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Insect growth inhibitors from asteraceous plant extracts Salloum, Gregory Stewart 1987

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INSECT GROWTH INHIBITORS FROM ASTERACEOUS PLANT EXTRACTS by GREGORY STEWART SALLOUM B.Sc, Macdonald College of Mc G i l l U n i v e r s i t y , 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Plant Science) We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1987 ^Gregory Stewart Salloum 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 P l a n t Science The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date A p r i l 28, 1987 Gregory Stewart Salloum A p r i l 1987 Department of Plant Science INSECT GROWTH INHIBITORS FROM ASTERACEOUS PLANT EXTRACTS ABSTRACT Pe t r o l and ethanolic extracts of s i x asteraceous weeds were added to a r t i f i c i a l d i e t and screened f o r i n h i b i t i o n of l a r v a l growth on variegated cutworm, Peridroma saucia (Hbn.). P e t r o l and ethanolic extracts of Artemisia t r i d e n t a t a and Chamomilla suaveolens and ethanolic extracts of Chrysothamnus nauseosus and Centaurea d i f f u s a were highly i n h i b i t o r y at f i v e times the n a t u r a l l y occurring concentrations. The two C. suaveolens extracts and the ethanol extract of _A. t r i d e n t a t a were active at the natural concentration (100%) and were further examined at 20, 40, 60, and 80% of t h i s l e v e l . I n h i b i t i o n of l a r v a l growth was d i r e c t l y related to concentration for each of the three extracts tested. EC^ Q'S ( e f f e c t i v e concentration to i n h i b i t growth by 50% r e l a t i v e to controls) for the three extracts were 36-42% of the n a t u r a l l y occurring l e v e l i n the plants. N u t r i t i o n a l i n d i c e s were calculated f o r second i n s t a r P_. saucia feeding on the ac t i v e ethanolic A^ . t r i d e n t a t a extract and the pe t r o l extract from C. suaveolens . The r e l a t i v e growth rate (RGR) of P_. saucia larvae fed the ethanolic extract of _A. t r i d e n t a t a i n a r t i f i c i a l d i e t was s i g n i f i c a n t l y lower than that i n larvae fed d i e t with the p e t r o l extract of C. suaveolens and larvae on co n t r o l d i e t . Dietary u t i l i z a t i o n was s i g n i f i c a n t l y lower for larvae fed the _A. t r i d e n t a t a extract. Results of a f i e l d t r i a l indicated that a s i n g l e treatment of _A. t r i d e n t a t a extract at the equivalent of 0.2 g/ml could protect cabbage s i g n i f i c a n t l y better than the c a r r i e r solvent (30% aq ethanol) or d i s t i l l e d water as measured by a v i s u a l damage estimate. An i n s e c t i c i d e standard, TM deltamethrin (17.9 ug/1 with 0.4% Superspred ), suppressed pest damage s i g n i f i c a n t l y better than the /A. tridentata-extract treatment. A r e s i d u a l o v i p o s i t i o n deterrency to P i e r i s rapae was found i n the f i e l d r e s u l t s . Caged experiments i n the laboratory confirmed the contact o v i p o s i t i o n deterrency of the A^ . t r i d e n t a t a extract at 0.2 g/ml. Offspring of f i e l d - c o l l e c t e d P_. saucia larvae grew 2.5-fold heavier than larvae from the laboratory colony. However, di e t with the _A. t r i d e n t a t a extract i n h i b i t e d both f i e l d - c o l l e c t e d and laboratory reared saucia larvae equally when compared to t h e i r respective controls fed untreated d i e t . In summary, these r e s u l t s i n d i c a t e the p o t e n t i a l benefit of using s p e c i f i c unrefined plant extracts for growth i n h i b i t o r s and o v i p o s i t i o n deterrents against insect pests. The contribution of i n d i v i d u a l phytochemicals i n the _A. t r i d e n t a t a ethanolic extract to growth i n h i b i t i o n or o v i p o s i t i o n deterrency i s currently speculative. i i i TABLE OF CONTENTS Page I. INTRODUCTION 1 A. Objectives of Thesis 2 I I . LITERATURE REVIEW 4 A. Chemical Basis for Plant Resistance to Insects 5 B. Insect Bioassays of Phytochemicals 8 C. Behavioral Response to Plant Defense Chemicals 10 D. Phytochemicals and Ph y s i o l o g i c a l Stress 13 E. Plant Extracts i n Crop Protection 15 I I I . MATERIALS AND METHODS 19 A. Plant Extracts 19 B. B i o l o g i c a l Screenings 19 1. I n i t i a l Screening 19 2. Bioassay of Unextracted and Extracted Plant Material 21 3. Second Screening 21 C. Dose-Response Bioassays 21 1. Dose-Response Bioassay on P_. saucia Neonates 21 2. Dose-Response Bioassay using the A l f a l f a Looper, Autographa c a l i f o r n i c a Speyer 22 3. S e n s i t i v i t y of Older P_. saucia Larvae 22 D. F i n a l Determination of Extract f o r F i e l d T r i a l 22 1. Detailed Growth Analysis of P. saucia Larvae Feeding on t r i d e n t a t a and C. suaveolens Extracts 22 2. Formulation of Active Extracts f o r F o l i a r A pplication and S t a b i l i t y of Crude Extracts 23 iv E. F i e l d T r i a l of the _A. t r i d e n t a t a extract on Cabbage 23 F. Laboratory Evaluation of the Oviposition Deterrence of _A. t r i d e n t a t a Extract on Caged P. rapae 25 G. Growth Comparison of Laboratory Reared and Wild Colonies of P_. saucia Larvae on Standard Diet and Diets with Addition of an Ethanolic A_. t r i d e n t a t a Crude Extract 26 H. Phytochemical Investigation 26 1. Chromatographic Separation of the Ethanolic _A. t r i d e n t a t a Extract 26 2. Chromatography of Sesquiterpene Lactones Previously Isolated from A^ tri d e n t a t a 28 IV. RESULTS 29 A. Laboratory Screenings 29 B. Detailed Growth Analysis of P_. saucia Larvae on the C. suaveolens and the k_. t r i d e n t a t a Extracts 42 C. F i e l d T r i a l of the _A. t r i d e n t a t a Extract on Cabbage 46 D. Oviposition of P_. rapae; Laboratory Experiment 54 E. Quality of Cabbage Heads from the F i e l d T r i a l 54 F. Comparison of Wild Versus Laboratory Reared P_. saucia 54 G. Preliminary Phytochemical Investigation 57 V. DISCUSSION 68 A. Screening Asteraceous Extracts for Insect Growth I n h i b i t o r s 68 B. Phytochemicals and Insect Growth In h i b i t o r s 72 C. F i e l d T r i a l s of Plant Extracts 76 VI. CONCLUSIONS 84 VII. BIBLIOGRAPHY 86 VIII. APPENDICES 99 v LIST OF TABLES Page Table I. Plant species, components extracted, harvest loca t i o n s i n B r i t i s h Columbia and dates of material bioassayed i n i n i t i a l screening 20 Table I I . E f f e c t s of weed extracts incorporated i n t o a r t i f i c i a l d i e t on growth and s u r v i v a l of neonate P. saucia i n an i n i t i a l screening bioassay 30 Table I I I . Growth and s u r v i v a l of P_. saucia neonate larvae fed on a r t i f i c i a l d i e t s with unextracted plant powder or on the extracted marcs 31 Table IV. E f f e c t s of selected weed extracts incorporated i n t o a r t i f i c a l d i e t at natural concentrations on the weight and s u r v i v a l of neonate P_. saucia fed f o r 11 days 33 Table V. E f f e c t s of dietary _A. t r i d e n t a t a extract (EtOH) and C_. suaveolens extract ( p e t r o l ) on 2nd i n s t a r P_. saucia d i g e s t i b i l i t y of food (AD), r e l a t i v e growth rate (RGR), r e l a t i v e consumption rate (RCR), and gross (ECI) and net (ECD) dietary u t i l i z a t i o n s A3 Table VI. Mean growth and percent s u r v i v a l of neonate P_. saucia larvae fed a r t i f i c i a l d i e t s incorporating fresh and eight month o l d ethanolic extracts, hot water, room temp water and 20% aq ethanol formulations of A^ . t r i d e n t a t a and C_. suaveolens for 16 days 45 Table VII. Mean v i s u a l q u a l i t y estimates of cabbage treated with an _A. t r i d e n t a t a ethanolic extract, deltamethrin, 30% aq ethanol, and water, recorded 25 days post-treatment 55 Table VIII.Mean P_. saucia l a r v a l weight of a F^ f i e l d c o l l e c t e d population compared to the laboratory colony fed the standard a r t i f i c i a l d i e t and d i e t containing a 50% _A. t r i d e n t a t a ethanolic extract (dwt/dwt) for 8 days 56 Table IX. Mean l a r v a l weight of neonate P_. saucia fed a r t i f i c i a l d i e t admixed with chromatographically separated f r a c t i o n s of an A^ . t r i d e n t a t a ethanolic extract compared to the o r i g i n a l extract at e c o l o g i c a l concentrations and the standard d i e t 58 Table X. Phytochemical constituents previously i s o l a t e d from Artemisia t r i d e n t a t a 74 LIST OF FIGURES Page Figure 1. Percent growth (o) r e l a t i v e to co n t r o l growth and percent t o t a l mortality (•) of P_. saucia neonates fed ethanolic extracts from A) _A. t r i d e n t a t a , and B) C. suaveolens t and a p e t r o l extract from C) C. suaveolens admixed to a r t i f i c i a l d i e t s (n=25 larvae per concentration ) 36 Figure 2. Percent growth (o) r e l a t i v e to c o n t r o l growth and percent t o t a l mortality (•) of s i x day-old £. saucia larvae fed ethanolic extracts from A) A_. t r i d e n t a t a , and B) C. suaveolens t and a p e t r o l extract from C) C. suaveolens admixed to a r t i f i c i a l d i e t s (n=25 larvae per concentration) 38 Figure 3. Percent growth (o) r e l a t i v e to c o n t r o l growth and percent t o t a l mortality (•) of neonate _A. c a l i f o r n i c a fed ethanolic extracts from A) _A. t r i d e n t a t a , and B) C. suaveolens, and a p e t r o l extract from C) C_. suaveolens admixed to a r t i f i c i a l d i e t s (n=25 larvae per concentration) 40 Figure 4. Percentage change i n cabbage looper equivalents (CLE) af t e r f i e l d spraying cabbage with a) 30% aq ethanolic so l u t i o n of A. t r i d e n t a t a (0.2 g/ml), b) 30% aq ethanol, c) deltamethrin 2.5 EC (17 ug/l a . i . ) with 0.1% Superspred or d) d i s t i l l e d water, July 24, 1985. 48 Figure 5. Percent change i n imported cabbageworm, JP. rapae l a r v a l populations before and a f t e r f i e l d spraying cabbage with a) 30% aq ethanolic s o l u t i o n of A^ . t r i d e n t a t a (0.2 g/ml), b) 30% aq ethanol, c) deltamethrin 2.5 EC (17 pg/l a . i . ) with 0.1% Superspred or d) d i s t i l l e d water, July 24, 1985. 50 Figure 6. Percent change i n imported cabbageworm, P_. rapae, o v i p o s i t i o n surveyed before and a f t e r f i e l d spraying cabbage with a) 30% aq ethanolic s o l u t i o n of _A. tri d e n t a t a (0.2 g/ml), b) 30% aq ethanol, c) deltamethrin 2.5 EC (17 pg/l a . i . ) with 0.1% Superspred or d) d i s t i l l e d water, July 24, 1985. 52 v i i Figure 7. Thin-layer chromatograph of f r a c t i o n s 2 (fr#2) and 4 (fr#4) from the separation of a crude ethanolic A.tridentata extract chromatographed with phytochemicals from Artemisia spp. The pure sesquiterpene lactones compared to the A^ . t r i d e n t a t a f r a c t i o n s were: Dehydroleucodin ( d h l ) , dihydrosantamarin (dhs), arbusculin A (abA), arbusculin C (abC), matricarin (mat), deacetoxymatricarin (dom), deacetylmatricarin (dam), and dehydroreynosin (dhr). Non-fluorescent colours occurred a f t e r developing the plate with a v a n i l l i n reagent and the arrows i n d i c a t e a colour s h i f t a f t e r 24 h. TLC developed with petroleum ether:CHCl2:Et20Ac (2:2:1) i n a non-saturated tank. 60 Figure 8. Thin-layer chromatograph of f r a c t i o n s 2 (fr#2) and 4 (fr#4) from the separation of a crude ethanolic _A. t r i d e n t a t a extract chromatographed with phytochemicals from Artemisia spp. The pure sesquiterpene lactones compared to the _A. t r i d e n t a t a f r a c t i o n s were: dehydroleucodin ( d h l ) , dihydrosantamarin (dhs), arbusculin A (abA), t a t r i d i n A ( t t A ) , matricarin (mat), deacetoxymatricarin (dom), and deacetylmatricarin (dam). Non-fluorescent colours occurred a f t e r developing the plate with a v a n i l l i n reagent and the arrows i n d i c a t e a colour s h i f t a f t e r 24 h. TLC developed with CHC13:acetone (6:1) i n a saturated tank. 62 Figure 9. Thin-layer chromatograph of f r a c t i o n s 2 (fr#2) and 4 (fr#4) from the separation of a crude ethanolic A^ . t r i d e n t a t a extract chromatographed with phytochemicals from Artemisia spp. The pure sesquiterpene lactones compared to the A_. t r i d e n t a t a f r a c t i o n s were: Dehydroleucodin ( d h l ) , arbusculin A (abA), arbusculin B (abB), arbusculin C (abC), t a t r i d i n A ( t t A ) , matricarin (mat), deacetoxymatricarin (dom), deacetylmatricarin (dam), and dehydroreynosin (dhr). Non-fluorescent colours occurred a f t e r developing the plate with a v a n i l l i n reagent and the arrows i n d i c a t e a colour s h i f t a f t e r 24 h. TLC developed with CHC1 3:acetone (6:1) i n a saturated tank. 64 Figure 10. Thin-layer chromatograph of f r a c t i o n s 2 (fr#2) and 4 (fr#4) from the separation of a crude ethanolic A^ . t r i d e n t a t a extract chromatographed with phytochemicals from Artemisia spp. The pure sesquiterpene lactones compared to the _A. t r i d e n t a t a f r a c t i o n s were: arbusculin A (abA), arbusculin B (abB), t a t r i d i n A ( t t A ) , matricarin (mat), deacetoxymatricarin (dom), and deacetylmatricarin (dam). Non-fluorescent colours occurred a f t e r developing the plate with a v a n i l l i n reagent and the arrows i n d i c a t e a colour s h i f t a f t e r 24 h. TLC developed with petroleum ether:CHClo:Et 90Ac (2:2:1) i n a non-saturated tank 66 v i i i ACKNOWLEDGEMENTS I s i n c e r e l y thank my supervisor, Dr. Murray B. Isman, for h i s guidance and continual support, throughout t h i s project, and i n my personal development. I earnestly thank my t h e s i s committee, s p e c i f i c a l l y , Dr. V. C. Runeckles f o r h i s advice i n the phytochemical i n v e s t i g a t i o n , Dr. G. H. N. Towers for h i s guidance on p o t e n t i a l plant species to extract, and Dr. R. S. Vernon for h i s assistance designing the f i e l d experiment. I thank Susan Schwab for e x c e l l e n t technical assistance, Rick Kelsey for sesquiterpene lactones, Gordon Ayer for P. saucia eggs, Roy Cranston r p w for C_. d i f f u s a plants, R. S. Vernon for providing cabbage seed and Decis , and G. H. N. Towers for use of h i s laboratory. This work was supported i n part by an NSERC operating grant (A2729) to M. B. Isman. I also acknowledge J . Carlson, D. Champagne, L. Gilkeson, C. Guppy, S. Johnson and the graduate students i n the Department of Plant Science for t h e i r assistance. I extend a s p e c i a l thanks to J i l l E. Evans f o r providing continual support during the arduous completion of t h i s manuscript. i x 1 I. Introduction Present day crop protection s t r a t e g i e s often r e l y heavily on the l i b e r a l use of broad-spectrum i n s e c t i c i d e s . Novel pest management t a c t i c s are beginning to reduce the use of these non-selective biocides (Huffaker 1980). Improvements to crop protection methods are neccesitated by health, environmental, and economic concerns associated with the use of synthetic i n s e c t i c i d e s (Luck et a l . 1977, Basol 1980, Metcalf 1980). Public concern regarding i n s e c t i c i d e s i s often the r e s u l t of chemophobia or the fear that widespread use of chemicals w i l l damage the environment and human health. The fear i s j u s t i f i a b l e , for example, where to x i c i n s e c t i c i d e s have been misused causing environmental damage, including groundwater contamination ( Z i t t e r 1984). A g r i c u l t u r a l agencies could better inform legitimate i n t e r e s t s ( i . e . , the public) of the r i s k s and benefits of i n s e c t i c i d e use (Czerwinski and Isman 1986) and be able to provide options and a l t e r n a t i v e s to the use of biocides. The cost of i n s e c t i c i d e usage i s increasing for several reasons. Where i n s e c t i c i d e s are used i n t e n s i v e l y , i n s e c t s often develop resistance to i n s e c t i c i d e s (Luck et a l . 1977). Almost i n v a r i a b l y , broad-spectrum i n s e c t i c i d e s increase the development of r e s i s t a n t pest genotypes. When i n s e c t i c i d e s are sprayed, major or minor pest species may become a serious problem i f t h e i r natural enemies are eliminated. When pest populations resurge or develop resistance to an i n s e c t i c i d e , farmers and other a p p l i c a t o r s , may use higher doses, increase the frequency of spraying, or change co n t r o l chemicals. New i n s e c t i c i d e s are thus needed to replace those no longer e f f e c t i v e . Insects that have aquired resistance to e x i s t i n g i n s e c t i c i d e s are often predisposed to develop cross-resistance to new i n s e c t i c i d e s (Devonshire and Moore 1982). Cross-resistance has 2 decreased the average l i f e s p a n of a new i n s e c t i c i d e to about two years (Metcalf 1980). Additional economic problems, such as cost (about $25 m i l l i o n ) and development time (8-10 years) for new products (Kinoshita 1985), suggest that innovative approaches are needed to control insect pests and manage extant i n s e c t i c i d e s (Croft 1982). Natural products from plants are p o t e n t i a l sources of useful crop protection agents and novel pest c o n t r o l s t r a t e g i e s . Plant products, such as vegetable o i l s , are known to have been used for crop protection i n early Greek and Roman a g r i c u l t u r e (Smith and Secoy 1975). More recently, plant extracts have been investigated for c o n t r o l of both v i r a l (Verma and Abid A l i Khan 1984) and fungal (Kuc and Shain 1977, El-Shazly et a l . 1981) crop diseases. Numerous plant extracts have also been examined for t h e i r acute i n s e c t i c i d a l properties (Mclndoo and Sievers 1924, Jacobson 1958). However, extensive screenings for acutely t o x i c phytochemicals have produced few commercially exploited botanical i n s e c t i c i d e s (Jacobson and Crosby 1971). A. Objectives of Thesis The primary objective of t h i s i n v e s t i g a t i o n was to screen crude extracts from asteraceous weeds as p o t e n t i a l i n s e c t c o n t r o l agents. Plants i n the Asteraceae were selected because of t h e i r richness i n secondary metabolites (Herout 1970). The i n s e c t used i n the extract screening was the variegated cutworm, Peridroma saucia (Hbn.), because i t i s a serious in s e c t pest of many crops i n Canada (Beirne 1971), and i s reared c o n s i s t e n t l y and a v a i l a b l e i n large q u a n t i t i e s i n Dr. M. B. Isman's laboratory. The plants chosen (Table I) were a l l weedy species with no current economic value. The dried plant powders were extracted with both polar and non-polar solvents to assess b i o l o g i c a l a c t i v i t y of f r a c t i o n s 3 containing d i f f e r e n t phytochemical mixtures. Dose-response of the most growth i n h i b i t i n g extracts showed the r e l a t i v e effectiveness of the extracts as l a r v a l growth i n h i b i t o r s and the nature of the growth i n h i b i t o r y response. To d i s t i n g i s h between gross behavioral and p h y s i o l o g i c a l e f f e c t s , growth i n h i b i t i o n was further studied by determination of n u t r i t i o n a l i n d i c e s for larvae feeding on a r t i f i c i a l d i e t admixed with extracts. The next objective was to prepare a formulation of the most active extract to assess the extract's f i e l d e f f i c a c y against cabbage insect pests. A short term phytotoxic t e s t on cabbage prior to the f i e l d test insured the s u r v i v a l of the cabbage plants f o r the f i e l d t r i a l . Two laboratory experiments were performed subsequent to the f i e l d t r i a l . One was to confirm the f i e l d observation of lowered P i e r i s rapae egg-counts i n the cabbage treated with Artemisia t r i d e n t a t a extract using caged b u t t e r f l i e s i n the laboratory. The other compared the e f f e c t s of the ethanolic _A. t r i d e n t a t a extract on f i e l d - c o l l e c t e d VC larvae r e l a t i v e to larvae from the laboratory colony. The f i n a l section of t h i s thesis involved an examination of the phytochemistry i n the most growth-inhibiting extract. Chromatographic separation i n conjunction with an i n s e c t bioassay investigated whether growth i n h i b i t i o n was a t t r i b u t a b l e to one or several groups of compounds. Chromatographic comparisons of pure compounds i s o l a t e d from c l o s e l y related plants with a c t i v e f r a c t i o n s allowed tentative i d e n t i f i c a t i o n of the chemical c l a s s of compounds responsible for the growth i n h i b i t i o n . 4 I I . L i t e r a t u r e Review The l i t e r a t u r e on the i n t e r a c t i o n s between plants and t h e i r insect herbivores i s large. This includes numerous reviews and books on the breeding of plants r e s i s t a n t or tole r a n t to insect attack (Painter 1951, Maxwell and Jennings 1980, Hedin 1983), i d e n t i f i c a t i o n of plant c h a r a c t e r i s t i c s conferring insect resistance (Thorsteinson 1960, Chapman 1974, Rosenthal and Janzen 1979, Dethier 1980, Hedin 1983), and the ph y s i o l o g i c a l factors that allow i n s e c t s to perceive and metabolize phytochemicals (Fraenkel 1959, Brattsten 1979, B e l l and Carde 1984). In addi t i o n , many classes of secondary compounds that influence insect behavior and physiology have been examined (Chapman 1974, Rosenthal and Janzen 1979, Hedin 1983, Whitehead and Bower 1983). Several studies have considered sublethal ways of manipulating insect populations with phytochemicals, including i n h i b i t i o n of feeding (Jermy et a l . 1981), growth (McMillian et a l . 1969) and o v i p o s i t i o n ( M i t c h e l l and Heath 1985). These chemicals are often termed 'allelochemics', defined by Whittaker (1970) as chemicals that mediate non-nutritional 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 . Many phytochemicals have been shown to reduce insect growth, and prolong the l i f e c ycle (Chapman 1974, Reese and Beck 1976). Extending an insec t ' s l i f e c ycle has been shown to increase exposure to predators (Wesloh et a l . 1983) and other environmental hazards (Courtney 1986). Phytophagous insect s that develop more slowly than normal are more l i k e l y to die of disease (Courtney 1981) and the early onset of winter (Chew 1975). Natural mortality of f i r s t i n s t a r O s t r i n i a n u b i l a l i s L. i s about 90% (Beck 1960). Introduction of new, and enhancement of e x i s t i n g mortality f a c t o r s at t h i s vulnerable stage may play an important r o l e i n i t s population biology and thus i t s e f f e c t i n a g r i c u l t u r a l systems. Oviposition interference i s another sublethal mode through which allelochemicals aid plants i n escaping insect herbivory. Both host and non-host plant extracts have been shown to deter o v i p o s i t i o n of several insect species (Tingle and M i t c h e l l 1984, Renwick and Radke 1985). Many authors have recognized the importance of using phytochemical disruptions of insect behavior to protect crops from herbivory (Munakata 1970, Chapman 1974, Jermy 1983). Plant allelochemicals that have sublethal e f f e c t s on insect pests are p o t e n t i a l l y useful for a g r i c u l t u r e as valuable breeding c r i t e r i a or as applied protectants, provided that they are non-toxic to humans, environmentally sound, economical to produce and convienient to apply. A. Chemical Basis for Plant Resistance to Insects Some insects feed on several d i f f e r e n t plant f a m i l i e s whereas others r e l y on a r e s t r i c t e d number of plants or even a single host species (Thorsteinson 1960). In addition, some plant species are r a r e l y attacked by i n s e c t s . Several factors influence the pattern of in s e c t attack on the av a i l a b l e plant species. Plant a r c h i t e c t u r e , including s i z e , shape, and co l o r , as well as plant habitat and d i s t r i b u t i o n , are important considerations i n host s e l e c t i o n by phytophagous i n s e c t s . However, the chemical content of the plant i s often considered the most important factor determining host-plant s p e c i f i c i t y (Haniotakis and Voyadjoglou 1978, Hardman and E l l i s 1978, Rosenthal and Janzen 1979). Many classes of phytochemicals have been shown to play an important r o l e i n insect-plant i n t e r a c t i o n s . Sugars, amino acids and proteins are 6 important for insect growth and development and can play a r o l e as feeding stimulants (Bernays and Simpson 1982, Dethier 1973). These ubiquitous constituents of plants are commonly known as primary metabolites. Their broad d i s t r i b u t i o n s i n the plant kingdom make them un l i k e l y candidates for insect host s p e c i f i c i t y or plant defensive chemistry (Bernays and Chapman 1978). The other major group of phytochemicals are c a l l e d "secondary" metabolites, either because they are poorly understood or because they are not involved i n primary metabolism, or both. Numerous secondary metabolites are known to mediate i n s e c t - p l a n t i n t e r a c t i o n s (Chapman 1974, Hedin 1983). These important phytochemicals are usually c l a s s i f i e d according to t h e i r structure or biosynthetic pathways. The e f f e c t s of a s i n g l e a l l e l o c h e m i c a l on insects may d i f f e r depending on a variety of b i o t i c and a b i o t i c f a c t o r s . The i n s e c t - a l l e l o c h e m i c a l i n t e r a c t i o n may be influenced by the i n s e c t species examined (Eisner 1964, Dethier 1973, Chew 1980), insect growth stage (Reese 1979, Chew 1980, Isman and Duffey 1982), previous exposure to allelochemicals (Jermy et a l . 1982), as well as concentration, route of entry and the simultaneous occurrence of other phytochemicals (Bernays and Chapman 1978). Isman and Duffey (1982) have shown that a phytochemical may e l i c i t a growth i n h i b i t o r y reponse at one concentration and a t o x i c reaction at a higher dose. Nepetalactone, a monoterpenoid, i s known to repel some in s e c t s but have l i t t l e or no e f f e c t on other i n s e c t s i n the same order (Eisner 1964). The d i s t r i b u t i o n of secondary chemicals i s frequently l i m i t e d to one plant taxon, while occurring s p o r a d i c a l l y i n systematically unrelated groups. Glucosinolates or mustard o i l glycosides provide a good example, occurring often i n the Brassicaceae and other Capparales f a m i l i e s , but occurring occasionally i n the unrelated plant family Caricaceae (Bjorkman 7 1976). Sesquiterpene lactones are another example, occurring widely i n the Asteraceae and l e s s frequently i n the Apiaceae and Magnoliaceae (Heywood et a l . 1977). S p e c i f i c allelochemicals (e.g., o v i p o s i t i o n cues) can have a b e n e f i c i a l e f f e c t on oligophagous insec t s ( i . e . , i n s e c t s having a r e s t r i c t e d range of food plants of related plant orders or even of a s i n g l e genus), while at the same time contributing to the plant's chemical defence against more general herbivores. A l l y l g l u c o s i n o l a t e , present i n many Brassicaceae plants, i s innocuous to the growth of the c r u c i f e r s p e c i a l i s t P i e r i s rapae, but i n h i b i t s the growth of the polyphagous Spodoptera  eridania , and i s acutely t o x i c when fed to the Apiaceae s p e c i a l i s t P a p i l i o  polyxenes (Blau et a l . 1978). Even compounds present i n t h e i r host plants may cause a n t i b i o s i s (sensu Painter 1951) or reduce the f i t n e s s and vigor of oligophagous i n s e c t s . The t r i t e r p e n o i d cucurbitacins of the Cucurbitaceae deter feeding by the c u c u r b i t - s p e c i a l i s t Epilachna  tredecimnotata, but act as attractants to the s t r i p e d cucumber beetle, Acalymma v i t t a t a ( C a r r o l l and Hoffman 1980). Very few i n s e c t s r e l y e n t i r e l y on s p e c i f i c chemical cues for feeding or o v i p o s i t i o n . Most oligophagous insects do not appear to r e l y on the presence or absence of a single compound for host a c c e p t a b i l i t y , but on t h e i r chemosensory response to the t o t a l phytochemical mixture (Dethier 1973). Studies of food consumption by cruciferous f l e a beetles, P h y l l o t r e t a spp., show that the amount consumed i s usually dependent on the balance of stimulant and deterrent chemicals (Nielsen 1978). Although the e f f e c t s of phytochemicals have often been investigated i n d i v i d u a l l y , an i n s e c t ' s sensory perception of t h e i r natural habitat must include the complexity of chemical mixtures i n non-host plants. Many 8 plants have compounds that, i n combination with other phytochemicals, increase insect a n t i b i o s i s more so than a s i n g l e compound alone (Adams and Bernays 1978, Kubo et a l . 1984, Berenbaum and Neal 1985). B. Insect Bioassays of Phytochemicals Several bioassays have been used to detect phytophagous pest c o n t r o l agents i n plant extracts. Bioassays may measure a variety of f a c t o r s , i n c l u d i n g mortality (Freedman et a l . 1979), feeding and o v i p o s i t i o n punctures, l a r v a l and egg counts (Jacobson et a l . 1978), growth, dietary u t i l i z a t i o n (Isman and Proksch 1985), reproductive p o t e n t i a l (Robert and B l a i s i n g e r 1978) and consumption (Bentley et a l . 1982). Laboratory evaluation of behaviorally a c t i v e allelochemicals may occur i n the form of choice or 'no-choice' experiments. Choice experiments o f f e r the t e s t i n s e c t a s e l e c t i o n of two or more substrates on which to feed or o v i p o s i t . In the so c a l l e d no-choice experiments the insect has a s i n g l e feeding or o v i p o s i t i o n a l substrate. Bioassays may be designed so that the i n s e c t s are evaluated by either an 'all-or-none' or a graded type of response. Larval growth, for example, i s usually a graded measure and mortality i s an ' a l l or none' response. Larval feeding and growth i n h i b i t i o n may be examined by incorporating plant powders or extracts i n a r t i f i c i a l d i e t s and feeding the d i e t s to t e s t i n s e c t s (Hsiao and Fraenkel 1968). After feeding, the surviving larvae are counted and weighed. Bernays (1983) and Smith (1978) discuss c r i t e r i a for choosing between d i f f e r e n t bioassay methods. The process of extracting allelochemicals from plant t i s s u e and incorporating them in t o a r t i f i c a l d i e t s could a l t e r a l l e l o c h e m i c a l s , p o t e n t i a l l y increasing or decreasing t h e i r effectiveness (Bernays and Chapman 1978). An a r t i f i c i a l d i e t may mask the presence of feeding 9 deterrents (Bernays and Chapman 1977). The masking may be a r e s u l t of increased feeding stimulation, absence of other fitness-reducing compounds or a better nutrient source than host plants. However, insects fed a r t i f i c i a l d i e t s provide a good r e l a t i v e measure of l a r v a l growth i n h i b i t i o n using extracted phytochemicals. The use of a r t i f i c i a l d iets a l l e v i a t e s problems inherent i n the use of l i v e plant materials and allows d i r e c t comparisons with other chemically defined food sources. Experimental error may increase with the use of l i v e plant material due to the p o t e n t i a l for large allelochemical differences (Risch 1985), and physical differences l i k e l e a f toughness (Reese 1983). Although many compounds i n d i f f e r e n t chemical classes have been shown to i n h i b i t insect growth and increase development time, bioassays do not often d i s t i n g i s h between grossly d i f f e r e n t modes of a c t i o n . Fecal p e l l e t counts, for example, may be excellent for detecting feeding i n h i b i t o r s but they do not d i s t i n g i s h between behavioral and p h y s i o l o g i c a l differences between treatments. Behavioral reasons for reduced f e c a l p e l l e t counts i n a treatment include reduced phagostimulation and feeding deterrency. Possible p h y s i o l o g i c a l causes for reduced f e c a l p e l l e t counts r e l a t e to t o x i c i t y , including reduced food u t i l i z a t i o n and i n h i b i t i o n of metabolism. Cockroaches are known to increase consumption to compensate for food d i l u t e d with a non-nutritive, non-toxic c e l l u l o s e f i l l e r ( B i g n e l l 1978). In addition, Risch (1985) has shown that feeding preferences can change, depending upon whether leaf disks or whole leaves are used i n feeding bioassays. Unless behavioral and p h y s i o l o g i c a l e f f e c t s can be separated, the mode of action at even a s u p e r f i c i a l l e v e l remains speculative. Dietary e f f i c i e n c y studies are used to d i s t i n g i s h between behavioral and p h y s i o l o g i c a l components of insect growth i n h i b i t i o n (Reese 1979, Isman and 10 Duffey 1982, Isman and Rodriguez 1984) as well as determining the adequacy of an in s e c t ' s food source (Soo Hoo and Fraenkel 1966, Waldbauer 1968, Kogan and Cope 1974). Indices of dietary u t i l i z a t i o n are calculated from measurements of food consumption, weight gain, and excreta production. The growth rate may then be separated i n t o i t s constituents, consumption rate and dietary u t i l i z a t i o n . Some of the t e c h n i c a l d i f f i c u l t i e s with t h i s bioassay method have recently been examined by Schmidt and Reese (1986). C. Behavioral Response to Plant Defence Chemicals Feeding and o v i p o s i t i o n i n phytophagous insec t s are regulated by several f a c t o r s , such as chemostimulants (Hsiao 1969) and deterrents (Shurr and Holdaway 1970, Renwick and Radke 1985) i n host and non-host plants (Jermy 1966, Bernays 1983, Thibout and Auger 1983). The term 'non-preference' has often been used to describe a behavioral non-event but i s l e s s than i d e a l due to i t s anthropocentric bias. 'Deterrent' i s used to specify a substance that when contacted prevents or in t e r r u p t s behavioral a c t i v i t y , i n c l u d i n g feeding or o v i p o s i t i o n (sensu Schoonhoven 1982). A 'stimulant' for the purpose of t h i s discussion i s the antonym of deterrent and i s a substance that, when p h y s i c a l l y contacted, i n c i t e s a p o s i t i v e behavioral response such as feeding or o v i p o s i t i o n . Two d i f f e r e n t neural events may produce the same behavioral response. A deterrent may act d i r e c t l y on a chemoreceptor (Schoonhoven 1982) or by masking the e f f e c t of a chemostimulant ( M i t c h e l l and S u t c l i f f e 1984). Chemoreceptors i n silkworm, Bombyx mori, larvae contain s p e c i a l i s t c e l l s that respond d i r e c t l y to both stimulants and deterrents (Ishikawa 1966). Sparteine, a phytoalkaloid feeding i n h i b i t o r , i s responsible f o r i n h i b i t i n g the response of the sugar-sensitive c e l l . A lack of response from the receptor detecting behavioral stimulants w i l l produce the same 1 1 response as a deterrent ( M i t c h e l l and S u t c l i f f e 1984). Both the cen t r a l and the peripheral nervous system have important r o l e s i n the feeding behavior of herbivorous insects (Dethier 1980). Many insect s examined do not have s p e c i f i c receptors for i n d i v i d u a l chemicals. Dethier (1973, 1980) hypothesizes that neuro-reception of feeding deterrents involves the processing of information c e n t r a l l y from several receptors. Herbivore g e n e r a l i s t s and s p e c i a l i s t s may have a s i m i l i a r capacity to detect chemicals but the information may be processed d i f f e r e n t l y (Dethier 1980). Phytochemicals may thus serve as feeding stimulants to some phytophagous insects while i n h i b i t i n g feeding and growth i n non-adapted species. Plant resistance to insects may often be traced to phytochemical defenses. Host-plant chemicals may aid plant breeders by focusing t h e i r a t t e n t i o n on factors contributing to arthropod resistance. Allelochemicals that may prevent arthropod attack have been i d e n t i f i e d i n several crops i n c l u d i n g tomatoes and cucumbers (Patterson et a l . 1975, de Ponti 1977). Gramine, an a l k a l o i d from barley, i s responsible for resistance to the aphid Schizaphis graminum (Zuniga et a l . 1985). Colorado potato beetles, Leptinotarsa decemlineata, are deterred from feeding by glycoalkaloids present i n Solanum chacoense (Sinden et a l . 1986). Oviposition deterrents, present i n many plants (Gupta and Thorsteinson 1960, Hsiao and Fraenkel 1968a), can occur as c u t i c u l a r components or as chemicals that are released upon feeding. F i e l d t e s t s with the melonworm, Diaphania hyalinata, and the pickleworm, Diaphania n i t i d a l a s , showed that the p r i n c i p l e mechanism of resistance i n two v a r i e t i e s of butternut squash, Curcurbita moschata, was o v i p o s i t i o n deterrency (Elsey 1985). European corn borer, 0_. n u b i l a l i s , females avoid o v i p o s i t i n g i n f i e l d s where damage to corn releases host v o l a t i l e s (Shurr and Holdaway 1970). 12 Oviposition and feeding deterrents are often present i n non-host plants. Spraying plant extracts as insect c o n t r o l agents has i n h i b i t e d mating and o v i p o s i t i o n of oligophagous (Robert and B l a i s i n g e r 1978, Dover 1985) and polyphagous insects (Burnett and Jones 1978). The non-host sesquiterpene lactone, glaucolide A, present i n Vernonia spp. (Asteraceae), deters feeding by larvae of several species of polyphagous lepidopterans (Burnett et a l . 1974). Phytochemicals may show behavioral deterrency only as n a t u r a l l y occurring mixtures. Woodhead and Bernays (1977) have shown that several non-toxic phenolic compounds at natural concentrations produce feeding deterrency only when combined. Even where dominant compounds have been i s o l a t e d , they seldom account for host discrimination even i n oligophagous insect s (Berenbaum 1985). Although glucosinulates are known to stimulate some c r u c i f e r feeding c a t e r p i l l a r s , the t o t a l response can be a t t r i b u t e d to more than one group of phytochemicals (Gupta and Thorsteinson 1960). Furthermore, Nielsen (1978a) states that the a c c e p t a b i l i t y of t h e i r c r u c i f e r host plants to f l e a beetles, P h y l l o t r e t a spp., could not be accounted for s o l e l y on the basis of glucosinolate content, but was l i k e l y due to a composite of allelochemicals. Plants commonly contain feeding deterrents (Woodhead and Bernays 1977, Isman and Duffey 1982). Insects, however, do not always avoid plants because of t o x i c phytochemicals. Insects may be deterred from feeding on harmless plants and conversely may be i n t o x i c a t e d by consuming poisonous plants. Certain tomato c u l t i v a r s that contain the t o x i c g l y c o a l k a l o i d , tomatine, are consumed with impunity by H e l i o t h i s zea larvae because of the a n t i d o t a l e f f e c t of f o l i a r s t e r o l s (Campbell and Duffey 1981). With cauterized chemoreceptors, tobacco hornworm larvae, Manduca sexta, r e a d i l y consume non-toxic plants previously avoided (de Boer et a l . 1977). 13 Phytochemicals, such as feeding deterrents, that protect a plant and allow s u r v i v a l of susceptible i n s e c t genotypes may protect plants longer than potent chemicals that quickly s e l e c t for r e s i s t a n t insect genotypes. Gould (1986), i n h i s simulation model to predict the d u r a b i l i t y of wheat germplasm r e s i s t a n t to the Hessian f l y , Mayetiola destructor, indicated that the most durable resistance would occur i f t o t a l l y susceptible c u l t i v a r s are planted with a r e s i s t a n t c u l t i v a r . This suggests that plant resistance w i l l be more durable when insect s are not under severe s e l e c t i o n pressure, such as would occur when a monoculture of a r e s i s t a n t c u l t i v a r i s planted. Plant extracts used i n the f i e l d may mimic the phytochemical p r o f i l e of a mixed cropping system. D. Phytochemicals and Ph y s i o l o g i c a l Stress Plant defense chemicals cause many adverse p h y s i o l o g i c a l e f f e c t s on in s e c t s , for example reduced digestion, suppression of microsomal enzymes, disruption of endosymbiotic organisms, interference with hormonal processes, reduction of reproductive capacity and death. Immature insects are confronted with chemical and physical plant defenses when obtaining v i t a l n u t r i e n t s . Dietary nitrogen and water, are often the most important factors l i m i t i n g l a r v a l growth (Mattson 1980, Scriber and Slansky 1981). Many adult female insects o v i p o s i t on or near p o t e n t i a l food plants. The gravid female must be g e n e t i c a l l y 'wired' to discriminate among po t e n t i a l l a r v a l food plants and the larvae must be able to detect and avoid ingestion of toxins. In f a c t , neonate lepidopteran larvae are highly susceptible to allelochemicals (Reese 1979) possibly because they have l e s s active or fewer d e t o x i f i c a t i v e enzymes, a t h i n p e r i t r o p h i c gut membrane or lack endosymbiotic organisms for xenobiotic d e t o x i f i c a t i o n . The ubiquitous mixed-function oxidase (MFO) system i s the major d e t o x i f i c a t i o n system i n insects (Ahmad 1986, Dauterman and Hodgson 1978). Adaptation to an allelochemical may r e s u l t from an increased e f f i c i e n c y of the nonspecific MFO system. Polyphagous ins e c t s , such as some lepidopteran larvae, are p o t e n t i a l l y exposed to a broad range of sublethal plant toxins and may be better able to detoxify ubiquitous fitness-reducing a l l e l o c h e m i c a l s than oligophagous i n s e c t s , as the former possess higher gut MFO l e v e l s (Krieger et a l . 1971). S p e c i f i c allelochemicals, however, may be better dealt with by s p e c i a l i s t insects feeding on t h e i r host plants (Blau et a l . 1978). How does the metabolism of xenobiotics a f f e c t i n s e c t r e s i s t a n c e to i n s e c t i c i d e s ? Insect resistance often involves increased enzymatic a c t i v i t y and when t h i s occurs, cross-resistance to chemically unrelated compounds i s quite common (Agosin and Perry 1974, Devonshire and Moore 1982). Spider mites bred for tolerance to an in s e c t r e s i s t a n t cucumber va r i e t y were, i n t e r e s t i n g l y , c r o s s - r e s i s t a n t to several i n s e c t i c i d e s and a va r i e t y of unrelated plants, but the mechanism of resistance was not investigated (Gould et a l . 1982). S p e c i a l i s t s and generalists may sequester a wide v a r i e t y of tox i c phytochemicals. Sequestration may e f f e c t i v e l y prevent the xenobiotic from causing damage to the i n s e c t . The monarch b u t t e r f l y , Danaus plexippus, which sequesters cardiac glycosides from i t s milkweed host, i s an excellent example (Roeske et a l . 1976). Nicotine i s highly toxic to many insect s but some i n s e c t pests of tobacco avoid t o x i c i t y by e f f i c i e n t metabolism and excretion ( S e l f et a l . 1964, Brattsten 1979). L-Canavanine i s an abundant non-protein amino acid found i n seeds of the Central American legume, Dioclea megacarpa, and i s highly toxic to most insects. However the bruchid, Caryedes b r a s i l i e n s i s , 15 not only feeds on J). megacarpa seeds but uses the arginine analog, L-canavanine, i n protein production (Rosenthal et a l . 1982). One must be cautious when drawing analogies between the arthropod response to i n s e c t i c i d e s , and t h e i r response to phytochemical mixtures. Insects r e s i s t a n t to a s i n g l e pyrethroid can develope strong cross-resistance to other pyrethroids ( P r i e s t e r and Georghiou 1980) and several other classes of i n s e c t i c i d e s (Funaki and Motoyama 1986). In contrast, d e t o x i f i c a t i o n of phytochemical mixtures i n plants has received scant atte n t i o n . However, Gould et a l . (1982) have shown that organophosphorous r e s i s t a n t mites are as s e n s i t i v e to a t o x i c host plant as are susceptible s t r a i n s . These r e s u l t s suggest that the d e t o x i f i c a t i o n mechanism of allelochemical mixtures i s d i f f e r e n t from organophosphorous i n s e c t i c i d e s and that other mechanisms may be involved. E. Plant Extracts i n Crop Protection Plant products have been used to c o n t r o l insects since man f i r s t began c u l t i v a t i n g plants. An extract from the flowers of Chrysanthemum  cine r a r i a e f o l i u m , c a l l e d pyrethrum, i s perhaps the most widely used i n s e c t i c i d a l plant product. Pyrethrum was f i r s t sold i n North America i n 1916 ( M a l l i s 1982). Rotenone, from D e r r i s spp. and Lonchocarpus spp., and n i c o t i n e , from Nicotiana r u s t i c a , are other commercially a v a i l a b l e i n s e c t i c i d a l plant constituents. Other botanical i n s e c t i c i d e s are used mainly where they are indigenous, i n c l u d i n g Ryania speciosa, tung seed ( A l e u r i t e s spp.) and s a b a d i l l a from Schoenocaulon spp.(Jacobson and Crosby 1971). Recently, crude extracts and i s o l a t e d phytochemicals from the neem tree, Azadirachta i n d i c a , have been investigated as ins e c t c o n t r o l agents. Azadirachtin, a limonoid i s o l a t e d from neem, completely i n h i b i t e d feeding 16 of the migratory locust, Schistocerca gregaria, at l e v e l s as low as 1 2 ng/cm on leaf disks (Kubo and Nakanishi 1977). The Environmental Protection Agency of the United States has registered a patented formulation (Larson 1985) of neem o i l for use on non-food crops, and r e g i s t r a t i o n on food crops i s pending (Jacobson 1986). Both pyrethrum and neem o i l contain more than one i n s e c t i c i d a l compound. The pyrethrum a c t i v i t y i s derived from s i x pyrethrin esters ( E l l i o t and Janes 1973) and the i n s e c t i c i d a l e f f e c t s of neem o i l are a r e s u l t of several t e t r a n o r t r i t e r p e n o i d s (Jacobson 1986). Botanicals may provide entomologists with novel crop protection agents. Mixtures of defensive chemicals have evolved i n plants and some evidence suggests plants mitigate damage by having combinations of phytochemicals (Berenbaum 1985). Adams and Bernays (1978) showed that fourteen phytochemicals i n n a t u r a l l y occurring concentrations did not produce a measurable e f f e c t on Locusta migratoria feeding when presented alone but deterred feeding when presented as a mixture. I f plants, having evolved over m i l l i o n s of years, use mixtures of chemicals to defend against herbivory perhaps we can also use t h i s 'novel' strategy. Naturally occurring insect growth i n h i b i t o r s (e.g., feeding deterrents) may provide e f f e c t i v e t o o l s for crop management by protecting crops from herbivory while avoiding destruction of b e n e f i c i a l i n s e c t s (Bernays 1983). Recent studies suggest that synthetic i n s e c t i c i d e s confer part of t h e i r benefit due to sublethal e f f e c t s . Aldicarb at sublethal doses reduces the a b i l i t y to f l y and probe, as well as the fecundity, of potato aphids, Macrosiphum euphorbiae (Boiteau et a l . 1985). Reduced f l y i n g and probing also decreased the a b i l i t y of t h i s aphid to transmit v i r a l diseases. The carbamate, methomyl, i n h i b i t s the growth and development of f a l l armyworm, Spodoptera frugiperda, larvae at sublethal 17 concentrations (Javid and A l l 1984). Some of the pyrethroids show promise at sublethal doses because they deter i n s e c t feeding and i n h i b i t development (Dobrin and Hammond 1985, Kumar and Chapman 1984). P h y s i o l o g i c a l or behavioral s t r e s s impeding optimal l a r v a l growth reduces i n s e c t s ' resistance to disease (Boucias et a l . 1984) and increases t h e i r s u s c e p t i b i l i t y to natural enemies. For example, feeding deterrents used i n conjunction with insect pathogens may increase l a r v a l i n f e c t i o n r a t e s . T r i c h o p l u s i a n i larvae i n the early i n s t a r s are more susceptible to i n f e c t i o n by the entomopathogenic fungus S p i c a r i a r i l e y i (Ignoffo et a l . 1975). If a deterrent can maintain an i n s e c t i n an early i n s t a r through growth i n h i b i t i o n , then other mortality f a c t o r s can play a greater r o l e i n population regulation. Laboratory studies provide evidence that chronic sublethal e f f e c t s may have an important but deferred impact on i n s e c t populations (Reese and Beck 1976). F i e l d use of feeding deterrents has been l i m i t e d to a few compounds. The best example of a feeding deterrent tested on a large scale i s the synthetic compound, 4'-dimethyltriazeno-acetanilide ( c i t e d i n Bernays 1983). This product was an e f f e c t i v e feeding deterrent i n f i e l d t e s t s against several herbivorous insects i n c l u d i n g , the cabbage looper, T. n i , the cotton leafworm, Alabama a r g i l l a c e a . and the b o l l weevil, Anthonomus grandis. However, no c o n t r o l was observed for several other pest i n s e c t s , such as the pink bollworm, Pectinophora g o s s y p i e l l a . and the codling moth, Cydia pomonella. These r e s u l t s emphasize that feeding and o v i p o s i t i o n deterrents are often species s p e c i f i c . The use of feeding and o v i p o s i t i o n deterrents integrate well with contemporary integrated pest management (IPM). IPM requires the monitoring of pests to determine when b i o c i d a l agents are needed. The a p p l i c a t i o n of deterrents could be prophylactic or applied when pest populations or crop damage reach an economic threshold. Advantages of applying phytochemical deterrents include s e l e c t i v e pest c o n t r o l and minimal environmental disturbances. Many host and non-host phytochemicals are known to i n h i b i t growth and o v i p o s i t i o n . Laboratory reports on the i s o l a t i o n of insect f i t n e s s -reducing phytochemicals are numerous (e.g., T r i a l and Dimond 1979, Delle Monache et a l . 1984). Most of the studies are not d i r e c t l y concerned with the a p p l i c a t i o n of the chemicals i n pest c o n t r o l s i t u a t i o n s , but deal with e c o l o g i c a l or p h y s i o l o g i c a l considerations. Most phytochemicals examined i n the laboratory have not been studied i n f i e l d t ests and r a r e l y with the ultimate aim of developing a useful a g r i c u l t u r a l product. The lack of experimental f i e l d data on the use of sublethal phytochemicals undermines the many laboratory studies on the subject. 19 I I I . MATERIAL AND METHODS A. Plant extracts The plants were a l l c o l l e c t e d from southern B r i t i s h Columbia, a i r -TM dried and f i n e l y ground i n a Wiley m i l l . Plant species, parts extracted, l o c a t i o n of harvest, and harvest dates are l i s t e d i n Table I. Powdered plant material (200 g) was thoroughly mixed with 1 l i t e r of e i t h e r p e t r o l (petroleum ether, b o i l i n g range 30-60°C) or 95% aq ethanol (EtOH) and soaked for 24 h at room temp (21°C). The s l u r r y was f i l t e r e d and r i n s e d , then the extracts were reduced under vacuum to 10-60 ml depending on t h e i r respective v i s c o s i t i e s . B. B i o l o g i c a l Screenings 1. I n i t i a l Screening Extracts 5-fold of those nat u r a l l y occurring, calculated as the dry weight of plant powder extracted to the dry weight of a r t i f i c i a l d i e t (dwt/dwt), were admixed with the dry portion of the a r t i f i c i a l d i e t (Bioserv Inc., Frenchtown, NJ, no. 9682) and the c a r r i e r solvent was removed i n a fume hood. Controls consisted of a r t i f i c i a l d i e t s i m i l a r l y treated with the c a r r i e r solvents alone ( p e t r o l and EtOH). Upon hatching, 2 neonate P_. saucia larvae from a laboratory colony were placed on about 2 g (wwt) a l i q u o t s of d i e t i n 30 ml p l a s t i c cups at room temperature. The rearing cups were placed i n p l a s t i c boxes with moistened paper towels to prevent desiccation of larvae and d i e t . Using l i v e l a r v a l weights (n=30), l a r v a l growth was measured as a percentage of the controls a f t e r 14 days. The l a r v a l gravimetric data was 1°8^Q transformed p r i o r to s t a t i s t i c a l a n a l y s i s i n each experiment. 20 Table I. Plant species, components extracted, harvest locations i n B r i t i s h Columbia and dates of material bioassayed i n i n i t i a l screening Plant Species Components Harvest Extracted Location date Artemisia t r i d e n t a t a stms, l v s , f l s Summerland 10--83 Centaurea d i f f u s a stms, l v s , f l s Kamloops 10--83 Chrysotharanus nauseosus stms, l v s Keromeos 05--84 Chamomilla suaveolens whole plant Vancouver 05--84 Senecio iacobaea stms, l v s Abbottsford 05--84 Tragopon dubius stms, l v s , f l s Hedley 05--84 stms=stems, lvs=leaves, fls=flowers 21 2. Bioassay of Unextracted and Extracted Plant Material The plant residue remaining from the i n i t i a l extraction, hereafter referred to as the 'marc', and t h e i r respective unextracted plant powders were assayed for b i o l o g i c a l a c t i v i t y against P_. saucia neonates to determine the e f f i c i e n c y of the extraction process. The dry ingredients of the a r t i f i c i a l d iet consisted of Bioserv no. 9682 with an equivalent portion of marc or plant powder (1:1 w/w). Control d i e t was prepared using one part powdered c e l l u l o s e (alphacel) to one part a r t i f i c i a l d i e t . An a d d i t i o n a l treatment consisted of the c o n t r o l diet without c e l l u l o s e . This treatment was used to determine the e f f e c t of d i l u t i n g the c o n t r o l d i e t with c e l l u l o s e on l a r v a l growth. The experimental design was the same as i n the previous experiment. 3. Second Screening The most i n h i b i t o r y extracts to P_. saucia l a r v a l growth were selected for a further bioassay. A r t i f i c i a l d i e t s were fr e s h l y prepared using natural concentrations (dwt/dwt) of the plant extracts. Control d i e t s were treated with the c a r r i e r solvent. P_. saucia neonates were i n d i v i d u a l l y placed on ca. 1 g of d i e t (n=25) and allowed to feed for 11 days and then weighed. A l l surviving larvae were placed on control diet on day 11 and allowed to continue feeding to determine the persistence of growth i n h i b i t o r y e f f e c t s through pupation and emergence. C. Dose-Response Bioassays 1. Dose-Response Bioassay on P_. saucia Neonates Four concentrations (20, 40, 60, and 80% of natural cone, dwt/dwt) of the ethanolic extract of _A. t r i d e n t a t a and both extracts of C_j_ suaveolens were assayed as above with ethanol and petroleum ether solvent c o n t r o l s . After 15 days P_. saucia larvae were counted, weighed, and then allowed to 22 feed on the c o n t r o l d i e t u n t i l pupation. Rates of pupation and emergence were recorded for each treatment. EC^Q S ( e f f e c t i v e concentrations i n h i b i t i n g l a r v a l growth by 50% r e l a t i v e to controls) were calculated using probit a n a l y s i s (Finney 1971). 2. Dose-Response Bioassay using the A l f a l f a Looper, Autographa  c a l i f o r n i c a Speyer Another dose-response bioassay was performed to determine i f the b i o l o g i c a l e f f e c t of extracts on P. saucia was also evident for other noctuid species. F i e l d c o l l e c t e d A., c a l i f o r n i c a larvae were reared to maturity on a r t i f i c i a l d i e t (Bioserv no. 9682). The r e s u l t i n g F^ neonates were used for t h i s experiment (n=25); the bioassay and data analysis were the same as described i n the neonate P_. saucia dose-response experiment. 3. S e n s i t i v i t y of Older P_. saucia Larvae To determine how the b i o l o g i c a l a c t i v i t y of the plant extracts was influenced by l a r v a l age, another dose-response experiment was i n i t i a t e d with older c a t e r p i l l a r s . Neonate P_. saucia larvae were fed for s i x days on the standard c o n t r o l d i e t . The r e s u l t i n g second i n s t a r larvae (ca. 7 mg)were then transferred to the treatment d i e t s (n=25). The bioassay and data a n a l y s i s were as previously described i n the neonate P_. saucia dose-response experiment. D. F i n a l Determination of Extract f o r F i e l d T r i a l 1. Detailed Growth Analysis of saucia Larvae Feeding on A. t r i d e n t a t a and C_. suaveolens Extracts To d i s t i n g u i s h between behavioral and p h y s i o l o g i c a l contibutions to l a r v a l growth i n h i b i t i o n , a d e t a i l e d growth analysis was i n i t i a t e d on second i n s t a r P_. saucia• Larvae (10.9 ± 1.5 mg, n=15) were fed d i e t s at t h e i r natural concentrations (100% dwt/dwt). An EtOH extract of k_. 23 t r i d e n t a t a and a p e t r o l extract of C_. suaveolens were compared with the standard d i e t treated with p e t r o l . The duration of the experiment was 48 h, although larvae were weighed at 24 h as well as 48 h to determine r e l a t i v e feeding and growth rates over the two 24 h periods. Except where otherwise ind i c a t e d , a l l measurements are based on dry weights. Growth ind i c e s were calculated as described by Scriber and Slansky (1981). 2. Formulation of Active Extracts for F o l i a r Application and S t a b i l i t y of Crude Extracts B i o l o g i c a l a c t i v i t y of two aqueous extracts and a 20% aq EtOH extract were compared to the o r i g i n a l EtOH extract (8 months old) and a f r e s h l y prepared EtOH extract from the o r i g i n a l plant material for both _A. t r i d e n t a t a and C_. suaveolens. The two aqueous extracts were prepared by adding 10 g of ground plant powder to 40 ml of d i s t i l l e d water. One of these s l u r r i e s was brought to a r o l l i n g b o i l and then both were kept at room temp f o r 24 hrs before being f i l t e r e d . The 20% aq EtOH extracts were prepared following the same procedure as the room temp water extracts. The aqueous extracts were then l y o p h i l i z e d to reduce t h e i r volume. Control larvae fed on a r t i f i c i a l d i e t s treated with water or EtOH. Neonate P. saucia larvae (n=25) were used for t h i s bioassay as described above. E. F i e l d T r i a l of the A. t r i d e n t a t a Extract on Cabbage An experiment was designed to t e s t the f i e l d e f f i c a c y of the _A. t r i d e n t a t a extract r e l a t i v e to i t s c a r r i e r (30% aq EtOH), water and the pyrethroid i n s e c t i c i d e , deltamethrin. Cabbage (cv. Early Marvel) was seeded on May 27, 1985. Plots of cabbage were assigned to four complete blocks and treatments were randomized within each block. P l o t s consisted 2 of a row of seven cabbage plants (11.7 m ) and were separated by an equivalent row of unsprayed cabbage. In addition, guard plants were 24 situated at both ends of each p l o t . Spray a p p l i c a t i o n s were made with a hand-held t r i g g e r sprayer and the nozzle was c a l i b r a t e d to deliver an equivalent volume (14 ml) of solu t i o n to each plant. The nozzle was ca l i b r a t e d by measuring 10 p u l l s of the tr i g g e r i n t o a graduated cylinder as a f i n e mist. Four t r i g g e r p u l l s were executed from d i r e c t l y over the plant and three p u l l s f o r coverage to the sides and three p u l l s for the lower side of the leaves. The A. t r i d e n t a t a extract was formulated i n 30% aq EtOH. This c a r r i e r gives an even f o l i a r coverage without the addition of a spreader or s t i c k e r . The r e s u l t i n g s o l u t i o n was the equivalent of 0.2 g/ml. The 3 other treatments consisted of the c a r r i e r solvent, 30% aq EtOH, d i s t i l l e d water, and the standard pest c o n t r o l agent, deltamethrin TM (Decis 2.5 EC, Hoechst) at 17.9 ug a i . / l plus 1 ml/1 of the spreader-TM TM TM s t i c k e r , Superspred (Decis and Superspred were provided courtesy of Dr. Robert S. Vernon, Ag r i c u l t u r e Canada, Vancouver, B.C). Spray a p p l i c a t i o n s were made to a l l pl o t s on July 24, 1985 at 6:30 am. with the cabbage at post-heading (59 days from seed). The above ground parts of a l l experimental plants were surveyed 2 days before spraying to e s t a b l i s h the baseline insect populations i n each of the experimental p l o t s . Once the t r i a l was i n i t i a t e d , a l l experimental plants were monitored 1, 6, 9, and 25 days a f t e r spraying to assess the e f f e c t s on the major insect pests of cabbage. The pre-count survey and the f i r s t three post-treatment counts were non-destructive v i s u a l observations of both sides of a l l non-head leaves. The f i n a l i n s e c t count at 25 days post-treatment was a destructive sampling of the above ground cabbage. The pests monitored were the cabbage looper (CL), T_. ni^, imported cabbageworm (ICW), P_. rapae, and diamondback moth larvae (DBM), P l u t e l l a x y l o s t e l l a L. Cabbage pests were analyzed separately where numbers warranted and together as cabbage looper 25 equivalents (CLE) : 1 CLE = 1 CL = 1.5 ICW = 20 DBM (Shelton et a l . 1982). Data from the f i e l d t r i a l was analyzed i n a completely randomized block design with repeated observations over time. The 'treatment' degrees of freedom were part i t i o n e d with i n d i v i d u a l treatment contrast comparisons. The 'day' e f f e c t was p a r t i t i o n e d with polynomial expansion c o e f f i c i e n t s . Then the sum of squares from the 'treatment X day' i n t e r a c t i o n was p a r t i t i o n e d for the l i n e a r , quadratic and r e s i d u a l v a r i a t i o n . S i g n i f i c a n t 'treatment X day' i n t e r a c t i o n s established differences i n v a r i a t i o n among pest population l e v e l s with the four spray treatments during the f i v e survey dates. A q u a l i t y estimate was obtained by r a t i n g each of the cabbage heads on the l a s t day of the experiment (25 days post-spray). The r a t i n g was based on the amount of v i s i b l e damage present i n and on each head a f t e r the wrapper leaves were removed (4=marketable cabbage, no e x t e r i o r damage; 3=sauerkraut grade, exterior damage only, no holes; 2=garden grade, one hole i n t o head; l=unmarketable, more than one hole; 0=no head remaining). An analysis of variance was performed on the v i s u a l damage estimates and means were compared by p a r t i t i o n i n g the treatment sum of squares. F. Laboratory Evaluation of the Oviposition Deterrence of A. t r i d e n t a t a  Extract on Caged P. rapae A f i e l d c o l l e c t e d colony of P_. rapae larvae was reared to pupation on cabbage (cv. Early Marvel). Eight adults of each sex were placed i n a 50 X 50 X 50 cm screened cage and fed a s o l u t i o n of 10% sucrose. The cage was on a laboratory bench and l a t e summer l i g h t from the south and west was supplemented with a 100 watt lamp f o r 16 h each day. On the following day s i x cabbage leaves i n 50 ml f l a s k s f i l l e d with water were offered to the b u t t e r f l i e s i n a choice o v i p o s i t i o n experiment. Two leaves from each of 26 the following treatments were included: A_. tridentata extract at the equivalent of 0.2 g/ml, 30% aq EtOH, and d i s t i l l e d water. The solutions were hand-painted onto the leaves. The leaves and the f l a s k s were arranged i n a c i r c l e i n a random order. Eggs were counted after 48 h and the experiment was repeated. Results from the 2 r e p l i c a t e s were pooled. G. Growth Comparison of Laboratory Reared and Wild Colonies of P. saucia  Larvae on Standard Diet and Diets with Addition of an Ethanolic A.  t r i d e n t a t a Crude Extract A chronic feeding bioassay was used to e s t a b l i s h the response of a natural P_. saucia population i n comparison with the £. saucia laboratory population on standard a r t i f i c i a l d i e t and diets with the a d d i t i o n of the EtOH-A. tr i d e n t a t a e x t r a c t . Neonate larvae from f i e l d c o l l e c t e d £. saucia and laboratory reared P_. saucia (>20 generations) were divided into two groups (n=30). One group from each colony was allowed to feed on e i t h e r the standard control d i e t or d i e t admixed with a ethanolic _A. t r i d e n t a t a extract (50% nat. cone, dwt/dwt). As i n previous experiments the neonates were each placed i n i n d i v i d u a l feeding containers with ca. 500 mg of d i e t . After 8 days the larvae were counted and weighed. The l a r v a l weights were log^Q transformed and analyzed by a two way analysis of variance. H. Phytochemical Investigation 1. Chromatographic Separation of the Ethanolic _A. t r i d e n t a t a Extract C e n t r i f u g a l t h i n layer chromatography was used to separate the a c t i v e component(s) of the crude ethanolic extract of A^ . t r i d e n t a t a . An a l i q u o t of extract equivalent to eight grams of plant powder was eluted on a 1.5 x 10 cm s i l i c a gel column (60-200 mesh) with EtOH and reduced to 3 ml. The TM reduced extract was loaded onto a Chromatatron plate (2 mm) and developed 27 with successively more polar solvents (hexane, hexanetCHCl^ (1:1) t CHCl 3:acetone (6:1), CHC1 3, EtOH, and MeOH) to give ten 10-ml f r a c t i o n s , 77 5-ml f r a c t i o n s and 2 50-ml f r a c t i o n s . The f i r s t 5 f r a c t i o n s were hexane eluates followed by 44 l a r g e l y acetone and CHCl^ eluates and 40 EtOH-MeOH f r a c t i o n s . The eighty-nine f r a c t i o n s were spotted on s i l i c a gel t h i n layer chromatography (TLC) plates containing a fluorescent i n d i c a t o r (Baker-TM f l e x , IB2-F). The plates were developed using a standard method for sesquiterpene lactones (Pieman et a l . 1980) with CHCl^-acetone (6:1). TLC plates were observed under short- and long- wave u l t r a v i o l e t l i g h t and then sprayed with a v a n i l l i n spray reagent. Fractions containing s i m i l a r compounds based on TLC observations were pooled i n t o 5 major f r a c t i o n s according to t h e i r phytochemical constituents and then reduced i n volume under vacuum. Pooled f r a c t i o n s consisted of: major f r a c t i o n no. 1 containing hexane eluates ( f r a c t i o n s 1-5), major f r a c t i o n nos. 2,3 and 4 were mainly CHCl^ eluates ( f r a c t i o n s 6-13,14-26,27-48) and major f r a c t i o n no. 5 contained EtOH and MeOH f r a c t i o n s 49-89. Major f r a c t i o n no. 3 was TM further separated on the Chromatatron as i t contained TLC spots common to both major f r a c t i o n s nos. 2 and 4. Major f r a c t i o n no. 3 was eluted with hexane:CHC13 (2:1,1:1,1:2,1:6) and CHCl^ giving 60 f r a c t i o n s that were spotted on TLC plates, developed and analyzed as above. The 60 f r a c t i o n s of major f r a c t i o n no. 3 were divided between major f r a c t i o n nos. 2 (1-35) and 4 (36-60). The r e s u l t i n g 4 major f r a c t i o n s were bioassayed with neonated P_. saucia larvae (n=20) using an 80% cone (dwt/dwt) of the extract major f r a c t i o n . A p o s i t i v e control c o n s i s t i n g of the o r i g i n a l extract was included as well as a solvent (CHC1-) c o n t r o l . 2 8 2. Chromatography of Sesquiterpene Lactones Previously Isolated from t r i d e n t a t a To determine the chemical nature of the highly active _A. t r i d e n t a t a extract, pure sesquiterpene lactone standards were chromatographed with the most ac t i v e major f r a c t i o n s from the previous experiment. Ten sesquiterpene lactones were obtained from Dr. Richard G. Kelsey, Dept. of Chemistry, University of Montana that had previously been i s o l a t e d from _A. t r i d e n t a t a and i t s c l o s e l y r e l a t e d subspecies (Kelsey and Shafizadeh 1979). Dehydroleucodin, dihydrosantamarin, arbusculin A, arbusculin B, arbusculin C, ( t a t r i d i n A - purity questionable), matricarin, deacetoxymatricarin, deacetylmatricarin, and dehydroreynosin were chromatographed with the major f r a c t i o n s obtained i n the previous experiment and the o r i g i n a l ethanolic A^ t r i d e n t a t a extract. The two solvent systems used consisted of CHCl 3:acetone (6:1) (Pieman et a l . 1980) and petroleum ether:CHCl 3:Et 20Ac (2:2:l)(Greissman and G r i f f i n 1971). Plates were developed i n either saturated or non-saturated TLC tanks and examined under long- and short-wave u l t r a v i o l e t l i g h t , and treated with the v a n i l l i n colour reagent as described above. IV. RESULTS 29 A. Laboratory Screenings Table I I shows the growth i n h i b i t o r y e f f e c t s on P_. saucia larvae of crude plant extracts added at 5 times the natural concentration (dwt/dwt) to a r t i f i c i a l d i e t . Six of the 12 extracts exhibited s u f f i c i e n t a n t i b i o s i s (sensu Painter 1951) to proceed to the next screening. Since the extract concentrations were high, only the s i x treatments that completely or severely i n h i b i t e d growth advanced to the second screening. No larvae survived on the d i e t s with ethanolic and p e t r o l extracts of t r i d e n t a t a and C. suaveolens; the ethanolic extract from C_. nauseosus and the ethanolic extract from C. d i f f u s a were s i m i l a r l y a c t i v e . The only other extract s i g n i f i c a n t l y d i f f e r e n t from the controls was the j>. jacobaea ethanol extract, but at the high concentration used i n the bioassay i t was not considered to be s u f f i c i e n t l y a c t i v e for further screening. The r e s u l t s of the unextracted plant powder and marc bioassay are shown i n Table I I I . The e f f i c i e n c y of the extraction process i n removing growth i n h i b i t o r y phytochemicals i s determined by t e s t i n g the extracted plant residue for l a r v a l growth i n h i b i t i o n . In cases where diet with the unextracted plant powder severely i n h i b i t e d P_. saucia l a r v a l growth, at l e a s t one of the respective marcs was shown to have had the growth i n h i b i t o r y agents removed. 30 Table I I . E f f e c t s of weed extracts^ incorporated into a r t i f i c i a l d i e t on growth and s u r v i v a l of neonate P_. saucia i n an i n i t i a l screening bioassay PLANT GROWTH i (% OF CONTROL) SURVIVORSHIP (% ) 2 SPECIES P e t r o l Ethanol P e t r o l Ethanol A. t r i d e n t a t a Oc 3 0c 0 0 C. d i f f u s a 106a 8c 63 3 C. nauseosus 80ab 0c 67 0 C. suaveolens 0c 0c 0 0 S. iacobaea 105a 18bc 90 37 T. dubius 107a 72ab 80 80 contr o l 100a 100a 77 73 Extract concentrations were f i v e times the natural cone.(dwt/dwt). 2N=30 3 Treatments followed by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t (Tukey's studentized range (HSD) t e s t , p=0.05). Table I I I . Growth and s u r v i v a l of P_. saucia neonate larvae fed on a r t i f i c i a l d i e t s with unextracted plant powder or on the extracted marcs PLANT POWDER ETHANOL MARC PETROL MARC SPECIES GROWTH2 SURVIVAL3 GROWTH SURVIVAL GROWTH SURVIVAL A. t r i d e n t a t a o g 4 0 121abc 50 19fg 53 C. d i f f u s a 47cdef 87 34defg 77 31efg 70 C. nauseosa 149ab 40 163ab 90 34bcde 50 C. suaveolens 18g 30 114abcd 77 190ab 80 S. iacobaea 15g 30 98abcd 73 145ab 77 T. dubius 96abcd 67 147ab 43 277a 73 Standard d i e t 272a 70 Control (with c e l l u l o s e ) 100abed 80 Plant material incorporated with a r t i f i c i a l d i e t (1:1 dwt/dwt). i Taken as the percentage of l a r v a l growth of the control treatment with c e l l u l o s e f i l l e r simulating the plant m a t e r i a l . 'Percentage of t o t a l l a r v a l survivors (N=30). Treatments followed by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t (Tukey*s studentized range (HSD) test;p=0.05). 32 S p e c i f i c a l l y , the d i e t containing the unextracted C_. suaveolens and £5. jacobaea powders produced s i g n i f i c a n t l y smaller larvae and higher mortality than both of t h e i r marcs. Larvae fed the marc die t s from these plants grew as w e l l , or better than control larvae. A n t i b i o s i s was s i m i l i a r l y high among the JP. saucia larvae fed the die t s with unextracted k_. t r i d e n t a t a powder, i n which no survivors were observed. Furthermore the weights of larvae fed the _A. t r i d e n t a t a ethanol marc di e t did not d i f f e r s i g n i f i c a n t l y from the controls. The extracts from _A. t r i d e n t a t a and C. suaveolens that produced these marcs were also shown to be the most a c t i v e P_. saucia l a r v a l growth i n h i b i t o r s (Table IV). These r e s u l t s demonstrate that i n nearly every case where P_. saucia growth i n h i b i t o r s were present i n the unextracted plant powders they were removed by one or both of the extracting solvents. P_. saucia larvae fed diet without c e l l u l o s e were, on the average, over two and a half times heavier than those fed d i e t containing c e l l u l o s e (50% dwt). The dietary addition of plant powders or marcs, however, did not always reduce l a r v a l growth. Larvae fed seven of the treatment d i e t s r e s u l t e d i n heavier larvae on average than the c o n t r o l larvae fed the standard d i e t with c e l l u l o s e . The r e s u l t s of the second screening experiment at natural concentrations are comparable to those of the plant powder and marc experiment. Even at the reduced concentration (100% dwt/dwt) the di e t containing the A. t r i d e n t a t a ethanolic extract resulted i n 100% P_. saucia l a r v a l mortality (Table IV). Table IV. E f f e c t s of selected weed extracts incorporated i n t o a r t i f i c a l d i e t at natural concentrations on the weight and s u r v i v a l of neonate P_. saucia fed for 11 days PLANT EXTRACT LARVAL WEIGHT SURVIVORSHIP SPECIES (% OF CONTROL) (% OF TOTAL) A. t r i d e n t a t a PETROL 63ab 2 51 A. t r i d e n t a t a ETHANOL Od 0 C. d i f f u s a ETHANOL 44ab 92 C. nauseosus ETHANOL 31bc 96 C. suaveolens PETROL 9d 36 C. suaveolens ETHANOL 12cd 8 control 100a 96 N=25 neonate P. saucia larvae Treatments followed by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t (p=0.05) using Tukey's studentized range (HSD) t e s t . 34 In addition, larvae fed d i e t s with both C. suaveolens extracts grew s i g n i f i c a n t l y l e s s and had reduced survivorship compared to larvae fed on control d i e t . These r e s u l t s agree with the plant powder and marc experiment showing that d i e t s incorporating plant powders of A_. t r i d e n t a t a and C_. suaveolens are the most b i o l o g i c a l l y a c t i v e towards J?. saucia larvae. Larvae surviving the second screening were transferred to c o n t r o l diet to examine lat e n t e f f e c t s of neonatal growth i n h i b i t i o n on l a t e r l a r v a l growth and development. Between 50 and 100% of the surviving JP. saucia larvae emerged as adults from a l l treatments. These treatments, therefore, do not appear to cause any obvious p h y s i o l o g i c a l damage which p e r s i s t s through the pupal to the adult stage. F i g . 1 shows the r e s u l t s of the dose-response experiment using ethanol and p e t r o l extracts of C_. suaveolens and the _A. t r i d e n t a t a ethanolic extract at four concentrations. I?, saucia l a r v a l weight was inversely r e l a t e d to the plant extract concentration for a l l 3 crude e x t r a c t s . The r e s u l t i n g EC^Q'S were 36, 39, and 42% for the C_. suaveolens EtOH- and pe t r o l extract, and _A. t r i d e n t a t a EtOH-extract, r e s p e c t i v e l y . Similar r e s u l t s were obtained when t h i s experiment was r e p l i c a t e d . EC^^'s for the p e t r o l extract of C_. suaveolens and the EtOH extract of _A. t r i d e n t a t a were 37 and 35%, r e s p e c t i v e l y . S u r v i v a l of P_. saucia larvae was mainly concentration-dependent for each ex t r a c t . Survivorship showed a l i n e a r response for the A^ . t r i d e n t a t a 2 ethanolic and C. suaveolens p e t r o l extracts (with r values of 0.84 and 0.87, rs p e c t i v e l y ) , b u t survivorship on the C_. suaveolens ethanolic extract 2 did not c o r r e l a t e well with the l i n e a r equation (r = 0.52) ( F i g . 1). Pooled r e s u l t s from two dose-response experiments suggest that low dietary 35 concentrations of the plant extracts (0-40% natural c o n e ) , had l i t t l e e f f e c t on s u r v i v a l . The highest dietary concentration (80%) usually resulted i n greatly reduced survivorship. F i g . 1 shows mortality i s l a r g e l y uneffected u n t i l the concentration increased between 40% and 60% for the three extracts. A n t i b i o s i s increased with concentration as shown by the r e s u l t s of both s u r v i v a l and growth i n h i b i t i o n ( F i g . 1). Dose-dependant a n t i b i o s i s was evident i n i t i a l l y as l a r v a l growth i n h i b i t i o n and at higher doses as both increased growth i n h i b i t i o n and m o r t a l i t y . The mode of a c t i o n of the bioactive plant extracts on P_. saucia larvae was examined further i n a l a t e r experiment measuring consumption and growth rates along with d i e t a r y u t i l i z a t i o n . As i n the previous experiment, s u r v i v i n g larvae placed on c o n t r o l d i e t , allowed to pupate and emerge, showed no obvious pe r s i s t e n t p h y s i o l o g i c a l e f f e c t s . Age-dependent e f f e c t s were also examined i n a dose-response experiment. Six-day o l d , second i n s t a r larvae appeared more to l e r a n t to the plant extracts than neonates when tested at the same concentrations. For example, even at an extract concentration of 80% the mortality was c o n s i s t e n t l y 15% or l e s s r e l a t i v e to the controls ( F i g . 2 ) . Furthermore, growth was 33, 52 and 72% of controls for the A_. t r i d e n t a t a EtOH and the C_. suaveolens EtOH- and p e t r o l extracts, r e s p e c t i v e l y , at t h i s concentration. The b i o l o g i c a l a c t i v i t y of these extracts i s not r e s t r i c t e d to JP. saucia larvae. Larvae of another polyphagous noctuid, the a l f a l f a looper, A_. c a l i f o r n i c a , were also tested i n a chronic feeding dose-response experiment ( F i g . 3). The ECL^'s of A_. c a l i f o r n i c a neonates were 10-20% 36 Figure 1. Percent growth (o) r e l a t i v e to c o n t r o l growth and percent t o t a l mortality (•) of £. saucia neonates fed ethanolic extracts from A) _A. t r i d e n t a t a , and B) C. suaveolens, and a p e t r o l extract from C) C. suaveolens admixed to a r t i f i c i a l d i e t s (n=25 larvae per concentration with each e x t r a c t ) . Error bars on the growth points are the standard deviation. 3 7 (-HAinviaoiAj % H - ) H 1 M 0 H 9 % 38 Figure 2. Percent growth (o) r e l a t i v e to control growth and percent t o t a l mortality (•) of six day-old P. saucia larvae fed ethanolic extracts from A) A. tridentata , and B) C_. suaveolens, and a p e t r o l extract from C) C. suaveolens admixed to a r t i f i c i a l d i e t s (n=25 larvae per concentration with each e x t r a c t ) . Error bars on the growth points are the standard deviation. % GROWTH M % MORTALITY (-•-) 6£ 40 Figure 3. Percent growth (o) r e l a t i v e to control growth and percent t o t a l mortality (•) of neonate A_. c a l i f o r n i c a fed ethanolic extracts from A) A^ tridentata , and B) C_. suaveolens, and a p e t r o l extract from C) C. suaveolens admixed to a r t i f i c i a l d i e t s (n=25 larvae per concentration with each e x t r a c t ) . Error bars on the growth points are the standard deviation. 20 4 0 60 8 0 0 20 4 0 60 8 0 % N a t u r a l C o n e , ( d w t ) 42 (natural cone.) for a l l three extracts. These E C ^Q ' S are about half the observed E C ^Q ' S for P_. saucia larvae and no A_. c a l i f o r n i c a larvae survived the 60 or 80% concentrations for any of the extracts. B. Detailed Growth Analysis of P. saucia Larvae on the C. suaveolens and  A. t r i d e n t a t a Extracts. The r e s u l t s of d e t a i l e d growth an a l y s i s of second i n s t a r P. saucia, fed d i e t s containing the A. t r i d e n t a t a EtOH extract and the C_. suaveolens p e t r o l extract, are shown i n Table V. The approximate d i g e s t i b i l i t y (AD) for the three l a r v a l cohorts did not d i f f e r s i g n i f i c a n t l y . The r e l a t i v e growth rate (RGR) i s a product of r e l a t i v e consumption rate (RCR) and d i e t a r y u t i l i z a t i o n . The RGR for larvae fed d i e t s with the C_. suaveolens p e t r o l extract was 70% of the c o n t r o l - d i e t fed larvae. The majority of t h i s growth i n h i b i t i o n appears to be associated with behavioral factors as in d i c a t e d by the s i g n i f i c a n t l y lower RCR, whereas dietary u t i l i z a t i o n s do 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 . The P_. saucia larvae fed d i e t s with the A_. t r i d e n t a t a EtOH extracts produced even lower growth rates than larvae fed the C. suaveolens ex t r a c t . This severe growth i n h i b i t i o n , however, appears l a r g e l y due to p h y s i o l o g i c a l e f f e c t s . This i s indicated by the extremely low net (ECI) and gross (ECD) dietary u t i l i z a t i o n even while the consumption rate remained about 60% of the c o n t r o l . Separate consideration of the RGRs over the 48 h experiment reveal an i n t e r e s t i n g phenomenon. The RGR for larvae fed the C_^  suaveolens p e t r o l extracts during the f i r s t 24 h period was severely retarded r e l a t i v e to the c o n t r o l s , but i n the second 24 h period the growth rate accelerated to equal the RGR of the c o n t r o l . A3 Table V. E f f e c t s of dietary _A. t r i d e n t a t a extract (EtOH) and C_. suaveolens extract (petrol) on second i n s t a r P_. saucia d i g e s t i b i l i t y of food (AD), r e l a t i v e growth rate (RGR), r e l a t i v e consumption rate (RCR), and gross (ECI)^ and net (ECD)''" dietary u t i l i z a t i o n s . N u t r i t i o n a l Dietary Supplement  Index C. suaveolens A. tr i d e n t a t a Control n= IA 13 15 AD ±SD 2 59.6 ± 15.2 (mg dwt/mg dwt-day x 100) 58.2 + 33.8 51.8 ± lA.6nsd 3 RGR ±SD O.AA ± 0.09 b 4 (mg dwt/mg dwt-day) 0.03 + 0.15 c 0.58 ± 0.10 a RCR ±SD 2.2 ± 0.6ab (mg dwt/mg dwt-day) 1.6 + 1.2 b 2.7 ± 0.A a ECI ±SD 20.A + 6.3 a (mg dwt/mg dwt-day x 100) -3.6 + 2A.6 b 22.0 ± 5.1 a ECD ±SD AA.9 ± A8.2 ab (mg dwt/mg dwt-day x 100) 0.8 + 59.5 b 52.9 ± AA.6 a ECI=efficiency of conversion of ingested food ECD=efficiency of conversion of digested food SD = standard deviation nsd = not s i g n i f i c a n t l y d i f f e r e n t Means i n a row followed by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t (Tukey's studentized range (HSD) t e s t , p=0.05). AA The s t a b i l i t y of crude extracts i s important for r e l i a b l e reproduction of laboratory experiments and for ins e c t c o n t r o l s i n the f i e l d . The growth i n h i b i t o r y response of P_. saucia to extracts kept at A°C for eight months was numerically though not s i g n i f i c a n t l y d i f f e r e n t (p<0.05) from f r e s h l y prepared extracts (Table VI). However, i n both treatments of the fresh as compared to the stored extracts, l a r v a l m o r tality was s i g n i f i c a n t l y reduced (orthogonal comparisons; p<0.05). Freshly prepared extracts were subsequently used i n further experiments. The r e s u l t s of the formulation t r i a l s of crude A_. t r i d e n t a t a and C. suaveolens extracts are shown i n Table VI. There was an almost complete loss i n a c t i v i t y of the C_. suaveolens extracts when d i l u t e d with water. The consistent growth i n h i b i t o r y a c t i v i t i e s of the A_. t r i d e n t a t a extracts formulated i n 20% aq EtOH and the added p h y s i o l o g i c a l component of growth i n h i b i t i o n determined i t s s e l e c t i o n for the f i e l d t r i a l . The undissolved tar and suspended p a r t i c u l a t e s i n the water formulated extracts did not present problems i n the laboratory bioassay because the extract was admixed with the a r t i f i c i a l d i e t . F i e l d spraying, however, required a more soluble medium, thus a 30% aq EtOH solution was used to dissolve most of the extract residues. 45 Table VI. Mean growth and percent s u r v i v a l of neonate P_. saucia larvae fed a r t i f i c i a l d i e t s incorporating fresh and eight month old ethanolic extracts, hot water, room temp water and 20% aq ethanol formulations of A_. tri d e n t a t a and C_. suaveolens for 16 days Treatment % Sur v i v a l Mean weight ± SD A. tridentata 95% ethanol fresh 24 2.6 + 2.5fg 2 aged 68 13.8 + 6.6def water 96 34.9 + 25.5cd Hot water 100 41.9 + 23.3bc 20% aq EtOH 40 8.7 + 5.3ef C. suaveolens 95% ethanol fresh 12 1.1 + 0.6g aged 52 5.8 + 5.3fg water 100 98.2 + 45.7a Hot water 100 121.5 ± 62.4a 20% aq EtOH 100 79.4 + 26.6ab Control 96 101.7 + 35.6a SD=standard deviation i Means followed by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t (Tukey's studentize range (HSD) t e s t , p=0.05). Extracts were kept at 4°C for eight months. 46 C. F i e l d T r i a l of the A. t r i d e n t a t a Extract on Cabbage The r e s u l t s of the f i e l d spraying are i l l u s t r a t e d i n Figures 4, 5, and 6. S i g n i f i c a n t differences between treatments were observed i n the aggregate number of cabbage pests computed as CLEs ( F i g . 4, Appendix I ) . When treatments were compared f o r the e n t i r e duration of the experiment, the cabbage treated with _A. t r i d e n t a t a extract had s i g n i f i c a n t l y (p<0.05) fewer CLEs compared with the c a r r i e r solvent (30% aq EtOH) treatment and the water sprayed cabbage. There was no s i g n i f i c a n t difference i n CLEs between the 30% aq EtOH and the water sprayed cabbage. In addition, the i n s e c t i c i d e treatment of deltamethrin was shown to give excellent c o n t r o l of the lepidopteran cabbage pests. The s i g n i f i c a n t l i n e a r e f f e c t indicates that there was a general increase i n the number of pest larvae on the cabbage over the duration of the experiment. The s i g n i f i c a n t r e s i d u a l i n the polynomial analysis shows that the f i r s t - o r d e r polynomial did not account for a l l the v a r i a t i o n i n the experiment. The 'treatment X day' i n t e r a c t i o n was s i g n i f i c a n t l y d i f f e r e n t (p<0.001) and the separation of the sum of squares showed that the l i n e a r i n t e r a c t i o n of the deltamethrin, i n contrast with the r e s t of the treatments, accounts for most (62%) of the v a r i a t i o n . The s i g n i f i c a n t quadratic component measures the a d d i t i o n a l improvement due to f i t t i n g the second-order polynomial. This shows that the v a r i a t i o n i n the CLE of the deltamethrin treatment contrasted against the r e s t of the treatments does not c l o s e l y follow the l i n e a r r e l a t i o n s h i p . Separate considerations of the key cabbage pests reveal highly s i g n i f i c a n t differences (p<0.001) i n the l a r v a l counts of ICW between the A^ . t r i d e n t a t a treatment and the 30% aq EtOH and water treated cabbage plots ( F i g . 5, Appendix I I ) . About 35% of the v a r i a t i o n i n ICW l a r v a l counts i s due to t h i s s i g n i f i c a n t d i f f e r e n c e . The plo t s treated with deltamethrin 47 had s i g n i f i c a n t l y (p<0.001) fewer ICW larvae than a l l the other treatment p l o t s . The deltamethrin contrast, however, accounted for 62% of the t o t a l v a r i a t i o n . ICW l a r v a l population counts were not s i g n i f i c a n t l y d i f f e r e n t between the water treated plants and the 30% EtOH treated p l o t s . Separation of 'treatment X day' i n t e r a c t i o n sum of squares for the deltamethrin versus the other treatments i n Appendix II again shows s i g n i f i c a n t l i n e a r and quadratic v a r i a t i o n . One of the most encouraging r e s u l t s comes from the analysis of the ICW egg counts. Low numbers of ICW eggs on the A. t r i d e n t a t a sprayed cabbage suggest an o v i p o s i t i o n deterring e f f e c t ( F i g . 6). The A^ t r i d e n t a t a sprayed cabbage had s i g n i f i c a n t l y (p<0.001) fewer ICW eggs than the 30% aq EtOH and water sprayed plants (Appendix I I I ) . I n t e r e s t i n g l y , plants exposed to the deltamethrin-spreader treatment had s i g n i f i c a n t l y (p<0.001) higher ICW egg counts than a l l other treatments. Reduced numbers of ICW eggs i n the _A. t r i d e n t a t a treated p l o t s r e l a t i v e to pl o t s treated with i t s c a r r i e r suggest one reason for the continued suppression of ICW larvae i n the A. t r i d e n t a t a treated p l o t s ( F i g . 5). 48 Figure 4. Percentage change i n cabbage looper equivalents (CLE) a f t e r f i e l d spraying cabbage with a) 30% aq ethanolic s o l u t i o n of _A. t r i d e n t a t a (0.2 g/ml), b) 30% aq ethanol, c) deltamethrin 2.5 EC (17 ug/1 a . i . ) with 0.1% Superspred or d) d i s t i l l e d water, July 24, 1985. ' 1 1 6 9 25 Days from spray Figure 5. Percent change i n imported cabbageworm, P_. rapae l a r v a l populations before and a f t e r f i e l d spraying cabbage with a) 30% aq ethanolic s o l u t i o n of t r i d e n t a t a (0.2 g/ml), b) 30% aq ethanol, c) deltamethrin 2.5 EC (17 ug/l a . i . ) with 0.1% Superspred or d) d i s t i l l e d water, July 24, 1985. Pier is larvae (% change) to ^ O CO O O O O o o o o o TS Figure 6. Percent change i n imported cabbageworm, P_. rapae, o v i p o s i t i o n surveyed before and a f t e r f i e l d spraying cabbage with a) 30% aq ethanolic sol u t i o n of k_. t r i d e n t a t a (0.2 g/ml), b) 30% aq ethanol, c) deltamethrin TM 2.5 EC (17 pg/l a . i . ) with 0.1% Superspred or d) d i s t i l l e d water, July 24, 1985. 54 D. Oviposition of P. rapae: Laboratory Experiment The o v i p o s i t i o n deterring e f f e c t of the k_. tridentata extract i n the f i e l d was confirmed i n a c o n t r o l l e d laboratory experiment. While a t o t a l of over 105 eggs were l a i d i n the two experimental t r i a l s only two eggs were l a i d on the cabbage leaves painted with the A^ . t r i d e n t a t a extract. Leaves with ethanol and water s o l u t i o n s received almost a l l of the eggs, 58 and 45 r e s p e c t i v e l y . Female J?. rapae were observed a l i g h t i n g on the extract sprayed cabbage leaves but without o v i p o s i t i n g either i n the laboratory or i n the f i e l d experiments. E. Quality of Cabbage Heads from the F i e l d T r i a l The v i s u a l q u a l i t y estimate of the f i e l d sprayed cabbage (Table VII) showed that the single spraying of the _A. t r i d e n t a t a extract produced cabbage of s i g n i f i c a n t l y (p<0.05) higher q u a l i t y than the 30% EtOH and water sprayed plants. The cabbage sprayed with deltamethrin, however, received the highest v i s u a l q u a l i t y estimate, which was s i g n i f i c a n t l y higher (p<0.001) than a l l the other treatments. No s i g n i f i c a n t difference (p=0.7) was detected between the EtOH and water sprayed treatments. F. Comparison of Wild Versus Laboratory Reared P. saucia Larvae A comparison of the growth response of two separate populations of I?. saucia fed diets with and without a growth i n h i b i t i n g extract are shown i n Table VIII. The F^ JP. saucia larvae from the f i e l d - c o l l e c t e d population grew s i g n i f i c a n t l y f a s t e r than larvae from the laboratory colony. After feeding on the standard d i e t f o r 8 days the P. saucia from the f i e l d population had grown an average of 137 mg versus 57 mg for the lab-reared larvae. The P_. saucia larvae fed d i e t with k_. t r i d e n t a t a extract grew, as 55 Table VII. Mean v i s u a l quality estimates of cabbage treated with an A^ tri d e n t a t a ethanolic extract, deltamethrin, 30% aq ethanol, and water, recorded 25 days post-treatment. Treatment Concentration Mean estimate ±SD^ 2 A., t r i d e n t a t a 0.2 g-eq 1.6 ± 0.7b (30% aq EtOH) Deltamethrin 17.9 pg/1 2.5 ± 0.7a Ethanol 30% aq 1.3 ± 0.8c d i s t i l l e d water 1.3 ± 0.6c  ^"Visual q u a l i t y estimates ± standard deviation; based on a scale from 0-4. The scale i s an estimate of market q u a l i t y i e . 0=no head remaining, l=unmarketable more than one hole i n the head, 2=garden grade, one hole i n t o head, 3=sauerdraut grade, exterior damage only, no holes, 4=marketable cabbage, no exterior or i n t e r i o r damage. 2 Means followed by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t (p>0.05, means separated by orthogonal c o n t r a s t s ) . 56 Table VIII. Mean P_. saucia l a r v a l weight of a f i e l d c o l l e c t population compared to the laboratory colony fed the standard a r t i f i c i a l d i e t and di e t containing a 50% _A. tr i d e n t a t a ethanolic extract (dwt/dwt) for 8 days. Diet Treatment Source of larvae % Su r v i v a l (n=30) Mean weight ±SD %R(T Standard d i e t Lab colony 90 F i e l d colony 100 3 _A. tri d e n t a t a diet Lab colony 90 F i e l d colony 80_ 56.9 ± 27.9 136.6 ± 43.6 13.5 ± 8.7 34.8 ± 14.7 standard deviation 2 % of respective control 3 50% (dwt/dwt) concentration Two-way analysis of variance 23.7 25.5 Source of Va r i a t i n DF SS F P r o b a b i l i t y Model 3 16.9 25.6 0.0001 between populations 1 8.3 a 37.8 0.0001 between d i e t s 1 7.5 34.0 0.0001 popul. * die t s 1 0.2 0.8 0.3833 Error 104 22.9 Sum of squares of l a r v a l growth are adjusted for mortality, expected, s i g n i f i c a n t l y l e s s than the larvae fed the standard d i e t . However, growth of the f i e l d P_. saucia larvae was 4-fold more than the l a b -reared P_. saucia on extract-treated d i e t . I n t e r e s t i n g l y , there was no s i g n i f i c a n t i n t e r a c t i o n between l a r v a l o r i g i n and response to dietary _A. t r i d e n t a t a e x t r a c t . In other words, the proportional growth i n h i b i t i o n (75%) was not s i g n i f i c a n t l y d i f f e r e n t between the two l a r v a l populations, thus i n d i c a t i n g that percentage of control l a r v a l growth i s a v a l i d expression for laboratory experiments. G. Preliminary Phytochemical Investigation The chromatographically separated ethanolic extract from A^ . t r i d e n t a t a was pooled into four major groups with generally d i s t i n c t chemical p r o f i l e s : a non-polar hexane f r a c t i o n , two groups from CHCl^ eluates, and a f i n a l group of EtOH and MeOH eluates. Table IX shows the growth and mortality of P_. saucia fed d i e t s incorporating these e l u t i o n s . The two CHClg f r a c t i o n s accounted for most of the growth i n h i b i t i o n and mortality i n J?. saucia larvae. There was no s i g n i f i c a n t difference among the other f r a c t i o n s or the c o n t r o l . The d i r e c t chromatography of sesquiterpene lactones with the two b i o l o g i c a l l y a c t i v e f r a c t i o n s i s shown i n F i g s . 7,8,9, and 10. The high b i o l o g i c a l a c t i v i t y of the two chromatographically separated major f r a c t i o n s of /U t r i d e n t a t a could not be c o n s i s t e n t l y correlated with any of the ten pure sesquiterpene lactones a v a i l a b l e . Although some of the values and colour reactions corresponded i n one solvent system, there were no unambiguous matches i n both solvent systems or tank arrangements. Table IX. Mean l a r v a l weight of neonate P. saucia fed a r t i f i c i a l d i e t admixed with chromatographically separated f r a c t i o n s of an _A. tri d e n t a t a ethanolic extract compared to the o r i g i n a l extract at e c o l o g i c a l concentrations and the standard d i e t ^ . TREATMENT % SURVIVAL (n=20) % LARVAL WEIGHT (of control) A. t r i d e n t a t a ELUANT #1-Hexane 95 63.5a 2 #2-CHCl 3 40 2.8b #A-CHC13 30 1.3b #5-MeOH,EtOH 95 74.2a O r i g i n a l extract 0 0.0c standard d i e t 95 100.0a The standard diet was treated with p e t r o l Larval growth followed by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t , Tukey's studentized range (HSD) t e s t (p=0.05). 59 Major f r a c t i o n no. 2 i s a highly complex phytochemical mixture containing seven major constituents, of which f i v e give a p o s i t i v e colour reaction with the v a n i l l i n reagent. Ten other spots were v i s i b l e i n short-and long-wave u l t r a v i o l e t l i g h t and using colour reactions with the v a n i l l i n reagent. Major f r a c t i o n no. 4 appears as a chemically simpler mixture of f i v e major TLC spots. Two of the spots fluoresced blue with long-wave u l t r a v i o l e t l i g h t and the other 3 spots gave p o s i t i v e colour reactions with v a n i l l i n reagent. Six other minor constituents were also detected i n major f r a c t i o n no. 4 . 60 Figure 7. Thin-layer chromatograph of f r a c t i o n s 2 (fr#2) and 4 (fr#4) from the separation of a crude ethanolic _A. tridentata extract chromatographed with phytochemicals from Artemisia spp. The pure sesquiterpene lactones compared to the J\. t r i d e n t a t a f r a c t i o n s were: Dehydroleucodin ( d h l ) , dihydrosantamarin (dhs), arbusculin A (abA), arbusculin C (abC), matricarin (mat), deacetoxymatricarin (dom), deacetylmatricarin (dam), and dehydroreynosin (dhr). Non-fluorescent colours occurred a f t e r developing the plate with a v a n i l l i n reagent and the arrows i n d i c a t e a colour s h i f t a f t e r 24 h. TLC developed with petroleum ether:CHCl 3:Et 20Ac (2:2:1) i n a non-saturated tank. R. 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 COLOR LEGEND o 0 P * * p -Br B Y Op** O N 9P?-P OBr o Bk* O P p * * o ** O B r ~ p p O P*I O Pp O fY-Bk C L o o * p** * P*Br** o P»Br** Bk Br B f N P PB Pp * Y black brown blue fluoresces longwave UV navy blue pink pale blue purple quenches longwave UV quenches shortwave UV yellow O Pp«»Bk O P*>Bk** frfil fr#4 abC abA dhl dom mat dhs dhr dam 62 Figure 8. Thin-layer chromatograph of f r a c t i o n s 2 (fr#2) and A (fr#A) from the separation of a crude ethanolic _A. t r i d e n t a t a extract chromatographed with phytochemicals from Artemisia spp. The pure sesquiterpene lactones compared to the A^ . t r i d e n t a t a f r a c t i o n s were: dehydroleucodin ( d h l ) , dihydrosantamarin (dhs), arbusculin A (abA), t a t r i d i n A ( t t A ) , matricarin (mat), deacetoxymatricarin (dom), and deacetylmatricarin (dam). Non-fluoresent colours occurred a f t e r developing the plate with a v a n i l l i n reagent and the arrows i n d i c a t e a colour s h i f t a f t e r 2A h. TLC developed with CHC13:acetone (6:1) i n a saturated tank. CD P B * O © 2 . efb v** COLOR LEGEND i * o f b O fb 0 Br PB - pale blue N - navy blue Br - brown P - pink fb - fluoresces blue, longwave UV Bk - black Pp - purple Y - yellow * - quenches long-wave UV quenches short-wave UV O P - B r j » o O fb O B r ** ~ quenches short- Pp-Brl £j Br "" ' o p O p~ pp 0 Bk 0 B r —J 1— 1 1 1 1 I , u_ f r / / 2 f r / M dhl dom mat abA dhs dam t t A 64 Figure 9. Thin-layer chromatograph of f r a c t i o n s 2 (fr#2) and 4 (fr#4) from the separation of a crude ethanolic t r i d e n t a t a extract chromatographed with phytochemicals from Artemisia spp. The pure sesquiterpene lactones compared to the A_. t r i d e n t a t a f r a c t i o n s were: Dehydroleucodin ( d h l ) , arbusculin A (abA), arbusculin B (abB), arbusculin C (abC), t a t r i d i n A ( t t A ) , matricarin (mat), deacetoxymatricarin (dom), deacetylmatricarin (dam), and dehydroreynosin (dhr). Non-fluorescent colours occurred a f t e r developing the plate with a v a n i l l i n reagent and the arrows i n d i c a t e a colour s h i f t a f t e r 24 h. TLC developed with CHCl^:acetone (6:1) i n a saturated tank. COLOR LEGEND Bk Br f PB B 0 Y N P PP * P-Br ** o P-Br** black brown fluoresceslong - U V pale blue blue orange yellow navy blue pink purple quenches long -UV quenches short - U V o Pp-Br o Pp-Bk O P . Br*. o Pp-Bk —I 1 I - J L _ fr//2 f r//4 abB abC - J u . dhl dom I i l 1 i mat abA dhr dam ttA 66 Figure 10. Thin-layer chromatograph of f r a c t i o n s 2 (fr#2) and 4 (fr#4) from the separation of a crude ethanolic tridentata extract chromatographed with phytochemicals from Artemisia spp. The pure sesquiterpene lactones compared to the _A. t r i d e n t a t a f r a c t i o n s were: arbusculin A (abA), arbusculin B (abB), t a t r i d i n A ( t t A ) , matricarin (mat), deacetoxymatricarin (dom), and deacetylmatricarin (dam). Non-fluorescent colours occurred a f t e r developing the plate with a v a n i l l i n reagent and the arrows indicate a colour s h i f t a f t e r 24 h. TLC developed with petroleum ether:CHClo:Et 90Ac (2:2:1) i n a non-saturated tank. COLOR LEGEND Bk Br B PB f N P PP Y * ** black brown blue pale blue fluoresces longwave UV l i g h t navy blue pink purple yellow quenches longwave UV l i g h t quenches longwave UV l i g h t D P * P P O ' B r C^Pp-Br Q P -** O P-Br** ^ * O pP*Bk fr//2 f r / / A abB abA dom mat dam ttA 68 V. DISCUSSION A. Screening Asteraceous Extracts for Insect Growth I n h i b i t o r s Plant extracts from many f a m i l i e s have been screened as insect c o n t r o l agents. Many large screenings for botanical i n s e c t i c i d e s occurred p r i o r to the advent of synthetic i n s e c t i c i d e s (Jacobson and Crosby 1971). Recent public i n t e r e s t i n a l t e r n a t i v e s to conventional i n s e c t i c i d e s has supported the s c i e n t i f i c e f f o r t to f i n d environmentally sound i n s e c t controls (Abivardi & Benz 1984, Bernays 1983). Screening for botanical i n s e c t controls has often focused on acute t o x i c i t y but the past ten years have seen a renewed i n t e r e s t i n materials with more subtle a c t i o n s , such as growth regulators, l a r v a l growth i n h i b i t o r s , and o v i p o s i t i o n deterrents. Extracts from c e r t a i n asteraceous plants are reported to i n f l i c t a variety of deleterious e f f e c t s on i n s e c t s ; some extracts from the Asteraceae have been shown to act as insect r e p e l l e n t s (Hwang et a l . 1985), feeding i n h i b i t o r s (Isman and Rodriguez 1984, Nawrot et a l . 1982) o v i p o s i t i o n deterrents (Lundgren 1975, Burnett and Jones 1978) and contact i n s e c t i c i d e s (Jacobson and Crosby 1971). The insect growth i n h i b i t o r y a c t i v i t y and chronic t o x i c i t y of ethanolic and p e t r o l extracts of s i x weeds i n the Asteraceae (Table I) have also been assessed. Many fa c t o r s determine an i n s e c t ' s response to plant a l l e l o c h e m i c a l s , such as insect species, stage of development, concentration of the a l l e l o c h e m i c a l , and the chemical context i n which the a l l e l o c h e m i c a l i s presented to the i n s e c t . Lepidopteran l a r v a l s e n s i t i v i t y to growth i n h i b i t o r s has been shown previously to be i n v e r s e l y c o r r e l a t e d with l a r v a l age (Reese 1983, Isman and Duffey 1982). These r e s u l t s follow t h i s pattern i n that younger P_. saucia larvae were more se n s i t i v e to e f f e c t s of the phytochemical growth i n h i b i t o r s than older larvae (Fig 1 & 2). Neonate P. saucia larvae grew l e s s , and suffered greater mortality than, second i n s t a r larvae fed a r t i f i c i a l d i e t containing the same l e v e l s of asteraceous extracts ( F i g 1 and 2). Younger lepidopteran larvae may possess fewer endosymbiotic microorganisms needed for d e t o x i f i c a t i o n (Jones et a l . 1981), or lower l e v e l s of c o n s t i t u a t i v e detoxifying enzymes (Ahmad 1986). Insect growth i n h i b i t i o n could r e s u l t from behavioral factors (e.g., feeding deterrency), p h y s i o l o g i c a l factors (e.g., microsomal enzyme suppression) or both. Schroeder (1976) has shown that decreases i n l a r v a l food u t i l i z a t i o n can be induced by food deprivation. Starvation or behavioral food aversion r e s u l t i n g i n a lower RCR may also decrease nutrient u t i l i z a t i o n . Table V shows that when JP. saucia larvae were fed the k_. t r i d e n t a t a e x t r a c t , even though the RCR was 60% of the c o n t r o l s , the ECI of those larvae was only 5% of the controls (Table V). Thus i t i s l i k e l y that the severely reduced food u t i l i z a t i o n of the A^ . t r i d e n t a t a fed larvae was due to p h y s i o l o g i c a l f a c t o r s rather than a lower consumption r a t e . Larval growth on C_^  suaveolens-petrol d i e t i s i n i t i a l l y i n h i b i t e d as shown by a RGR of 51% of that of the controls f o r the f i r s t 24 hrs of the 48 hr n u t r i t i o n a l experiment. In the second h a l f of the experiment these larvae recovered from the p r i o r i n h i b i t i o n and attained an RGR equal to the co n t r o l fed larvae. In contrast, the RGR for P_. saucia larvae fed an _A. tri d e n t a t a - e t h a n o l i c d i e t was s i g n i f i c a n t l y lower (Tukey's studentized range (HSD) t e s t ; p=0.05) than the controls for both 24 hr periods and remained e s s e n t i a l l y the same at 27 and 29% of control fed l a r v a l RGR, re s p e c t i v e l y . This implies that growth i n h i b i t o r s that function mainly as behavioral feeding deterrents can be r e a d i l y overcome by i n s e c t s , whereas growth i n h i b i t o r s that decrease nutrient u t i l i z a t i o n may protect plants better because of t h e i r more per s i s t e n t a c t i v i t y . The a b i l i t y of the extraction process to remove insect growth i n h i b i t o r s i s an important step i n an e f f i c i e n t screening process. Table I I I shows that solvent extraction removed inse c t growth i n h i b i t o r s i n nearly every case where i n h i b i t o r s were present i n the unextracted plant powders. Increases i n l a r v a l growth on the marc-diets i n d i c a t e that the extraction process was e f f i c i e n t i n removing i n s e c t growth i n h i b i t o r s from the plant material. The d i l u t i o n of i n s e c t d i e t with a non-nutritive, non-deterrent substance has been shown to increase food consumption i n a number of inse c t s (Dadd 1970). Cockroaches have been shown to increase feeding i n response to dietary d i l u t i o n s of c e l l u l o s e ( B i g n e l l 1978). In contrast, growth i s reduced i n P_. saucia larvae by dietary additions of c e l l u l o s e (Table I I I ) . This may r e s u l t from a decrease i n a v a i l a b l e n u t r i t i o n , phagostimulation, or both. Increased consumption does not necessarily imply increased f i t n e s s but may lead to decreased dietary u t i l i z a t i o n , ultimately r e s u l t i n g i n growth reduction. Results i n Table I I I show that P_. saucia larvae fed many of the d i e t s (e.g., the ethanolic marc-diet from CJ. nauseosa and the p e t r o l marc-diet from C. suaveolens and T_. dubius) grew more than did larvae fed the c e l l u l o s e containing control d i e t . A r t i f i c i a l d i e t s i n c l u d i n g plant material may possess a d d i t i o n a l nutrients or phagostimulants lacking i n the c e l l u l o s e containing control d i e t . Insect growth i n h i b i t i o n and feeding deterrent a c t i v i t y has previously been reported i n asteraceous p l a n t s . The growth i n h i b i t i o n and feeding deterrency, however, appears to be species s p e c i f i c rather than broad spectrum. Nawrot et a l . (1982) screened 23 extracts from asteraceous plants against three coleopteran pests of stored products and found that 7 extracts possessed strong feeding deterrency. I n t e r e s t i n g l y , there was no consistency between the extracts' a c t i v i t y amongst the insect species tested. These authors l a t e r confirmed that sesquiterpene lactones were i n part responsible for the feeding deterrency of the asteraceous extracts (Nawrot et a l . 1984, Harmatha and Nawrot 1984). Table IV shows that of the extracts examined, C. suaveolens and A_. t r i d e n t a t a had the strongest i n h i b i t o r y a c t i v i t y on P_. saucia l a r v a l growth. An ethanolic extract of A. t r i d e n t a t a was chosen for f i e l d evaluation because of superior performance against A. c a l i f o r n i c a larvae ( F i g . 3) and because i t s i g n i f i c a n t l y lowered the RGR and ECI of the P. saucia larvae (Table V). Several i n v e s t i g a t o r s have chosen extracts of Artemisia species as potent i n s e c t growth and feeding i n h i b i t o r s . V i l l a n i and Gould (1985) investigated crude extracts from twelve Asteraceae and found that two, Artemisia dracunculus and Santolina virens, deterred feeding by corn wireworm, Melanotus communis. Suomi and associates (1986) examined eleven Asteraceae (and 14 other plants) for feeding deterrency to l a r v a l codling moth, Cydia pomonella. They found the strongest deterrency i n the Asteraceae extracts from Artemisia absinthium, Chrysothamnus nauseosus and Tanacetum vulgare. Yang (1983) has reported that two phenlylalkynes from the buds of A_. c a p i l l a r i s were feeding deterrents for the imported cabbageworm, P i e r i s rapae. In choice experiments Jermy et a l . (1981) reported that Colorado potato beetle (Leptinotarsa decemlineata) larvae were i n h i b i t e d from feeding on l e a f disks coated with an ethanolic extract from _A. t r i d e n t a t a . In my study I have shown that an ethanolic extract of A. t r i d e n t a t a strongly i n h i b i t s l a r v a l growth of two lepidopteran larvae, A_. c a l i f o r n i c a and P. saucia, i n feeding bioassays. The above r e s u l t s i n d i c a t e that extracts of Artemisia species have broad spectrum a c t i v i t y on phytophagous inse c t pests. 72 B. Phytochemicals and Insect Growth I n h i b i t i o n Research on insect growth i n h i b i t o r s and feeding deterrents has often focused on the i s o l a t i o n of s p e c i f i c phytochemicals. Many investigators have emphasized i n d i v i d u a l compounds and s i n g l e classes of phytochemicals as the key to insect-plant i n t e r a c t i o n s . In nature however, phytophagous insects are always exposed to complex mixtures of phytochemicals. Considering the within plant d i v e r s i t y of chemicals, i n t e r a c t i o n s among phytochemicals may be a common determining f a c t o r i n i n s e c t / p l a n t r e l a t i o n s (Berenbaum 1985). Plant defense strategies using chemical mixtures probably occur more frequently than defensive strategies using s i n g l e a l l e l o c h e m i c a l s . Most plants contain more than one defensive phytochemical (Berenbaum 1985, Harborne 1982), but, few studies have examined the growth i n h i b i t o r y a c t i v i t i e s among co-occurring phytochemicals. In the l i m i t e d number of cases where co-occurring chemicals have been examined, the r e s u l t s underscore the importance of phytochemical i n t e r a c t i o n s . Adams and Bernays (1978) examined the e f f e c t s of fourteen simple phenolic chemicals from Sorghum b i c o l o r fed to Locusta migratoria at n a t u r a l l y occurring concentrations. These phytochemicals produced a measurable feeding deterrency only when combined. When feeding deterrents from unrelated chemical groups were combined i n binary combinations (e.g., s i n i g r i n and tomatine) deterrent e f f e c t s were often a d d i t i v e (Adams and Bernays 1978). Phytochemicals presented as a mixture may have a greater than a d d i t i v e e f f e c t on i n s e c t s . Berenbaum and Neal (1985) report the s y n e r g i s t i c e f f e c t s of the methylene dioxyphenyl compound, m y r i s t i c i n and the co-occurring furanocoumarin, xanthotoxin, at n a t u r a l l y occurring concentrations. Insect growth may be reduced more e f f e c t i v e l y by a 73 chemical mixture causing d i f f e r e n t behavioral and p h y s i o l o g i c a l a c t i v i t y than a s i n g l e deterrent chemical. Polyphagous pests are generally more r e s i s t a n t to growth i n h i b i t o r s than i n s e c t s with a narrow host range (Bernays 1983) and thus may provide evidence of a broader spectrum of growth i n h i b i t o r y a c t i v i t y . In the present t h e s i s , the growth of the highly polyphagous P_. saucia larvae was s i g n i f i c a n t l y reduced r e l a t i v e to the c o n t r o l s by ethanolic extracts from f i v e of the s i x plants investigated but only two of s i x p e t r o l extracts tested at f i v e times the natural concentration. This i n d i c a t e s that the growth i n h i b i t o r s i n the plants chosen (Table I) contain mostly polar compounds. The greater proportion of the ethanol extracts e x h i b i t i n g potent a c t i v i t y support the r e s u l t s of Freedman et a l . (1979). A_. t r i d e n t a t a i s known to contain many phytochemicals (Table X) and some are reported to have inse c t growth and feeding i n h i b i t o r y a c t i v i t y . Kelsey and Shafizadeh (1979) have i s o l a t e d several sesquiterpene lactones from A. t r i d e n t a t a and i t s subspecies. Jermy et a l . (1981) bioassayed one of these, deacetylmatricarin. They reported good feeding deterrent a c t i v i t y against l a r v a l Colorado potato beetle but noted that s i g n i f i c a n t feeding deterrent a c t i v i t y remained i n the extract even a f t e r removal of deacetylmatricarin. Wisdom et a l . (1983) tested f i v e sesquiterpene lactones against H_. zea and found that only a guaianolide from _A. t r i d e n t a t a , dehydroleucodin, s i g n i f i c a n t l y reduced growth. Sesquiterpene lactones from other plants have been shown to a f f e c t i n s e c t growth and feeding. Isman and Rodriguez (1983) found that several sesquiterpene lactones extracted from Parthenium species (Asteraceae) i n h i b i t e d l a r v a l growth of H_. zea. Burnett and co-workers (1974) reported that of s i x lepidopteran l a r v a l species examined, four were deterred from feeding on Vernonia spp. (Asteraceae) containing sesquiterpene lactones. TABLE IX: Phytochemical constituents previously i s o l a t e d from Artemisia t r i d e n t a t a Monoterpenes camphor 1,8-cineole delta-3-carene s a n t o l i n y l ester alpha-pinene camphene Flavonoids Sesquiterpene lactones' 1 matricarin t a t r i d i n A, B, C deacetoxymatricarin deacetylmatricarin r i d e n t i n detatin A, B dehydroleucodin arbusculin A, B, C quercetagetin 3,6-dimethyl ether quercetagetin 3,6,7-trimethyl ether kaempferol 3,6,7-trimethyl ether l u t e o l i n luteolin-7-0-glucoside 6-methoxy l u t e o l i n a x i l l a r i n Coumarins e s c u l i n umbelliferone c i c h o r i i n i s o s c o p o l e t i n scopoletin scoparon e s c u l e t i n a r t e l i n Buttkus et a l . (1977) The l i s t e d monoterpenes comprise 80% of the e s s e n t i a l o i l s 2Seaman (1982) 3Brown et a l . (1975)., Murray et a l . (1982) These compounds are 80% of the phenolic f r a c t i o n of an A^ . t_. spp. vaseyana extract. 4Rodriguez et a l . (1972) However, Jones et a l . (1979) reported that cabbage looper, T. nd^ , and yellow woollybear, Spilosoma v i r g i n i c a , were not i n h i b i t e d from feeding on d i e t containing the sesquiterpene lactones, glaucolide-A. The above r e s u l t s i n d i c a t e that several sesquiterpene lactones have i n s e c t growth i n h i b i t o r y and feeding deterrent properties. However, not a l l sesquiterpene lactones are e f f e c t i v e and those that are do not show a c t i v i t y against a l l i n s e c t species tested. In the present study four f r a c t i o n s of a chromatographically separated ethanolic extract of _A. t r i d e n t a t a were assayed, and two f r a c t i o n s accounted for nearly a l l of the growth i n h i b i t o r y a c t i v i t y of the i n i t i a l extract (Table IX). Thin layer chromatographic separations (Figs. 8-11) showed that several major spots reacted to a v a n i l l i n reagent, suggesting that these were sesquiterpene lactones (Pieman et a l . 1980). Camphor and 1,8-cineole, major monoterpenes i n the e s s e n t i a l o i l of _A. t r i d e n t a t a have previously been shown to be highly a c t i v e against i n s e c t s . Camphor i s reported to be a mosquito re p e l l e n t (Hwang et a l . 1985), and 1,8-cineole has been shown to repel the American cockroach, Periplaneta  americana (Scriven and Meloan 1984). Thin layer chromatographic (TLC) study of the two major f r a c t i o n s i n t h i s t h e s i s revealed several spots that quenched u l t r a v i o l e t l i g h t that may be monoterpenes (Croteau and Ronald 1983) i n the most growth i n h i b i t o r y f r a c t i o n s (Figs. 7-10). Jermy et a l . (1981) examined the feeding deterrency of several coumarins reported from A^ . t r i d e n t a t a and found that none of these reduced feeding of Colorado potato beetle larvae. Coumarin has, however, been shown to i n h i b i t l a r v a l growth and development as well as adult f e r t i l i t y i n the cotton leafworm, Spodoptera l i t t o r a l i s (Mansour 1982). In Figures 7-10 several TLC spots showed a weak blue fluorescence i n the a c t i v e f r a c t i o n s of A^ . t r i d e n t a t a that could be coumarins. Isman and Rodriguez 76 (1983) reported that quercetagetin 3,7-dimethy ether, a flavonoid from guayule (Parthenium argentatum) was a l a r v a l growth i n h i b i t o r of l\_. zea and J3. exigua whereas a c l o s e l y related 6-hydroxykaempferol 3,6,7-trimethyl ether was stimulatory to both ins e c t species. The other phenolic compounds i n A. tridentata have not been investigated for i n s e c t a c t i v i t y . The a e r i a l parts of A^ . t r i d e n t a t a are known to possess a wide range of b i o l o g i c a l l y a c t i v e compounds. Indigenous peoples of B r i t i s h Columbia used A^ . t r identata f o l i a g e as a d i s i n f e c t a n t , i n s e c t r e p e l l e n t and as a deodorant when handling corpses (Turner 1979). V o l a t i l e (Nagy and Tengerdy 1967) and non-volatile (Ramirez 1969) components of the leaves are known to possess a n t i b a c t e r i a l a c t i v i t y . In a d d i t i o n , a l l e l o p a t h i c a c t i v i t y has been reported from v o l a t i l e and n o n - v o l a t i l e l e a f f r a c t i o n s (Groves and Anderson 1981). Seasonal (Kelsey et a l . 1982) and i n t r a s p e c i f i c (Shafizadeh et a l . 1971) v a r i a t i o n s i n the terpenoid content of _A. t r i d e n t a t a have been reported; u n t i l the active ingredients are known and bioassayed with co-occurring compounds, one must be cautious i n i n t e r p r e t i n g i n s e c t growth i n h i b i t o r y and o v i p o s i t i o n deterrence a c t i v i t y . Kelsey and co-workers (1983) suggest that the b i o l o g i c a l a c t i v i t y of A_. t r i d e n t a t a may be a r e s u l t of synergism between the v o l a t i l e e s s e n t i a l o i l s and other secondary compounds l i k e sesquiterpene lactones and phenolics. Although t h i s hypothesis i s i n t r i g u i n g i t i s nonetheless speculative. C. F i e l d T r i a l s of Plant Extracts The proper s e l e c t i o n of botanicals as f i e l d - a c t i v e c o n t r o l agents requires the evaluation of extracts i n the target area. Since laboratory and greenhouse studies have not always predicted the e f f e c t s i n the f i e l d 77 (Obrycki and Tauber 1984, Haverty and Robertson 1982), f i e l d studies are an e s s e n t i a l part of a complete screening procedure. While laboratory screenings of crude plant extracts for growth i n h i b i t o r s and feeding deterrents are not uncommon, reports of f i e l d t r i a l s on extracts i s scarce, save for work on neem e x t r a c t s . Figures 4-6 show the r e s u l t s of a f i e l d t r i a l on cabbage using an ethanolic extract of _A. t r i d e n t a t a selected i n the laboratory. The suitable bioassay for both the pest and intended a p p l i c a t i o n should be c a r e f u l l y chosen when screening plant extracts against pest i n s e c t s . For example, the a c r i d i d , Locusta migratoria, was s u b s t a n t i a l l y more s e n s i t i v e to a wider range of compounds than four lepidopteran pest species tested (Simmonds et a l . 1985). The anthranoid, harunganine, was a phagodeterrent to the polyphagous cotton leafworm Spodoptera l i t t o r a l i s when presented on cabbage, whereas the same compound was i n e f f e c t i v e when the host plant was wheat, despite cabbage being a preferred plant (Simmonds et a l . 1985). The difference i n response to a treatment can depend more on the t e s t method than on the product tested. For example, larvae fed supra-optimal d i e t s treated with moderate amounts of allelochemicals may exaggerate the l a r v a l growth response of treated versus c o n t r o l larvae. Another important factor to consider when using bioassays to screen p e s t i c i d a l or i n h i b i t o r y products i s the number of choices given an i n s e c t . A dual choice feeding bioassay resulted i n 100% deterrency of P i e r i s brassicae compared with only 19% deterrence i n a s i n g l e choice bioassay when fed an equivalent concentration of an Artemisia absinthium extract applied to l e a f discs (Abivardi and Benz 1984). Results from laboratory screenings should be 78 substantiated i n the target area (e.g., greenhouse or f i e l d ) . F i e l d t e s t i n g candidates selected i n the laboratory may determine the relevance of the bioassay for screening s u i t a b l e control agents. The relevance of using r e s u l t s from laboratory reared i n s e c t s to c a l c u l a t e concentration l e v e l s f or f i e l d t r i a l s should be addressed when screening i n s e c t c o n t r o l products. Laboratory reared i n s e c t s may respond d i f f e r e n t l y than f i e l d populations of the same species (Brattsten et a l . 1986), j u s t as products that function well i n the laboratory may not be stable under f i e l d conditions. I have shown (Table VIII) that JP. saucia larvae from an F^ generation of a f i e l d c o l l e c t e d population were 2.5 times heavier than larvae from a two-year-old laboratory reared colony fed the same a r t i f i c i a l d i e t with 50% (dwt/dwt) of an ethanolic _A. t r i d e n t a t a extract. Therefore, a r e a l i s t i c estimation of the extract concentration needed should be performed on f i e l d c o l l e c t e d i n s e c t s or t h e i r o f f s p r i n g . Another part of t h i s experiment showed the accuracy of the laboratory bioassay f o r reporting r e l a t i v e growth i n h i b i t i o n . There was no s i g n i f i c a n t growth differences (p=0.05) between the laboratory reared or f i e l d c o l l e c t e d populations of JP. saucia larvae (Table VIII) fed d i e t containing _A. t r i d e n t a t a extract when compared to t h e i r respective c o n t r o l s . Natural plant defenses may be of use i n a g r i c u l t u r e for the management of pest populations. Several researchers have noted that the spraying of crude plant extracts should not present insurmountable problems (Jacobson 1983, Jermy et a l . 1981), p a r t i c u l a r l y for underdeveloped countries where va r i a b l e e f f i c a c y i s acceptable. The r e s u l t s reported herein show the f e a s i b i l i t y of f i e l d spraying _A. t r i d e n t a t a extracts formulated i n 30% ethanol. 79 My f i e l d t r i a l with an _A. t r i d e n t a t a extract against cabbage insect pests using a s i n g l e a p p l i c a t i o n resulted i n a higher q u a l i t y cabbage y i e l d and lower l a r v a l pest counts than the solvent treated controls (Table VII). TM The standard i n s e c t i c i d e spray of deltamethrin and Superspred gave the highest quality cabbage. The benefits of the _A. t r i d e n t a t a extract were evident for the f i r s t week a f t e r spraying, but i n t e g r a t i n g other control options such as spraying more frequently or combining c o n t r o l strategies may make the _A. t r i d e n t a t a extract more e f f e c t i v e . Cabbage looper equivalents (CLEs) have been used for measuring pest damage on cabbage from the three major lepidopteran cabbage pests (Shelton et a l . 1982). In the present t h e s i s , _A. t r i d e n t a t a sprayed cabbage was s i g n i f i c a n t l y l e s s damaged by major i n s e c t pests, estimated as CLEs, than the solvent treated controls ( F i g . A). Deltamethrin was shown to be a good choice as an i n s e c t i c i d e standard as i t resulted i n s i g n i f i c a n t l y lower CLEs than any of the other treatments. Deltamethrin sprays, however, are reported to decrease the populations of several predatory arthropods such as carabids, s t a p h i l i n i d s , spiders and phytoseiid mites (Matcham and Hawkes 1985, Basedow et a l . 1985, Samsoe-Petersen 1985). Bernays (1983) suggests that one of the p o t e n t i a l advantages of spraying plants with growth i n h i b i t o r y compounds i s that they may avoid damage to non-target organisms. An important r e s u l t of the f i e l d experiment was observed i n the s i g n i f i c a n t l y lower l a r v a l ICW counts on the _A. t r i d e n t a t a extract sprayed cabbage ( F i g . 6). The lower counts of l a r v a l P_. rapae may be due to an i n d i r e c t mode of action of the _A. t r i d e n t a t a extract. Reduced P_. rapae egg counts could explain most, i f not a l l , of the reduced l a r v a l populations i n f e s t i n g the cabbage f o l i a g e . In plants, phytochemicals that deter o v i p o s i t i o n are the f i r s t l i n e of chemical defense against herbivorous i n s e c t s . When females o v i p o s i t on plants, they determine l a r v a l food choice and s u r v i v a l of the succeeding generation. To optimize l a r v a l survivorship, adult o v i p o s i t i o n preference and l a r v a l food s u i t a b i l i t y should be synchronized. Non-host plant extracts applied to crop plants may deter o v i p o s i t i o n , and thus protect the crop from herbivory, i f larvae have l i m i t e d mobility as i n the case of P_. rapae. Lundgren (1975) tested several plant extracts, i n c l u d i n g Artemisia absinthium and A^ . abrotanum, for t h e i r a b i l i t y to deter o v i p o s i t i o n of three P i e r i s species i n two-choice t e s t s , and found s i g n i f i c a n t l y fewer eggs l a i d on extract treated cabbage leaves. In t h i s t h e s i s an _A. tri d e n t a t a extract applied to f i e l d grown cabbage r e s u l t e d i n s i g n i f i c a n t l y fewer (p<0.05) P_. rapae eggs compared to cabbage treated with the c a r r i e r solvent alone and the deltamethrin-surfactant treatment ( F i g . 7). The deltamethrin-surfactant treated cabbage received the most P_. rapae eggs. TM (The surfactant, Triton-X-100 , has been shown to increase P l u t e l l a  x y l o s t e l l a o v i p o s i t i o n on Brussel sprouts [Perrin and P h i l l i p s 1978], but the surfactant e f f e c t was not i s o l a t e d i n t h i s experiment.) Laboratory experiments confirmed the o v i p o s i t i o n deterrency of the _A. t r i d e n t a t a extract. Observations indicated the mode of o v i p o s i t i o n deterrency of the A- t r i d e n t a t a treated cabbage was due to a contact chemoreception rather than repellency, because cabbage b u t t e r f l i e s were not i n h i b i t e d from a l i g h t i n g on the treated plants. However, whether the P_. rapae females were deterred from o v i p o s i t i n g , or i f the plants were unrecognizable as host plants, i s not c l e a r . For example, c u t i c u l a r components i n tobacco have been shown to stimulate o v i p o s i t i o n of R. virescens (Cutler et a l . 1986). T a r s a l contact with cabbage f o l i a g e was found to have important influences on the o v i p o s i t i o n behavior of P_. rapae, whereas host-plant odor, o v i p o s i t o r t i p contact and previously l a i d eggs showed no influence (Traynier 1979). In the f i e l d and laboratory experiments reported i n t h i s t hesis i t remains to be determined whether spraying the cabbage masked o v i p o s i t i o n stimulating c u t i c u l a r chemicals, blocked P_. rapae chemoreceptors, or ac t i v a t e d deterrent receptors. Non-host plant extracts and s p e c i f i c phytochemicals have been examined as o v i p o s i t i o n deterrents to i n s e c t pests of cabbage. Non-host cruciferous and non-cruciferous extracts have been reported as o v i p o s i t i o n deterrents fo r P. rapae (Renwick and Radke 1985). Tabashnik (1985) showed that coumarin sprayed cabbage deterred o v i p o s i t i o n by P_. x y l o s t e l l a . In the f i e l d experiment reported herein ( F i g . A), the large population of £. x y l o s t e l l a larvae on A., t r i d e n t a t a sprayed plants i n d i c a t e d that i f coumarins were present i n the extract, they were not f a c t o r s i n the f i e l d at the concentration sprayed. F i e l d t r i a l s of o v i p o s i t i o n deterrents without the a i d of f i e l d cages are rare. Most in v e s t i g a t o r s that have ventured i n t o the f i e l d have resorted to the use of cages and laboratory reared i n s e c t s (e.g., Williams et a l . 1986). The e f f e c t s of the c o n t r o l l e d environments and laboratory reared insects may have l i t t l e bearing on the a c t u a l f i e l d s i t u a t i o n . Nonetheless, Burnett and Jones (1978) using cage experiments with Vernonia plants, with and without sesquiterpene lactones, showed that o v i p o s i t i o n preference depended on the species of moth. Yellow woollybear, Spilosoma  v i r g i n i c a , showed no o v i p o s i t i o n preference, the cabbage looper, T_. n i , showed a preference for the two plants containing sesquiterpene lactones and the other three lepidopterans, ( f a l l , southern and yellowstriped armyworms, Spodoptera frugiperda, S^. e r i d a n i a , and S^ . o r n i t h o g a l l i ) showed a preference for the sesquiterpene lactone lacking Vernonia species. 82 Burnett and Jones (1978) a l s o showed that the f a l l armyworm was s i g n i f i c a n t l y i n h i b i t e d from o v i p o s i t i n g on the Vernonia species lacking sesquiterpene lactones when 1% glaucolide-A was applied to the f o l i a g e . The sesquiterpene lactones i n the A^ . t r i d e n t a t a extract could therefore be responsible for the o v i p o s i t i o n deterrency reported i n t h i s thesis on £. rapae. Combining co n t r o l s t r a t e g i e s ( i e . i n s e c t i c i d e and plant extract) could have advantages over the use of either agent alone. Combinations of i n s e c t i c i d e s may prolong the use of e x i s t i n g and novel c o n t r o l techniques by slowing the rate of i n s e c t resistance (Georghiou 1983). Reduced synthetic i n s e c t i c i d e use would lower the i n s e c t i c i d e load on the crop and i n the environment and may permit the return of b e n e f i c i a l organisms. Growth i n h i b i t o r s and o v i p o s i t i o n deterrents may enhance the action of natural enemies i f they are not adversely affected by these compounds. The use of growth i n h i b i t o r s could, for example, be used i n conjunction with the release of i n s e c t parasites and predators. Weseloh et a l . (1983) have shown that release of the p a r a s i t i c wasp, Apanteles melanoscelus, and f i e l d sprays of the lepidopteran pathogen, B a c i l l u s t h u r i n g i e n s i s , acted s y n e r g i s t i c a l l y to c o n t r o l gypsy moth because the bacteria maintained the larvae longer i n the second i n s t a r , which i s the host stage preferred by the parasite. Velvet bean c a t e r p i l l a r , A n t i c a r s i a gemmatalis, and soybean looper, Pseudoplusia includens, are more susceptible to the entomophagous pathogen, Nomuraea r i l e y i , i n the e a r l y i n s t a r s (Boucias et a l . 198A). If growth i n h i b i t o r s maintain i n s e c t s i n stages vulnerable to predators and parasites, they may provide another t o o l for protecting crops i n i n t e n s i v e l y managed a g r i c u l t u r a l systems. 83 Mammalian t o x i c i t y i s an important factor when considering the merits of a p e s t i c i d e . While there are no studies on mammalian t o x i c i t y of ethanolic ti. t r i d e n t a t a extracts, big sagebrush i s used as a major winter forage by pronghorn antelope (Cronin et a l . 1978), mule deer (Hansen and Reid 1975), and pygmy rabbits (White et a l . 1982). In addition, sesquiterpene lactones, one of the p r i n c i p l e classes of secondary compounds i n _A. t r i d e n t a t a , have been investigated as antitumor agents (Lee et a l . 1977). Plant-derived chemicals are most often biodegradable and thus they might prove to be preferred a l t e r n a t i v e s to synthetic chemicals that p e r s i s t i n the environment. _A. t r i d e n t a t a i s l i s t e d as one of four weeds i n the United States with the most p o t e n t i a l for crop development and commercialization for sources of ins e c t a t t r a c t a n t s , r e p e l l e n t s or toxicants (Jacobson 1983). This species i s drought t o l e r a n t (Rickard and Warren 1981) and can support a winter shoot removal of about 50% (Fetcher 1981). The r e s i l i e n c e of _A. t r i d e n t a t a shrubs adds to t h e i r p o t e n t i a l as a new crop for e x p l o i t i n g semi-arid marginal lands (Jacobson 1983). Insects that are r a p i d l y developing resistance to synthetic i n s e c t i c i d e s create problems for pest c o n t r o l , while plants, with t h e i r multichemical defenses, may contain solutions to some of these problems. Reliance on mono-chemical pest c o n t r o l i s inadequate and thus i t i s an opportune time to study pest c o n t r o l methods that have evolved i n plants. 84 VI. CONCLUSIONS The objectives of t h i s thesis were to s e l e c t a potent growth i n h i b i t o r y extract from an asteraceous weed, to assess the extract for deleterious e f f e c t on i n s e c t s , to evaluate the f i e l d e f f i c a c y of the most potent growth i n h i b i t o r and to explore the chemistry of insect growth i n h i b i t o r y a c t i v i t y . The r e s u l t s show that: 1. Of six asteraceous weeds extracted i n EtOH and p e t r o l , s i x of the 12 extracts i n h i b i t e d P_. saucia l a r v a l growth by more than 90% compared to the growth of c o n t r o l larvae (at f i v e times n a t u r a l l y occurring concentrations). 2. Naturally occurring concentrations of an ethanolic extract from the leaves and flowers of _A. t r i d e n t a t a and two of i t s chromatographic f r a c t i o n s s i g n i f i c a n t l y i n h i b i t e d early l a r v a l growth of P_. saucia. 3. Feeding bioassays showed a s i g n i f i c a n t dose-response by P_. saucia and _A. c a l i f o r n i c a larvae to _A. t r i d e n t a t a and C_. suaveolens extracts, but, no s i g n i f i c a n t differences were found between these e x t r a c t s . Extracts from both plants i n h i b i t e d growth more i n A_. c a l i f o r n i c a larvae than i n P_. saucia larvae and the _A. t r i d e n t a t a ethanolic extract i n h i b i t e d growth more i n A. c a l i f o r n i c a larvae than the C. suaveolens e x t r a c t s . 4. Second i n s t a r P_. saucia larvae were les s s e n s i t i v e to the growth i n h i b i t o r y e f f e c t s of the extracts than neonatal P_. saucia. 5. Results obtained from the f i e l d t r i a l suggest that the 30% aq A_. t r i d e n t a t a ethanolic extract at 0.2 g/ml protected cabbage from ins e c t pest damage s i g n i f i c a n t l y better than the water or EtOH c o n t r o l s . Insect pest damage to cabbage, however, was s i g n i f i c a n t l y l e s s with the deltamethrin spray at 17.9 ug/l than i n a l l other treatments. 6. Residual o v i p o s i t i o n deterrency to P_. rapae was suggested from r e s u l t s obtain i n the f i e l d t r i a l . Laboratory experiments with caged J?. rapae appear to confirm a contact o v i p o s i t i o n deterrency due to the A^ . t r i d e n t a t a ethanolic extract at 0.2 g/ml on cabbage. 7. An F-^  generation of f i e l d - c o l l e c t e d P. saucia grew s i g n i f i c a n t l y better than the larvae from the laboratory colony. However, the growth i n h i b i t i o n of P_. saucia larvae by the _A. t r i d e n t a t a extract was not s i g n i f i c a n t l y d i f f e r e n t between the two populations r e l a t i v e to t h e i r respective c o n t r o l s . My findings revealed that using i n s e c t growth i n h i b i t o r y extracts, selected i n laboratory bioassays, should c o n s t i t u t e only the f i r s t stage i n the development of novel botanical pest c o n t r o l s . The next stage should i n v a r i a b l y be t e s t i n g the products on target plants and insect s i n the f i e l d . Further i n v e s t i g a t i o n s using more inte n s i v e phytochemical techniques may help elucidate the s p e c i f i c chemicals or chemical mixtures responsible for both the growth and o v i p o s i t i o n i n h i b i t o r y a c t i v i t y . The extraction and screening of asteraceous weeds has advanced the p o t e n t i a l use of these natural products i n inse c t pest c o n t r o l programmes. 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APPENDICES APPENDIX I Analysis of Variance Table including the separation of i n d i v i d u a l degrees of freedom using orthogonal contrasts for Cabbage Looper Equivalents, for the four treatments, four blocks and f i v e survey days from the f i e l d t r i a l on cabbage, Ju l y 25, 1985 SOURCE DF SUM SQ F-VALUE PROBABILITY BLOCKS 3 0.928 0.897 0.4487 TREATMENTS 3 27.646 26.715 <0.0001 Deltamethrin vs. OTHERS* 1 25.596 74.203 <0.0001 A. t r i d e n t a t a vs. CONS 1 1.744 5.055 0.0284 30% aq EtOH vs. H20 1 0.306 0.886 0.3506 DAYS 4 45.650 33.085 <0.0001 TREATMENT * DAYS 12 14.464 3.494 <0.0007 Deltamethrin vs. OTHERS * LIN 1 8.524 24.712 <0.0001 Deltamethrin vs. OTHERS * QUA 1 2.819 8.173 0.0059 Deltamethrin vs. OTHERS * DEV 1 0.141 0.409 0.5251 A. t r i d e n t a t a vs. CONS * LIN 1 0.324 0.937 0.3367 A. t r i d e n t a t a vs. CONS * "QUA 1 0.213 0.619 0.4348 A. t r i d e n t a t a vs. CONS * DEV 1 0.031 0.091 0.7646 30% aq EtOH vs. H20 * LIN 1 0.541 1.569 0.2155 30% aq EtOH vs. H20 * QUA 1 0.353 1.024 0.3159 30% aq EtOH vs. H20 * DEV 1 0.754 2.187 0.1447 ERROR 57 19.662 TOTAL 79 108.35 OTHERS = the three other treatments, namely, _A. tr i d e n t a t a extract i n 30% aq EtOH, 30% aq EtOH, and H 20 2 CONS = the two controls, the c a r r i e r solvent 30% aq EtOH, and H 90 100 APPENDIX II Analysis of Variance Table i n c l u d i n g the separation of i n d i v i d u a l degrees of freedom using orthogonal contrasts f o r imported cabbageworm, ]?. rapae, l a r v a l counts for four treatments, four blocks and f i v e survey days from the f i e l d t r i a l on cabbage, July 25, 1985 SOURCE DF SUM SQ F-VALUE PROBABILITY BLOCKS 3 2.475 2.986 0.0386 TREATMENTS 3 19.790 23.880 <0.0001 Deltamethrin vs. OTHERS* 1 12.173 44.067 <0.0001 A. tri d e n t a t a vs. CONS 1 6.896 24.964 <0.0001 30% aq EtOH vs. H20 1 0.721 2.609 0.1118 DAYS 4 34.009 30.779 <0.0001 TREATMENT * DAYS 12 10.984 3.314 <0.0007 Deltamethrin vs. OTHERS * LIN 1 4.283 15.505 0.0002 Deltamethrin vs. OTHERS * QUA 1 2.001 7.245 0.0093 Deltamethrin vs. OTHERS * DEV 1 0.031 0.111 0.7403 A. t r i d e n t a t a vs. CONS * LIN 1 1.319 4.775 0.0330 A. t r i d e n t a t a vs. CONS * QUA 1 0.784 2.840 0.0974 A. t r i d e n t a t a vs. CONS * DEV 1 0.012 0.045 0.8327 30% aq EtOH vs. H20 * LIN 1 0.129 0.468 0.4967 30% aq EtOH vs. H20 * QUA 1 0.546 1.964 0.1653 30% aq EtOH vs. H20 * DEV 1 0.593 2.148 0.1482 ERROR 57 15.746 TOTAL 79 83.003 OTHERS = the three other treatments, namely, A^ . t r i d e n t a t a extract i n 30% aq EtOH, 30% aq EtOH, and H„0 CONS = the two controls, the c a r r i e r solvent 30% aq EtOH, and H„0 101 APPENDIX III Analysis of Variance Table i n c l u d i n g the separation of i n d i v i d u a l degrees of freedom using orthogonal contrasts for imported cabbageworm, P. rapae, egg counts, for the four treatments, four blocks and f i v e survey days from the f i e l d t r i a l on cabbage, July 25, 1985 SOURCE DF SUM SQ F-VALUE PROBABILITY BLOCKS 3 9.363 4.270 0.0094 TREATMENTS 3 31.476 14.353 <0.0001 Deltamethrin vs. OTHERS* 1 17.901 24.488 <0.0001 A. tri d e n t a t a vs. CONS 1 12.287 16.809 0.0002 30% aq EtOH vs. H20 1 1.288 1.763 0.1906 DAYS 4 77.694 26.572 <0.0001 TREATMENT * DAYS 12 23.006 2.6227 0.0089 Deltamethrin vs. OTHERS * LIN 1 0.008 0.010 0.9194 Deltamethrin vs. OTHERS * QUA 1 8.195 11.211 0.0016 Deltamethrin vs. OTHERS * DEV 1 0.599 0.819 0.3700 A. tr i d e n t a t a vs. CONS * LIN 1 0.563 0.771 0.3843 A. tr i d e n t a t a vs. CONS * QUA 1 5.515 7.544 0.0085 A. tr i d e n t a t a vs. CONS * DEV 1 3.808 5.209 0.0269 30% aq EtOH vs. H20 * LIN 1 0.675 0.924 0.3413 30% aq EtOH vs. H20 * QUA 1 0.149 0.204 0.6536 30% aq EtOH vs. H20 * DEV 1 0.037 0.051 0.8230 ERROR 57 35.087 TOTAL 79 191.43 OTHERS = the three other treatments, namely, _A. tri d e n t a t a extract i n 30% aq EtOH, 30% aq EtOH, and H o0 CONS = the two controls, the c a r r i e r solvent 30% aq EtOH, and H 90 

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