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Ihrwll....4..l.tsh .... .4 . 4 .. 111-4. 551.... . . .1 I} 4 , ... y. rra'rzfiél Ir . .11... 4! 4.9.5.! 30-! 0&1. 5.1.; :11). 4 . ‘El .. .......... .. 4 . ‘r.1.v§,..,oe.$vu .4! \v 3‘73? 1:55 . 4 . 1.2.6.}. .y "I... r. l ..4.4.II.uI l:.h’l;l|uo . 4a! .4 . ... . 4 LIBRARY 0mm This is to certify that the . ‘ " r thesis entitled Barley Leaf Chemistry and the Cereal Leaf Beetle Feeding Response presented by Robert T. Kon has been accepted towards fulfillment of the requirements for Ph. D. degeein Entomology W 2% Major progsor Date February 132 1976 0-7639 - "' ' ABSTRACT BARLEY LEAF CHEMISTRY AND THE CEREAL LEAF BEETLE FEEDING RESPONSE By Robert T. Kon Extracts of seedling barley, Hordeum vulgare L., a host for the cereal leaf beetle, Oulema melanopus (L.), and seedling pea plants, Pisum sativum L., a non-host, were fractionated and bioassayed in three per cent agar for beetle feeding responses. One response estimate was the count of beetles in contact with the bioassay medium after one, two, or three hours. The estimate considered most reliable was a visual examination of the bioassay medium after the test period. Feeding damage to the agar was graded from 0-6 in units of O.l. Greater sensitivity was demonstrated toward the hydrophobic compounds of barley than to the hydrophilic compounds. Numerical response to barley hydrophilic compounds was low from 20-300 ppm, but increased rapidly above this level to become equal with that produced by hydrophobic compounds. Statistically, the maximum numerical response to hydrophobic compounds occurred from about 300-2,000 ppm. Response was good from about l0-300 ppm. Determination of hydrophobic feeding stimulants became the prime objective of this study. Pea extract was a repellent/deterrent in its crude form. The deterrence was found to reside partly with the surface wax, but was Robert T. Kon strongest in the dewaxed apolar fraction of the hydrophobic compounds. Despite the deterrence of pea crude extract, incorporation of barley crude extract with it at a ratio of 1:1 or greater (barley:pea,wt/wt) renewed the beetle feeding behavior. Thus, a host—specific, chemical quality of barley overcame the effect of deterrents when the proper ratio between the two factors was achieved. These facts support the opinion that feeding deterrents and host-specific sign stimulants inter- act so that plant selection or rejection represents the net effect. The cereal leaf beetle feeding response was based on a multicom- ponent stimulant system. The type of agar damage observed was, to a large extent, characteristic of the fraction being bioassayed and gave clues to their respective functions. Primary alcohols were the only active fraction in the epicuticular wax. l-Hexacosanol was the most effective alcohol bioassayed alone and was active at 1.0 ppm. Little response to concentration above the threshold level was seen with the alcohols, and the agar damage was domi- nated by biting and rashing which indicated that stimulation of the biting response was the function of these compounds. Some indication was seen that l-hexacosanol combined with l-docosanol at 20:l (wt/wt) was more effective than l-hexacosanol alone. Apolar hydrophobic compounds of dewaxed barley seedlings were non- stimulative. Glycolipids and phospholipids were each active. Agar damage with these fractions contained a larger proportion of channels in the agar than found with the alcohols. It was concluded that these fractions reinforced the biting response and lowered the threshold to hydrophilic stimulants. The polar hydrophobic compounds, together, counteracted the deterrents in pea extract. The glycolipids and Robert T. Kon phospholipids from pea seedlings did not stimulate feeding, but neither were they highly deterrent to the beetles. Individual glycolipids of pea were also inactive. Barley monogalactosyldiglyceride and digalactosyldiglyceride were active in equal measure above 20 ppm, although there was little dose-dependent response. Barley sulfolipid was active at l-2 ppm. It was subsequently found that most of the activity of barley phospholipids was due to the neutral phospholipids. The acid phospho- lipids were stimulants of low effectiveness. The activity of the neutral phospholipids was, in turn, found to be due mostly to interaction with the alkaloid, gramine. Some indication was found, but not confirmed, that the fatty acid composition of phosphatidyl choline may have influenced the cereal leaf beetle response to that compound. Gramine was a stimulant at the lowest level tested, 3 ppm. It converted formerly rejected glycolipids and phospholipids of pea seedlings into palatable substrates, and when mixed with barley hydrophilic com- pounds, counteracted the deterrents of pea apolar hydrophobic compounds. It was concluded that gramine acted as a sign stimulant in this study. Little work was conducted with barley hydrophilic compounds. How- ever, the cationic compounds were active. Sucrose evoked a low, consistent response at 0.002M (776 ppm). Agar damage with this complete fraction consisted primarily of channels which was interpreted to indicate highly directed efforts toward continued feeding. The only pea fraction to stimulate the feeding response was the hydrophilic fraction. It was concluded that these compounds acted beyond the sensory level of host recognition and served to forge the final link in the chain of Robert T. Kon responses which result in continued feeding. A model of cereal leaf beetle host selection and feeding response was suggested from the results presented. BARLEY LEAF CHEMISTRY AND THE CEREAL LEAF BEETLE FEEDING RESPONSE By Robert T? Kon A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Entomology T976 TABLE OF CONTENTS Page INTRODUCTION ............................ 1 LITERATURE REVIEW ......................... 4 I. Terminology ...................... . . 4 11. Historical Period (1910-1953) ............... 6 III. Current Period (l953-l974) ................. 8 A. Insect Perception of Nutrient and Secondary Chemicals of Plants ....................... 8 B. Interactions Between Chemicals ............. 15 c. Electrophysiological Studies .............. 17 D. The Physiological Basis of Insect Feeding Patterns. . . 19 MATERIALS AND METHODS ....................... 23 I. Beetles .......................... 23 II. Plants ........................... 24 A. Barley ......................... 24 B. Pea ...................... . . . . 24 III. The Bioassay ........................ 24 A. Physical Considerations ................ 24 8. Temporal Considerations ............. . . . 26 C. Experimental Design .................. 25 0. Treatment Composition ................. 27 E. Scoring ........................ 27 F. Validity of the Bioassay ................ 28 IV. Isolation of Plant Biochemicals . . . ........... 28 A. Crude Extract ..................... 29 B. The Hydrophobic Fraction ................ 29 l. Isolating the Total Fraction ............ 29 2. Isolating Hydrophobic Subfractions ......... 30 a. Epicuticular Wax ............. . . . 30 (1) Total Wax .............. . . . 30 (2) Wax Fractions ............... 31 ii Page b. Hydrophobic Compounds Minus Wax, (H-W). . . . 36 (1) Separation into Polar and Apolar Fractions ............... 35 (2) Separation of the Polar Fraction . . . . 37 (a) Isolation of Individual Glycolipids ............ 37 (b) Isolation of Neutral and Acidic Phospholipids ........... 38 (c) Isolation of Individual Neutral Phospholipids ........... 41 (d) Isolation of Gramine ........ 42 C. The Hydrophilic Fraction ............... 43 l. Extraction of Hydrophilic Compounds ....... 43 2. Fractionation of the Hydrophilic Compounds. . . . 43 3. Non-Extracted Hydrophilic Compounds ....... 47 RESULTS ........................... . . 48 I. Validity of the Bioassay ................. 48 II. Crude Extract .................... . . 48 III. Hydrophobic Compounds Vs. Hydrophilic Compounds ..... 58 IV. Hydrophobic Compounds . . . . .............. 62 A. Complete Epicuticular Wax ............. . 62 B. Epicuticular Wax Fractions .............. 67 C. Hydrophobic Compounds Minus Wax, (H—W) ........ 77 D. (H-W) Apolar Fraction VS. (H-W) Polar Fraction. . . . 77 E. (H-W) Polar Compounds ............. . . . 84 l. Individual Glycolipids .............. 84 2. Phospholipids .................. 91 a. Acidic Phospholipids ............. 91 b. Neutral Phospholipids . . . . ........ 95 F. Gramine ....................... 100 V. Hydrophilic Compounds . . . . . . . . . . ..... . . . 109 A. Commercially Obtained Chemicals . . . ........ 109 B. Extracted Chemicals ................. ‘13 DISCUSSION .................... . . . . . . . . 119 I. Validity of the Bioassay ............... . . 119 II. The Parallel Study of Pea Seedling Extracts ....... 119 III. Estimation of Beetle Response .............. ‘20 IV. Functions of Barley Stimuli, and Corresponding Pea Fractions ............. . . . . ....... 12] Page A. Hydrophobic Compounds ................. lZl l. Epicuticular Wax ................. 121 2. Internal Compounds ................ l24 a. Total Hydrophobic Fraction Minus Wax ..... 124 b. Glycolipids .................. l26 (l) Mono- and Di-galactosyldiglycerides of Barley .................. l26 (2) Sulfolipids of Barley .......... 126 (3) Total Glycolipids of Pea Seedlings. . . . l26 (4) Mono- and Di-galactosyldiglycerides of Pea ................... 127 (5) Pea Sulfolipids ............. 127 c. Phospholipids ................. 128 (l) Acid Phospholipids of Barley ....... 128 (2) Neutral Phospholipids of Barley ..... 129 (3) Acid Phospholipids of Pea ........ 130 (4) Neutral Phospholipids of Pea ....... 130 d . Grami ne ................... 130 B. Hydrophilic Compounds ................. l33 l. Sucrose ...................... l33 2. Amino Acids .................... l34 3. Saponarin ..................... 135 V. A Model of Cereal Leaf Beetle Host Selection and Feeding Response ......................... l35 VI. Suggested Areas of Future Research ............ l36 VII. Closing Statements .................... l38 SUMMARY .............................. l39 BIBLIOGRAPHY ........................... T42 iv LIST OF TABLES Table Page l. CLASSIFICATION OF RESPONSES AND STIMULI ASSOCIATED WITH THE FEEDING BEHAVIOR OF PHYTOPHAGOUS INSECTS ......... 6 2. EXAMPLES OF SECONDARY PLANT SUBSTANCES KNOWN T0 STIMULATE FEEDING IN SPECIES OF COLEOPTERA ............... 15 3. THIN-LAYER CHROMATOGRAPHIC COMPARISON OF ELUTION SERIES USED TO SEPARATE EPICUTICULAR WAXES OF BARLEY AND WHEAT LEAVES ON SILICIC ACID COLUMNS ................ 33 4. RESPONSE OF FIELD-COLLECTED, ADULT CEREAL LEAF BEETLES TO CRUDE EXTRACT OF BARLEY AND NON-HOST PLANTS INCORPORATED INTO THREE PER CENT AGAR ................... 49 5. ANOVA TABLE FOR NUMERICAL RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES T0 CONCENTRATIONS OF BARLEY CRUDE EXTRACT IN THREE PER CENT AGAR ................ 55 6. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO PEA SEEDLING HYDROPHILIC COMPOUNDS IN THREE PER CENT AGAR. 6] 7. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO BARLEY CRUDE EXTRACT ADDED WITH EXTRACTS OF PEA SEEDLINGS TO THREE PER CENT AGAR .................... 53 8. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO BARLEY EPICUTICULAR WAX INCORPORATED INTO THREE PER CENT 64 AGAR ............................. 9. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES T0 EPICUTICULAR WAX 0F PEA SEEDLINGS, ALONE AND WITH BARLEY CRUDE EXTRACT IN THREE PER CENT AGAR ............. 68 10. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES T0 SUCROSE IN THE PRESENCE OF PEA SEEDLING EPICUTICULAR WAX IN THREE PER CENT AGAR .................... 69 ll. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO BARLEY EPICUTICULAR WAX FRACTIONS ELUTED FROM SILICIC ACID COLUMNS AND INCORPORATED INTO THREE PER CENT AGAR WITH OR WITHOUT SUCROSE ...................... 70 Table 12. l3. l4. l5. l6. l7. l8. T9. 20. 21. 22. 23. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO BARLEY EPICUTICULAR WAX FRACTIONS OBTAINED BY PREPARA- TIVE THIN-LAYER CHROMATOGRAPHY AND INCORPORATED INTO THREE PER CENT AGAR ........................ RESPONSE OF FIELD-COLLECTED, ADULT CEREAL LEAF BEETLES TO FRACTIONS OF BARLEY EPICUTICULAR WAX ELUTED FROM SILICIC ACID COLUMNS AND INCORPORATED INTO THREE PER CENT AGAR . . . RESPONSE OF FIELD-COLLECTED, ADULT CEREAL LEAF BEETLES TO EPICUTICULAR WAX FRACTIONS OF BARLEY OBTAINED BY PREPARA— TIVE THIN-LAYER CHROMATOGRAPHY AND INCORPORATED INTO THREE PER CENT AGAR ........................ RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO COMMERCIALLY OBTAINED PRIMARY ALCOHOLS IN THREE PER CENT AGAR WITH AND WITHOUT SUCROSE ................ RESPONSE OF FIELD-COLLECTED, ADULT CEREAL LEAF BEETLES TO COMMERCIALLY OBTAINED PRIMARY ALCOHOLS INCORPORATED INTO THREE PER CENT AGAR WITH AND WITHOUT SUCROSE ........ RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO DEWAXED PEA SEEDLING HYDROPHOBIC COMPOUNDS INCORPORATED WITH AND WITHOUT BARLEY CRUDE EXTRACT INTO THREE PER CENT AGAR ............................ RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO DEWAXED PEA SEEDLING APOLAR AND POLAR HYDROPHOBIC COM- POUNDS WITH AND WITHOUT BARLEY CRUDE EXTRACT IN THREE PER CENT AGAR .......................... RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO BARLEY EXTRACT FRACTIONS INCORPORATED WITH PEA SEEDLING CRUDE EXTRACT INTO THREE PER CENT AGAR ........... RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO BARLEY GLYCOLIPIDS AND PHOSPHOLIPIDS IN THREE PER CENT AGAR ............................ RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO VARIOUS RECOMBINATION RATIOS OF BARLEY GLYCOLIPID TO PHOSPHOLIPID IN THREE PER CENT AGAR ............. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO GLYCOLIPIDS INDIVIDUALLY ISOLATED FROM BARLEY AND INCOR- PORATED INTO THREE PER CENT AGAR .............. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO PEA SEEDLING GLYCOLIPIDS INDIVIDUALLY ISOLATED AND INCOR- PORATED INTO THREE PER CENT AGAR .............. vi Page 71 73 74 75 76 78 82 83 85 86 89 9O Table 24. 25. 26. 27. 28. 29. 30. 3T. 32. 33. 34. 35. 36. Page RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO BARLEY AND PEA SEEDLING PHOSPHOLIPIDS INCORPORATED INTO THREE PER CENT AGAR ...................... 92 RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO BARLEY SEEDLING NEUTRAL AND ACID PHOSPHOLIPIDS INCORPORA- TED INTO THREE PER CENT AGAR ................. 93 RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO BARLEY ACID PHOSPHOLIPIDS TREATED TO ALTER ASSOCIATED CATIONS AND INCORPORATED INTO THREE PER CENT AGAR ....... 94 RESPONSE OF NEWLY EMERGED, UNFED ADULT CEREAL LEAF BEETLES TO PEA SEEDLING ACID AND NEUTRAL PHOSPHOLIPIDS INCORPORATED INTO THREE PER CENT AGAR ................... 95 RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO BARLEY PHOSPHATIDYL SERINE AND THE REMAINING ACID PHOSPHOLIPIDS INCORPORATED INTO THREE PER CENT AGAR ...... 97 RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO BARLEY NEUTRAL PHOSPHOLIPIDS IN THREE PER CENT AGAR . . . . 98 RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO COMBINATIONS OF TWO BARLEY NEUTRAL PHOSPHOLIPIDS IN THREE PER CENT AGAR ......................... 99 RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO THREE OR MORE NEUTRAL PHOSPHOLIPIDS OF BARLEY IN THREE PER CENT AGAR WITH GRAMINE PRESENT .............. 10] RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO THREE SPECIES OF COMMERCIALLY OBTAINED PHOSPHATIDYL CHOLINE WITH AND WITHOUT GRAMINE IN THREE PER CENT AGAR. . . .102 A SELECTED SERIES OF MASS INTENSITIES FROM THE MASS SPECTRUM OF GRAMINE, 3-(DIMETHYLAMINOMETHYL)-INDOLE .......... l03 RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO GRAMINE IN THREE PER CENT AGAR ............... 105 RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES T0 COMBINATIONS 0F GRAMINE WITH OTHER PLANT BIOCHEMICALS IN THREE PER CENT AGAR ...................... ‘07 RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO BARLEY NEUTRAL PHOSPHOLIPIDS WITH AND WITHOUT GRAMINE IN THREE PER CENT AGAR ...................... 108 vii Table 37. 38. 39. 40. 4l. 42. 43. 44. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO PEA SEEDLING GLYCOLIPIDS AND PHOSPHOLIPIDS WITH AND WITH- OUT GRAMINE IN THREE PER CENT AGAR .............. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO A DETERRENT FRACTION OF PEA SEEDLING EXTRACT IN BARLEY HYDROPHILIC EXTRACT WITH AND WITHOUT GRAMINE IN THREE PER CENT AGAR .......................... RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO SELECTED AMINO ACIDS IN THREE PER CENT AGAR WITH AND WITHOUT SUCROSE OR GRAMINE .................. RESPONSE OF NEWLY EMERGED AND FIELD-COLLECTED, ADULT CEREAL LEAF BEETLES TO PLANT-STIMULATED AMINO ACID MIXTURES IN THREE PER CENT AGAR ..................... RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO FRACTIONS OF BARLEY HYDROPHILIC COMPOUNDS SEPARATED ON A SEPHADEX G-lO COLUMN AND INCORPORATED INTO THREE PER CENT AGAR ............................. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO BARLEY HYDROPHILIC COMPOUNDS SEPARATED BY GEL FILTRATION AND ION EXCHANGE COLUMN CHROMATOGRAPHY AND INCORPORATED INTO THREE PER CENT AGAR ..................... RESPONSE OF NEWLY EMERGED AND FIELD-COLLECTED, ADULT CEREAL LEAF BEETLES TO SAPONARIN-CONTAINING EXTRACT FROM BARLEY SEEDLINGS, INCORPORATED INTO THREE PER CENT AGAR ....... THE SURFACE LIPIDS OF BARLEY AND PEA LEAVES ......... viii Page llO lll ll2 ll4 llS ll7 ll8 123 LIST OF FIGURES Figure Page 1. Diagram of a paper chromatogram of barley seedling hydrophilic compounds eluted from a Sephadex G-lO column. . . 45 2. Numerical response of newly emerged, unfed, adult cereal leaf beetles to the concentration of barley seedling crude extract in three per cent agar - I .............. 50 3. Numerical response of newly emerged, unfed, adult cereal leaf beetles to the concentration of barley seedling crude extract in three per cent agar - II ............. 52 4. Response of newly emerged, unfed, adult cereal leaf beetles to the concentration of barley and pea seedling crude extract, separate and combined, in three per cent agar. . . . 56 5. Response of newly emerged, unfed, adult cereal leaf beetles to the concentration of total hydrophobic and hydrophilic compounds of barley imcorporated into three per cent agar . . 59 6. Numerical response of newly emerged, unfed, adult cereal leaf beetles to the concentration of dewaxed barley hydro- phobic compounds with and without the readdition of epicuticular wax ....................... 55 7. Response of newly emerged, unfed, adult cereal leaf beetles to the concentration of dewaxed barley hydrophobic polar and apolar compounds in three per cent agar ......... 80 8. Response of newly emerged, unfed, adult cereal leaf beetles to the concentration of barley and pea seedling glycolipids incorporated into three per cent agar ............ 87 9. Major ions represented in the mass Spectrum of gramine, 3-(dimethylaminomethyl)-indole ................ 104 ix INTRODUCTION The cereal leaf beetle, Oulema melanopus (L.), (CLB) is an introduced species to the United States (21). Castro et_al, (22) have discussed the systematics and natural history of this chrysomelid pest of the plant family, Gramineae. In a study of its New-World hosts, Shade and Wilson (149) found that CLB developed best on wheat, Triticum vulgare Vill., oats, Avena sativa L., barley, Hordeum vulgare L., Spelt, I, spelta L., and rye, Secale cereale L., all members of the subfamily, Festucoideae (130). AS a result, this pest quickly became the object of extensive research aimed at limiting its potential damage to the small grain industry of the United States. Despite early quarantine efforts, the CLB now occupies a portion of the Province of Ontario, and several Northeastern and Mid-Western states (58). Connin et_al, (25) developed a system to rear the CLB in mass which enabled laboratory studies to become allied and coordinated with field studies. A published bibliography (184) and a Michigan State Univer- sity Research Report (3), reveal how extensive the literature related to this pest had become up to 1970. These publications cover such areas aS natural history and bionomics, population dynamics, economic impact and chemical control, biochemistry, physiology, and resistant crop varieties. The use of control by introduced egg and larval parasitoids has been inaugurated as a factor in an integrated control approach (95, 156, 157). Efforts are underway to represent the pest-parasite-crop ecosystem by mathematical models which would be predictive of pest populations based on current environmental and biological data (59). Control recommen— dations will be based upon the predicted degree of interaction of all components of the ecosystem model. If it were possible to reduce the overall interaction between the plant and its pest, the resultant control costs would be reduced. The most effective means of reducing host-plant interactions by any given amount is by an appropriate degree of plant resistance. Currently, the only well defined form of host resistance to the CLB is derived from a physical basis. Pubescent wheat leaves offer a physical deterrent both to oviposition and subsequent larval survival (138, 183), resulting in decreased damage to such protected seedlings (181). Closely Spaced vascular bundles have been correlated with resistance to larval feeding on unfavored members of the Gramineae (150). Recently, it was reported that wheat lines having thin leaves were more resistant to the CLB than lines with thicker leaves (4). Some varieties of barley seem to Show a low level of resistance (52), but the actual basis is uncertain save for the designation "non- preference for oviposition" (180). An understanding of the chemical basis of CLB preference for a susceptible variety of barley might provide a means of developing a more effective varietal resistance by selecting against any feeding stimulants amenable to genetic manipulation. USDA researchers have screened several hundred compounds for CLB attractant properties in field tests with little success (unpublished data, ARS, Entomology and Small Grain Laboratory, East Lansing, Michigan). Haynes gt_al, (59) pointed out that it is not known why the CLB feeds on wheat and oats (nor on any host for that matter). Knowledge of CLB nutritional requirements should assist plant breeders toward directed selection of varieties possessing a nutritional imbalance and thus add another degree of resistance against this insect. An artificial diet has been under development, but has not yet allowed egg production and successive generations to be reared (182, personal communication). The addition of natural feeding stimulants to present diet mixtures might allow research in this area to proceed at a faster rate. The work of Panella et_al, (129) failed to demonstrate any signif- icant olfactory response by the CLB to extracts of barley seedlings. However, strong responses were derived with crude extracts of susceptible barley seedlings incorporated into an agar medium as a bioassay. The objective of the present study is to increase the understanding of the chemical basis of host selection by the cereal leaf beetle once the plant is physically contacted by the insect. The method will be to isolate and identify from barley seedlings, a number of biochemicals which stimulate CLB feeding as determined by a modified form of the Panella et_al, bioassay. LITERATURE REVIEW This review deals primarily with the chemical basis of host-plant selection or rejection by gustation once an insect has physically encoun- tered a plant. Orientation to a plant from a distance by an insect often involves visual and olfactory perception. For consideration of visual orientation, see Prokopy and Haniotakis (131), Moericke et_al, (116), Meyer (106) or Meyer and Raffensperger (107). Many authors have reported on the olfactory responses of insects to plant volatiles. Papers by Trayiner (168), Kennedy and Moorhouse (89) and Schwinck (146) ought to be reviewed by anyone wishing to pursue such research. Dethier and Schoonhoven (35) reported an electrophysiological investigation of the neuronal basis of olfaction. Also, examples exist where the initial encounter of a host-plant appears to be a random happening (88, 119, 189). I. Terminology Normal feeding behavior by phytophagous insects has been divided into three components by Dethier (30): a) orientation to the food, b) a biting response, and c) continued feeding. Thorsteinson (165) viewed feeding broadly in terms of two antagonistic neuroregulatory systems in constant opposition. One system caused a settled state favoring feeding, the other, a dispersing drive when the thresholds for feeding became too high to hold the insect on a food source. Consequently, he proposed a fourth element for Dethier's framework, "dispersal." To answer a need for a more descriptive terminology relating chem- icals to aspects to insect feeding behavior, Dethier §t_al, (33) suggested the following terms and summarized definitions: 1. attractant - a chemical causing oriented movement toward the source. 2. arrestant - a chemical causing insects to aggregate in contact with it. 3. stimulant a chemical eliciting feeding or oviposition. 4. repellent source. a chemical causing oriented movement away from the 5. deterrent a chemical which inhibits feeding or oviposition. The term “phagostimulant” was proposed by Thorsteinson (163) for those chemicals to which insects respond by feeding. It has become part Of the literature, although it was viewed by Kennedy (86) as an "etymolo- gical chimaera." Thorsteinson (164) classified those nutrients detected by an insect as "sapid nutrients." It has been shown more clearly with larvae of the silkworm, Bombyx mori L., that each sequential step in feeding may be under the influence of different chemicals. Attractants were found to be citral, terpinyl acetate, linalyl acetate, and linalol. Biting required/8 -sitosterol and isoquercitrin. Cellulose was required for proper swallowing and sucrose, inositol, inorganic phosphate and silica were co-factors (53). The chemicals responsible for such discrete compoments Of feeding behavior would not be adequately described by the classification of Dethier et_al, just presented. Beck (13) felt that a new system was needed to provide a versatile terminology for students of the insect feed- ing response. Table 1 represents his effort toward this end. Hsiao (66) used the term ”sign stimulant" for botanically restricted substances releasing biting and feeding response. Table l. CLASSIFICATION OF RESPONSES AND STIMULI ASSOCIATED WITH THE FEEDING BEHAVIOR OF PHYTOPHAGOUS INSECTS - BECK (l3). Evoking Stimulus Response POSITIVE NEGATIVE Orientation Attractant Repellent Orientation Arrestant Repellent Biting or piercing Incitant Suppressant Maintenance of feeding Stimulant Deterrent II. Historical Period (l9lO—l953) The Dutch botanist, Verschaffelt (175) was the first investigator to put the study of host preference by insects, particularly phytophagous insects, on a sound chemical basis. He observed that mustard oil gluco- sides [now termed glucosinolates (159)] were common to the plants (chiefly Crucifereae) forming the host range of Pieris rapae L. and P, Brassicae L., the lesser and the greater cabbage butterfly, respective- ly. Verschaffelt applied solutions of glucosinolate, sinigrin, to non- host leaves and only after such treatment were these leaves readily eaten by the Ejgrj§_larvae. McIndoo (99, 100) demonstrated that odors emanating from a plant could attract a natural insect pest. He introduced the concept of an olfactometer. Using a Y-tube device, he found that 62.7% of the time, adult Colorado potato beetles, Leptinotarsa decemlineata (Say), would select the tube leading to the host odor. These insects failed to respond to odors from non-host plants. Dethier (28) confirmed the principle of insect response to host Odors. Larvae of the monarch butterfly, Danaus (=Anosia) plexippus (L.), recognized host leaves separated from them by a screen. Recognition was indicated by searching, turning movements when they were over host leaves. Rather straight movements occurred over non-host leaves. Ten years later, Dethier (29) reviewed what had been learned re- garding the chemical basis of host preference. From hindsight, it is clear that he saw host selection as a simple chemical phenomenon: "In every case, in the final analysis, odor is the organisms index, regardless of food." "Essentially, the problem of plant choice resolves itself into a study of attractants and repellents and vice versa." These odors originated with the essential oils, and plant selection could be "largely divorced from nutritional requirements." A significant event in this field was the Insect/Plant Relationship Symposium in 1951 at the IXth International Congress of Entomology in Amsterdam. Only four papers were published, but they represented such polarized opinions that the resulting controversy inspired a great deal of research. Dethier (30) continued to support the importance of plant volatiles as the ultimate determinant of a preferred host. These substances would cause biting and continued feeding by most monophagous and oligophagous species. Nevertheless, some insects seemed to require contact chemore- ception to release their feeding behavior. For instance, Thorpe et_al, (161) had shown that wireworms, Agriotes sp,, were attracted by asparagine glutamine and amides of short chain fatty acids, but for biting, they re- quired sugars, lipids and polypeptides. Fraenkel (41), like Dethier, felt that the "Odd" chemicals or secondary substances of plants were responsible for host selection among leaf feeding insects. He emphasized two points: a) among those insects studied, the nutritional requirements were very similar, and b) the Chemical composition Of the green leaves studied was similar. Therefore, he felt that good nutrition for any phytophagous insect could be achieved if a sufficient quantity of leaves of any non-toxic plant species were eaten; nutrition could not be a factor in the host Specificity of insects. Painter (l28) considered a possible role for nutritional factors in host preference by insects. Previously, he had defined one mechanism of resistance as antibiosis (126,127) and suggested that required nutrients might be deficient or lacking. Kennedy (84) restated his theory of "dual discrimination" (87), invoked to explain observations that Aphis fabae Scop. preferred growing and senescing leaves over mature leaves, and that the surrmer form of the alDl'l‘id preferred to feed on potted, growing specimens of the winter host, Spindle, Euonymus europaeus L., than on growing summer host, Beta vulgare L- 3 under greenhouse conditions. One type of discrimination would fulfill the ecological need to distinguish between plants in a similar growth State and another would be associated with materials nutritionally good For the aphids. This latter sense allowed them to select among leaves on the same plant for their stage of physiological development. III. The Current Period (1953—1975) A~ Insect Perception of Nutrient and Secondary Chemicals of Plants To a great extent, research into host-plant selection since 1953 has Feiated to these two positions, the "dual disrimination" theory and selection based on the ”odd" chemicals of plants. Kennedy had, of course, only a deduction based on circumstantial evidence that nutrients might affect the feeding behavior of phytophagous insects. Some direct evidence for subterranian insects had been provided by Thorpe et_gl, (161) An agar medium was used by Thorsteinson (162) to Show that neither nutrients nor sinigrin, alone, were very stimulating to larvae of the diamond-back moth, Plutella maculipennis (Curt.). Yet, as little as 2 ppm of sinigrin blended with the nutrients (2.0% pea leaf powder) evoked a marked response. He later found that larvae of P, maculipennis and L, decemlineata were stimulated to feed by ascorbic acid (163). Thorsteinson (163) also reported that thiamine stimulated a feeding response by L, decemlineata larvae. Feeding was evoked in a grasshopper, Chorthippus longicornus Lat., by sucrose, glucose, betaine, and monosodium glutamate. He concluded that nutrients could stimulate feeding in oligophagous and polyphagous insects. Elsewhere, he suggested that the perception of nutrients by plant feeding insects had not received due attention (164). Mittler (110, 111) found that the sap exuded from stylets severed from aphids, Tuberolachnus salignus (Gemlin), feeding on potted willows, Sglix_§p,, had a higher amino-nitrogen content when analyzed from growing leaves than they did on mature leaves. Kennedy (85) felt that this work better established the relationship between nutrition and host-plant selection. Fraenkel (42) was unconvinced by these results and during this time he made a famous declaration that host-plant specificity depended entirely upon the presence of the secondary plant substances to which the insect would respond positively or negatively. Kennedy retained his conviction in the "dual discrimination" capabilities of insects, but was conservative lO in his appraisal of results germane to the subject. He pointed out (86) that no direct evidence had yet been presented to link host selection to the overall quality of available nutrients acting as feeding stimulants. A breakthrough in bioassay procedures for aphids occurred when Mittler and Dadd (113, 114) developed a means of supplying artificial test solutions to aphids via a parafilm sachet. Mittler (112) determined that growth and feeding rate of the aphid, Myzus persicae (Sulzer), could be influenced by the ratio of sucrose to a mixture of 20 amino acids. Sucrose ranged from O - 40% while amino acids were held at 2.4%. Then sucrose was held at 15% while amino acids varied from 0 - 4.8%. Uptake was poor on diets with less than 5% sucrose or when total amino acid was below 1%. Optimum concentration range for sucrose was l0 - 20% and for amino acids, the optimum level was 3%. Declines occurred above either optimum, emphasizing the "importance of the behavioral aspects of nutrition." A similar study was reported earlier by Auclair (7) which agrees with Mittler's conclusions. van Emden (174) reported a complex study of M, persicae (polyphag- ous) and Brevicoryne brassicae (L.) (oligophagous on curcifers). These aphids were grown on two crucifers and two non-crucifers. The plant leaves were analyzed at different physiological ages for allyl isothiocya- nate and total free amino acid content. Using multiple regression analysis, he concluded that both secondary substances and nutrients played a role in host susceptibility, but the oligophagous B, brassicae was less influenced by amino acids than the polyphagous M, persicae. Regression equations indicated that the amino acids correlating with good growth performance by B, brassicae would tend to remain relatively constant over age and growth condition differences and this aphid may not select for their ll presence. Concentration changes for amino acids correlated with good performance by M, persicae were significant with changes in the host- plant leaves allowing the aphid to select for these plants on the basis of the physiological state of the host. It appears that for aphids, the dual discrimination theory does have some basis in fact. Dethier (31) credited the theory with having "brought the nutritional aspects of the plant into a picture that had become unbalanced" at the time of its proposal. Another attack on Fraenkel's hard-line "secondary substances“ theory came, in part, from Waldbauer (176). It had been shown by Wald- bauer and Fraenkel (178) that maxillectomized larvae would feed on normally rejected plants. Waldbauer analyzed the growth and reproduction of maxillectomized larvae of the tobacco hornworm, Manduca (=Protoparce) sexta (Johan.) fed upon a normal host, tomato, Lycopersicon esculentum Mill., and on four nonhosts. Success on dandelion, Taraxacum officinale Weber, was equal to tomato and reasonably good on burdock, Arctium minus (Hill). However, mullein, Verbascum thapsus L., was unsuitable. Mullein- fed hornworms had longer larval periods, 45% mortality, mean weight gain per day was less than one-half that on tomato, and females laid fewer eggs, all unviable. Catalpa, Catalpa speciosa Warder, was also a very poor growth medium. As a result, Waldbauer (176) stood in opposition to Fraenkel's view that all green leaves should satisfy the nutritional needs of all insect species. His belief was that nutritional considerations could, indeed, restrict the host-plant range of a phytophagous insect species. In a further study of consumption, digestion and utilization of non-host leaves by maxillectomized M, sexta, Waldbauer (177) confirmed that l2 mullein was nutritionally inferior for this insect. Likewise, Mehta and Saxena (103) found that growth of larval cotton spotted bollworms, Eagles fable, was very poor on two non—hosts, Pisum_sativum L. and Brassica oleraceae botrxtis L., even though the index of consumption and adsorp- tion was higher in each case than for plants providing better growth. Additional studies correlating the nutritional composition of a diet with insect feeding behavior have been made. Auclair §t_al. (9) reported a lower concentration of amino acids and amides in three pea varieties susceptible to the aphid, Acyrthosiphon pisum (Harr.). Another report (6) revealed that A, pjsum_had a lower feeding rate on the resis— tant varieties than on the susceptible plants tested. Sugars had been known to elicit a feeding response from insects (44). Feeding by polyphagous larval European corn borers, Ostrinia (=Pyrausta) nubilalis (HUbn.) was correlated with plant parts having the higher concentrations of sugars (12). A monophagous insect, the sweet clover weevil, Sitgna_cylindricollis Fahraeus, was highly influenced by the glucose, fructose and sucrose in its host plant (1). When the appeal of several sugars to phytophagous insects has been tested, sucrose has generally been preferred to any other (1, 49, 61, 70, 74, 129). Larvae of Clerig euphorbiae L. showed a tendency to eat less and to gain less weight on an unbalanced diet compared to a balanced diet (64). When comparisons were made between diets diluted to 85, 70, and 50% of the nutrients in a control, weight gains were not significantly different, but the amount eaten definitely increased with dilution. In a later review, House (65) concluded that the quantitative aspects of nutrition, particu- larly the balance of nutrients, could affect insect food selection. Ma (95) tested 3, brassicae larvae with an agar—cellulose medium and found 13 2M while ascorbic acid was increased, 2 that when sucrose was held at 10' feeding also increased until ascorbic acid reached 10' M, after which a deterrent reaction was observed. This deterrence was neutralized by 1M. increasing the sucrose to 10' Auclair (8), too, felt that not only overall amino acid concentra- tion, but also the relative concentrations of individual amino acids would weigh heavily on the susceptibility/resistance of plants to aphids. The pink bollworm, Pectinophora gossypiella (Saunders), was found by Vanderzant (172) to develop well on an artificial diet when the amino acid composition Simulated cottonseed protein, but did not survive when the composition resembled that of casein. Within a class of chemicals, some may stimulate positively, some not at all, and others may be deterrent. Beck and Hanec (14) found that L-alanine, L-serine, L-threonine, and L-methionine were stimulants for Q, nubilalis larvae, while A9-alanine, L-tryptOphan, L-phenylalanine and L-arginine were deterrents. Larvae of L, decemlineata were highly stimulated by L-alanine, 3 -aminobutyric acid and L-Serine and moderately by other amino acids (70). In a study where eight amino acids, considered essential to the aphid, A, fabae, were omitted from a synthetic diet, Leckstein and Llewellyn (93) concluded that alanine and proline were phagostimulants. Amino acids were non-stimulatory to larvae of the alfalfa weevil, Hypgga postica (Gyll.) (66), but adenine and adenosine isolated from alfalfa, Medicago sativa L. were powerful stimulants (67). Other purines, pyrimidines and their nucleotides were inactive. Phosphatilyl choline, phosphatidyl inositol, and to a lesser extent, phosphatidyl serine were good stimulants for the grasshopper, l4 Melanopus bivittatus (Say) and Camnula pellucida (Scudder) (166). These same phospholipids plus phosphatidyl ethanolamine were active stimulants for larvae of L, decemlineata (70). Hsiao (69) reported on the sensitivi- ty of five species of Leptinotarsa to several nutrients. Only L, haldermani Rogers and L, decemlineata responded to vegetable lecithin. Phospholipids as a group evoked a greater response from Schistocerca gregaria Forsk. than from Locusta migratoria L. (102). Choline phospho- lipids were the only commercially obtained phospholipids which stimulated larval cabbage loopers, Trichoplusia nj_(HUbn.) (49). Other lipid nutrients have been determined to be feeding stimulants for plant feeding insects. Triglycerides and a mixture of free sterols and fatty acids from wheat germ oil elicited feeding from S, gregaria (102). Ag-Sdtosterol was a stimulant for larvae of B, mggi (54) In Spite of growing evidence that nutrients could strongly influ- ence the feeding behavior of insects, Fraenkel (43) was willing to make only a small concession as to their importance in determining host specificity. He did recognize that all leaves would not equally well serve phytophagous insects as food, but if nutrients played any role in host selection, he felt that it was a minor one. Indeed, there are many examples of "odd" chemicals which have been Shown to be feeding stimulants for insects. Hsiao (68) and Schoonhoven (142), in particular have compiled extensive lists, not only of those that stimulated feeding, but also of many that were deterrent. So far, the latter outnumber the former. Table 2 is modified after Schoonhoven (142). It lists only coleopteran species and associated feeding stimu- lants. 15 Table 2. EXAMPLES OF SECONDARY PLANT SUBSTANCES KNOWN TO STIMULATE FE ING IN SPECIES OF COLEOPTERA - modified from Schoonhoven (14 —_I Stimulating Chemical Insect Species Refere (+) Catechin-7-<$--D- Scolytus multistriatus (Marsham) (37 xylopyranoside* Cucurbitacin Aulacophora foveicollis Lucas (152 Cucurbitacin Diabrotica undecimpunctata Barb.** (23 Gossypol Anthonomus grandis Bohe. (97 p-Hydroquinone S, multistriatus (125 Hypericin Chrysolina brunsvicensis Grav.** (133 Isoquercitrin A, grandis (6O Linamarin Epilachna varivestis Mulsant (121 Lotaustrin L, varivestis (121 Lupeyl cerotate S, multistriatus (36 Oxalic acid Gastroidea viridula Deg. (134 Phaseolutanin S, varivestis (121 Quercetin A, grandis (6O Quercitrin A, grandis (60 7- $-L-rhamnosyl-6- Agasicles SR.“ (191 methoxyluteolin Salicin Plagiodera versicolora (Laich.) (96 Sinigrin* Phyllotreta cruciferae (Goeze)** (62 Sinigrin Phaedon cochleariae Fab. (158 * Additions by the present author. **Chrysomelidae - indicated by the present author. 16 B. Interactions Between Chemicals A highly significant phonomenon evident from the years of research is that many forms of interactions between plant chemicals occur in the sensory systems of phytophagous insects. One type of interaction is be- tween nutrients. The effect may be additive or synergistic. Beck and Hanec (14) found that serine and glucose had an additive effect on the feeding response of larval European corn borers. However, synergistic interactions seem to be more common. For the grasshopper, S, pellucida, Thorsteinson (165) reported that KH2P04 alone was ineffective, but at 0.004M with sucrose at 0.02M, the response was appreciably enhanced over sucrose alone. He also reported that the amino acids, serine, alanine, and B-aminobutyric acid, each at 0.008M, syner- gistically interacted and addition of 0.02M sucrose increased the response even more. The aphid, M, persicae, reacted in a synergistic way to a mixture of amino acids and sucrose compared to sucrose alone (115). Larvae of the Spruce budworm, Choristoneura fumiferana (C1em.), Showed a synergistic response to 0.03M each of glucose, fructuse and sucrose compared to 0.09M sucrose alone (61). Gothlif and Beck (49) found synergism between K+ salts (the anion had little or no effect) and the neutral lipids of wheat germ oil for I, 21: Ma (95), working with A, brassicae, found that vitamin C was ineffective alone, but synergized with sucrose. L-Proline also syner- gized the feeding response to sucrose by S, fumiferana larvae as did hydroxyproline and glutamate (61). It is evident that sucrose was often involved in reported syner- gisms of the feeding response of insects. Thorsteinson (165) suggested that the gustatory effects Of saccharides interacting with other plant 17 substances may influence food selection by phytophagous insects. Auclair (8) pointed out the apparent specific requirement for sucrose as a feeding stimulant for many aphids. Another class of interactions involves nutrients with secondary substances. In fact, Thorsteinson (165) advised researchers to consider secondary substances as "synergizers." In that review, he reported that larvae of E, maculipennis responded very little to either sinigrin alone or to sucrose at any concentration, but the addition of 0.1% Sinigrin to 0.2M sucrose was highly stimulatory. Regarding these ”Odd" chemicals which are known to stimulate insect feeding, Schoonhoven (142) indicated that they often require the presence of a sugar to be an effective stimulant. Heron (61) made a similar statement. C. Electrophysiological Studies Since host selection had been accepted as primarily a chemically based response, it was desirable to study as directly as possible, the chemoreceptors involved. Hodgson and Roeder (63) improved existing electrophysiological techniques for insect material and reported a study of the labellar setae of the blowfly, Phormia regina (Meigen). Since an electrolytic solution was required to be in contact with the insect's sensory apparatus, only water-soluble materials could be tested with this tip-recording method. Morita (118) introduced side-wall recording which enabled hydrophobic materials to be tested. Torii and Morii (167), Ito eL_Sl, (73) [both according to Dethier, (31)] and Waldbauer and Fraenkel (178) demonstrated indirectly, by removal of the maxillae, that these structures were regions of gustatory sensory abilities. An olfactory ability of much less significance was found on 18’ the maxillae (145). Ishikawa (72) was the first worker to demonstrate the gustatory function of the sensilla styloconica on the maxillae of S, mg:i_larvae. Two such structures arise from each maxilla (one lateral, one medial) and each is innervated by four contact chemoreceptor cells. Schoonhoven (144) has reviewed the known sensory abilities of Lepidoptera sensilla styloconica. Each cell therein has a range of sensitivity, qualitatively and quantitatively somewhat unique to itself. In the lateral sensillum, for example, one cell is responsive to amino acids while another responds to sucrose. Broadly, the medial sensillum is Often the location of deterrent recognition. The most extensive study of chemoreceptor spectra to date seems to be that made by Dethier and Kuch (34), who studied ten species of lep— idopterous larvae. Lateral and medial styloconica were exposed to 15-53 compounds from the following classes: salts, acids, sugars, amino acids, polyhydric alcohols, glucosides, sterols, P04"2 buffer, and quinine. A major purpose of this work was to counter an idea which had gotten into the literature that the receptor neurons of these structures had a narrow range of specificity. They were successful and confirmed a previous hypothesis (145) that ”each of the eight cells is sensitive to a number of compounds.” Recent investigation Of_L. decemlineata larvae revealed amino acid receptors in the lateral sensillum of the galea and on the maxillary and labial palps (109). The medial sensillum did not respond to any chemical tested in a manner similar to the lateral sensillum, although low-frequency impulses were detected. The amino acids giving the greatest effect were those determined by Hsiao and Fraenkel (70) to be the most effective in behavioral studies. A similar correlation between behavior and 19 electrophysiological observations exists for M, £2522: The antennae of this insect responded less to odors from Nicotiana Sp, than to Odors from other hosts (145). Jermy §L_gl, (80) found Manduca to be less inducable by Nicotiana than by other hosts. For discussions of the phenomenon of induced preference, also see Wicklund (187), Waldbauer and Fraenkel (178) and Schoonhoven (141). D. The Physiological Basis of Insect Feeding Patterns Another unsettled question relates to the basis for monophagous, Oligophagous, and polyphagous food habits. The terms themselves are somewhat vague regarding the level Of plant taxonomy at which they should be applied (165). Mechanistically, however, Dethier (29) believed that these patterns could be defined relative to the number of chemicals which were attractive to an insect. Monophagy resulted from attraction [this term is pre-Dethier §L_gl,, (33)] to one compound or to several confused as one by the insect. Oligophagous insects reacted to several distinct compounds while polyphagous insects did not require specific attractants, but fed on all plants not containing repellents. Thorsteinson (162) suggested that in oligophags, positive and negative influences might underlie host selection. He later assumed a broader position by stating that "a variety of mechanisms probably underlie oligophagy" (165). Jermy (78) opted for oligophagy based more on avoiding deterrents than by responding to specific stimulants. Using single leaf—disc tests and sandwich tests, a disc of a non—host leaf between discs of a host leaf, he found that most non-host plants of nine insect species contained feeding deterrents. Both slightly and highly restricted feeders were very sensitive to the deterrents. A tendency existed for the highly restricted feeders to Show the greater sensitivity. The author felt that 20 the importance of botanically restricted, specific stimulants would be reduced if substances more widely distributed could replace them by virtue of possessing a similar stereochemistry or configuration. Noteworthy here is a paper by Meyer and Norris (105) where the molecular shapes of hydroquinone and p-hydroxybenzaldehyde were said to be similar. These two compounds were the best of six substances tested as feeding stimulants for the smaller European elm bark beetle, Scolytus multistriatus (Marsham). The topic of molecular structure and stimulating effectiveness was given some consideration by Schoonhoven (142). Gupta and Thorsteinson (51) applied larval A, maculipennis to 62 non-host plants (37 families) not containing the glucosinolate stimulants of normal hosts and found that nine species were fed upon, untreated, during an 18 hour period. Twelve Species became acceptable when coated with sinigrin solution and 41 remained unacceptable following treatment with sinigrin. Because a normal host, black mustard, Brassica nigra Koch, was rejected when coated with aqueous extract of various fully rejected plants, feeding inhibitors were suggested as being potentially as Signif- icant as feeding stimulants in circumscribing an insect's host range. However, of the nine non-host plants acceptable to E, maculipennis, only pea supported successive generations from the egg stage. First instar larvae transferred from black mustard to sweet clover, Melilotus officin— gljS_Lam., or to coumino clover, M, §19§_Boiss., produced some adults, but sinigrin applied to the two clovers did not increase survival. In these cases, some nutritional considerations would seem to be involved in host selection. In fact, botanically restricted compounds are not required to release the feeding behavior of many insects. None have yet been conclusively demonstrated for M, sexta or L, decemlineata (43). A 21 list of 15 insect species was prepared by Schoonhoven (142) of instances where one or more generations were reared on meridic diets containing no secondary substances. The alkaloids in non-host solanaceous plants apparently prevent colonization of these plants by the Colorado potato beetle (78). Kogan and Goeden (90) compared behavior of larval Lema trilineata daturaphila (Oliv.) with published data-for the tobacco hornworm and the Colorado potato beetle, all of which feed on Solanaceae. These species reacted differently to the various alkaloids contained in this family, and no positive stimulation has been attributed to these alkaloids, nor to alkaloids generally. Some steroidal alkaloids did not harm the Colorado potato beetle at high concentrations, while a tropane alkaloid, scopola— mine, was toxic at 1%. Contrary to this fact, L, L, daturaphila avoided plants containing steroidal alkaloids, but suffered no ill effects from the tropane alkaloids of QEE!!§.§E: Injection of this insect with scop— olamine at 12.5 mg/g of larval fresh weight did not prevent normal devel- opment. Kogan and Goeden (90) concluded that the range of these insects within the Solanaceae would be determined by the alkaloids deterrent to feeding activity, and that feeding excitants would exist generally throughout this family. Regarding the negative influence of many "odd" chemicals on insect feeding preferences, Fraenkel (43) described their possible effects as feeding deterrents, toxins, and hormone-mimetic substances. Gordon (48) suggested that these protective secondary substances may sometimes act as antibiotics to microbial commensals which might help insects to overcome nutritional imbalances in their food. 22 It was once held that polyphagous insects did not require specific feeding stimulants (attractants), but would consume any leaf which did not contain repellent substances (28). However, Thorsteinson (164) stated that polyphagous insects did depend on specific gustatory stimu- lants for the expression of their feeding activities. Mehrotra and Rao (101) reported differences in phagostimulant requirements for L, migggg Lg:ig_and S, gregaria. Subsequently, Mehrotra and Rao (102) reported on the components of edible oils (six types) as phagostimulants for these two species. No feeding was induced by hydrocarbons, sterol esters, diglycerides, or monoglycerides. Active fractions were triglycerides, a free sterol-fatty acid mixture, and phospholipids. Triglycerides were stronger stimulants for Locusta than for Schistocerca. The influence of deterrents in determining the feeding pattern of insects has been emphasized in many of the preceding examples, but Kennedy (86) seemed to feel that a more complete understanding of maxillary input to an insect's central nervous system was required. He referred to the suggestion of Waldbauer and Fraenkel (178) that maxillary palps may spontaneously provide inhibitory inputs which must be overcome be the presence of adequate positive stimuli. In agricultural entomology, it is not entirely a matter of host selection between different plant Species that demands our concern. The real consideration often is preference between varieties of a crop (86). In these instances, it seems very likely, based on the foregoing behavior— al and electrophysiological studies and others, that selection would be a net—effect result, produced by both positive and negative stimuli. Several authors have subscribed to this opinion (11, 32, 73, 93, 95, 105, 144, 174. MATERIALS AND METHODS I. Beetles The cereal leaf beetles used in this study were provided by the Entomology and Small Grains Laboratory of the USDA, ARS at East Lansing, Michigan. They were reared under the regime described by Connin §L_gl, (25). Adults, newly emerged from the pupal cell, were collected at about 5:00 p.m. daily. They were maintained overnight under plastic refrigerator boxes (fewer than 200 per box) inverted over, but separated from a clean glass plate by a piece of nylon screen. An 11 cm circle of Whatman No. 2 paper was placed on the screen beneath the box and a slight excess of distilled-deionized water was applied to the box-paper inter- face at 5:30 p.m. and again at 9:45 a.m. the following day. A 75-watt incandescent bulb controlled by a 24-hour automatic timer from Sears, Roebuck and Co. (model 796.6445) was used to provide a 16 hour photophase when room lights were off overnight. Normal fluorescent lighting prevailed during the daytime. The CLB were laboratory reared from about mid-October to about mid- July. The breeding stock was renewed with field-collected, pre-diapause adults each July. A number of bioassays were conducted with these field- collected beetles, and, when used, the results have been clearly distin— guished from those employing cultured beetles. 23 24 II. Plants A. Barley (Hordeum_vulgare L., cultivar 'Lakeland') All seeds and greenhouse facilities were supplied by the USDA, ARS, Entomology and Small Grains Laboratory. Approximately 60 barley seeds were sown per plastic pot (3.5 in. diam.) containing a mixed soil of three parts field soil, three parts peat, and one part sand, which had been sterilized for two hours by injected steam. In the greenhouse, the pots were held under a 16 hour daily photophase at 17°/21° day/night temperature. The seedlings were taken to the laboratory when the second leaf was ca. 1.0-1.5 cm in length. All harvests were made at 11:00 a.m. F 5 min., just prior to extraction. 8. Pea (Pisum sativum L., cultivar 'Yellow Wonder') Five seeds were planted per pot (3.5 in. diam.) and grown under the same conditions as the barley. The seedlings were removed when 6-8 cm in height and harvested just prior to extraction. Identical procedures were used to fractionate the barley and pea plants. III. The Bioassay A. Physical Considerations The original report of a bioassay for CLB feeding responses was that by Panella gL_§l, (129). The present bioassay was derived from that bioassay through various modifications. Each experiment in this study was composed of one or more treatments. A treatment consisted of an extracted plant fraction after it had been incorporated into an agar medium at a known concentration. Some fractions were too light for weight determination and have been recorded as weightless. Samples were taken 25 from these treatments to provide test units which became the immediate source of all bioassay data. To prepare a treatment, 1.5 g of Bacto Agar (Difco Labs., Detroit, Mich.) was added to 52 ml of distilled-deionized water at about 90°. While on the hot plate, the test fraction was added in solution to the boiling agar. Two ml of water were assumed to vaporize during preparation and cooling of the agar medium so that the final weight, less the added fraction, was considered to be 51.5 g per treatment. If the test solution were aqueous, an equivalent volume of water was deleted from the agar to maintain the treatment weight Of 51.5 9. When organic solvents were used, intermittent heating and stirring was used until no solvent odor could be detected. The hot treatments were poured into glass Petrie dishes (15x100 mm) and immediately covered with the top reversed to prevent condensation from collecting on the inside of the cover. After the agar had cooled, the covers were positioned normally and the dishes regrigerated at 4°-6°. Treatments generally were made up two or three hours before using, occasion— ally on the evening before. Control agar was prepared by adding the pure solvent equal to the greatest volume of test solvent used in that particu- lar experiment. A test unit consisted Of a plastic Petrie dish (13x90 mm) inside of which two agar strips (ca. 6.5x0.15x0.6 cm) were positioned in the center to form a closed elliptical circle. Generally, one agar strip was a control and the other was a test strip, although two strips of the same treatment were sometimes used if deemed desirable. Three ”X" marks were applied with a black marking pen to the lower exterior of the plastic dish 26 and the test strip was placed over these marks. This was done just before the bioassay was begun for the day. The bioassay was started when 25 newly emerged, unfed, unsexed, adult CLB were placed in the center of the agar ellipse and the cover positioned. All test units were placed in a shallow cardboard box (capacity of 6 test units) and the box closed and sealed by a weight lengthwise along the cover seam. The lights of the windowless room were turned off and the doors closed. Room temperature was approximately 23°. B. Temporal Considerations The bioassays were started daily at 12:50 p.m. * 5 min. At the end of each hour for three consecutive hours, a tally was made of beetles with at least the head in contact with the control or the test strip. Counts were recorded separately for the test and control strips. The first count was made in the darkened room by light from a 25- watt bulb in a dark—room lamp equipped with a Kodak Safelight No. 2 filter which passed only light above 640 nm. The CLB is attracted by white light, and Wigglesworth (188) pointed out that such insects are less responsive to longer wavelengths than to those approaching the ultra- violet. The remaining two counts were quickly made in normal room light followed by a return of the room to darkness. C. Experimental Design Each experiment followed one of three bioassay formats: a) several concentrations of a single fraction, b) a comparison of different fractions at one or more concentrations, c) two or more fractions combined at various concentrations. At the outset of the study, data were collected from five or ten completely randomized test units (reps) per day from a single 27 concentration (treatment) of crude extract. Several concentrations of crude extract were tested in this manner. Subsequently, a randomized complete block design was employed where several different treatments were bioassayed with one test unit per day over a four day period (four reps). When possible, two control strips were included as a test unit in an experiment. Due to daily fluctuations in the number of beetles available, por— tions of an experiment were now and then deleted after they were initiated. Occasionally, whole experiments had to be abandoned for that reason. Only treatments with three or more replicates were given serious consideration during data interpretation. 0. Treatment Cgmposition The composition of a treatment agar took one of three general forms: a) The test fraction only was admixed with the agar. b) The test fraction was agumented with sucrose in the agar. c) The test fraction was combined with another fraction from barley or pea plants or commercially obtained biochemicals in the agar. E. Scoring Two methods were used to rate the response in each bioassay. To obtain a numerical response index for a treatment, the average hourly count of beetles on a test strip was calculated, based on the total number of hourly counts made over the duration of the experiment. If two identical test strips were used for an experiment, the sum of beetles responding to both strips was divided by twice the total number of hourly counts. At the end of each day's test, a visual analysis of damage to the agar was made using a dissecting microscope to provide a second estimate 28 of feeding response. The strips were then rated as 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). Four daily values for the control and the test strips were averaged by treatment to give each an activity index. F. Validity of the Bioassay To demonstrate the reliability of the bioassay, extracts of barley seedlings were compared to an extract of several non—host plants. The latter, except pea plants, were obtained from an organic gardener and had not been treated with any type of pesticide. The fresh leaves were frozen within 10 minutes of harvesting the entire plant. All extractions were in benzene:methanol (1:1, v/v) followed by a second extraction in 25 ml distilled-deionized water. Ten replicates of each test were bioassayed in completely randomized tests. No visual analysis was performed. Under certain circumstances, deviations from the normal procedure detailed above were made. Departures such as incomplete experiments, altered number of beetles used, and other experimental details are noted in the RESULTS in appropriate tables or figures. IV. Isolation of Plant Biochemicals In the following procedures, all organic solvents used were analy- tical grade which had been glass-distilled. Methanol at 0.5% was added to redistilled chloroform as a stabilizer. All water was distilled, then deionized by passage through a bed of charcoal followed by a mixed resin bed (Barnstead, Boston, Mass.). Each fraction obtained was weighed until its series of bioassays had been completed after which weights were not taken to conserve material for subfraction bioassays. Extracts in 29 organic solvents were stored under nitrogen below O’until analyzed. Aqueous samples were refrigerated at 4°-6°. A. Crude Extract A sample of seedling barley (10-12 g) was weighed to the third decimal place and then homogenized for two minutes in a blendor with isopropanol (120 ml) to deactivate lipolytic enzymes (82). The homogenate was filtered through Whatman No. 1 paper and 250 ug of butylated hydroxy- toluene added as an antioxidant (81). Successively, the residue plus filter paper were extracted with 120 ml of chloroformzmethanol, 1:1 (v/v) for two minutes (83), then chloroformzmethanol, 2:1 (v/v) for two minutes (24). The three combined extracts were filtered through a “c“ sintered glass funnel and taken to near dryness jg_!§gg9_on a BUCHI Rotovapor-R at 30°-35°. Compounds were dissolved in 25 ml of chloroformzmethanol, 2:1 (v/v). This fraction was the crude extract. B. The Hydrophobic Fraction l. Isolating the Total Fraction The above method did not provide for separation of the hydrophobic compounds from the hydrophilic compounds. A variation of procedure IV.A was therefore developed. At the third homogenization, the system, chloroformzmethanol:water, 4:2:1 (v/v/v), was used (120 ml) and a fourth extraction with methanol:water, 1:1 (v/v) followed. To these combined extractions in a 1 L. separatory funnel, ca. 150 m1 of chloroform were added to develop two phases. The lower organic layer was removed to another separatory funnel where it was washed three times with 40 ml of water and these washes were added to the aqueous layer. Repeated chloroform washes of the aqueous were made and combined in a 30 third separatory funnel and backwashed with 2x20nfl water washes which were discarded. Both chloroform washes were then combined. The organic and aqueous phases were concentrated jg_gggg9_at 30°-35° and 40°-45° respec— tively. In addition to the procedure above, a column of Sephadex G-25 (Pharmacia Fine Chemicals, Inc., Piscataway, N. J.) was used to separate the hydrophobic and hydrophilic compounds (190). Chloroformzmethanol: water (200 m1, 100 ml, 75 ml, respectively) were mixed in a separatory funnel. Two phases were formed and collected separately into aluminum foil-wrapped Erlenmeyer flasks which were then stoppered. Part Of the upper phase was used to imbibe the gel. The swollen gel was then pipetted into a column to make a bed Of 1x5 cm. Approximately 50 m of the lower phase were used to displace the upper phase from around the gel beads in the column and to equilibrate with the upper phase within the beads before applying the crude extract. Aliquots totalling 8 ml of the lower phase were used to take up the soluble materials from the concentrated extract in the near-dry state and to filter them through a "c" sintered glass funnel. This 8 ml of extract was applied to the Sephadex column and rinsed onto it with 2 ml followed by 30 ml of lower phase to remove the hydrophobic compounds. Hydrophilic materials had partitioned within the beads, giving a yellow color to the top of the bed, and were eluted with 30 ml of the upper phase. 2. Isolating Hydrophobic Subfractions a. Epicuticular Wax (1) Total Wax This external group of hydrophobic compounds was isolated by a Simple chloroform wash (92), but two approaches were used: (1) If waxes 31 were to be bioassayed, the seedlings were sealed in their pots with plas- ter of Paris. The pots were inverted and Shaken to dislodge loose dirt and the seedlings were immersed and swirled in chloroform twice for periods of 3-5 seconds. The plants were discarded and the chloroform concentrated ig_ygggg at 30°-35° to produce a clean, white wax solution- suspension. (2) When hydrophobic compounds other than epicuticular waxes were to be bioassayed, the wax was removed from harvested seedlings by two 10 second washes in separate chloroform baths. The dewaxed seed- lings were then extracted for further separations described later. (2) Wax Fractions von Wettstein-Knowles (185, 186) and Jackson (76) have reported on the composition of barley surface waxes. Tulloch and Weenink (171) and Tulloch and Hoffman (169, 170) have reported on wheat, Triticum compactum Host., oats, A, Snggg, and rye, S, cereale. Surface waxes of these species showed qualitative similarity to barley, thus, isolation of barley wax components was first based on the preliminary siliCic acid column separation described by Tulloch and Weenink (171). Efficiency of the column separation was monitored by thin—layer Chromatography (TLC) on 5x20 cm silica gel G F—254 plates (Merck) devel- oped in chloroform and visualized with iodine vapor and then 50% H2S04 at 110°. Modification of initial procedures was required which resulted in three different columns being used. The conditions of each column are presented below and the separations achieved are compared in Table 3 to those reported by Tulloch and Weenink (171) for I, compactum. Column 1. A hexane slurry of 6 ml of unactivated Unisil (Clarkson Chemical Co., Williamsport, Pa.) was poured into a 1.8 cm (i.d.) glass column having a frittered glass drip tip. A wax sample of 55 mg was 32 applied as a hexane suspension. Elution was performed as described in Table 3. A yellow color appeared at the column top as development progressed. This colored portion was eluted and collected separately in the last 62 m1 of the hexanezchloroform, 4:1 (v/v) eluent. The color was shown to be a contaminant of the distilled hexane as described under Column 2. Column 2. A Slurry of 6 ml of Unisil, activated at 130° for 5 hours was poured into a 1.8 cm (i.d.) glass column fitted with a fritted glass drip tip. A wax sample of 45 mg was applied in hexane. The elutropic series Shown in Table 3 was employed. To determine if the yellow color eluting from the first two columns was a contaminant of the hexane, 250 ml of the distilled hexane were con- centrated to ca. 1-2 ml and subjected to TLC in chloroform. Several spots appeared in iodine vapor and with 50% H2S04 spray. These spots correspond- ed with those found in the yellow fractions of columns 1 and 2. It was found that passing hexane through a Short column of Unisil would remove these impurities. Silverstein gL_§l, (151) confirm that aromatic impuri- ties can be removed from hexane by silicic acid. Hexane purified in this manner was used in succeeding separations. Column 3. A slurry of 12 ml of Unisil activated for 17 hours at 120° was poured in hexane into a 1.8 cm (i.d.) glass column fitted with a fritted glass drip tip. A wax sample of 112 mg was applied in hexane. The elution series shown in Table 3 was used. The epicuticular alcohols were identified as a class from their blue color reaction after TLC when visualized with a vanillin-sulfuric acid spray at 110°. This chromogenic reagent was prepared by dissolving 1.0 g vanillin in 100 m1 conc. H2504 (94). At a later time, co-chromato- graphy with a standard alcohol, l—tetracosanol, (Applied Science 33 Table 3. THIN-LAYER CHROMATOGRAPHIC COMPARISON OF ELUTION SERIES USED TO SEPARATE EPICUTICULAR WAXES 0F BARLEY AND WHEAT LEAVES 0N SILICIC ACID COLUMNS. Eluents Column Number and Type Activated Non-Activated . . . Biosil Aa Unisil Act1vated Un1s1l None 1 2 3 Rf Values and Compositionb Hexane 0.74 (HC) 0.67 (HC, Es) 0.72 (HC) 0.72 (HC) 0.52 (Ca) 250 ml 375 ml 100 m1 Hex:Chl. Tube No.C 7:1 N.U. N.U. N.U. Blank - 0.72 (Es) 4-5 100 ml 5:1 N.U. N.U. 0.67 (Es) 0.70 (Es) 1-3 0.55 (Ca) 0.55 (Ca) 112 ml 200 m1 4:1 N.U. 0.17 (A1)d 0.16 (A1)d 0.72(tr.Es)d’e 125 ml 120 ml O.55(tr.Ca) 0.20 (A1) 300 ml 4:1 N.U. 0.14 (Al)f 0.16 (A1)f N.U. 62 ml 120 ml 10-20% Chl. in Hex. 0.70 (Es) N.U. N.U. N.U. 1.5:l N.U. N.U. N.U. 0.03-0.17 Streak 200 ml 1:1 0.45 (fl-Dik) 0.15 (A1) 0.16 (Al) N.U. 100 ml 0.00 (Ac) 160 ml Chl. 0.15 (A1) 0.15 (Al) 0.00 (Ac) 0.21 (A1) 0.00 (Ac) 200 ml 0.06 (?) 100 ml 0.00 250 ml 20% Ethanol in Chl. 0.06 (0H-fl-Dik)0.00-0.03 (?) 0.00 (Ac) 0.21 (A1) 0 00 (Ac) 100 ml 100 ml 0 0 (Ac) 34 Table 3. (Cont'd.) aInitial procedure and results reported by Tullock and Weenink (171) for I, compactum. bIdentification of barley wax composition as described in MATERIALS AND METHODS and by comparison with data of Tulloch and Weenink (171). Development in chloroform on silica gel F-254 plates from Merck (Brink- man Instruments, Ins.) C20 ml collected per tube. dThis fraction did not contain yellow impurities from hexane. e . . . . . . . . . Hexane 1mpur1t1es removed by s111c1c ac1d pr1or to use in column No. 3. fThis fraction contained the yellow impurities from hexane. Hex. (hexane); Chl. (chloroform); HC (hydrocarbons); Es (esters); Al (alcohol); Dik (diketone); Ca (carbonyl); Ac (acids); tr (trace amount); N.U. (not used) 35 Laboratories, State College, Pa.) confirmed the identity. To verify that the spot at Rf 0.67 in the hexane eluent of Column 1 contained esters along with hydrocarbons, a pair not always separated by TLC in chloroform (171), an aliquot of the hexane fraction was placed in a test tube with ca. 10 ml methanol and 10-15 mg of NaOH. This tube was placed into boiling water for 30 minutes and methanol was added to counter vaporization losses. TLC of this saponification milieu along with an untreated sample showed that the spot at 0.67 had largely disappeared from the treated sample. However, spots at Rf 0.18 and the origin appeared in the treated sample Where none were found in the untreated sample. This indicated that alcohols and acids, respectively, had been freed from their esterified form. Treatment with acetyl chloride (39) of a spot from the chloroform fraction of Column 1 on a TLC plate followed by development in chloroform along with an untreated spot, caused the spots at 0.16 and the origin to move to a higher Rf value. The alcohol had been esterified and the origin presumably contained free acids which migrated as acid anhydrides after the treatment. The Nilles and Schuetz (124) table of solvent properties indicated that an acid anhydride may be less polar than free acids, and, therefore, could rise off the origin in chloroform. To establish the chromatographic behavior of the carbonyl reported by Jackson (76) for barley wax, a sample of whole wax was treated with Girard's "T” reagent (Fisher Chemical, Fairlawn, N. J.) according to Fieser and Fieser (40). Less than 10 mg of sample were combined with 0.5 g of the "T" reagent and 0.5 ml of conc. acetic acid in 5 ml of 95% ethanol. Following a 30 minute reflux, the mixture was transferred to a separatory funnel. Ethyl ether and saturated NaCl, 5 ml each, were 36 added. As revealed by TLC of the ether phase, the carbonyls had been derivatized and had partitioned into the water. The spot seen at Rf 0.52 remained in the untreated sample. In some instances, whole waxes were separated into fractions by preparative TLC on silica gel H plates having a 500"coating (Prekotes from Applied Science, Ann Arbor, Mich.). Approximately 20 mg of wax were applied in chloroform and the plates developed in chloroform. The desired fractions were: alcohols; hydrocarbons, esters and carbonyls; and acids plus other. The alcohol band was located by applying vanillin- sulfuric acid reagent along one edge of the developed plate and heating only that edge on a hot plate to produce a blue color in the alcohol zone. The respective zones were eluted from the silica gel with chloroform: methanol, 2:1 (v/v) in centrifuge tubes which were then spun at 1,000xg for five minutes to sediment the silica gel. Three such elutions were given to each fraction. b. Hydrophobic Compounds Minus Wax, (H—W) Before extraction, the surface wax was removed from barley seed- lings as described in section IV.A.2.a.(l)(b). The crude extract was then passed through the Sephadex column described in section IV.B.1. (1) Separation into Polar and Apolar Fractions A slurry of Unisil in chloroform was degassed by water vacuum and poured into a 1.4 cm (i.d.) glass column fitted with a fritted glass drip tip. Approximately 1 ml of silicic acid was used per 10 mg of (H-W) applied and ca. 20 ml of chloroform were passed through the column before addition of the sample. The (H-W) apolar fraction was eluted with chloro- form until the dark green pigments were eluted and a Slower moving yellow band neared the bottom of the column. Methanol, 100 ml, was then used to elute the polar materials. 37 (2) Separation of the Polar Fraction The polar lipids of plants are comprised chiefly of glycolipids and phospholipids (122). The system used to separate these two major groups was that described by Rouser gL_§1, (136). A 10 9 portion of Unisil (200-325 mesh) and later, Adsorbosil-CAB (200-250 mesh) from Applied Science, was slurried in chloroform into a 2.0 cm (i.d.) column fitted with a teflon stopcock. Elution was by chloroform, 80-100 ml, to remove apolar materials; acetone, 300 ml, to elute glycolipids, and methanol, 200 ml, to remove phospholipids. It was noted that the phospho- lipids contained an unknown compound later identified as gramine (see RESULTS, section IV.F.) which reacted with the vanillin-sulfuric acid reagent at room temperature to produce a light pink color and at 120 to produce a purple color. The phospholipids were bioassayed with this compound until a method could be devised to remove it. Separations were monitored by TLC on 5x20 cm Merck Pre-Coated Sili- ca gel F-254 plates (250”coating) (Brinkman Instruments, Inc., Westbury N. Y.) developed in chloroform:methanol:7N ammonium hydroxide, 60:35:5, (v/v/v) (Skidmore and Enteman, 1962). Phospholipids were identified by reaction with a phosphomolybdic acid spray (Applied Science) to produce a blue color at room temperature. Glycolipids, mono- and di-galactosyldi- glycerides and sulfolipid, were visualized by the vanillin-sulfuric acid spray with which they produced a light red color at room temperature. (a) Isolation of Individual Glycolipids Preparative TLC was used to obtain purified glycolipids which were identified by co-chromatography with standards from Applied Science. The 2 TLC plates were 20x20 cm , Silica gel H Prekotes (500/‘coating). Approxi- mately 20 mg of glycolipid were applied to each plate followed by 38 development in the system of Skidmore and Enteman (153). A 1.5 cm strip of coating was isolated along one edge of the developed plate with a pen- cil point and the vanillin-sulfuric acid reagent applied with a Pasteur pipett. Selective heating of this strip with a hot plate disclosed the location of the desired glycolipid classes. These bands were carefully scraped and eluted as described for wax fractions (section IV.B.2.a(2).). (b) Isolation of Neutral and Acidic Phospholipids Cellex D, a diethylaminoethyl cellulose anion exchanger (DEAE) from Bio-Rad Laboratories (Richmond, Calif.) was prepared according to Rouser §L_gl, (135). The fine particles were first decanted several times from an aqueous suspension. The cellulose was washed 3 times in a BUchner funnel. Each wash consisted of 1N HCl (100 ml), deionized water until neutral, O.lN KOH, and deionized water until neutral. Following further washes of acetone, methanol and then ethanol, the cellulose was oven dried at 100° to a constant weight. A 7.5 9 portion of cleaned and dried Cellex D was mixed with excess conc. acetic acid and left overnight with stirring by a magnetic rod to remove the clumps. The resultant slurry was carefully poured down a glass rod into a 2.0 cm (i.d.) glass column fitted with a teflon stopcock. The final bed was ca. 20-22 cm high. After eluting the excess acetic acid with 100 ml of methanol at 3 ml per minute, mixtures of chloroform/methanol were passed through the column with gradual enrichment in chloroform until the solvent ratio was reached in which the phospholipid sample would be applied. To separate the neutral from the acidic phospholipids, the column was stabilized in chloroform:ethanol, 2:1 (v/v). 39 Following the scheme of Rouser gL_gl, (135), all of the neutral phospholipids (phosphatidyl choline, phosphatidyl ethanolamine, lysophos— phatidyl choline and lysophosphatidyl ethanolamine) were eluted with 200 ml of chloroform:methanol, 2:1. Next followed 100 ml of methanol to remove acetates and other salts in the sample. TLC of this fraction showed only traces of phospholipid and it was routinely discarded. Acidic phospholipids were generally not identified individually, although there was some evidence for the presence of phosphatidyl serine. As a group, the acidic phospholipids were eluted with 200 m1 of chloroform:methanol, 4:1 (v/v) that was 0.05M in ammonium acetate. Another 100 ml of methanol were passed through the column and discarded, thereby removing ammonium acetate from the column. The column was then reactivated with 60 m1 of conc. acetic acid. Excess acetic acid was eluted with 100 m1 methanol which then served to maintain the column until it was prepared for the next separation. Fractions were concentrated jfl_y§ggg_at 30°-35° and taken up in 2.0, 5.0, or 10.0 ml of chloroform. To prepare the ammonium acetate solution for acidic phospholipid elution, 4 ml of 28% ammonia (concentrated NH4OH) were added to the chloroform:methanol, 4:1 followed by 0.6 g conc. acetic acid. The NH4OH was first filtered with a Millipore filtering system (Gellman filter pad, Metricel, Type VM-l, 5.0 ) to remove crystalline impurities. With the possible exception of phosphatidyl serine, discussed later in this section, the acidic phospholipids were not eluted individu— ally from the DEAE column, nor were they further isolated by preparative TLC. Four of the first silicic acid columns used to separate glycolipids from phospholipids were smaller than those described in section IV.B.2.b. (2), with the result that a glycolipid having the chromatographic (TLC and 4O DEAE anion exchange) and the chromogenic reaction (vanillin-sulfuric acid) of the sulfolipid was a contaminant of the acid phospholipids derived from those columns. These phospholipids were bioassayed with the contaminant present and the results interpreted with this fact in mind. The ammonium acetate was removed from the acid phospholipids part- ly by means of the Sephadex G-25 column of section IV.B.l. Further salt removal was effected during jg_ggggg_concentration, since ammonium acetate is slightly volatile (135). Repeated addition of chloroform:methanol, 2:1, to the sample aided in salt removal. In some instances, the acid phospholipids were partitioned between chloroform and the upper phase used for the Sephadex G-25 column. Since an exchange of positive metal ions occurs between acid phos- pholipids and silicic acid (123), the possible influence of these ions on CLB behavior in the bioassay was investigated. The acid phospholipids from the DEAE column were partitioned against a saturated NaZ-EDTA solution to exchange sodium ions for those cations present with the phospholipids. In another case, acid phospholipids from two barley samples were combined and one-third remained untreated while two-thirds were washed with a saturated CaCl2 solution to impart a heavy calcium concentration (19). From this two-thirds portion, one-half was further treated with Naz-EDTA to replace the calcium with sodium. Three different classes of ion composition were thus available for bioassay. It is not known for certain whether phosphatidyl serine was found in this study. After TLC in the chloroform:methanol:7N ammonium hydroxide system [section IV.B.2.b.(l)(a)], a ninhydrin positive spot was observed. This same compound eluted from the DEAE column with cone. acetic acid as 41 phosphatidyl serine did according to Rouser §L_Sl, (135). The Rf of that spot in this study (0.14-0.17) was similar to that for phosphatidyl serine reported by Skidmore and Enteman (153) for the same TLC system (0.19). In the RESULTS, this fraction has been called phosphatidyl serine, but is accompanied by a question mark (?). Benson and Mauro (15) did not find phosphatidyl serine in barley seedlings. The neutral phospholipids were easily eluted from the DEAE column by chloroform:methanol, 2:1. It was in this fraction that gramine also eluted. (c) Isolation of Individual Neutral Phospholipids The elution scheme presented by Rouser gL_Sl, (137) to isolate individual neutral phospholipids from a DEAE column was seldom totally successful in this study. Even after considerable modification, some degree of fraction overlap usually resulted. Since several different ratios of chloroform:methanol were used from one DEAE column to the next, and given that eight columns were developed in the attempt to isolate in- dividual neutral phospholipids, the qualitative results of these separa- tions are presented only with the tabulation Of results of their bioassay. As a last resort, preparative TLC was twice used to obtain an indi- vidual class of neutral phospholipid. The concern for oxidation of the lipids as a likely result of TLC prevented a more extensive use of this technique in this area. Another question which seemed open to bioassay was that of the influence of the fatty acid composition in a particular class of phospho— lipid. Three species of phosphatidyl choline were obtained from Applied Science Laboratories. These species were L-<»-l-stearoyl-2-oleoyl leci- thin, L-<»-dilinoleoyl lecithin, and L-<%-distearoyl lecithin. 42 Identification of neutral phospholipids was made through co- chromatography with standards from Applied Science (phosphatidyl choline and phosphatidyl ethanolamine). A ninhydrin reagent (0.3 g ninhydrin in 100 ml of ethanol) aided in identification of the ethanolamine phospho- lipids and Dragendorf's reagent (obtained from Applied Science) was used to visualize the choline phospholipids. (d) Isolation of Gramine Through observation of the chromatographic behavior on silicic acid of the phospholipid contaminant, it was found that much of it trailed the entire phospholipid fraction off silicic acid with the methanol eluent. It could then be collected pure in the last 50 ml of the 200 ml used. It was also found that this unknown could be removed from neutral phospholipids on the DEAE column with 75 ml of chloroform: methanol, 35:1 (v/v), although some phosphatidyl choline tended to elute with it. Infrared data on the unknown was obtained in micropellet form (KBr), using the material eluted pure from the silicic acid column. A Perkin-Elmer 337 Grating Infrared Spectrometer was used. The IR data were compared to those contained in the Spec Finder volume of the Stadtler Index System. The best fitting compound was an indole, 5-amin0-3-(dimethyl- aminomethy1)-indole. Mass spectral data were obtained by a direct probe on a Bell and Howell 21-490 mass spectrometer which was interfaced to a Digital POP 12 computer to provide summarized data for mass and mass intensity. The results indicated a molecular weight of 178 for the unknown. Since an alkaloid seemed a possibility, a check of library references on that topic led to Raffauf's work (132) on alkaloids in plants. Barley, 43 indeed, possessed an alkaloid called gramine, 3-(dimethylaminomethyl)- indole, a derivative of tryptophan (16). A quantity of gramine was obtained from Sigma Chemical Co. and its mass Spectrum was found to be identical to that for the unknown isolated from barley. Similar congru- ence was found between the two IR spectrums; also, the TLC behavior and chromogenic reaction with vanillin-sulfuric acid reagent were alike for commercial and isolated gramine. C. The Hydrophilic Fraction l. Extraction of the Hydrophilic Compounds When bioassays of the complete hydrophilic fraction were conducted, these compounds were obtained by the methods described in section IV.B.l. Another method, outlined in the next section, was used when subfractions of this fraction were bioassayed. 2. Fractionation of the Hydrophilic Compounds Work by Seikel and Geissman (148) demonstrated the presence in barley leaves of a glycoflavone, saponarin. Harborne (55) stated that the leaves of wheat, oats, and barley contained glycoglavones, compounds otherwise rarely found in monocots and therefore, a characteristic chem- ical feature of the Gramineae. Thus, saponarin seemed a likely compound to bioassay as a botanically restricted secondary plant compound. Another barley glycoflavone, lutonarin, was only present in plants grown out-of- doors (147) and was not investigated in this study. Gross extraction of saponarin followed the procedure of Harborne and Hall (56). Approximately 25 g of freshly cut barley leaves were refluxed in 400 ml of methanol for 2 hours. The extract was concentrated to ca. 2 ml jg ygggg_at 50°-55°, then transferred with deionized water and petroleum ether, 8:1 (v/v) to a 50 ml centrifuge tube fitted with a 44 glass stopper. Hydrophobic materials were partitioned into the pet ether followed by centrifugation at 1,000xg for 3 minutes. The result- ing aqueous solution was taken to dryness jg_ygggg_at 45°-50° and taken up in 5 m1 of 10% methanol in water. It was found rather serendipitously that a small column of Sepha- dex G-lO (fine grade) packed in water separated the saponarin quickly from most other hydrophilic materials present due to an adsorption effect which caused it to "hang up" on the column after most other substances had been eluted with water. To construct the column, ca. 10 g of Sepha- dex G-lO beads were swollen in water and poured into a 2.0 cm (i.d.) glass column having a teflon stopcock. The void volume as measured by a run of dextran blue dye was 9.5 ml and total volume (VT) was 25 m1. A 2 ml volume of extract was found to be satisfactory for applica- tion to the column. The first effort was collected as fractions in the volumes: 9.5 ml (void), 1.5 ml, 4.0 ml, 2x5.0 ml, 3x10.0 ml, 7.5 m1, and 62.5 ml. Initial characterization was by paper Chromatography as described by Seikel and Geissman (148). Aliquots from each fraction were spotted onto Whatman No. 3 paper and developed in a mixture of equal volumes of n-butanol and 27% acetic acid in water. Under UV light, (254 nm), patterns of fluorescence were marked on the paper by pencil and the chromatogram exposed to ammonia vapors to visualize the saponarin which turns bright yellow under alkaline conditions (Seikel and Geissman, 1957). Results from this column showed that the second column should be collected in fractions of 25.0 ml (including void volume), 37.5 ml and 75.0 ml. A diagramatic representation of these developed fractions is shown in Figure 1. Later columns may have had different elution volumes, 151. 25 ml next 37ml next 75 ml 065 -> 9 BW PUT, PH- PH- . Figure 1. Diagram of a paper chromatogram of barley seedling hydrophilic compounds eluted from a Sephadex G-10 column. System: Whatman No.3 paper developed in n—butanolz27Z acetic acid, 1:1 (v/V)- D (dark appearance in UV light - 254 nm) BW (blue—white fluoresence in UV light - 254 nm) YL (light yellow in ammonia vapors) HY (deep yellow in ammonia vapors) N: (ninhydrin positive, ninhydrin negative) 46 but the qualitative patterns were Similar. All of the ninhydrin reaction due to amino acids was found in the first 25 m1 (V = 25 ml). The 2nd T and 3rd fractions contained a substance which had an Rf of 0.48 and appeared dark under the UV lamp and bright yellow in ammonia vapor. Above these spots were areas which fluoresced blue-white under UV light. Concentrated samples were dissolved in 10% methanol in water and refrig- erated at 4°-6°. In the 2nd and 3rd fractions, material was seen to precipitate after a few days of refrigeration, an event not seen in the "whole" sample. Rinsing the precipitate in its volumetric flask several times with deionized water removed non-precipitates. The addition of 95% ethanol followed by sonication yielded a sample for UV analysis. The UV scan in 95% ethanol was performed on a Beckman DB-G spectrophotometer using a 1 cm quartz cell. The peaks obtained at 334 nm and 272 nm agreed with those reported by Seikel and Geissman (148) for saponarin. The precipitate also gave the bright yellow reaction with ammonia vapor on paper. This precitatate was bioassayed as indicated in RESULTS. Analysis of an aliquot of the total methanol extract for gramine was accomplished by the method of Audette gL_gl, (10). The extract was adjusted to pH 10 with KOH (2N), then extracted with chloroform into which gramine would partition. The test for gramine was positive. Subse- quent TLC of the fractions from Sephadex G-10 on silica gel G in the system of Skidmore and Enteman (153) Showed that gramine was predominantly in fraction No. 2, light in No. 3 and absent from NO. 1. A similar examination of preserved hydrophilic materials obtained as in section IV.B.l also revealed gramine to be present and, thus, probably present in the hydrophilic fractions bioassayed from other extracted samples. 47 An aniline-phosphoric reagent (20), was used to locate sugars in the fractions. TLC of these along with a sucrose standard showed that sugars were present only in fraction NO. 1. The elution volumes and general composition of each fraction in the bioassays conducted have been included in the tabulated bioassay results. To fractionate the first 25 m1 sample from the Sephadex G-10 column, a Dowex 50-X12 cation exchange resin, 40-80 mesh, was used (Bio. Rad). A column of the resin (l.4x6.0 cm) was converted to the H+ form by 10 ml of 2N HCl, then washed to neutrality with deionized water. The concentrated sample (5 ml) from the Sephadex column was applied to the Dowex-50 column and 30 ml of deionized water was used to elute neutral and anionic compounds. A 50 ml volume of 10% NH40H was used next to elute cations. Paper chromatography in the respective systems described, showed nearly all of the ninhydrin reaction to be with the basic fraction, but a faint ninhydrin reaction was seen in the neutral-anionic fraction. This latter ninhydrin positive zone was also positive to a sugar detecting reagent (aniline-phosphoric acid). The identity of the compound(s) was not established. 3. Non-Extracted Hydrophilic Compounds Another approach to the elucidation of CLB feeding stimulants involved the bioassay of nutrient type chemicals available as shelf chemi- cals in the laboratory. Such compounds as sucrose, and various amino acids were tested in several concentrations and combinations. Synthetic mixtures of amino acids corresponding to those reported by Fauconneau (38) for a CLB host, orchard grass, Dactylis glomerata L. and a non-host, alfalfa, Medicago sativa L., were bioassayed at different concentrations. Though not a nutrient, indole-3-acetic acid was also bioassayed. RESULTS 1. Validity of the Bioassay The CLB did not respond to crude extracts of non-host plants when an extract of barley was present. Nor were these non-host extracts effective stimulants in the presence of a blank agar strip (Table 4). II. Crude Extract The crude extract was bioassayed to gain experience with the bio- assay and to otbain an estimate of the range of subfraction concentrations to be used in later experiments. Figure 2 summarizes the data from the completely randomized experiments where one concentration at a time was studied. Figure 3 represents subsequent randomized complete block experi- ments where several concentrations were bioassayed at once, one test unit per day over four days. Computer analysis of the data produced a series of coefficients for polynomial equations up through the 5th degree. A 4th degree and a 5th degree equation provided the best fit for the data in Figure 2 and Figure 3 respectively. The responses to all controls for analysis of the crude extract have been summarized in the lowermost curve of Figure 3. This curve was fitted by eye, and the responses were typical of those obtained throughout the remainder of the study for the control strips. Consequent- ly, no other count data for controls has been presented graphically. 48 49 Table 4. RESPONSE OF FIELD-COLLECTED, ADULT CEREAL LEAF BEETLES TO CRUDE EXTRACT OF BARLEY AND NON-HOST PLANTS INCORPORATED INTO THREE PER CENT AGAR*. Test Unit Combination PPM Countsa Barley (S, vulgare) 7969.0 6.3 Peppzr (Capsicum annum L.) 9211.0 0.5 Blank 0.0 1.8 vs _C_. 911.0110. 9211.0 1.5 S, vulgare 7969.0 5.4 Brozzoli (Brassica oleraceae italica L.) 5903.0 1.0 Blank 0.0 0.8 vs S, 9, italica 5903.0 1.3 .A. vulgare 6289.0 3.3 13, :ztivum 7082.0 1.0 S, vulgare 6289.0 3.0 Kohliabi (S, 9, caulo-rapa L.) 5777.0 1.3 A, vulgare 6289.0 3.8 SuniTower (Helianthus annuus L.) 6233.0 0.3 S, vulgare g 4469.0 3.1 Tomzio (Lycopersicon esculentum Mill.) 5648.0 0.1 *25 beetles (fasted 24 hours) per test unit. aAverage number of beetles responding to each strip after one hour. Ten replicates. 50 _.o- N.Fm m.wmmi N.oem No.m u> do» mpmewumw mo Loggm nemucwpm n.m_mi Om.o "cowpmcwELopmo mo xmucH mpcmwuwwwwoo ”cowpmzcm mesoczFOQ mmgmmv sue a An vmnwLUmmu we; pew #mmn mo m>ezu esp .pcmewcwaxm some :04 mmFwan mo meowpm>gwmno z—Lso; 03p gone emmme>m mew; mpcsoo .pwcs pmmu Lea mmemmn venom; >L0pwgoan mm ”A o v mmpmowramg m go A o V mmpmovFQmL m mo mpcwevewaxm umeEovceL armprQEou mo mmegmm a Eoew empow__ou mam; open .H - Lame pcmo emu wean“ cw pomeuxm «cage ace—uwmm xmrimn mo :owpmeucmocoo esp ow monmon meoF Femiwo szue .cww:: .uwmemem xrzo: mo wmcoamme FmowLmE:z .m wizard M HGURE2 11111 I 1000 l l l l 100 11111 l l l l l l L l l l I l l 10 ‘l 03 N 10 I") '— dlUlS 1531 213d $311339 :10 'ON '97“! LOG PPM EXTRACT 52 .mxm >3 umppwm >Paewm mm; mpmn Focucoo esp Low m>c=u wee o.m w.mm a.mom- m.moo ~.mm©1 mo.m “> Low mpmswpmw mo Loggm newncmpm N.omm m¢.o ”cowpmcvsgmpmo mo xmccH . mpcmwowwwmou "cowpmzcm Persochoa madman :pm m an cmnweummn we: come pomguxw esp com “we pmmn mo w>e=o ace .m weaned Cw gmcummOp czocm mam m weaned use N wizard now even Pogpcoo .mxmu Lsom Lm>o awe emu mmemmn mo mcovmm>gmmno x_gso; 03p Eoem cmmmem>m mam; mpczou .xmu Lea cowpmgpcmucou Lea pee: pmmp mco ”peg: pmmp Lea mappwwn coded; zeopegonw— mm "mpcwewemaxm xuo_n mmeanu umeEoucmL mo mwvemm m seem cmpom__oo mam: memo .HH - Lema ucmu emu mmcsp c? pumgpxm musno mcwycwwm am—Lmn mo cowpmgpcwocoo mcp op mmemmn mam? megmo sznm .nmmc: .ummLmEm zpzm: mo mmcoamme Fmowcmszz .m mcsmwm 53 hum_mmn -.m ummgpxm mcwpummm emu -.m .Apcmmeuxmv m .Aeoom Amm>v m .Auoom marscv e .Auoomv m .Amwmmv N .Apzmw_v "Fin .n pcmswcmuxm Low mmmoom x—wmu meow seem use ._-m .m pcmewmmuxm com mmmoom mmmEmu Lema upwmn 03p Eomm vmmmmm>m we; Apcmvm pmmp .pmmF Fomucoo mmmmmgucmmmu crv Nuw>wpm< '—— .mxmu Luow Lm>o zen Lmu mcovpm>mmmno >szo; mmmgp Eomm ummmmm>e mum; mpcuoo .xmv emu cowpmmpcmucou emu pen: pmmp mco News: pmmp emu mmemma vmmmmm Amopmmoan mm "mpcme -wmmuxm xuopn mumFquu nvaEoucmm 03» Scam umcwmpao memo .mmmm pcmu emu mmcgp cw .umcwnEom new mpmmmumm .pommpxm muzmo m:w_nmmm emu ecu xmrmmu mo cowpmmucmucoo mgp op mmrpmmn mum? Pmmmmu prawn .cmmcmEm xpzm: mo mmcoummm .e mmumwu 57 .—U<~:xm SE a 00.. 000. 00. o. _ ________ _ :2____ _ 5.0.6.9 .1 m. .\61.w..o.9 mofiflf/ J _ .. $3.9 . 1 2.0.0.9 .. m .. m a.mmoe 1 .. N. 86.9 L m #550: I: (11815 1531 213d $311338 :10 'ON '91“! 58 attractive power of the combined extracts was enhanced at a 1:1 ratio over that of the pea extract alone, based on the higher CLB counts on the mixtures. III. Hydrophobic Compounds VS. Hydrophilic Compounds Several bioassays of the hydrophobic and hydrophilic fraction each were performed. The data were analyzed by computer to derive coefficients for the polynomial equation of best fit (Figure 5). Greater sensitivity was shown by the beetles to the hydrophobic materials at the lower concen- trations (up to 300 ppm), but the average count and activity relating to the hydrophilic compounds increased rapidly beyond this point to approxi- mate equality with the hydrophobic fraction. Selected activity scores for control and test strips are given in parentheses. Based on the data of Figure 5, it was decided to emphasize the determination of hydrophobic feeding stimulants. A similar bioassay for pea plants was performed for only the hydro- philic compounds (Table 6) because bioassays, to be reported later, indicated that the major deterrence of the pea extract came with the hydrophobic materials. The values for both count and feeding activity were lower for pea hydrophilic compounds than for Similar concentrations of barley hydrophilic materials, but it was Clear that the beetles fed upon this fraction from pea plants. To determine the relative deterrent influence of the hydrophobic and hydrophilic fractions of pea seedlings to CLB feeding, a test was made. A 485 ppm portion of barley crude extract was added to 388 ppm of pea crude extract, to 227 ppm of pea hydrophobic compounds, to 161 ppm of pea hydrophilic compounds (each equivalent to 333 ppm of whole crude 59 mm.p H> eom mpmswpmm mo eoeem uemmcmpm Nm.o “cowpmceEempmo mo xmucH mesmeueeemou "coepmzcm Feweocxrou mmemmc vem m An empreUmmu we; muczoqum m__w;uoeu>; mew eoe ewe ummn mo m>e=m mew mm.e ”e e6 mpeseemm e6 eoeem eeeeeaem oe.o "cowmmcweemmmo mo xmucH mesmeuvmemou "cowumzcm Feweocxpou mmemmu new m >3 nmneemmmu mm; mecuoueou menozuoenzz mew eoe “we pmmn mo m>e=o mge .ApcmFquxmv o .Acoom xem>v m .Auoom mpwzcv e .Avoomv m .Aewmev N .Augme_v P "mmeOUm mmmsme emmm x—wmc euoe Eoee ummmem>m mm; weave pmmp .pemF Foepcoo mmmmmspcmemu cw mm:_m> umpummev pw>wpu< .mxmm esoe em>o xmu emu mmrummn mo meowpm>emmno zeezo; mmeem eoee ummmem>m mezzou .xmu emu cowpmeucmucoo emu “we: ummu mco mews: pmmu emu mmppmmn umemme aeopmeonm_ mN ”mecmseemuxm emopn mpm—qumcw new mmequu umeEoucme Eoee umeem223m mpmo .emmm memo emu mmegp one? vmpmeoueomcw amFemn mo mezzoueou oe—wcuoevxg ecu ownocu -oenx; Fmpom mo coepmepcmucom om mmppmmu emm_ Pmmemu p_:vm .vmmc: .cmmemam >F3m: mo mmcoumma .m mesmeu 60 hu<~3xm SE a 00.. 000. 00. o. _ _ Z. _ _ _ _ _ _ q: _ _ a _ _ _ 03.503»: . m. 1 _ AN.N.N.OVo I . a.m.oQ N. NONE. 1 m 8m .09. a.m..mé a.m.movo o 0 3b .59 Améfii .... m U_mOIuO¢o>I m 2.30.“.— l __ 'SAV dllliS 1831 213:! 5311338 :10 'ON 61 Table 6. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO PEA SEEDLING HYDROPHILIC COMPOUNDS IN THREE PER CENT AGAR*. Concentration of Average Countsa Average Activityb Extract 7 ppm Control Test Control Test 0.0 0.7 1.3 0.7 0.9 0.0 1.8 0.8 1.5 0.6 291.0 1.2 1.7 0.4 1.8 1164.0 1.0 2.5 0.2 1.3 1164.0 1.0 3.4 0.5 3.6 1746.0 0.7 2.2 0.2 2.0 1746 0.6 4.4 0.6 2.6 *25 laboratory reared beetles per test unit; one test unit per treatment per day; one control and one test agar strip per test unit. 6Average of three hourly counts of beetles per day over four days. bAverage of four daily agar damage scores: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). 62 extract), and to both fractions recombined. Activity was compared to a blank test and to 485 ppm of barley crude extract in agar. The results (Table 7), confirmed that the hydrophilic materials were not the major source of deterrents detected in pea extract by the CLB. Yet, there was some indication that they did possess a small degree of deterrence, although this matter was not investigated further. Again, it was found that barley crude extract partially overcame the effect of pea crude extract when both were combined at approximately a 1:1 ratio. IV. Hydrophobic Compounds A. Complete Epicuticular Wax Data from five experiments, some incomplete due to a lack of beetles, are presented in Table 8. These data revealed that feeding activity was stimulated by barley epitucular wax, but no consistent dose response was found when either average counts or average activity were considered. Further evidence that the barley epicuticular wax was involved in the CLB feeding response is presented in Figure 6. After removal of the wax, the resulting hydrophobic compounds minus wax, (H-W), were extracted and bioassayed with the wax readded at 0.0, 10.0 or 16.0%. The epicuticu- lar wax amounted to 15.3% of the total barley hydrophobic compounds. Both epicuticular wax and other hydrophobic factors clearly were CLB feeding stimulants. Response to the recombined (H-W) and wax fractions was consistently better than that for corresponding (H-W) concentrations alone. Investigation of pea epicuticular wax was also made. Simultaneously, the influence of pea wax upon CLB response to barley crude extract was 63 Table 7. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO BARLEY CRUDE EXTRACT ADDED WITH EXTRACTS OF PEA THREE PER CENT AGAR*. SEEDLINGS TO Barley Extract Pea Extract Average Counta Average Activityb (ppm) (ppm) Control TestC Control Test 0.0 0.0 1.3 1.1 (K) 0.4 0.7 485.0 0.0 1.2 8.0 (0) 0.6 4.6 485.0 R, 161.0 1.1 7.0 (N) 0.1 3.5 485.0 S, 388.0 1.1 3.3 (M) 0.1 1.9 485.0 T, 227.0 0.8 3.8 (M) 0.3 2.8 485.0 R+T=388.0 1.5 2.6 (L) 0.2 1.7 *25 laboratory reared beetles, one control, one test agar unit; one test unit per concentration per day. strip per test aAverage of three hourly counts of beetles per day over four days. bAverage of four daily agar damage scores: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). CMeans sorted by Duncan's MRT. Values opposite the same letter were not Significantly different at the 5% level. Transformation: (0’2 R (pea hydrophilic compounds), S (pea crude extract), T (pea hydrophobic compounds). 64 Table 8. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO BARLEY EPICUTICULAR WAX INCORPORATED INTO THREE PER CENT AGAR.* Experimental Type Wax ConigggrationAveraé: Countc Average CountC Average Activityd Test Control Test Control Test 0.0 1.97 1.8 1.212 0.9 0.78 1.0 1.55 --- --- --- --- 4.8 --- 1.6 2.5 0.9 2.4 9.7 2.77 2.3 . 4.74 1.6 5.02 19.4 3.33 1.9 2.9 1.1 3.16 38.8 --- 1.4 3.98 0.8 3.46 58.0 2.21 --- --— --- --- 87.0 --- 1.9 3.1 1.0 1.02 97.0 2.4 --- --- --- --- 116.0 --- 1.5 3.7 1.0 4.7 126.0 --- 3.2 3.8 1.7 3.62 155.0 --— 2.9 2.2 1.4 3.1 *25 beetles per test unit; one test unit per concentration per day. aType 1 test units contained two agar strips from the same treatment per test unit; average is for two hourly counts per day; no visual analysis. bType 2 test units contained one control and one test agar strip per test unit. CAverage of three hourly counts of beetles per strip per day over four days. dAverage of four daily agar damage scores: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). 1-12 Superscript applies to all values to its left until superseded indica- ting the number of days in the computed average if not four days. 65 o._- e.m FN.N H> eoe mmeepmm mo eoeem eemecmpm “.0- ON.o ”cowpmcesempme mo xmece mpcmeeweemou ”cowpmucm meEocxrou mmemme.mmm m xe emewemmme mmz meme =emeemme xm3= eoe pee mmme eo m>e=m mee N.Nu N.e~ o._Ni Fe.r n> eoe mpmaepmm mo eoeem eemecmpm e.NP eN.o "cowpmceeempme mo xmece mucmemwemmou .11. ”coepmucm Fmesocxpou mmemme eem m me emeeeemme mm: mpme =xmz mazes: eoe “we pmme eo m>ezo mee .emeeoeemu mm; mwmapmcm qumw> oz .mzme ence em>o zme emu meowpm>emmeo »_e:o; mmeep Eoee emmmem>m memz mpceou .xme emu coeumepcmecom emu pee: pmmp mco News: pmmp emu mmppmme ememme xeopmeoemp mN "mesmewemuxm eeope mmeueouse ecm mumFqum emNeEoecme Eoee meme emNeemEe:m .mmxmz empsmweemwum mo cowpweemme mew pzogpwz ecm new: mecuoquu meeoeuoeexe xmpemeiemxmzme mo coepmepcmocou me» on mm—pmme emm— Pmmemo p—zem .emec: .emmemsm >F3mc mo mmcoumme FmUWemE:z .e meemwu 66 FIGURE 6 1000 MINUS WAX 100 PLUS WAX #- 1111 l d 141 I 9 ll~ (D I\ 10 PO "" dlllIS 1531 83d $311338 :10 °°N °9AV LOG PPM EXTRACT 67 tested. The results of both experiments are presented in Table 9. Re- sponse to pea wax was very low when barley extract was absent from the agar. These waxes also Significantly reduced the numerical response and feeding activity of the CLB to crude barley extract which indicated a deterrent effect for the pea wax. However, there was no increased deterrence with increased pea wax content. The deterrent quality of pea wax was confirmed by adding it to 40 mg of sucrose (0.002M) (Table 10), which preliminary work had shown would stimulate a consistent, low level of activity. One ppm of pea wax significantly reduced the CLB response to the sucrose. B. Epicuticular Wax Fractions To locate the activity of the barley epicuticular wax, silicic acid columns were first used to separate the wax. Various degrees of fraction purity resulted. It was apparent (Table 11) that the major portion, per- haps all, of the activity found was due to the alcohols. The effect was seen with fractions bioassayed alone or in combination with sucrose. The three fractions obtained by preparative TLC, the alcohols; the hydrocarbons, esters, and carbonyls; and the acids plus other, were bio- assayed (Table 12). To avoid induced activity on the control strip by the test strip, only strips from the same treatment were used in a test unit. The experiment was designed to determine whether any interaction of wax fractions might produce increased activity. No interaction of non-alcohol fractions was observed, while any combination containing the alcohols was an effective stimulant. A 38.8 ppm sample of silica gel H from a prepara— tive TLC plate was added to a whole wax fraction (38.8 ppm) and it reduced the response to the wax. 68 Table 9. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO EPICUTICULAR WAX OF PEA SEEDLINGS, ALONE AND WITH BARLEY CRUDE EXTRACT IN THREE PER CENT AGAR*. Treatment Mixture Average Counta Average Activityb Barley - ppm Pea wax - ppm Control Test Control Test 0.0 0.0 1.4 2.8 0.7 0.9 0.0 9.7 1.0 2.0 0.1 0.2 0.0 19.4 0.8 1.7 0.4 0.23 0.0 48.5 1.3 0.9 0.3 0.1 291.0 0.0 1.0 7.6x 0.6 4.7 291.0 29.0 0.1 4.1x 0.3 2.6 291.0 58.0 0.9 5.6x 0.6 3.1 291.0 87.3 1.4 4.9x 0.3 3.2 *25 laboratory reared beetles, one control and one test agar strip per test unit;:one test unit per treatment per day. aAverage of three hourly counts of beetles per day over four days. bAverage of four daily agar damage scores: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). x [no significant difference at the 5% level among those means of count data analyzed. Transformation: (Y)%] 3Superscript applies to all values to its left, indicating the number of days in computed averages if not four days. 69 Table 10. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO SUCROSE IN THE PRESENCE OF PEA SEEDLING EPICUTICULAR WAX IN THREE PER CENT AGAR*. Treatment Mixture Average Counta Average Activityb Sucrose (ppm) Pea wax (ppm) Control Test Control Test 0.0 0.0 0.3 1.7 0.7 1.81 776.0 0.0 0.4 3.1 . 0.6 2.4 776.0 1.0 0.0 2.0 0.0 0.61 776.0 1.9 0.3 2.3 0.4 1.21 776.0 9.7 0.5 1.5 0.0 0.92 776.0 27.2 0.8 3.4 0.3 0.6 *25 laboratory reared beetles, one control and one test agar strip per test unit; one test unit per treatment per day. aAverage of three hourly count of beetles per day over four days. bAverage of four daily agar damage scores: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). 1-2 Superscript applies to all values to its left, indicating the number of days in the computed averages if not four days. 70 Table 11. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO BARLEY EPICUTICULAR WAX FRACTIONS ELUTED FROM SILICIC ACID COLUMNS AND INCORPORATED INTO THREE PER CENT AGAR WITH OR WITHOUT SUCROSE*. Wax Fraction With or Without Sucrose Sucrose Controls . . b a . . b Average Counta Average Act1v1ty Count Act1v1ty Content PPM Control Test Control Test Test Test n w 7.8 1.2 1.3 3.0 1.82 --— --— n,o,p 5.4 1.5 0.9 2.1 0.93 --— --- n,0,p 10.9 0.9 1.3, 1.4 1.5 —-- --- n,s 0.8 --- 3.4. 4 --- --- 2.11 1 --— n,s 0.8 --- 1.7‘: --- ---2 1.3 . --- o,p 4.7 1.7 1.0. 1 0 1.9. --—. --- o,s 8.5 --- 0.71 ——— 1.0} 2 1.51 1.1‘ p,s 1.9 --- 1.01 --— 1.6 , 1 41 2.01 q 0.8 1.6 1.6 0.9 3.4 --- --- q 12.4 0.5 2.0. 1 1 3.0. --—. -— q,s 17.5 --- 3.11 --- 2.61 1.4. 1.1. q,s 33.0 --- 3.51 -—— 4.4} 2 1.51 1.11 q,s 34.9 --- 5.31 -—- 3.9 . 1.4‘ 1.11 ql 2.3 1.5 2.7. 2 1 3.2. 3 --- --- q1,s 23.3 --- 4.31 --- 4.31:2 ---. --- q1,s 46.6 --- 3.91 --- 3.5‘: 1.41 1.1' q,r 0.4 0.9 2.5. 0 8 3.6. ---. ---. q,r,s 11.6 --- 3.61 --- 3.81 1.91 1.91 r 1.0 1.4 1.3. 0.9 0.7. ---. ---. r,s x --- 2.01 —-— 1.11:2 1.61 0.91 r,s 2x --- 2.31 --- 1.5‘ 1.61 0.91 *25 laboratory reared beetles, one control, one test agar strip per test unit, except "i" units; one test per concentration per day. aAverage of three hourly counts of beetles per day over four days. bAverage of four daily agar damage scores: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). 1Test units contain two strips from the same treatment. n (hydrocarbons); o (esters); p (carbonyl); q (alcohol); ql (alcohol plus yellow contaminants from hexane); r (acid, other); s (776 ppm sucrose). X (weightless sample). 1"3Superscript applies to all values to its left until superseded, indica- ting the number of days in the computed average if not four days. 71 Table 12. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO BARLEY EPICUTICULAR WAX FRACTIONS OBTAINED BY PREPARATIVE THIN- LAYER CHROMATOGRAPHY AND INCORPORATED INTO THREE PER CENT AGAR*. Wax Fraction PPM Average Countsa’] Average Activityb Control 0.0 1.8 (D) 1.1 U 7.0 1.3 (C) 0.9 25.0 3.4 (H) 3.9 Lr 1.2 2.0 (DE) 1.1 U, R 7.0, 25.0 2.7 (EF) 2.4 U, Lr 7.0, 1.2 1.8 (D) 0.9 R, L 25.0, 1.2 2.4 (DE) 1.9 U, R, L 7.0, 25.0, 1.2 3,3 (H) 2.8 Whole wax 38.4 2.9 (G) 3.3 Whole wax, ge1 H 38.4, 38.4 2.7 (EF) 2.1 LS 1.2 2.0 0.9 R, LS 25.0, 1.2 3.4 2.6 *25 laboratory reared beetles, two agar strips from the same treatment per test unit; one test unit per concentration per day. aAverage of three hourly counts of beetles per strip over four days. b Average of four daily agar damage scores: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). rSlight indication of alcohols by TLC. SAfter removal of trace alcohol from (L) by preparative TLC. U (hydrocarbons, esters, carbonyl), R (alcohols), L (acids, other). 1Means sorted by Duncan's MRT. Values opposite the same letter were not significantly different at the 5% level. Transformation: (Y)% 72 When only field-collected beetles were available, several bioassays of wax fractions were also performed with samples obtained using either silicic acid columns or preparative TLC. These beetles had fed on barley or native grasses as adults and were of unknown age and they were less responsive to the bioassay than the newly emerged, unfed adults produced in the laboratory. Two similar agar strips from one treatment were pro- vided in each test unit and either a blank test unit or a sucrose test unit provided the control. Activity was rated as to how much above the respective control each test unit scored. The column of activity values in both Table 13 and Table 14 showed again that only the alcohol fraction stimulated a Significant response, with or without the presence of sucrose. Samples of the primary alcohols present in barley epicuticular wax were obtained commercially and bioassayed (Table 15). When comparable concentrations were compared in the absence of sucrose, the progression was from low or no activity for 1-docosanol (C-22), to better activity for 1-tetracosanol (C-24), to still better activity for l-hexacosanol (C-26). The differences were considered significant. Those tests con- taining sucrose were too incomplete to be conclusive. The field-collected beetles were tested with the commercially obtained alcohols, both with and without sucrose (Table 16). Without sucrose, the response beyond a blank control was very light for each alcohol bioassayed alone. When the C-26 and C-22 alcohols were mixed in a ratio of 20:1 there was a significant increase in activity beyond the blank control. Field-collected beetles did not seem to prefer any sucrose-alcohol mixture to the sucrose control (Table 16), but mixtures of the C-26 and C-22 alcohols at ratios from 20:1 to 1:1 (C-26:C—22, wt/wt), showed 73 Table 13. RESPONSE OF FIELD-COLLECTED, ADULT CEREAL LEAF BEETLES TO FRACTIONS OF BARLEY EPICUTICULAR WAX ELUTED FROM SILICIC ACID COLUMNS AND INCORPORATED INTO THREE PER CENT AGAR*. Wax Fraction Sucrose Blank Component PPM Counta Activityb Counta Counta U U“ U U U MMOOOMMM U 'D‘U'O \0 (11mm N N U U U U CDCDNNNbph I I I want mmm \I .U U 0 11034040033130) (A) (D U U U 0 o 60 mm NNdNN—JwN—ld—Jd—JNN—JHN—Ju—l—JNNN I WWI WWW-PODKDKOKDI DWWI I I I ow—aoouwnowuem-bmoowooooo-hoooN-h OOON—‘LflN—‘N-‘NN-d—‘OOOOOOOOOO wooomwwooomoomuowwowoooooooo '1 '5 'S-DDDQQDQDD‘U'D'C'OO O 3 3 3 3 3 :3 (A) CD mmwmmoooooooo-boowoouowomummeomp NNI NNON—JOOOI —-'l\)l\)I -—'-'—'-'-—'I\)|'\)|'\) U 0 mm *25 beetles, fasted, but watered for two days prior to test, and two agar strips from the same treatment per test unit; one test unit per concentra- tion per day. aAverage of three hourly counts of beetles per day over four days. bAverage of four daily scores of a ar damage relative to control: 1 (light), 2 (fair), 3 (good), 4 (quite good?, 5 (very good), 6 (excellent). n (hydrocarbons), o (esters), p (carbonyl), q (alcohol), r (acid), s (776 ppm sucrose). 74 Table 14. RESPONSE OF FIELD-COLLECTED, ADULT CEREAL LEAF BEETLES TO EPICUTICULAR WAX FRACTIONS OF BARLEY OBTAINED BY PREPARATIVE THIN-LAYER CHROMATOGRAPHY AND INCORPORATED INTO THREE PER CENT AGAR*. Component PPM Average Counta Average Activityb None 0.0 1.3 0.0 (The base score) Whole Wax 38.0 0.6 1.5 U 0 1.2 .3 R 38.8 0.5 2.0 L O 0.5 0.8 U, R O, 38.8 1.3 2.3 U, L Q. Q 1.0 0.8 R, L 38.8, 0 1.6 2.8 *25 beetles, fasted, but watered for two days prior to test, and two agar strips from the same treatment per test unit; one test unit per concen- tration per day. aAverage of three hourly counts of beetles per day over four days. bAverage of four daily scores of agar damage relative to control: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). 0 equivalent to 38.8 ppm alcohol (equal volumes taken from equal volumes). U (hydrocarbons, esters, carbonyl), R (alcohols), L (acids, other). 75 Table 15. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO COMMERCIALLY OBTAINED PRIMARY ALCOHOLS IN THREE PER CENT AGAR WITH AND WITHOUT SUCROSE*. Alcohol Sucrosef Chain length PPM Counta Activityb Counta Activityb C-22 19.4 0.9 0.6 --- --- 38.8 1.3 0.7 -—— --- C-24f 1.9 2.7 1.42 1.0 0.5; 9.7 2.2 3.3 2.0 2.8 C-24 19.4 1.7 1.1 --_ --- C-24f 19.4 2.8 3.14 0.8 2.0; 29.0 2.5 1.9 2 O 2.8 38.0 2.0 2.0 --- --- C-26 0.2 1.3 1.3 --- --- 19.4 1.8 2.8 --- --- 19.4 2.5 2.1 --- --- 38.8 2.9 3.0 ——- --- *25 laboratory reared beetles, two agar strips from the same treatment per test unit; one test unit per concentration per day. aAverages of three hourly counts of beetles per strip per day over four days. bAverage of four daily scores of agar damage: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). f(776 ppm sucrose added to test agar). 1’ZSuperscript applies to all values to its left until superseded, indi- cating the number of days in computed averages if not four days. 76 Table 16. RESPONSE OF FIELD-COLLECTED, ADULT CEREAL LEAF BEETLES TO COMMERCIALLY OBTAINED PRIMARY ALCOHOLS INCORPORATED INTO THREE PER CENT AGAR WITH AND WITHOUT SUCROSE*. Alcohol Sucrosef Chain length PPM Counta Activityb Counta C-22f 1.9 1.2 0.0 1.8 1.9 2.4 0.0 2 8 C-22 1.9 2.0 0.0 --- C-22f 3.8 1.2 0.0 1 8 C-22 28.1 1.4 0.3 --- C-22f 38.8 1.5 0.3 1.8 38.8 2.1 0.0 2.8 C-24f 1.9 2.3 0.0 C-24 28.1 1.5 0.0 --- C-24f 38.8 2.6 1.3 2.8 C-26 28.1 1.7 0.8 --- C-26f 38.8 1.8 0.5 1.8 38.8 1.8 0.0 2.8 38.8 3.0 0.7 2.8 C-22, 26 1.9, 38.8 1.8 1.5 --- C-22, 26f 1.9, 38.8 1.8 2.3 1.8 1.9, 38.8 2.6 0.3 2.8 3.8, 38.8 2.7 2.3 1.8 38.8, 38.8 2.0 2.5 1.8 C-24, 26f 1.9, 38.8 2.3 0.0 2.8 C-22, 24, 26f 1.9, 1.9, 38.8 2.6 1.3 2.8 * 25 beetles, fasted, but watered, for two days prior to test, and two agar strips from the same treatment per test unit; one test unit per concentration per day. aAverage of three hourly counts of beetles per day over four days. bAverage of four daily agar damage scores: 4 (quite good), 5 (very good), 6 (excellent). f(776 ppm sucrose added to the treatment). 1 (light), 2 (fair), 3 (good), 77 significant activity relative to the sucrose control. This result was in agreement with the similar test without sucrose. The combination of C-26 and C-24 alcohols showed no activity over control, but was tested only once. The three alcohols together did not show any greater response than the C-22 and C-26 combinations. C. Hydrophobic Compounds Minus Wax, (H-W) The results of bioassay of the hydrophobic compounds minus wax, (H-W), from barley is presented in Figure 6 where barley epicuticular wax data have been presented also. The (H-W) fraction possesses stimulant qualities by itself. To determine whether the pea epicuticular wax was the sole deterrent source to the CLB seen in Table 9 and Figure 4, pea seedlings were dewaxed before extraction. The resulting (H-W) fraction was bioassayed alone and at two levels in combination with 482 ppm of barley crude extract (Table 17). The pea (H-W) fraction caused overall activity to be less within the test units (control plus test) at both concentrations bioassayed alone than was found for the blank test unit. This fraction, in combination at 169 ppm with 482 ppm of barley crude extract, Significantly reduced the CLB feeding response toward the barley agar. As the amount of pea (H-W) added was increased and then supplemented with 33 ppm of pea surface wax, there was a significant trend to greater reduction in feeding response to the barley extract. 0. (H-W) Apolar Fraction Vs. (H-W) Polar Fraction Having determined that the barley (H-W) fraction was an effective stimulant (Figure 6), this fraction was further separated to more closely isolate the active principles. Initially, the (H-W) apolar compounds 78 Table 17. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO DEWAXED PEA SEEDLING HYDROPHOBIC COMPOUNDS INCORPORATED WITH AND WITHOUT BARLEY CRUDE EXTRACT INTO THREE PER CENT AGAR*. Treatment Mixture Average Counta Average Activityb Barley (ppm) Pea (ppm) Control TestC Control Test 0.0 0.0 0.8 1.1 0.7 0.9 0.0 291.0 0.1 1.1 0.2 0.0 0.0 482.0 1.0 0.5 0 2 0.1 482.0 0.0 0.6 8.2 (L) 0.7 4.8 482.0 169.0 0.2 4.3 (K) 0.4 2 1 482.0 337.0 0.5 2.4 (K) 0 3 l 8 482 0 337.0, 33.0d 0.6 3.3 (K) 0.3 1 8 *25 laboratory reared beetles, one control and one test agar strip per test unit; one test unit per concentration per day. aAverage of three hourly counts of beetles per day over four days. bAverage of four daily agar damage scores: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). CMeans sorted by Duncan's MRT. Values opposite the same letter were not Significantly different at the 5% level. Transformation: (Y+1)% dPea epicuticular wax. 79 were separated from the (H-W) polar compounds by silicic acid columns and each fraction was bioassayed at concentrations which made each equivalent to the same amount of total (H-W). A positive dose-response was found with the (H-W) polar, but none was found among the (H-W) apolar bioassays (Figure 7). The (H-W) apolar and (H-W) polar fractions of pea seedlings were bioassayed alone and combined with barley crude extract (Table 18). Neither class of compounds was able to stimulate feeding behavior by itself. The (H-W) apolar group was strongly deterrent at the levels tested and significantly reduced response toward the barley extract. Pea (H-W) polar compounds also reduced the activity (but not the counts) toward barley extract, but not nearly as much as did (H-W) apolars and then only at much greater concentrations than required of (H-W) apolar compounds. To answer the reciprocal question for barley, location of fractions effective in overcoming the deterrence of pea extract was determined. Two experiments were directed to this question (Table 19). The pea extract significantly reduced the response to both hydrophobic and to hydrophilic compounds of barley. Yet, the hydrophobic compounds Slightly, but sig- nificantly, more effectively counteracted the deterrence of the pea extract despite being at half the concentration of the hydrophilic com- pounds. Since barley crude extract at a 1:1 ratio with pea crude extract was able to overcome the deterrence of pea extract (Figure 4), various hydrophobic fractions of barley were readded to the barley hydrophilic materials in the presence of 582 ppm of pea crude extract (Table 19). Each was readded in such amounts that it was equivalent to its level in 80 m.eu m.wN «.mm- eN.F H> eoe mpmeepmm mo eoeem eemecmum o.Ne e_.o "cowpmcweempme eo xmecH mpcmemwemmou ”cavemecm FmeEochou mmemme.mum m an emeeemmme mm; mezzoque emPoum eom are mmme mo m>e=o mew em._ u> eom memewpmm mo eoeem eemecmum em.o ”cowmmcwEempme eo xmecH mpcmwmmemmoo "cowpmecm _meEo:>—ou mmemme.mmw m xe emeeeumme mm; meme meceoueom emFou eoe pee pmme mo m>e=u me» .Aucmfipmuxmv e .Aeoom >em>v m .Aeoom mpeeav e .Aeoomv m .Aeemev N .Apemerv P "mmeoem momsme emmm >Feme ence seem emmmem>m mm: Apemwe pmmp .emmp Foepcoe mmmmmgpcmemu cry pr>muu< .mxme eeoe em>o Ame emu mmrpmme mo mcoepm>emmeo zpezo; mmeee Eoem emmmem>m memz mucsou .xme emu cowpmepcmucou emu ewe: pmmu mco News: pmmp emu mmppmme ememme xeoemeoemp mN “mesmewemuxm eeope mpmpueoece ecm mmequm emesoecme Eoem emNeemEE:m memo .emmm meme emu mmecp c? mecsousou emFoum ecm emFou eweoeuoeexe mcwpemmm xmreme -emxm3me mo coepmeecmeeou mew om mm—pmme emm_ meeme ppuem .emecs .emmemem xpzmc mo mmcoummm .e meumwu 81 .64:th 2.. u 00.. 000. 00_ ......4. . 2... «30.2 38.9 6m 99 em .mo. 6 o 0 /\ meme. .2. .90. . “(1.0m 6.3.: O 6.8.9 n 3.30.”. 10 PO [e 03 (“815 1.53]. 83:! $311338 :10 'ON '07“! 82 Table 18. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES T0 DEWAXED PEA SEEDLING APOLAR AND POLAR HYDROPHOBIC COMPOUNDS WITH AND WITHOUT BARLEY CRUDE EXTRACT IN THREE PER CENT AGAR*. Pea Extra t b Pea extgact plus barle R Avg. Count Avg. Activity Count Activity Fraction PPM Cont. Test Cont. Test Cont. Test Cont. Test 14 13 Controls 0.0 1.3 0.8 0.8 0.4 0.9 7.6 0.8 4.3 Ap 43.6 1.5 1.9 0.4 0.3 0.6 4.5 0.4 2.2 Ap 73.7 0.3 0.7 0.0 0.0 0.9 3.1 0.1 1.0 Ap 97.0 1.6 0.3 0.4 0.2 1.7 3.7 0.4 1 13 P 19.4 1.0 1.4 0.9 1.23 --- --- —-- --- P 43.6 1.9 1.2 0.5 0.23 -—- --- --- —-— P 131.0 1.3 2.8 0.6 1.310 1.7 7.8 1.2 3.57 P 229.0 0.6 1.4 0.3 0.8 --- --- --- --- P 291.0 2.7 1.1 0.5 0.43 --- --- --- --- P 411.0 1. 1.7 0.2 0.6 0.7 5.6 0.4 3.3 P 547.0 1.6 0.2 0.1 0.2 0.8 4.1 0.7 2.63 *25 laboratory reared beetles, one control, one test agar strip per test unit; one test unit per concentration per day. aAverage of three hourly counts of beetles per strip per day over four days. b Average of four daily agar damage scores: 4 (quite good), 5 (very good), 6 (excellent). 1 (light), 2 (fair), 3 (good), Ap (dewaxed apolars), P (dewaxed polars), R (482 ppm barley crude extract). 3-14 Superscript applies to all values to its left until superseded, indicating the number of days in the computed average if not four days. 83 Table 19. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO BARLEY EXTRACT FRACTIONS INCORPORATED WITH PEA SEEDLING CRUDE EXTRACT INTO THREE PER CENT AGAR*. Treatment Mixture Average Counta Average Activityb Barley Fraction PPM PPM Pea Extract Cont. Test Cont. Test. None 0.0 None 1.9 1.6 (K) 0.6 0.7 Philic 1260.0 None 1.1 4.7 (L) 0.4 2.9 Philic 1260.0 291.0 0.9 3.6 (KL) 0.1 0.5 Philic 1260.0 582.0 1.9 2.1 (K) 0.0 0.3 Phobic 582.0 None 2.5 7.8 (M) 1.2 3.3 Phobic 582.0 291.0 1.8 3.4 (KL) 1.1 0.8 Phobic 582.0 582.0 2.4 2.7 (KL) 0.3 1.1 None 0.0 None 1.6 0.8 (P) 1.0 0.5 Philic 1260.0 None 1.8 3.0 (OR) 0.7 2.6 Philic 1260.0 582.0 1.0 1.5 (P0) 0.0 0.8 Philic+W 1260.0+70.0 582.0 1.1 4.6 (R) 0.2 2.6 Philic+P 1260.0+586.0 582.0 1.4 4.2 (R) 0.0 1.8 Philic+A 1260.0+196.0 582.0 1.8 1.5 (P0) 0.0 1.0 Philic+W+P+A 1260.0+852.0 582.0 2.0 3.1 (OR) 0.3 3.0 *25 laboratory reared beetles, one control and one test agar strip per test unit; one test unit per concentration per day. aAverage of three hourly counts of beetles per strip per day over four days. bAverage of four daily agar damage scores: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). 1Means sorted by Duncan's MRT. Values opposite the same letter were not significantly different at the 5% level. Transformation: (Y+l)% Philic (hydrophilic compounds); Phobic (total hydrophobic compounds); A (dewaxed apolar compounds); P (dewaxed polar compounds); W (surface wax). 84 a whole sample of hydrophobics. Barley (H-W) apolar compounds were significantly less effective than the others at overcoming the deterrence of pea extract. Barley epicuticular wax was the most effective fraction. Barley (H-W) polars were significantly better than the (H-W) apolars, but significantly less effective than the wax. All three fractions added together resulted in good recovery of activity. E. (H-W) Polar Compounds The (H-W) polar materials were separated into glycolipids and phospholipids on silicic acid columns. The phospholipid fraction con- tained a non-phosphorous compound, gramine, for which a means of separa- tion was not immediately available. It was found that both (H-W) subfractions were stimulants (Table 20). Different combinations of glycolipids and phospholipids were made and bioassayed at various times in the study (Table 21). There was no consistent pattern of either count or activity data due to changes in the glycolipid/phospholipid ratio. The CLB response to glycolipids from barley and pea seedlings is compared in Figure 8. The barley data Clearly Showed a dose-dependent response, while the pea data indicated only a low response at all levels tested. The middle curve of Figure 8 represents pea glycolipids minus monogalactoslydiglyceride. A Slightly better response was found in this instance than for other pea total-glycolipid bioassays, but it remained a low response. The monogalactosyldiglyceride was bioassayed separately in that case and is reported below. 1. Individual Glycolipids The mono- and di-galactosyldiglycerides and the sulfolipid of both barley (Table 22) and pea (Table 23) were isolated and bioassayed. For 85 Table 20. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES T0 BARLEY GLYCOLIPIDS AND PHOSPHOLIPIDS IN THREE PER CENT AGAR*. Treatment Average Counta Average Activityb Fraction PPM Control Test Control Test PL 97.0 0.6 3.6 0.4 3.2 PLt 97.0 0.8 2.2 0.5 3.2 PLt 155.0 1.1 3.2 0.5 3.63 6Lt 194.0 1.2 2.8 0.6 3.1 GL 242.0 1.9 3.0 0.5 3.5 * 25 laboratory reared beetles, one control, one test agar strip per test unit; one test unit per concentration per day. aAverage of three hourly counts of beetles per day over four days. bAverage of four daily agar damage scores: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). t20 beetles per test unit. 3Superscript applies to all values to its left, indicating the number of days in computed average if not four days. PL (phospholipid); GL (glycolipid). 86 Table 21. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO VARIOUS RECOMBINATION RATIOS OF BARLEY GLYCOLIPID TO PHOSPHOLIPID IN THREE PER CENT AGAR*. GL/PL GL PL Average Counta Average Activityb Ratio PPM Sum-PPM Control Test Control Test 0.8t 164 205 369 1.0 5.2 1.0 4.5 1.9t 243 126 369 0.7 7.1 0.6 3.9 2.0t 194 97 291 1.3 4.3 0.8 3.81 2.5 242 97 339 0.9 7.3 0.3 4.6 4.2 242 58 300 1.6 6.7 0.5 4.53 4.6t 299 70 369 0.7 4.2 0.7 3.8 *25 laboratory reared beetles, one control, one test agar strip per test unit; one test unit per concentration per day. aAverage of three hourly counts of beetles per day over four days. bAverage of four daily agar damage scores: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). t20 beetles per test unit. PL (phospholipid); GL (glycolipid). 1,3 days in computed average if not four days. Superscript applies to all values to its left, indicating the number of 87 o.— m.e- op.. H> eoe memewpmm mo eoeem eemecmum m.m mo.o "cowpmceeempme mo xmec. mpcmwuweemoo "cowpmzcm mesocxpou mmemme mmM.m an emeeeemme mm: mewuVFOUme _mpop mmu eom pee pmme mo m>e=m mee N¢N.N Ne.. H> eom mpmeepmm eo eoeem eemecmum «No.0- mN.o ”cowpmcwsemmme mo xmece mpcmeoeeemou .111 "coepmecm .mVEocxpou mmemme mm. m xe emeweUmme mm; mewuwFom>Fm xmreme eoe pee pmme eo m>e=u mew .mzme ezoe mo: e. mmmem>m emuzquu meg cw mxme mo eme53c mmu mmummwecw muweomemuem ..ucm—quxm. e .Aeoom xem>v m ..eoom memes. e ..eoom. m ..ewme. N .AeLDPP. — ”mmeoum mmmEme emmm prme eeoe Eoee emmmem>m mm; Augmee pmmp .uem. Foeucou .mmmmzecmemu cw mm=Pm> emeumfimm. pr>wpe< .mxme eeoe em>o xme emu mm.pmme mo meowpm>emmeo >Fezo; mmeep Eoee emmmem>m memz mpczou .zme emu coepmepemueom emu ewe: “mmu mco ”pee: pump emu mmpumme ememme zeoumeoemF mN ”mpcmaeemuxm emcee mmequece ecm mumpueou emNTEoecme Eoee emNeemEE=m memo .emmm acme emu mmegp one. empmeoueoec. meeuepoQAPm mcmpemmm mmu ecm szeme mo coeemepcmmcom mew om mmppmme emmF .mmeme p.mem .emec: .emmemem arzm: mo mmcoummm .m me=m_u like» 88 ~01;th 2a... 00.. 000. 00. o. . . . .....J. . ._.o.o«o.o. ...... . . . N8 6.8. mm. 11111111111 6.8.0.8. _ 333850 .59 3.. N. ...... _. 1 . . u- c. .0 AN. 0.0.9.- AN.N.®.OV A m . 35359350853on2 Mime 1 m 32.2 ”9.38:0 <2 . ememme. 33.0. 1 0 8.3.8.. . 1 . 8.39 1 N. 3330050 .59 >23; 1 L m L m £50: 1: 'OAV (11815 1531 83d $311338 30 'ON 89 Table 22. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO GLYCOLIPIDS INDIVIDUALLY ISOLATED FROM BARLEY AND INCORPOR- ATED INTO THREE PER CENT AGAR*. Average Counta Average Activityb G1yco1ipid PPM Contro1 Test Contro1 Test MDG 1.9 0.9 1.2 1.4 1.6 19.4 0.3 2.4 0.7 2.3 19.4 0.6 2.1 0.4 2.5 97.0 1.8 2.7 1.1 4.41 136.0 0.0 3.7 1.5 3.7 177.0 0.5 2.4 0.4 1.8 214.0 0.4 2.8 1.0 3.4 061) 1.9 2.7 1.1 1.2 0.53 19.4 0.4 2.2 0.2 2.4 19.4 0.8 2.9 0.3 3.4 97.0 0.4 2.4 0.3 4.4 97.0 0.0 3.6 0.3 3.0 194.0 1.3 3.2 0.2 3.1 SUL 0.2 1.2 2.7 2.2 2.0 0.2 1.2 1.8 0.8 0.7 1.9 1.7 2.8 1.1 1.9 1.9 2.6 2.0 1.9 2.2 19.4 0.7 3.1 0.5 4.43 19.4 1.2 4.0 1.5 4.3 19.4 1.3 2.0 1.1 2.0 *20 1aboratory reared beet1es, one contro1, one test agar strip per test unit; one test unit per concentration per day. aAverage of three hour1y counts of beet1es per day over four days. bAverage of four dai1y agar damage scores: 1 (1ight), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (exce11ent). MDG (monoga1actosy1dig1yceride); DGD (diga1actosy1dig1yceride); SUL (su1fo1ipid). 1’BSuperscript app1ies to a11 va1ues to its 1eft, indicating the number of days in computed average if not four days. 9O TabIe 23. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO PEA SEEDLING GLYCOLIPIDS INDIVIDUALLY ISOLATED AND INCORPORATED INTO THREE PER CENT AGAR*. Average Counta Average Activityb Glycolipid PPM Control Test Control Test Complete 142.0 0.9 2.2 0.6 0.83 M06 29.0P 0.6 4.1 0.5 4.23 97.0$ 0.5 2.8 0.5 1.7 $ 3 060 97.0 0.8 2.7 0.3 1.4 SUL x$ 0.9 1.3 0.3 1.43 22.0# 3.2 1.3 1.0 1.12 *20 laboratory reared beetles, one control, one test agar strip per test unit; one test unit per concentration per day. aAverage of three hour1y counts of beetles per day over four days. bAverage of four daily agar damage scores: 1 (Tight), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). PFirst isolated from silicic acid column with chloroformzacetone, 1:1 (v/v) followed by preparative TLC on silica gel H, and then eluted with chloro— form:methanol, 2:1 (v/v). $Complete glycolipids applied to second silicic acid column (5 ml Unisil). E1ution: 50 m1 chloroform:acetone (90:10, v/v) (M06); 50 ml chloroform: acetone (25:75, v/v) (D60); 75 m1 acetone (SUL). #E1uted from a silicic acid column in last 200 ml of 300 ml used. MGD (monogalactosyldiglyceride); DGD (digalactosyldiglyceride); SUL (sulfo- 1ipid). 2’3Superscript applies to all values to its left, indicating the number of days in computed average if not four days. 91 each class of glycolipid, the dose-response relationship was relatively flat. Based on activity scores, there seemed to be a stronger dose- response for sulfolipid than for mono- or di-galactosy1diglyceride. The CLB also seemed more sensitive to the former than to the latter two classes. On one occasion, the monogalactosyldiglycerides of pea seedlings did stimulate a high level of feeding activity by the CLB. A possible explanation for this unique event is presented in the DISCUSSION. 2. Phospholipids The phospholipids of pea and barley seedlings were bioassayed (Table 24). The data confirmed earlier observations that pea-derived, (H-w) polar compounds did not stimulate CLB feeding behavior to a signif- icant extent. The data for the barley phospholipids in Table 24 is the same as that in Table 20. a. Acidic Phospholipids Barley acidic phospholipids, without further treatment after elution from the DEAE column, showed a low stimulatory effect (Table 25). Three experiments were performed to determine whether the change of associated cations such as occurs with the acid phospholipids during silicic acid column chromatography (123), could alter CLB response in this study (Table 26). When washed with deionized water, a remarkable positive effect on response to the acidic phospholipids was seen compared to any other wash. A slight positive dose-response was indicated when the 2 or Na+1 form compared to what- acid phospholipids were in either the Ca+ ever their state was directly from the DEAE column, but many more tests would be required to confirm this indication. Acid phospholipids of pea seedlings were bioassayed without alter- ation of their cation content (Table 27). Like nearly all previous 92 Table 24. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO BARLEY AND PEA SEEDLING PHOSPHOLIPIDS INCORPORATED INTO THREE PER CENT AGAR*. a . . b Phospholipid PPM Average Count Average Act1V1ty Source Control Test Control Test Pea Seedlings 56.0 1.9 1.8 0.5 0.33 72.0 1.2 0.8 1.6 1.4 151.0 1.6 2.1 0.6 1.2 Barley SeedlingsR 97.0 0.6 3.6 0.4 3.2 97.0 0.8 2.2 0.5 3.2 155.0 1.1 3.2 0.5 3.63 *20 laboratory reared beet1es, one control, one test agar strip per test unit; one test unit per concentration per day. aAverage of three hourly counts of beetles per day over four days. bAverage of four daily agar damage scores: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). RData taken from Table 20. 3 Superscript applies to all values to its left, indicating the number of days in computed average if not four days. 93 Table 25. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO BARLEY SEEDLING NEUTRAL AND ACID PHOSPHOLIPIDS INCORPORATED INTO THREE PER CENT AGAR*. Average Counta Average Activityb Treatment PPM Control Test Control Test APL 0.0 0.4 0.4 0.5 0.9 19.4 0.3 1.8 0.4 1.8 136.0 1.9 1.4 0.4 1.3 NPLC 19.4 0.7 2.5 0.6 3.6 34.2 1.1 2.6 0.4 0 9 136.0 0.5 4.6 0.2 5 0 180.0 1.6 6.8 0.2 3 0 291.0 0.9 3.1 0.3 2 3 APL+NPL 15.5+7l.8 1.6 4.1 0.3 4.7 *20 beetles, one control, one test agar strip per test unit; one test unit per concentration per day. aAverage of three hour1y counts of beetles per day over four days. bAverage of four daily agar damage scores: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). APL (acid phospholipids); NPL (neutral phospholipids). c . . Contained gramine. 94 Table 26. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES T0 BARLEY ACID PHOSPHOLIPIDS TREATED TO ALTER ASSOCIATED CATIONS AND INCORPORATED INTO THREE PER CENT AGAR*. a . . b Washing APL Average Count Average Act1v1ty SOIUt1°n (PPM) Control Test Control Test NaZ-EDTA 19.4 0.2 1.6 0.3 1.2 58.0 0.1 1.2 0.1 2.6 Deionized Water 19.4 0.8 4.3 0.8 5.3 58.0 1.0 3.0 0.5 5.3 Unwashed 9.7 1.7 1.2 2.4 1.2 19.4 1.0 1.8 0.6 2.0 55.0 1.0 1.5 0.8 1.4 CaCl2 9.7 1.1 1.3 2.4 1.5 19.4 0.6 1.2 0.6 1.4 55.0 0.2 2.2 0.7 2.7 CaClz, then NaZ-EDTA 9.7 0.9 2.2 0.6 1.4 19.4 0.7 1.0 1.2 1.1 40.7 0.7 0.9 0.9 1.2 *20 laboratory reared beet1es, one control, one test agar strip per test unit; one test unit per concentration per day. aAverage of three hour1y counts of beetles per day over four days. bAverage of four daily agar damage scores: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). APL (acid phospholipids). 95 Table 27. RESPONSE OF NEWLY EMERGED, UNFED ADULT CEREAL LEAF BEETLES TO PEA SEEDLING ACID AND NEUTRAL PHOSPHOLIPIDS INCORPORATED INTO THREE PER CENT AGAR*. a . . b Phospholipid PPM Average Count Average Act1v1ty Fract1on Control Test Control Test APL 0.0 1.5 1.1 0.6 0.8 21.0 1.2 2.0 0.4 1.1 132.0 1.6 2.7 0.6 1.1 NPL 34.0 0.7 1.1 0.7 0.9 180.0 1.5 2.4 0.8 0.6 APL, NPL 21.0+34.0 2.1 2.1 0.7 0.8 *20 laboratory reared beetles, one control, one test agar strip per test unit; one test unit per concentration per day. aAverage of three hour1y counts of beetles per day over four days. bAverage of four daily agar damage scores: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). APL (acid phospholipids); NPL (neutral phospholipids). 96 bioassays of pea hydrophobic compounds, there was only a marginal response which was consistent with earlier observations that no strong deterrence was associated with (H-W) polar compounds of pea seedlings. It was hoped that elution of the DEAE column with conc. acetic acid would separate the suspected phosphatidyl serine from the remaining acid phospholipids. However, an absolutely pure sample of phosphatidyl serine was not obtained. The remaining acid phospholipids and the phos- phatidyl serine were bioassayed separately at several concentrations and together at one concentration (Table 28). Based on the activity scores, it can only be repeated that the acid phospholipids of barley stimulated a light to fair feeding response by the CLB. b. Neutral Phospholipids When bioassayed simultaneously, the neutral phospholipids of barley were far more effective than the acid phospholipids (Table 25). Gramine was present in the neutral phospholipids during these bioassays. Bioassay of the neutral phospholipids of pea plants showed them to be ineffective (Table 27). The individual neutral phospholipids of barley were obtained from the DEAE column in various degrees of purity and bioassayed without further separation attempted. In general, barley neutral phospholipids stimulated an inconsistent, low level of feeding behavior when bioassayed separately (Table 29). No combination of two barley neutral phospholipids was found to act as a strong stimulant (Table 30). All combinations of two were, on the average, significantly more effective than their respective blank controls when gravimetric amounts were available for bioassay. The combination of phosphatidyl ethanolamine with acid phospho— lipids (Table 30) was very effective, having produced a degree of response 97 Table 28. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO BARLEY PHOSPHATIDYL SERINE AND THE REMAINING ACID PHOSPHO- LIPIDS INCORPORATED INTO THREE PER CENT AGAR*. a . . b Phospholipid PPM Average Count Average Act1v1ty Fraction Control Test Control Test PS(?) 4.8; 0.8 2.2 0.4 2.3 5.22 1.4 3.1 0.3 2.8 10.51 1.0 1.2 0.5 0.8 12.04 0.5 2.6 0.4 2.4 l2.01 1.1 1.3 1.1 0.7 13.01 0.8 1.5 0.4 1.2 17.02 0.8 2.0 0.0 2.5 21.04 1.1 2.6 0.8 0.9 25.0 1.6 0.9 1.4 0.7 R 20.0 1.8 2.8 l.0 1.4 22.0 0.6 2.2 0.5 2.2 27.0 1.4 2.1 0.6 1.7 60.0 1.2 2.1 0.3 2.2 68.0 2.0 1.1 1.1 1.0 PS, R 5.2,22.0 0 5 2.1 0 3 2.4 *20 laboratory reared beetles, one control, one test agar strip per test unit; one test unit per concentration per day. aAverage of three hour1y counts of beetles per day over four days. bAverage of four daily agar damage scores: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). PS(?) Believed to be phosphatidyl serine. R (remaining acid phospholipids). 1By TLC, contained one unidentified phospholipid. ZBy TLC, contained lysophosphatidyl ethanolamine plus unidentified acid phospholipid. 3By TLC, contained lysophosphatidyl ethanolamine. 4By TLC, contained several unidentified acid phospholipids. 98 Table 29. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO BARLEY NEUTRAL PHOSPHOLIPIDS IN THREE PER CENT AGAR*. Average Counta Average Activityb Phospholipid PPM Control Test Control Test PE X 0.6 1.5 0.6 1.9 X 1.1 2.8 0.8 1.7 5.8 0.9 1.8 0.5 1.4 9.7 0.8 0.5 0.5 0.82 13.6 1.3 0.8 0.4 0.5 PC X 1.2 1.4 0.3 1.4 13.0 0.4 1.1 0.3 1.42 23.3 0.0 0.8 0.4 0.9 LPE x 0.2 0.3 0.2 0.33 LPC X 1.4 0.6 1.0 0.8 1.7 1.9 2.0 1.1 1.5 3.5 1.7 2.0 0.8 1.7 6.7 0.6 3.1 0.5 3.2 *20 laboratory reared beet1es, one control, one test agar strip per test unit; one test unit per concentration per day. aAverage of three hourly counts of beetles per day over four days. bAverage of four daily agar damage scores: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). X (weightless sample). 2Superscript applies to all values to its left, indicating the number of days in computed average if not four days. PE (phosphatidyl ethanolamine); PC (phosphatidyl choline); LPE (lysophatidyl ethanolamine); LPC (lysophosphatidyl choline). 99 Table 30. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO COMBINATIONS OF TWO BARLEY NEUTRAL PHOSPHOLIPIDS IN THREE PER CENT AGAR*. Average Counta Average Activityb Phospholipids PPM Control Test Control Test PE + PC 12.0 + 32.0 0.8 0.8 0.7 1.3 1.9 + 13.0 0.9 1.6 0.6 1.32 13.6 + 23.3 0.6 1.6 0.6 1.2 PE + LPE 12.1 + X 1.4 2.8 0.6 2.5 PE + LPC X 0.5 1.4 0.4 0.8 6.0 1.0 1.4 0.3 1.2 8.4 1.2 2.0 0.9 1.3 12.0 + X 1.6 2.5 0.9 2.2 14.2 0.8 1.3 1.3 0.8 16.5 0.8 1.8 0.7 1.6 23.0 0.8 2.0 0.2 1.8 PE + APL 23.0 + 44.0 1.4 3.4 0.6 3.2 PC + LPE X .5 1.4 1.1 1.1 32.0 + X 1.3 1.7 0.5 1.9 PC + LPC 36.9 + x 0.8 1.3 0.4 1.72 32.0 + X 0.5 2.0 0.3 1.3 PC + U 9.7 1.2 1.2 01 0.62 27.0 1.6 2.5 l 2.7 LPE + LPC X 1.0 1.5 0.4 2.1 *20 laboratory reared beetles, one control, one test agar strip per test unit; one test unit per concentration per day. aAverage of three hourly counts of beetles per day over four days. bAverage of four daily agar damage scores: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). 2Superscript applies to all values to its left, indicating the number of days in computed average if not four days. PE (phosphatidyl ethanolamine); PC (phosphatidyl choline); LPE (lysophospha- tidylethanolamine); LPC (lysophosphatidyl choline); APL (acid phospholipids); U (non-phosphorous compound later identified as gramine). 100 that indicated an interaction had occurred. Similarly, phosphatidyl choline, which had been a poor stimulant alone or in dual combination, produced a response of 2.7 when bioassayed with the gramine at a combined concentration of 27 ppm. When the bioassay mixture approached the composition of the native neutral phospholipid fraction, the response became consistently signifi- cant (Table 31). A positive dose—response was indicated, but not strongly. The combination of neutral phospholipid mixture and phospha- tidyl serine or acid phospholipid minus phosphatidyl serine gave a consistently good response. While interaction was indicated, it appeared to be of an additive nature. To investigate the possibility that the fatty acid composition might affect the response of the CLB to phospholipid, three species of lecithin were bioassayed (Table 32). One species, L-c*-l-stearoly- 2—oleoyl lecithin, was significantly more effective than the other two lecithins. F. Gramine Since the non-phosphorous contaminant of neutral phospholipids was involved in every bioassay reported in Table 31, it became necessary to isolate, identify and bioassay this substance. A combination of infrared and mass spectrometry, as well as available literature, helped to identify the isolated compound as gramine. The selected series of masses from the mass spectrum for the isolated and commercial gramine were identical and are presented in Table 33. The proposed fragmentation patterns of major interest are shown in Figure 9. A definite positive response was obtained with isolated and commercial gramine (Table 34), lOl Table 3l. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO THREE OR MORE NEUTRAL PHOSPHOLIPIDS OF BARLEY IN THREE PER CENT AGAR WITH A NON-PHOSPHOROUS COMPOUND*. Average Counta Average Activityb Phospholipids PPM Control Test Control Test PC + PE + LPE + U 19.4 1.8 1.7 0.9 1.7 38.8 1.0 3.0 0.8 2.1 52.4 0.9 3.0 0.5 3.1 PC + PE + LPE + LPC + U 53.0 0.9 4.6 0.5 2.4 58.0 1.1 3.7 0.3 3.4 60.0 0.8 2.6 0.5 2.1 83.0 1.7 2.8 0.9 2.4 96.0 1.2 4.0 0.9 2.8 102.0 0.5 5.5 0.5 4.8 PC + PE + LPE + U + (G) 60.0 + (20.0) 1.2 3. 0.7 3.4 58.0 + (22.0) 1.4 3.5 0.3 3.4 PC + PE + LPE + U + (PS?) 58.0 + (5.0) 0 6 4.2 O 3 4 2 *20 laboratory reared beet1es, one control, one test agar strip per test unit; one test unit per concentration per day. aAverage of three hour1y counts of beetles per day over four days. bAverage of four daily agar damage scores: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). PE (phosphatidyl ethanolamine); PC (phosphatidyl choline); LPE (lysophatidy ethanolamine); LPC (lysophosphatidyl choline); PS (phosphatidyl serine) G (acid phospholipids minus phosphatidyl serine); U (non-phosphorous com- pound later identified as gramine). l02 Table 32. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO THREE SPECIES OF COMMERCIALLY OBTAINED PHOSPHATIDYL CHOLINE WITH AND WITHOUT GRAMINE IN THREE PER CENT AGAR*. Treatment Mixture Average Counta Average Activityb Phospholipid (PPM) Gramine PPM Control Test Control Test l-s-2-o 155.2 0.0 2.0 2.3 1.4 2.1 Dilin. 97.0 0.0 2.1 1.3 1.2 1.4 155.2 0.0 1.2 1.0 1.2 1.2 97.0 58.2 1.5 4 2 0.5 3.4 0.0 58.2 1.1 4.4 0.5 3.0 Distear. 155.2 0.0 0.9 2.5 0.4 1.1 0.0 38.8 1.7 4.6 0.8 1.9 155.2 38.8 2.3 2.8 1.2 2.4 *20 laboratory reared beet1es, one control, one test agar strip per test unit; one test unit per concentration per day. aAverage of three hourly counts of beetles per day over four days. bAverage of four daily agar damage scores: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). l-s-2-o (L-ok-l-stearoyl-Z-oleoyl lecithin). Dilin. (L-ok-dilinoeloyl lecithin). Distear. (L-aL-distearoyl lecithin). 103 Table 33. A SELECTED SERIES OF MASS INTENSITIES FROM THE MASS SPECTRUM OF GRAMINE 3-(DIMETHYLAMINOMETHYL)-INDOLE. Mass/e Relative Intensity Mass/e Relative Intensity 41 5.0 102 13.6 43 21.1 103* 9.2* 44 8.9 104 1.2 50 5.5 127 0.8 77* 10.0* 128 6.5 78 2.8 129 35.6 79 1.3 130* 100.0* 81 1.5 131 32.4 83 3.6 132 3.5 85 4.5 142 1.3 86 2.1 156 2.0 87 7.8 173 8.2 89 1.1 174 (M+) 60.6* 100 0.6 175 8.9 101 2.2 *Peaks corresponding to fragments shown in Figure 9. 104 m/e I74 m/e 130 m/e 77 Figure 9. Major ions represented in the mass spectrum of gramine, 3—(dimethylaminomethyl)-indole. 105 Table 34. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO GRAMINE IN THREE PER CENT AGAR*. Average Counta Average Activityb Gramine Source PPM Control Test Control Test Extracted from barley 3.9 2.0 2.0 1.9 1.9 9 7 l 2 3.0 0 9 2 4 Commercial 9.7 2 2 2 6 0.7 1.3 19.4 1 2 l 0.8 l 9 19.4 15 1.5 0.3 1.62 38.8 2 o 2 8 1 3 1 72 97.0 0.9 l 5 0.7 1 9 116.0 2 1 1.6 l l l 4 *20 laboratory reared beetles, one control, one test agar strip per test unit; one test unit per concentration per day. aAverage of three hour1y counts of beetles per day over four days. bAverage of four daily agar damage scores: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). 2Superscript applies to all values to its left, indicating the number of days in computed average if not four days. 106 but like that of the epicuticular wax, it was a fairly flat response with little concentration effect. Because gramine can be classified as a secondary plant compound, it was bioassayed in combination with other compounds present in barley leaves to look for interactions (Table 35). Gramine produced a signifi- cant increase in response to the barley glycolipids, but only a slight interaction was seen with l-hexacosanol. Indole-3-acetic acid (IAA) was bioassayed alone and combined with gramine due to its similar structure. Judged by TLC, gramine was more abundant than other indoles in barley. IAA was, therefore, tested at lower concentrations than was gramine. It showed marginal activity by itself and, in every combination with gramine, reduced the response relative to the gramine control. This reduction was judged to be insignificant overall. To assess the effect of gramine on CLB response to the neutral phospholipids, gramine was separated from that fraction during elution from the DEAE column. Two bioassays of neutral phospholipids (143 ppm), gramine (38.8 ppm), and the two combined were performed (Table 36). With- out gramine, the neutral phospholipids evoked a significantly reduced response from the CLB from that usually observed. With gramine readded, the response became more typical of that found for the neutral phospho- lipids plus the former unknown contaminant; cf. Table 25 and Table 31. Since the response to the combination was greater than to either alone, an interaction was clearly established. The effect of gramine with glycolipids and phospholipids of pea seedlings was investigated. Neither fraction had been an effective CLB stimulant (Figure 8, Tables 23 and 24). Gramine transformed the two pea 107 Table 35. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO COMBINATIONS OF GRAMINE WITH OTHER PLANT BIOCHEMICALS IN THREE PER CENT AGAR*. Treatment Mixture Average Counta’] Average Activityb Gramine ppm Other ppm Control Test Control Test 0.0 GL 112.0 1.7 4.5 0.2 3.8 31.0 GL 112.0 0.8 6.2 0.1 5.2 0.0 C-26 19.4 0 8 3 6 1.1 4.1 19.4 C-26 19.4 1 1 4 7 1.0 4.7 38 8 --- 0.0 1.3 3.7 0.2 2.3 38 8 IAA 13.6 .5 2.1 0.4 1.8 38.8 --- 0.0 0.8 4.2 (M) 0.6 3.3 38.8 IAA 1.9 1.4 2.4 (L) 1.0 2.8 38.8 IAA 3.8 2.4 2.4 (L) 1.0 3.1 38.8 IAA 7.6 1.2 3.1 (LM) 0.7 3.03 0.0 IAA 1.9 1.9 1.4 0.8 1.3 0.0 IAA 3.8 1.3 1.0 (K) 1.3 1.7 0.0 IAA 7.6 1.4 2.6 (L) 0.7 1.4 *20 laboratory reared beet1es, one control, one test agar strip per test unit; one test unit per concentration per day. aAverage of three hourly counts of beetles per day over four days. bAverage of four daily agar damage scores: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). GL (barley total glycolipids); C-26 (l-hexacosanol); IAA (indole-3-acetic acid 3Superscript applies to all values to its left, indicating the number of days in computed average if not four days. 1Means associated with letters have been sorted by Duncan's MRT. Means opposite the same letter were not significantly different at the 5% level. Transformation: (Y+l)’2 l08 Table 36. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO BARLEY NEUTRAL PHOSPHOLIPIDS WITH AND WITHOUT GRAMINE IN THREE PER CENT AGAR*. Treatment Mixture Average Counta’1 Average Activityb NPL - ppm Gramine - ppm Control Test Control Test 143.6 0.0 0.8 1.3 (K) 0.5 1.4 0.0 38.8 1.7 4.6 (L) 0.8 1.9 143.6 38.8 0.9 3.6 (L) 0.8 3.9 143.6 0.0 0.5 1.6 (S) 0.3 2.1 0.0 38.8 0.0 0.5 (S) 0.3 0.9 143.6 38.8 0.5 1.4 (S) 0.3 3.8 *20 laboratory reared beetles, one control, one test agar strip per test unit; one test unit per concentration per day. aAverage of three hourly counts of beetles per day over four days. bAverage of four daily agar damage scores: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). NPL (neutral phospholipids). 1Means sorted by Duncan's MRT. Means opposite the same letter wege not significantly different at the 5% level. Transformation. (Y+l) 109 (H-W) polar fractions from unpreferred substrates to a degree of palata- bility previously found for barley extracts only (Table 37). Gramine was bioassayed in combination with two of the three species of commercially obtained phosphatidyl choline which differed in their fatty acid composition (Table 32). There was no significant increase of response compared to gramine alone. Further investigation of the influence of gramine was made by intro- ducing the highly deterrent pea (H-W) apolar fraction to barley hydrophilic extract and three concentrations of gramine (Table 38). The pea compounds deterred the usual CLB feeding response to the barley hydrophilic com- pounds. Addition of gramine enabled the test agar to again stimulate the response to a level comparable to that of the barley hydrophilic compounds alone. A slight inconsistency of this effect was seen at the 58.2 ppm concentration of gramine, but the effect was irrefutable. V. Hydrophilic Compounds A. Commercially Obtained Chemicals Several bioassays of individual amino acids and amino acid combined with sucrose or gramine were performed (Table 39). Tryptophan, from which gramine is derived, was not active at the levels tested either by itself or combined with gramine, relative to gramine alone. £3-Alanine was not active as a stimulant by itself, while it significantly increased the response with sucrose compared to the sucrose control. Additional study would be required to confirm this apparent interaction. Proline gave only a slight response when bioassayed alone and did not significantly increase the response to sucrose compared to a sucrose control. 110 Table 37. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO PEA SEEDLING GLYCOLIPIDS AND PHOSPHOLIPIDS WITH AND WITHOUT GRAMINE IN THREE PER CENT AGAR*. Treatment Mixture Average Counta’1 Average Activityb Lipid Class-ppm Gramine-ppm Control Test Control Test Glycolipid 194.0 0.0 0.6 1.1 0.5 0.82 0.0 57.0 2.0 2.9 0.8 2.8 194.0 57.0 2.4 7.0 0.9 3.4 PL - PC 151.3 0.0 1.4 2.1 (S) 0.6 1.2 0.0 48.0 1.4 2.3 (S) 1.0 2.6 151.3 48.0 2.2 4.1 (S) 0.9 3.4 *20 laboratory reared beetles, one control, one test agar strip per test unit; one test unit per concentration per day. aAverage of three hour1y counts of beetles per day over four days. bAverage of four daily agar damage scores: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). 1Means sorted by Duncan's MRT. Means opposite the same letter were not significantly different at the 5% level. Transformation: (Y+1)2 2Superscript applies to all values to its left, indicating the number of days in computed average if not four days. 111 Table 38. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO A DETERRENT FRACTION OF PEA SEEDLING EXTRACT IN BARLEY HYDRO- PHILIC EXTRACT WITH AND WITHOUT GRAMINE IN THREE PER CENT AGAR*. Treatment Mixture Average Counta’] Average Activityb B.H. P.A. Gramine ppm ppm ppm Control Test Control Test 660.0 0.0 0.0 0.7 2.2 (S) 0.5 1.9 660.0 38.8 0.0 0.7 0.8 (S) 0.4 0.8 660.0 38.8 9.7 0.5 2.6 (S) 0.5 1.5 660.0 38.8 29.0 1.6 2.3 (S) 0.7 1.8 660.0 38.8 58.2 1.3 2.0 (S) 0.3 1.4 *20 laboratory reared beet1es, one control, one test agar strip per test unit; one test unit per concentration per day. aAverage of three hourly counts of beetles per day over four days. bAverage of four daily agar damage scores: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). B.H. (barley hydrophilic fraction); P.A. (dewaxed pea apolar compounds). 1Means sorted by Duncan's MRT. Means opposite the same letter wege not significantly different at the 5% level. Transformation: (Y+l) ll2 Table 39. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO SELECTED AMINO ACIDS IN THREE PER CENT AGAR WITH OR WITHOUT SUCROSE OR GRAMINE*. Sucrose Gramine Average Counta Average Activityb Amino Acid (PPM) (PPM) (PPM) Control Test Control Test L-Tryptophan 97.0 0.0 0.0 1.6 0.8 0.5 0.2% 199.0 0.0 0.0 1.3 0.4 1.1 0.3 0.0 0.0 38.8 1.1 2.1 0.6 2.8 97.0 0.0 38.8 1.0 1.9 0.7 2.1 199.0 0.0 38.8 1.6 1.9 0.9 2.0 /3-Alanine 691.0 0.0 0.0 2.6 0.5 1.8 1.2 432.0 0.0 0.0 1.3 0.6 1.3 0.6 346.0 776.0 0.0 0.3 6.0 0.5 3.6 0.0 776.0 0.0 2.0 3.1 0.8 2.83 259.0 0.0 0.0 2.1 1.9 1.4 1.0 L-Proline 55.8 0.0 0.0 2.1 1.3 0.9 1.5 111.6 0.0 0.0 0.8 2.1 0.5 0.5 892.0 0.0 0.0 1.1 2.0 1.2 1.9 0.0 776.0 0.0 1.0 3.4 0.8 2.12 97.0 0.0 0.0 1.0 1.1 1.3 1.5 97.0 776.0 0.0 3.1 4.6 0.5 2.5 *20 laboratory reared beetles, one control, one test agar strip per test unit; one test unit per concentration per day. aAverage of three hourly counts of beetles per day over four days. bAverage of four daily agar damage scores: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). 2’3Superscript applies to all values to its left, indicating the number of days in the computed average if not four days. ll3 Amino acid mixtures corresponding to the free amino acids of a CLB host-plant, orchard grass, and a non-host, alfalfa (38) were bioassayed. Accommodating to a reduced number of laboratory reared beetles, the number of beetles used per test unit was reduced to 20. Field-collected beetles were also used. Results under both conditions are presented in Table 40. There was no evidence of a positive or a negative response to either plant-simulated amino acid mixture presented to the laboratory beet1es. Nor was there any indication that the amino acids interacted with sucrose when field beet1es were used. There seemed to be a greater response to gramine when the orchard grass amino acid mixture was combined with it than to gramine alone, but further tests with laboratory reared beetles would be appropriate. B. Extracted Chemicals Most portions of the bioassays of the fractions derived from the Sephadex G-10 column (MATERIALS AND METHODS, section IV.C.2.) had to be deleted after one, two, or three days since the laboratory beetles were in the last days of their seasonal production and the number available became erratic. Results of the first experiment are presented in Table 41. Fraction No. 1 contained all compounds not held back by adsorption effects on the column; it was active at both concentrations tested. Fraction No. 2 was considered inactive and fraction No. 3 was active. Addition of sucrose (485 ppm) to fraction No. l (1145 ppm) did little to increase CLB response to this fraction compared to 563 ppm of No. 1 alone. Gramine (38.8 ppm) added to 1145 ppm of fraction No. 1 significantly increased the response compared to 2,250 ppm of this fraction alone. 114 Table 40. RESPONSE OF NEWLY EMERGED AND FIELD COLLECTED ADULT CEREAL LEAF BEETLES TO PLANT-SIMULATED AMINO ACID MIXTURES IN THREE PER CENT AGAR*. Plant Average Counta Average Activityb Mixture PPM Add1t1ve PPM Control Test Control Test NEW BEETLES Dac 369.0 ---- ---- 2.3 0.8 1.7 1.22 Med 369.0 ---- —--- 1.2 0.6 0.3 0.93 FIELD COLLECTED BEETLESC ---- ---- Gramine 38.8 1.3 2.0 0.8 1.0 Dac 369.0 Gramine 19.4 0.6 2.7 0.9 1.8 ---- ---- Sucrose 776.0 0.8 1.2 0.4 0.9 Dac 369.0 Sucrose 776.0 0.4 0.4 0.0 0.32 Dac 369.0 --—- ---- 0.1 0.8 1.0 0.22 ---- ---- Sucrose 776.0 1.2 3.1 0.7 2.33 Dac 369.0 Sucrose 776.0 0.8 3.5 1.0 2.5 *20 beetles, one control, one test agar strip per test unit; one test unit per concentration per day. aAverage of three hour1y counts of beetles per day over four days. bAverage of four daily agar damage scores: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). cBeetles fasted, but watered, two days prior to test; daily exposure to test materials was 23.5 hours after which visual analysis was made. 2’3Superscript applies to all values to its left, indicating the number of days in computed average if not four days. Dac (Dactylis glomerata L.); Med (Medicage sativa L.). Reference: (38). 115 Table 41. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO FRACTIONS OF BARLEY HYDROPHILIC COMPOUNDS SEPARATED ON A SEPHADEX G-lO COLUMN AND INCORPORATED INTO THREE PER CENT AGAR*. Average Counta Average Activityb Fraction No. PPM Control Test Control Test 1st: 25 m1C 563.0 1.0 2.0 0.1 2.12 1st: 25 m1C 2250.0 0.8 2.6 0.6 2.6 2nd: 37 m1d 21.3 1.1 1.0 0.5 0.32 2nd: 37 m1d 171.0 2.0 3.8 0.2 0.72 3rd: 75 m1e 13.2 0.3 2 4 0.0 2.22 1st plus S 1145.0 + 485.0 2.2 2.2 0.5 2.72 1st plus Gr 1145.0 + 38.8 1.1 4.6 0.7 3.4 *20 laboratory reared beet1es, one control, one test agar strip per test unit; one test unit per concentration per day. aAverage of three hourly counts of beetles per day over four days. bAverage of four daily agar damage scores: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). CContained sugars, amino acids, other. dContained gramine, other. eContained majority of saponarin, other. S (sucrose); Gr (gramine). 2Superscript applies to all values to its left, indicating the number of days in the computed average if not four days. 116 From another Sephadex column, only two fractions were collected. The first 25 ml were separated into a cation fraction and a neutral plus anion fraction by a cation exchange column. Due to lack of beetles, the bioassay results of these fractions (Table 42) were inconclusive for the neutral and anionic compounds. The cations were surely a source of stimulation as was the last fraction (200 ml) from the Sephadex column. The activity of the saponarin-containing fraction of barley seed- lings was examined (Table 43). There was little indication among field beetles that the saponarin may have had much influence either alone or combined with sucrose. In one instance where laboratory beetles were used, the saponarin had been obtained as a precipitate from 10% methanol in water after the remainder of the hydrophilic compounds had been sep- arated by the Sephadex column. The test generated a very significant response on both the test and control agar strips even though successive- ly fewer beetles were used each day. 117 Table 42. RESPONSE OF NEWLY EMERGED, UNFED, ADULT CEREAL LEAF BEETLES TO BARLEY HYDROPHILIC COMPOUNDS SEPARATED BY COMBINED GEL FILTRA— TION AND ION EXCHANGE COLUMN CHROMATOGRAPHY AND INCORPORATED INTO THREE PER CENT AGAR*. Average Counta Average Activityb Fraction Column PPMc Control Test Control Test Last 200 m1d G-lO ---- 1.1 2.4 0.8 2.43 Cations (9%) Dowex—50 ---- 3.2 1.3 0.4 1.62 Anions, 1 Neutrals (91%) Dowex-50 ---- 0.3 0.0 0.7 0.0 *20 laboratory reared beetles, one control, one test agar strip per test unit; one test unit per concentration per day. aAverage of three hourly counts of beetles per day over four days. bAverage of four daily agar damage scores: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). CNo weight recorded. Equivalent volumes of each were used. dContained saponarin. 1'3Superscript applies to all values to its left, indicating the number of days in computed average if not four days. 118 Table 43. RESPONSE OF NEWLY EMERGED AND FIELD-COLLECTED, ADULT CEREAL LEAF BEETLES TO SAPONARIN-CONTAINING EXTRACT FROM BARLEY SEEDLINGS INCORPORATED INTO THREE PER CENT AGAR*. Fraction Mixture ‘ Average Countb Average ActivityC Sap. ppm Suc. ppm Control Test Control Test NEW BEETLESa 38.8 ---- 2.0 4.0 1.5 2.92 38.8 776.0 0.7 4.7 1.7 3.01 87.3d ---- 0.8 0.6 3.2 2.9 FIELD BEETLESe 155.0 ---- 0.3 0.4 0.6 0.9 0.0 776.0 0.8 0.9 0.5 1.6 155.0 776.0 0.7 1.6 0.3 1.6 155.0 ---- 0.1 0.6 0.5 0.5 0.0 776.0 0.1 0.8 0.5 1.4 155.0 776.0 0.1 1.2 0.3 2.0 *One control, one test agar strip per test unit; one test unit per concentration per day. a20 laboratory reared beetles per test unit. bAverage of three hour1y counts of beetles per day over four days. cAverage of four daily agar damage scores: 1 (light), 2 (fair), 3 (good), 4 (quite good), 5 (very good), 6 (excellent). dThis saponarin obtained from a washed precipitate. Remaining saponarin fractions contain unidentified substances. e15 beet1es per test unit; 23.5 hour exposure per day to test materials. 1’ZSuperscript applies to all values to its left, indicating the number of days in computed average if not four days. DISCUSSION 1. Validity of the Bioassay The agar medium employed in this study stimulated a feeding response by the CLB only when extract of a host—plant was incorporated with it, but not when extract of non-hosts were used (Table 4). This result suggests that conclusions drawn from the laboratory studies reflected the molecular ecology of the insect/host relationship in the field, and, therefore, have increased the understanding of the chemical basis of CLB host selection. 11. The Parallel Study of Pea Seedling Extracts Leaves of non-host plants have commonly been used in studies of the feeding response of phytophagous insects. There are few examples, however, in which extracts of non-hosts have been used. Jermy (79) used leaves and the juice from non-hosts applied to host leaves of eight phytophagous insects. Gilbert et_al, (47) used extract fractions of host and non-hosts of the smaller European elm bark beetle, S. multjs; triatus. The bioassay of pea fractions was an asset to this study. Some extract fractions of this plant deterred the CLB, i.e., Figure 4 and Tables 7 and 9, while other fractions stimulated feeding (Table 6). The deterrence was partly overcome by certain barley fractions, i.e., Figure 119 4 and Tables 17 and 19. These observations made two facts apparent. First, pea is not a host of the CLB, in part, because it contains deterrent chemicals. This conclusion lends support to the contention of Jermy (79) that the host range of a phytophagous insect is strongly limited by the distribution of deterrent chemicals. Second, there must have been a host—Specific, recognizable quality about the barley extract which caused the CLB to feed, at reduced activity, in the presence of the deterrent(s). This conclusion supports those who believe that host selection depends on both the presence of specific stimulants and the absence of strong deterrents (11, 73, 162). III. Estimation of Beetle Response For many of the early experiments in this study, CLB response was assayed only by count data for the time periods indicated in the Tables and Figures. Later, it was found that response measurement could be improved by visually examining and scoring the damage to the agar strips on a daily basis. The value of a visual analysis is readily apparent. Two means of the count response were not significantly different at the 5% level in Table 7 (means indicated by "M"), while their respective activity scores were significantly different (1.9 and 2.8). Count means found significantly different at the 5% level (L, M) are paired with activity scores not significantly different (1.7, 1.9). A difference of 0.7 in activity was considered significant. In another instance (Table 9), crude extract from barley was bioassayed by itself and combined with three concentrations of pea seedling epicuticular wax. No significance was found among those count means analyzed statistically (bottom four). Yet, activity was Significantly reduced in this group when pea surface 121 lipids were present. In Table 12, insignificant differences in count data were associated with activity differences considered Significant to the observer. Based on the considerations presented above, it was concluded that activity estimates by an experienced observer were a valid basis of judging CLB response. IV. Functions of Barley Stimuli,_and Corresponding_ Pea Fractions A. Hydrophobic Compounds l. Epicuticular Wax Little has been done to relate insect feeding behavior to the nonvolatile plant cuticular chemicals. Yet, secondary substances are found free on leaf surfaces in amount easily detected by insects (143). Barley; The leaf alcohols of barley, 1-hexacosanol in particular, stimulated feeding by the laboratory reared beetles and field-collected beetles (Tables 15 and 16). Other wax fractions bioassayed alone were ineffective, and they did not interact in a detectable manner with the alcohols (Tables 11, 12, l3, 14). Due to the low numerical response obtained in the alcohol bioassays shown in those Tables and from samples of whole barley wax (Table 8), it was concluded that the function of these alcohols was neither to attract CLB through olfaction nor to arrest their movement pgr_§g_after contact with the test agar had been made. The function of the leaf alcohols of barley was to incite a biting response. Evidence for this conclusion came from visual analysis of the test agars after the test period. Several categories of agar damage were recognizable: bites, nibbles, channels, and rashes of close nibbling over a variable amount of surface area. When whole surface lipids or purified 122 alcohols were bioassayed, a great amount of random biting, nibbling and rashing predominated. There were some channels of short to medium length. If a blank agar strip were in the same test unit, the blank often showed greater damage than usually seen in other test situations. This effect probably resulted because the beetles were incited to bite by the alcohols, but, not finding other chemicals to stimulate continued feeding, they continued to seek these chemicals. Their movements brought them to the control strip where biting also occurred as a carry—over response. An indication of the strength of the influence of the leaf alcohols is found in Table 19. Barley wax in combination with barley hydrophilic compounds overcame some of the deterrence of pea seedling crude extract. The waxes also produced a pronounced effect by increasing the response to hydrophobic compounds of barley from which the wax had been previously removed (Figure 4). (Note: Inclusion of the silicic acid coating from a preparative TLC plate, silica gel H, with barley wax significantly reduced the response compared to untreated wax (Table 12). Such materials Should be excluded from the bioassay medium.) Although other reports of non-volatile, primary alcohols as feeding stimulants have not been made, volatile alcohols have been shown to serve as attractants for such insects as Epilanchna fulvosignata (120), for B, mgri (179), and for A, grandis (108). Primary alcohols of C-28 and C-30 chain length did not arrive from their commercial source in time to be bioassayed. 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