WO2018042023A1

WO2018042023A1 - Use of nootkatone as a larvicide

Info

Publication number: WO2018042023A1
Application number: PCT/EP2017/072026
Authority: WO
Inventors: Jean Amick
Original assignee: Evolva AG
Current assignee: Evolva Holding SA
Priority date: 2016-09-02
Filing date: 2017-09-01
Publication date: 2018-03-08
Anticipated expiration: 2019-03-02

Abstract

Compositions and methods for treating and preventing infestations of larval-stage insects are disclosed herein.

Description

USE OF NOOTKATONE AS A LARVICIDE

BACKGROUND OF THE INVENTION

Field of the Invention

[0001] This disclosure relates to the use of nootkatone and nootkatone-containing compositions to treat and prevent infestations by larval-stage insects and to treat, pretreat, or reduce infestations of insects with larval stages of life cycle.

Description of Related Art

[0002] Aedes species mosquitoes, including Ae. aegypti and Ae. albopictus, are known vectors for multiple human diseases, including those caused by dengue, chikungunya, and Zika viruses (i.e., arthropod-borne viruses or "arboviruses"). Similarly, Anopheles mosquitoes that carry the Plasmodium parasite which causes malaria continue to pose an enormous health risk in tropical regions and sub-Saharan Africa.

[0003] Based on the dangers posed by mosquito-borne pathogens, reduction in the number of mosquito bites is a global concern.

[0004] Current approaches to prevent mosquito bites and associated diseases often focus on repelling and killing adult mosquitoes with chemical repellents and pesticides. But repellents and pesticides have considerable limitations, such as having a strong "chemical" smell or that mosquitoes are developing resistance to those typically used. Other methods of repelling or killing mosquitoes, such as bug zappers, etc. also have limited efficacy.

[0005] In addition to mosquitoes, there are many other insects and pests such as gnats, cockroaches, chiggers, black flies, blow flies, screwflies, sandflies, stable flies, tsetse flies, etc. that present significant challenges to humans. Such insects and pests can bite, carry diseases, ruin crops, infest houses, and otherwise cause physical discomfort, etc. to humans and domestic animals. Similarly, certain members of the order Lepidoptera (i.e., the larvae of moths and butterflies) present significant economic challenges to humans by damaging or ruining crops, trees, fabrics, furs, etc. For example, species of the Tortricidae, Noctuidae, and Pyralidae families, such as armyworms, corn earworms, Pieris brassicae, cloth moths, and cotton bollworms are particularly problematic pests. [0006] Prior to becoming adults, many insects and pests pass through three life cycle stages: egg, larva, and pupa. For example, mosquitoes lay eggs in a body of water, which hatch to release larvae into the water, and then mature into pupa. Pupa mature into adults, eclose and emerge from the water to complete the cycle. Other insects and pests have similar life cycles, where adults lay eggs, for example on moist surfaces, such as soil, the floors of animal sheds, etc., the eggs hatch releasing larvae, which then mature into pupae, and then become adults.

[0007] Because the first three stages of the mosquito life cycle occur in water, methods to combat mosquitoes have also focused on killing larvae in the water with mixed success. One approach to killing larvae or preventing their maturation includes removing breeding sites (i.e., disposing of cans, old tires, etc.). But, many breeding sites are water sources that have an aesthetic value or serve a functional purpose and thus removal is undesirable. For these breeding sites, mosquito larvae can be killed by treating the water with larvicides, such as certain oils, for example, soy oil or mineral oil, the spores or metabolites of Bacillus thuringiensis israelensis, and insect growth regulators, such as, methoprene. However, current larvicides have limited effectiveness against mosquito larvae and some raise concerns regarding animal and child safety or environmental impact. For other insects and pests, other larvicides are similarly employed such as cyromazine (a modified triazine similar to melamine), which may be applied to directly soils and to animal feed to combat fly larvae. Yet, cyromazine has been identified as an aquatic ecotoxin that affects fish.

[0008] Therefore, there is a need for new compositions and methods that effectively control mosquitoes and other insects and pests at the larval-stage in an efficient, safe, and environmentally friendly manner.

SUMMARY OF THE INVENTION

[0009] Provided herein are effective natural compositions and methods of their use to treat and prevent larval-stage insect infestations.

[0010] In a first aspect, the invention provides a method of killing a larval-stage insect, including applying an effective amount of a nootkatone-containing larvicide composition to a larval- stage insect breeding site. In one embodiment of the first aspect, the larval-stage insect breeding site includes a body of water or a moist surface. In another embodiment of the first aspect, the nootkatone-containing composition creates a film at an air-liquid interface of the body of water or on the moist surface. In one embodiment of the first aspect, the film comprises nootkatone. In one embodiment of the first aspect, the effective amount of the nootkatone-containing larvicide composition is an amount sufficient to render a final surface concentration of at least about 0.09 mg/cm² at the larval-stage insect breeding site. In one embodiment of the first aspect, the larval- stage insect includes a pathogen-carrying or a pathogen-free insect. In one embodiment of the first aspect, the larval-stage insect includes a member of the genera Anopheles, Aedes, Culex, or Haemagogus or a member of the families Tortricidae, Noctuidae, or Pyralidae. In another embodiment of the first aspect, the larval-stage insect is a gnat, a cockroach, a chigger, a black fly, a deer fly, a sandfly, a stable fly, a tsetse fly, a myiatic fly, an armyworm, a corn earworm, Pieris brassicae, a cloth moth, or a cotton bollworm. In one embodiment of the first aspect, the larval- stage insect is a member of the genus Anopheles. In a preferred embodiment of the first aspect, the member of genus Anopheles is Anopheles gambiae or Anopheles quadrimaculatus. In a particular embodiment of the first aspect, the larval-stage insect is a member of genus Aedes. In a preferred embodiment of the first aspect, the member of genus Aedes is Aedes aegypti.

[0011] In a second aspect, the invention provides a method of treating or preventing a larval- stage insect infestation including (a) providing a nootkatone-containing larvicide composition, (b) optionally diluting the composition to a working concentration with a carrier, and (c) applying the composition to a larval-stage insect breeding site. In one embodiment of the second aspect, the larval-stage insect breeding site is a connected water system. The connected water system can be at least one of a watershed, a tributary, an irrigation system, a sprinkler system, a pool, a water fountain, a drainage system, an animal watering system, a bird path, a fish pond, an aqueduct, or any other part of a water system that may serve as a larval-stage insect breeding site. In a particular embodiment of the second aspect, the connected water system is an irrigation system.

[0012] In a third aspect, the invention provides a larvicide composition including (a) about 0.01 % to about 10% nootkatone, (b) an optional additional active ingredient, and (c) an optional carrier. In one embodiment of the third aspect, the larvicide composition when applied to a larval- stage insect breeding site has a final concentration of about 0.01 % nootkatone (v/v). In another embodiment of the third aspect, the composition when applied to a larval-stage insect breeding site has a final working surface concentration of at least about 0.09 mg / cm². In a further embodiment of the third aspect, the composition includes an additional active ingredient, wherein the additional active ingredient is a soy oil, a mineral oil, spores or metabolites of Bacillus thuringiensis israelensis, an insect growth regulator, and combinations thereof. In one embodiment of the third aspect, the insect growth regulator is methoprene or cyromazine. In one embodiment of the third aspect, the composition kills at least about 90% larvae one week after application to a larval-stage insect breeding site. In another embodiment of the third aspect, the composition kills at least about 100% larvae exposed to the composition two weeks after application to a larval-stage insect breeding site. In a further embodiment of the third aspect, the composition is about 5X, about 10X, about 20X, about 30X, or about 60X concentrate.

[0013] In a fourth aspect, a method of killing a larval-stage insect includes applying an effective amount of a nootkatone-containing larvicide composition to a larval-stage insect food source. In one embodiment of the fourth aspect, the food source comprises a food crop. In one embodiment of the fourth aspect, the food crop includes one or more of lettuce, cabbage, beans, corn, tomatoes, and cotton. In one embodiment of the fourth aspect, the food source is a grass or other plant leaf. In one embodiment of the fourth aspect, the larval-stage insect includes a species of the Tortricidae, Noctuidae, and Pyralidae families. In one embodiment of the fourth aspect, the insect food source is a tree or a part of a tree. In one embodiment of the fourth aspect, the food source is a root. In one embodiment of the fourth aspect, the insect food source is a grain, a fruit, or a nut, a shoot, a bud, or a flower.

[0014] Other aspects, embodiments, and implementations will become apparent to those of ordinary skill in the art by reading the following detailed description, with reference where appropriate to the accompanying drawings.

DESCRIPTION OF DRAWINGS

[0015] Figure 1 illustrates a biosynthetic pathway for nootkatone;

[0016] Figure 2 shows the results of treating Aedes aegypti larvae with 0.01 % and 0.03% nootkatone (v/v) in uncovered beakers compared to an ethanol control. Results were measured after 24 and 48 hr;

[0017] Figure 3 shows the results of a comparison of 0.03% nootkatone versus soy oil and mineral oil for effectiveness of killing Aedes aegypti larvae. A 0.03% nootkatone (v/v) concentration was more effective than either soy oil or mineral oil after 24 hr treatment. Ethanol- treated larvae were unaffected;

[0018] Figure 4 shows the results of an experiment examining the effect of surface area on the efficacy of nootkatone compositions for larvicide activity. A correlation between nootkatone concentration and treatment surface area (i.e., air-liquid interface) was observed. As surface area increased, the effectiveness of nootkatone at lower concentrations diminished. However, increasing nootkatone concentrations restored the treatment effect against mosquito larvae to overcome the effect of increased surface area. Ethanol had marginal effect on larvae regardless of surface area size; [0019] Figure 5 shows that nootkatone is an effective larvicide over an extended period of time. Larvae added 0, 24, 48, and 78 hours after application of a 0.03% nootkatone (v/v) concentration all experienced 100% mortality. Ethanol-treated controls showed marginal differences in mortality over 72 hours;

[0020] Figure 6, similar to Figure 5, shows that nootkatone is an effective larvicide over an extended period of time. Larvae added 0, 24, 48, and 78 hours after application of a 0.01 % or 0.03% nootkatone (v/v) concentration all experienced 100% mortality through 48 hours. While the 0.03% nootkatone composition showed 100% mortality through 72 hours, the mortality rate of the 0.01 % nootkatone composition fell to about 10%. Ethanol-treated controls showed marginal differences in mortality over 72 hours;

[0021] Figure 7 further illustrates the effectiveness of nootkatone in killing larvae over an extended period of time, as shown in Figures 5 and 6. Here, a 0.03% nootkatone (v/v) concentration maintained greater than 90% mortality rates through one week of treatment;

[0022] Figure 8 further illustrates the effectiveness of nootkatone in killing larvae over an extended period of time. Here, larvae exposed to a 0.03% nootkatone (v/v) solution for twenty four hours exhibited 100% mortality rates through one week of treatment. Similarly, twenty-four hours of exposure to 0.01 % nootkatone (v/v) produced over 80% larvae mortality rates through one week of treatment;

[0023] Figure 9 further illustrates the effectiveness of nootkatone in killing larvae over an extended period of time. Here, forty-eight hours of exposure to 0.03% nootkatone or 0.01 % nootkatone (v/v) produced 100% larvae mortality rates through one week of treatment; and

[0024] Figure 10 further illustrates the effectiveness of nootkatone in killing larvae over an extended period of time. Here, a 0.03% nootkatone (v/v) concentration maintained a 100% mortality rate upon 24 hour exposure of larvae two weeks after water treatment.

[0025] Figure 1 1 shows typical results of treating a caterpillar on a bush in the open air with a 0.25% nootkatone (v/v) aerosol spray. The caterpillars were observed frequently over 6 hours. All displayed the following timeline of response. Figure 1 1 a shows a caterpillar immediately prior to treatment. Figure 1 1 b shows the caterpillar immediately after treatment. Figure 1 1 c shows the caterpillar 1 hr after treatment. All caterpillars became inactive within the first hour after treatment and most died within 30 to 45 minutes following treatment. Figure 1 1 d shows the caterpillar 6 hr after treatment, at which point all treated caterpillars were dead. Dead caterpillars were often observed to darken within 6 hs after treatment. [0026] Figure 12 shows typical effects of feeding caterpillars with collected leaves sprayed with a 0.06% nootkatone formulation. Figure 12a shows caterpillars at the time of introduction to treated leaves. All caterpillars were active. Figure 12b shows the caterpillars of the negative control 6 hr after introduction to untreated leaves. All caterpillars were active and feeding. Figure 12c shows the caterpillars introduced to treated leaves 6 hr after introduction to treated leaves. All caterpillars displayed restricted motility and many had become completely inactive. Figure 12d shows the caterpillars of the negative control 24 hr after introduction to untreated leaves. All caterpillars were active and feeding. Figure 12e shows the caterpillars introduced to treated leaves at 24 hr after introduction to treated leaves. All caterpillars on treated leaves were clearly dead.

DETAILED DESCRIPTION

[0027] All publications, patents and patent applications cited herein are hereby expressly incorporated by reference in their entirety for all purposes.

[0028] Before describing the present invention in detail, a number of terms will be defined. As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "an active ingredient" means one or more active ingredients.

[0029] It is noted that terms like "preferably," "commonly," and "typically" are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention.

[0030] For the purposes of describing and defining the present invention it is noted that the term "substantially" is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term "substantially" is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.

[0031] As used herein, the term "about" refers to ±10% of any particular value.

[0032] As used herein, the terms "or" and "and/or" are utilized to describe multiple components in combination or exclusive of one another. For example, "x, y, and/or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z." [0033] As used herein, the term "active ingredient" refers to a chemical compound or mixture of chemical compounds that kills larval-stage insects.

[0034] As used herein, the term "larval-stage insect" refers to any insect or non-insect pest in a larval stage or insect or non-insect pest that has a larval life cycle stage. Examples of such larval- stage insects include but are not limited to pathogen-carrying and pathogen-free insects and non- insect pests, such as mosquitoes, gnats, cockroaches, chiggers, black flies, deerflies, sandflies, stable flies, and tsetse flies, and for example, members of the genera Anopheles, Aedes, Culex, Haemagogus, Chrysomya, and Lucilia. Further examples include certain members of the order Lepidoptera including species of the Tortricidae, Noctuidae, and Pyralidae families, such as armyworms, corn earworms, Pieris brassicae, cloth moths, and cotton bollworms, among others.

[0035] As used herein, the term "pathogen" refers to any disease causing substance or life- form (e.g., virus, arbovirus, Flavivirus, bacteria, fungus, parasite, or microbe).

[0036] As used herein, the term "mosquito" refers to any mosquito species. Examples include members of the genera Anopheles, Aedes, Culex, and Haemagogus.

[0037] As used herein, the term "larval-stage insect breeding site" refers to a location where eggs of a larval-stage insect hatch into larvae and often includes food sources for the newly hatched larvae. Examples typically preferred by insects with waterborne larval stages include stagnant or fresh water sources, an inland body of water (e.g., ponds, lakes, canals, creeks, ditches, irrigation channels, or marshy areas), and objects that create a pool of water (e.g., cans, old tires, animal troughs, ornamental ponds, swimming pools, puddles, catch basins, paddling pools, rain barrels, gutters, or sewers - such small volume water sources as bottle caps and the like are similarly contemplated). Other examples of larval-stage insect breeding sites include moist surfaces, such as soil, animal feces/manure, animal sheds or pens, rotting vegetation and plant material such as plant stems, fruits, or wood, or funguses. Further examples include food crops before or after harvest, such as lettuce, cabbage, beans, corn, tomatoes, cotton, ornamental plants grown commercially or domestically for display or harvest, trees, tree parts, roots, shrubs, bushes, as well as grasses and other plant leaves. Additional breeding sites or food sources include grains, fruits, nuts, shoots, bud, and flowers.

[0038] Treatment, pre-treatment, or control of a specific species can be performed by selecting a specific larval-stage insect breeding site that is suited to the insect to be treated.

[0039] As used herein, the terms "treatment of larval-stage insects" and "treating a larval-stage insect" refer to a process by which at least one larval-stage insect is at least one of contacted with a composition including an active ingredient, killed, or repelled from a surface. Further, treatment of larval-stage insects may include directly or indirectly contacting a larval-stage insect breeding site with a composition including an active ingredient. In these contexts, treatment of larval-stage insects may include any manner of treatment performed to reduce the population of larval-stage insects. Examples of treatments include applying a nootkatone-containing composition including a conventional larvicide, such as an oil, such soy oil or mineral oil, the spores or metabolites of Bacillus thuringiensis israelensis, or an insect growth regulator, such as, methoprene, or a modified triazine, such as, cyromazine. Treatment of larval-stage insects may include a second or subsequent treatment to prevent recovery of the larval-stage insect population.

[0040] As used herein, the terms "surface" and "object to be treated" interchangeably refer to any larval-stage insect, a connected water system, any larval-stage insect breeding site, a portion of a larval-stage insect breeding site, a surface area and/or material that larval-stage insects may attempt to traverse or inhabit, and surfaces and objects on which larval-stage insects may be observed or that could act as vectors for the transportation of larval-stage insects. Examples of surfaces include, without limitation, water surfaces (e.g., of ponds, lakes, canals, creeks, ditches, irrigation channels, or marshy areas), the edges of water bodies (e.g., shorelines, pool liners and/or covers, banks, etc.), and the surfaces of objects that can create a pool of water (e.g., animal troughs, ornamental ponds, swimming pools, catch basins, paddling pools, rain barrels, gutters, or any surface of equipment, tools used in conjunction with any of the aforementioned objects, or moist surfaces, such as soil, rotting vegetation, animal feces, animal enclosures, such as pens, sheds, and barns, plant and fungal materials.

[0041] As used herein, the term "effective concentration" refers to a concentration of an active ingredient (such as nootkatone) within a composition such that when the composition is applied to a larval-stage insect or to a relevant surface, a larval-stage insect that is contacted by the composition is repelled and/or experiences paralysis, poisoning, neuro-muscular damage, or death.

[0042] As used herein, the term "effectively treat" refers to at least one of directly (e.g., by contacting a larval-stage insect or its immediate surroundings) or indirectly (e.g., by contacting a larval-stage insect breeding site or other surface that a larval-stage insect will be affected by) repelling, paralyzing, poisoning, damaging neuro-muscular tissue of, killing, or preventing the maturation of a larval-stage insect.

[0043] As used herein, the term "nootkatone" refers to a compound seen in Figure 1 that may be synthesized, isolated, and purified from of a mixture of products produced in a host modified to express enzymes of the nootkatone biosynthetic pathway or that can be produced from naturally occurring sources, such as, but not limited to, citrus plants. "Nootkatone" further refers to derivatives and analogs analogs thereof. For example, the nootkatone compound contemplated for use herein may be produced in vivo through expression of one or more enzymes involved in the nootkatone biosynthetic pathway in a recombinant yeast or in vitro using isolated, purified enzymes involved in the nootkatone biosynthetic pathway, such as those described in U.S. Patent Application Publication Nos. 2015/0007368 and 2012/0246767. Therefore, nootkatone as defined herein can differ chemically from other sources of nootkatone, such as extracts from plants and derivatives thereof, or may include such plant extracts and derivatives thereof.

OVERVIEW

[0044] Disclosed herein are nootkatone-containing compositions and methods that effectively treat larval-stage insects.

[0045] Some aspects of the current disclosure aim to kill larval-stage insects, such as mosquitoes, cockroaches, chiggers, black flies, deerflies, sandflies, tsetse flies, etc., or to reduce the frequency or prevelance of larval-stage insect maturation into adulthood. In further aspects of the invention, larval-stage insect breeding sites can be treated with nootkatone-containing compositions so as to reduce the frequency or prevelance of larval-stage insect maturation into adulthood.

[0046] More generally, it is highly desirable to treat insects or non-insect pests during a larval stage because during that stage of the life cycle they are less mobile and therefore may be controlled by application of smaller quantities of an active ingredient contemplated herein to a more localized surface or area suitable for larvae. In addition, application of an active ingredient to a larval-stage insect breeding site preferred by the species to be treated has the significant benefit of reducing the chance of affecting other beneficial insects.

[0047] In another aspect, the current disclosure provides methods and uses for a composition comprising nootkatone suitable for treating a surface, a larval-stage insect breeding site, or an environment rich in larval-stage insects for preventing or delaying the onset of maturation into adulthood.

[0048] Additional aspects of the current disclosure are intended to reduce or prevent the occurrence of disease transmission by larval-stage insects that mature into adults.

[0049] In one embodiment, the use of nootkatone provides a sustainable and biodegradable alternative to current insect repellents and pesticides for combatting larval-stage insects in an efficient, safe, and environmentally friendly manner. [0050] In some embodiments, compositions containing nootkatone may be administered alone to effectively treat larval-stage insects. In other embodiments, nootkatone-containing compositions are used in combination with other insecticides or other treatments disclosed herein to effectively treat larval-stage insects. For example, compositions including nootkatone may be administered in combination with or successively with the application of natural predators of larval-stage insects. For example, natural predators of mosquitoes include dragonfly nymphs and frogs. Some pesticides used against larval-stage insects are also effective in killing their natural predators, thus reducing the long term biological control available in the area in which pesticide has been applied. However, at lower concentrations, nootkatone is not believed to have such a broad specificity on common insects, fish, nymphs, and frogs.

[0051] In some embodiments, treatment for larval-stage insects can be through administration of a contemplated composition to any part of a connected water system, such as a watershed, a tributary, an irrigation system, a sprinkler system, a pool, a water fountain, a drainage system (such as a gutter), an animal watering system, a bird path, a fish pond, an aqueduct, or any other part of a water system that may serve as or contact a larval-stage insect breeding site. Further, administration of contemplated compositions for effective treatment of larval-stage insects can be within any part of a connected water system that is in fluid communication with the remainder of the connected water system to be treated, meaning that such application will result in an added treatment composition being distributed to the remainder of the connected water system.

[0052] In some embodiments, irrigation systems are contemplated that apply nootkatone- containing compositions during the process of watering plants. Examples of such irrigation systems include small systems, such as those used in private gardens and lawns and commercial systems used for commercial scale crop production facilities, such as farm fields and hydroponic facilities.

COMPOSITIONS

[0053] Nootkatone-containing larvicide compositions contemplated herein can be formulated for direct application to a surface to effectively treat existing larval-stage insect populations or as a prophylactic to prevent the growth or spreading of a larval-stage insect population to new larval- stage insect breeding site.

[0054] Generally and without limitation, larvicide compositions contemplated herein can be in the form of an aqueous liquid, an oil-based liquid, a concentrated liquid, a gel, a foam, an emulsion, a slurry, a paint, a clear coat, a wax, a block, a pellet, a puck, a dunk, a granule, a powder, a capsule, a vesicle, an effervescent tablet, slow release tablet, an impregnated dissolvable sheet or film, an impregnated material, and combinations thereof. Further larvicide compositions may be configured for immediate release, delayed release, intermittent release, or extended release by inclusion of excipients and/or packaging structures and/or materials that enable such release profiles.

[0055] In certain aspects, a larvicide composition may be formulated as a liquid or aerosol formulation suitable for application in a spray, a roll on, a dip, detergents, durable water repellent formulations.

[0056] In certain aspects, a larvicide composition may be formulated for application by dispensing into or onto an area of a connected water system to be distributed throughout the system. In this context, the larvicide composition can be provided as a solution, an emulsion, an oil, a spray, a gel, a powder, a foam, a block, a pellet, a dunk, a puck, a composition-filled dissolvable pouch, a granule, a vesicle, a capsule, and combinations thereof.

[0057] In certain aspects, a larvicide composition may be formulated including a portion of material such as a tissue, pad, cloth, sponge or sheet impregnated, immersed or coated with a liquid composition comprising nootkatone at a concentration of between 0.01 - 10% by volume of the liquid composition. In certain aspects, the portion of material is a disposable thin sheet of material such as a tissue, a wet wipe, or a wet pad, similar to those sold under the Swiffer®, Pledge®, Windex®, Clorox® brands.

[0058] In other embodiments of the invention, larvicide compositions contemplated herein can contain any amount of nootkatone. In another embodiment, larvicide compositions contemplated herein can contain a carrier and at least about 0.001 %, or at least about 0.005%, or at least about 0.01 %, or at least about 0.02%, or at least about 0.03%, or at least about 0.04%, or at least about 0.05%, or at least about 0.06%, or at least about 0.07%, or at least about 0.08%, or at least about 0.09%, or at least about 0.1 %, or at least about 0.2%, or at least about 0.3%, or at least about 0.4%, or at least about 0.5%, or at least about 0.6%, or at least about 0.7%, or at least about 0.8%, or at least about 0.9%, or at least about 1 %, or at least about 2%, or at least about 3%, or at least about 4%, or at least about 5%, or at least about 6%, or at least about 7%, or at least about 8%, or at least about 9%, or at least about 10%, or greater than about 10%, or greater than about 15%, or greater than about 20%, or greater than about 25%, or greater than about 30%, or greater than about 35%, or greater than about 40%, or greater than about 45%, or greater than about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 99% by weight nootkatone. [0059] In one embodiment, the provided larvicide compositions contain nootkatone in an amount at or about 0.001 % to at or about 2%, or about 0.01 % to at or about 5%, or about 0.01 % to at or about 75% by weight of the composition. In another example, a larvicide composition may contain nootkatone in an amount of from at or about 1 % to at or about 50% by weight of the composition. In another example, a larvicide composition may contain nootkatone in an amount of from at or about 5% to at or about 40% by weight of the composition. In another example, a larvicide composition may contain nootkatone in an amount of from at or about 10% to at or about 30% by weight of the composition. In another example, a larvicide composition may contain nootkatone in an amount of from at or about 15% to at or about 25% by weight of the composition. In another example, a larvicide composition may contain nootkatone in an amount of from at or about 1 % to at or about 90% by weight of the composition. In another example, a larvicide composition may contain nootkatone in an amount of about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 50% by weight of the composition. In another example, a larvicide composition may contain nootkatone in an amount of up to about 99% or more by weight of the composition.

[0060] In one particular embodiment, a contemplated nootkatone-containing larvicide composition is provided as a concentrate. For example, a nootkatone-containing larvicide composition may be provided as a 20X, or a 10X, or a 5X, or a 3X concentrate that can be diluted by an end user with an appropriate solvent or by application to a connected water system or larval- stage insect breeding site to achieve a 1 X (or other desired) working concentration. Alternatively, a nootkatone-containing larvicide composition may be provided to an end user at a 1 X working concentration. However, any concentration is contemplated for use herein. For example, compositions provided as concentrates can be used without dilution at all or may be diluted from a highly concentrated concentrate {e.g., about 20X to about 100X, or about 30X to about 60X, or about 30X, or about 60X) to some multiple of concentration higher than 1X, such as 2X, 2.5X, 3X, etc. or can be used at a more dilute concentration, such as 1/2X, 1/4X, 1/10X, etc.

[0061] In one embodiment, a final working concentration of nootkatone applied to a connected water system or larval-stage insect breeding site can be about 0.01 % to about 0.03% or higher.

[0062] In one embodiment, a desired final working concentration of nootkatone applied to a connected water system or other larval-stage insect breeding site can be determined by calculating the relative surface area of the water system or breeding site to be treated, wherein the relative surface area refers to an air-liquid interface where a larval-stage insect would surface to breathe or the approximate surface area of the site. For example, a final working concentration can be based on percent coverage of the relative surface area, the relative thickness of nootkatone at the air- surface interface over a relative surface area, or a combination of both. Specific final working concentration examples are about 5 mmol/m², or about 10 mmol/m², about 15 mmol/m², about 25 mmol/m², about 50 mmol/m², about 60 mmol/m², about 70 mmol/m², about 80 mmol/m², about 90 mmol/m², about 100 mmol/m², or higher.

[0063] In another embodiment, a contemplated larvicide composition may be seen in Table No. 1 , where ingredients can be measured in percent volume per volume, percent weight per volume, or percent by weight.

[0064] Table No. 1. Contemplated larvicide composition formulation.

[0065] In certain embodiments, larvicide compositions contemplated herein may include nootkatone and one or more additional active ingredients. The one or more additional active ingredients may be effective against larval-stage insects. In another embodiment, a contemplated larvicide composition may include one or more active ingredients against larval-stage insects and one or more active ingredients against adult stage insects (i.e., a larval-stage insect that has matured to an adult). In some aspects, an additional active ingredient can be highly selectively toxic for a specific larval-stage insect. In another embodiment, an additional active ingredient can have broad specificity for larval-stage insects. In another embodiment, an additional active ingredient can have a different effective treatment profile than nootkatone.

[0066] Additional active ingredients can include one or more biopesticides or biopesticide active ingredients, such as one or more of those registered with the United States Environmental Protective Agency. Additional active ingredients can also include attractants that lure larval-stage insect adults to lay eggs in a larval-stage insect breeding site that has been treated with a contemplated larvicide composition of the present disclosure. Further examples include pyrethroids, neem oil, natural plant extracts, soy oil, mineral oil, spores or metabolites of Bacillus thuringiensis israelensis, or an insect growth regulator, such as, methoprene, pyriproxyfen, or a modified triazine, such as, cyromazine, and combinations thereof. [0067] Further examples of additional active ingredients include plant essential oil compounds or derivatives thereof. Examples include aldehyde C16 (pure), oterpineol, amyl cinnamic aldehyde, amyl salicylate, anisic aldehyde, benzyl alcohol, benzyl acetate, cinnamaldehyde, cinnamic alcohol, carvacrol, carveol, citral, citronellal, citronellol, p-cymene, diethyl phthalate, dimethyl salicylate, dipropylene glycol, eucalyptol (cineole) eugenol, is-eugenol, galaxolide, geraniol, guaiacol, ionone, menthol, methyl salicylate, methyl anthranilate, methyl ionone, methyl salicylate, opheliandrene, pennyroyal oil perillaldehyde, 1 - or 2-phenyl ethyl alcohol, 1 - or 2- phenyl ethyl propionate, piperonal, piperonyl acetate, piperonyl alcohol, D-pulegone, terpinen-4-ol, terpinyl acetate, 4-tert butylcyclohexyl acetate, thyme oil, thymol, lavender oil, rosemary oil, peppermint oil, neem oil, clove extract, metabolites of trans-anethole, vanillin, and ethyl vanillin.

[0068] In another embodiment, a contemplated larvicide composition can include a nootkatone to additional active ingredient ratio of about 1 :10, or about 1 :8, or about 1 :6, or about 1 :4, or about 1 :2, or about 1 :1 , or about 2:1 , or about 4:1 , or about 6:1 , or about 8:1 , or about 10:1 .

[0069] In a further example, nootkatone-containing larvicide compositions can also include an additional active ingredient effective for repelling or killing other non-larval-stage insects or pests or for killing nuisance plants.

[0070] In other embodiments, larvicide compositions contemplated herein can include nootkatone in combination with one or more additives, such as a fragrance, a preservative, a propellant, a pH buffering agent, a UV blocker, a pigment, a dye, a surfactant, an emulsifier, a solvent, a salt, an acid, a base, an emollient, a sugar, and combinations thereof. Additional additives include disinfectants, larval-stage insect attractants or chemical lures and detergents. Contemplated disinfectants include quaternary ammonium compounds, phenol-based antimicrobial agents, and botanical oils with disinfectant properties. In one embodiment, a nootkatone-containing composition contemplated includes an organic material that a larval-stage insect would consume.

[0071] In other embodiments, nootkatone-containing larvicide compositions can include a carrier, such as an aqueous liquid carrier, water, a saline, a gel, an inert powder, a zeolite, a cellulosic material, a microcapsule, an alcohol such as ethanol, a hydrocarbon, a polymer, a wax, a fat, an oil, a protein, a carbohydrate, and combinations thereof. Some carriers include time release materials where a nootkatone-containing composition may be released over a period of hours, or days, or weeks.

[0072] Carriers may be added to a composition in an amount of about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 50% by weight of the composition. In some applications, a carrier can be present in an amount that is at or greater than about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% by weight of the composition. In another embodiment, a carrier can be included in an amount up that when added to the amount of nootkatone included in the composition amounts to 100% by volume.

METHODS

[0073] According to some aspects of the current invention, nootkatone-containing larvicide compositions can be directly applied to larval-stage insects and larval-stage insect breeding sites. According to other aspects of the current invention, nootkatone-containing larvicide compositions may be applied to any larval-stage insect, a connected water system, any larval-stage insect breeding site, a portion of a larval-stage insect breeding site, a surface area and/or material that larval-stage insects may attempt to traverse or inhabit, or surfaces and objects on which larval- stage insects may be observed or that could act as vectors for the transportation of larval-stage insects. Examples of such surfaces include, without limitation, water surfaces (e.g., of ponds, lakes, canals, creeks, ditches, irrigation channels, or marshy areas), the edges of water bodies (e.g., shorelines, pool liners and/or covers, banks, etc.), surfaces of objects that can maintain a pool of water (e.g., animal troughs, ornamental ponds, swimming pools, catch basins, paddling pools, rain barrels, gutters, or any surface of equipment, tools used in conjunction with any of the aforementioned objects, or moist surfaces, such as soils, rotting vegetation, animal feces, animal enclosures, such as pens, sheds, and barns, plant and fungal materials.

[0074] Treatment for larval-stage insect infestation may be routine or prophylactic based on changing environmental conditions (such as raised humidity or temperature), seasonal changes (such as transitions from spring to summer to fall to winter to spring), or in response to large numbers of adult insects or non-insect pests. In some embodiments, contemplated methods include treatment with a larvicide composition including nootkatone may be performed at a temperature between 0 and 50 C, or during a season of high breeding activity of larval-stage insects. Alternatively or in addition, contemplated methods include application of nootkatone- containing larvicide compositions to connected water systems or other larval-stage insect breeding grounds prior to freezing during winter, so that these environments are pre-treated for the following season at the time of thawing.

[0075] According to some aspects of the current invention, nootkatone-containing larvicide compositions may be applied to a surface or larval-stage insect breeding site once the temperature of said surface or larval-stage insect breed site reaches a temperature range suitable for the hatching from eggs and survival of larval stage insects. For example, according to some aspects of the current invention, a first treatment of nootkatone-containing composition may be applied once the water temperature of a body of water suitable for acting as a habitat for mosquito larvae reaches greater than 16 C, greater than 17^°C, greater than 18 C, or greater than 19 C.

[0076] According to some aspects of the current invention, nootkatone-containing larvicide compositions may be applied about once per day, about once every 3 days, about once per week, about twice per week, about once per two weeks, about once per month, about once per two months, or about once per three months, or about once per season.

[0077] According to some aspects of the current invention, nootkatone-containing larvicide compositions may be applied with a frequency calculated such that if a first treatment is applied to the same surface or larval-stage insect breeding site, a second treatment may be applied to the same surface or larval-stage insect breeding site before the end of the adult stage of the insect (target species specific) to be treated as counted from the day before the first treatment was applied. In this manner, the first treatment is effective against larvae present at that time, and the second treatment is effective against larvae resulting from eggs laid by mature insects of the last generation immediately prior to the first treatment that would have been in adult form during the first treatment. If the larval stage of the insect life cycle is shorter than the adult stage, several treatments may be applied until the maximum time for adult stage has passed. For example, according to some aspects, when a first application of nootkatone-containing larvicide compositions is applied to a surface or larval-stage insect breeding site on d=0, a second application of nootkatone-containing larvicide compositions may be applied at d=15, 15 days later to treat any larvae newly hatched from eggs laid by insects that were adults when the first nootkatone-containing larvicide compositions were applied. Optionally, additional treatments of nootkatone-containing larvicide composition may be applied to the surface or larval-stage insect breeding site approximately every subsequent 15 days (e.g., d=30, d=45, d=60) and so on until the maximum adult life of the insect that emerged from a pupa the day before treatment (d=-1 ) has expired. In some aspects, the repetition of the treatment may continue beyond the timeline of the adult life stage to prevent insects from untreated larval stage insect breeding sites laying eggs in the previously treated insect breeding site. This aspect is preferable when there are multiple larval- stage insect breeding sites, at least one inaccessible larval-stage insect breeding site, some difficult to treat larval-stage insect breeding sites, and/or if the insect is highly mobile (e.g., airborne) so able to travel during the timeline during the mature stage of the insect life cycle from one untreated larval-stage insect breeding site to the previously treated larval-stage insect breeding site.

[0078] According to a further embodiment of the present invention, methods of preventing larval infestation of animals and/or humans are contemplated including myiasis caused by myiatic flies, including for example, the botfly, blowfly, and screwfly. Such methods include application of nootkatone-containing compositions directly to larvae of myiatic flies or to plants and surfaces that harbor myiatic fly larvae or to vectors of myiatic fly eggs and larvae, such as mosquitoes, ticks, or other vectors.

[0079] Various methods according to some aspects of the current invention may be employed to contact larval-stage insects, surfaces, and environments rich in larval-stage insects with nootkatone-containing larvicide compositions.

[0080] Nootkatone can be applied, such as by directly pouring the composition into the water or placing a composition dispenser within a connected water system or larval-stage breeding site such that the surface or environment to be treated comes into contact with the nootkatone at an effective concentration of, for example, between 100 and 2,000 ppm, preferably between 200 and 400 ppm, most preferably approximately 300 ppm. The applied nootkatone-containing composition can be left without active removal to degrade naturally.

[0081] In contrast to many active agents against larval-stage insects in the art, nootkatone is able in some compositions of the current invention to form a film on the surface of water. Without wishing to be bound by theory, it is believed that the results disclosed herein indicate that the mechanism of action for killing larval-stage insects in water is tied, at least in part, to the fact that nootkatone floats on the surface of the water in a thin layer. Mosquito larvae, for example, must approach the surface of the water to breathe, but when they come into contact with the nootkatone they are killed. The nootkatone layer eventually evaporates from the surface of the water and prevents accumulation of nootkatone in a stagnant water system. Further, if the nootkatone enters running water unsuitable for mosquito larvae, the inherent movement of the water increases evaporation rates and dilutes the nootkatone below effective larvicidal concentrations.

DISPENSERS/APPLICATORS

[0082] In some embodiments, dispensers or applicators for dispensing or applying a larvicide composition contemplated herein are intended to be reused. For example, upon dispensing a nootkatone-containing larvicide composition, the dispenser or applicator can be refilled. In other embodiments, a dispenser or applicator is a single-use device or substance that functions as a nootkatone-containing larvicide composition carrier that is, itself, dispensed or degraded. For example, a dispenser or applicator can be a dissolvable vehicle such as a pouch, a puck, a pellet, a block, a granule, a vesicle, or a capsule that contains at least one additional substance (i.e., carrier) that contributes to at least one of the structure of the dispenser or application or a controlled release of the nootkatone from the dispenser or applicator.

[0083] Topical larvicide compositions are also contemplated herein that may be dispensed using a dispenser or applicator including one or more of a spray bottle, a brush, a dropper, a sponge, a soft-tipped marking device with reservoir, pressurized dispenser, an aerosol can, a roll on bottle, a wipe, a tissue, and other devices suitable for application to surfaces, objects, or larval- stage insect rich environments.

[0084] In one embodiment, compositions contemplated herein may be applied to one or more surfaces using an applicator having a reservoir for carrying a composition in a wet form and/or a dry form. Examples of applicators that may be used include an aerosol container with a spray nozzle with or without a spray straw to focus delivery of the composition, a spray gun, an impregnated sheet, film, and/or matrix where the composition is released onto the surface by a releasing agent, such as water or other carrier. Additional examples include a pump sprayer, a trigger sprayer, a pressurized spraying device, a sponge, a squeegee, an airbrush, a brush, or a roller. The composition may alternatively be applied by spraying or dispersing over at least a portion of an area susceptible to infestation by larval-stage insects, including but not limited to spraying from a tractor, boat, irrigation spray, helicopter, crop duster or airplane.

[0085] In a further embodiment, methods for treating connected water systems and larval- stage insect breeding grounds may include utilization of nootkatone-containing larvicide composition dispensers that release a contemplated composition into a body of water (treatment locale) over a period of time of minutes, hours, days, or weeks. Contemplated dispensers include floating dispensers that float and dispense at the surface of a body of water. For example, contemplated floating dispensers include those that are used for chlorine dispensing in swimming pools. Floating dispensers may float on the surface of a locale either freely or may be anchored. Further, when anchored, floating dispensers may float subsurface at a predetermined and adjustable depth.

[0086] Other dispensers include sink-floats that may be immersed within a treatment area and sink to or near the bottom of a treatment locale until such time as the treatment composition is completely released, at which time the dispenser floats to the surface to be recharged. In another embodiment, a weighted and buoyed dispenser may be used that includes a weighted composition dispenser connected to a floating buoy that suspends the dispenser at a predetermined depth at which depth the composition is dispensed.

[0087] In another embodiment, it is contemplated that a "use up cue" may be included in the contemplated dispensers, such as, for example, a beacon that gives off light and/or sound or changes color when a treatment composition has been nearly or completely used up. The use up cue may be based on a timer, in that, after a predetermined length of time that coincides with the time when the treatment composition is nearly or fully dispensed, the use up cue is triggered by the timer.

[0088] In the context of the sink float, the use up cue may function based on the rate of solubility of the treatment composition, such that when the treatment composition is fully dissolved, the weight loss from the sink float causes the float to rise to the surface. Alternatively, the use up cue of the sink float may be based on the rate of solubility of a companion substance within the sink float that dissolves at a rate corresponding to the rate of dispensing of the treatment composition.

[0089] In a further embodiment, a dispensor for use at the edge of a body of water may be in the form of a spike or similar device that can be driven into the bed of the body of water or at the edge of the body of water. The nootkatone composition within the device can then leach out into the body of water to effectively treat any present larval-stage insects and prevent further infestation of the connected water system or larval-stage breeding site.

[0090] Further, when treating an indoor space, such as the floor of an animal enclosure, metered timed dispensers may be used, such as those that incorporate a container, such as a trigger sprayer or an aerosol container and trigger spraying of a metered amount of a composition into the indoor space periodically based on a timer or a sensor (e.g., light, movement, heat, moisture). Examples of such dispensers include active fragrance dispensers available from Rubbermaid®, Lexpon®, and Airwick®.

[0091] Another aspect of the current invention includes pretreatment of surfaces, objects, environments prone to infestation with in larval-stage insects. This may be accomplished by coating the surfaces or objects with compositions that resist removal from the surface and contain an amount of a nootkatone, such as a paint, a clear coat, a wax, an oil, an adhesive, a resin, a cleaning solution, and combinations thereof. Another approach includes lining the surfaces, objects, environments prone to infestation with in larval-stage insects with one or more nootkatone- impregnated materials, such as thermoplastic or thermoset sheets, paperboard, or cardboard impregnated with nootkatone.

[0092] A further treatment approach is to construct surfaces or objects with nootkatone- impregnated or nootkatone-coated materials, such as plastics, wood, cloth, textiles, composites, or porous materials to prevent infestation of connected water systems and other areas where larval- stage insects may breed. The approaches disclosed herein can be used alone or in any combination. EXAMPLES

[0093] The Examples that follow are illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only and are not taken as limiting the invention. In particular, these examples demonstrate the susceptibility of larval-stage insects to nootkatone-containing larvicide compositions.

Example No. 1 : Susceptibility of mosquito larvae to treatment with nootkatone formulations.

[0094] This example describes a laboratory bioassay in which groups of mosquito larvae were exposed to a nootkatone-containing composition to determine larval susceptibility to nootkatone.

[0095] The organisms used for testing are shown in Table No. 2 below.

[0096] Table No. 2. Organisms used for testing.

Treatment

[0097] One milliliter of a solution of nootkatone in ethanol was applied directly to 100 milliliters of water containing larvae, using a micropipette. One treatment condition used 3% (v/v) nootkatone in ethanol, and a second treament condition used 1 % (v/v) nootkatone in ethanol, for final concentrations of 0.03% or 0.01 % (v/v), respectively. One milliliter of pure ethanol was used as a control. Four replicates of 25 larvae were tested per treatment. Clean, glass 600 ml_ laboratory beakers were used as test containers such that the surface area of the water to be treated was 54 cm².

Assessments

[0098] Larvae were observed after 24 hours post-introduction to the test containers. The larvae were scored according to the following criteria: Morbidity (M): does not swim to/from the water surface to feed and breathe or otherwise initiate directional movement, but still exhibits movement with or without tactile stimulation; or

Dead (mortality): exhibits no movement, even with tactile stimulation.

[0099] Readings were discontinued if treated mortality reached 100%, or control mortality exceeded 20%.

Results

[00100] The results for 0.03% nootkatone treatment are shown in Table No. 3 below, and complete results are shown in Figure 2.

[00101] Table No. 3. Effects of Nootkatone Treatment on mosquito larvae.

[00102] None of the control larvae that were exposed to 1 % ethanol were affected after 24 hours. In the four replicates that were exposed to 0.03% nootkatone (final concentration, v/v), at the end of 24 hours of exposure, 100% were killed, on average. In the four replicates that were exposed to 0.01 % nootkatone (final concentration, v/v), at the end of 24 hours of exposure, 80% were killed, on average, but mortality rose to 100% after 48 hours.

[00103] The test results indicate very high mortality rates for mosquito larvae exposed to low concentrations of nootkatone compared to control after 24 hours of exposure. Therefore, mosquito larvae are highly susceptible to low concentrations of nootkatone, and nootkatone-containing compositions can effectively treat and prevent mosquito larvae infestations. These data further suggest that infestations by other larval-stage insects can be effectively treated with nootkatone- containing compositions. Example No. 2: Susceptibility of mosquito larvae to treatment with formulations that contain oils without nootkatone.

[00104] This example describes a laboratory bioassay in which groups of mosquito larvae were exposed to compositions that contain oils without nootkatone, to determine whether larval mortality was due to suffocation by oils in general, or due to a specific property of nootkatone.

[00105] The organisms used for testing were mosquito larvae, as shown in Table No. 2 above.

Treatment

[00106] One milliliter of a solution of oil in ethanol was applied directly to 100 milliliters of water containing larvae, using a micropipette. One treatment condition used 3% (v/v) soybean oil in ethanol, and a second treatment condition used 3% (v/v) mineral oil in ethanol, for final concentrations of 0.03% (v/v), respectively. One milliliter of pure ethanol was used as a control, and 0.03% (v/v) nootkatone in ethanol was included as a comparison of efficacy. Two replicates of 25 larvae were tested per treatment in clean, glass 600 mL beakers.

Assessments

[00107] Larvae were observed at +24, +48, and +72 hours post-introduction to the test containers. At each observation period, larvae were scored according to the following criteria:

Morbidity (M): does not swim to/from the water surface to feed and breathe or otherwise initiate directional movement, but still exhibits movement with or without tactile stimulation; or

Dead (mortality): exhibits no movement, even with tactile stimulation.

[00108] Readings were discontinued if treated mortality reached 100%, or control mortality exceeded 20%.

Results

[00109] The results are shown in Figure 3. As in previous experiments, addition of 0.03% nootkatone killed 100% of larvae in less than 24 hours, and the larvae were unaffected by an ethanol-only treatment. Mineral oil was somewhat effective, killing an average of 84% of larvae in 24 hours. Soy oil was not effective as a larvicide, as only 14% of larvae were killed in 24 hours. These test results indicate that soy oil and mineral oil caused lower mortality rates and slower mortality than replicates of larvae that were treated with the same concentration (0.03% v/v) of nootkatone. Therefore, the data demonstrate that nootkatone is unique in the rapidity and efficacy of its killing larvae compared to the other tested oils. Example No. 3: Determining the effect of surface area on the efficacy of nootkatone compositions for larvicide activity

[00110] In this example, concentration titration was performed to determine effective concentrations of nootkatone in nootkatone-containing protective compositions that maintain larvicidal activity, and to determine the effect of surface area on larvicidal activity of nootkatone- containing compositions.

Treatment

[00111] One milliliter of a solution of nootkatone in ethanol was applied directly to 200 milliliters of water containing larvae, using a micropipette. One treatment condition used 3% (v/v) nootkatone in ethanol, a second treament condition used 1 % (v/v) nootkatone in ethanol, and a third treatment used 0.5% (v/v) nootkatone in ethanol, for final concentrations of 30, 10, or 5 mg of nootkatone added per container. One milliliter of pure ethanol was used as a control. Three replicates of 25 larvae were tested per treatment in small- (250 mL flasks (23.8 cm² surface area) and medium-sized (600 mL beakers (54 cm² surface area)) containers, but only two replicates were made in large 1000 mL beakers (86.6 cm² surface area).

[00112] Containers were lightly covered with paper towels. Indicated surface areas were calculated based on the inside diameter of the container at the surface level when 200 mL water was added to the container.

Assessments

[00113] Larvae were observed after 24 hours post-introduction to the test containers. The larvae were scored according to the following criteria:

Dead (mortality): exhibits no movement, even with tactile stimulation.

[00114] Readings were discontinued if treated mortality reached 100%, or control mortality exceeded 20%.

Results

[00115] The surface concentrations tested were 5 mg/ 23.8 cm² (about 0.2 mg/cm²), 10 mg/ 23.8 cm² (about 0.42 mg/cm²), 30 mg/ 23.8 cm² (about 1 .3 mg/cm²), 5 mg/ 54 cm² (about 0.09 mg/cm²), 10 mg/ 54 cm² (about 0.19 mg/cm²), 30 mg/ 54 cm² (about 0.56 mg/cm²), 5 mg/ 86.6 cm² (about 0.06 mg/cm²), 10 mg/ 86.6 cm² (about 0.12 mg/cm²), and 30 mg/ 86.6 cm² (about 0.35 mg/cm²). The results are shown in Figure 4. A few larvae died in the 250 mL control flasks, perhaps due to the stress of being in a deeper water column, but these control deaths averaged only 4% of the larvae in the control flasks. The presence of 30 mg of nootkatone was sufficient to kill all larvae in 24 hours, no matter which container size was used. Ten milligrams of nootkatone killed all larvae in the flasks with the smallest surface area (at a concentration of about 0.42 mg/cm²), but the mortality rate decreased as surface area increased, even though total volume, and therefore volumetric concentration of nootkatone, was held constant. The same effect was even more pronounced when only 5 mg of nootkatone was introduced into each container, as larval mortality dropped from 68% in the flasks to only 16% in the medium sized containers. This lowest concentration of nootkatone did not kill in the largest containers, even though the volumetric concentration of nootkatone, was the same in all cases. Therefore, at a surface concentration of at least about 0.09 mg/cm² nootkatone exhibits larvicidal properties. It is believed that this surface concentration would be equally effective on water and a solid surface. It is further contemplated that even lower surface concentrations of nootkatone can affect larvae development to hinder progression of the larvae to adulthood.

Example No. 4: Determining duration of protection from larvae by nootkatone- containing compositions

[00116] In this example, nootkatone-containing larvicide compositions were formulated to maximize duration of protection by at least one of killing or repelling larvae.

[00117] Method 1: Clean, glass 600 mL beakers were set up as test containers (such that the surface area of water to be treated was 54 cm²), and were treated with 1 mL of 3% nootkatone in ethanol (v/v), or 1 mL of ethanol alone, as in Example No. 1 , to achieve a final concentration of 0.03% nootkatone (v/v). After 24 hours, 100% of larvae were dead in the treated beakers, but 100% were alive in the control beakers, as in Example No. 1 . An additional cohort of 25 larvae was added to each treatment beaker in the smallest practical volume of additional water, estimated at 1 mL per beaker. After an additional 24 hours, total live and dead larvae were counted and recorded. Testing continued by addition of new larval cohorts (25 per beaker, per day) for additional 24 hour periods until either there were no available 3rd/4th instar larvae, or until the rate of larval killing decreased.

[00118] In two replicate experiments, larvae cohorts were added to test containers in the presence of 0.03% nootkatone, 1 % ethanol, starting 24 to 72 hours after addition of nootkatone and ethanol to the test containers (see Figures 5 and 6). In the second experiment, additional additional larval replicates were added simultaneously to test containers in the presence of 0.01 % nootkatone, 1 % ethanol (see Figure 6).

[00119] In a separate experiment with the same experimental set-up, test containers were inoculated with nootkatone in ethanol 48 hours prior to addition of larvae (see Figure 7). In these experiments, 100% of larvae were killed within 24 hours up to a week after nootkatone treatment. One week after addition of nootkatone, larval mortality was still at 94%.

[00120] Method 2: Clean, glass 600 mL beakers were set up as test containers (such that the surface area of water to be treated was 54 cm²), and were treated with 1 mL of 1 % or 3% nootkatone in ethanol (v/v), or 1 mL of ethanol alone, to achieve a final concentration of 0.01 % or 0.03% nootkatone (v/v), respectively. Beakers were lightly covered with Kimwipes to prevent contamination, and to reduce evaporation. Beakers treated with 1 mL of 100% ethanol were used as controls. Four cohorts of 25 3rd and 4th instar larvae were added to individual beakers per day, 3-14 days after treatment with nootkatone, and mortality of larvae was recorded 24 and 48 hours after addition of larvae to beakers.

Results

[00121] For the experimental data shown in Figures 5-7, the method used included addition and removal of water, and potentially, removal of nootkatone, on each succeeding day. In a new experiment, individual beakers were set up in advance, so that all beakers were used one and only one time to test the longevity of nootkatone, with the only possible loss due to evaporation. The results shown in Figure 8 demonstrate that a concentration of 0.03% nootkatone killed 100% of larvae within 24 hours when larvae were added to treated water 3-7 days after the water was treated. Treatment with 0.01 % nootkatone resulted in a lower level of killing at 24 hours, and killing at 24 hours in the presence of 0.01 % nootkatone gradually decreased from 98% 3 days after water treatment to 81 % killing 7 days after water treatment. However, when the same beakers were scored 48 hours after addition of larvae (Figure 9), the 0.01 % nootkatone treatment resulted in 100% larval mortality for 3-7 days after water treatment.

[00122] In a separate experiment using the same setup, nootkatone-treated beakers were held for two weeks prior to addition of larvae. Due to the extended time delay, more than 50% of the original liquid volume had evaporated. To avoid having a potentially higher concentration of nootkatone than the desired 0.03%, sterile water was added to bring the total volume back to 100 mL before larval cohorts were added. As shown in Figure 10, 100% of the larvae were killed within 24 hours, demonstrating a nootkatone killing duration of at least two weeks at the 0.03% concentration. Example No. 5: Evaluation of nootkatone's larvicidal activity against caterpillars.

Overview

[00123] Some of the most damaging and therefore commercially relevant larvae are those of Lepidopteran species. There are over 1 10,000 different species in this order, many of which are significant pests. The aim of this experiment was to confirm that the larvicidal activity of nootkatone extended to caterpillar larvae.

METHODOLOGY

[00124] Test Formulations: Nootkatone ("NK") solutions at 0.25% concentrations were obtained by dissolving 98% pure NK in 100% EtOH (250 mg/mL) and diluted to achieve 0.25% solutions with filtered butane to permit application via an aerosol spray. For negative controls, 4 ml of 100% ethanol was added to filtered butane to give an appropriate volume of solution for the aerosol sprays used for each test.

[00125] Selection of specimens: All caterpillars selected for use in these experiments were 5- 6 cm long and actively moving. Caterpillars in the treatment groups were sprayed outside (in the open air) on bushes with 100 mg NK aerosol per caterpillar or with the same amount of negative control aerosol.

[00126] Treatment conditions: The whole experiment was carried out at 26±1 °C. During and after treatment, caterpillars were permitted to move freely over the branches of the bushes.

[00127] Assessment / Observations: Caterpillars of the nootkatone-treated and negative control groups were frequently monitored over the 6 hour course of the experiment for whole body movements and categorised as sinuously motile, sluggishly motile (intermittent jerky movements on touch), coiling, other movements, immotile/paralysed, or dead.

RESULTS

[00128] Most nootkatone-treated caterpillars were noted to be dead by 30 to 45 minutes after treatment. Of the surviving caterpillars, all caterpillars exhibited restricted movement and became inactive within 1 hour of treatment. By 6 hours post-treatment, all caterpillars were dead and many had darkened. In contrast, caterpillars exposed to negative controls were alive, exhibited normal motility, and were feeding normally. Example No. 6: Evaluation of nootkatone residue on larval food source for feeding reduction and larvicidal activity.

Overview

[00129] Some methods of preventing and/or treating larvae disclosed herein include the application of nootkatone to a larval food source or hatching location. The aim of this experiment was to determine whether the larvicidal activity of nootkatone extends to untreated larvae coming into contact with and/or eating larval food sources sprayed with nootkatone.

METHODOLOGY

[00130] Collection of specimens: Live caterpillars were collected from the same bush.

[00131] Test Formulations: Nootkatone ("NK") solution at a concentration of 600 ppm was obtained by dissolving 98% pure NK in 100% EtOH and diluting in filtered Butane (LPG) to permit application via an aerosol spray. For negative control, 4 ml of 100% ethanol was added to filtered butane (LPG) to give an appropriate volume of solution for the aerosol sprays used for each test.

[00132] Selection of specimens: All caterpillars selected for use in these experiments were 5- 6 cm long and actively moving. Eight caterpillars were placed in each treatment group and kept in individual plastic trays with 100 g leaves. The leaves in one treatment group were sprayed with 300 μg NK aerosol. The leaves in the negative control group were sprayed with the same quantity negative control aerosol.

[00133] Treatment conditions: The entire experiment was carried out at 26±1 °C, which was well within the natural temperature range commonly experienced by the species of caterpillar tested. During treatment, caterpillar larvae were maintained in identical clean plastic trays containing the relevant leaves. The trays were oversized such that the caterpillars could crawl away from the leaves if their instinct was to do so.

[00134] Assessment / Observations: Caterpillars were under continuous observation and scored for motility, immobility and death at 0 hours, 6 hours and 24 hours following introduction to NK treated or control leaves. Typical displays of motility included whole body movements, sinuous motility, coiling, and leg movements. Cessation of feeding, sluggish motility, and intermittent jerky movements on touch stimulation were typical early symptoms of immotility and/or paralysis.

RESULTS

[00135] Greatly reduced rates of feeding were observed in trays containing leaves treated with nootkatone. The caterpillars were observed trying to move away from the leaves. Within 6 hours post-exposure, all caterpillars introduced to nootkatone-treated leaves exhibited very restricted movement and became inactive. By 24 hours post-exposure, all caterpillars in the trays containing nootkatone-treated leaves were seen to be dead, whilst all the caterpillars in the negative control were alive and feeding normally.

Example No. 7: Susceptibility of stable fly larvae to treatment with nootkatone formulations.

[00136] This example describes a laboratory bioassay in which groups of stable fly larvae were exposed to a nootkatone-containing composition to determine larval susceptibility to nootkatone.

[00137] The organisms used for testing are shown in Table No. 4 below.

[00138] Table No. 4. Organisms used for testing.

Treatment

[00139] One milliliter of treatment was applied directly to fly media in a 100 millimeter Petri dish using a Paasche air sprayer from 12" distance. Media were allowed to air dry for 1 hour before introduction of larvae. One treatment was a 1 % (w/v) solution of nootkatone in ethanol. Pure ethanol was used as a control treatment. The two treatments were compared to an untreated control. Four replicates of 10 larvae were tested per treatment or control.

Assessments

[00140] Larvae were observed after 4 hours and 24 hours post-introduction to the test containers. The larvae were scored according to the following criteria:

Dead (mortality): exhibits no movement, even with tactile stimulation.

Results

[00141] The results are shown in Tables No. 5 and 6 below.

[00142] Table No. 5. Effects of Nootkatone Treatment on stable fly larvae.

0% 15% 45%

Untreated

0% 38% 90%

Ethanol

1 % 0% 75% 100%

Nootkatone

[00143] The control larvae that were not exposed to treatment exhibited 15% mortality after 4 hours, and 45% mortality after 24 hours ("untreated" in Table No. 5). Due to this high rate of control mortality, Abbott's formula was used to recalculate 4 hour treatment mortality rates, and the corrected values are shown in Table No. 6. The 24 hour mortality values were discarded, as control mortality exceeded 20%.

[00144] Abbott's correction = 1 - (1-uncorrected treatment mortality)/(l-untreated mortality) [00145] Table No. 6. Effects of Nootkatone Treatment on stable fly larvae.

[00146] Although treatment of fly media with 100% ethanol was associated with (corrected) 26% mortality after 4 hours of exposure, 71 % of larvae placed on fly media treated with 1 % nootkatone in ethanol were killed at 4 hours, nearly a three-fold higher rate.

[00147] The test results indicate high mortality rates for maggots, such as stable fly larvae, exposed to low concentrations of nootkatone compared to control after 4 hours of exposure. Therefore, maggots are believed to be highly susceptible to low concentrations of nootkatone, and it is believed that nootkatone-containing compositions can be used to effectively treat and prevent maggot infestations.

[00148] Having described the invention in detail and by reference to specific aspects and/or embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims. More specifically, although some aspects of the present invention may be identified herein as particularly advantageous, it is contemplated that the present invention is not limited to these particular aspects of the invention. Percentages disclosed herein may vary in amount by ±10, 20, or 30% from values disclosed and remain within the scope of the contemplated invention.

Claims

WHAT IS CLAIMED IS:

1 . A method of killing a larval-stage insect, comprising:

applying an effective amount of a nootkatone-containing larvicide composition to a larval- stage insect breeding site.

2. The method of claim 1 , wherein the larval-stage insect breeding site comprises a body of water or a moist surface.

3. The method of claim 2, wherein the nootkatone-containing composition creates a film at an air-liquid interface of the body of water or on the moist surface.

4. The method of claim 3, wherein the film comprises nootkatone.

5. The method of claim 1 , wherein the effective amount of the nootkatone-containing larvicide composition is an amount sufficient to render a final surface concentration of at least about 0.09 mg/cm² at the larval-stage insect breeding site.

6. The method of claim 1 , wherein the larval-stage insect includes a pathogen-carrying or a pathogen-free insect or a non-insect pest.

7. The method of claim 1 , wherein the larval-stage insect includes a member of the genera Anopheles, Aedes, Culex, Chrysomya, Lucilia, or Haemagogus or a member of the families Tortricidae, Noctuidae, or Pyralidae.

8. The method of claim 1 , wherein the larval-stage insect is a gnat, a cockroach, a chigger, a black fly, a deer fly, a sandfly, a stable fly, a tsetse fly, a myiatic fly, an armyworm, a corn earworm, Pieris brassicae, a cloth moth, or a cotton bollworm.

9. The method of claim 7, wherein the larval-stage insect is a member of the genus

Anopheles.

10. The method of claim 9, wherein the member of genus Anopheles is Anopheles gambiae or Anopheles quadrimaculatus.

1 1 . The method of claim 7, wherein the larval-stage insect is a member of genus Aedes.

12. The method of claim 1 1 , wherein the member of genus Aedes is Aedes aegypti.

13. A method of treating or preventing a larval-stage insect infestation, comprising:

(a) providing a nootkatone-containing larvicide composition;

(b) optionally diluting the composition to a working concentration with a carrier; and

(c) applying the composition to a larval-stage insect breeding site.

14. The method of claim 13, wherein the larval-stage insect breeding site is a connected water system.

15. The method of claim 14, wherein the connected water system includes at least one of a watershed, a tributary, an irrigation system, a sprinkler system, a pool, a water fountain, a drainage system, an animal watering system, a bird path, a fish pond, an aqueduct, or any other part of a water system that serves as a larval-stage insect breeding site.

16. The method of claim 15, wherein the connected water system is an irrigation system.

17. A larvicide composition, comprising:

(a) about 0.01 % to about 10% nootkatone;

(b) an optional additional active ingredient; and

(c) an optional carrier.

18. The larvicide composition of claim 17, wherein the composition when applied to a larval- stage insect breeding site has a final concentration of about 0.01 % nootkatone (v/v).

19. The larvicide composition of claim 17, wherein the composition when applied to a larval- stage insect breeding site has a final working surface concentration of at least about 0.09 mg / cm²

20. The larvicide composition of claim 17 further comprising an additional active ingredient, wherein the additional active ingredient comprises a soy oil, a mineral oil, spores or metabolites of Bacillus thuringiensis israelensis, an insect growth regulator, and combinations thereof.

21 . The larvicide composition of claim 20, wherein the insect growth regulator comprises methoprene or cyromazine.

22. The larvicide composition of claim 17, wherein the composition kills at least about 90% larvae exposed to the composition one week after application to a larval-stage insect breeding site.

23. The larvicide composition of claim 22, wherein the composition kills at least about 100% larvae exposed to the composition two weeks after application to a larval-stage insect breeding site.

24. The larvicide composition of claim 17, wherein the composition is about 5X, about 10X, about 20X, about 30X, or about 60X concentrate.

25. A method of killing a larval-stage insect, comprising:

applying an effective amount of a nootkatone-containing larvicide composition to a larval- stage insect food source.

26. The method of claim 25, wherein the food source comprises a food crop.

27. The method of claim 26, wherein the food crop comprises one or more of lettuce, cabbage, beans, corn, tomatoes, and cotton.

28. The method of claim 25, wherein the food source is a grass or other plant leaf.

27. The method of claim 25, wherein the larval-stage insect includes a species of the

Tortricidae, Noctuidae, and Pyralidae families.

28. The method of claim 25, wherein the food source is a tree or a part of a tree.

29. The method of claim 25, wherein the food source is a root.

30. The method of claim 25, wherein the food source is a grain, a fruit, or a nut, a shoot, a bud, or a flower.