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Acute, sublethal and synergistic effects of some essential oil constituents against the Asian armyworm,… Hummelbrunner, Laurin Arthur 2000

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A C U T E , SUBLETHAL AND SYNERGISTIC E F F E C T S OF S O M E ESSENTIAL OIL CONSTITUENTS AGAINST THE ASIAN A R M Y W O R M , SPODOPTERA LITURA (LEPIDOPTERA: NOCTUIDAE). by LAURIN ARTHUR HUMMELBRUNNER B.Sc. McMaster University, 1987 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science in THE FACULTY OF AGRICULTURAL SCIENCES (Department of Plant Science) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA APRIL 2000 (S)Laurin A. Hummelbrunner, 2000 Abs t rac t II Public concerns over synthetic pesticides have stimulated the search for alternative methods of control. Plant essential oils are the odorous components and secondary metabolites that can be separated from other plant tissues through steam distillation. Generally regarded as plant defenses against herbivory, they have demonstrated acute toxicity to various insect species and are of interest for the development of 'ecologically friendly' pesticides. After identifying three main active ingredients in several samples of commercial fragrance oils against the Asian armyworm, Spodoptera litura, a number of related compounds were also tested for acute toxicity (measured as LD 5 0). The most toxic was thymol (LD50=25.4 jj.g/larva). Compounds were then tested for sublethal effects. Growth inhibition after topical application and feeding deterrence using leaf-disc choice experiments were determined. An LD 1 0 dose reduces growth by 20% on average three days after topical application, and the most deterrent compound to feeding was thymol, with a D C 5 0 of 85.6 u.g/cm2 leaf disc area. Since minor constituents in complex essential oils are thought to act as synergists, binary mixtures of the compounds were tested for synergy vis a vis acute toxicity and feeding deterrence. Trans-anethole acts synergistically with thymol, citronellal and a-terpineol. Based on these findings, several complex mixtures were developed and tested as leads for effective control agents. Candidate mixtures demonstrated good synergistic effects. The observed LD 5 0 of Mixture 3 was 40.6 u.g/larvae compared to an expected value of 74.6 (.ig/larvae. The result of this research is a proprietary product soon to enter commercial production. I" Table of Contents Abstract ii List of Tables iv List of Figures v List of Equations vi Acknowledgments vii 1. INTRODUCTION 1 l . l . PUBLIC PERCEPTIONS OF SYNTHETIC PESTICIDES 2 1.2. PLANT DERIVED INSECTICIDES 4 1.3. POTENTIAL FOR DEVELOPMENT OF OTHER BOTANICALS INSECTICIDES 6 1.4. ESSENTIAL OILS 8 1.5. ESSENTIAL OILS A s INSECTICIDES 10 1.5.1. Acute Toxicity to Insects 10 1.5.2. Stored-Product And Greenhouse Pests 12 1.5.3. Sublethal Effects of Essential Oils 14 1.5.4. Deter rency and Repellency of Secondary Compounds 15 1.6. OTHER USES OF ESSENTIAL OILS IN AGRICULTURE '. 2 0 1.6.1. Miticides 20 1.6.2. Nematicides 23 1.6.3. Fungicides and Antibacterials 23 1.6.4. Crop Protectants 24 l .7. EXPERIMENTAL RATIONALE 2 5 2. EXPERIMENTAL METHODS 29 2 . 1 . INSECTS 2 9 2.2 . CHEMICALS 2 9 2 .3 . BIOASSAYS 3 0 2.3.1. Acute Toxicity of Pure Compounds 30 2.3.2. Sublethal Growth Effects of Pure Compounds 31 2.3.3. Antifeedant Effects of Pure Compounds 32 2.3.4. Acute Effects of Binary Mixtures 34 2.3.5. Antifeedant Effects of Binary Mixtures 35 2.3.6. Acute and Synergistic Effects of Complex Mixtures 36 3. RESULTS 37 3.1 . ACUTE TOXICITY OF PURE COMPOUNDS 3 7 3.2. SUBLETHAL GROWTH EFFECTS OF PURE COMPOUNDS 3 9 3.3. ANTIFEEDANT EFFECTS OF PURE COMPOUNDS 4 0 3.4. ACUTE TOXICITY OF BINARY MIXTURES 4 2 3.5. ANTIFEEDANT EFFECTS OF BINARY MIXTURES 43 3.6. ACUTE TOXICITY OF COMPLEX MIXTURES 45 4. DISCUSSION 46 5. CONCLUSION 53 6. REFERENCES 55 List of Tables Table 1: Monoterpenoids in plant essential oils. 9 Table 2: Main constituents of four plants tested against three stored-product pests (after Sarac and Tunc, 1995a&b). 13 Table 3: Acute toxicities of essential oils (u.g/larva) to early 4 t h instar Spodoptera litura larvae 37 Table 4: Acute effects of binary mixtures of essential oils to early 4 t h instar larvae of Spodoptera litura, and measures of interactions. 42 Table 5: Comparison of D C 5 0 ' S for binary mixtures with pure compounds. 44 Table 6: Composition and acute toxicity of some complex mixtures of essential oil compounds 45 V List of Figures Figure 1: Chemical structures of some of the essential oils tested. 29 Figure 2: Cuticular damage manifested after the moult following topical application of a-terpineol. 38 Figure 3: Growth deterrence at 3 and 6 days after topical application of pure compounds at sublethal doses. 39 Figure 4: Feeding deterrence of essential oils against 5 t h instar larvae of Spodoptera litura. 41 Figure 5: Feeding deterrence of binary mixtures and pure compounds To 5 t h instar larvae of Spodoptera litura 43 vi List of Equations Equation 1. Calculation of deterrence index (percent)(lsman et al., 1990)... 33 Equation 2: Calculation of expected mortalities from binary mixtures of known dosages (Trisyano and Whalon, 1999) 34 Equation 3: Calculation of values for determining additive, synergistic or antagonistic behaviour of binary mixtures. (Trisyano and Whalon, 1999).35 Equation 4: Calculation of expected additive mortality in complex mixtures based on LD 5 0 ' s of components (Hewlett and Plackett, 1979 in Don Pedro, 1996). 36 Vll Acknowledgements I would like to thank my supervisor, Dr. M. Isman, for his faith, direction and patience; Steve Bessette, of Ecosmart Technologies Inc., for his infectious enthusiasm; Ecosmart Technologies Inc. for funding the research and my wife, Jackie, for her love, support and patience mixed with a solid dose of 'get on with it!' 1 1. Introduction Plants produce a vast array of substances that have been labeled as secondary metabolites, with over 20 000 identified to date (Berenbaum and Seigler, 1992), and probably many more still undescribed. All phytophagous insects will encounter them in the course of their lives and these substances are frequently the mediators in plant-insect relationships. They may be used by insects as semiochemicals functioning in host recognition in a variety of manners, including suitability of oviposition sites or food sources. They can function in a multitrophic manner, such as when parasitoids utilize them as signals that suitable host insects may be found. Physiologically, some are utilized by the insects themselves and stored in specialized cells as chemical defenses against predators or as pheromones to attract others of their species. More recently, they have been studied as the causative agents in complex insect/plant co-evolutionary pathways in what amount to plant-herbivore arms races. Evidence that many of these chemicals play a major role in the palatability of plants to insects has led to the hypothesis that the majority of the secondary compounds are intrinsic to the plant's defense against natural enemies. 2 1.1. Public Perceptions of Synthetic Pesticides In 1995, annual U.S. pesticide expenditures totaled $11.3 billion US, and 31 new active ingredients were registered as pesticides. World wide sales totaled $37.7 billion, of which insecticides accounted for 33% (Aspelin, 1997). It is clear that the quantity and quality of food and fiber production currently produced in North America could not be maintained without the aid of pesticides to control weeds, fungi, nematodes, and insects. Indeed, it was the development of synthetic pesticides in the 1940's that eventually allowed farming on the vast monoculture scales that we have become used to. Home and garden use of pesticides is also extensive. Insecticides can be extremely effective in controlling pest levels, rapidly reducing infestations to manageable or insignificant levels, and maintenance programs can be used to keep populations at acceptable levels. They are generally easy to use and provide excellent cost/benefit ratios under most circumstances. When label instructions and proper safety measures are taken, the human health risks are mitigated. Given the widespread use and value of pesticides, the number of accidental deaths is low, accounting for only 0.2% of accidental poisonings or 14 deaths in the US in 1988, and these primarily from misuse. Compare this to 48 deaths from naturally occurring compounds in the same year. There is, however, a real toxicity problem in 3rd world countries, with approximately 200 000 deaths annually (WHO, 1992). In other parts of the world, polychlorinated hydrocarbon pesticides are 3 still in widespread use where economic and health benefits outweigh perceived risks (Coats 1994). However, indiscriminate use, improper application and poor planning have lead to an extremely poor perception of current pesticide usage, some of this quite justified. The public is generally familiar with tales of ecological disasters, from devastating effects on non-target species such as birds and fish, to poisoned water tables and finally, human mortalities. Most of this distrust stems from the widespread use and misuse of first generation synthetics such as DDT (dichlorodiphenyltrichloroethane) and various polychlorinated compounds. For various reasons, long term effects were not considered or were poorly documented, and there still remains great suspicion over these concerns. In the case of DDT, although the acute toxicity was very low for vertebrates, bio-accumulation and long half-lives of metabolites combined with public perceptions of misuse and some notable environmental effects eventually forced an end to its usefulness. People have a poor understanding of pesticides and are concerned over involuntary exposures in the form of residues (Coats, 1994), despite stringent controls, yet we discount hazards from everyday substances such as benzene from gasoline. Fears are fueled by media inaccuracies and even technological advances, where contamination can now be measured in parts per trillion as opposed to per million. These public fears have translated into legislation and a bureaucracy that has resulted in the withdrawal of an increasing number of 4 pesticides. Couple these losses with those due to the development of insect resistance, and there is the prospect of several insect 'crises', where there will be no effective controls available. All of these fears have effectively created a burgeoning market for "natural" pesticides. There has been a concomitant increase in awareness and development of alternative control methods encompassed by the catch-all phrase 'integrated pest management', within which natural pesticides may play an important role. The recent strong emergence of the "organic" food market presents another impetus. In the U.S., the National Organic Standards Board (NOSB) was created to help oversee the implementation of the Organic Foods Production Act of 1990, in which lists of permitted substances for use in organic farming were created. These include citrus products, herbal preparations, neem and its extracts, plant extracts, botanical insecticides, and wood wastes (Quarles, 1998). Further, a number of least-toxic ingredients for pesticides are exempted from E P A registration, including eugenol and various plant essential oils (Quarles, 1996). 1.2. Plant Derived Insecticides Botanical insecticides are plant extractives that are directly utilized as insecticides. They have a long history of use. Of the few currently registered for use, there is generally low vertebrate toxicity and very little environmental persistence. 5 Pyrethrum, probably the best known, dates back to Persia for known use and was introduced into Europe in the early 1800's. Derived from flowers of Chrysanthemum cinerariaefolium, it accounts for the vast majority of the world botanical insecticide market, and generated extensive research into synthetic pyrethroids because of its biodegradability and low mammalian toxicity. The active principles are pyrethrin esters which act as neurotoxins that blocki sodium channels in neural membranes, producing fast knockdown in insects. Produced primarily in Kenya and Tanzania, there is currently a world-wide shortage of this product. Because ultraviolet light rapidly degrades it, pyrethrum is generally limited to indoor or greenhouse uses, and is mixed with a synergist piperonyl butoxide to inhibit detoxicative enzymes in insects. Rotenone, derived from tubers of the tropical legumes Derris and Lonchocarpus species, has been used since the 1600's as a piscicide. As an insecticide, although slow acting, it causes immediate cessation of feeding, acting as a mitochondrial poison blocking electron transport. It is primarily used in home and garden care as a broad spectrum dust but also enjoys use in the organic food industry. The ground wood from the tropical shrub Ryania speciosa yields the muscle poison ryania while sabadilla, whose active ingredients, veratrum alkaloids, 6 are axonic poisons, comes from the powdered seeds of a tropical lilly, Schoenocaulon officianale. Neem from the seeds of the large shade tree Azadirachta indica native to India contains the principal active ingredient azadirachtin, a triterpene. It acts as an insect growth regulator, blocking the release of ecdysones in moulting, acts as an anorexic and is the most potent insect antifeedant discovered to date. Used in a variety of manners including indigenous medicine, as a spermicide, in toothpastes and as an antiviral in treatment of cervical cancer, it is essentially non-toxic to vertebrates. 1.3. Potential for Development of Other Botanicals Insecticides Combined with current cultural pressures, the development of neem has spurred research into sources of other natural pesticides. Worldwide, laboratories screen thousands of plant species in search of bioactive compounds. The majority of work has been centered on the family Meliaceae. The genera Azadirachta (to which neem belongs), Melia, Aglaia and Trichilia, have produced a significant number of bioactive compounds, with the likelihood of a considerable number more (Isman, 1995). Other research screens flowering plants: for instance, senescent flowers of the yellow azalea, Rhododendron molle japonicum, produce an insecticide used on a local basis in China (Isman, 1997). 7 Other useful compounds may be derived from waste by-products of the food and agriculture industries, particularly as seeds, often quite rich in secondary metabolites, are generally a significant part of this waste. Citrus limonoids, for example, have demonstrated antifeedant properties (Alford et al. 1987) and acetogenins from paw paw wood, Asimina triloba, are toxins (McLaughlin et al. 1996). Similarly, waste products such as wood chips are a potential source of bioactive compounds. Bark from chinaberry trees, Melia toosendan and Melia azedarach yields an antifeedant and insecticidal product (Chen et al. 1995), while bark of the ornamental shrub Aglaia odorata yields rocaglamide, an antifeedant and slow-acting toxin (Isman, 1995). One of the most active areas of research is plant essential oils. Various essential oils have demonstrated efficacy as repellents, particularly citronella which is registered for use as a mosquito repellent, and limonene in animal flea shampoos. This group of compounds appears to offer great potential as fumigants against stored-product pests, as well as nematicidal and fungicidal uses, and more recently, research includes field crop protection. There are significant complications in developing botanicals as viable products (Isman, 1997). First, efficacy under lab conditions frequently does not translate under field conditions. Selectivity is a two-fold concern: toxicity to non-target insects (pollinators and predators) and vertebrates is a concern, but the spectrum of activity against pests must be broad enough to justify the 8 costs of development and production. Persistence must strike a balance between efficacy and environmental contamination. Although they may be effective, compounds that are not widely available, either from raw plant material or as biosynthetics, have little future. As many botanical insecticides contain a complex mixture of active ingredients (e.g. neem has a dozen azadirachtin analogues), there is a potential problem in standardizing the amount of active ingredients, hence quality control becomes difficult, as does government regulatory approval. On the other hand, complex mixtures are likely to be more efficacious than the same amount of pure compound given the likelihood of synergy amongst the constituents. There are likely to be difficulties in patent applications for numerous reasons. Finally, given that the large multinational corporations have no interest in botanicals other than as leads to new synthetics, the major costs of introducing a new product that satisfies all of the regulatory requirements, potentially running into the millions, can be prohibitive. 1.4. Essential Oils Plant essential oils are the odorous components and secondary metabolites that can be separated from other plant tissues through steam distillation. Most are mixtures of terpenoids, some quite complex, including mono- and sesquiterpenes (such as a-terpineol and pulegone) and biogenically related phenolics or monophenols (such as thymol, carvacrol and eugenol). They are commonly used as fragrances and as flavouring food additives, and more 9 recently, in aromatherapy. There may be a hundred or more constituents forming a complex mixture. Oil of mint, for example, has more than 200 compounds found in low concentrations (Harwood et al., 1990). There are, however, usually one or a few that account for the majority of the essential oil by weight. Herbs and spices are particularly noted for their essential oils (see Table 1). Table 1: Monoterpenoids in plant essential oils. Compound Plant Source Scientific Name frans-anethole anise, fennel Pimpinella anisum a-terpineol cardamom, marjoram rosemary Amomum subulatum, Origanum vulgare, Rosmarinus officinalis carvacrol marjoram, oregano, thyme, American horsemint 0. vulgare, Satureja thy rubra, Coridothymus capitatus, Monarda punctata cinnamaldehyde cinnamon Cinnamonum spp. cinnamic acid vanilla Vanilla spp. 1,8-cineole bay, cardamom, pennyroyal, sage, eucalyptus Laurus nobilis, A.subulatum, Hedeoma pulegioides, Salvia fruticosa, Eucalyptus camaldulensis citronellal geranium, lemongrass Geranium spp., Cymbopogon citrata eugenol allspice, basil, bay P.dioica, Ocimum spp., Lnobilius limonene anise, coriander, celery, pines, rosemary, peppermint P.anisum, Coriandrum sativum, Apium graveolens, Pinus spp., R. officinalis, Mentha piperita pulegone Pennyroyal, peppermint H. pulegioides (Mentha pulegium) , Mentha piperita terpinen-4-ol false indigo Amorpha fruticosa thymol marjoram, eucalyptus, thyme 0. vulgare and O.majorana, E.camaldulensis, Coridothymus capitatus, The uses of essential oils are quite varied. They have been documented as medicinal agents, as insect repellents, insecticides, fungicides, miticides, piscicides, and even as bird repellents (Avery et al., 1996), and rodent repellents (Ahn et al., 1995). Most have minimal vertebrate toxicity, and most of the essential oils of potential use are found on the U.S. Food and Drug Administration's GRAS (Generally Regarded as Safe) list and are exempt from data requirements for toxicity (Isman, 1999). 10 1.5. Essential Oils As Insecticides 1.5.1. Acute Toxicity to Insects The biological properties of essential oils against insects have been known for decades as demonstrated by R.C.Bushland in a paper from 1939 entitled "Volatile oils as ovicides for control of the screw worm, Cochliomyia americana" in the Journal of Economic Entomology, (pp 430-431). Herbivorous insects are almost entirely dependent on their host plants for nutrition, which means the insects are constantly exposed to dietary toxins or other plant chemical defenses. Any compound may have deleterious effects on insects that come into contact, but toxicity is defined broadly as the outcome of an interaction between a toxin and sensitive target sites in an organism that results in some physiological lesion or membrane disruption (Isman, 1992). Secondary metabolites can act as toxins in a variety of manners. Some will interact with specific receptors such as acetylcholine while others interact with macromolecules present in a wide variety of organisms, with general cellular targets such as cellular membranes, DNA or proteins (Berenbaum, 1991). The insect nervous system, endocrine system, heart, gonads and epidermis have all been demonstrated as targets of plant allelochemicals. 11 There is no rule that allows one to accurately predict the effects of any compound against any single insect. Indeed, compounds that act as toxins or deterrents to some, have no effect or even act as attractants to others. As Janzen (1979) puts it: "with respect to a particular organism a toxin...is to be defined by its effect rather than by some intrinsic characteristic of the molecule." This is due to the remarkable ability of insects to develop resistance to plant toxins. Resistance is the pre-adaptive ability of a population to overcome the effects of a toxin over time. The life history traits of high fecundities and rapid generational times allow insects to respond to toxins quite rapidly in evolutionary time, depending on the degree of selection pressures. Experience with pesticides has demonstrated that resistance may develop within ten to fifteen generations. For example, in the case of aldrin, the onion root maggot fly, Hylemya antiiqua, corn seed worm, Hylemya platura, and cabbage maggot worm, Hylemya brassicae all developed resistance within this generational span; for the multivoltine root maggot fly (three or four generations per year) it occurred within five years (Harris, 1972). The literature is replete with examples of the acutely toxic effects of essential oils on insects, including such economically important pests as the western corn rootworm, two-spotted spider mite, various bruchid beetles, cockroaches, and fire ants (Lee et al., 1997; Regnault-Roger et al., 1993; Ngoh et al., 1998; Karr and Coats, 1988). Household pests such as the 12 housefly and cat flea are also adversely effected. Tests of d-limonene against a wide range of insects indicated variable toxicities with further variation in the routes of exposure, including topical and fumigant action (Coats etal., 1991). Essential oils generally produce acute toxicity in a dose-dependent manner, although non-lethal doses can reduce fecundity or increase developmental times. Such responses were demonstrated against the confused flour beetle, Tribolium confusum (Sarac and Tunc, 1995b), the maize weevil, Sitophilus zeamais, the lesser grain borer, Rhyzopertha dominica, and the angoumois grain moth, Sitotroga cerealella (Bekele et al., 1996). 1.5.2. Stored-Product And Greenhouse Pests There is a considerable body of literature documenting the efficacy of essential oils in controlling stored-product pests (e.g. Shaaya et al., 1991; Sarac and Tunc, 1995a&b). Losses of food grains to insect pests during storage is a serious problem - a survey in 1985 indicated that 20-25% of stored wheat in North America was infested (White et al., 1985 in Singh et al., 1989). In Africa, the bulk of grain production comes from small-scale farm holdings that traditionally use various plant materials mixed in with stored grain products for protection against pests (Bekele et al., 1996). The fumigant action of volatile essential oils is what makes them so effective in closed-in storage conditions. 13 Trans-anethole is the principal constituent of star anise, llicium verum. It has demonstrated contact toxicity and fumigant toxicity to Tribolium castaneum, Sitophilus zeamais, Rhyzopertha dominica and Tenebrio molitor (Xu et al, 1996; Ho et al., 1997). Sarac and Tunc (1995 a&b) were interested in oils from plant species that harbour few insects in the wild. Anise, eucalyptus, and two species of thyme were tested against the confused flour beetle, Tribolium confusum, the rice weevil, Sitophilus oryzae, and the Mediterranean flower moth, Ephistia kuehniella. Anise was an effective fumigant against all three pests, but the flour beetle was unaffected by the other three plant oils. Fifty percent residual activity was demonstrated after four weeks. Table 2 shows the primary constituents of the essential oils in order of greatest concentrations. Table 2: Main constituents of four plants tested against three stored-product pests (after Sarac and Tunc, 1995a&b). Plant Main Constituents anise Pimpinella anisum anethole, >cineole, >carvacrol eucalyptus Eucalyptus camaldulensis cineole, >limonene, >terpinen-4-ol, > carvacrol thyme Thymbra spicata carvacrol thyme Satureja thymbra thymol and carvacrol Essential oils have demonstrated persistence as controls: oil of bergamot mint, Mentha citrata, with major constituents linalool and linalyl acetate, was effective against rice weevils, Sitophilus oryzae, thirty days after application and oil of chir pine, Pinus longifolia (a-pinene, p-pinene and carene), sixty days later (Singh et al. 1989). 14 Greenhouse pests can be even more sensitive to vapours than stored-product pests. Anethole, carvacrol, cineole, pulegone and terpinen-4-ol were all effective as controls against the carmine spider mite, Tetranychus cinnabarinus, and the cotton aphid, Aphis gossypii in greenhouse trials, with demonstrated growth effects and fecundity effects at sublethal concentrations (Tuni and Sahinkaya, 1998). Thyme oil and rosemary oil have been demonstrated to be strong repellents of several aphid species (Hori, 1998). Individual constituents of those oils include 1,8-cineole which repels Neotoxoptera formosana and linalool which repels the green peach aphid, Myzus persicae. 1.5.3. Sublethal Effects of Essential Oils Sublethal effects of terpenoids are likely to be more important and more stable as evolutionary strategies in terms of plant defense against herbivory than acute toxicity. Toxic allelochemicals generally are found in sublethal concentrations in plants such that selection pressure from them is likely to be diffuse (Isman 1992). Increased feeding times, longer developmental periods and various developmental complications can substantially affect herbivore life histories. If Bernays is correct, more than 90% of insect mortality occurs during feeding (Bernays, 1998). For example, the variegated cutworm, Peridroma saucia, is subject to several adverse effects from terpenoids in the essential oil of peppermint. Severe feeding deterrence, delayed growth and 15 failed eclosion from the pupa have been noted. Behavioural effects include initial hyperactivity followed by a substantial period of quiescence (Harwood et al., 1990). The sublethal behavioural effects of monoterpenoids are similar to those produced by chlordimeform, a formamidine insecticide, which causes profound behavioural changes, including dropping off host plants and severe anorexia. Life history traits such as fecundity may also be affected: adults of the southern armyworm, Spodoptera eridania, had lower fecundity when fed pulegone as larvae (Gunderson et al., 1985). 1.5.4. Deterrency and Repellency of Secondary Compounds Taste in insects is more accurately described as contact chemoreception, in which specialized sensory cells sheathed in hairs or cones of cuticle (collectively the sensillum) transmit receptor potentials generated from chemical cues. The antennae house olfactory sensilla, while contact chemoreceptors may be found on the tarsi, antennae and ovipositor as well as on the mouthparts. Substances that do not cause movement away from plants but still prevent feeding or oviposition are termed deterrents while repellents actually cause herbivores to orient movement away from the source (Pedigo 1989). Examples of insect feeding deterrence are well documented, and there are suggestions that feeding deterrents are more important in plant recognition than are phagostimulants (Schoonhoven et al, 1992). This conclusion is also supported by the observation that despite the staggering number of insects in the world and their potential for population 16 growth, the biota is dominated by plants, suggesting that most plants must contain deterrents for most insects. Gunderson et al. (1985) note that the concentrations of compounds in mint are well below those required for acute toxic effects to various potential herbivores, but are found at concentrations which are effective for feeding deterrence. Toxins will often fulfill the role of deterrents, and their effectiveness can span different orders of insects. Although few extensive studies have been done, a single insect species may be deterred by hundreds of different deterrents. Of the small fraction of known secondary metabolites tested, over 100 compounds have been shown to be deterrent to Locusta migratoria (Bernays and Chapman 1978). But, just as toxic effects differ between insect species, so do deterrents, and even though deterrents may be present, a plant can still be acceptable as a host, depending on the balance of positive and negative information from all sources. This will include chemo-information, the internal status of the insect and various environmental factors (Miller and Strickler, 1984; Isman, 1992; Renwick and Radke, 1987; van Loon, 1990). The co-existence of deterrents and stimulants in a plant is demonstrated by wormseed mustard, Erysiumum cheiranthoides. The stimulatory activity of glucosinolates is outweighed by the potent deterrence of cardenolides for the cabbage butterfly Pieris rapae, whereas the related butterfly, P. napi oleracea accepts the mustard as an oviposition host (Huang et al, 1993). Similarly, P. rapae rejects candytuft, Iberis amara, which contains attractant cardenolides 17 as a host because of the potent antifeedant cucurbitacins it contains (Sachdev-Gupta et al., 1993). Terpenoids are widely documented as feeding deterrents, and in North America, citronellal is commonly used in insect repellents, as a component of sprays, lotions and candles. A single species of insect may be deterred by a number of different compounds. The northern fowl mite, Ornithonyssus sylviarum, for instance, considered one of the most important arthropod pests of poultry in the U.S., feeds on the blood of its host. It is deterred by a number of terpenoids, including citronellal, limonene, pulegone, 1,8-cineole and terpinen-4-ol. Citronellal was effective as a systemic deterrent when incorporated into the chicken feed, while other compounds were suitable as cage treatments (Carroll, 1994). Deterrence or repellence, in addition to fumigant toxicity has been demonstrated repeatedly against stored-product pests (Sarac and Tunc, 1995a). Foliage of the pepper tree, Schinus molle, is traditionally used in Ethiopia as a housefly repellent, where the leaves are spread on eating tables and the branches are draped overhead. Powder from the berries can protect stored bean seeds from bruchid beetle (Acanthoscelides spp.) infestation for up to three months (Wimalaratne, et al., 1996). 18 Monoterpenoids have been demonstrated to act as oviposition deterrents. Pine oil containing carene, cymene and limonene reduced oviposition by female onion maggots, Delia antiqual, responding to onion oils by 72% (Ntiamoah et al., 1996). The essential oil of the perennial mint, Tetradenia riparia, is repellent to ovipositing females of bruchids. Although the concentrations found in plants are not high enough to cause adult bruchid mortality, when used in stored-products, repellency provides protection against developing populations, with significant reductions in emerging adults (Harwood et al., 1990). As with toxicity, repellency cannot be reliably predicted. One species may be attracted to compounds that repel closely related species. For many species of beetles that attack conifers, the initial attraction to host trees is often due to volatile monoterpenes emitted by the trees. The volatility of terpenes contributes to their effectiveness as long range kairomones. It is likely that the terpenes which now function as attractants originally functioned as defensive chemicals or repellents. It is hypothesized that through time, various herbivores were able to overcome the plant defenses and then utilize the characteristic tree chemical as a cue for a suitable host. The turpentine beetles, Dendroctonus terebrans and D. valens, appear to locate suitable hosts primarily by means of host terpenes (Renwick, 1988). Terpenes may also 'alert' insects to stressed or injured trees. Pine shoot 19 beetles, Tomicus piniperda, detect host susceptibility by means of terpenoids released from the oleoresin exuding from damaged host tissues (Byers et al., 1985; Schroeder, 1988). Terpenes play an important role in the mass attack of bark beetles in conifers. Once the initial beetle colonists have landed on a suitable host, attracted by host cues or otherwise, they produce volatile aggregation pheromones to attract other beetles for a synchronized mass attack. These aggregation pheromones are frequently allylic oxidation products of monoterpenes found in the host tree: i.e. several Ips species synthesize (+)-ipsdienol, an allylically hydroxylated derivative of the conifer monoterpene myrcene. The actual mode of production may be directly by the beetles themselves, or indirectly by microbial gut symbionts in the beetles, or through abiotic processes (Pickett, 1991). When the beetles or their symbionts carry out the synthesis, the reaction is usually a detoxification reaction, the products having been evolutionarily selected as pheromones (Raffa etal., 1993). The mountain pine beetle, Dendroctonus ponderosae, detoxifies a-pinene into the aggregation pheromone frans-verbenol, and other host terpenes act as synergists for this attraction. The aggregation pheromones frans-verbenol and verbenone of the western pine beetle, Dendroctonus brevicomis, are oxidation products of the conifer terpene a-pinene. 20 Predators and parasitoids can also utilize host terpenes as chemical cues for locating prey. Parasitoids of larval southern pine beetle, Dendroctonus frontalis, are attracted to host tree terpenoids. Other examples exist where host terpenes act in synergy with prey pheromones to attract predators (Dixon and Payne, 1980). The same compound can even have different effects on an insect depending on its concentration. Pine oil is an effective repellent to various Diptera, including the housefly, at high concentrations, but can act as an attractant at low concentrations (Maganga et al., 1996). 1.6. Other Uses of Essential Oils in Agriculture 1.6.1. Miticides The honeybee, Apis mellifera, enjoys widespread use for honey production and as crop pollinators in North America. Colonies here and in Europe are being infested with Varroa jacobsoni, an ectoparasitic mite, and the tracheal mite, Acarapis woodii. Mortality in infected colonies can approach 100% (Calderone et al., 1997). The efficacy of the only existing registered varroacide, the pyrethroid fluvalinate, has decreased due to the development of resistance in the mites. It also has the significant drawback of wax and honey contamination. With both lethal and sublethal effects on mites, significant efforts to develop controls based on monoterpenoids have been made in the industry. 21 Seven monoterpenoids tested for toxicity to tracheal mites and to their hosts demonstrate that potencies vary considerably and minor structural differences can elicit major differences in toxicity. Menthol, currently registered for use against tracheal mites, was more toxic to bees than other monoterpenoids except thymol. Although thymol controls adult and larval mites, it was also the most toxic to bees, with a low margin for safety between controlling mites and killing bees. It is also particularly dependent on temperature,, with . increased bee mortality at higher temperatures, presumably from increased volatility. Citral, which functions as an alarm pheromone in several mite species, killed all stages of tracheal mites with a consistent margin of safety at all temperatures (Ellis and Baxendale, 1997). A product, Apilife VAR, composed of 76% thymol, 16.4% eucalyptol and 3.8% camphor was developed for use in Europe against the varroa mite. Under optimal conditions (ambient temperature between 15-20°C), 95% control was achieved over an eight week treatment. Further, there were no residues found in the honey or wax (Imdorf et al., 1995). It was noted that an overdose resulting in high concentrations of thymol in the air (thymol is volatile) causes high bee mortality or repels the bees from the hive. A more stringent testing of Apilife VAR yielded somewhat lower efficacies, in the range of 70% over 22 five weeks (Gregorc and Jelenc, 1996). A blend of citronellal and thymol was demonstrated to be highly active against the tracheal mite, but also varied with environmental conditions (Calderone et al, 1997). Kraus et al., (1994) attempted to avoid non-target toxicity to bees by using lower doses of essential oils to inhibit the odour orientation used by the mites to complete their reproductive cycle. Oils of citronella, lavender, geranium and rosemary were effective, and bee mortality could be avoided at concentrations under 10%. A cautionary lesson arises from eugenol. Although acutely toxic to varroa, it is noted that oils of clove and cinnamon, which are high in eugenol, were found to be mite attractants (Kraus et al., 1994). However, this type of attraction might be exploited in terms of drawing a pest to effective control agents. A product called Cinnacure™, has received U.S. EPA approval for use in greenhouses for control of western flower thrips, Franklinella occidentalis, greenhouse whiteflies, Trialeruodes vaporariorum , two-spotted spider mites, Tetranychus urticae, and various aphid species. Derived from cinnamon oil, high in cinnamic aldehyde, it is advertised to be fast-acting with low mammalian toxicity and a short residual period. It does come with the warning that it is phytotoxic to poinsettas and roses (Anonymous, 1998). 23 1.6.2. Nematicides Nematodes are microscopic roundworms that live in soil or water. Parasitic nematodes can cause significant losses to fruits, vegetables and field crops. Various species are root parasites that leave plants susceptible to other pathogens. They tend to have an impermeable cuticle which makes penetration characteristics important in nematicides. Normally applied by soil fumigation, synthetic nematicides can be very toxic to vertebrates, are persistent, have a broad spectrum of action, and are often highly flammable or explosive. The application method makes water contamination a serious issue. Synthetic nematicides are being phased out in increasing numbers, creating an urgent need for safe and environmentally benign products. Monoterpenoids have been tested against nematode pests with good results (Tsao and Yu, unpublished data; Sangwan et al., 1990). 1.6.3. Fungicides and Antibacterials The advent of systemic organic fungicides in 1967 offered eradication of extant infections as well as the protection against future infections offered by existing inorganic fungicides. There is recent evidence, however, that organic fungicides pose more of a carcinogenic risk than other pesticides. Further impetus for new products is that several existing synthetics are no longer viable given the development of resistance to them. The fungicidal properties of essential oils have been known for a while, going back to the observation that garlic extracts contain potent fungicides (Wilson et al. cite Ark and 24 Thompson, 1959; and Singh et al.,1980). Muller-Riebau et al. (1995) tested essential oils of wild growing herbs against several fungal species. The oil of anise, Pimpinella anisum, high in frans-anethole content, was strongly inhibitory, as was thymol, while carvacrol and eugenol were moderately effective in comparison to six synthetic fungicides. They suggest that the "possibility of using essential oils of aromatic plants... as natural fungicides approaching realistic control levels depends on their phenolic content of thymol and carvacrol". In addition to thymol and eugenol, cinnamaldehyde, pulegone and carvacrol have been found to be effective, badly damaging fungal mycelia (Kurita et al., 1981; Economou and Nahrstedt, 1991; Arras et al., 1997). Synthetic versions of frans-anethole, carvacrol, eugenol and thymol demonstrated effective control of a food spoilage yeast (Curtis et al, 1996). Wilson et al. (1997) suggest that plant essential oils are likely to provide a wide variety of alternatives to synthetics. Essential oils from Ocimum spp. in Congo were more effective than several reference antibiotics against common bacteria Staphylococcus aureus, Streptococcus faecalis, Escherichia coli, Salmonella spp., Klebsiella pneumonia and Candida albicans (Ndounga and Ouamba, 1997). 1.6.4. Crop Protectants There has been some debate on the practicality of using feeding deterrents as crop protectants (Bernays, 1983), but, all else being equal, compounds 25 with significant deterrence may be effective as protectants. One complication in utilizing essential oils as crop protectants is their phytotoxicity. Emulsions containing greater than two percent oils can be phytotoxic, although this is true of vegetable and mineral oils as well (Isman, 1999). This may be overcome in proper formulation or if the essential oils are effective at lower concentrations. 1 .7. Experimental Rationale Stemming from a fortuitous discovery that fragrance oils added to a common household pesticide significantly increased the product's efficacy, Dr. Isman entered into a proprietary arrangement with a US company, Ecosmart Technologies Inc., to help develop natural pest control products. Initially, we evaluated the acute toxicity of several different fragrance oils supplied by the firm. These samples were of varying complexity, with up to 71 identified compounds. Tests of the most likely active ingredients demonstrated three acutely toxic compounds, and three other major ingredients of lesser toxicity. Early experiments indicated comparable toxicity for these ingredients to the Asian armyworm (also known as the tobacco cutworm), Spodoptera litura, and several other insect species, including the variegated cutworm, Peridroma saucia, the migratory grasshopper, Melanoplus sanguinipes, the milkweed bug, Oncopeltus fasciatus, and the two-spotted spider mite, Tetranychus urticae. 2 6 This information led to acute toxicity tests of other closely related compounds. Acute toxicity is measured as LD 5 0: the dose required to kill 50% of a sample population. This is an accepted method that has been standardized by both the FAO (Food and Agriculture Organization) and the ESA (Entomological Society of America). These values are used for comparing efficacy of compounds and as guidelines in considering public health safety. This testing resulted in a suite of compounds that have become the focus of subsequent studies. The Asian armyworm, Spodoptera litura is an economically important pest of vegetable and tobacco crops in southeast Asia, India, China and Japan. This lepidopteran is particularly suited to research because of its ease of rearing and the ability to obtain large numbers of larvae of synchronous size and age. For insecticide discovery, we believe it to be a conservative model, in that this species seems to require higher doses for acute toxic effects relative to other insect species, including German cockroaches, houseflies and diamondback moths. Data from Ahn et al. (1995) in tests of carvacrol against a range of species including S. litura supports this conclusion. Largely as a result of registration difficulties, synthetic control agents have been developed and marketed as pure compounds, yet there are a variety of reasons to suggest that complex mixtures would be more effective, including synergy, desensitization and resistance. Since plants usually present 27 defenses as a suite of compounds, not as individual ones, it is thought that . the minor constituents found in low percentages may act as synergists, enhancing the effectiveness of the major constituents through a variety of mechanisms. It has been demonstrated that repeated exposure to antifeedants can result in desensitization: an increasing acceptability to insect herbivores. This appears to be more likely when a single antifeedant is applied, especially if it is a weak stimulus. Complex mixtures are more likely to prevent this. Because of their short generation times, insects demonstrate a remarkable ability to develop resistance to pure compounds, but it is unlikely that an individual would possess multiple genes for resistance. The status of many essential oil compounds as EPA registration exempt, either as least-toxic ingredients or on the GRAS list (Generally Regarded as Safe) makes them good candidates for use in developing complex mixtures. For these reasons and others, synergy amongst compounds was a key factor in the development of such mixtures. Initial tests for synergy were carried out in binary mixtures, moving to more complicated tertiary ones, and finally, to a number of even more complex mixtures, upon which a control product was based. 28 The present work was carried out as part of the experimental regime conducted in association with other laboratories supported by Ecosmart Technologies to develop natural pesticide products based on complex mixtures of essential oil constituents. 29 2 . Experimental Methods 2.1 .Insects Bioassays were conducted using larvae of the Asian armyworm, Spodoptera litura (Fab), obtained from an established laboratory colony (more than 50 generations; out-crossed once). Insects were reared on an artificial diet (No. 9795, BioServ Inc., Frenchtown, NJ) supplemented with finely ground alfalfa to improve acceptability, and vitamins (No. 8045, BioServe Inc.). The colony was reared at 25°C under a L16:D8 photoperiod. 2 . 2 . Chemicals Initial fragrance oils produced by ArrylEssence Inc., Atlanta, were received from EcoSmart Technologies, Atlanta, Georgia. Pure compounds were purchased from Sigma Chem. Co., St.Louis, Mo., and Aldrich Chem. Co. Analytical grade acetone was used as the carrier. Figure 1: Chemical structures of some of the essential oils tested. Monoterpenes O l imonene a - te rp ineo l pulegone terpinen-4-ol citronellal Phenolics eugenol thymol carvacrol cinnamic alcohol trans -anethole 30 2.3 .B ioassavs 2.3.1. Acute Toxicity of Pure Compounds Acute toxicity (measured as mortality after 24 hr) of essential oils was determined by topical application to early 4 t h instar larvae (15-20 mg bodyweight). Initial screening to approximate the active dose range determined a range of five doses that were used to establish the LD 5 0 . Four replicates of ten larvae were tested per dose. Larvae were individually weighed prior to treatment. Essential oil compounds were prepared using acetone as a carrier such that each larva received 1 uJ of oil solution per treatment, with acetone alone as the control. Doses were applied to the dorsum using a repeating topical dispenser attached to a 50 of syringe. Since acetone dissolves some plastics, insects were treated in glass petri dishes, with larvae left for two minutes to allow the carrier to evaporate. All ten treated larvae from each replicate were then transferred onto a 2 cm3 block of diet placed in a 5 cm diameter plastic petri dish (each replicate was transferred to a separate dish). Treatment groups were then placed in sealed plastic boxes lined with moistened paper towels and held for 24hrs in a growth chamber (L16:D8, 26°C). Mortality was recorded after 24hrs. Death was recorded if larvae did not respond to prodding with forceps. Dishes were returned to the growth chamber and re-checked after 48 hrs to confirm mortality. 31 Probit analysis (Finney, 1971) was used to determine LD 5 0 , LD 9 0 , and the corresponding 95% confidence intervals. Experiments were repeated at least twice. 2.3.2. Sublethal Growth Effects of Pure Compounds Sublethal growth effects of essential oil compounds were investigated using 4 t h instar larvae (15-20mgbw) treated topically, then placed on standard artificial diet and weighed at three and six days post-treatment. Treatment groups were held in large plastic boxes lined with moistened paper towels and placed in a growth chamber (L16:D8, 22°C). Sublethal dosages of LD 1 0 and LD 3 0 were calculated based on the LD 5 0 / LD 9 0 values determined through probit analysis. For each treatment, twenty-five larvae were weighed, then treated in a glass petri dish using acetone as a carrier. Controls received acetone only. Larvae were then placed on standard artificial diet placed in individual cells of injection-molded plastic trays. Diet was removed and replaced after three days. Larvae were weighed after three and six days. Data were analysed for mean, variance and standard deviation using Microsoft Excel and values for control, LD 1 0 , and LD 3 0 were then compared using single factor ANOVA. 32 2.3.3. Antifeedant Effects of Pure Compounds Antifeedant effects were investigated using leaf-disc choice bioassays. The amount of food eaten under test conditions was compared to controls with untreated leaf discs to establish a feeding deterrence index. Leaf disc choice experiments are relevant to mobile insects, since they are free to move to an acceptable host. Koul (1993) suggests that disc size can effect such experiments because it alters the ratio of chemical signals from the cut leaf edge to those of the intact leaf centre. Since S. litura ate as likely to eat from the center of a leaf as from the edge, it is unlikely that this is a factor in these experiments. Fresh cabbage leaf discs (c.v. 'Stonehead' Hybrid Cabbage 61A, Stokes Seeds Ltd, St. Catherines, Ontario) were cut from cabbage plants grown in the greenhouse. Leaf discs were made using hole punch #8, yielding discs approximately 1.1 cm in diameter. Increasing amounts of essential oil compounds were 'painted' on to one side of leaf discs with a pipetter using acetone as a carrier. The dose of essential oil was determined as the amount of essential oil per cm2 of leaf disc area. Controls were treated with acetone alone. The dose range was determined from pilot trials which indicated the upper and lower limits for the concentrations of the most active compounds. Twenty larvae were tested per dose. 33 Late 4 t h instar larvae were placed on fresh cabbage over night to eliminate any effects of novel foods. Insects were removed from the cabbage and starved for 4 hrs prior to testing. Freshly moulted 5 t h instar larvae (2-4 hrs post-moult) were then placed in cells of injection molded plastic trays with treated and untreated leaf discs and small pieces of moistened cotton to protect leaf discs from dessication. Larvae were allowed to feed for 4-6 hours, after which the leaf discs were removed for analysis. Leaf discs were placed on plate glass slides and a digital picture was captured using an imaging system (IS-500 Digital Imaging System, Alpha Innotech Corporation) and saved as electronic files. NIH software (Scion Image for Windows, Beta 3b Release) was utilized to determine amounts of treated leaf discs consumed versus control discs, and a feeding deterrence index was calculated according to the formula from Isman et al. (1990): Equation 1. Calculation of deterrence index (percent)(lsman et al., 1990). deterrence = (control - treated)/(control + treated) * 100 For each compound tested, a DC 5 0 (concentration required to produce 50% feeding deterrence compared to untreated discs) was determined using Probit analysis (Finney, 1971). A control experiment was conducted to establish any effects of acetone on feeding deterrence. Leaf discs were painted with similar amounts of acetone 34 as were used in essential oil deterrence experiments and feeding deterrence was compared to untreated discs. With the low amounts of acetone used as a carrier, no effects were seen. 2.3.4. Acute Effects of Binary Mixtures The acute effects of binary mixtures of essential oil compounds were determine as in the LD 5 0 experiments described earlier. Three test groups were run concurrently for each binary tested: the binary mixture and each of the pure compounds. The compounds were combined in a 1:1 ratio. Initially, the LD5o value of the most active compound of the pair was chosen as the concentration for each in the mixture. Further experimentation included variation of the dosage around this value, with values somewhat lower and higher than the LD 5 0 of the most active compound. Owing to the large numbers of larvae required to run concurrent trials, larvae were chosen by age (early 4 t h instar) and approximate size, such that weight varied between 15 to 35 mg per larvae (compared to the earlier method used to determine LD 5 0 of pure compounds, where each individual larvae was weighed, adhering to the strict 15-20 mgbw). Actual mortalities were compared to expected mortalities based on the formula: Equation 2: Calculation of expected mortalities from binary mixtures of known dosages (Trisyono and Whalon, 1999). E = Oa + Ob(1-Oa) where E is expected mortality, Oa and Ob are observed mortalities of pure compounds at the given concentration 35 The effects of mixtures were designated as either antagonistic, additive or synergistic by analysis using %2 comparisons: Equation 3: Calculation of values for determining additive, synergistic or antagonistic behaviour of binary mixtures. (Trisyono and Whalon, 1999). x 2 = (Om-E)2/E where Om is observed mortality from the binary mixture and E is expected mortality jl with df=1 and a=0.05 is 3.84 A pair with %2 values greater than 3.84 and having greater than expected mortality were considered to be synergistic, with yl values less than 3.84 representing additive effects. 2.3.5. Antifeedant Effects of Binary Mixtures Compounds were mixed in a 1:1 (w:w) ratio, such that each represented one-half of the total dose tested (i.e. 50 u,g eugenol and 50 u.g citronellal would be, found in a 100 u.g dose). Choice tests were then carried out and analyzed as described for pure compounds, again using 20 larvae per dose. DC 5 0 ' S were determined using Probit analysis as per pure compounds. 36 2.3.6. Acute and Synergistic Effects of Complex Mixtures Complex mixtures were prepared from a number of essential oil constituents that previous experiments indicated would be appropriate for development of a control product. Ratios of compounds were manipulated based on efficacy in binary mixtures and several other factors, including cost and input from associated laboratories. Actual LD 5 0 values were compared to expected mortalities (assuming additive mortality) calculated from: Equation 4: Calculation of expected additive mortality in complex mixtures based on LD 5 0 's of components (Hewlett and Plackett, 1979 in Don Pedro, 1996). E=(A1*Z1) + (A2*Z2) + (A3*Z3) + (A4*Z4) + (A5*Z5) where Ai is the proportion of compound A in the mix and Zi is the LD5o of compound 1 37 3. Results 3.1. Acute Toxicity of Pure Compounds Table 3: Acute toxicities of essential oils (ug/larva) to early 4 t h instar Spodoptera litura of 15-20 mgbw. L D 5 0 9 5 % c . i . L D 9 0 9 5 % c . i . pyrethrum 1.6 1.3-1.9 3.0 2.2-6.1 thymol 25.4 23-28 46.8 38-75 carvacro l 42 .7 38-48 73.8 56-142 pu legone 51.6 49-54 69.7 62-91 f rans-anethole 65.5 62-70 98.8 88 -129 citronel lal 111.2 104-119 153.4 131-224 terpinen-4-ol 130.4 122-140 205.8 180-284 a- terp ineol 141.3 128-155 206.4 190-250 eugeno l 157.6 150-166 212.9 195-263 d- l imonene 273.7 234-320 744.1 584 -1336 c innamic a lcoho l 311.4 242-299 1590 758 -9000 thyme oil 43 .7 41-47 60 .9 55-77 "conta ins 2 0 % pyrethrins as act ive ingredients c i . deno tes con f idence interval Of the essential oil compounds tested for acute toxicity, the most potent were thymol, carvacrol, pulegone and frans-anethole. Citronellal, terpinen-4-ol, a -terpineol, and eugenol were of intermediate toxicity. The activities of all tested compounds were significantly less than that of pyrethrum. Thyme oil, a complex mixture of thymol and carvacrol, obtained from garden thyme, Thymus vulgaris (Labiatae), was similar in toxicity to the most active pure compounds tested. There were notable behavioural effects following topical application to larvae. Most of the essential oils elicited symptoms diagnostic for neurotoxicity: extreme agitation and hyperactivity, the larvae immediately engaging in 38 vigorous writhing and rolling around, followed by tremors, forced diuresis, and convulsions, ending finally in paralysis, and death. Citronellal was notable for causing the most extreme examples of hyperactivity. In individuals that survived, recovery from paralysis took from a few hours to more than eight hours. A small percentage of individuals exhibited morphological effects from topical applications. The essential oils caused some form of cuticular damage, leaving an opaque, whitish area of unsclerotized cuticle (Fig.2). This was likely some form of inhibition of cuticle formation since it was manifested after the subsequent moult. Figure 2: Cut icular damage manifested after the moult fol lowing topical application of a-terpineol. CM 39 3.2. Sublethal Growth Effects of Pure Compounds Chronic effects of topical application were demonstrated in growth assays. A good dose response was obtained at both LD 1 0 and LD 3 0 , with a greater degree of growth inhibition occurring at the higher dosage (Fig. 3). At the ILDio, growth was approximately 80% of control weights for most of the compounds, and significantly less than respective controls after 3 days. Figure 3: Growth deterrence at 3 and 6 days after topical application of pure compounds at sublethal doses. eugenol frans-anethole —800 u> B ~ 600 a o> §400 c n o S200 Day 0 cS800 E B>600 15 400 £200 o>800 CT600 ro400 E200 -jh h d e e i n i Day 3 citronellal Day 6 Day 0 Day 3 thyme oil Day 6 600 400 200 1 control • L D 1 0 ] L D 3 0 a-terpineol ( CO I d e f I 800 600 400 200 thymol •9-g h d e f „ — I V, 800 600 400 200 -9-9-1 d 1 ' ——• ! ™ 1 DayO Day 3 Day 6 h i d e f \ DayO Day 3 Day 6 9 9 n d d c DayO Day 3 Day 6 DayO Day 3 Day 6 *different letters denote significant differences between larval weights of the same day (p<0.05; Duncan's new multiple range test, n=25) 40 Although significant differences between LD10 or LD3o and controls occurred at three days, larvae in the LD10 treatments had often recovered to approach the weights of the controls by the sixth day. At the higher dose (LD30), although recovery isn't as complete as at LD 1 0 , over time, larval growth appears to increase between day 3 and day 6. 3.3. Antifeedant Effects of Pure Compounds Most compounds demonstrated a good dose response in the leaf disc choice bioassay (see Fig.4). There was minor deterrence at 50 u.g/cm2, approaching or reaching 100% deterrence at 200 u.g/cm2. Thymol and trans -anethole were the most effective feeding deterrents with the lowest DC 5 0 's. Citronellal did not demonstrate any significant feeding deterrence. At the lowest dose, some of the compounds, such as eugenol and carvacrol may act as attractants or feeding stimulants. 41 Figure 4 : Feeding deterrence and DC 5 0 's of pure compounds against 5 instar larvae of Spodoptera litura. Comparison of Feeding Deterrence DC 5 0 (|ig/cmz) 95% c i . a-terpineol 130.2 104.4-162.3 carvacrol 115.1 109.3-121.2 eugenol 141.8 122.8-163.8 thymol 85.6 69.2-105.8 frans-anethole 103.1 82.3-129.2 ci. denotes confidence interval 42 3.4. Acute Toxicity of Binary Mixtures Table 4: Acute effects of binary mixtures of essential oils to early 4 instar larvae of Spodoptera litura, and measures of interactions. Larval Mortality (%) Pure Compounds Binary Mix Compound a Compound b Dosage (ng/larvae) observed a observed b expected observed X2 Effect thymol trans-anethole 35+35 37.5 12.5 45.3 100 66.0 synergy thymol trans-anethole 17.7+17.7 5 2.5 7.4 55 307.5 synergy thymol a-terpineol 35+35 32.5 5 35.9 32.5 0.3 additive thymol citronellal 40+40 80 0 80.0 90 1.3 additive citronellal a-terpineol 110+110 10 15 23.5 65 73.3 synergy citronellal trans-anethole 70+70 15 60 66.0 100 17.5 synergy a-terpineol trans-anethole 60+60 32.5 37.5 57.8 95 23.9 synergy a-terpineol trans-anethole 30+30 • 0 5 5.0 47.5 361.3 synergy Expected Mortality: E = Oa + Ob(1-Oa) where E is expected mortality, Oa and Ob are observed mortalities of pure compounds yfl : x2 = (Om-E)2/ E where Om is observed mortality from binary mixture and E is expected mortality X2 with df=1 and a=0.05 is 3.84 n=10 LD 5 0 values reported here (Table 4) for pure compounds are somewhat higher than reported in Table 3. This is explained by the broader size range of larvae tested as per the experimental methods. Larger, heavier larvae will require a larger dose for lethal effects. Further bioassays using binary mixtures of compounds revealed that one of the ingredients, frans-anethole, strongly synergized the toxicity of the other ingredients. The citronellal/a-terpineol mix also demonstrated synergy, 43 whereas the rest operated in an additive fashion only. Tests conducted at one-half concentration continued to demonstrate synergistic effects. 3.5. Antifeedant Effects of Binary Mixtures Similar to the results for acute toxicity, frans-anethole demonstrated synergistic effects for feeding deterrence when combined with other compounds (Fig. 5). Citronellal also demonstrated this in combination with Figure 5: Feeding deterrence of binary mixtures and pure compounds to 5 t h instar larvae of Spodoptera litura. thymol. Leaf Disc Choice Feeding Deterrence 100 -, Irans-anethole +thymol A trans-anethole + a-terpineol -- ©^.-trans-anethole + citronellal M thymol + citronellal a-terpineol + citronellal —#- .thymol — ^ -a-terpineol —SK -citronellal - > itrans-anethole * Binary mixes are 1:1 0 J 50 100 150 200 Dose (pg/leaf disc) Comparison of DC 5 0's (Table 5) for pure compounds used in binary mixtures demonstrates synergism for several of the pairs, including frans-anethole with thymol and a-terpineol. Table 5: Compar ison of D C 5 0 for binary mixtures with pure compounds . D C 5 0 95% ci. frans-anethole + thymol* 66.77 56.42-79.01 t-anethole 103.13 82.34-129.16 thymol 85.59 69.24-105.79 frans-anethole + a-terpineol 94.59 81.24-116.13 t-anethole 103.13 82.34-129.16 a-terpineol 130.18 104.40-162.3 a-terpineol + eugenol 115.78 96.38-139.09 a-terpineol 130.18 104.40-162.3 eugenol 141.81 122.78-163.79 thymol + citronellal 77.77 63.83-94.75 thymol 85.59 69.24-105.79 citronellal none bold indicates deterrence for binary mixtures italic indicates deterrence of pure compounds * 1:1 mixtures totalling each dose (i.e. 25\Ag + 25 ng = 50 ^ g dose) 45 3.6. Acute Toxicity of Complex Mixtures Due to proprietary restrictions, results from only 3 of the many potential complex mixtures are presented (Table 6). Table 6: Composition and acute toxicity of some complex mixtures. Essential oil constituent* Composition (% by weight) Mixture 1 Mixture 2 Mixture 3 1 15 15 25 2 — 13 — 3 10 10 10 4 10 10 10 5 — 15 15 6 15 — — 7 — 37 40 8 50 ~ --Expected LD 5 0 78.2 72.8 74.6 (ug/larvae) Observed LD 5 0 61.0 45.8 40.6 (u.g/larvae) il 3.78 10.01 15.5 effect additive synergistic synergistic "identities of compounds cannot be revealed for proprietary reasons Expected Mortality: E = Oa + Ob(1-Oa) where E is expected mortality, Oa and Ob are observed mortalities of pure compounds X2 : x2 = (Om-E)2/ E where Om is observed mortality from binary mixture and E is expected mortality yl with df=1 and a=0.05 is 3.84, n=10 Synergy was clearly demonstrated in two of the complex mixtures, with observed mortality significantly greater than expected for additive effects. 46 4. D iscuss ion Many of the individual essential oil constituents proved acutely toxic to larvae of Spodoptera litura to some degree. In general, the pure compounds tested were considerably less efficacious than those of other commercially available botanical compounds, notably pyrethrum. Unlike many insecticides currently in use, however, essential oils generally have quite favourable vertebrate toxicities, with rat oral LD5 0's ranging from 2 to 5 g/kg. A mixture of a-terpineol, eugenol and thyme oil was found to be around 300 times less toxic to fish than either azadirachtin, rotenone or pyrethrum as well as some of the more common synthetic insecticides (Stroh et al., 1998). This could be due in part to differing pharmacokinetics and detoxicative metabolism, but may also be a result of a biorational mode of action. Several essential oil compounds have been demonstrated to block octopamine (Enan et al., 1998), a neurotransmitter unique to insects that functions similarly to epinephrine (adrenaline) and norepinephrine found in vertebrates. Octopamine affects all insect organs, including the corpus cardiacum; is intimately involved in flight (especially as a neurotransmitter mediating the release of 'flight' hormones); modulates learning and memory (Candy et al. 1997) and is of primary importance in generating specific behaviours, such as 'fight or flight' (Orchard and Lange, 1987). Since it is unique to insects, the octopaminergic system is of considerable interest as a target site for control agents. There also appears to be selectivity between 47 different insect species, depending on the ability to activate adenylase cyclase (Downer, 1988). Octopamine agonists and antagonists act as antifeedants and can have profound adverse effects on insect behaviour, with symptoms including quick knockdown, agitation, hyperactivity, tremors, forced diuresis, convulsions, and death (Nathanson et al., 1993). The behavioural effects of sublethal topical doses of the tested compounds are significant, and are consistent with octopamine agonists. The hyperactivity manifested as extreme agitation and rolling would likely result in larvae dropping off the plant. When this happens, the probability of mortality increases greatly, particularly as a result of predation from ground beetles and ants, although other factors such as wandering and exposure would contribute. These behavioural effects are similar to those of formamidine insecticides, which have been referred to as 'pestistatic' since they function at doses far below the acute lethal doses of conventional insecticides. Synergistic effects within complex mixtures are thought to be important in plant defenses against herbivory. Plants usually present defenses as a suite of compounds, not as individual ones, and it is thought that the minor constituents may act as synergists, enhancing the effect of the major constituents through a variety of mechanisms. It is frequently noted that the 'original' complex essential oils are considerably more efficacious than are applications of the pure compounds derived from them. Examples include oil 48 of anise (from which frans-anethole is derived), rosemary oil and various citrus oils (Ho et al.,1977; Hori, 1998; and Don-Pedro, 1996). Identifying such 'synergizing' compounds within complex mixtures may allow for the development of more effective control agents as well as allowing the use of smaller absolute amounts in the mixture to achieve satisfactory levels of control. Among the most active compounds, it was found that frans-anethole strongly synergized the toxicity of other essential oil monoterpenes and phenols, whereas the others (with the exception of a-terpineol and citronellal mixed) operated in an additive fashion only. In the search for new pesticides, acute toxic effects, as demonstrated by LD 5 0 's, are usually the yardstick by which products are measured, yielding a spectrum of fast-acting, potent products. Concomitantly, most such compounds (to date) are axonic or muscular poisons, which, because of the gross similarities between insect and vertebrate neuromuscular physiology (with the notable exception of octopamine receptors), are also quite toxic to mammals. However, compounds with little immediate toxicity may still confer protection to crops through a reduction of fitness in insect herbivores, and, combined with other effects such as feeding deterrence, may be sufficient to protect a crop through its vulnerable stages of growth. This is borne out by the situation in nature, in which chemical defenses are usually present only in sublethal concentrations (Isman, 1992). Prolonging the duration of the developmental stages of insect herbivores likely exposes them to increased 49 mortality, particularly if the bulk of larval mortality occurs during feeding (Bernays, 1998). Topical exposure to several of the tested compounds delayed larval development through decreased growth rates. Although the effect appears to be transitory, with recovery occurring over the duration of six to eight days, this may still represent a significant period that could enhance abiotic and biotic mortality factors. Life table studies using lepidopterans indicate that 80% of larval mortality occurs before the third instar (reached in this species by the sixth day of development), with parasitoids and predators accounting for the majority of deaths (Isman, 1994). The growth effects of binary mixtures were not tested, but thyme oil, comprised largely of thymol and carvacrol, demonstrated greater sub-lethal effects on growth than either of the pure compounds. The preponderance of chemical cues that act as feeding deterrents is suggestive evidence that insects rely far more on phagodeterrents in host plant recognition than on stimulants (Schoonhoven et al., 1992). As previously noted, many secondary plant compounds are present in sub-acute concentrations which are none the less effective concentrations for feeding deterrence of potential herbivores. Several of the essential oil compounds tested demonstrated feeding deterrence in a dose-dependent manner, including frans-anethole, eugenol and a-terpineol. Under no-choice 50 laboratory conditions, test insects may actually starve to death because of the absence of a perceived acceptable food source, whereas, in the field, insects are able to leave unacceptable hosts to seek viable food sources. However, increased search times for acceptable food would significantly increase exposure to mortality factors. Repeated exposure to antifeedants can result in desensitization, with previously rejected food sources rendered acceptable (Jermy et al, 1982). This appears to be more likely when a single antifeedant is applied, while complex mixtures are apt to prevent this. Bomford and Isman (1996) provide a clear demonstration of this using pure azadirachtin, described as one of the most potent antifeedants known, and an extract from the seeds of the neem tree, Azadirachta indica, from which azadirachtin was originally isolated. Larvae of Spodoptera litura became desensitized to the pure compound with repeated exposures but not to the chemically complex extract with the same absolute amounts of azadirachtin. There was notable synergy in terms of feeding deterrence in some of the binary mixtures. Trans-anethole plus thymol, thymol plus citronellal and ct-terpineol plus citronellal were more deterrent than the pure compounds. A further advantage of using complex mixtures stems from the ability of insects to rapidly develop resistance to pure compounds. Experiments with 51 pyramiding resistant traits in a single crop cultivar demonstrate that the presence of multiple factors can delay the development of resistant insects. Sachs et al. (1996) demonstrated that elevated levels of terpenoids in cotton delayed resistance to the transgenic CrylA(b) insecticidal protein. Delayed resistance is even more likely if the compounds have different target sites within the insect, since the chance occurrence of multiple resistance genes in the same insect is exceedingly low (Tabashnik, 1994). Comparison of LD5o's to feeding deterrence values yields a generally positive correlation (r=0.75). Thymol, carvacrol and frans-anethole were the most active in terms of LD 5 0 and also demonstrated the greatest degrees of feeding deterrence. This is a positive relationship in terms of effectiveness as control agents. Simple feeding deterrents can lack effectiveness either by allowing for desensitization or by loss of efficacy (many known antifeedants are highly degradable) or they may simply redirect herbivores to the highly vulnerable, unprotected new growth of plants (Koul, 1993 and Isman, 1994). However, the acute toxic effects would help to overcome these weaknesses. Vice versa, surviving insects are exposed to great increases in mortality because of the unacceptability of the food source. 52 Given the above observations and rationales, several candidate compounds were chosen from which a unique complex control agent could be developed. Some served as effective acute toxicants, others as effective growth inhibitors, and others still as potent feeding deterrents and synergizers. The inclusion of a number of compounds is more desirable in that the insecticidal spectrum of action is increased, since different species have different responses to individual compounds. A number of complex mixtures based on these compounds were tested. Candidate mixtures demonstrated good synergistic effects, with acute toxicities at much lower doses than those expected for additive effects. Concentrations of individual compounds were varied with several factors in mind, including cost of individual compounds and any potential phytotoxicity issues, and additional compounds were inserted based on previous experimentation. The resulting proprietary product (Mixture No. 3) is an effective control agent for a variety of insects and is undergoing further development and refinement before entering commercial production. Tests with additional essential oil compounds and other acceptable organics, adjustments in absolute amounts and varying delivery formulations are ongoing. 53 5. Conc lus ion Certain plant essential oils and their major chemical constituents are known to be toxic to insects and to deter feeding. Essential oil constituents are good candidates for developing 'environmentally friendly' insecticides since they are selective for insects and basically non-toxic to vertebrates. After evaluating the acute toxicity of several fragrance oils of varying complexity, tests of the most likely active ingredients and various closely related compounds led to a refined list of candidates for the development of a blended formulation for control of insect pests. Based on acute toxicity to the Asian armyworm, Spodoptera litura, an optimal blend of compounds was proposed. This blend demonstrated synergistic action in that the observed toxicity was greater than that expected through additive toxicity. Along with exploiting possible synergisms, complex mixtures are advantageous as control agents because they decrease the chances of resistance and desensitization. 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