t r i o l i n l a r g e  amounts; i n c o n t r a s t , o n l y a g l a i o n d i o l was found i n A.. odorata from Taiwan, and i n A., o d o r a t i s s i m a .  Neither  dammarane was p r e s e n t i n A., a r g e n t i a . but o t h e r major compounds which g i v e s i m i l a r c o l o r r e a c t i o n s on TLC may represent  o t h e r dammaranes.  151  Dammarane t r i t e r p e n e s may  s e r v e as p r e c u r s o r s i n the  b i o s y n t h e s i s of euphane and apo-euphane compounds, which are themselves 1972;  Nes  p r e c u r s o r s o f the l i m o n o i d s (Devon and S c o t t , and McLean, 1977).  The p r o d u c t i o n of l a r g e  amounts of dammaranes, and apparent  absence of l i m o n o i d s  (except p o s s i b l y Compound 6 ) , c o n t r a s t s w i t h most o t h e r M e l i a c e a e and i n p a r t i c u l a r w i t h o t h e r members of the  tribe  A g l a i e a e , and suggests t h a t the pathway t o l i m o n o i d synthesis i s blocked i n A g l a i a . The bis-amide  o d o r i n e was  p r e s e n t i n a l l A a l a i a samples  examined i n t h i s study, but d i f f e r e n c e s i n the d i a s t e r e o m e r s produced  were noted.  specific  In a l l A., odorata  samples examined o d o r i n e B predominates;  l e s s e r amounts of  o d o r i n e A are a l s o p r e s e n t o n l y i n the sample from  Hawaiii  In c o n t r a s t A. o d o r a t i s s i m a and A., a r g e n t i a produce the A and B forms i n approximately equal amounts. observed t o i s o m e r i z e i n MeOH i n v i t r o .  Odorine was  not  These r e s u l t s  i n d i c a t e t h a t the p r o d u c t i o n of each diastereomere  is likely  r e g u l a t e d by a s e p a r a t e enzyme.  O d o r i n o l was  t h r e e samples of A., o d o r a t a . was  p r e s e n t i n v e r y low amounts  i n A., o d o r a t i s s i m a O d o r i n o l A was  f  and was  found i n a l l  absent from A. a r g e n t i a .  the o n l y isomer observed  i n the T h a i and  Taiwan samples of A. o d o r a t a ; i n the Hawaiian sample odorine A predominated t h i s argues  but some o d o r i n e B was  f o r s e p a r a t e enymatic  a l s o present.  c o n t r o l f o r each  Again isomer.  The p r e v a l e n c e of the o d o r i n e s w i t h i n the genus i s u n c l e a r : all  s p e c i e s examined t o date c o n t a i n o d o r i n e , but i n g e n e r a l  152  surveys of p l a n t s f o r a l k a l o i d s many A g l a i a s p e c i e s gave a negative t e s t  (Farnsworth  e t a l . , 1954).  C e r t a i n l y the  o d o r i n e s appear t o be unique t o A g l a i a , and d i f f e r markedly from a l k a l o i d s p r e s e n t i n o t h e r Meliaceae s p e c i e s (Aladesanmi and 1988)  .  I l e s a n m i , 1987;  such as Dysoxylum  Aladesanmi e t a l . ,  The b i o s y n t h e t i c o r i g i n of these compounds i s  u n c l e a r but they c o u l d be d e r i v e d from o r n i t h i n e , p h e n y l a l a n i n e , and i s o l e u c i n e .  B Insecticidal  Activity  E x t r a c t s of A a l a i a odorata f o l i a g e from T h a i l a n d were as e f f e c t i v e as neem o r c h i n a b e r r y l e a f e x t r a c t s a t the growth of £. s a u c i a neonates. phytochemical  inhibiting  The degree of  defense appeared t o v a r y between p o p u l a t i o n s  (or i n d i v i d u a l s ) of A., odorata, as samples from Taiwan  and  Hawaii were somewhat l e s s i n h i b i t o r y .  other  E x t r a c t s o f two  A a l a i a s p e c i e s , A,, o d o r a t i s s i m a and A., a r g e n t i a . were even l e s s a c t i v e , i n d i c a t i n g i n t e r s p e c i f i c v a r i a t i o n i n the p r o d u c t i o n of defense compound(s). v a r i a t i o n was  noted  Similar interspecific  i n the a c t i v i t y of A g l a i a seed e x t r a c t s  a g a i n s t Spodoptera f r u g i p e r d a ( M i k o l a j c z a k e t a l . ,  1987,  1989) . S e v e r a l secondary m e t a b o l i t e s were p r e s e n t i n A.. odorata f o l i a g e i n h i g h c o n c e n t r a t i o n s ; however none of them exhibited i n h i b i t o r y a c t i v i t y against E.  s a u c i a when t e s t e d  as pure compounds a t e c o l o g i c a l l y r e l e v a n t c o n c e n t r a t i o n s . Flavones  and f l a v o n o l s , e s p e c i a l l y those w i t h c a t e c h o l  153  s u b s t i t u t i o n s , are known t o i n h i b i t the growth of some Waiss g t a i . ,  phytophagous i n s e c t s (Chan e t a l . , 1978; E l l i g e r et a i - / and  Waage, 1986)  1980, and  1981;  Isman and  Duffey, 1982;  5-methoxy f l a v o n e s  flavonoids  However the 5-methoxy and  f l a v a n o n e s i s o l a t e d from h.  5-hydroxy  odorata were i n a c t i v e a g a i n s t  saucia at n a t u r a l l y occurring concentrations, pure compounds or as m i x t u r e s .  P.  when t e s t e d  as  (-)-Odorinol i s c y t o t o x i c  to  P-388 leukemia c e l l s j j i v i t r o and i n v i v o 1988), but none o f the  Hedin  have r e c e n t l y been  shown t o be more i n h i b i t o r y than 5-hydroxy (Mahoney e t a l . , 1989).  1979;  ai.,  (Hayashi g t  isomers o f o d o r i n e o r o d o r i n o l ,  whether n a t u r a l or s y n t h e t i c , had  any  e f f e c t on £.  saucia  growth. Dammarane aglycones have a p p a r e n t l y tested for a c t i v i t y against glycosides  not p r e v i o u s l y been  insect herbivores,  but dammarane  such as the saponins do i n h i b i t growth of some  i n s e c t s when p r e s e n t i n d i e t a t high c o n c e n t r a t i o n s  (1-5%  fwt)  the  (Applebaum and  ginsenocides, vertebrates,  apparently  as a r e s u l t of d i r e c t a c t i o n on  p i t u i t a r y r e s u l t i n g i n s t i m u l a t i o n of  glands ( S h i b a t a ,  t e s t e d i n d i v i d u a l l y and 1,000  Other g l y c o s i d e s ,  produce a v a r i e t y of p h y s i o l o g i c a l e f f e c t s i n  hypothalamus and adrenal  B i r k , 1979).  1986).  A g l a i o n d i o l and  i n h i b i t o r y to E.  the  aglaitriol,  as a mixture a t combinations up  ppm/component, were not  the  to  saucia  growth. I t appears t h a t the growth i n h i b i t o r y a c t i v i t y of A.. o d o r a t a may  be a s c r i b e d almost e n t i r e l y t o a s i n g l e compound  154  (Compound 6 ) .  T h i s c o n t r a s t s with most other p l a n t s , where  i n s e c t r e s i s t a n c e may  be a s c r i b e d t o s e v e r a l  compounds (McKey, 1979).  co-occurring  Feeding c h o i c e t e s t s i n d i c a t e d  t h a t the g r o w t h - i n h i b i t i n g a c t i v i t y of Compound 6 was due  t o a n t i f e e d a n t e f f e c t s and must r e f l e c t t o x i c i t y .  was  s i m i l a r t o the a c t i v i t y of some limonoids  cedrelone of IGR  and  anthothecol  (Chapter 4 ) .  not This  including  Although no  e f f e c t s on m o l t i n g were seen, the known IGR  evidence compound  a z a d i r a c h t i n a l s o f a i l s t o produce obvious e f f e c t s on m o l t i n g when f e d t o E . The  s a u c i a (Chapter 4 ) .  c o n c e n t r a t i o n o f Compound 6 i n the E t 0 e x t r a c t 2  c o u l d not be determined a c c u r a t e l y by HPLC, as i t cochromatographed w i t h the  3-hydroxy-5,7,4'-methoxyflavanone.  However, the i s o l a t e d y i e l d  (3 ng/g  l e a f dwt)  much l e s s than the expected c o n c e n t r a t i o n estimated  (98  appeared t o fig/g)  from the a c t i v i t y o f the crude MeOH e x t r a c t .  discrepancy  be  The  suggested the p o s s i b i l i t y of s y n e r g i s t i c  i n t e r a c t i o n s between phytochemical c o n s t i t u e n t s i n A.. odorata.  S e v e r a l s t u d i e s have now  i n d i c a t e d the  occurrance  o f a d d i t i v e o r s y n e r g i s t i c i n t e r a c t i o n s between phytochemicals i n o t h e r p l a n t s (Berenbaum, 1985); f o r i n s t a n c e an a r r a y of p h e n o l i c s i n sorghum s i g n i f i c a n t l y d e t e r r e d f e e d i n g by Locusta  m i g r a t o r i a . even though  i n d i v i d u a l compounds were not d e t e r r e n t  (Adams and  1971).  mixed i n p r o p o r t i o n s  Combinations of i s o b u t y l a m i d e s ,  Bernays,  s i m i l a r t o those o c c u r r i n g i n the p l a n t , were more t o x i c than would be expected from simple  a d d i t i o n of  the  155  a c t i v i t i e s o f t h e i n d i v i d u a l compounds (Miyakado e t a l . , 1989), and N-isobutylundecylenamide s y n e r g i s e s t h e a c t i v i t y of n a t u r a l p y r e t h r o i d s ( M e t c a l f , 1967; Matsui and Yamamoto, 1971).  In some p l a n t s , methylenedioxyphenyl  (MDP) compounds  may s y n e r g i s e t o x i c i t y by i n h i b i t i n g m i x e d - f u n c t i o n based o x i d a t i v e metabolism o f x e n o b i o t i c s i n the i n s e c t . example, t h e MDP compounds m y r i s t i c i n , s a f r o l e ,  For  isosafrole,  and fagaramide s y n e r g i s e t h e t o x i c i t y o f c o - o c c u r r i n g furanocoumarins i n p a r s n i p (Berenbaum and N e a l , 1985; N e a l , 1989).  However, when v a r i o u s combinations o f t h e A a l a i a  compounds here i s o l a t e d were t e s t e d f o r s y n e r g i s t i c i n t e r a c t i o n s , no combination was more a c t i v e than would be expected from t h e simple a d d i t i o n o f t h e a c t i v i t y o f t h e components i n i s o l a t i o n . be  The p u t a t i v e s y n e r g i s t remains t o  identified. Although t h e s t r u c t u r e o f t h e a c t i v e compound was not  determined, t h e pronounced  growth i n h i b i t o r y a c t i v i t y o f A..  odorata e x t r a c t s suggests t h a t they may have some a p p l i c a t i o n i n p e s t management, p a r t i c u l a r l y i n areas where the p l a n t o c c u r s n a t u r a l l y o r as a r e s u l t o f c u l t i v a t i o n . The phytochemical b a s i s o f t h i s a c t i v i t y r e q u i r e s f u r t h e r a t t e n t i o n ; t h e work p r e s e n t e d i n t h i s Chapter s h o u l d f a c i l i t a t e f u t u r e attempts t o i s o l a t e and i d e n t i f y t h e a c t i v e compound and e l u c i d a t e t h e p a r t i c i p a n t s i n t h e synergistic  interaction.  156  Chapter 4 : E f f e c t s o f Limonoids from the R u t a l e s  on  Peridroma s a u c i a and Oncopeltus f a s c i a t u s  Introduction  Limonoids are the most c h a r a c t e r i s t i c secondary i n the Meliaceae, (reviewed Over 300  and  metabolites  are a l s o prominent i n the Rutaceae  i n the I n t r o d u c t i o n t o t h i s t h e s i s , pg  16-27).  l i m o n o i d s t r u c t u r e s have been e l u c i d a t e d t o date  ( T a y l o r , 1987), but o n l y seventy such compounds have been examined f o r b i o l o g i c a l a c t i v i t y a g a i n s t i n s e c t s . summarizes the r e p o r t e d e f f e c t s of limonoids  Table  on i n s e c t  f e e d i n g and growth; s t r u c t u r e s f o r these compounds are i n F i g u r e 4-1.  given  Examination of t h i s c o m p i l a t i o n r e v e a l s  impediments t o d e v e l o p i n g  4-1  two  a q u a n t i t a t i v e understanding of  s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s amongst  limonoids.  F i r s t l y , very few o f the p u b l i s h e d s t u d i e s u t i l i z e the same bioassay  s p e c i e s ; as a r e s u l t i t i s d i f f i c u l t t o  separate  e f f e c t s owing t o s t r u c t u r a l d i f f e r e n c e s between compounds from e f f e c t s due  t o i n t e r s p e c i f i c d i f f e r e n c e s i n the  response of the t e s t i n s e c t s .  Secondly, the m a j o r i t y  s t u d i e s were designed t o d e t e c t f e e d i n g d e t e r r e n c e , not i n d i c a t e non-behavioral  and  effects including toxicity  i n s e c t growth r e g u l a t i n g (IGR)  effects.  or  function  For example, T a y l o r  commented t h a t "the limonoids  do  This s i t u a t i o n  r e f l e c t s the p r e v a i l i n g b e l i e f t h a t limonoids p r i m a r i l y as a n t i f e e d a n t s .  of  (1987)  seem t o be remarkably b e r e f t  157  o f p h y s i o l o g i c a l p r o p e r t i e s ; we f i n d i n g anything  have examined many without  beyond the c h a r a c t e r i s t i c b i t t e r t a s t e . . . " .  In the most comprehensive survey t o date, Kubo and (1987) assayed seventeen limonoids lepidopteran species. concentrations  against  Klocke  three  However, as they r e p o r t e d o n l y  EC  5 0  f o r growth i n h i b i t i o n , the r o l e s of  chemosensory ( a n t i f e e d a n t ) and p o s t - i n g e s t i v e e f f e c t s i n producing  the observed growth i n h i b i t i o n cannot  separated.  Despite  such l i m i t a t i o n s , Table 4-1  i n d i c a t i n g general q u a l i t a t i v e trends  be i s of use  in  in structure-activity  relations. The  b i o s y n t h e s i s and  e v o l u t i o n of the limonoids  been d e s c r i b e d r e c e n t l y by Das Biosynthesis apparently  et a i .  (1984, 1987).  proceeds along f o u r major pathways,  a l l dominated by t r e n d s of i n c r e a s i n g o x i d a t i o n and rearrangement ( F i g u r e 4-2). f o r the p r o d u c t i o n  I f the primary r a i s o n  of limonoids  skeletal d'etre  i s to gain protection  a g a i n s t i n s e c t h e r b i v o r y , one may evolutionary trends  has  expect t o f i n d t h a t  ( i . e . i n c r e a s i n g o x i d a t i o n and  rearrangement) correspond w i t h i n c r e a s i n g a c t i v i t y  skeletal against  insects. In the present  study I bioassayed  ten  limonoids,  i n c l u d i n g r e p r e s e n t a t i v e s of a l l the major c l a s s e s , f o r growth and  f e e d i n g i n h i b i t i o n a g a i n s t neonates of  v a r i e g a t e d cutworm, Peridroma s a u c i a , and molting  (IGR  milkweed bug,  e f f e c t s ) and r e p r o d u c t i o n Oncopeltus f a s c i a t u s .  f o r i n h i b i t i o n of  a g a i n s t the  The  the  large  r e s u l t s of these  158  assays,  and a c o m p i l a t i o n of the l i t e r a t u r e t o date,  a p p l i e d t o t e s t the h y p o t h e s i s  were  that there i s a c o r r e l a t i o n  between e v o l u t i o n a r y advancement o f t h e compounds (as d e f i n e d by Das e i a i . , 1984,1987) and t h e i r a c t i v i t y phytophagous i n s e c t s .  against  159  T a b l e 4-1.  E f f e c t s o f limonoids on i n s e c t f e e d i n g and growth.  Abbreviations  a r e : E C , c o n c e n t r a t i o n producing 5 0  50% growth  i n h i b i t i o n ; F l , f e e d i n g i n h i b i t i o n ; GI, growth i n h i b i t i o n ; MI, molt i n h i b i t i o n ; IGR, i n s e c t growth r e g u l a t o r ; MI, molt i n h i b i t i o n ; IA, i n a c t i v e .  Limonoid  Test Insect  Group 1.  E f f e c t i v e cone.  Ref.  Protolimonoids  Meliantriol Schistocerca gregaria  FI  1 0 0  = 8 ug/cm  2  1  Melianone Epilachna v a r i v e s t r i s Azedarachol A g r o t i s seietum  F l §0.05% FI  1 0 0  §500 PP  Group 2. I n t a c t apoeuphol s k e l e t o n  2 m  3  limonoids  Azadiron Epilacna v a r i v e s t i s  FI =0.66%  4  Azadiradione H e l i o t h i s zea Heliothis virescens Spodoptera f r u g i p e r d a Pectinophora g o s s y p i e l l a Epilacna varivestis  EC EC EC EC FI  5 6 5 5 4  14-Epoxyazadiradione Epilacna v a r i v e s t i s  FI =0.14%  4  7-Deacetylazadiradione H e l i o t h i s zea Heliothis virescens Spodoptera f r u g i p e r d a Pectinophora g o s s y p i e l l a  EC EC EC EC  5 6 5 5  50  50 50 50 50 50  =250 ppm =560 ppm =130 ppm =42 ppm =0.033%  50  50 50 50 50  =3500 ppm =1600 ppm =5000 ppm =290 ppm  7-Deacetyl-17 -hydroxyazadiradione Heliothis virescens EC =240 ppm 50  6  160  Sendanal H e l i o t h i s zea Heliothis virescens Spodoptera f r u g i p e r d a Pectinophora g o s s y p i e l l a  EC EC EC EC  Diacetyoxyvilasinine Epilachna v a r i v e s t r i s  FI  8  Cedrelone Ostrinia nubilalis Spodoptera l i t u r a Spodoptera f r u g i p e r d a Pectinophora g o s s y p i e l l a H e l i o t h i s zea  50 ppm F I , GI 0.1% FI EC o=2ppm EC Q=3ppm EC =8ppm  9 10 5 5 5  Anthothecol Ostrinia nubilalis Spodoptera f r u g i p e r d a Pectinophora g o s s y p i e l l a H e l i o t h i s zea  50 ppm F I , GI EC =3ppm EC o=8ppm EC =24ppm  9 5 5 5  Nimocinolide Aedes a e g y p t i  IGR, LC =0.625ppm  11  Isonimocinolide Aedes a e g y p t i  IGR, LC =0.74ppm  11  Sendanin H e l i o t h i s zea Heliothis virescens Spodoptera f r u g i p e r d a Pectinophora g o s s y p i e l l a  EC EC EC EC  ppm ppm ppm ppm  7 7 7 7  Trichirokanin H e l i o t h i s zea Heliothis virescens Spodoptera f r u g i p e r d a Pectinophora g o s s y p i e l l a  EC =41 ppm EC =50 ppm EC =11 ppm E C = 9 ppm  7 7 7 7  20 ppm FI  12  FI§ 300ppm  13  FI§ 200ppm  13  inactive  13  50 50 50  =400 ppm =400 ppm =70 ppm = 200 pp  5 0  5  5  50  50  5  50  50  50  50 50 50  =45 =60 =11 = 9  5 0  50  5Q  50  5 0  7 7 7 7  Toosendanin Ostrinia furnacalis Trichilin A Spodoptera e r i d a n i a Trichilin B Spodoptera e r i d a n i a Trichilin C Spodoptera e r i d a n i a  Trichilin D Spodoptera e r i d a n i a Meliatoxin A  FI§ 400ppm  13  F l , GI@ 300 ppm  14  GI@ 400 ppm  14  2  Spodoptera l i t u r a Meliatoxin Spodoptera l i t u r a  Group 3. D - r i n g seco l i m o n o i d s Gedunin Ostrinia nubilalis Epilachna v a r i v e s t i s Pectinophora g o s s y p i e l l a Spodoptera f r u g i p e r d a H e l i o t h i s zea  50 ppm, 50%FI § EC =32 EC o=47 EC =50 50  5  50  DS 0.1% ppm ppm ppm  6 4 5 5 5  7-Deacetylgedunin Spodoptera f r u g i p e r d a EC =60ppm Pectinophora g o s s y p i e l l a EC =22ppm H e l i o t h i s zea EC =165ppm  5 5 5  7-Ketogedunin Spodoptera f r u g i p e r d a EC =800ppm Pectinophora g o s s y p i e l l a EC o=51ppm H e l i o t h i s zea EC =900ppm  5 5 5  5o  50  50  50  5  50  Group 9. A.D-ring seco l i m o n o i d s Limonin Spodoptera l i t u r a Spodoptera f r u g i p e r d a Spodoptera f r u g i p e r d a H e l i o t h i s zea H e l i o t h i s zea Choristoneura fumiferana Spodoptera exempta Eldana s a c c h a r i n a Maruca t e s t u l a l i s  0.5% F l 50 756ppm PC =6.12ug/disk EC =900ppm PC c=60.8ug/disc IA @ l,000ppm IA § lOOug/disc 61%FI @l00ug/disc 58%FI § l O u d / d i s c  E C  =  95  50  9  5  10 7 5 7 15 16 16 16  162  Nomilin Spodoptera f r u g i p e r d a Spodoptera f r u g i p e r d a Spodoptera f r u g i p e r d a H e l i o t h i s zea H e l i o t h i s zea Trichoplusia n i Ostrinia nubilalis Earias insulana  50 PP PC =0.66ug/disk Fl EC =95ppm PC =6.6ug/disk IA F l § 50ppm ED =0.05%  E C  = 7 2  i n  95  50  95  50  5  7 17 5 7 17 9 18  Deacetylnomi1in Spodoptera f r u g i p e r d a IA § 2000ppm H e l i o t h i s zea IA § 2000ppm Pectinophora g o s s y p i e l l a EC =950ppni  5 5 5  Obacunone Ostrinia nubilalis Spodoptera exempta Eldana s a c c h a r i n a Maruca t e s t u l a l i s Spodoptera f r u g i p e r d a Spodoptera f r u g i p e r d a H e l i o t h i s zea H e l i o t h i s zea  50 ppm F l , GI 50%FI§100ug/disc 79%FI§1 u g / d i s c 61%FI@1 u g / d i s c EC =70ppm PC =0.60ug/disk EC =97ppm PC =6.5ug/disk  9 16 16 16 5 7 5 7  PC =125ug/disc  7  55%FI§100ug/disc 66%FI@100ug/disc 500 ppm GI IA § lOOug/disc  16 16 15 16  32%FI§100ug/disc 74%FI@ l u g / d i s c 69%FI§ l O u g / d i s c 20 ppm F l  16 16 16 19  50  50  50  50  95  Rutaevin Heliothis  zea  Citrolin Eldana s a c c h a r i n a Maruca t e s t u l a l i s Chorisoneura f u m i f e r a n a Spodoptera exempta Harrisonin Spodoptera exempta Eldana s a c c h a r i n a Maruca t e s t u l a l i s Spodoptera exempta  95  12 - A c e t o x y h a r r i s o n i n Spodoptera exempta Eldana s a c c h a r i n a Maruca t e s t u l a l i s Spodoptera exempta  ,500 ppm F l <50%FI@100ug/disc >75%FI§100ug/disc IA § lOOug/disc  Pedonin Eldana s a c c h a r i n a Maruca t e s t u l a l i s Spodoptera exempta  >75%FI@lug/disc >75%FI@10ug/disc <50%FI§100ug/disc  19,20,7 21 21 21 21 21 21  163  Groups 4,5. B D - r i n g f  seco  limonoids  Methylangolensate H e l i o t h i s zea ED =60 ppm Spodoptera f r u g i p e r d a ED =40 ppm Pectinophora g o s s y p i e l l a ED =15 ppm  5 5 5  50  50  50  Entandrophragmin Ostrinia nubilalis  FI§500 ppm,DS@50 ppm 9  Bussein Ostrinia nubilalis  FI§500 ppm  9  Methyl 3 - i s o b u t y r y l o x y - l - o x o m e l i a c - 8 ( 3 0 ) - e n a t e Spodoptera f r u g i p e r d a FI  Group 6. A - r i n g seco  22  limonoids  Evodulone H e l i o t h i s zea EC =80 ppm Spodoptera f r u g i p e r d a EC o=120 ppm Pectinophora g o s s y p i e l l a EC =96 ppm  5 5 5  Tecleanine Spodoptera f r u g i p e r d a EC =320 ppm Pectinophora g o s s y p i e l l a EC =210 ppm  5 5  7-Deacetylproceranone H e l i o t h i s zea EC =740 ppm Spodoptera f r u g i p e r d a EC =350 ppm Pectinophora g o s s y p i e l l a EC =175 ppm  5 5 5  50  5  50  50  50  50  50  50  Group 7. A B - r i n g seco f  limonoids  Prieurianin H e l i o t h i s zea Spodoptera f r u g i p e r d a Epilachna v a r i v e s t i s  60-90%FI§6ug/cm IA §19.8ug/cm 60-90%FI§19.8ug/cm  P r i e u r i a n i n acetate H e l i o t h i s zea Spodoptera f r u g i p e r d a Epilachna v a r i v e s t i s  60-90%FI § 6ug/cm 60-90%FI @19.8ug/cm 60-90%FI @1.5ug/cm  23 23 23  Rohitiukin H e l i o t h i s zea Spodoptera f r u g i p e r d a Epilachna v a r i v e s t i s  IA @19.8ug/cm IA §19.8ug/cm IA §19.8ug/cm  23 23 23  23 23 23  2  2  2  2  2  2  2 2 2  164  Rohitiuka-7 H e l i o t h i s zea Spodoptera f r u g i p e r d a Epilachna v a r i v e s t i s  "Tr—A"  60-90%FI ei9.8ug/cm IA §19.8ug/cm IA §19.8ug/cm 2  2  2  23 23 23  A g r o t i s sejetum  FI § 200 ppm  24  "Tr-B" A g r o t i s sejetum  FI @ 200 ppm  24  "Tr-C" Agrotis  FI @ 200 ppm  24  seietum  Group 10. B - r i n g seco  limonoids  Toonacilin Epilachna v a r i v e s t i s  FI  25  6-Acetyoxytoonaci1in Epilachna v a r i v e s t i s  FI  25  21-(R,S)-hydroxytoonacilid Epilachna v a r i v e s t i s  FI§  25-50 ppm  2 3-(R,S)-hydroxytoonac i 1 i d Epilachna v a r i v e s t i s  FI§  2000 ppm  Group 8. C - r i n g seco  limonoids  Azadirachtin Epilachna v a r i v e s t i s Epilachna v a r i v e s t i s H e l i o t h i s zea Spodoptera f r u g i p e r d a Pectinophora g o s s y p i e l l a  FI =0.0014% MI o=1.66 ppm EC =0.7 ppm EC =0.4 ppm EC =0.4 ppm  4 26 5 5 5  7-Acetylazadirachtin Rhodnius p r o l i x u s Rhodnius p r o l i x u s  MI =0.45ug/ml FI =30.Oug/ml  27 27  5o 5  50  50 50  50  50  22,23-dihydro-23B-methoxyazadirachtin Epilachna v a r i v e s t i s IGR 8 Mythimna s e p a r a t a FI,IGR@ 0.01%, 0.1/ig/larva 28 3 - T i g l o y l a z a d i r a c h t o l ( = A z a d i r a c h t i n B) ( = D e a c e t y l a z a d i r a c h t i n o l ) Epilachna v a r i v e s t i s MI Q=1.30 ppm 26 Heliothis virescens ED =0.17 ppm 5 5  50  165  l-Cinnamoyl-3-feruloyl-ll-hydroxymeliacarpin Epilachna v a r i v e s t i s IGR Epilachna v a r i v e s t i s MI =1.57 ppm 50  ( = A z a d i r a c h t i n D) 8 26  l-Cinnamoyl-3-feruloyl-ll-hydroxy-22,23-dihydro-23methoxymeliacarpin Epilachna v a r i v e s t i s IGR 8 l-Tigloyl-ll-methoxy-20-acetylmeliacarpinin Epilachna v a r i v e s t i s IGR  8  l-Tigloyl-3-acetyl-ll-methoxyazadirachtinin Epilachna v a r i v e s t i s IGR  8  Azadirachtin C Epilachna v a r i v e s t i s  MI =1 .57 ppm  26  Azadirachtin F Epilachna v a r i v e s t i s  MI =1 .57 ppm  26  Azadirachtin G Epilachna v a r i v e s t i s  MI =1 .57 ppm  26  Nimbinen Epilachna  FI  4  varivestis  50  50  50  = .018% 0  5 0  6-Deacetylnimbinen Epilachna v a r i v e s t i s  F I = 0 .0082%  4  Nimbandiol Epilachna  F I = 0 .01%  4  6-Acetylnimbandiol Epilachna v a r i v e s t i s  F I = 0 .011%  4  Salannin Spodoptera f r u q i p e r d a Spodoptera l i t t o r a l i s Earias insulana Acalymma v i t t a t a Musca domestica Epilachna v a r i v e s t i s Epilachna v a r i v e s t i s  50%FI@ 13ug/cm 0.01% F l 0.01% F l Fl 100%FI@0.1% FI =0.0082% no MI§ 100 ppm  3-Deacetylsalannin Epilachna v a r i v e s t i s  F I = 0 .0027%  4  Salannol Epilachna  Fl  8  varivestis  varivestis  Salannolacetate Epilachna v a r i v e s t i s  5 0  5 0  5 0  50  5 0  F I = 0 .00085% 5 0  2  29 30 30 31 32 4 26  4  166  Salannolactame-(21) Epilachna v a r i v e s t i s  FI  8  Salannolactame-(23) Epilachna v a r i v e s t i s  FI  8  Ochinolide B Heliothis virescens EC =1500 ppm Spodoptera f r u g i p e r d a EC =600 ppm Pectinophora g o s s y p i e l l a E C = 700 ppm  7 7 7  Ochinal H e l i o t h i s zea Heliothis virescens Spodoptera f r u g i p e r d a Pectinophora g o s s y p i e l l a  IA IA IA EC  7 7 7 7  Volkensin Spodoptera  50%FI@3.5ug/cm  50  50  5 0  frugiperda  V o l k e n s i n hydroxylactone Spodoptera f r u g i p e r d a  § 1000 ppm @ 1000 ppm § 1000 ppm = 1800 ppm  5 0  50%FI@6ug/cm  2  2  29 29  Unknown s t r u c t u r e Nkolbisonin H e l i o t h i s zea EC =71 ppm Spodoptera f r u g i p e r d a EC =65 ppm Pectinophora g o s s y p i e l l a EC =20 ppm 50  50  50  1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11) 12) 13) 14) 15) 16) 17) 18) 19) 20) 21) 22) 23)  L a v i e e t a l . , 1967. Kraus and Grimminger, 1980. Nakatani g t a l . , 198 Schwinger g t a l . , 1984. Kubo and Klocke, 1986. Lee g t a l . , 1988. Kubo and Klocke, 1981. Kraus g t a l . , 1987. Arnason g t a l . , 1987. K o u l , 1983. Naqui, 1987. Chiu and Zhang, 1984. Nakatani e t a l . , 1981. Koul g t a l . , unpublished data A l f o r d and B e n t l e y , 1986. H a s s a n a l i g t a l . , 1986. A l t i e r i g t a l . , 1984. Weissenburg g t al., 1986. Kubo e t a l - , 1976. L i u g t a l . , 1981. H a s s a n a l i and B e n t l e y , 1987. M i k o l a j c z a k g t a l . , 1988. L i d e r t g t a l « , 1985.  (1989).  5 5 5  167 24) 25) 26) 27) 28) 29) 30) 31) 32)  Nakatani e t a l . , 1984. Kraus e t a l . , 1978. Rembold, 1988. G a r c i a e t a l . , 1984. Sankaram e t a i - , 1987. Rajab e t a i . , 1988. Meisner e t a l . , 1981. Reed e t a l . , 1981. Warthen e t a l . , 1978.  168  Group 1: Proiolimoaoidi  Meliaaoae  Croup 2: Apo-Esphol LiaoBokU  H OH  7-DeaccryIaxadiradioM 7-Deac«yl-17hydrexjrazadindioM  OAc Azadiradione  >OJU  or  I  14-epoxyazadiradioaa  Orriunia  "A F>Ac  Srnriania  R - C O C H C H J C H J Trichirokaaia  Figure 4 - 1 .  12 - O H O Trichilia A 12 - O H O Trichilia B  Structures of limonoids included i n Table 3  169  170  Tecleanin 7-DeacetyIproceranone  Evodulone  OAe  CH CH Tr-A" CH Tr-C" 2  3  3  "O  o  Rohiiuka-7 H OAc  Toonacilin 6-AcetoxytoonaciIin  Ac H  Prierianin accute Prieriania  171  oco  Eniandrophragmin  172  OR R=Ac R=H R=Ac R=H  Nimbinen 6-Deacetylnimbinen Salannolactame-(21)  Salannin 3-Deacetylsalannin  O JO Sallanolactame-(23)  R=H Salannol R=Ac Salannolacetate  '—6  R = OH Volkensin R = O  Volkensin hydroxylactone  OTig Ochinolide B  R,0  R , 0 ^ > ^ > Ri  R2  Ac Ac Tig Fer Fer  Tig Tig H Cin Cin  R3 COOCH3 COOCH3 COOCH3  CH  CH3  3  X  " ' O H  R4  Rs  H 2H H H 2H  H Azadirachtin OCH3/H 3-Tigloylazadirachtol H l-Cinnamoyl-3-feruloy 1-11 -hydroxy meliacarpin H OCH3/H  173  F i g u r e 4-2. Major b i o s y n t h e t i c r o u t e s of limonoids i n the Meliaceae.  174  175  M a t e r i a l s and Methods  A. Sources o f Chemicals Cedrelone, a n t h o t h e c o l , gedunin, n o m i l i n ,  entandrophragmin,  and b u s s e i n were o b t a i n e d from Dr. J.T. Arnason, of Ottawa, Canada.  H a r r i s o n i n , obacunone, and pedonin,  e x t r a c t e d from H a r r i s o n i a a b y s s i n i c a 1986,  University  (Hassanali e t a l . ,  1987) were p r o v i d e d by Dr. A. H a s s a n a l i , ICIPE,  Nairobi.  The p u r i t y o f a l l compounds was a s s e s s e d by HPLC,  and compound i d e n t i t y and p u r i t y were a l s o confirmed by H 1  NMR (400 MHz).  S t r u c t u r e s o f a l l compounds a r e shown i n  F i g u r e 4-3. In many o f t h e experiments t h e a z a d i r a c h t i n used was i s o l a t e d by Dr. J . Kaminski, U n i v e r s i t y o f Ottawa. i n i t i a l experiments were done w i t h a z a d i r a c h t i n  However,  isolated  a c c o r d i n g t o an a d a p t a t i o n o f t h e methods o f Uebel g t a l (1979) and Yamasaki g t a_l. (1986).  Azadirachta indica o i l  (100 ml) ( a l s o s u p p l i e d by Dr. J.T. Arnason) was d i l u t e d t o 250 ml i n MeOH, d e f a t t e d w i t h t h r e e p a r t i t i o n i n g s a g a i n s t equal volumes o f hexane, and then e x t r a c t e d t h r e e times w i t h e q u i v a l e n t volumes o f E t 0 . 2  The E t 0 phase was c o n c e n t r a t e d 2  under vacuum, and chromatographed  i n 0.5 g l o t s by s p i n n i n g -  p l a t e p r e p a r a t i v e TLC (Chromatotron, Model 7924, H a r r i s o n Research, P a l o A l t o , C a l i f o r n i a ) u s i n g a 2mm p l a t e ( S i g e l 60 P F  2 5 4  , Merck).  The s o l v e n t system c o n s i s t e d o f 200 mis  each o f E t 0 , E t 0 : A c e t o n e 2  Et 0:Acetone 2  2  (95:5), E t 0 : A c e t o n e 2  (3:1),  (1:1), and f i n a l l y 100% MeOH; flow was 4 m i s /  176  Figure 4-3.  Structures  of l i m o n o i d s examined i n t h i s  study.  177  AZADIRACHTIN  BUSSEIN  OCOCHMe,  178  min.,  and 8 ml f r a c t i o n s were c o l l e c t e d .  F r a c t i o n s were  monitored f o r t h e presence of a z a d i r a c h t i n by a n a l y t i c a l HPLC ( V a r i a n Model 5000), u s i n g a MCH-10 5 x 250 mm column, MeOH:H 0 (1:1), flow = l ml/ min. 2  Peak d e t e c t i o n was  monitored a t 217 nm, u s i n g a V a r i a n S e r i e s 634 d e t e c t o r and a Spectra-Physics  SP4100 r e c o r d i n g  integrator.  Under these  c o n d i t i o n s t h e r e t e n t i o n time o f pure a z a d i r a c h t i n was 6.5 min.  F r a c t i o n s c o n t a i n i n g a z a d i r a c h t i n were pooled and  rechromatographed u s i n g a MCH-10 1.0 x 30 cm  semipreparative  column w i t h i s o c r a t i c ACHN:H 0 67:33, flow = 3 ml/ min; 6 ml 2  f r a c t i o n s were c o l l e c t e d . a z a d i r a c h t i n so o b t a i n e d  The i d e n t i t y and p u r i t y o f t h e  was e s t a b l i s h e d by c o -  chromatography ( a n a l y t i c a l HPLC) and H-NMR s p e c t r o s c o p y . 1  The low,  y i e l d o f a z a d i r a c h t i n from t h i s o i l sample was q u i t e about 1 mg/ 100 ml. S o l v e n t s were purchased from BDH and a l l except MeOH  were r e d i s t i l l e d p r o i r t o use.  HPLC grade s o l v e n t s were  degassed p r i o r t o u s e .  B. I n s e c t s Peridroma s a u c i a  (Lepidoptera:  Noctuidae) was o b t a i n e d  a l a b o r a t o r y colony maintained as d e s c r i b e d  from  previously  (Chapter 2 ) . Oncopeltus f a s c i a t u s (Hemiptera: Lygaeidae) nymphs were o b t a i n e d  from a colony maintained a t room  temperature on d r y seeds o f A s c l e p i a s s p e c i o s a . s u p p l i e d from a moist c o t t o n r o l l , as an o v i p o s i t i o n s i t e .  Water was  and c o t t o n was p r o v i d e d  179  C. Growth S t u d i e s For growth s t u d i e s , compounds i n methanol were added t o t h e d r y components o f t h e a r t i f i c i a l  diet  (Velvetbean  c a t e r p i l l a r d i e t , B i o s e r v e I n c . , Frenchtown, N.J. # 9682), the s o l v e n t was evaporated prepared  i n a fume hood, and t h e d i e t was  i n t h e u s u a l manner.  a z a d i r a c h t i n were assayed d i e t fwt.  A l l compounds  except  a t 0.01, 0.05, and 0.5 nmol/  q  Two grams o f d i e t and 10 neonate £ . s a u c i a were  p l a c e d i n each o f t h r e e 30 ml p l a s t i c cups p e r treatment; t h i s d e s i g n made t h e most e f f i c i e n t use o f s c a r c e s u p p l i e s of  most o f t h e compounds, and p r e l i m i n a r y experiments  i n d i c a t e d t h a t both l a r v a l growth and s u r v i v o r s h i p up t o seven days were independent o f l a r v a l d e n s i t y . b i o a s s a y was repeated t h r e e times except  The e n t i r e  i n t h e case o f  n o m i l i n , where l i m i t e d amounts o f compound allowed o n l y two replications.  Larvae were r e a r e d under 16L:8D and 26° C, i n  a c l e a r p l a s t i c box l i n e d w i t h moistened paper towels t o m a i n t a i n h i g h humidity. were counted and weighed. transformed  A f t e r seven days s u r v i v i n g l a r v a e L i v e l a r v a l weights were  log  1 0  t o c o r r e c t f o r h e t e r o s c e d a s t i c i t y before  a n a l y s i s by ANOVA and Tukey's S t u d e n t i z e d  (HSD) Range T e s t .  The e f f e c t o f a z a d i r a c h t i n on growth and s u r v i v a l o f neonate E - s a u c i a was assessed  i n two experiments.  In the  f i r s t , neonate E- s a u c i a were r e a r e d f o r seven days on artificial  d i e t c o n t a i n i n g 0.5, 1.5, 4.5, 15, o r 45 nmol/g  d i e t fwt (0.36, 1.08, 3.24, 10.8, and 32.4 fxq/q d i e t f w t ) .  180  C o n t r o l d i e t was t r e a t e d w i t h s o l v e n t were r e a r e d ,  (MeOH) alone.  Larvae  t h r e e t o a cup (10 cups/ treatment) i n 30 ml  cups w i t h 2 g fwt d i e t , as p r e v i o u s l y d e s c r i b e d .  Surviving  l a r v a e were counted and weighed a f t e r seven days. experiment was r e p l i c a t e d  The  t h r e e times; l a r v a l weights were  r e c a l c u l a t e d as % o f c o n t r o l and l o g l O transformed p r i o r t o a n a l y s i s by l i n e a r r e g r e s s i o n a g a i n s t l o g l O dose.  In t h e  second experiment, neonate l a r v a e (30 per treatment) were r e a r e d on d i e t c o n t a i n i n g a z a d i r a c h t i n / g fwt d i e t respectively).  0, 0.15, 0.5, o r 1.5 nmol  (0, 0.11, 0.36, o r 1.08  Conditions  ng/g  were t h e same as i n t h e f i r s t  experiment, except t h a t l a r v a e were r e a r e d  individually  a f t e r t h e f i r s t seven days, as they tend t o become cannibalistic  i n later instars.  Larvae were checked every  2-3 days and f r e s h d i e t was p r o v i d e d  ad l i b . S i x t h - i n s t a r  l a r v a e were t r a n s f e r r e d t o p l a s t i c cups c o n t a i n i n g moistened s o i l t o f a c i l i t a t e p u p a t i o n .  sterile  At t h i s point  larvae  were checked f o r p u p a t i o n every two days. Parameters measured i n c l u d e d % o f l a r v a e pupating, time t o p u p a t i o n , pupal weights, and % a d u l t emergence.  D. Feeding Assays Antifeedant test.  e f f e c t s were examined u s i n g a simple  P e t r i dishes  choice  (5 cm diameter) were marked i n t o  quadrants, and c o n t r o l and t r e a t e d d i e t cubes were p l a c e d i n alternating  quadrants.  Ten neonate £• s a u c i a were r e l e a s e d  i n t h e c e n t e r o f t h i s f e e d i n g arena; the d i s h e s were then  181  put  i n t o an opaque box  The  number of l a r v a e on the d i e t and  determined a f t e r 24 h. presence of f r a s s . 4.5  to eliminate phototactic  Feeding was  A z a d i r a c h t i n was  effects.  i n each quadrant confirmed by  the  t e s t e d a t 0.5,  1.5,  nmol/g d i e t fwt; other compounds were t e s t e d a t  /imol/ g d i e t , and  the assay was  replicated  and  0.5  s i x times.  Responses were compared t o a random d i s t r i b u t i o n of expected i n the absence of a n t i f e e d a n t  was  larvae,  e f f e c t s , using a  G-  test.  E. N u t r i t i o n a l  Analyses  Growth, consumption, and d i e t a r y use by t h i r d i n s t a r saucia larvae  ( s t a r t i n g weight, 11.2  treatment) were determined. (individually) 0.5,  or 1.5  mg,  n=20/  Larvae were allowed t o  feed  on about 1 g fwt of d i e t c o n t a i n i n g 0,  nmol a z a d i r a c h t i n / g d i e t fwt.  the l a r v a e , f r a s s , and to constant  + 0.9  £.  A f t e r three  uneaten d i e t were separated  weight a t 60° C.  and  I n i t i a l dry weights of  l a r v a e and d i e t s were c a l c u l a t e d from fwt/dwt r a t i o s by d e t e r m i n i n g the fwt/dwt r a t i o of 10 l a r v a e and a l i q u o t s a t the s t a r t o f the experiment. r a t e (RGRi) and  0.15, days dried  the derived  5 diet  R e l a t i v e growth  r e l a t i v e consumption r a t e (RCRi) were  c a l c u l a t e d r e l a t i v e t o the i n i t i a l r a t h e r than the mean weight of the l a r v a e , as t h i s measure i s independent of ECI  (Farrar et a l . ,  ingested  (ECI)  and  1989). digested  The  e f f i c i e n c y of c o n v e r s i o n  (ECD)  d i e t , and the  the of  approximate  182  digestibility  (AD), were c a l c u l a t e d  a c c o r d i n g t o Reese and  Beck (1976).  RGRi = ( F i n a l l a r v a l weight - I n i t i a l I n i t i a l l a r v a l weight  l a r v a l weight /D  RCRi = Weight d i e t consumed /D I n i t i a l l a r v a l weight  ECI  = ( F i n a l l a r v a l weight - i n i t i a l l a r v a l weight) X 100 Weight d i e t consumed  ECD  = ( F i n a l - i n i t i a l l a r v a l weight) X 100 (Weight d i e t consumed - f r a s s )  AD  = (Weight d i e t consumed - f r a s s ) X 100 Weight d i e t consumed  F. Molt I n h i b i t i o n  Assays  The e f f e c t o f l i m o n o i d s on m o l t i n g was a s s e s s e d by t o p i c a l l y a p p l y i n g compounds i n acetone t o t h e d o r s a l abdominal t e r g a e of staged (< 24 h) f i f t h i n s t a r Q_. f a s c i a t u s  nymphs.  compound was t e s t e d t w i c e (10 nymphs per r e p l i c a t e ) 25, and 50 pg/  nymph.  a t 10,  Cedrelone was t e s t e d t h r e e times a t  5, 10, 15, 25, and 50 ng/  nymph.  A z a d i r a c h t i n was assayed  a t 1, 2, 3, 5, 8, 10, 15, and 20 ng/nymph. application  Each  After  o f t h e t e s t compound, nymphs were maintained i n  10 cm diameter g l a s s p e t r i d i s h e s a t 27° C, 16L:8D, and p r o v i d e d w i t h d r y seeds o f A s c l e p i a s s p e c i o s a and a  183  moistened  cotton r o l l .  M o l t i n g was s c o r e d a c c o r d i n g t h e  f o l l o w i n g s c a l e : c a t e g o r y I was a normal molt, c a t e g o r y I I was a molt t o a deformed a d u l t (crumpled wings o r i n a b i l i t y to  completely shed the e x u v i a ) , category I I I was m o r t a l i t y  d u r i n g a molt attempt, initiating  and c a t e g o r y IV was death  a molt attempt.  without  Responses were compared by  p r o b i t a n a l y s i s (SAS, 1988) w i t h c a t e g o r i e s I and I I pooled as s u r v i v o r s and c a t e g o r i e s I I I and IV pooled as mortalities.  F o l l o w i n g t h e molt, a d u l t s were maintained as  d e s c r i b e d f o r the nymphs, but w i t h c o t t o n as an o v i p o s i t i o n site,  f o r a f u r t h e r two weeks and the occurrence o f mating  b e h a v i o r , eggs, and neonates was r e c o r d e d .  G. C o r r e l a t i o n Between E v o l u t i o n a r y Advancement and A c t i v i t y Against Insects EC  5 0  v a l u e s ( i n /imol/g d i e t fwt) o f a l l limonoids t e s t e d  here were c o r r e l a t e d w i t h measurements o f m o l e c u l a r o x i d a t i o n ( O ) and rearrangement ( S ) g i v e n by Das e t a l . (1984, 1987).  The same a n a l y s i s was a l s o a p p l i e d t o E C  5 0  v a l u e s f o r 17 l i m o n o i d s and t h r e e l e p i d o p t e r a n s p e c i e s g i v e n by Kubo and Klocke  (1987).  To e l i m i n a t e E C  5 0  differences  due t o d i f f e r e n c e s i n the m o l e c u l a r weight o f compounds, a l l EC  5 0  v a l u e s were converted from ppm t o /imol/g.  184  Results  A. Growth and  Feeding S t u d i e s : Limonoids Other than  Azadirachtin Of the compounds t e s t e d , other than a z a d i r a c h t i n , and  cedrelone  anthothecol  were the most i n h i b i t o r y t o neonate E-  s a u c i a growth.  Both compounds reduced growth t o about  of c o n t r o l growth when i n c o r p o r a t e d d i e t fwt 0.05  (Table 4-2);  /xmol/g.  The  n e i t h e r compound had  any  effect  /mol/g at  ( C o n t r o l l a r v a e mean weights were 45-70 mg  a f t e r seven days.) rtmol/g.  i n t o d i e t a t 0.5  10%  S u r v i v o r s h i p was  growth i n h i b i t i o n was  not a f f e c t e d a t  apparently  not due  0.5 to a  chemosensory a n t i f e e d a n t e f f e c t as n e i t h e r compound s i g n i f i c a n t l y i n f l u e n c e d neonate d i e t c h o i c e i n a 24 h assay (Table  4-2).  Gedunin, s i m i l a r t o cedrelone epoxylactone D r i n g , had or f e e d i n g a t 0.5  except f o r an  no e f f e c t on growth, s u r v i v o r s h i p ,  /imol/g.  The A,D-seco limonoids  obacunone  and n o m i l i n a l s o d i d not a f f e c t d i e t c h o i c e or growth of Esaucia.  H a r r i s o n i n had  an a n t i f e e d a n t e f f e c t  against  neonate l a r v a e , but t h i s must have been temporary as  the  l a r v a l weights d i d not d i f f e r s i g n i f i c a n t l y from the c o n t r o l s a t the end of seven days of growth. s p i r o l a c t o n e compound pedonin was  The  not a n t i f e e d a n t  related and  s t i m u l a t e d growth, as the l a r v a e weighed s i g n i f i c a n t l y more than the c o n t r o l s by day  seven.  185 T a b l e 4-2. E f f e c t o f limonoids on growth and d i e t c h o i c e of neonate Peridroma s a u c i a . A l l compounds were a d m i n i s t e r e d a t 0.5 /xraol/g d i e t fwt; c o n c e n t r a t i o n s i n ppm a r e a l s o g i v e n . L a r v a l growth i s g i v e n as % o f c o n t r o l growth; numbers f o l l o w e d by t h e same l e t t e r a r e not s i g n i f i c a n t l y d i f f e r e n t (Tukey's S t u d e n t i z e d [HSD] Range T e s t ) . In t h e c h o i c e t e s t s , l a r v a l d i s t r i b u t i o n i s compared t o a n u l l h y p o t h e s i s d i s t r i b u t i o n o f 50:50 u s i n g a Gt e s t ; d i s t r i b u t i o n s d i f f e r i n g from 50:50 a r e i n d i c a t e d by an asterisk.  Compound  Cone.  Growth (% o f C o n t r o l )  (ppm)  (mean +  S.D.)  Percentage o f l a r v a e on C o n t r o l (C) o r T r e a t e d (T) d i e t . C T  Cedrelone  211  10.8 +  3.2  d  57  43  Anthothecol  248  12.9 +  7.5  d  57  43  Harrisonin  259  89.2 + 22.6  b  80  20*  Obacunone  227  110.8 + 11. 8  b  61  39  Nomilin  265  118.3 + 18.3  63  37  Gedunin  249  96.8 + 23.7  43  57  Pedonin  250  63  37  Entandrophragmin  416  66  34  67  33  Bussein  420  133.3 +  b  8.6  96.8 + 18.3  b  a  b  64.5 + 19.5°-  186  Of the B,D-seco l i m o n o i d s t e s t e d , entandrophragmin d i d not a f f e c t P_. s a u c i a f e e d i n g or growth a t 0.5 fwt.  Bussein s i g n i f i c a n t l y reduced  of the c o n t r o l s ) , than was  jzmol/g d i e t  l a r v a l growth ( t o  although t h i s e f f e c t was  64.5%  much l e s s marked  the case w i t h c e d r e l o n e o r a n t h o t h e c o l .  Bussein  d i d not s i g n i f i c a n t l y d e t e r f e e d i n g by neonates i n the choice t e s t .  B. Growth S t u d i e s w i t h A z a d i r a c h t i n Of the l i m o n o i d s examined i n t h i s study, a z a d i r a c h t i n produced  the g r e a t e s t i n h i b i t i o n of growth and s u r v i v o r s h i p ;  as a r e s u l t i t s e f f e c t on E .  s a u c i a growth was  more d e t a i l than were o t h e r compounds.  examined i n  In the seven-day  assay, both growth and s u r v i v o r s h i p were reduced dependent manner ( F i g u r e 4-7); t h e E C fwt (0.29 M9/g fwt (3.7 /ig/g).  d i e t fwt) and the L C  5 0  was  was  5 0  0.4  5.2  i n a dosenmol/g d i e t  nmol/g d i e t  Although a few l a r v a e were seen w i t h  s l i p p e d head c a p s u l e s o r a h a l f - s h e d band of c u t i c l e , i n most cases m o r t a l i t y was  not o b v i o u s l y due t o molt f a i l u r e .  These e f f e c t s c o n t i n u e d throughout the i n s e c t .  the l i f e c y c l e of  Regression a n a l y s i s i n d i c a t e d a s i g n i f i c a n t  n e g a t i v e e f f e c t o f a z a d i r a c h t i n ( a t 0.15, d i e t fwt) on the % of l a r v a e pupating  and 1.5  (r =0.94, p< 2  and on pupal weight (r =0.94, p< 0.01) 2  0.5  (Table 4-3).  nmol/g, o n l y 13% o f the l a r v a e pupated; o n l y one s u r v i v e d t o pupation a t 1.5  nmol/g.  nmol/g  0.01), At  larva  Pupal weight a t the  0.5  Figure 4 - 4 . E f f e c t o f d i e t a r y a z a d i r a c h t i n on growth and s u r v i v o r s h i p of Peridroma s a u c i a neonates. Growth and s u r v i v o r s h i p were determined a f t e r seven days o f f e e d i n g on a z a d i r a c h t i n - t r e a t e d d i e t ; each p o i n t r e p r e s e n t s the mean o f t h r e e r e p l i c a t i o n s , n o r m a l i z e d t o % of c o n t r o l .  Azadirachtin Cone (nmot/g diet fwt)  189 Table 4 - 3 . Effect of azadirachtin on Peridroma saucia pupation and adult emergence. Concentration (nmol/g d i e t ) Control 0.15 0.5 1^  % Pupation 75 60 13 3  Time to Pupation 24.8 ± 1.9 26.3 ± 3.2 30.2 ± 4.8 22  Pupal wgt (mg) 319.9 +44.1 300.8 39.9 282.3 ± 42.1 25Q.7 T  % Adult Emergence 75 40 0 Q  190  h i g h e r dose was was  increased  78%  of the c o n t r o l s .  0.05),  2  l a r v a e f e d d i e t w i t h 0.5  1.5  time t o p u p a t i o n  i n a dose-dependent manner (r =0.83, p<  w i t h c o n t r o l s r e q u i r i n g 24.8  days.  The  ± 1.9  days f o r development,  nmol/g a z a d i r a c h t i n r e q u i r i n g  30.2  None of the l a r v a e r e a r e d on d i e t c o n t a i n i n g 0.5  nmol a z a d i r a c h t i n / g completed development t o the  stage;  a d u l t emergence was  o n l y 40%  a t 0.15  and  or  adult  nmol  a z a d i r a c h t i n / g d i e t , compared t o 69% emergence f o r the controls.  The  data i n d i c a t e an E C  5 0  f o r pupation o f  0.16  nmol/g.  C. Feeding Choice T e s t s w i t h A z a d i r a c h t i n In c h o i c e t e s t s , a z a d i r a c h t i n i n h i b i t e d f e e d i n g neonate P.. s a u c i a  (Table 4-4).  A f t e r o n l y one  by  hour t h e r e  were s i g n i f i c a n t l y more l a r v a e on c o n t r o l d i e t than on d i e t t r e a t e d w i t h 1.5  o r 4.5  nmol a z a d i r a c h t i n / g fwt.  neonates were s i g n i f i c a n t l y d e t e r r e d by 0.5  A f t e r 24  h  nmol  a z a d i r a c h t i n / g d i e t as w e l l . As the s e n s i t i v i t y of l a r v a e t o a n t i f e e d a n t s t o d e c l i n e w i t h age Duffey, 1982;  Isman e t a l . ,  repeated w i t h 6 day which had  i n some cases (Reese, 1977;  i s known  Isman  and  1989), these c h o i c e t e s t s were  o l d second i n s t a r P. s a u c i a  larvae,  not p r e v i o u s l y been exposed t o a z a d i r a c h t i n .  A f t e r 24 h, l a r v a e were d e t e r r e d by a z a d i r a c h t i n o n l y a t 4.5  nmol/g l e v e l ; a t 1.5  nmol/g t h e r e was  e i t h e r t r e a t e d or c o n t r o l d i e t .  no p r e f e r e n c e  the for  191 T a b l e 4-4. E f f e c t o f a z a d i r a c h t i n on d i e t c h o i c e by neonate and s i x day o l d , t h i r d i n s t a r Peridroma s a u c i a . Values which d i f f e r from a 50:50 d i s t r i b u t i o n (G-test) a r e i n d i c a t e d w i t h an a s t e r i s k . Azadirachtin concentration fnmol/a d i e t fwt) 0 0.15 0.5 1.5 4.5  % of l a r v a e on c o n t r o l [C] o r t r e a t e d [T] d i e t a f t e r 24 h Neonates Third Instar _C T_ C T 50 50 53 47 36 64 59 41 70 30* 89 11* 47 53 86 14* 72 28*  192  D.  Diet U t i l i z a t i o n  Experiments  In a d e t a i l e d study of growth, f e e d i n g , and  dietary  u t i l i z a t i o n , a z a d i r a c h t i n reduced the r e l a t i v e growth r a t e (RGRi) of t h i r d - i n s t a r p_. s a u c i a i n a dose-dependent manner (r =0.90, p<0.01) (Table 4-5).  T h i s e f f e c t was  2  owing t o a dose-dependent decrease i n the consumption r a t e (RCRi) (r =.85, p<0.05). 2  e f f i c i e n c y of c o n v e r s i o n and  ECD  of i n g e s t e d  largely  relative E f f e c t s on  and d i g e s t e d food  (ECI  r e s p e c t i v e l y ) were not s i g n i f i c a n t l y c o r r e l a t e d w i t h  a z a d i r a c h t i n dose; subsequent ANOVA and a n a l y s i s (Tukey's S t u d e n t i z e d  means-comparison  Range T e s t ) i n d i c a t e d t h a t  both i n d i c e s were s i g n i f i c a n t l y lower than the c o n t r o l s o n l y a t the h i g h e s t a z a d i r a c h t i n dose t e s t e d . digestibility  (AD)  was  approximate  a l s o not c o r r e l a t e d w i t h  a z a d i r a c h t i n dose, but was c o n t r o l a t 1.5  The  the  s i g n i f i c a n t l y h i g h e r than  the  nmol a z a d i r a c h t i n / g .  A n a l y s i s of the r e l a t i o n s h i p between RCRi and  RGRi  showed t h a t a l l treatment groups f i t a r e g r e s s i o n l i n e of RGRi = 3.244*RCRi + 4.450  r =0.85, df=2, p<.05 2  although t h e r e were between-treatment d i f f e r e n c e s i n the maximum v a l u e s  f o r RCRi ( F i g u r e 4-8).  i n d i c a t e t h a t , i n P. i s due  to reduction  (cf. B l a u e t a l . ,  These r e s u l t s  s a u c i a , the observed growth i n h i b i t i o n i n f e e d i n g and  1978).  not t o d i r e c t t o x i c i t y  193 Table 4-5. E f f e c t o f a z a d i r a c h t i n on t h i r d i n s t a r Peridroma s a u c i a growth and n u t r i t i o n . Means i n a column w i t h t h e same l e t t e r a r e not s i g n i f i c a n t l y d i f f e r e n t (Tukey's S t u d e n t i z e d Range (HSD) T e s t , a=0.05). An a s t e r i s k f o l l o w i n g a column heading i n d i c a t e s t h a t index has a s i g n i f i c a n t o v e r a l l negative r e l a t i o n s h i p with a z a d i r a c h t i n dose. Treat RGRi* RCRi* ECI ECD AD Control 2.90 10.53 27.65 59.16 46.74 0.15 2.42 10.20 23.74 56.65 41.91 0.5 1.44 5.77 24.89 60.77 40.96 1.5 0.45 3.76 12.08^ 20.13^ 60.00^ Treat=treatment (/ig/g i n s e c t fwt), RGRi=Relative Growth Rate based on i n i t i a l wgt, RCRi=Relative Consumption Rate based on i n i t i a l wgt, E C I = E f f i c i e n c y of Conversion of Ingested Food, E C D = E f f i c i e n c y of Conversion o f Digested Food, and AD=Approximate Digestibility. Values were c a l c u l a t e d a c c o r d i n g t o Reese and Beck (1976) except f o r t h e RGRi and RCRi, which were c a l c u l a t e d a c c o r d i n g t o F a r r a r e_£ a l . (1989). a  a  a  a  a  a  a  a  a  194 F i g u r e 4-5. P l o t o f RGR a g a i n s t RCR f o r l a r v a e o f Peridroma saucia f e d d i e t containing various concentrations of azadirachtin. F o r c l a r i t y , o n l y every second data p o i n t i s p l o t e d f o r each treatment. A l l treatments do not d i f f e r s i g n i f i c a n t l y from t h e r e g r e s s i o n e q u a t i o n : RGRi = 3.244*RCRi + 4.450  r  2  = 0.85 p<0.05  20  15 -  tr o  a  Control  •  0.5 nmol/g  •  1.5 nmol/g  •  4.5 nmol/.g  10  r— 10  —i— 20  RCRI  —I—  30  40  196  E. Molt I n h i b i t i o n  Assays  At 50 ug/nymph, o n l y a z a d i r a c h t i n and c e d r e l o n e had e f f e c t on m o l t i n g o f f i f t h  i n s t a r O.  fasciatus.  any  Tests with  c e d r e l o n e were r e p e a t e d a t 5, 10, 15, 25, and 50  ^g  cedrelone/nymph; these i n d i c a t e d a dose-dependent range of e f f e c t s which were d i v i d e d i n t o response c a t e g o r i e s as described previously.  Cedrelone doses as low as  /ig/nymph had a marked e f f e c t on m o l t i n g success  five ( F i g u r e 4-  5 ) ; a t t h i s dose most nymphs molted t o a d u l t s w i t h c u r l e d wings ( c a t e g o r y I I ) , but some d i e d a f t e r i n i t i a t i n g e c d y s i s ( c a t e g o r y I I I ) . At 10 and 15 pg cedrelone/nymph,  an  i n c r e a s i n g p r o p o r t i o n of the t r e a t e d nymphs showed c a t e g o r y I I I and IV responses; a t 25 /ig/nymph, a l l responses i n t o these two nymphs).  fell  c a t e g o r i e s (each a c c o u n t i n g f o r 50% o f the  At the h i g h e s t dose t e s t e d , 50 Mg/nymph, 70% o f  the nymphs d i e d without m o l t i n g , and molt attempt.  30% d i e d i n a f a i l e d  I f a l l c a t e g o r y I and I I responses a r e pooled  as s u r v i v o r s , and a l l c a t e g o r y I I I and IV responses  are  c o n s i d e r e d as m o r t a l i t i e s , the MD  molt  50  (dose producing  i n h i b i t i o n i n 50% o f the t r e a t e d nymphs) o f t o p i c a l l y a p p l i e d c e d r e l o n e was 9.29-16.09  12.24  ng/nywph  (95% f i d u c i a l  limits  jig/nymph).  A z a d i r a c h t i n was  about f i v e thousand  than c e d r e l o n e i n the milkweed bug assay the same spectrum  of e f f e c t s was  times more a c t i v e ( F i g u r e 4-6),  produced.  but  Even a t 1  ng/nymph, most l a r v a e showed a c a t e g o r y I I response,  and a  197 F i g u r e 4-6. E f f e c t o f c e d r e l o n e on m o l t i n g success i n Oncopeltus f a s c i a t u s . Responses were s c o r e d as: Category I, normal molt t o an undeformed a d u l t ; Category I I , s u c e s s f u l molt t o a deformed a d u l t ( c u r l e d wings o r l e g s ) ; Category I I I , death d u r i n g a f a i l e d molt attempt; Category IV, death without i n i t i a t i n g ecdysis.  Category I  HI  Category Ii  III  Category IV  Category III  % of treated population in category  tegjamwmw III 11  III III11 11  Control  if  1  25 cedrelone dose ug/nymph  sit ;M  50  199 Figure 4-7. E f f e c t o f a z a d i r a c h t i n on m o l t i n g s u c c e s s i n Oncopeltus f a s c i a t u s . Responses were s c o r e d a s : Category I, normal molt t o an undeformed a d u l t ; Category I I , s u c e s s f u l molt t o a deformed a d u l t ( c u r l e d wings o r l e g s ) ; Category I I I , death d u r i n g a f a i l e d molt attempt; Category IV, death without i n i t i a t i n g 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  Azadirachtin dose ng/ nymph  100  500 N5 O O  201  few d i e d with and without i n i t i a t i n g e c d y s i s . /xg/nymph, no normal molts were recorded,  At  10  and most nymphs  d i e d without i n i t i a t i n g e c d y s i s . Again pooling; category and  I I responses, the M D  of a z a d i r a c h t i n was  50  3.0  I  ng/nymph.  To check a g a i n s t the p o s s i b i l i t y t h a t the r e s u l t s of the assays with c e d r e l o n e the cedrelone  c o u l d be due  t o c o n t a m i n a t i o n of  sample with t r a c e s of a z a d i r a c h t i n , I examined  the p u r i t y of the cedrelone  sample by HPLC and NMR.  Neither  t e c h n i q u e i n d i c a t e d the presence of a z a d i r a c h t i n or contaminants i n the cedrelone A d u l t s obtained maintained f o r two  sample.  from the molt i n h i b i t i o n assays were  weeks t o check f o r p o s s i b l e long-term  e f f e c t s on f e c u n d i t y and t r e a t e d w i t h cedrelone  fertility.  and  m o l t i n g without mating. began mating 3-4  other  A d u l t s which had  been  a z a d i r a c h t i n d i e d w i t h i n 48 h of  A d u l t s from a l l other  days a f t e r m o l t i n g ,  treatments  and by the end o f  the  o b s e r v a t i o n p e r i o d a l l treatments had produced v i a b l e eggs, i n d i c a t e d by the presence of f i r s t - i n s t a r nymphs i n the r e a r i n g arenas.  F. R e l a t i o n s h i p of A n t i - i n s e c t A c t i v i t y t o O x i d a t i o n  and  S k e l e t a l Rearrangement No r e l a t i o n s h i p was  found between the degree of b i o l o g i c a l  a c t i v i t y of the limonoids  studied herein  ( i n terms of growth  i n h i b i t i o n a g a i n s t £. s a u c i a ) and the degree of o x i d a t i o n or rearrangement of the o r i g i n a l apo-euphol s k e l e t o n , d e f i n e d by Das g t a l (1984).  as  However, o n l y f o u r compounds  202  e x h i b i t e d a p p r e c i a b l e b i o a c t i v i t y , an inadequate sample f o r comparison.  The a n a l y s i s was t h e r e f o r e extended t o the  comparative E C  5 0  d a t a f o r seventeen l i m o n o i d s and t h r e e  l e p i d o p t e r a n s p u b l i s h e d by Kubo and Klocke (1986). v a l u e s were r e c a l c u l a t e d i n terms o f nmol/g  A l l EC  5 0  d i e t before  comparison by r e g r e s s i o n a n a l y s i s t o measurements o f o x i d a t i o n and s k e l e t a l rearrangement (1984).  g i v e n by Das e t a l  Separate r e g r e s s i o n s were performed u s i n g E C  from each l e p i d o p t e r a n s p e c i e s . None o f these  5 0  data  comparisons  i n d i c a t e d a s i g n i f i c a n t r e l a t i o n s h i p between the growth i n h i b i t o r y a c t i v i t y o f l i m o n o i d s and the degree o f m o l e c u l a r o x i d a t i o n and rearrangement  ( F i g u r e 4-9); indeed the most  a c t i v e l i m o n o i d s were those w i t h the most ( a z a d i r a c h t i n ) o r the l e a s t ( c e d r e l o n e ) d e r i v a t i o n from the o r i g i n a l apoeuphol s k e l e t o n . rearrangement  Compounds w i t h i n t e r m e d i a t e degrees o f  and o x i d a t i o n appeared t o be the l e a s t  active.  203  F i g u r e 4-8. Comparison of i n s e c t growth i n h i b i t i n g a c t i v i t y of l i m o n o i d s w i t h measurements of o x i d a t i o n and s k e l e t a l rearrangement. The v a l u e s f o r S and O used are from Das e t a l (1984); the v a l u e s f o r i n s e c t growth i n h i b i t i o n are E D data f o r Spodoptera f r u g i p e r d a , g i v e n by Kubo and Klocke (1987), c o r r e c t e d t o /imol/g d i e t and l o g transformed. N e i t h e r S nor O are s i g n i f i c a n t l y r e l a t e d t o the i n h i b i t o r y a c t i v i t y of the limonoids. 5 0  Log ED50  (S. frugiperda)  Log ED50  (S.  frugiperda)  205  Discussion  For purposes of d i s c u s s i o n , compounds are grouped a c c o r d i n g t o s k e l e t a l c l a s s as g i v e n i n F i g .  4-2.  A. Group 2 Limonoids Cedrelone  and a n t h o t h e c o l are simple l i m o n o i d s which  r e t a i n an i n t a c t apoeuphol s k e l e t o n ( F i g . 4-3). occurs i n a v a r i e t y  of Meliaceae  including  Cedrelone  s p e c i e s of  C e d r e l a , C h u c k r a s i a , Khaya, and Toona; a n t h o t h e c o l d i f f e r s o n l y i n p o s s e s s i n g an a c e t a t e s u b s t i t u t i o n  a t C l l of the C  r i n g , and i s found i n s p e c i e s of Khaya (Das e t a l . , 1984). N e i t h e r compound a f f e c t e d d i e t , but a t 0.5 reduced  /imol/ g d i e t both c e d r e l o n e and  growth by almost  e f f e c t was  £. s a u c i a growth a t 0.05  /imol/ g anthothecol  90% compared t o the c o n t r o l s .  This  e v i d e n t l y not due t o a chemosensory a n t i f e e d a n t  e f f e c t as n e i t h e r compound had any e f f e c t on d i e t c h o i c e by neonate E .  s a u c i a i n the a n t i f e e d a n t assays.  Cedrelone  and a n t h o t h e c o l were a l s o h i g h l y e f f e c t i v e  growth i n h i b i t o r s a g a i n s t the European corn b o r e r , nubilalis  (Arnason  e t ajL. , 1987).  f e e d i n g i n h i b i t i o n a t h i g h doses, l i m o n o i d s reduced  Ostrinia  This a c t i v i t y included 50 and 500 ppm.  Both  growth by r e d u c i n g the e f f i c i e n c y of  c o n v e r s i o n of i n g e s t e d and d i g e s t e d d i e t a t lower doses a t which consumption was  actually  r e s u l t which p a r a l l e l s my  enhanced (10 and 30 ppm),  a  o b s e r v a t i o n of growth i n h i b i t i o n  206  without a n t i f e e d a n t e f f e c t s i n E- s a u c i a .  In another study,  c e d r e l o n e and a n t h o t h e c o l were the most i n h i b i t o r y o f 17 l i m o n o i d s (other than a z a d i r a c h t i n ) , r e d u c i n g the growth of t h r e e l e p i d o p t e r a n s , Spodoptera f r u g i p e r d a . H e l i o t h i s zea, and P e c t i n o p h o r a g o s s y p i e l l a  (Kubo and K l o c k e , 1986).  Cedrelone a l s o had growth i n h i b i t o r y a c t i v i t y Spodoptera  l i t u r a a t 0.1%;  against  i n t h i s case the observed  i n h i b i t i o n c o u l d be e n t i r e l y a s c r i b e d t o reduced consumption,  due e i t h e r t o b e h a v i o r a l o r p h y s i o l o g i c a l  e f f e c t s (Koul, 1983).  R.  s a u c i a appears t o be  relatively  i n s e n s i t i v e t o c e d r e l o n e and a n t h o t h e c o l , as growth i n h i b i t i o n o c c u r r e d o n l y a t doses 100 times the E C  5 0  levels  r e p o r t e d by Kubo and Klocke (1986) and 10 times the i n h i b i t o r y concentrations f o r Ostrinia n u b i l a l i s  (Arnason e t  a i . , 1987). A marked d i f f e r e n c e was  noted i n the response o f 0.  f a s c i a t u s nymphs t o the two compounds.  Cedrelone a f f e c t e d  m o l t i n g a t doses as low as 5 ug/nymph, and had an L D 16.4  ug/nymph.  5 0  of  Nymphs which molted s u c c e s s f u l l y a t the  lower doses a l l e x h i b i t e d d e f o r m i t i e s o f the wings and d i e d w i t h i n 48 h of m o l t i n g , without mating.  These e f f e c t s were  s i m i l a r t o those r e s u l t i n g from much lower doses o f azadirachtin (LD  5 0  = 3.0 ng/nymph).  Although c e d r e l o n e was  not observed t o i n h i b i t the m o l t i n g of e i t h e r P. ( t h i s study) o r o f O. n u b i l a l i s  saucia  (Arnason e t a i . , 1987),  Spodoptera f r u g i p e r d a , o r H e l i o t h i s zea (Kubo and K l o c k e , 1986), i t has been r e p o r t e d t o i n h i b i t e c d y s i s i n the p i n k  207  bollworm Pectinophora and Klocke,  1986).  g o s s y p i e l l a a t 150 ppm i n d i e t  (Kubo  The a c e t a t e s u b s t i t u t i o n a t C l l o f t h e C  r i n g a b o l i s h e d t h e m o l t - i n h i b i t i n g a c t i v i t y , as nymphs t r e a t e d t o p i c a l l y w i t h anthothecol  doses up t o 50 ug/nymph  molted t o undeformed a d u l t s which susequently  mated and  produced v i a b l e eggs. The  nature o f s u b s t i t u e n t s a t C12 o f t h e C r i n g a r e  known t o markedly a f f e c t t h e a n t i f e e d a n t a c t i v i t y o f limonoids.  F o r example, t h e a n t i f e e d a n t a c t i v i t y o f  v i l a s i n i n e d e r i v a t i v e s i s reduced by s u b s t i t u e n t s a t C12 (Pohnl,  1985).  S i m i l a r l y , a c e t y l a t i o n a t C12 reduces t h e  a n t i f e e d a n t a c t i v i t y o f h a r r i s o n i n 2 5 - f o l d (Kubo e t a l . , 1976).  T r i c h i l i n s r e q u i r e an OH s u b s t i t u t i o n a t C12;  a l t e r a t i o n t o a ketone o r a c e t a t e markedly reduce t h e antifeedant a c t i v i t y  (Nakatani  gt a l . , 1981)  The r e s u l t s  r e p o r t e d here suggest t h a t s u b s t i t u t i o n s a t t h e C l l p o s i t i o n may a l s o be important i n determining  IGR a c t i v i t y .  r e s u l t s a l s o i n d i c a t e t h a t even simple  limonoids  These  can have  marked p h y s i o l o g i c a l e f f e c t s , and cannot be c o n s i d e r e d "harmless d e t e r r e n t s " (sensu Bernays and Graham, 1988).  B. D-seco Limonoids Other comparisons suggest t h e importance o f t h e epoxide s u b s t i t u t i o n on an i n t a c t D r i n g . anthothecol,  Cedrelone and  t h e most a c t i v e o f t h e limonoids  have such a s u b s t i t u t i o n .  here t e s t e d ,  Gedunin, which d i f f e r s i n t h a t  the D r i n g i s o x i d i z e d t o a ^ - l a c t o n e , as w e l l as having  208  d i f f e r e n t s u b s t i t u t i o n s on a s a t u r a t e d B r i n g ,  lacks  a n t i f e e d a n t o r IGR a c t i v i t y a g a i n s t £. s a u c i a and Q_. fasciatus. or  Gedunin was  a l s o much l e s s a c t i v e than c e d r e l o n e  a n t h o t h e c o l a g a i n s t O. n u b i l a l i s  (Arnason e_£ a l . , 1 9 8 7 ) ,  P. g o s s y p i e l l a . S_. f r u g i p e r d a , and H. zea (Kubo and K l o c k e , 1987).  T h i s reduced a c t i v i t y i s not due t o s a t u r a t i o n i n  the B r i n g s i n c e n i m o c i n o l , comparable for  the B r i n g s a t u r a t i o n , has IGR,  t o c e d r e l o n e except  antifertility,  i n h i b i t i n g a c t i v i t y a g a i n s t Musca domestica a l . , 1988)  and  molt  (Siddiqui et  and Aedes a e g y p t i (Naqui, 1 9 8 7 ) .  B ring  s u b s t i t u e n t s can, however, a f f e c t a n t i f e e d a n t a c t i v i t y as  7-  d e a c e t y l g e d u n i n and 7-ketogedunin are both l e s s a c t i v e than gedunin  (Kubo and K l o c k e , 1 9 8 7 ) .  O v e r a l l i t seems c l e a r  t h a t o x i d a t i o n o f the D r i n g epoxide t o an epoxylactone r e s u l t s i n l o s s o f IGR a c t i v i t y and a marked decrease i n antifeedant a c t i v i t y .  N e v e r t h e l e s s t h i s type o f o x i d a t i o n  i s c h a r a c t e r i s t i c o f a l l known rutaceous and  simaroubaceous  l i m o n o i d s , and i s t y p i c a l o f most meliaceous l i m o n o i d s as (Das e t a l . , 1 9 8 4 ) .  well  C. A D-seco f  In  Limonoids  addition to t h e i r c h a r a c t e r i s t i c D ring  t y p i c a l l y l i m o n o i d s from the Rutaceae  structure,  and Simaroubaceae have  undergone B a y e r - V i l l a g e r o x i d a t i o n of the A r i n g l i m o n o i d s ) (Dreyer, 1 9 8 3 ) .  None o f the A,D-seco l i m o n o i d s  t e s t e d here (obacunone, n o m i l i n , h a r r i s o n i n , and produced  (A seco-  IGR o r a n t i f e r t i l i t y  e f f e c t s i n O.  pedonin)  fasciatus.  209  H a r r i s o n i n had a n t i f e e d a n t a c t i v i t y i n a c h o i c e t e s t neonate P. s a u c i a , but d i d not s i g n i f i c a n t l y a f f e c t growth i n a seven-day no-choice  bioassay.  against larval  The a n t i f e e d a n t  a c t i v i t y of h a r r i s o n i n i s known t o vary w i d e l y between s p e c i e s . H a s s a n a l i e t a l (1986) found i t t o be d e t e r r e n t t o Eldana  s a c c h a r i n a and Maruca t e s t u l a l i s a t ( r e s p e c t i v e l y ) 1  and 10 ug/cm  2  d i s c , but i t had o n l y marginal  i n h i b i t i o n a g a i n s t Spodoptera exempta a t 100  feeding ug/disc.  Obacunone and n o m i l i n had no a n t i f e e d a n t o r IGR  activity  a g a i n s t £. s a u c i a a t c o n c e n t r a t i o n s up t o 0.5 /nmol/g fwt. Again,  these compounds a r e f e e d i n g d e t e r r e n t s a t low  concentrations M« t e s t u l a l i s  f o r some i n s e c t s , i n c l u d i n g JSj. s a c c h a r i n a and ( H a s s a n a l i e t a l - , 1986), have o n l y  moderate  a c t i v i t y a g a i n s t o t h e r s , i n c l u d i n g Q. n u b i l a l i s  (Arnason e t  a l . , 1987), £5. f r u g i p e r d a , and H.  Klocke,  zea (Kubo and  1986), and a r e i n a c t i v e a g a i n s t y e t o t h e r s p e c i e s i n c l u d i n g T r i c h o p l u s i a n i ( A l t i e r i et a i . , 1984).  Similar Citrus  limonoids were a l s o i n a c t i v e a g a i n s t the spruce budworm, Choristoneura  fumiferana  ( A l f o r d and B e n t l e y ,  1985).  In the r e l a t e d compound pedonin, the D r i n g has been opened t o a y - k e t o f u r a n ,  and the r e s u l t i n g c a r b o n y l  group  methylated t o a carbomethoxy group; as w e l l the A r i n g has undergone  1,2  s k e l e t a l rearrangement t o a s p i r o s t r u c t u r e  ( H a s s a n a l i et a i . , 1987).  Pedonin was s t i m u l a t o r y t o £.  s a u c i a a t 0.5 /nmol/g fwt, and was fasciatus.  i n a c t i v e against  Q.  T h i s compound was a l s o i n a c t i v e a g a i n s t t h e  polyphagous £3. exempta, but was a s t r o n g a n t i f e e d a n t t o the  210  more oligophagous  M.- t e s t u l a l i s and E_. s a c c h a r i n a ( H a s s a n a l i  and B e n t l e y , 1987).  D. B,D-seco Limonoids W i t h i n t h e Swieteniodeae,  D-seco limonoids may be f u r t h e r  o x i d i z e d i n t h e B r i n g ; t h i s pathway produces a g r e a t e r variety  o f l i m o n o i d s than any o t h e r (Das e t a l . , 1985;  C o n n o l l y , 1983).  Bussein and entandrophragmin a r e t y p i c a l  of t h i s group o f B,D r i n g seco l i m o n o i d s , and a r e characteristic  p a r t i c u l a r l y o f s p e c i e s o f Khaya and  Entandrophraoma ( T a y l o r , 1988).  Entandrophragmin had no  e f f e c t on e i t h e r E. s a u c i a o r O. f a s c i a t u s .  Bussein  the growth o f E« s a u c i a neonates, b u t was much l e s s than e i t h e r  reduced active  c e d r e l o n e o r a n t h o t h e c o l . The growth i n h i b i t i o n  c o u l d n o t be a s c r i b e d t o an a n t i f e e d a n t e f f e c t , as b u s s e i n d i d n o t have a s i g n i f i c a n t e f f e c t on d i e t c h o i c e . e f f e c t s were observed  i n t h e Q. f a s c i a t u s  No IGR  assays.  In t h e o n l y o t h e r study t o examine t h e e f f e c t s o f entandrophragmin and b u s s e i n , both reduced  f e e d i n g and  s u r v i v o r s h i p o f Q. n u b i l a l i s a t 500 ppm i n d i e t  (Arnason gt  a l . . 1987), s l i g h t l y h i g h e r than t h e maximum c o n c e n t r a t i o n used i n my assays.  S e v e r a l B-seco limonoids a r e known t o  have a n t i f e e d a n t a c t i v i t y (Table 3-1), although t h i s can v a r y w i d e l y w i t h r e l a t i v e l y minor changes i n s t r u c t u r e . Although  prieurianin  and i t s a c e t a t e had a n t i f e e d a n t  a c t i v i t y a g a i n s t H.. z e a  f  S_. f r u g i p e r d a ( a c e t a t e o n l y ) , and  E. v a r i v e s t i s . t h e r e l a t e d  r o h i t i u k i n and r o h i t u k a - 7 , which  211  d i f f e r i n t h a t the B r i n g c a r b o x y l fragment i s c y c l i z e d C29 al.,  t o form a new 1985).  et  l a c t o n e r i n g , were i n a c t i v e ( L i d e r t  Three compounds r e l a t e d t o p i e r i a n i n are known  t o have a n t i f e e d a n t a c t i v i t y a g a i n s t A g r o t i s (Nakatani  with  et al.,  1984).  T o o n a c i l i n and  seietum  6-  a c e t o x y t o o n a c i l i n , which are s i m i l a r t o c e d r e l o n e  except f o r  an o x i d a t i v e l y c l e a v e d B r i n g , d e t e r f e e d i n g by E_. varivestis  (Kraus e t a l . ,  E. C-seco Limonoids  1978).  (Azadirachtin)  A z a d i r a c h t i n had the most marked e f f e c t on £. s a u c i a growth and  f e e d i n g of any of the limonoids t e s t e d i n t h i s  and was  study,  the o n l y compound t o s i g n i f i c a n t l y reduce  survivorship.  Choice t e s t s with neonate l a r v a e i n d i c a t e d  t h a t an a n t i f e e d a n t e f f e c t may  be an important  the observed growth i n h i b i t i o n , as these  component of  larvae avoid d i e t  t r e a t e d w i t h a z a d i r a c h t i n c o n c e n t r a t i o n s as low as nmol/g fwt.  0.5  Although i n s e c t s are a b l e t o l e a r n a v e r s i o n t o  t o x i c d i e t s ( D e t h i e r , 1980), and can s e l e c t  nutritionally  o p t i m a l d i e t s based on changes i n b r a i n s e r o t o n i n (Cohen e t al.,  1988)  o r catecholamine n e u r o t r a n s m i t t e r s  (Wurtman,  1981), the r a p i d i t y o f the response t o a z a d i r a c h t i n ( s i g n i f i c a n t a f t e r o n l y 1 h) suggests a chemosensory b a s i s f o r the avoidance.  A "deterrent receptor", s e n s i t i v e to  a z a d i r a c h t i n , i s present polyphagous l e p i d o p t e r a n s  i n a number of oligophagous (Simmonds and Blaney,  Schoonhoven and Jermy, 1977).  1984;  T h i s r e c e p t o r does not  and  212  interfere  w i t h the a c t i v i t y of other n u t r i e n t r e c e p t o r s ,  r a t h e r a l t e r s the c e n t r a l nervous p r o c e s s i n g information  (Simmonds and  Sensitivity  of chemosensory  Blaney, 1984).  t o the a n t i f e e d a n t  a c t i v i t y of a z a d i r a c h t i n  d e c l i n e d markedly by the t h i r d i n s t a r .  Ostrinia  nubilalis  l a r v a e a l s o show a d e c l i n e i n the d e t e r r e n t a c t i v i t y a z a d i r a c h t i n between the f i r s t and e t a l . , 1985).  The  the t h i r d i n s t a r  r e l a t i v e l y greater s e n s i t i v i t y  neonate c a t e r p i l l a r s t o secondary m e t a b o l i t e s previously phenolics  (Reese, 1973), and (Isman and  (Isman e t  lactones The  been noted  includes s e n s i t i v i t y  aJL., 1986)  and  (Arnason  to and  sesquiterpene  a l . , 1989).  growth i n h i b i t i n g a c t i v i t y of a z a d i r a c h t i n  continued  throughout the l a r v a l development of E.  d e s p i t e the decrease i n a n t i f e e d a n t effect  has  of  of  Duffey, 1982), p o l y a c e t y l e n e s  thiophenes (Champagne e t  but  of a z a d i r a c h t i n c o n c e n t r a t i o n  significant,  the e x t e n t  /nmol/g the few  activity.  While  the  on pupal weight  was  of i n h i b i t i o n was  not l a r g e : a t  pupae which were formed weighed 88%  c o n t r o l pupae.  saucia,  of  0.5  the  While even s m a l l pupal weight r e d u c t i o n s  may  c o r r e l a t e w i t h reduced a d u l t f e c u n d i t y , the most biologically  significant  long-term e f f e c t of a z a d i r a c h t i n i s  a marked decrease i n p u p a t i o n and results  a d u l t emergence.  have been noted w i t h H e l i o t h i s zea  Klocke, 1987), O s t r i n i a the l e p i d o p t e r a n  nubilalis  (Barnby  (Arnason e t  r i c e p e s t s Mythimna separata  Similar and  a l . , 1985), and  213  Cnaphalocrocis  medinialis  (Schmutterrer e t a i . , 1983)  the f a c e f l y Musca autumnalis (Gaaboub and  and  Hayes, 1984).  F N u t r i t i o n a l Indices A z a d i r a c h t i n markedly reduced growth and third-instar E.  consumption i n  saucia at dietary concentrations  not a f f e c t the ECI,  ECD,  o r AD.  The  reduced consumption i s  u n l i k e l y t o r e f l e c t a chemosensory a n t i f e e d a n t consumption was  may  e f f e c t as  reduced even a t low c o n c e n t r a t i o n s  not a f f e c t d i e t c h o i c e by s e c o n d - i n s t a r reduced RCR  which d i d  larvae.  which d i d  Rather,  r e f l e c t a d i r e c t a c t i o n of a z a d i r a c h t i n  the gut o r on n e u r a l r e g u l a t i o n of f e e d i n g .  r  and  on  Mordue e t a l .  (1985) have shown t h a t a z a d i r a c h t i n reduces the r a t e of p e r i s t a l s i s i n Locusta  the  gut  indeed i n h i b i t s a l l p r o c t o l i n -  mediated muscular a c t i v i t i e s  (Mordue and  Plane, 1988).  Such  a mechanism c o u l d w e l l account f o r the observed decrease i n the RCR;  c o n s i s t e n t w i t h t h i s i s the o b s e r v a t i o n  t h a t the  AD  tended t o i n c r e a s e w i t h i n c r e a s i n g a z a d i r a c h t i n concentration.  T h i s e f f e c t c o u l d r e s u l t from a prolonged  exposure qf the food b o l u s t o d i g e s t i v e enzymes (Slansky S c r i b e r , 1985).  The  decrease i n the ECI  the h i g h e s t a z a d i r a c h t i n c o n c e n t r a t i o n  and  ECD  and  found a t  here t e s t e d  could  r e f l e c t i n c r e a s e d m e t a b o l i c c o s t s of d e t o x i f i c a t i o n . However, reduced consumption can a l s o r e s u l t d i r e c t l y i n a reduction  i n ECI and  ECD,  as the f i x e d c o s t s of m e t a b o l i c  a c t i v i t y w i l l consume a h i g h e r p r o p o r t i o n of energy i f t h a t i n t a k e i s s m a l l r a t h e r than l a r g e .  intake  In p a r t i c u l a r ,  214  consumption a t t h e h i g h e s t a z a d i r a c h t i n c o n c e n t r a t i o n  may be  j u s t s u f f i c i e n t t o meet t h e m e t a b o l i c requirements o f t h e l a r v a e , w i t h no excess t o support growth.  This  i n t e r p r e t a t i o n i s supported by t h e o b s e r v a t i o n  t h a t , when  RGRi i s p l o t t e d a g a i n s t RCRi, a l l treatments f i t t h e same regression  l i n e ( F i g . 4-7). T h i s r e s u l t s t r o n g l y suggests  that the growth-inhibiting a c t i v i t y of azadirachtin i s r e l a t e d t o reduced consumption and n o t t o m e t a b o l i c t o x i c i t y (cf. few  Blau e t a i . ,  1978).  I t i s a l s o noteworthy t h a t  larvae died while molting;  Arnason e £ a i .  s i m i l a r r e s u l t w i t h 0. n u b i l a l i s .  very  (1985) noted a  The m o l t - i n h i b i t i n g  a c t i v i t y o f a z a d i r a c h t i n appears t o be most prominent i n s p e c i e s which f a i l t o respond t o a z a d i r a c h t i n as an antifeedant  ( i . e . G a r c i a and Rembold, 1983; Chapter 5 o f  t h i s t h e s i s ) , or i n bioassays  where i n s e c t s a r e a r t i f i c i a l l y  exposed ( v i a t o p i c a l a p p l i c a t i o n , i n j e c t i o n , o r g u t cannulation) be  t o doses o f a z a d i r a c h t i n which would o r d i n a r i l y  avoided. The  r e s u l t s o f t h e n u t r i t i o n a l index study a r e  comparable t o r e s u l t s obtained  w i t h Melanoplus s a n g u i n i p e s  (Chapter I V ) , H e l i o t h i s v i r e s c e n s and  Crocidolomia  (Barnby and Klocke, 1987),  b i n o t a l i s (Fagoonee, 1984).  In t h e l a t t e r  study, ECI and ECD were found t o i n c r e a s e w i t h a z a d i r a c h t i n concentration.  In c o n t r a s t t o these r e s u l t s , Arnason e t a i  (1985) found no r e d u c t i o n  i n consumption when 0. n u b i l a l i s  l a r v a e were exposed t o a z a d i r a c h t i n ; r a t h e r , a decrease i n the RGR was r e l a t e d t o decreases i n t h e ECI and ECD.  Rao  215  and Subramantham (1985) found t h a t a z a d i r a c h t i n reduced consumption, ECI, and ECD Schistocerca gregaria.  i n a dose-dependent manner i n  T h i s r e s u l t may  r e f l e c t the extreme  s e n s i t i v i t y of t h i s i n s e c t t o a z a d i r a c h t i n (Blaney,  1980).  G. Limonoid E v o l u t i o n and S t r u c t u r e - A c t i v i t y R e l a t i o n s h i p s The  i n s e c t i c i d a l a c t i v i t y of l i m o n o i d s does not appear t o  c o r r e l a t e with the e v o l u t i o n a r y t r e n d s of i n c r e a s i n g o x i d a t i o n and s k e l e t a l rearrangement d i s c u s s e d by Das (1984).  T h i s c o n c l u s i o n i s supported  a n a l y s i s i n F i g u r e 4-8, 4-1  not o n l y by  et  the  but a l s o by a c o n s i d e r a t i o n of  and the r e s u l t s of the b i o a s s a y s presented here.  dammarane p r e c u r s o r s of the limonoids are i n a c t i v e a g a i n s t i n s e c t s (Chapter protolimonoids  The  the  and the simple  euphol type l i m o n o i d s  (Group 2) are a c t i v e ; i n the  case the a c t i v i t y may  i n c l u d e both t o x i c and  ( i . e . i n the case o f c e d r e l o n e ) .  Table  apparently  3), but both  (Group 1 i n F i g . 4-2)  al  IGR  apolatter  effects  However, the dominant  e v o l u t i o n a r y pathways w i t h i n most Meliaceae  and a l l  Rutaceae, g i v i n g r i s e t o D-seco, A,D-seco, and B,D-seco l i m o n o i d s , l e a d t o compounds with reduced a p p a r e n t l y no IGR may  activity.  antifeedant  In C i t r u s , the reduced  be t o some e x t e n t compensated f o r by the very  c o n c e n t r a t i o n s of A,D-seco limonoids produced (> 1,000  ppm)  (especially  (Rouseff and Nagy, 1982).  these pathways are the o n l y ones expressed Swietenioideae  and  activity  high limonin) Curiously,  i n members of the  ( T a y l o r , 1981), c o n s i d e r e d t o be the most  216  advanced s u b f a m i l y o f the Meliaceae 1975).  The  (Pennington and S t y l e s ,  o n l y pathway which appears t o l e a d t o compounds  with increased a c t i v i t y against g e n e r a l i s t i n s e c t s i s that g i v i n g r i s e t o the C-seco l i m o n o i d s ; these compounds may  be  d e r i v e d v i a a d i f f e r e n t pathway from the o t h e r  limonoids,  i n v o l v i n g an e x t r a e p o x i d a t i o n s t e p  (Siddiqui et  al.,  1988).  ( F i g . 1-2)  T h i s pathway i s expressed  M e l i e a e , which i n c l u d e s the genera A z a d i r a c h t a and  o n l y i n the  tribe  (morphologically p r i m i t i v e )  Melia.  D e s p i t e the absence of an obvious r e l a t i o n s h i p between l i m o n o i d e v o l u t i o n and a c t i v i t y a g a i n s t phytophagous i n s e c t s , the p o s s i b i l i t y of a r o l e f o r limonoids  in a  c o e v o l u t i o n a r y r e l a t i o n s h i p between p l a n t s and i n s e c t s remains.  A l l of the b i o a s s a y  s p e c i e s used t o date are  polyphagous or oligophagous s p e c i e s which do not Meliaceae  as host p l a n t s .  specialist  The  utilize  p o s s i b i l i t y remains t h a t  i n s e c t s , adapted t o t o l e r a t e exposure t o  c l a s s o f l i m o n o i d s , may  one  be d e t e r r e d o r i n t o x i c a t e d by  exposure t o another c l a s s .  For example, p a p i l i o n i d  larvae  a b l e t o t o l e r a t e l a r g e doses of l i n e a r furanocoumarins are susceptable  t o angular  furanocoumarins (Berenbaum,  1981), even though the l a t t e r are c o n s i d e r e d  1978,  l e s s t o x i c as  they are o n l y a b l e t o form monofunctional adducts with The  r o l e of limonoids  DNA.  i n a putative coevolutionary  r e l a t i o n s h i p between the Meliaceae  and  i n s e c t s would have t o  be examined i n the c o n t e x t of the adapted i n s e c t fauna found f e e d i n g on meliaceous s p e c i e s .  For i n s t a n c e , the shoot  217  b o r e r s H y p s i p y l a spp. a t t a c k a l l s p e c i e s o f C e d r e l a and S w i e t e n i a , t o t h e p o i n t o f l i m i t i n g t h e i r u s e f u l n e s s as p l a n t a t i o n crops w i t h i n t h e i r n a t u r a l area o f d i s t r i b u t i o n (Grijpma, 1973).  These i n s e c t s must be adapted t o cope w i t h  Group 2 compounds such as c e d r e l o n e , which occur i n t h e shoots and heartwood as w e l l as f o l i a g e o f C e d r e l a . and may a l s o be exposed t o l i m o n o i d s o f groups 1, 3 and 4, known from t h e seeds o f C e d r e l a and S w i e t e n i a s p e c i e s ( T a y l o r , 1981).  C e d r e l a and Swietenia o f t e n co-occur w i t h s p e c i e s o f  Guarea (Pennington  and S t y l e s , 1 9 8 1 ) , which a r e not a t t a c k e d ;  i s t h e r e s i s t a n c e due t o l i m o n o i d s o f c l a s s e s 5 and 7, found i n Guarea but not i n t h e o t h e r genera?  Azadirachta  indica  has a s m a l l entomofauna o f twelve s p e c i e s , almost a l l o f which a r e monophagous (Warthen, 1979).  Has t h i s  specialized  fauna r e s u l t e d from a c o e v o l u t i o n a r y a s s o c i a t i o n d u r i n g t h e e v o l u t i o n o f t h e C-seco type limonoids?  C l e a r l y the  s i g n i f i c a n c e o f l i m o n o i d s i n mediating such  interactions  requires further investigation.  H. C o r r e l a t i o n o f Phytochemistry The  and Crude E x t r a c t Bioassays  s t r u c t u r e / a c t i v i t y c o n c l u s i o n s drawn here a r e supported  by a c o r r e l a t i o n o f t h e r e s u l t s o f t h e crude e x t r a c t b i o a s s a y s r e p o r t e d i n Chapter o f limonoids i n t h e M e l i a c e a e .  2 w i t h t h e known d i s t r i b u t i o n T a b l e 4-6 l i s t s t h e c l a s s e s  of l i m o n o i d s known t o occur i n t h e v a r i o u s genera o f Meliaceae  (updated  from T a y l o r , 1981).  Genera w i t h group 8  l i m o n o i d s were p r e d i c t e d t o be h i g h l y a c t i v e a g a i n s t E«  218 T a b l e 4 - 6 . Comparison of E C v a l u e s of crude e x t r a c t s of M e l i a c e a e w i t h p r e d i c t i o n s of a c t i v i t y based on c l a s s e s of l i m o n o i d s r e p o r t e d t o occur i n the genera examined. E x t r a c t s were p r e d i c t e d t o have h i g h a c t i v i t y i f group 8 l i m o n o i d s had been r e p o r t e d , moderate a c t i v i t y i f group 2 or 10 limonoids were known, low a c t i v i t y i f o t h e r l i m o n o i d s had been r e p o r t e d , and no a c t i v i t y i f limonoids had not been s a u c i a , those w i t h group 2 limonoids were p r e d i c t e d t o be moderately a c t i v e , those w i t h o t h e r c l a s s e s of l i m o n o i d s found. Phytochemical d a t a i s taken from T a y l o r (1981), updated as noted. Limonoids known from s p e c i e s o t h e r than the one(s) b i o a s s a y e d a r e i n parentheses. Observed E C s are from T a b l e 2-2 and are mg/g; where more than one s p e c i e s i n a genus was b i o a s s a y e d the range of r e s u l t s i s shown. 5 0  5 0  Predicted Limonoid Groups Activity Reported Aglaia inactive dammaranes Azadirachta 1,2,3,8 high Carapa 3,4,(5,6) low Cedrela 1,2,3,4 moderate Chuckrasia 5 low (9) low Dysoxylum Ekeberaia 4 low Entandrophragma (1,3,4),5 low Guarea (3,4,7) low Khaya (2),3,4 moderate Lansium none inactive Melia 1,2,3,8 high Sandoricum none inactive Swietenia 4 low Toona 2,6,10 moderate moderate Trichilia 2,(7) 2 moderate Turreae R e f e r e n c e s : l ) El-Shamy g t a i . , 1988. 2) J o g i a and Andersen, i n p r e s s . 3) Kraus and Grimminger, 1978,1980. Genus  1  2  3  Observed ^•^50 2.U7-11.88 0.69-0.89 29.68 24.66 31.13 inactive 102.94 29.83 53.32 67.05 inactive 1.58-2.10 22.79 23.64-47.36 22.93-47.63 18.88 2.80-45.40  219  were p r e d i c t e d t o have low a c t i v i t y , and the genera without l i m o n i d s were p r e d i c t e d t o be i n a c t i v e .  G e n e r a l l y the  c o r r e l a t i o n i s good: a l l s p e c i e s p r e d i c t e d t o have h i g h a c t i v i t y do, and those s p e c i e s found t o be i n a c t i v e had been p r e d i c t e d t o have l i t t l e or no a c t i v i t y .  Most s p e c i e s  p r e d i c t e d t o have moderate l e v e l s o f a c t i v i t y had EC50 v a l u e s c l o s e t o 20 mg/g, low a c t i v i t y had E C  5 0  and most of those p r e d i c t e d t o have  v a l u e s a t o r above 30  mg/g.  Noteworthy e x c e p t i o n s were seen, however: A g l a i a and Turreae were much more a c t i v e than was p r e d i c t e d based on t h e i r known phytochemistry, and Sandoricum had a moderate l e v e l o f a c t i v i t y d e s p i t e the r e p o r t e d absence o f l i m o n o i d s . s p e c i e s should be i n v e s t i g a t e d f u r t h e r .  These  On the o t h e r hand,  Khaya was l e s s a c t i v e than expected, but the s p e c i e s i n v e s t i g a t e d , K. s e n e g a l e n s i s . has not been r e p o r t e d t o c o n t a i n group 2 compounds, although o t h e r members of the genus do.  I . Comparison o f I n s e c t i c i d a l and C y t o t o x i c A c t i v i t y The i n s e c t i c i d a l a c t i v i t y of l i m o n o i d s i s t o some e x t e n t p a r a l l e l e d by t h e c y t o t o x i c a c t i v i t y o f these compounds a g a i n s t murine P-388 lymphocytic leukemia c e l l s . al  (1983) found t h a t a 14-15  P e t t i t et  epoxide on an i n t a c t D r i n g  was r e q u i r e d f o r c y t o t o x i c i t y ; they suggested t h a t the epoxide may be r e q u i r e d f o r a l k y l a t i o n o f bioamines o r t h i o l s , but I found t h a t a z a d i r a c h t i n does not spontaneously form adducts w i t h the s u l f h y d r y l c y s t e i n e i n v i t r o  (Chapter  220  5).  Limonoids  w i t h a 19-28  l a c t o l group were most a c t i v e ,  but those w i t h a 3-oxo-l-ene A r i n g s t r u c t u r e (as i n c e d r e l o n e and a n t h o t h e c o l ) were a l s o h i g h l y a c t i v e ; A r i n g s a t u r a t i o n a b o l i s h e d the c y t o t o x i c i t y . al.  Conversely, Kraus g t  (1987) concluded t h a t A - r i n g s a t u r a t i o n , w i t h oxygen  f u n c t i o n s a t C-2  and C-3,  was  required for antifeedant  a c t i v i t y a g a i n s t E p i l a c h n a v a r i v e s t i s , based on a comparison o f the a c t i v i t y of v i l a s i n i n e d e r i v a t i v e s w i t h a z a d i r a d i o n e . Compounds w i t h a D r i n g epoxylactone,  and the s e c o - r i n g  A,D  c i t r u s l i m o n o i d s , were mostly not c y t o t o x i c ( P e t t i t g t a l . , 1983). An i n t e r e s t i n g o b s e r v a t i o n from t h i s and s t u d i e s (Kubo and Klocke, 1987; Naqui,  1987)  i s the IGR  previous  Siddiqui gt a l . ,  a c t i v i t y of c e d r e l o n e and  1988;  1988;  related  compounds w i t h a 14-156-epoxide on an otherwise i n t a c t D r i n g , a n u n s u b s t i t u t e d C r i n g , and a 3-oxo-l-ene A r i n g . These r e l a t i v e l y simple s t r u c t u r e s may  be amenable t o  s y n t h e s i s or m a n i p u l a t i o n , and so c o u l d p r o v i d e l e a d s f o r the development of s y n t h e t i c limonoid-based  insecticides.  221  Chapter 5: E f f e c t s o f a z a d i r a c h t i n on the n u t r i t i o n development o f the m i g r a t o r y sanguinipes  and  grasshopper, Melanoplus  Fab.  Introduction  Among the few  i n s e c t s r e p o r t e d t o be r e s i s t a n t t o  a z a d i r a c h t i n are the New migratory  World grasshoppers, i n c l u d i n g the  grasshopper, Melanoplus sanguinipes  (Orthoptera:  Acrididae)  Fab.  (Mulkern and M o n g o l k i t i , 1975).  In  i n i t i a l experiments I confirmed the absence of a chemosensory-based a n t i f e e d a n t e f f e c t a g a i n s t t h i s i n s e c t , but observed marked subsequent d i s r u p t i o n of m o l t i n g . allowed  me  This  t o compare the t o x i c i t y of a z a d i r a c h t i n f o l l o w i n g  a p p l i c a t i o n o r a l l y , t o p i c a l l y , and v i a i n j e c t i o n , and e v a l u a t e the s i g n i f i c a n c e of the gut and  so  integument as  f a c t o r s l i m i t i n g the b i o a v a i l a b i l i t y of t h i s compound t o p u t a t i v e t a r g e t s i t e s w i t h i n the i n s e c t . Azadirachtin-induced  i n h i b i t i o n of m o l t i n g has  been  shown, i n s e v e r a l i n s e c t s , t o i n v o l v e a d e l a y i n the appearance o f the e c d y s t e r o i d peaks which r e g u l a t e a p o l y s i s (reviewed occurs  i n Chapter 1); however the mechanism by which t h i s  is still  unclear.  Mordue and  suggested t h a t such e f f e c t s may  Evans (1987) have  r e s u l t from a d i r e c t a c t i o n  on the gut r a t h e r than a d i r e c t a c t i o n on s y n t h e s i s or metabolism.  ecdysteroid  222  The  t o x i c o l o g y of a z a d i r a c h t i n i s i n many  respects  p a r a l l e l e d by the a z a s t e r o l s , which b l o c k the c o n v e r s i o n B - s i t o s t e r o l and inhibiting  2 4  other phytosterols and  22,24 s t e r o l r e d u c t a s e s o f  (Svoboda e t a l . , 1972; Svoboda, 1973).  to cholesterol  by  insects  Svoboda and Robbins, 1971;  Walker  S i m i l a r i t i e s i n c l u d e the i n h i b i t i o n  growth, m o l t i n g  and  low  As a z a s t e r o l t o x i c i t y can be r e v e r s e d  as 3 /ig/g.  of  and  of  oogenesis a t d i e t a r y c o n c e n t r a t i o n s  as by  d i e t a r y supplementation w i t h c h o l e s t e r o l , I i n v e s t i g a t e d e f f e c t of c h o l e s t e r o l and toxicity.  other phytosterols  on  I a l s o i n v e s t i g a t e d the p o s s i b i l i t y  a z a d i r a c h t i n may  the  azadirachtin that  i n t e r f e r e w i t h s t e r o l t r a n s p o r t through  the  hemolymph, a p r o c e s s dependent on the c a r r i e r l i p o p r o t e i n lipophorin  (Chino and  G i l b e r t , 1971;  Chino, 1985).  F i n a l l y , as both the t r a n s d u c i n g chemoreception and  the n e u r o s e c r e t o r y m a t e r i a l  pars i n t e r c e r e b r a l i s are u n u s u a l l y residues  proteins involved  ( N o r r i s , 1986;  F r i e d e l and  both s i t e s have been p o s t u l a t e d  in  formed i n the  rich in sulfhydryl Loughton, 1980),  t o be p u t a t i v e  and  molecular  t a r g e t s f o r a z a d i r a c h t i n a c t i v i t y , I t e s t e d the a b i l i t y  of  a z a d i r a c h t i n t o form adducts w i t h the s u l f h y d r y l amino a c i d cysteine  in vitro.  223  M a t e r i a l s and Methods  A. Experimental I n s e c t s F i f t h i n s t a r Melanoplus s a n g u i n i p e s nymphs,  non-diapause  s t r a i n , were o b t a i n e d from a l a b o r a t o r y c o l o n y r e a r e d on f r e s h l y c u t s e e d l i n g wheat, d r y wheat bran, and chickweed, Cerastium s t e l l a t a , fifth  (Isman, 1985).  Synchronized groups o f  i n s t a r nymphs were o b t a i n e d by c l e a r i n g cages of f i f t h  i n s t a r i n d i v i d u a l s 24 h p r i o r t o s t a r t i n g an experiment, then u t i l i z i n g nymphs which molted o v e r n i g h t .  In some cases  i n d i v i d u a l s so o b t a i n e d were s t o r e d a t 4° C f o r 24 h p r i o r to  s t a r t i n g a bioassay, t o allow c o l l e c t i o n of s u f f i c i e n t  numbers o f nymphs f o r the b i o a s s a y .  P r e l i m i n a r y experiments  e s t a b l i s h e d t h a t grasshoppers c o u l d be s t o r e d a t 4° C f o r up to  72 h without i n f l u e n c i n g subsequent growth r a t e , d u r a t i o n  of  the i n s t a r , or molting success.  B. Source o f Chemicals A z a d i r a c h t i n used i n t h i s study was i s o l a t e d from A z a d i r a c h t a i n d i c a seeds by Dr. J . Kaminski, and was k i n d l y made a v a i l a b l e Dr. J . T. Arnason, U n i v e r s i t y of Ottawa. C h o l e s t e r o l and B - s i t o s t e r o l were purchased from Sigma Chemical Company, S t . L o u i s , Mo. and were used without further p u r i f i c a t i o n . 4- C-B-sitosterol 1 4  purchased from Amersham.  (56 mCi/mmol) was  224  C. A n t i f e e d a n t A c t i v i t y Assays In  no-choice a n t i f e e d a n t assays, a z a d i r a c h t i n i n acetone  (0.5 o r 0.05 mg/ml) was a p p l i e d t o both s u r f a c e s of f r e s h l y punched and weighed  1 cm diameter cabbage ( B r a s s i c a o l e r a c e a  cv.  E a r l y Copenhagen) l e a f d i s c s t o a c h i e v e c o n c e n t r a t i o n s  of  5, 10, 15, 20, 25, 50, 100, 200, 300, and 500 ug/ g l e a f  f r e s h weight ( f w t ) .  C o n t r o l l e a f d i s c s were t r e a t e d w i t h 10  / i l acetone, a volume e q u i v a l e n t t o t h a t a p p l i e d a t t h e highest azadirachtin concentration. p r e s e n t e d t o each f i f t h  One l e a f d i s c was  i n s t a r grasshopper i n a 4 oz unwaxed  paper cup capped w i t h a p l a s t i c p e t r i d i s h l i d ; grasshoppers were s t a r v e d f o r two hours p r i o r t o t h e t e s t .  Ten nymphs  were used f o r each c o n c e n t r a t i o n , and t h e t e s t was r e p l i c a t e d three times.  The number o f i n d i v i d u a l s i n each  treatment group which had completely consumed t h e d i s c  after  15, 30, and 60 minutes was r e c o r d e d .  D. D i e t a r y U t i l i z a t i o n  Experiments  The e f f e c t s o f a z a d i r a c h t i n on r e l a t i v e growth,  consumption,  and d i g e s t i v e performance of nymphs were determined. fifth  Staged  i n s t a r nymphs (20 per treatment) were f e d a s i n g l e  dose of 10 o r 15 /ig/g i n s e c t fwt o f a z a d i r a c h t i n a p p l i e d t o a 1 cm diameter cabbage l e a f d i s c ; these doses were chosen as they produce markedly d i f f e r e n t e f f e c t s on m o l t i n g success i n U. s a n g u i n i p e s .  Subsequently t h e nymphs were f e d  d a i l y w i t h weighed a l i q u o t s o f f r e s h l y c u t s e e d l i n g wheat.  225  I n i t i a l d r y weight of the wheat was e s t i m a t e d by d r y i n g samples of wheat and c a l c u l a t i n g a dwt/fwt r a t i o .  After  48  h the nymphs were weighed, then nymphs, f r a s s , and remaining wheat were d r i e d t o c o n s t a n t weight a t 70 C and weighed. The i n i t i a l dw o f the nymphs was c a l c u l a t e d based on t h e dw/fw r a t i o o f a sample of 10 nymphs. I n d i c e s determined i n c l u d e d the approximate d i g e s t a b i l i t y  (AD), e f f i c i e n c y o f  c o v e r s i o n of i n g e s t e d food (ECI), and e f f i c i e n c y o f c o n v e r s i o n o f d i g e s t e d food (ECD), c a l c u l a t e d a c c o r d i n g t o Reese and Beck  (1976).  In a d d i t i o n growth arid consumption  r a t e s were c a l c u l a t e d i n r e l a t i o n t o t h e weight of t h e nymphs a t t h e s t a r t of the experiment (RGRi and RCRi respectively) (Farrar et a l ,  1989).  Formulas f o r  c a l c u l a t i n g these i n d i c e s are g i v e n i n Chapter 4.  E. M o l t I n h i b i t i o n Assays To a s s e s s the growth r e g u l a t i n g a c t i v i t y o f a z a d i r a c h t i n f o l l o w i n g o r a l a d m i n i s t r a t i o n , staged (<24 h) f i f t h nymphs were weighed  instar  (114.3 ± 1 2 . 1 mg/nymph), then f e d  s u f f i c i e n t a z a d i r a c h t i n a p p l i e d i n acetone t o a l e a f d i s c t o a c h i e v e a dose of 3, 5, 8, 10, 13, 15, o r 25 fig/ fwt. only.  g insect  C o n t r o l s were f e d l e a f d i s c s t r e a t e d w i t h acetone A f t e r the s i n g l e dose of a z a d i r a c h t i n was consumed,  u s u a l l y i n l e s s than 1 h, grasshoppers were maintained i n 4 oz paper cups , i n a c o n t r o l l e d environment chamber a t 30 + 1° C, about 40 % RH  (ambient a i r RH), a 16L:8D p h o t o p e r i o d ,  and f e d u n t r e a t e d wheat, bran, and chickweed ad l i b ,  until  226  m o l t i n g o r death o c c u r r e d .  D u r a t i o n of the i n s t a r , m o l t i n g  s u c c e s s , and weight a t m o l t i n g were r e c o r d e d . per dose were used i n each of f o u r r e p l i c a t e s . (the  Ten nymphs MD  50  values  dose which i n h i b i t e d m o l t i n g i n 50% of t h e t r e a t e d  nymphs) were determined by p r o b i t a n a l y s i s , w i t h c a t e g o r y 3 and 4 responses ( d e f i n e d i n R e s u l t s s e c t i o n ) combined as mortalities. A z a d i r a c h t i n i n acetone was a l s o a p p l i e d t o p i c a l l y t o the to  d o r s a l abdominal t e r g a e o f staged f i f t h  instar  nymphs,  a c h i e v e doses o f 2, 4, 6, 8, and 10 /ig/g i n s e c t fwt.  C o n t r o l s were t r e a t e d w i t h 5 / i l acetone, e q u i v a l e n t t o t h e volume used a t t h e h i g h e s t a z a d i r a c h t i n dose.  A f t e r the  s i n g l e treatment i n s e c t s were maintained as d e s c r i b e d above. Ten nymphs were used f o r each c o n c e n t r a t i o n i n each o f t h r e e r e p l i c a t e s . For i n j e c t i o n experiments, staged f i f t h  instar  nymphs were weighed, c h i l l e d t o 4° C, then i n j e c t e d midl a t e r a l l y between abdominal t e r g a 3 and 4 , u s i n g a Hamilton s y r i n g e f i t t e d w i t h a 26-gauge needle a t t a c h e d t o a Hamilton repeating dispensor.  A z a d i r a c h t i n i n acetone (0.5 mg/ml)  was a p p l i e d a t 3, 5, 8, 10, and 15 fig/  g i n s e c t fwt;  c o n t r o l s were i n j e c t e d w i t h 3 / i l acetone a l o n e .  Mortality  i n the c o n t r o l s was about 6% (2 o f 30 nymphs) w i t h t h i s solvent.  Three r e p l i c a t e s o f t e n i n s e c t s per dose were  performed.  F. P i p e r o n y l Butoxide Synergism  Assay  227  The p o s s i b l e r o l e of m i x e d - f u n c t i o n o x i d a s e s (MFOs) i n the metabolism  of a z a d i r a c h t i n by M.. s a n g u i n i p e s  was  i n v e s t i g a t e d by f e e d i n g staged f i f t h i n s t a r nymphs a z a d i r a c h t i n a t 2, 4, 6, 8, and 10 ng/  g i n s e c t fwt,  c o a d m i n i s t e r e d w i t h 500 /xg p i p e r o n y l butoxide (PBO) 1:1  w i t h acetone, on a cabbage l e a f d i s c .  diluted  Control insects  were f e d l e a f d i s c s t r e a t e d w i t h 500 ng PBO  only.  Insects  were subsequently maintained as d e s c r i b e d above, and d u r a t i o n of the i n s t a r and molt success were r e c o r d e d . experiment  i n v o l v e d t h r e e r e p l i c a t e s of t e n nymphs per  G. F e c u n d i t y  The dose.  Experiment  Staged t e n e r a l a d u l t female M.. s a n g u i n i p e s (<24 m o l t i n g ) were weighed, then f e d s u f f i c i e n t  h from  azadirachtin,  a p p l i e d t o a cabbage l e a f d i s c , t o a c h i e v e an o r a l dose o f 0, 5, 10, 15, 25, o r 50 pg/g  i n s e c t fwt.  Thereafter,  females were maintained i n d i v i d u a l l y i n 4 oz unwaxed paper cups a t 30° C, 18L:6D, and f e d s e e d l i n g wheat, and bran ad l i b . arenas: a 1.5  chickweed,  The cups were m o d i f i e d as o v i p o s i t i o n  cm diameter h o l e , punched i n the bottom o f the  cup, gave access t o a 2 oz p l a s t i c cup f i l l e d w i t h s i e v e d , sterilized soil. was  One male, not t r e a t e d w i t h a z a d i r a c h t i n ,  added t o each cup 24 h a f t e r the female.  Five  females  were used f o r each a z a d i r a c h t i n c o n c e n t r a t i o n t e s t e d ; the experiment  was  repeated t w i c e .  The cups of s o i l were  removed, examined f o r egg masses, and r e p l a c e d w i t h f r e s h soil  every t h r e e days.  Egg masses were disassembled t o  228  count i n d i v i d u a l eggs; t h i s p r e c l u d e d d e t e r m i n i n g egg f e r t i l i t y . six  weeks.  The b i o a s s a y  Data were analysed  H. E f f e c t o f D i e t a r y  the p o s s i b i l i t y of  by l i n e a r  was continued f o r regression.  Sterols  To determine t h e e f f e c t o f supplementing t h e d i e t w i t h s t e r o l s on a z a d i r a c h t i n t o x i c i t y , I e s t a b l i s h e d s i x treatment groups: (1) s o l v e n t c o n t r o l , (2) a z a d i r a c h t i n alone,  (3) c h o l e s t e r o l alone,  azadirachtin,  (4) c h o l e s t e r o l p l u s  (5) 8 - s i t o s t e r o l alone,  plus azadirachtin.  and (6) B - s i t o s t e r o l  I n s e c t s were i n i t i a l l y  f e d 15 /ig/g  i n s e c t fwt a z a d i r a c h t i n (treatments 2, 4, and 6) o r a s o l v e n t c o n t r o l (treatments 1, 3, and 5 ) .  For the duration  of t h e i n s t a r they were f e d wheat dipped i n CHC1 o r a 10 mg/ml CHC1 sitosterol  3  3  (1 and 2 ) ,  s o l u t i o n o f c h o l e s t e r o l (3 and 4) o r B -  (5 and 6 ) .  success were r e c o r d e d .  Duration  o f t h e i n s t a r and m o l t i n g  Ten nymphs p e r c o n c e n t r a t i o n  were  used i n each o f two r e p l i c a t e s .  I.  S t e r o l Transport  Experiment  I tested the hypothesis that azadirachtin i n t e r f e r e s with the t r a n s p o r t o f s t e r o l s i n t h e hemolymph by f e e d i n g sitosterol  14  C-B-  (10,000 dpm/nymph) t o c o n t r o l and a z a d i r a c h t i n  (15 /ig/g i n s e c t f w t ) - t r e a t e d f i f t h - i n s t a r nymphs.  Insects  (36/treatment) were f e d a l e a f d i s c t r e a t e d w i t h a z a d i r a c h t i n o r acetone 24 h b e f o r e t h e s i n g l e p u l s e o f radiolabelled sterol.  At hourly  i n t e r v a l s f o r 12 h, t h r e e  229  i n s e c t s from each treatment were randomly s e l e c t e d , a c u t was made l a t e r a l l y  along the abdominal  w a l l , and 10 f i l  hemolymph was c o l l e c t e d w i t h a m i c r o c a p i l l a r y tube.  The  samples were i n d i v i d u a l l y d i g e s t e d f o r 1 h i n 1 ml P r o t o s o l , then 4 ml Aquasol was added i n p l a s t i c s c i n t i l l a t i o n  tubes,  and t h e c o c k t a i l was allowed t o e q u i l i b r i a t e f o r 24 h t o quench chemiluminescense experiment  before counting.  (24 h) t h e nymphs from each treatment were  pooled, homogenized i n CHC1 , f i l t e r e d , 3  c o n c e n t r a t e d t o 4 ml. for  Following the  scintillation  and t h e e x t r a c t was  A 1 ml a l i q u o t was d r i e d and prepared  c o u n t i n g as d e s c r i b e d above.  The  remaining 3 mis were d r i e d , d e r i v a t i z e d w i t h 30% trifluroacetic for  a c i d (TFA) i n a c e t o n i t r i l e (ACHN) a t 80° C  1 h, then chromatographed by reverse-phase TLC developed  3 times i n a c e t i c acid:ACHN.  Spots were v i s u a l i z e d w i t h 3.0  M H S 0 , scraped from the p l a t e , e l u t e d w i t h MeOH:H 0 (1:1), 2  4  2  and prepared f o r s c i n t i l l a t i o n c o u n t i n g i n Aquasol. Standards o f TFA d e r i v a t i v e s o f c h o l e s t e r o l and 6 - s i t o s t e r o l were a l s o prepared and chromatographed a l o n g s i d e the hemolymph e x t r a c t s .  J . J n vitro  assay f o r t h e f o r m a t i o n o f adducts  Azadirachtin  (0.02 /imoles = 1.8 mg) and c y s t e i n e  ( f r e e base)  (0.02 /xmoles = 0.3 mg) were mixed i n 1 ml o f pH 7.0 phosphate  b u f f e r a t room temperature.  A l i q u o t s were removed  a t t e n minute i n t e r v a l s f o r t h e f i r s t hour, and t h e r e a f t e r h o u r l y f o r f i v e hours, s p o t t e d on c e l l u l o s e TLC p l a t e s , and  developed  i n t h e upper phase o f n - b u t a n o l : a c e t i c acid:H20  (4:1:5) (Pieman e t a l . , 1979).  Spots were v i s u a l i z e d by  s p r a y i n g t h e p l a t e w i t h n i n h y d r i n reagent  ( S t a h l , 1972)  f o l l o w e d by 2.0 M H S 0 , w i t h h e a t i n g a f t e r each spray 2  reagent.  4  231  Results  A. A n t i f e e d a n t Azadirachtin  assays  had  no a n t i f e e d a n t  s a n g u i n i p e s nymphs a t any  e f f e c t against  M..  of the c o n c e n t r a t i o n s  Leaf d i s c s were u s u a l l y consumed w i t h i n s i n g l e f e e d i n g bout; on o c c a s i o n  two  tested.  30 minutes i n a  f e e d i n g bouts were  required.  B. Growth and  Dietary  Utilization  Treatment w i t h both 10 and  15 /ig/g a z a d i r a c h t i n r e s u l t e d i n  a s i g n i f i c a n t decrease i n the r e l a t i v e growth r a t e (RGRi: and  40%  of c o n t r o l s r e s p e c t i v e l y , p<.0001, Tukey's  •i t e s t ) (Table  5-1).  T h i s was  10 and  (RCRi: 34 and  41%,  There were no d i f f e r e n c e s between the e f f e c t s a t  15 /ng/g  ingested  (HSD)  almost e n t i r e l y owing t o a  decrease i n the r e l a t i v e consumption r a t e p<.0001).  38  (ECI)  azadirachtin. and  i n s e c t biomass was  digested  The (ECD)  e f f i c i e n c y w i t h which food was  converted to  not s i g n i f i c a n t l y decreased.  approximate d i g e s t a b i l i t y was  Tukey's (HSD)  test).  The  s l i g h t l y increased  a z a d i r a c h t i n treatment a t the h i g h e r dose o n l y  new  following  (p<.017,  232  Table 5 - 1 .  E f f e c t of a z a d i r a c h t i n on Melanoplus  growth and n u t r i t i o n .  Means i n a column w i t h the same  l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t Range (HSD) Treat  (Tukey's  Studentized  T e s t , oc =0.05). RCRi  RGRi  C o n t r o l 0.243 10  sanguinipes  a  0.150°  0.843  ECI a  0.569°  ECD  30.2  a  30. 5  a  60.3  AD a  54.0 ° a  53. 2  a  59. 4  a  0.145^ 25.9 44. 6^ 59. B 15 0.500^ Treat=treatment (/ig/g i n s e c t f w t ) , RGRi=Relative Growth Rate based on i n i t i a l wgt, RCRi=Relative Consumption Rate based on i n i t i a l wgt, E C I = E f f i c i e n c y of Conversion of Ingested Food, E C D = E f f i c i e n c y of Conversion of D i g e s t e d Food, and AD=Approximate Digestability. Values were c a l c u l a t e d a c c o r d i n g t o Reese and Beck ( 1 9 7 6 ) except f o r the RGRi and RCRi, which were c a l c u l a t e d according to Farrar et a l . ( 1 9 8 9 ) . s  3  233  C. Molt I n h i b i t i o n S t u d i e s Melanoplus s a n g u i n i p e s nymphs t h a t consumed a z a d i r a c h t i n subsequently showed a range o f dose-dependent are  here a r b i t r a r i l y  response.  effects  which  divided into four categories of  R e s u l t s f o r males and females d i d not d i f f e r and  were t h e r e f o r e combined.  C o n t r o l s and nymphs  consuming  a z a d i r a c h t i n a t 3 jxg/g i n s e c t fwt molted n o r m a l l y t o a d u l t s (=Category I ) . of  A t 5, 8, and 10 \xq/q, i n c r e a s i n g p r o p o r t i o n s  t h e t r e a t e d nymphs molted t o a d u l t s w i t h deformed  and, a t h i g h e r doses, deformed  l e g s (=Category I I ) .  i n d i v i d u a l s took l o n g e r t o complete t h e molt.  wings These  The  d e f o r m i t i e s may have r e s u l t e d from t h e nymphs i n i t i a t i n g s c l e r o t i z a t i o n o f t h e a d u l t s t r u c t u r e s b e f o r e e c d y s i s was complete.  The d u r a t i o n o f t h e i n s t a r was a l s o  significantly  i n c r e a s e d a t 8 and 10 nq/q a z a d i r a c h t i n (12.0 + 1.4 days c f . 8.0 + 1.0 days f o r t h e c o n t r o l s ) .  A t 8 and 10 nq/q, a s m a l l  p r o p o r t i o n (<10%) o f t r e a t e d nymphs d i e d d u r i n g an incomplete molt attempt  (=Category I I I ) .  A notable  t r a n s i t i o n was seen between 10 and 13 nq/q: a t t h e lower dose most nymphs showed a c a t e g o r y I I response, but a t 13 nq/q the  a l l nymphs d i e d i n t h e molt.  A t 15 nq/q about 25% o f  nymphs e v e n t u a l l y d i e d without i n i t i a t i n g any molt  attempt  (Category I V ) , and a t 25 nq/q 80% o f the nymphs  showed t h i s response.  In some cases c a t e g o r y IV i n s e c t s  were observed t o l i v e f o r over 60 days without i n i t i a t i n g the  molt.  A t day 13 post-treatment t h e weight o f c a t e g o r y  IV nymphs exceeded t h e weight o f t h e c o n t r o l s a t m o l t i n g  234  Figure 5-1.  Morphogenic e f f e c t s of o r a l l y a d m i n i s t e r e d  a z a d i r a c h t i n on f i f t h - i n s t a r nymphs of Melanoplus sanguinipes.  Response c a t e g o r i e s i n c l u d e : Category I : no  m o r p h o l o g i c a l e f f e c t ; Category I I : molt t o an a d u l t w i t h deformed wings o r l e g s ; Category I I I : death a t a f a i l e d molt attempt; Category IV: death without i n i t i a t i n g a molt attempt.  236  F i g u r e 5-2.  E f f e c t of o r a l l y  a d m i n i s t e r e d a z a d i r a c h t i n on  m o l t i n g success o f f i f t h - i n s t a r nymphs of Melanoplus sanguinipes.  Responses are d i v i d e d i n t o f o u r c a t e g o r i e s : I,  no response except d e l a y of molt; I I , molt t o an a d u l t w i t h deformed wings o r l e g s ; I I I , death a t a f a i l e d molt attempt; and IV, death without i n i t i a t i n g a molt attempt.  Percent of treated population  Control  3  5  8  10  13  15  25  Azadirachtin dose ug/g insect fwt Category 1  Category 2  Category 3  Pill Category 4  238  (232.1 + 17.6 mg v s 207.6 ± 25.4 mg r e s p e c t i v e l y ) .  Grouping  c a t e g o r y 1 and 2 responses as s u r v i v o r s and c a t e g o r y 3 and 4 as m o r t a l i t i e s , t h e o r a l MD f i d u c i a l l i m i t s 10.34-11.44  50  was 10.83 /ig/g i n s e c t fwt (95% pg/g).  T o p i c a l a d m i n i s t r a t i o n o f a z a d i r a c h t i n produced t h e same range o f e f f e c t s , but a t lower doses.  A t 2 /ig/g o n l y  55% o f t h e nymphs molted t o normal a d u l t s , and a t 4 pg/g no nymphs molted n o r m a l l y .  Category 3 responses dominated  at 6  /ig/g, and a t 8 and 10 /ig/g category 4 responses were most prevalent.  The t o p i c a l MD  50  was 3.80 /ig/g i n s e c t fwt (95%  f i d u c i a l l i m i t s 3.196-4.353 /ig/g). When a z a d i r a c h t i n was i n j e c t e d d i r e c t l y i n t o t h e hemocoel, t h e same spectrum o f e f f e c t s was produced 5-3).  (Figure  Even a t 3 ng/g o n l y 20% o f t h e nymphs molted  n o r m a l l y ; c a t e g o r y I I and I I I responses were each 40%. A t doses o f 5, and p a r t i c u l a r l y 8 and 10 /ig/g, c a t e g o r y I I I responses were most common.  A t 8 and 10 /ig/g, some nymphs  f a i l e d t o molt; t h i s respose accounts f o r 80% o f t h e treatment group a t 15 /ig/g.  The MD  50  was 3.01 /ig/g (95%  f i d u c i a l l i m i t s 2.49-3.50 /i/g).  D. Synergism by p i p e r o n y l butoxide C o a d m i n i s t r a t i o n o f p i p e r o n y l butoxide (PBO) s i g n i f i c a n t l y i n c r e a s e d t h e o r a l t o x i c i t y o f a z a d i r a c h t i n i n M« s a n g u i n i p e s nymphs.  A t 4 ng/g, 100% o f t h e t r e a t e d  nymphs  showed a c a t e g o r y I o r I I response, but a t 6 /ig/g o n l y 57%  Figure 5-3.  E f f e c t of i n j e c t e d a z a d i r a c h t i n on m o l t i n g  success o f f i f t h i n s t a r nymphs o f Melanoplus s a n g u i n i p e s . Responses are d i v i d e d i n t o f o u r c a t e g o r i e s : I, no response except d e l a y of molt; I I , molt t o an a d u l t w i t h deformed wings o r l e g s ; I I I , death a t a f a i l e d molt attempt; and IV, death without i n i t i a t i n g a molt attempt.  Percent of treated population  Injected  Control  3  5  8  10  15  Azadirachtin dose ug/g Insect fwt Category 1  i i i l Category 2  Category 3  Category 4 ro o  241  F i g u r e 5-4.  E f f e c t of t o p i c a l l y a p p l i e d a z a d i r a c h t i n on  m o l t i n g success of f i f t h - i n s t a r nymphs of Melanoplus sanguinipes.  Responses are d i v i d e d i n t o f o u r c a t e g o r i e s : I,  no response except d e l a y o f molt; I I , molt t o an a d u l t w i t h deformed wings o r l e g s ; I I I , death a t a f a i l e d molt attempt; and IV, death without i n i t i a t i n g a molt attempt.  Percent of treated population  Control  2  4  6  8  Azadirachtin dose ug/g Insect fwt Category 1  i l l Category 2  Category 3  Category 4  -F  243  Figure 5 - 5 . (PBO)  E f f e c t of co-administered  piperonyl  on the molt i n h i b i t o r y a c t i v i t y of o r a l l y  azadirachtin.  In t h i s f i g u r e c a t e g o r y I and  are combined t o g i v e s u r v i v o r s h i p curves.  butoxide administered  I I responses  The  e f f e c t of  i n j e c t e d and t o p i c a l l y a p p l i e d a z a d i r a c h t i n i s a l s o shown.  D O M (ug/g Intact fwt)  245  molted s u c c e s s f u l l y . III  A t 8 /ig/g most nymphs showed a type  response and a t 10 ng/g  response. /xg/g  The o r a l MD  i n s e c t fwt.  50  most showed a c a t e g o r y IV  of azadirachtin  p l u s PBO was 6.5  PBO alone had no e f f e c t on i n s t a r  length  or molt s u c c e s s .  E. F e r t i l i t y  Experiment  Azadirachtin  decreased the number o f egg masses and eggs  produced over t h e f i r s t s i x weeks o f t h e a d u l t stage, i n a dose-dependent manner ( F i g u r e  5-6).  O v e r a l l t h e r e was a  h i g h l y s i g n i f i c a n t dose-response, w i t h an F I fertility  5 0  i n h i b i t i o n ) o f 35 /ig a z a d i r a c h t i n / g  (50% insect.  This  appeared t o be due t o an a z a d i r a c h t i n - i n d u c e d dose-dependent d e l a y i n t h e time t o p r o d u c t i o n and s i z e o f t h e f i r s t egg mass.  Survivorship  t o t h e end o f t h e 6-week assay was 100%  i n a l l treatment groups.  F. S t e r o l supplementation assays Supplementing t h e d i e t w i t h c h o l e s t e r o l o r B - s i t o s t e r o l d i d not  s i g n i f i c a n t l y influence  the t o x i c i t y of o r a l l y -  administered a z a d i r a c h t i n .  The s t e r o l s alone had no e f f e c t  on molt success o r d u r a t i o n  of the i n s t a r .  Treatment w i t h a z a d i r a c h t i n  d i d not a b o l i s h t h e  movement o f r a d i o l a b e l l e d s t e r o l i n t o t h e hemolymph from t h e gut  (Figure  5-7).  In both c o n t r o l and t r e a t e d  nymphs  r a d i o l a b e l f i r s t appeared i n t h e hemolymph 4 h a f t e r with  1 4  C-B-sitosterol.  In c o n t r o l s , t h e amount o f  feeding  Figure 5-6.  Effect  of o r a l l y administered  a d u l t female f e c u n d i t y .  azadirachtin  Each p o i n t r e p r e s e n t s  f i v e females; c o n t r o l s produced 4 2 + 1 1  on  the mean of  eggs/female.  Fedundity (% o f C o n t r o l ) = 1 1 0 - 1 . 7 [ A a z d i r a c h t i n ]  r =.96 2  cn  F i g u r e 5-7.  Pharmacokinetics o f r a d i o l a b e l l e d s t e r o l s i n  the hemolymph o f c o n t r o l and a z a d i r a c h t i n  treated  nymphs.  Each p o i n t r e p r e s e n t s t h e mean o f t h r e e i n d i v i d u a l nymphs.  250  r a d i o a c t i v i t y increased  t o a peak a t 10 h  whereas, i n a z a d i r a c h t i n - t r e a t e d  post-feeding  nymphs, the i n c r e a s e  more g r a d u a l and d i d not peak d u r i n g  was  the course o f the  experiment. Chromatography o f TFA d e r i v a t i v e s of the s t e r o l f r a c t i o n from c o n t r o l and a z a d i r a c h t i n t r e a t e d  insects  i n d i c a t e d t h a t / 3 - s i t o s t e r o l was p r i m a r i l y m e t a b o l i z e d t o c h o l e s t e r o l i n M - sanguinipes. r e l a t i v e proportion  No d i f f e r e n c e s i n t h e  of c h o l e s t e r o l , 6 - s i t o s t e r o l , and  desmosterol were e v i d e n t  between the two treatment groups  (Table 5-2).  G. In v i t r o assay f o r adduct f o r m a t i o n Chromatography o f a l i q u o t s of an equimolar mixture o f a z a d i r a c h t i n and c y s t e i n e  i n pH 7.0 phosphate b u f f e r d i d not  i n d i c a t e the f o r m a t i o n of adducts between these Azadirachtin detected  (Rf=0.98) and c y s t e i n e  compounds.  (Rf=0.37) c o u l d be  i n a l i q u o t s taken a t a l l time i n t e r v a l s ; a f t e r one  hour c y s t i n e (Rf=0.31), the o x i d a t i o n product o f c y s t e i n e , was a l s o  detected.  251  T a b l e 5-2.  Radioladelled  azadirachtin-treated  Treatment Control 15 ug/g  aza  %  s t e r o l composition of c o n t r o l  M. s a n g u i n i p e s f e d  Cholesterol  and  4- C-B-sitosterol. 1 4  % B-Sitosterol  %  Desmosterol  72.7  26.1  1.2  68.7  29.9  1.4  Discussion  A. A n t i f e e d a n t  and n u t r i t i o n a l e f f e c t s  A z a d i r a c h t i n had no chemosensory-based a n t i f e e d a n t a g a i n s t M. s a n g u i n i p e s nymphs a t c o n c e n t r a t i o n s  activity  up t o 500  /ig/g, as t r e a t e d l e a f d i s c s were u s u a l l y consumed i n a s i n g l e f e e d i n g bout.  However, t h e RCRi was s i g n i f i c a n t l y  reduced f o l l o w i n g a z a d i r a c h t i n i n g e s t i o n , i n d i c a t i n g an a c t i o n a g a i n s t t h e gut o r on t h e n e u r a l r e g u l a t i o n o f feeding. RGRi.  T h i s e f f e c t r e s u l t e d i n a s i g n i f i c a n t decrease i n  Reduced consumption f o l l o w i n g a z a d i r a c h t i n  administration,  i n t h e absence o f chemosensory  has been noted w i t h other  inhibition,  i n s e c t s (Rembold e t a l . , 1980;  Redfern e t a l . , 1981; Schmutterrer, 1985; Mordue e t a l . , 1985; Simmonds and Blaney, 1984) and i n L o c u s t a may be due t o a decreased r a t e o f gut p e r i s t a l s i s 1985).  (Mordue e t a l . ,  These r e s u l t s suggest t h a t b i o a s s a y s  extending  beyond a s i n g l e f e e d i n g bout cannot d i s t i n g u i s h between a t r u e chemosensory response (deterrency)  and t o x i c i t y w i t h  t h i s compound; a t l e a s t some o f t h e many r e p o r t s o f t h e antifeedant  e f f e c t o f a z a d i r a c h t i n may be due t o p o s t -  ingestive effects.  However, e l e c t r o p h y s i o l o g i c a l s t u d i e s  and c h o i c e t e s t s show unambiguously t h a t a z a d i r a c h t i n does have a chemosensory-based a n t i f e e d a n t  e f f e c t a g a i n s t some  i n s e c t s (Schoonhoven, 1982; Simmonds and Blaney, 1984).  In  S c h i s t o c e r c a g r e g a r i a a z a d i r a c h t i n may a c t i v a t e a s p e c i f i c " l a b e l l e d l i n e " deterrent receptor  (Blaney, 1980).  Neither  253  the ECI  nor the ECD  increased  were reduced s i g n i f i c a n t l y ; the AD  o n l y a t the h i g h e r  concentration.  Similar results  have been noted i n s t u d i e s w i t h the l e p i d o p t e r a n s Klocke, 1987)  and  was  Heliothis  virescens  (Barnby and  Crocidolomia  binotalis  (Fagoonee, 1984); i n both cases consumption  was  decreased by a z a d i r a c h t i n without concomitant r e d u c t i o n s e f f i c i e n c y of d i e t a r y u t i l i z a t i o n , and l a t t e r i n s e c t both the ECI fora reduction  and  i n food volume.  ECD  i n f a c t f o r the  i n c r e a s e d t o compensate  In c o n t r a s t , Arnason e t a l .  (1985) found t h a t dose-dependent r e d u c t i o n s O s t r i n i a n u b i l a l i s were due consumption r a t e was concentrations  to a reduction  unaffected  in  by  i n growth of i n ECI  and  ECD;  azadirachtin  i n a r t i f i c i a l d i e t up t o 30 ppm.  r e s u l t s c o u l d be owing t o the o b s t r u c t i o n of the  Their sensillar  pores on the mouthparts by the agar-based d i e t (Arnason, p e r s . comm. 1988).  The  limonoids  a l s o reduce the ECI  and  ECD  c e d r e l o n e and  at concentrations  anthothecol  which a c t u a l l y  i n c r e a s e the consumption r a t e i n O s t r i n i a n u b i l a l i s e t a l . , 1987).  Rao  and  Subramanyam (1986) found t h a t  a z a d i r a c h t i n lowers the RCR, Schistocerca gregaria. unaffected  (Arnason  ECI,  and  ECD  in fifth-instar  In a l l s t u d i e s t o date the AD  is  or i n c r e a s e s w i t h i n c r e a s i n g a z a d i r a c h t i n  concentration.  Such a phenomena would r e s u l t from a  decreased r a t e of gut p e r i s t a l s i s  (Mordue §t  a l . , 1985),  i n c r e a s i n g the amount of time d u r i n g which the food b o l u s i s exposed t o d i g e s t i v e enzymes (Slansky  and  S c r i b e r , 1985).  254  Most n o t a b l e i s the absence o f any d i f f e r e n c e i n growth, consumption, o r n u t r i t i o n a l u t i l i z a t i o n between 10 and 15 nq/q  indices  i n s e c t , as these doses produce  remarkably d i f f e r e n t e f f e c t s on m o l t i n g .  A t the lower dose,  most nymphs molted s u c c e s s f u l l y , a l b i e t t o malformed  adults,  whereas a t the h i g h e r dose a l l nymphs d i e d i n the molt.  The  l a c k o f c o r r e l a t i o n between n u t r i t i o n a l and presumably endocrine-mediated e f f e c t s suggests t h a t s e p a r a t e p h y s i o l o g i c a l t a r g e t s may  be i n v o l v e d .  B. O r a l , t o p i c a l , and i n j e c t i o n experiments M. s a n g u i n i p e s nymphs which consumed a z a d i r a c h t i n a t t h e b e g i n n i n g of t h e i n s t a r subsequently d i s p l a y e d a dosedependent range o f e f f e c t s .  Low doses (<10 nq/q  r e s u l t e d i n molt d e l a y and d e f o r m i t y o f a d u l t i n t e r m e d i a t e doses (13-15 nq/q)  appendages,  r e s u l t e d i n death a t a  f a i l e d molt attempt, and h i g h doses (>25 nq/q) permanent  produced  f i f t h i n s t a r nymphs i n which t h e m o l t i n g response  was a b o l i s h e d .  These e f f e c t s p a r a l l e l those seen w i t h  Locusta (Mordue e t a l . , al-,  insect)  1985, 1986), Oncopeltus (Redfern e t  1982), and o t h e r i n s e c t s (Gaaboub and Hayes,  Koul, 1984; Ladd e t a l . ,  1984;  1984).  Molt f a i l u r e has been a t t r i b u t e d t o d i s r u p t i o n of the normal e c d y s t e r o i d t i t r e s i n s e v e r a l s p e c i e s (Koul e t a l . . 1987; Rembold and S i e b e r , 1981; Rembold g t a l . , Schluter et a l . , Shin-Foon, 1987).  1984;  1985; Mordue and Evans, 1987; M i n - L i and However, the p h y s i o l o g i c a l b a s i s of t h i s  255  d i s r u p t i o n i s not known.  Mordue e t aJL.(1986) suggested t h a t  f a i l u r e t o a t t a i n c r i t i c a l weight was r e s p o n s i b l e e c d y s i s i n Locusta  for failed  m i g r a t o r i a , but t h e lowest dose a t which  they measured growth ( 7 . 5 /ig) was almost t h r e e times t h e dose r e q u i r e d t o i n h i b i t m o l t i n g .  I n t h i s study f a i l u r e t o  molt was not due t o a f a i l u r e t o reach  critical  body mass as  the weight o f Category IV nymphs a t d 13 post-treatment ( 2 3 2 . 1 + 1 7 . 6 mg) exceeded t h e weight o f t h e c o n t r o l s a t molting  ( 2 0 7 . 6 ± 2 5 . 4 mg).  T h i s i m p l i e s a more d i r e c t  a c t i o n on the endocrine system, r a t h e r than an i n d i r e c t e f f e c t o p e r a t i n g through i n t e r n a l measures o f growth. A p p l i c a t i o n o f a z a d i r a c h t i n t o p i c a l l y o r by intrahemocoelic  i n j e c t i o n a l s o r e u l t e d i n dose-dependent  i n h i b i t i o n of molting. i n M. s a n g u i n i p e s . for  Locusta  The M D  50  f o r injected azadirachtin  3.01 /ig/g, was h i g h e r than t h a t  migratoria,  reported  2.0 /ig/g (Mordue e t a l . , 1985) o r  S c h i s t o c e r c a g r e g a r i a , 1.66 /ig/g (Rao and Subrahmanyam, 1986).  A z a d i r a c h t i n i s a l s o a c u t e l y t o x i c t o some i n s e c t s  when i n j e c t e d a t h i g h doses: t h e 24 h L D  5 0  f o r Locusta  m i g r a t o r i a i s 80 /ig/g (Mordue ej£ a l . , 1985? Cottee e t a l . . 1 9 8 8 ) , and f o r S c h i s t o c e r c a g r e g a r i a t h e L D (Cottee  e t a l . , 1988).  5 0  i s 330 /ig/g  Acute t o x i c i t y t o M. sanguinipes was  not observed a t t h e h i g h e s t dose t e s t e d i n t h i s study, 25  The  MD  50  v i a oral administration  t h r e e times t h a t v i a i n j e c t i o n  (10.83 /ig/g) was over  ( 3 . 0 1 /ig/g), i n d i c a t i n g t h a t  the gut poses a p h y s i c a l o r p h y s i o l o g i c a l b a r r i e r t o  256  azadirachtin bioavailability.  The gut presents  a similar  b a r r i e r t o s e v e r a l other n a t u r a l products i n Locusta m i g r a t o r i a and S c h i s t o c e r c a g r e g a r i a , i n d i c a t e d by s i m i l a r r a t i o s o f o r a l t o i n j e c t e d acute t o x i c i t y 1988).  The o b s e r v a t i o n  t h a t the o r a l t o x i c i t y o f  a z a d i r a c h t i n can be s y n e r g i s e d coadministration  (Cottee ej: a l . ,  i n M- s a n g u i n i p e s by  o f t h e m i x e d - f u n c t i o n oxidase  inhibitor  p i p e r o n y l butoxide suggests t h a t t h e b a r r i e r i s l a r g e l y owing t o MFO based o x i d a t i v e metabolism.  The s l o p e o f t h e  dose-response curve was t h e same i n t h e o r a l and i n j e c t i o n assays.  The t o x i c i t y curve f o r t h e o r a l dose-response i s  s h i f t e d by 10 /ig/g r e l a t i v e t o t h e i n j e c t e d dose-response suggesting  t h a t t h e gut MFO's a r e a b l e t o m e t a b o l i s e up t o  10 ng a z a d i r a c h t i n / g  i n s e c t , a l l o w i n g a z a d i r a c h t i n i n excess  of t h i s dose t o penetrate  t o target s i t e s within the i n s e c t .  In c o n t r a s t , a z a d i r a c h t i n was e q u i t o x i c when a p p l i e d topically  (MD = 3.80 /ig/g) o r v i a i n j e c t i o n (95% f i d u c i a l 50  l i m i t s overlap), i n d i c a t i n g that i n t h i s insect the integument does not s i g n i f i c a n t l y l i m i t p e n e t r a t i o n and therefore b i o a v a i l a b i l i t y of azadirachtin t o putative s i t e s within the i n s e c t . have c o n s i d e r a b l e  target  In each case a z a d i r a c h t i n must  s t a b i l i t y once i n s i d e t h e i n s e c t , as i t s  e f f e c t s on m o l t i n g  a r e expressed 8 t o 14 days a f t e r exposure  t o a s i n g l e dose.  Rembold e t a l (1984) found t h e h a l f - l i f e  of i n j e c t e d H - d i h y d r o a z a d i r a c h t i n 3  i n Locusta  migratoria.  t o be g r e a t e r than 1 week  257  The  response o f M.. s a n g u i n i p e s t o a z a d i r a c h t i n  c o n t r a s t s w i t h i t s response t o sesquiterpene b i t t e r p r i n c i p l e s from Asteraceous p l a n t s Parthenin, LD  5 0  lactones,  (Isman, 1985).  t h e most a c t i v e o f s i x compounds t e s t e d , had a  o f 1.5 /imol/g i n s e c t fwt when i n j e c t e d i n t o t h e  hemocoel, compared t o a L D azadirachtin.  5 0  o f 4.4 nmol/g (= 3.2 /ig/g) f o r  However, a d u l t males were a b l e t o t o l e r a t e  exposure t o doses up t o 12 /imol/g, o r a l l y , without t o x i c symptoms. therefore provide sesquiterpene azadirachtin.  applied t o p i c a l l y or  The gut and integument  e f f e c t i v e barriers to b i o a v a i l a b i l i t y of  lactones, unlike the s i t u a t i o n with The f a c t o r s a l l o w i n g p e n e t r a t i o n o f some  compounds and e x c l u s i o n o f o t h e r s  a r e not w e l l understood i n  t h i s i n s e c t : although l i p o p h i l i c i t y may p l a y a r o l e i t i s u n l i k e l y t o be a dominant f a c t o r as both h y d r o p h i l i c ( i . e . a z a d i r a c h t i n ) and s t r o n g l y hydrophobic compounds (eg at e r t h i e n y l , a thiophene) ( S m i r l e , Champagne, Isman, u n p u b l i s h e d data) a r e absorbed  C. F e c u n d i t y  efficiently.  Experiment  A z a d i r a c h t i n , f e d t o a d u l t females, reduced egg p r o d u c t i o n d u r i n g the f i r s t s i x weeks o f t h e a d u l t stage. i s s i m i l a r t o the reported Locusta migratoria  This e f f e c t  c h e m o s t e r i l i z a t i o n o f female  (Rembold and S i e b e r ,  1981), Oncopeltus  f a s c i a t u s (Dorn e t a l . , 1986), Dysdercus k o e n i g i i  (Koul,  1984), and Rhodnius p r o l i x u s (Feder e t a l . , 1989).  In  L o c u s t a t h i s e f f e c t has been a s c r i b e d t o d i m i n i s h e d  JH and  258  e c d y s t e r o i d t i t r e s , and i n Rhodnius a z a d i r a c h t i n lowered hemolymph e c d y s t e r o i d and v i t e l l o g e n i n t i t r e s , and impared ecdysteroid synthesis  i n the o v a r i e s .  As a d u l t female  Melanoplus s a n g u i n i p e s s u r v i v e i n t h e f i e l d f o r o n l y 25-33 days on average, t h e e f f e c t s r e p o r t e d here c o u l d r e s u l t i n a significant decline i n f i e l d  populations.  D. S t e r o l s t u d i e s Molting  i s t h e end r e s u l t o f a complex s e r i e s o f  p h y s i o l o g i c a l events dependent on proper f u n c t i o n i n g o f s e v e r a l organ systems i n c l u d i n g t h e gut ( N i j h o u t , 1981).  As  suggested by Mordue e t a l . (1985, 1986; Mordue and Evans, 1987), t h e observed endocrine e f f e c t s o f a z a d i r a c h t i n be due t o t h e d i s r u p t i o n o f some aspect  o f gut f u n c t i o n  necessary f o r the i n i t i a t i o n of the molting Azasterols i n h i b i t the conversion  process.  o f p h y t o s t e r o l s such as 6-  s i t o s t e r o l t o c h o l e s t e r o l by i n h i b i t i n g t h e A  2 2  and A  s t e r o l reductases, intermediate  could  22,24  r e s u l t i n g i n t h e accumulation o f t h e  desmosterol (Svoboda and Robbins, 1971; Svoboda  e t a l . , 1972; Walker and Svoboda, 1973; Svoboda and Thompson, 1985).  As c h o l e s t e r o l i s r e q u i r e d f o r t h e  assembly o f normal membranes and as a p r e c u r s o r f o r ecdysteriod synthesis, azasterol t o x i c i t y i s characterized by i n h i b i t i o n o f growth and m o l t i n g , c h e m o s t e r i l i z a t i o n o f a d u l t females.  and by These e f f e c t s may be  a l l e v i a t e d by p r o v i d i n g a source o f c h o l e s t e r o l i n t h e d i e t (Walker and Svoboda, 1973).  The molt i n h i b i t i n g e f f e c t s o f  259  a z a d i r a c h t i n were not a l l e v i a t e d by supplementation w i t h d i e t a r y c h o l e s t e r o l , i n d i c a t i n g that the target involved  i s probably not t h e A  2  2  and  site  ^22,24 s t e r o l  reductases. As  i n s e c t s a r e unable t o s y n t h e s i z e  ( C l a r k e and B l o c h , 1959), t r a n s p o r t of a b s o r b t i o n  s t e r o l s de novo  o f s t e r o l from t h e s i t e  to t a r g e t t i s s u e s i s of c r i t i c a l  This transport  importance.  i s accomplished v i a a hemolymph l i p o p r o t e i n  termed l i p o p h o r i n  (Chino, 1985), which loads c h o l e s t e r o l  d i r e c t l y from t h e midgut (Chino and G i l b e r t , 1971). Lipophorin  i s also involved  i n the transport  of f a t t y acids,  mainly i n t h e form o f d i a c y l g l y c e r o l , and i s able t o b i n d l i p o p h i l i c p e s t i c i d e s and a l l e l o c h e m i c a l s Bowers, 1986).  (Haunerland and  In M. s a n g u i n i p e s nymphs, r a d i o l a b e l l e d B-  s i t o s t e r o l was observed t o c r o s s t h e gut and appear i n t h e hemolymph, r u l i n g out i n h i b i t i o n o f s t e r o l t r a n s p o r t mechanism o f a c t i o n f o r a z a d i r a c h t i n  i n this insect.  as a The  three-hour time l a g between consumption and the f i r s t appearance o f r a d i o l a b e l l e d s t e r o l i n t h e hemolymph suggests that s t e r o l adsorbtion M. s a n g u i n i p e s . in  probably o c c u r r s  In g e n e r a l ,  from t h e midgut i n  the s i t e of s t e r o l  absorbtion  i n s e c t s appears t o be r e l a t e d t o t h e type o f food used:  c a r n i v o r o u s and omnivorous i n s e c t s adsorb from the crop, whereas phytophagous i n s e c t s absorb s t e r o l s from the midgut (Clayton  e t a l . , 1964; J o s h i and Agarwal, 1977; K u t h i a l a and  R i t t e r , 1988).  In p a r t i c u l a r , i n t h e few phytophagous  o r t h o p t e r a n s s t u d i e d t o date, i n c l u d i n g  Schistocerca  260  g r e g a r i a , s t e r o l a b s o r b t i o n o c c u r s from t h e g a s t r i c ( J o s h i and Agarwal, 1977).  caceae  The decreased r a t e o f appearance  of r a d i o l a b e l i n t h e hemolymph o f a z a d i r a c h t i n - t r e a t e d nymphs i s c o n s i s t a n t w i t h a decreased r a t e o f gut peristalsis.  The decrease i n r a d i o l a b e l content o f c o n t r o l  hemolymph a t 11 and 12 h i s probably due t o s t e r o l  unloading  a t t h e f a t body and o t h e r t a r g e t t i s s u e s ; i n H e l i o t h i s zea a s i m i l a r decrease i n hemolymph s t e r o l was a s s o c i a t e d w i t h an i n c r e a s e i n the amount o f r a d i o l a b e l a s s o c i a t e d w i t h t h e f a t body ( K u t h i a l a and R i t t e r , 1988). experiment  Unfortunately t h i s  d i d not extend f o r l o n g enough t o observe a  s i m i l a r decrease i n r a d i o l a b e l content o f hemolymph  from  a z a d i r a c h t i n - t r e a t e d nymphs, so t h e e f f e c t o f a z a d i r a c h t i n on s t e r o l u n l o a d i n g remains unknown.  I t i s not known i f  c h o l e s t e r o l u n l o a d i n g i s p a s s i v e o r an a c t i v e , r e c e p t o r mediated process i n i n s e c t s (Chino, 1985). The i d e n t i t y o f r a d i o l a b e l l e d s t e r o l s i n t h e i n s e c t 24 h a f t e r feeding with  1 4  C - B - s i t o s t e r o l i s g i v e n i n T a b l e 5-2.  The s t e r o l composition o f c o n t r o l and a z a d i r a c h t i n - t r e a t e d i n s e c t s does not d i f f e r , c o r r o b o r a t i n g the r e s u l t s o f t h e s t e r o l supplementation  experiment  and r u l i n g out s t e r o l  r e d u c t a s e i n h i b i t i o n as a mechanism o f a c t i o n .  The s t e r o l  composition o f M. s a n g u i n i p e s i s dominated by c h o l e s t e r o l , w i t h a l e s s e r amount o f B - s i t o s t e r o l and < 2% o f t h e i n t e r m e d i a t e desmosterol.  T h i s p a t t e r n i s common t o most  phytophagous i n s e c t s , which a r e a b l e t o d e a l k y l a t e C Q and 2  C g 2  phytosterols to cholesterol  (Svoboda and Thompson,  261  1985).  Although some i n s e c t s accumulate l a r g e amounts of  cholesterol esters  (Svoboda and  Thompson, 1985), these were  not observed i n the M.. s a n g u i n i p e s e x t r a c t s ; however d e r i v i t i z a t i o n w i t h t r i f l u o r o a c e t i c a c i d may ester  function.  E.  vitro  In  The  remove the  f o r m a t i o n of adducts  t o x i c a c t i o n of a number of p l a n t products  the f o r m a t i o n of c o v a l e n t a l l e l o c h e m i c a l and  M i c h a e l adducts between the  s u l f h y d r y l residues  i n c l u d e the s e s q u i t e r p e n e l a c t o n e s warburganal (Ma,  1977).  involves  of p r o t e i n s ;  examples  (Pieman e t a l . , 1979)  Such a mechanism of a c t i o n has  proposed t o account f o r the c y t o t o x i c i t y of  Two  tissues possibly involved  s u l f h y d r y l residues 1980).  ( N o r r i s , 1988;  pars  Loughton,  spontaneously  form adducts w i t h c y s t e i n e , a t l e a s t a t n e u t r a l suggesting that non-specific binding  the  rich in  F r i e d e l and  I found t h a t a z a d i r a c h t i n does not  (Pettit  in  a z a d i r a c h t i n t o x i c i t y , mouthpart chemoreceptors and i n t e r c e r e b r a l i s , contain protein unusually  been  several  l i m o n o i d s t o murine P-388 lymphocytic leukemia c e l l s e t a l . , 1983).  and  pH,  to s u l f h y d r y l - r i c h  p r o t e i n i s u n l i k e l y t o p l a y an important r o l e i n azadirachtin t o x i c i t y . observation extracted  This i s consistant with  t h a t unbound a z a d i r a c h t i n may  the  be r e a d i l y  from L o c u s t a b r a i n s as much as one  week a f t e r  i n j e c t i o n of the compound (H. Rembold, pers comm 1988). w e l l , a z a d i r a c h t i n t r e a t e d s e n s i l l a e r e t u r n t o normal  As  262  f u n t i o n i n g w i t h i n 2-5 min (Simmonds and Blaney, 1984), i n d i c a t i n g that the receptors  have not s u f f e r e d i r r e v e r s a b l e  damage such as i s produced by s u l f h y d r y l reagents (Ma. 1977). The  p o s s i b i l i t y remains t h a t o t h e r t a r g e t s i n t h e gut  can be a f f e c t e d by a z a d i r a c h t i n , f o r example t h e r e l e a s e o f factors involved synthesis  i n stimulating elevated  r a t e s of p r o t e i n  i n the f a t body, r e c e n t l y demonstrated i n L o c u s t a  migratoria  (Laughton e t a l . , 1987).  between f e e d i n g  As the r e l a t i o n s h i p  and endocrine events has been p a r t i c u l a r l y  w e l l s t u d i e d i n Jf- s a n g u i n i p e s (Dogra and G i l l o t t , 1971; E l l i o t t and G i l l o t t ,  1977a,b), t h i s i n s e c t may p r o v i d e a  convenient model system f o r f u r t h e r s t u d i e s on t h e mode o f action of azadirachtin. F. A g r i c u l t u r a l i m p l i c a t i o n s Grasshoppers p e r i o d i c a l l y i n f l i c t severe damage on c e r e a l c r o p s and rangeland f o r a g e (Bierne, Onsager, 1983).  1971; Hewitt and  Melanoplus s a n g u i n i p e s i s always a major  c o n t r i b u t o r t o grasshopper outbreaks, and o v e r a l l i s considered agriculture  the f o u r t h most damaging p e s t i n s e c t t o Canadian (Bierne,  a e r i a l spraying al.,  1971).  C u r r e n t l y , c o n t r o l r e l i e s on  of the pyrethroids  deltamethrin  1986), cypermethrin, and c a r b a r y l  (Mukerji  (Johnson e t and Ewen,  1984); dimethoate, m a l a t h i o n , and methamidophos a r e a l s o used ( H a r r i s , 1985).  As t h i s method o f a p p l i c a t i o n may have  severe impact on non-target i n s e c t s , e s p e c i a l l y p o l l i n a t o r s ,  263  recent  work has focussed  on use o f b a i t , p a r t i c u l a r l y wheat  bran, impregnated w i t h i n s e c t i c i d e (Onsager e t a i . , Mukerji e t a l . ,  1980a,b;  1981; M u k e r j i and Ewen, 1984; Johnson and  Henry, 1986) o r t h e pathogen Nosema l o c u s t a e Johnson and P a v l i k o v a ,  (Henry, 1972;  1986; Johnson and Henry, 1986).  At  s u b l e t h a l doses N. l o c u s t a e reduces f e e d i n g and reproduction.  The a p p l i c a t i o n o f bran b a i t i s f a c i l i t a t e d  by t h e d i s t r i b u t i o n o f grasshoppers, which tend t o c o n c e n t r a t e w i t h i n 10-15 m of r o a d s i d e s  (Bierne, 1971;  Johnson and Henry, 1986). As a z a d i r a c h t i n does not produce an a n t i f e e d a n t response i n M. s a n g u i n i p e s . i t would appear t o have p o t e n t i a l as an i n s e c t i c i d e a p p l i e d t o b a i t s .  Application  would have t o be timed t o c o i n c i d e w i t h t h e presence o f e a r l y i n s t a r s , b e f o r e t h e nymphs a r e capable o f economic l e v e l s o f damage, as m o r t a l i t y would occur o n l y a t m o l t i n g , some days a f t e r a p p l i c a t i o n .  Application l a t e r i n the l i f e  c y c l e c o u l d produce permanent nymphs. of a z a d i r a c h t i n c o u l d residue  Advantages t o t h e use  i n c l u d e minimal non-target impact, low  l e v e l s due t o t h e r a p i d photodegradation o f  azadirachtin i n sunlight possibly compatability i n c l u d i n g N. l o c u s t a e .  (Yamasaki e t a l . ,  1988), and  w i t h b i o l o g i c a l c o n t r o l measures Even a t s u b l e t h a l doses a z a d i r a c h t i n  based treatments c o u l d be expected t o reduce f e e d i n g and reproduction. A l l t h e c u r r e n t l y a v a i l a b l e i n s e c t i c i d e s share a common mode o f a c t i o n , n e u r o t o x i c i t y ; a z a d i r a c h t i n c o u l d p r o v i d e an  264  a l t e r n a t i v e w i t h a r a d i c a l l y d i f f e r e n t mode o f a c t i o n , m i n i m i z i n g the chances o f c r o s s - r e s i s t a n c e . Where a z a d i r a c h t i n has a n t i f e e d a n t  activity  In i n s e c t s ( i e P.. s a u c i a ) ,  the development o f r e s i s t a n c e would i n v o l v e overcoming both the chemosensory and t h e p h y s i o l o g i c a l e f f e c t s . two  F o r example  s t r a i n s o f P l u t e l l a x y l o s t e l l a showed no evidence o f  r e s i s t a n c e i n f e e d i n g and f e c u n d i t y t e s t s a f t e r 35 g e n e r a t i o n s o f exposure t o neem seed e x t r a c t ; t h e same two s t r a i n s developed r e s i s t a n c e f a c t o r s o f 20-35 t o deltamethrin  i n t h e same time p e r i o d  ( V o l l i n g e r , 1986).  P o p u l a t i o n s which became r e s i s t a n t t o d e l t a m e t h r i n  d i d not  show c r o s s - r e s i s t a n c e t o neem ssed e x t r a c t .  However, M -  sanguinipes already  activity  posseses s i g n i f i c a n t MFO  a g a i n s t a z a d i r a c h t i n and l a c k s an a n t i f e e d a n t  response, and  so c o u l d be expected t o develop r e s i s t a n c e f a i r l y r a p i d l y i n the presence o f s t r o n g  s e l e c t i o n pressure.  A sucessful  pest  management s t r a t e g y w i l l have t o r e l y on a v a r i e t y o f c o n t r o l measures, employing a v a r i e t y o f modes o f a c t i o n , j u d i c i o u s l y a p p l i e d t o minimize t h e chance o f d e v e l o p i n g resistance.  265  Chapter 6: General Summary  The work d e s c r i b e d i n the p r e v i o u s f o u r c h a p t e r s examined s e v e r a l a s p e c t s of the p u t a t i v e defenses a g a i n s t h e r b i v o r o u s i n s e c t s found i n members of the p l a n t f a m i l y M e l i a c e a e .  The  s t u d i e s began w i t h a p r e l i m i n a r y examination of the r e l a t i o n s h i p between defense s t r a t e g i e s and p l a n t  life-  h i s t o r y c h a r a c t e r i s t i c s , moved t o an attempt t o i d e n t i f y the phytochemicals i n v o l v e d , and concluded w i t h d e t a i l e d i n v e s t i g a t i o n s of the e f f e c t s and mode of a c t i o n of the major group of phytochemicals i n v o l v e d , the l i m o n o i d s , i n t h r e e model i n s e c t s p e c i e s . In the f i r s t study, r e l a t i v e investment i n phytochemical-based was  defenses i n t h i r t y s p e c i e s of M e l i a c e a e  e s t i m a t e d , by i n v e s t i g a t i n g t h e response of an  unadapted, g e n e r a l i s t h e r b i v o r e , Peridroma s a u c i a e n t i r e s u i t e of phytochemicals produced, methanolic e x t r a c t of mature f o l i a g e .  P h y s i c a l defenses i n  f a c t o r s ; consequently l e a f toughness was Leaf l i f e t i m e was  t o the  as i n c l u d e d i n the  the Meliaceae are l a r g e l y c o n f i n e d t o l e a f  species.  f  toughness  measured on s i x t e e n  not measured d i r e c t l y , but a l l  s p e c i e s were c l a s s e d as deciduous or evergreen, w i t h the assumption  t h a t l e a f l i f e t i m e s would be l o n g e r f o r the  evergreen s p e c i e s .  Large d i f f e r e n c e s were found between  s p e c i e s i n the r e l a t i v e investment i n phytochemical defenses.  E x t r a c t s of some s p e c i e s , p a r t i c u l a r l y i n the  t r i b e M e l i e a e , were i n h i b i t o r y t o E .  s a u c i a growth a t  266  c o n c e n t r a t i o n s o n l y 1% of those o c c u r r i n g n a t u r a l l y i n the foliage.  The most a c t i v e e x t r a c t s were a l l from members of  the s u b f a m i l y M e l i o i d e a e , whereas e x t r a c t s from the s u b f a m i l y Swietenioideae  were on average l e s s a c t i v e .  t h r e e s p e c i e s appeared t o l a c k phytochemical  Only  defenses  a g a i n s t g e n e r a l i s t h e r b i v o r e s , assuming £. s a u c i a i s a v a l i d model s p e c i e s . The p l a n t apparency h y p o t h e s i s of Feeny (1976) and Rhoades and Cates (1976) p r e d i c t s s i m i l a r defenses  in a l l  the s p e c i e s s t u d i e d here, as they are a l l "apparent", perennial tree species. by my  T h i s p r e d i c t i o n was  not  supported  d a t a , as l a r g e d i f f e r e n c e s between s p e c i e s i n  d e f e n s i v e a t t r i b u t e s were found.  The  resource  availability  h y p o t h e s i s of Coley e_t a l . (1985), on the other hand, p r e d i c t s that species with short l e a f l i f e t i m e s ( i . e . deciduous s p e c i e s i n t h i s study) should be s e l e c t e d f o r phytochemically-based  defenses,  l i f e t i m e s ( i . e . evergreen defenses  and s p e c i e s with l o n g l e a f  s p e c i e s ) should e l a b o r a t e p h y s i c a l  i n c l u d i n g l e a f toughness.  In t h i s study, e x t r a c t s from deciduous s p e c i e s were found t o be s i g n i f i c a n t l y more i n h i b i t o r y t o Egrowth.  saucia  P r e v i o u s attempts t o q u a n t i f y investment  chemical defenses  in  have r e l i e d on c o l o r i m e t r i c assays f o r  p h e n o l i c s o n l y , and these d i d not c o r r e l a t e w i t h  leaf  l i f e t i m e or show an i n v e r s e r e l a t i o n s h i p w i t h h e r b i v o r y (Coley, 1983,  1988), l e a d i n g Coley  (1988) t o suggest  the importance of p l a n t chemistry as a defense had  that  been  267  overestimated.  As g e n e r a l i s t or unadapted  herbivores ( i . e .  P.saucia) have been p o s t u l a t e d t o be the t a r g e t of t o x i c or a n t i f e e d a n t n a t u r a l products (Feeny,1976; Rhoades and Cates, 1976;  Coley e t a l . ,  1985), b i o a s s a y s w i t h such s p e c i e s "may  r e p r e s e n t the most r e l e v a n t method f o r a s s e s s i n g r e l a t i v e investment i n phytochemical-based  defenses.  T h e r e f o r e , my  r e s u l t s i n d i c a t e t h a t Meliaceae w i t h s h o r t l e a f l i f e t i m e s  do  i n v e s t more i n secondary-metabolite based defenses than do the evergreen s p e c i e s .  Leaves of evergreen s p e c i e s were  almost t w i c e as tough as l e a v e s o f deciduous  species.  However, the r e l a t i o n s h i p of i n c r e a s i n g l e a f toughness  with  i n c r e a s i n g l e a f l i f e t i m e i s w e l l e s t a b l i s h e d from p r e v i o u s s t u d i e s (Coley, 1983, l e a f toughness  1985), as i s the r e l a t i o n s h i p between  and reduced r a t e s of h e r b i v o r y .  As w e l l ,  t h e r e i s evidence i n my data t o suggest an i n v e r s e r e l a t i o n s h i p between l e a f t o x i c i t y and l e a f  toughness,  s u g g e s t i n g t h a t s p e c i e s w i t h tough l e a v e s r e q u i r e l e v e l s of p r o d u c t i o n of phytochemical defenses. t h i s h y p o t h e s i s needs f u r t h e r  lower However,  examination.  S p e c i e s not p r e v i o u s l y known f o r the p r o d u c t i o n of i n s e c t i n h i b i t o r y n a t u r a l p r o d u c t s and i d e n t i f i e d here f o r the f i r s t time i n c l u d e d A g l a i a odorata and Turreae  holstii.  The t h r e e a v a i l a b l e A g l a i a s p e c i e s showed a range of b i o a c t i v i t y a g a i n s t P. s a u c i a , and so t h i s genus was  chosen  f o r an i n v e s t i g a t i o n i n t o the phytochemical b a s i s of resistance to herbivory.  The n a t u r a l product c h e m i s t r y of  A., odorata proved complex; compounds i s o l a t e d and  identified  268  u s i n g s p e c t r o s c o p i c methods i n c l u d e d the known dammaranes a g l a i t r i o l and odorine,  a g l a i o n d i o l , and the bis-amides  (S,R)-odorine (a new  o d o r i n o l , and  natural product),  (S,R)-odorinol.  methylated flavanones,  (S,S)(S,S)-  As w e l l a s e r i e s of  p r e v i o u s l y unknown i n the  Meliaceae,  were i d e n t i f i e d , i n c l u d i n g 3-hydroxy-5,7,4'trimethoxyflavanone trimethoxyflavanone,  ( a l s o a new and  natural product),  5,7,4'-  5-hydroxy-7,4'-dimethoxyflavanone.  These compounds, however, a l l proved t o be i n a c t i v e a g a i n s t P. s a u c i a , when t e s t e d s i n g l y or i n combination.  Rather,  the a c t i v e c o n s t i t u e n t appeared t o be a s i n g l e compound, t e n t a t i v e l y i d e n t i f i e d as a l i m o n o i d .  T h i s compound  i n h i b i t s P. s a u c i a growth, with an E C and  an L C  5 0  of 11.2  f e e d i n g behaviour.  /ig/g, c o n c e n t r a t i o n s The  f  I-  4  /ig/g d i e t  which do not  compound, 3 /ig/g l e a f dwt,  was  The  fwt  affect  a c t i v i t y t h e r e f o r e appears t o be  to post-ingestive t o x i c effects.  concentration,  o 5 0  i s o l a t e d y i e l d of  due the  l e s s than the expected  98 /ig/g, based on the a c t i v i t y of  methanolic e x t r a c t ; the d i s c r e p a n c y  the  suggests the  possibility  of a s y n e r g i s t i c i n t e r a c t i o n , but combinations of the a c t i v e compound and the o t h e r phytochemicals i s o l a t e d from A. odorata The  produced o n l y a d d i t i v e e f f e c t s . t h i r d study began with a review of the  l i t e r a t u r e on the e f f e c t s of limonoids insects.  Most s t u d i e s have focussed  current  on phytophagous  on assays f o r f e e d i n g  i n h i b i t i o n , r e f l e c t i n g the p r e v a i l i n g b e l i e f t h a t f u n c t i o n mostly t o d e t e r i n s e c t f e e d i n g , but  limonoids  pre-ingestive  269  chemosensory-based e f f e c t s are not c l e a r l y separated  from  p o s t - i n g e s t i v e t o x i c e f f e c t s , i n c l u d i n g growth r e g u l a t i n g effects.  As w e l l , the a c t i v i t y of these compounds has  been e v a l u a t e d  i n r e l a t i o n t o t h e i r proposed e v o l u t i o n .  Consequently, I i n v e s t i g a t e d t e n representing  e f f e c t s on m o l t i n g  and  The  anthothecol,  and  molting,  against  cedrelone  w i t h an i n t a c t s t e r o i d s k e l e t o n , were s a u c i a growth a t 0.5  umol/g d i e t  which d i d not a f f e c t f e e d i n g i n a  As w e l l , c e d r e l o n e i n h i b i t e d Q.  w i t h an L D  simple limonoids  for  Oncopeltus  simple apo-euphol type limonoids  a concentration  choice t e s t .  f e e d i n g a g a i n s t £. s a u c i a , and  reproduction  highly i n h i b i t o r y to E. fwt,  limonoids,  a l l the major b i o s y n t h e t i c c l a s s e s , f o r  i n h i b i t i o n of growth and  fasciatus.  not  may  5 0  of 12.2 have IGR  fasciatus  /ig/nymph, i n d i c a t i n g ' t h a t even activity.  Anthothecol,  which  d i f f e r s from c e d r e l o n e i n having an acetoxy s u b s t i t u t i o n a t C - l l , was  i n a c t i v e i n the Oncopeltus assay, i n d i c a t i n g the  importance of C - r i n g s u b s t i t u t i o n s i n d e t e r m i n i n g b i o l o g i c a l a c t i v i t y , and  suggesting  t h a t growth and molt  i n v o l v e separate p h y s i o l o g i c a l t a r g e t s . precursors E-  of these simple limonoids  The  inhibition dammarane  were i n a c t i v e a g a i n s t  s a u c i a , so t o t h i s p o i n t b i o s y n t h e t i c e v o l u t i o n  w i t h an i n c r e a s e i n i n s e c t i c i d a l a c t i v i t y .  coincides  However,  o x i d a t i v e opening o f t h e D r i n g , as i n gedunin, leads t o a pronounced drop i n a c t i v i t y .  P a r a d o x i c a l l y , t h i s i s a major  s t e p i n the b i o s y n t h e t i c e v o l u t i o n of limonoids, c h a r a c t e r i z e s a l l limonoids  found i n the Rutaceae  and and  270  Simaroubaceae, and most limonoids  found i n the  Swietenioideae. F u r t h e r o x i d a t i o n t o produce the A,D-seco  limonoids  l e a d s t o compounds, i n c l u d i n g obacunone, n o m i l i n , h a r r i s o n i n , and pedonin, which were a l s o i n a c t i v e a g a i n s t p.. s a u c i a and O.  fasciatus.  entandrophragmin was  Of two  i n a c t i v e and b u s s e i n weakly i n h i b i t e d  P. s a u c i a growth; such limonoids Swietenioideae  B,D-seco l i m o n i d s t e s t e d ,  are c h a r a c t e r i s t i c of many  i n c l u d i n g Khaya and Entandrophragma ( T a y l o r ,  1981), c o n s i d e r e d t o be advanced genera on grounds (Pennington and S t y l e s , 1975). l i m o n o i d e v o l u t i o n i n most Meliaceae,  morphological  The main l i n e s of t h e r e f o r e , do  appear t o c o r r e l a t e w i t h i n s e c t i c i d a l a c t i v i t y generalist herbivores.  T h i s c o n c l u s i o n was  against  supported  attempt t o c o r r e l a t e measures of s k e l e t a l o x i d a t i o n rearrangement, i d e n t i f i e d by Das e t a l .  (data from t h i s study and Kubo and Klocke,  an  and as  the  inhibitory  a c t i v i t y a g a i n s t f o u r s p e c i e s of polyphagous  The  by  (1984, 1987)  dominant themes i n l i m o n o i d e v o l u t i o n , with  c o r r e l a t i o n was  not  lepidopterans  1986).  No  found.  C-seco l i m o n o i d s ,  including azadirachtin,  may  r e p r e s e n t a p o s s i b l e e x c e p t i o n t o the above c o n c l u s i o n . These limonoids may  be d e r i v e d from euphol or  p r e c u r s o r s independently al.,  1988), or they may  tirucallol  of the other limonoids  (Siddiqui et  be formed from an i n t a c t apo-euphol  type l i m o n o i d w i t h o x i d a t i o n a t C-12  (Jones e t a l . , 1988).  T h i s c l a s s of l i m o n o i d i s the most a c t i v e , and the most  271  advanced C-seco l i m o n o i d ,  a z a d i r a c h t i n , i s known t o  a c t i v e a g a i n s t more than two  hundred s p e c i e s  be  of  a g r i c u l t u r a l l y important pest i n s e c t s (Warthen, 1979,  1989;  Saxena, 1989). A z a d i r a c h t i n was w i t h an E C nmol/g.  5 0  of 0.4  h i g h l y i n h i b i t o r y a g a i n s t P.. s a u c i a ,  nmol/g d i e t fwt and  Chemosensory-based a n t i f e e d a n t  an L C  5 0  of  5.2  e f f e c t s were most  pronounced a g a i n s t neonate c a t e r p i l l a r s , but became much reduced by the e a r l y t h i r d i n s t a r . growth i n h i b i t i o n and l i f e cycle.  However, pronounced  m o r t a l i t y continued  A n a l y s i s of d i e t a r y use  and  throughout  the  efficiency  demonstrated t h a t a z a d i r a c h t i n l e d t o decreased consumption at concentrations  which d i d not a f f e c t measures of d i e t a r y  e f f i c i e n c y or f e e d i n g  i n a choice t e s t , suggesting  post-  i n g e s t i v e e f f e c t s , perhaps i n v o l v i n g the gut d i r e c t l y n e u r a l r e g u l a t i o n of f e e d i n g . was  As w e l l , the  i n c r e a s e d a t the h i g h e s t c o n c e n t r a t i o n  or  digestability tested.  Together  these r e s u l t s concur w i t h the mechanism of a c t i o n proposed by Mordue e t a l . (1985), who  suggested t h a t i n h i b i t i o n  gut p e r i s t a l s i s c o u l d l i m i t f e e d i n g and growth by  limiting  the r a t e a t which a food b o l u s c o u l d move through the A z a d i r a c h t i n was  gut.  a l s o h i g h l y i n h i b i t o r y t o O. f a s c i a t u s .  d i s r u p t i n g molting  (MD  50  = 3.8  ng/nymph) and  decreasing  a d u l t s u r v i v a l , i n agreement w i t h e a r l i e r s t u d i e s 1983,  of  (Dorn,  1987). The  f i n a l study focussed  Melanoplus s a n g u i n i p e s .  on the m i g r a t o r y grasshopper,  as t h i s i n s e c t had  been r e p o r t e d  to  272  be r e s i s t a n t t o a z a d i r a c h t i n 1975).  (Mulkern and M o n g o l k i t t i ,  I n i t i a l experiments confirmed a complete l a c k o f an  antifeedant  response when nymphs were f e d l e a f d i s k s  w i t h up t o 500 ppm a z a d i r a c h t i n .  treated  However, nymphs f e d  a z a d i r a c h t i n were subsequently unable t o molt. response experiment i n d i c a t e d a dose-dependant  A doserange o f  e f f e c t s , from d e l a y o f molt a t doses below 5 /ig/g i n s e c t fwt,  t o deformation o f a d u l t s t r u c t u r e s a t doses up t o 10  /ig/g, t o death d u r i n g an incomplete molt attempt a t 13 and 15 /ig/g, c u l m i n a t i n g i n complete blockage o f t h e molt a t doses o f 15 /ig/g and above.  These e f f e c t s were s i m i l a r t o  those r e p o r t e d e a r l i e r i n L o c u s t a (Rembold and S e i b e r , 1981, Mordue e t a l . , 1985; Mordue, 1988), but a r e noteworthy as they f o l l o w e d o r a l a p p l i c a t i o n , whereas i n e a r l i e r  studies  the a z a d i r a c h t i n was a p p l i e d by i n j e c t i o n , b y p a s s i n g t h e normal chemosensory  mechanisms and gut d e f e n s e s .  The consumption o f p h y s i o l o g i c a l l y a c t i v e doses o f a z a d i r a c h t i n by M.- s a n g u i n i p e s nymphs allowed me t o e v a l u a t e the  importance o f t h e gut and integument t o b i o a v a i l a b i l i t y  of a z a d i r a c h t i n , by comparing o r a l and t o p i c a l a c t i v i t y w i t h the a c t i v i t y o f i n j e c t e d a z a d i r a c h t i n .  There was no  s i g n i f i c a n t d i f f e r e n c e between t o p i c a l and i n j e c t e d azadirachtin  (MD  50  3.8 and 3.01 /ig/g r e s p e c t i v e l y ) ,  i n d i c a t i n g the absence o f b a r r i e r s t o a z a d i r a c h t i n b i o a v a i l a b i l i t y i n t h e integument.  However, t h e o r a l MD , 50  10.8 /ig/g, was s i g n i f i c a n t l y h i g h e r than t h e i n j e c t e d MD . 50  The b a r r i e r a s s o c i a t e d w i t h t h e gut l i k e l y i n v o l v e s t h e MFO  273  system and o x i d a t i v e metabolism, as t h e a c t i v i t y of o r a l l y administered a z a d i r a c h t i n i s s i g n i f i c a n t l y increased a p p l i c a t i o n of t h e MFO i n h i b i t o r p i p e r o n y l  by c o -  butoxide.  A n a l y s i s o f growth, food consumption, and measures o f d i e t a r y e f f i c i e n c y f o l l o w i n g consumption of a z a d i r a c h t i n showed t h a t t h e consumption r a t e was  significantly  decreased, d e s p i t e t h e l a c k o f an a n t i f e e d a n t  response,  a g a i n i n d i c a t i n g an e f f e c t on t h e gut o r on t h e n e u r a l r e g u l a t i o n of f e e d i n g .  However, t h e r e was no d i f f e r e n c e i n  n u t r i t i o n a l performance between 10 and 15 /ig/g, although t h e s e doses produced markedly d i f f e r e n t e f f e c t s on molt s u c c e s s , s u g g e s t i n g t h a t e f f e c t s on endocrine events  leading  t o molt i n h i b i t i o n a r e not d i r e c t l y r e l a t e d t o t h e e f f e c t s on gut p e r i s t a l s i s . Two hypotheses f o r t h e mechanism o f a c t i o n of a z a d i r a c h t i n were t e s t e d .  The symptoms o f a z a d i r a c h t i n  treatment c l o s e l y p a r a l l e l t h e symptoms produced by treatment w i t h a z a s t e r o l s , compounds known t o i n h i b i t t h e conversion  of phytosterols  Thompson, 1985).  t o c h o l e s t e r o l (Svoboda and  However, a z a d i r a c h t i n t o x i c i t y c o u l d not  be rescued by supplementing t h e d i e t w i t h c h o l e s t e r o l , and a z a d i r a c h t i n treatment d i d not a f f e c t t h e a b i l i t y o f M. s a n g u i n i p e s nymphs t o m e t a b o l i z e B - s i t o s t e r o l t o desmosterol and  cholesterol.  As w e l l , t h e a b i l i t y o f hemolymph  lipophbrin to transport  s t e r o l s was not reduced, although  the time course of s t e r o l pharmacokinetics was  slightly  a l t e r e d , presumably due t o a slower r a t e o f gut p e r i s t a l s i s .  274  Secondly, P e t t i t e t a l . (1983) suggested t h a t c y t o t o x i c i t y of some limonoids  might be due  the  t o the  ability  t o form M i c h a e l adducts w i t h s u l f h y d r y l r e s i d u e s , and two  the  most conspicuous t a r g e t s f o r a z a d i r a c h t i n ,  neurosecretory  m a t e r i a l and t r a n s d u c i n g  chemosensillae, ( N o r r i s , 1988;  are u n u s u a l l y F r i e d e l and  protein i n  r i c h i n cysteine  Laughton,1980).  residues  However, I found  t h a t a z a d i r a c h t i n d i d not spontaneously form adducts w i t h c y s t e i n e FB,  suggesting  that non-specific binding  to  s u l f h y d r y l - r i c h p r o t e i n i s u n l i k e l y t o be i n v o l v e d i n the mechanism of a c t i o n . These s t u d i e s suggest t h a t a z a d i r a c h t i n does have a pronounced e f f e c t on gut p h y s i o l o g y , Mordue (1988) (Mordue e t al.,  as has been claimed  by  1985), but t h i s e f f e c t i s  probably not d i r e c t l y r e l a t e d t o the d i s r u p t i o n of e n d o c r i n e events l e a d i n g t o the molt.  The  r e s u l t s are c o n s i s t a n t w i t h  a mechanism i n v o l v i n g s p e c i f i c i n h i b i t i o n of the r e l e a s e o f neurosecretory  m a t e r i a l from neurohaemal organs, as has  suggested by Rembold (Subrahmanyam e t a l - ,  1989).  mechanism by which t h i s occurs i s not y e t known.  The  been  275 References Adams, CM. and E.A. Bernays, 1978. The e f f e c t of combinations of d e t e r r e n t s on the f e e d i n g b e h a v i o r of Locusta migratoria. Entomol. exp. a p p l . 23:101-109. Ahmad, S., 1986. 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E f f e c t s o f t h r e e d i f f e r e n t neem seed k e r n e l e x t r a c t s and a z a d i r a c h t i n on l a r v a e of d i f f e r e n t mosquito s p e c i e s . J . A p p l . Ent. 102:455-463. Zittear, T.A. 1984. E f f e c t s of c i t i z e n chemophobia on pathology. P l a n t D i s . 68:655.  plant  Appendix 1: Host p l a n t s of Peridroma s a u c i a 1972; B i e r n e , 1972). Abies balsamea (Linn) s e e d l i n g s Abies g r a n d i s (Dougl.) Acer sp. Acer negundo L i n n . Acer saccharinum L i n n . A l l i u m cepa L i n n . Alnus r u b r a Bong. A l t h a e a rosea Cav. Ambrosia a r t e m i s i i f o l i a L i n n . Amelanchier f l o r i d a L i n d l . Anthemis c o t u l a L i n n . Asparagus medeoloides Thunb. Asparagus o f f i c i n a l i s L i n n . Asparagus plumosa Baker Beta v u l g a r i s L i n n . B r a s s i c a n i g r a (Linn.) B r a s s i c a h i r t a Moench Brassica oleracea Linn. B r a s s i c a rapa L i n n . C a p s e l j a b u r s a - p a s t o r i s (Linn.) Capsicum annum L i n n . ( p e p p e r s ) Chamecyparis t h y o i d e s (Linn.) s e e d l i n g s Chrysanthemum sp. C i r s i u m sp. C i r s i u m v u l a a r e (Savi) C i t r u s limon (Linn.) lemon C i t r u s s i n e n s i s (Linn.) orange Conyza canadensis (Linn.) Cucumus s a t i v u s L i n n . Datura stramonium L i n n . Daucus c a r o t a v. s a t i v u s H o f f . Dianthus c a r y o p h y l l u s L i n n . E p i l o b i u m a n a u s t i f o l i u m (Linn.) Eupatorium sp. F r a g a r i a c h i l o e n s i s (Linn.) Geranium sp. G l e d i t s i a triacanthos Linn. Gossypium herbaceum L i n n . H e l i a n t h u s annuus L i n n . Humulus l u p u l u s L i n n . Lactuca s a t i v a L i n n . L a t h y r u s odoratus L i n n . L y c o p e r s i c o n esculentum (Linn.) Maclura pomifera (Raf.) Maius pumila M i l l . Medicago s a t i v a L i n n . M e l i l o t u s a l b a Desr. Morus sp. Nasturtium sp. N i c o t i a n a tabacum L i n n . Parthenium araentatum Gray  (after Tietz,  314 Persea americana M i l l . Phieuro pratense L i n n . Plantago l a n c e o l a t a L i n n . Plantago sp. Polygonum a v i c u l a r e L i n n . Portulaca oleracea Linn. Prunus americana Marsh. Prunus cerasus L i n n . Prunus armeniaca L i n n . Prunus domestica L i n n . Prunus domestica v. g a l a t e n s i s ex Hook Prunus emarginata Dougl. Prunus p e r s i c a (Linn.) P t e r i d i u m l a t i u s c u l u m (Desv.) Ouercus sp. Ouercus a l b a L i n n . Raphanus s a t i v u s L i n n . Rheum rhaponticum L i n n . Rhus sp. Rhus c o p a l l i n a L i n n . Ribes l a c u s t r e (Pers.) Ribes sanouineum Pursh. Ribes sativum Syme Rosa sp. Rubus a l l e g h e n i e n s i s P o r t e r Rubus o c c i d e n t a l i s L i n n . Rubus idaeus v. s t r i o o s u s (Michx.) Rumex c r i s p u s L i n n . S a l i x sp. S a l i x hookeriana B a r r . S a l i x xcouleriana Barr. Salvia o f f i c i n a l i s Linn. Smilax r o t u n d i f o l i a L i n n . Solanum tuberosum L i n n . Solidago leavenworthii T.&G. S t e l l a r i a media (Linn.) T r i f o l i u m sp. T r i t i c u m aestivum L i n n . Tropaelum majus L i n n . Tsuga canadensis (Linn.) s e e d l i n g s U r t i c a sp. Vaccinium a n g u s t i f o l i u m A i t . Vaccinium corymbosum L i n n . Vaccinium m y r t i l l o i d e s Michx. V i o l a sp. Viola t r i c o l o r Linn. V i t i s sp. V i t i s v i n i f e r a Linn. Xanthium strumarium L i n n . Zea mays L i n n . Ferns, g e n e r a l f e e d e r on f i e l d c r o p s , f o r e s t t r e e s , f r u i t t r e e s , g r a s s , low p l a n t s , most anything, shrubs, vegetables.  315 Appendix 2: H-NMR and mass s p e c t r a o f compounds i s o l a t e d from A a l a i a o d o r a t a . 1  Compound 3:  3-hydroxy-5,7,4'-trimethoxyflavanone  00  NHSS  SMMPLEI  BASE H'Ei 134 RICi 33213288.  OATAi octei t33  SPECTRUM  ej-21 -as i J i e n e e • 1140 HD-2  •98 TO I I M SUMMED - «3 TO «10 XI.80 134  lea.e - i  3330710. 10.  tee  132  91  30.0-  29?  63 7?  33  41  96  3d  90  100.0•  l,J,iJklL,, tJl,J 1111^4 ,, Jpiln  iiiJllpiL JL  jjj] M'E  189  130  r,T, , T .  LL.I,ji>llly I l tu\%  230  r 3330710. 10.  3 4 se.e -  Compound 4 :  S^^'-trimethoxyflavanone  t— 1  286  29?  U3  I  rvE  388  •  *  3:>» I  -  341 I  ' i 363 • *i  3 ^  • •I i 338  .13? jae .lag . , 430  I  '  I  '  1  I  •  I  '  I  DATAi PC 102 »39 •^•i TO «6? SUMMED - «I0 TO »I3 XI.«e 134  IOP.O -,  BASE M'El 134 PICi 3345Sieg. p 2617349. ie.  91  50.9-1  166 193  63  77  5.1  103  41  M-E  lee.e  -U  181  lull 156  58  243 —f 2se  29?  272  r 2617340.  ie.  300 50.PH  Compound 5:  5-hydroxy-7,4/-di.Heth.oxyflavanone  1*1 to to  Compound 7:  (S,R)-odorine  MtiSS S P E C T R U M u-t i ; 8 3 9 I 4 7 I 6 6 * 1102 S H U P L E I FLrtU • S 3 T O « 6 3 SUMMED - #10 T O 1 1 3 M l . 8 6  BASE M^El 134 RICi 33436160.  DATAI 0C1B2 639  134  iae.0 - i  2617340. 10.  1?»  9.1  90.0 H  166 193  63 91 41 M'E 166.6  I  77  163  -Ik-Li 36  129  -I  In.*.']!! >,.Jin|Ui>  2 2  101  T,l.. f .^, -gt  I*  2  9  1 200  130  3  297  272  ...if.  290  2617340. 10.  366  36.6  Compound 7 :  H  (S,R)-odorine  ro M^E  366  3>9 ,  , 3j6,  , 336  ^  377;  jB? ,186,  t  442,  I  430  •  I '  I  • I ' s i . '  '  '  '  Compound 8 :  (S,S)-odorine  MMiS S P E C T R U M 6412/89 I 8 H 4  I 0 0  •  SHMPIEI AMIOE •118 T O « 1 1 4 SUMMED  iee.e-i  DATAI  1sse  DC183 1112  BASE RICi  M'E I1 3 1 17836980.  - «7 T O « 1 4 XI.00 131  1966710. 10.  103 73 201  30.0-  109  77  39  83 49 9  91  1 Jj.lu  63  100.0-  T  Ililly.lt.J..  ^4  30  4  3  I  'I  190  J I-  ,  ™ t  290  I- 1 9 C 6 7 1 0 . 10.  se.e -  M-E  244  T  3  Compound 9: ( S , S ) - o d o r i n o l  388  .1.  36? 338  , , 3 8 3 , ,399, 408  1U  ,  .128  • ,11? , 490  I  •  I  •  I  Compound 1 0 :  (S,R)-odorinol  

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