Host plant resistance of maize, Zea mays L., to the European corn borer (ECB), Ostrinia nubilalis Hubner, has been largely attributed to the hydroxamic acid DIMBOA. However, DIMBOA does not consistently account for observed resistance in both field and greenhouse environments. It was hypothesized that phenolic acids, a major secondary metabolite in plants, make an important contribution to resistance, through cell wall carbohydrate complexes. Preliminary studies incorporating ferulic acid into the meridic diet of the ECB did not show any adverse effects until 10 mg/g, a dosage that is approximately 30 times that found in maize. Semi-preparative samples of sugar conjugates of ferulic and p-coumaric acids were tested using leaf-feeding bioassays that showed phagostimulant activity at ecologically relevant dosages. These observations suggested that soluble phenolics did not make a major contribution to host plant resistance but may be used for host plant recognition by the ECB. Bound phenolics can form dimers with adjacent phenolic acids. It was hypothesized that these dimers, cross-linked phenolic acid-carbohydrate complexes, could constitute a quantitative defence against the ECB. Developmental studies of a resistant maize synthetic using leaf bioassays and standard phytochemical procedures showed ECB larvae prefer to feed on immature tissue within the whorl and on plants between the 3- and 7-leaf stages of development. These preferred tissues had elevated levels of DIMBOA and lower levels of cell wall phenolics and lower levels of detergent fibre than more mature leaf tissue. Leaf toughness related inversely to leaf consumption. Microspectrophotometer readings of abaxial, epidermal cell walls best predicted insect preference for leaf tissues of different maturity. These studies showed that leaf toughness, vis a vis phenolic acid-carbohydrate complexes, is one of the major resistance factors which can account for larval feeding behavior. Environmental factors also influence maize resistance, with light quality having a significant impact. Under reduced UV light conditions, maize resistance in both greenhouse and field environments was reduced as were the levels of cell wall bound cyclobutane dimers. Cyclobutane dimers appear to increase the mechanical strength of leaf tissue by cross-linking hemicelluloses. The changes in maize phytochemistry and nutritional quality that accompanied increased resistance were monitored for leaf(mature and immature), sheath, stalk, node and pith tissues. DIMBOA levels increased with selection as did fibre content and the cell wall phenolics p-coumaric acid and dehydrodiferulic acid. Cultivars developed at the International Center for Maize and Wheat Improvement have resistance to several lepidopteran borer species but the mechanism of this resistance is unknown. Correlations of field resistance were highest for fibre, dehydrodiferulic acid content and protein content. A nutritional model for host plant resistance in maize based on these three plant components was proposed that accounts for 78 to 87 percent of the variation in field damage ratings observed over two years. According to Feeny's hypothesis of plant apparency, maize appears to rely heavily on quantitative defences. This is logical given the extensive areas planted in maize, with many areas employing no-till practices and continuous cropping which elevate the apparency of maize to insect pests. Phenolic acid-carbohydrate complexes have been demonstrated in this study to be a major component in this defence strategy. (Abstract shortened by UMI.)

To prevent serious post-harvest losses in corn by the Larger Grain Borer, Prostephanus truncatus (Coleoptera: Bostrichidae) in the sub-saharan Africa, kernel resistance mechanisms to this insect were analyzed. Kernels of seven cultivars from the International Centre for Corn and Wheat Improvement (CIMMYT, Mexico) were analyzed for their physical characteristics and their biochemical properties. Also, grain damage and biology of the borer were recorded on intact kernels. Excellent correlations were observed between hardness and adult mortality and oviposition. The quality of the endosperm appeared to be determinant of levels of susceptibility. Unlike what was noted with Sitophilus zeamais (Serratos, 1987), lipids appear to stimulate Prostephanus truncatus activities and development, and the estimated protein content did not present significant correlations. Among the three endogeneous phenolic acids in corn kernels, ferulic, sinapic and p-coumaric, the latter presented very significant and negative correlations with damage and with oviposition rate. (Abstract shortened by UMI.)

In this study I identified and characterized a storage protein in the European corn borer, Ostrinia nubilalis. The protein was identified as a storage protein by its native molecular weight of approximately 500 kDa and the observation that it increased in abundance throughout larval development and diminished with adult development. The protein had the characteristics of an arylphorin-like storage protein because it is a heterohexamer and is the most abundant protein in the haemolymph, increasing to the intermoult period with a decline just prior to pupation. Larval weight accounts for much of the variation in storage protein levels in the fat body (90%) and intact larvae (80%). Similar results were found with larval age and storage protein levels (80% for both fat body and intact larvae). Since nutrition has been proposed as a regulator of storage protein levels, I examined the relationship between storage protein levels, protein quality in the diet and larval weight. There was no significant effect of protein quality in the diet on storage protein levels after correcting for the weight of the larvae. Finally, I examined how development was affected by protein quality in the insect's diet. Corn borers fed poorer diets could compensate by consuming more and increasing their larval duration, however, they did so at lower pupal weights than those corn borers on the standard diet. Logistic regression analysis showed that pupal weight had a significant effect on survival, suggesting that heavier pupae are more likely to survive to the adult stage than lighter pupae. (Abstract shortened by UMI.)

Over 400 tree species are primary or secondary hosts of gypsy moth. Interactions between host plant compounds and a viral pathogen such as nuclear polyhedrosis virus (NPV) in vivo may explain gypsy moth host preference in later instars. Phenolic extracts from pines negatively affected growth of 2nd and 3rd but not 4th instars when incorporated into artificial diet at low concentrations. The aglycone quercetin, and two of its glycosides found in pines, rutin and quercetin-3-O-glucoside, also reduced growth for 2nd and 3rd instars. When 3rd instars were dosed with NPV (60,000 PIBs/larva) and fed pine extract (0.1%) in diet, growth was reduced and mortality increased. Survivorship to adulthood was not significantly higher than control for 3rd instars fed pine extracts over a range of viral doses, nor was it higher for 4th instars dosed at 60,000 PIBs/larvae. The three flavonoids also tended to have negative and synergistic effects on growth and survivorship of 3rd and 4th instars does with 60,000 PIBs/larva. These results suggest that early instars of gypsy moth are quite sensitive to pine phenolics in diet, which may explain why they do not feed on pines in those instars. Gypsy moth is more tolerant of pine phenolics in later instars but these results do not support the hypothesis that gypsy moth later instars which feed on plant hosts such as pine, gain some protection from infectivity of NPV due to the phenolics present in those hosts.

The phenomenon of multi-drug resistance in tumor cells is mediated by the overexpression of a membrane protein called P-glycoprotein. Since its initial discovery in tumor cells, P-glycoprotein-like molecules have been found to transport a wide range of substrates in organisms from microorganisms to humans. To test the hypothesis that a P-glycoprotein-like mechanism is operating in insects, three insect species were used: Manduca sexta, Periplaneta americana and Rhodnius prolixus, the nicotine-resistant M. sexta was the model toxin-resistant insect. Immunostaining with a monoclonal antibody, C219, which recognizes a highly conserved epitope in all P-glycoprotein molecules, demonstrated positive immunolabelling of the Malpighian tubules of M. sexta and R. prolixus and at the blood-brain barrier of the central nervous systems of M. sexta and P. americana. To assess the extent of P-glycoprotein involvement at the blood-brain barrier of M. sexta, I took advantage of the three different stages of the insect's life cycle, the actively feeding larval stage and the non-feeding pupal and adult stages. Using an extracellular recording technique to monitor levels of neural activity in isolated abdominal nerve cords, I demonstrated that, in contrast with larval M. sexta, the CNS of both the pupal and adult stages are both highly nicotine-sensitive. Immunostaining for P-glycoprotein in the metamorphosing CNS illustrated that the distribution of P-glycoprotein in the barrier region changes dramatically when the need for a barrier to dietary neurotoxins is lost. M. sexta Malpighian tubules provided an ideal in vitro assay system to directly test our hypothesis that a P-glycoprotein-like molecule is involved in nicotine transport. Using an isolated Malpighian tubule preparation, the tubules were assayed for their ability, first, to concentrate nicotine in the tubule lumen (confirming the presence of an active transporter in the tubules) and, second, to determine if various drugs interfere with nicotine transport. Tubules bathed in 0.5 mM nicotine were found to concentrate nicotine in the secreted fluid at least 9-fold. Nicotine transport was inhibited with verapamil, a known inhibitor of P-glycoprotein. In addition, nicotine transport was competitively inhibited by another alkaloid, atropine, suggesting that the M. sexta pump may be a non-selective alkaloid pump. To further confirm that the insect pump is a P-glycoprotein-like multi-drug pump, the tubules were tested for their ability to transport a known P-glycoprotein substrate, vinblastine. The tubules were not only able to transport radiolabelled vinblastine, but this transport was also inhibited by verapamil. P-glycoprotein is localized to the digestive tracts of mammals and of invertebrates, where it is thought to act as a defective mechanism to protect organisms from dietary toxins. Immunohistochemical staining for P-glycoprotein demonstrated that it is also expressed in the M. sexta gut. In a parallel study, a monoclonal antibody against a P450 from a DDT-resistant housefly (Waters et al., 1989) was used to localize a cytochrome P450 monooxygenase enzyme (P450 or PSMO) to the M. sexta midgut. The co-localization of P450 and P-glycoprotein to the midgut suggests that larval M. sexta use a combination of P450-mediated metabolism coupled with transport to process dietary toxins.

The dark-sided cutworm, Euxoa messoria (Harr.) was reared under laboratory conditions on 8 artificial diets, a meridic diet containing various concentrations of one of the alkaloids, veratrine, berberine, nicotine, or atropine, and on two varieties of Nicotiana tabacum, Lonibow and Delhi-34. Observations of the development times, larval and pupal weights, survival, and number and widths of head capsules were recorded. Feeding preference studies were carried out to look at possible food choice mechanisms used by the larvae. Larvae showed quantitative requirements for ascorbic acid and cholesterol. Soybean protein was unsuitable as the sole protein source but it did supplement casein. Wheat germ provided an unknown nutritional quality to the diet which improved developmental characteristics. Chronic exposure to alkaloid diets demonstrated that survival and developmental characteristics are variously affected by the source and concentration of alkaloid. Larvae demonstrated a tolerance to levels of 0.01%, 0.03%, and 0.1%, berberine; 0.01%, and 0.03% nicotine; and 0.01%, 0.03% and 0.1% atropine. Atropine at all levels tested enhanced survival and larval size. Detrimental effects on growth, development rates survival and/or number of instars were observed in larvae fed dietary levels of 0.3% and 1.0% berberine; 0.1%, 0.3%, and 1.0% nicotine; 0.3% and 1.0% atropine; and 0.01%, 0.03%, 0.1%, and 0.3%, and 1.0% veratrine, indicating that this insect is susceptible to these compounds at these levels. Larvae were most sensitive to the detrimental (or antibiosis) effects of the alkaloids berberine and nicotine in the early stages of development. There was a degree of plasticity observed in the duration of the prepupal period which Allowed the development rates of individuals fed various levels of an alkaloid to become synchronized, thus ensuring that the adults emerged at roughly the same time. The developmental characteristics of larvae fed two varieties of N. tabacum, Lonibow and Delhi-34, indicated that Delhi-34 shows some resistance to the larvae by decreasing survival, larval and pupal sizes, and increasing the development times. E. messoria demonstrates a definite preference for some food sources over others, and responds to both nutritive and secondary plant compounds in making a choice. Sucrose, nicotine, and atropine are phagostimulants to the larvae while berberine (at 0.03%, 0.1%, 0.3%, and 1.0%) and veratrine (at all levels tested) are feeding deterrents. The threshold level for the antifeedant effect of berberine appears to be 0.01%. Response to the alkaloids seems to be dependant upon the presence of nutrient compounds. Sucrose mixed with atropine had a synergistic effect on the feeding response. Larvae show a preference for some varieties and species of Nicotiana, and strongly preferred Atropa belladona over tobacco. Both the antennae and maxillary palps are involved in host selection. Responses by maxillectomized larvae suggest that sucrose receptors are located on the maxillary palps and in the buccal cavity, and that the receptors responsible for the synergistic effect of sucrose and atropine are also on the maxillary palps. The presence of the maxillary palps appears to be essential for feeding to occur. Larval preference for a food source does not guarantee the choice of a diet which is suitable for normal growth and development.

The nocturnal calling behavior of three closely related, interfertile species of cutworm moths, Euxoa declarata, E. campestris, and E. rockburnei, was studied in the laboratory at four different temperatures. The temporal pattern of calling activity by virgin females was found to differ for each species, with peak periods of pheromone release by each species remaining distinct at each of the experimental temperatures. The calling period of declarata was found to be exclusive from that rockburnei and nearly exclusive from that of campestris at. all temperatures. An overlap between the calling periods of campestris and rockburnei was found to occur, however the initial and peak phases, which under natural conditions with males present, would be by far the most important, were found to be separated by a minimum of 2 hours. Decrease in dark phase temperature was found to cause a shift to earlier calling by females of all three species, however such shifts did not eliminate the differences in the peak periods of release of pheromones occurring among the three species. Although differences in the periods of pheromone release may not be the only mechanism involved in the reproductive isolation of the species belonging to the declarata group, differences in the calling periods are sufficient to account for the strong conspecific mating tendency exhibited by these species in laboratory tests and are certainly major components of reproductive isolation. The calling period of E. declarata (female) X E. rockburnei (male)(F1) hybrid females was found to correspond almost exactly with that of the maternal declarata stock. The calling period of the reciprocal cross was found to be extended, intersecting the calling periods of both parental species, however, the major activity peak occurred early in the scotophase. The female terminalia, including abdominal segments 8 and 9 + 10 of Euxoa were found to be similar to the typical type of terminalia described for higher Lepidoptera by Mutuura (1972) and have evolved to enable the females to oviposit in the soil. Structural modifications adapted for pre-oviposition digging include: the heavily sclerotized 8th tergite, anterior position of the copulatory opening on the 8th segment, the reduced 8th sternite, the elongate and highly flexible cuticle of the 8th intersegmental membrane, the heavily sclerotized dorsal valves, the eversible ovipositor, and extensive musculature throughout the ovipositor. The functional aspects of these features in oviposition are discussed and a description of the mechanics of oviposition extension, digging and retraction is given. Epidermal gland cells of the ovipositor were found in two specialized areas. One area is located in the ventral intersegmental membrane between segments 8 and 9 + 10. The "Class I" epidermal gland cells of this area were found to undergo progressive vacuolation for several days after adult emergence and comprise the sex pheranone gland. Glandular cells were also found to occur on the dorsal and lateral surfaces of the dorsal valves. These gland cells are typical trichogenous cells communicating with tubular setae. The function of large "droplets" within these cells and their presumed secretion is unknown at present.

The banded woollybear, Pyrrharctia isabella (J. E. Smith) was reared under laboratory conditions from egg to adult. Groups of individually reared larvae were kept at one of 8 photoperiods and in combination with one of 4 temperatures and were fed one of 6 artificial diets and fresh plantain. The times of moulting, head capsule widths, larval and pupal weights, and adult characteristics (fertility, sex) were recorded. Larvae were easily reared on bean, wheat germ, casein-wheat germ and fresh plantain, while casein, alfalfa, and plantain diets proved unsatisfactory. Development time increased at photoperiods longer than 14 hours. This increase was characterized by extra instars. The results suggest that scotophase may be more important than photophase in influencing development. Temperature did not affect overall larval development time although higher temperatures did slow the growth rate. Mature larvae were found to undergo an obligatory diapause in the Montreal-Ottawa area. Chilling was not an absolute requirement for diapause development, although mortality was high at higher temperatures. Diapausing larvae at 30°C were found not to be cold hardy. Increments in head capsule widths at each successive instar did not follow Dyar's rule. The relationships between voltinism and obligatory diapause, cold-hardiness and diapause, photoperiod, temperature and development, chilling and diapause development as well as Dyar's rule are discussed.

The present study highlights the practical application of a Piper-based botanical insecticide for controlling insect pests of the home and garden in urban areas in eastern Canada and northeastern North America. Biopesticides, including botanicals, can offer a safe and effective alternative to conventional insecticides for controlling major insect pests within an IPM program. Secondary compounds from the Piperaceae family, specifically the abundant isobutyl amides and lignans, have shown promise for insecticidal applications. A method for extraction and HPLC-MS analysis of Piper spp. was developed in order to allow quick and accurate measure of piperamide levels in P. nigrum, P. tuberculatum, West African Guinea pepper, P. guineense Schum and Thonn, and in less recognized species from Central America. Extraction of leaf and peppercorn material with 50:50 ethyl acetate and water provided a greater than 80% recovery of spiked piperine. HPLC analysis using a binary gradient of acetonitrile and water provided a clean separation of the major amide peaks between 5 and 12 min. The use of APCI-MS improved the detection limit 10 fold below the 2-ng limit of the HPLC-DAD method. Extracts from P. nigrum, P. guineense and P. tuberculatum were tested for efficacy against insects from five orders. Among the insect pests tested, the most sensitive species were, in order of increasing lethal concentration: Eastern tent caterpillar, Malacosoma americanum F. < European pine sawfly larvae, Neodiprion sertifer Geoffroy < spindle ermine moth larvae, Yponomeuta cagnagella Hubner < Viburnum leaf beetle larvae, Pyrrhalta viburni Paykull < stripped cucumber beetle adult, Acalymma vittatum Fabrcius < Colorado potato beetle adult, Leptinotarsa decemlineata (Say) < Japanese beetle adult, Popillia japonica Newman < hairy chinch bug, Blissus leucopterus hirtus Montandon. Greenhouse trials revealed that the pepper formulations also had a repellent activity thus protecting plant leaves from: (1) herbivory by lily leaf beetles, Lilioceris lilii (Scopoli) adults and larvae, and striped cucumber beetle, Acalymma vittatum F. adults and (2) oviposition by European corn borer, Ostrina nubilalis (Hubner). When an insecticide resistant strain of potato beetle larvae was tested with the P. tuberculatum extract, there was less than a two fold tolerance ratio compared to the 22-fold tolerance ratio to cypermethrin, a pyrethroid. An in vitro polysubstrate monoxygenase (PSMO) enzyme assay, using the substrate methoxyresorufin O-demethylation (MROD), determined that piperine, is responsible for inhibition of that specific enzyme. A subsequent toxicokinetic study determined that piperine is quickly eliminated from the exoskeleton (t1/2 = 16.5 h) and hemolymph (t 1/2 = 12 h) of the adult American cockroach Periplaneta americana L. after a topical application. (Abstract shortened by UMI.)

I examined transport characteristics of isolated Malpighian (excretory) tubules from the tobacco hornworm (Manduca sexta), a Lepidopteran which tolerates high levels of dietary nicotine, to test the hypothesis that alkaloid (e.g. nicotine) transport in the tubules is the consequence of a multidrug transport system similar to that mediated by P-glycoprotein in multidrug resistant cancer cells. P-glycoprotein substrates were applied to the basal (blood) side of the tubule and their subsequent distribution monitored. First, using cannulated tubules in a modification of the Ramsay assay, [3H]-vinblastine levels were measured in samples of bathing and luminal solutions. After 1 h, the tubules had concentrated vinblastine in the lumen 3-fold (from 1 muM). This accumulation was independent of a transepithelial potential, and was inhibited by verapamil, a P-glycoprotein inhibitor, and by nicotine. Second, fluorescent drugs, including daunomycin, rhodamine 123 and acridine orange, were applied to living tubules in a well of saline on a coverslip and examined by confocal microscopy. All of these drugs stained the cells of the tubule within 1 min, but, contrary to expectation, none of the drugs appeared in the lumen even after 1--2 h of incubation. Neither verapamil nor nicotine altered the pattern of daunomycin staining. The fast and intense Malpighian tubule staining was in sharp contrast to that of other tissues, which (for most drugs) stained lightly and only after prolonged exposures. Fluid-phase markers appeared to penetrate the tissue, but more slowly, and remained largely extracellular. The results suggest that the Malpighian tubule clears hemolymph of xenobiotics by passive filtration, active transepithelial transport involving a P-glycoprotein-like pump which handles alkaloids, and a scavenger-like absorption of compounds.