WO2024178039A2

WO2024178039A2 - Bottle bioassay methods of pesticide assessment

Info

Publication number: WO2024178039A2
Application number: PCT/US2024/016610
Authority: WO
Inventors: Laeticia Leroy Rene-Boisneuf; Andrea LEVEY; Joanna Maria Tyszko
Original assignee: Clarke Mosquito Control Products Inc
Current assignee: Clarke Mosquito Control Products Inc
Priority date: 2023-02-24
Filing date: 2024-02-21
Publication date: 2024-08-29
Anticipated expiration: 2025-08-24
Also published as: WO2024178039A3

Abstract

Methods for determining the susceptibility of pests to pesticides are described herein, including pesticides that, with assistance, penetrate a pest's cuticle, such as solid pesticides. Further disclosed are methods of determining resistance of pests to a pesticide and selecting a surfactant for a pesticide composition to be used in the foregoing methods. Exemplary methods may be applicable to novel pesticides with new modes of action.

Description

BOTTLE BIOASSAY METHODS OF PESTICIDE ASSESSMENT

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63/486,885, filed February 24, 2023, which is incorporated herein by reference in its entirety.

FIELD

[0002] This disclosure relates to methods for determining the susceptibility of pests to pesticides. Exemplary methods may be particularly suited for solid pesticides. The disclosure also relates to methods of determining resistance of pests to a pesticide and selecting a surfactant for a pesticide composition to be used in the foregoing methods.

INTRODUCTION

[0003] Resistance is a heritable change in the susceptibility of a pest population that causes the repeated failure of a pesticide to reach an appropriate level of pest control. As a result of continuous applications of the same pesticide, naturally resistant pests prevail (namely, vector population), which mate leaving offspring also resistant and becoming predominant in the population.

[0004] Monitoring resistance in the vector population is extremely valuable in determining the possible causes of pest control failures. To help delay or prevent the development of pesticide resistance in vector populations, annual monitoring of resistance in target populations allows for early determination of resistance and proper selection of pesticides.

[0005] The Centers for Disease Control (CDC) has formulated an assay trial to determine whether a specific active ingredient in an insecticide can kill mosquito vectors. The technique is known as the “CDC Bottle Bioassay”, published as CONUS Manual for Evaluating Insecticide Resistance in Mosquitoes Using the CDC Bottle Bioassay Kit, CDC 2020, incorporated herein by reference in its entirety. The CDC Bottle Bioassay includes coating a bottle with a diagnostic dose of insecticide, placing mosquitoes into the bottle, and observing the mosquitoes for two hours. Resistance is determined by the percentage of mosquitoes that die (namely, the mortality rate) at a pre-determined threshold time during those two hours. [0006] Accordingly, the CDC Bottle Bioassay methodology and technique will need to be modified for control products with new modes of actions (MOAs), new active ingredients, and new formulations. The CDC Bottle Bioassay protocol involves diluting an active ingredient in acetone, coating the test bottles, and then introducing mosquitoes after the bottles are dry.

SUMMARY

[0007] In one aspect, a method of determining susceptibility of pests to a pesticide is disclosed. An exemplary method may comprise collecting a first sample of pests inside of a first container, wherein the inside of the first container is coated with acetone, collecting a second sample of pests inside of a second container, wherein the inside of the second container is coated with a composition comprising the pesticide, one or more nonionic surfactants, and acetone; monitoring the first sample of pests and the second sample of pests for mortality within one or more predetermined periods of time; determining a mortality ratio for each predetermined period of time for the first sample of pests; determining a mortality ratio for each predetermined period of time for the second sample of pests; and when the mortality ratio is higher for the second sample of pests as compared to the first sample of pests, determining the pests are susceptible to the pesticide.

[0008] In another aspect, a method of determining resistance of pests to a pesticide is disclosed. An exemplary method may comprise collecting a first sample of pests inside of a first container; wherein the inside of the first container is coated with a composition comprising the pesticide, one or more nonionic surfactants, and acetone; and wherein the first sample of pests are susceptible to the pesticide; collecting a second sample of pests inside of a second container, wherein the inside of the second container is coated with the composition comprising the pesticide, the one or more nonionic surfactants, and acetone; and wherein susceptibility of the second sample of pests to the pesticide is unknown; monitoring the first sample of pests and the second sample of pests for mortality within one or more predetermined periods of time; determining a mortality ratio for each predetermined period of time for the first sample of pests; determining a mortality ratio for each predetermined period of time for the second sample of pests; and when the mortality ratio is low for the second sample of pests, determining the second sample of pests is resistant to the pesticide. [0009] The disclosure provides for other aspects and embodiments that will be apparent in light of the following detailed description and accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. l is a graph showing results from the standard CDC Bottle Bioassay using imidacloprid as a pesticide that was dissolved in acetone. Mosquitoes were exposed to 0.2 mg/mL, 1 mg/mL, 2 mg/mL, and 4 mg/mL of imidacloprid.

[0011] FIG. 2 shows images of surfactants tested in a bottle bioassay described herein. FIG. 2 shows images of an ethoxylated lauryl alcohol surfactant dissolved in acetone at a concentration of 1 mg/mL, coated, and dried onto the inside of a bottle. The images show that the mosquitos do not stick to the side of the glass bottle (that is, neither surfactant has a sticking effect).

[0012] FIG. 3 is a graph showing results from a bottle bioassay described herein using three different compositions: a composition comprising 0.2 mg/mL imidacloprid alone, a composition comprising 1 mg/mL polyoxyethylene-polyoxypropylene block copolymer (a surfactant) alone, and a composition comprising 0.2 mg/mL imidacloprid and 1 mg/mL polyoxyethylenepolyoxypropylene block copolymer, where each composition was dissolved in acetone.

[0013] FIG. 4 is a graph showing results from a bottle bioassay described herein using compositions comprising 0.2 mg/mL imidacloprid alone dissolved in acetone or 0.2 mg/mL imidacloprid in combination with 1 mg/mL polyoxyethylene-polyoxypropylene block copolymer dissolved in acetone. Each composition was tested against Aedes aegypti Rockefeller (ROCK) and Puerto Rico (PR) strains.

DETAILED DESCRIPTION

[0014] The CDC Bottle Bioassay does not lend itself to formulations comprising solid active ingredients or other formulations that work at least in part by penetrating the cuticle of the pest. For example, one such active ingredient is imidacloprid, a neonicotinoid active ingredient. As a solid, imidacloprid requires a formulation that includes solvents and surfactants to effectively penetrate a pest’s cuticle and deliver a lethal dose of imidacloprid to the pest. Therefore, diluting solid active ingredients in acetone alone according to the CDC Bottle Bioassay protocol will not provide effective penetration and administration of the solid active ingredient to a mosquito. Thus, there is a need for a method of determining the susceptibility and resistance in pests to pesticides that include solid active ingredients as well as other pesticides that penetrate a pest’s cuticle.

[0015] The instant disclosure relates to a modified bottle bioassay for testing for solid active ingredients, new modes of action, or new active ingredient products for pest control. As described in greater detail below, exemplary methods expose pests to a composition comprising a pesticide and one or more nonionic surfactants diluted with acetone. Not all surfactants at all possible concentrations provide acceptable performance, particularly with all active ingredients. Accordingly, exemplary surfactants and exemplary amounts described herein are particularly suited for improving solubility in the bioassay solvent and optimizing transport across the pest cuticle, and for adhering to the bottle surface and transporting the active ingredient to the pest while not causing the pest to stick to the wall of the bottle.

[0016] Exemplary methods may include determining susceptibility of pests to a pesticide. Exemplary methods allow the pesticide to penetrate the pest’s cuticles while the effect on the pests can be observed in new ways compared to traditional methods. Exemplary methods may allow for more rapid assessments and may provide better predictability of performance in the field.

I. Definitions

[0017] Unless otherwise defined, all technical and scientific terms used here have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described here are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event however of any latent ambiguity, definitions provided here take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. [0018] All publications, patents, patent applications, and/or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and/or other document were individually indicated to be incorporated by reference for all purposes. The materials, methods, and examples disclosed are illustrative only and not intended to be limiting.

[0019] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used here, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and,” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented here, whether explicitly set forth or not.

[0020] For the recitation of numeric ranges, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0021] The term “about” or “approximately” as used here as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In certain aspects, the term “about” refers to a range of values that fall within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Alternatively, “about” can mean within three or more than three standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, preferably within five-fold, and more preferably within two-fold, of a value.

[0022] As used herein, the term “active ingredient” refers to a pesticide. [0023] As used herein, “coated” refers to a surface where at least about 90% of the surface area is covered by a composition described herein.

[0024] The terms “control,” “reference level,” and “reference” are used interchangeably. The reference level may be a predetermined value or range, which is employed as a benchmark against which to assess the measured result. “Control group” as used refers to a group of control organisms. The predetermined level may be a cutoff value from a control group. The predetermined level may be an average from a control group. The normal levels or ranges for a lethal dose or for pesticide activity may be defined in accordance with standard practice. A control may be an organism not exposed to an insecticide. A control may be an organism, or a sample therefrom, whose condition is known. The organism, or sample therefrom, may be exposed to an insecticide or not exposed to an insecticide.

[0025] “Effective amount” refers to a dosage of a compound or composition sufficient for eliciting a desired effect, commensurate with a reasonable benefit/risk ratio. This term as used here may also refer to an amount effective at bringing about a desired in vivo effect in a subject, preferably, an insect, such as mortality after exposure to an effective amount of an insecticide.

[0026] “Mosquito” as used here refers to any species of the roughly 3,500 species of the insect that is commonly associated with and given the common name “mosquito.” Mosquitoes span 41 insect genera, including the non-limiting examples of Aedes, Culex, Anopheles (vector of malaria), Coquillettidia, and Ochlerotatus.

[0027] “Pest” refers to small organisms that are capable of destroying crops, food, causing diseases, and attacking other organisms. The pest may be of the order Diptera such as: mosquitoes, Nematocera (e.g., crane flies, midges, gnats), Brachycera (e.g., horse flies, robber flies, bee flies), and Cyclorrhapha (e.g., flies that breed in living or dead vegetable or animal material).

[0028] As used here, the term “pesticide” includes insecticides. A pesticide may also be referred to as a “toxin.” Pesticides may be any compound that is capable of acting as such.

[0029] As used here, “pesticide resistance” or “resistance” refers to an overall reduction in the ability of a pesticide to kill a pest. Meaning that, when a pesticide is used as directed, the pesticide no longer works or only partially works. Pesticide resistance can be pesticide specific, or it can develop to certain class(es) of pesticide(s). Over time, the repeated use of pesticide(s) can lead to pesticide resistance in pest populations.

[0030] “Sample” or “test sample” as used here can mean any sample in which the efficacy or susceptibility of a pesticide is to be detected or determined. The sample may be a biological sample. Samples may include pests, liquids, solutions, emulsions, or suspensions. Samples can be obtained by any means known in the art. The sample can be used directly as obtained from an organism or can be pre-treated, such as, by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, and addition of reagents, to modify the character of the sample in some manner or otherwise as is known in the art.

[0031] “Subject” and “organism” as used here interchangeably refer to any insect or pest, including, but not limited to, any insect or pest which is known to offer at least some resistance to a pesticide or insecticide, and which is considered necessary to disable and/or kill. Examples include those that attack or damage or otherwise reduce the commercial or other value of a substrate, such as, crops, particularly arable crops, such as, food and material crops, such as, cotton. Other pests include those that are a nuisance to or an adversary of other living organisms, including mammals, such as humans. For example, the insect may be a mosquito. The subject may be at any stage of development, for example, egg, larva, pupa, or imago (adult) stages.

[0032] “Treatment” or “treating” as used here refers to applying a toxin or pesticide to a subject as a means of inducing damage or death due to exposure to the toxin or pesticide. A treatment may be either performed in an acute or chronic way.

[0033] Described herein are exemplary containers, compositions, and methods. Exemplary containers and compositions are described in section II, below. Exemplary methods are described in section III, below.

II. Containers and Compositions

[0034] Exemplary compositions and exemplary containers for housing the compositions are described in greater detail below. A. Exemplary Containers

[0035] An exemplary container may be a container in compliance with standards developed and promulgated by a governing organization, such as the Centers for Disease Control and/or the World Health Organization. A container may be made of a substrate such as glass. A container may be made of a substrate such as a plastic material. In some embodiments, the container may be a glass bottle with a lid. The glass bottle may be a 250 mL glass bottle with a plastic cap such as, but not limited to, a phenolic cap.

[0036] A composition described herein may be compatible with a substrate, such as glass. For example, the composition may adhere to a substrate described herein, but does not bind too strongly to the substrate to allow for a pesticide to be taken up by a pest. The composition may adhere to a substrate described herein, but does not bind too weakly to the substrate. Weak binding results in droplet formation on the substrate.

B. Exemplary Compositions

[0037] Described herein are compositions comprising one or more pesticides and one or more surfactants. Not all possible surfactants provide improved solubility and optimized transport of the active ingredient across the pest cuticle. Various surfactants may require specific concentrations in acetone such that the surfactant concentration alone is responsible for pest mortality. Certain surfactants may be particularly suited for a specific active ingredient.

[0038] Exemplary pesticides may be solid or liquid. Exemplary pesticides may be an insecticide including, but not limited to, pyrethrin, a pyrethroid, a neonicotinoid, a carbamate, an organophosphate, an organochlorine, a sulfoximine, a foramidine, an organosulfur, an organotin, an avermectin, an oil, a soap, a botanical extract such as nicotine, sabadilla, rotenone, ryanodine, pyrethrum, meta-diamides (e.g., broflanilide), or combinations thereof. The one or more pesticides may be one or more new pesticides with new modes of action.

[0039] Compositions described herein may comprise various amounts of active ingredient. In some instances, exemplary compositions may comprise one or more active ingredients at an amount from about 0.00005 mg/mL to about 100 mg/mL, about 0.0001 mg/mL to about 50 mg/mL, about 0.001 mg/mL to about 25 mg/mL, about 0.01 mg/mL to about 20 mg/mL, about 0.01 mg/mL to about 10 mg/mL, about 0.1 mg/mL to about 15 mg/mL, about 0.5 mg/mL to about 10 mg/mL, about 0.00005 mg/mL to about 5 mg/mL, about 0.00005 mg/mL to about 1 mg/mL, about 0.00005 mg/mL to about 0.1 mg/mL, about 0.00005 mg/mL to about 0.01 mg/mL, or about 0.00005 mg/mL to about 0.001 mg/mL.

[0040] Surfactants are compounds that lower the surface tension between two liquids, between a gas and a liquid, or between a liquid and a solid. To identify surfactants that are compatible with the bottle bioassay, the physical and chemical compatibility of a surfactant with the bottle and with a pesticide need to be determined. In addition, the surfactants individually diluted in acetone in a bottle bioassay screen need to be free of any detrimental effects on the mosquitoes or the integrity of the study.

[0041] Exemplary surfactants aid in the penetration of a pesticide through a pest cuticle. Exemplary surfactants adhere to a surface of a substrate, but do not bind too strongly to the substrate to allow for a pesticide to be taken up by a pest. The substrate may be a material of which a container described herein is made. Exemplary surfactants by themselves are non-toxic to a pest and do not alter behavior of a pest.

[0042] Exemplary surfactants may be nonionic. Exemplary surfactants may be ethoxylated. Exemplary surfactant may have a hydrophilic-lipophilic balance (HLB) of at least 5 on a scale of 0 to 18, or a combination thereof. The HLB may be at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, or at least 17. The HLB may be at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most

12, at most 13, at most 14, at most 15, at most 16, at most 17, or at most 18. The HLB may be from about 5 to about 18, from about 6 to about 18, from about 7 to about 18, from about 8 to about 18, from about 9 to about 18, from about 10 to about 18, from about 11 to about 18, from about 12 to about 18, from about 13 to about 18, from about 14 to about 18, from about 15 to about 18, from about 16 to about 18, from about 17 to about 18, from about 5 to about 17, from about 5 to about 16, from about 5 to about 15, from about 5 to about 14, from about 5 to about

13, from about 5 to about 12, from about 5 to about 11, from about 5 to about 10, from about 5 to about 9, from about 5 to about 8, from about 5 to about 7, or from about 5 to about 6. [0043] Exemplary surfactants may be an ethoxylated lauryl alcohol, a polyoxyethylene - polyoxypropylene block copolymer, an ethoxylated oleyl alcohol, a monobutyl ether copolymer, an alkoxylated alcohol, or combinations thereof.

[0044] Compositions described herein may comprise from about 0.01 mg/mL to about 20 mg/mL of the one or more surfactants. The amount of surfactant does not over-saturate a substrate described herein and, therefore, does not result in a sticky effect that traps pests to the substrate. In various implementations, exemplary compositions may comprise surfactant at an amount of at least 0.01 mg/mL; at least 0.1 mg/mL; at least 1 mg/mL; at least 2.5 mg/mL; at least 5 mg/mL; at least 10 mg/mL; at least 15 mg/mL; or at least 20 mg/mL. In various implementations, exemplary compositions may comprise surfactant at an amount of no more than 20 mg/mL; no more than 15 mg/mL; no more than 10 mg/mL; no more than 5 mg/mL; no more than 2.5 mg/mL; no more than 1.5 mg/mL; no more than 0.5 mg/mL; or no more than 0.05 mg/mL.

[0045] The ratio of pesticide to surfactant may depend on the pesticide and surfactant combination, the specific pest species of interest, and/or the solubility of the pesticide in the surfactant.

[0046] Also described herein are control compositions. An exemplary control composition may be a composition that comprises acetone alone, acetone and surfactant, or acetone and pesticide. A control composition may be used to assess the effect of a surfactant for a pesticide on a pest such as on mortality of the pest or the effect of a surfactant on the ability of a pesticide to increase penetration of the pesticide through a pest cuticle.

III. Methods

[0047] Exemplary methods described herein relate to methods of determining susceptibility of pests to a pesticide and methods of determining resistance of pests to a pesticide. In some instances, exemplary methods may be used to identify appropriate surfactants for use in pesticidal formulations and/or optimize surfactant amounts in pesticidal formulations. As described in greater detail below, exemplary methods expose pests to a composition comprising one or more pesticides and one or more surfactants diluted with acetone. A. Methods of Determining Susceptibility of Pests to a Pesticide

[0048] Described herein are methods of determining susceptibility of pests to an exemplary pesticide. Exemplary methods may include collecting a first sample of pests inside of a first container and collecting a second sample of pests inside of a second container. In some embodiments, the pests in the samples of pests may be mosquitoes.

[0049] In some embodiments, the inside of the first container may be coated with acetone. A container may comprise from about 0.5 m to about 1.2 mL, about 0.8 mL to about 1.2 mL, about 1 mL to about 1.2 mL, about 0.5 mL to about 1 mL, or about 0.5 mL to about 0.8 mL of acetone.

[0050] In some embodiments, the inside of the second container may be coated with a composition comprising an exemplary pesticide, one or more exemplary surfactants, and acetone.

[0051] Containers described herein may comprise various amounts of pesticides. In various instances, containers may comprise an amount of pesticide from about 0.00005 mg/container to about 100 mg/container, about 0.0001 mg/container to about 75 mg/container, about 0.001 mg/container to about 50 mg/container, about 0.01 mg/container to about 25 mg/container, about 0.1 mg/container to about 20 mg/container, about 0.5 mg/container to about 15 mg/container, about 0.00005 mg/container to about 10 mg/container, about 0.00005 mg/container to about 5 mg/container, about 0.00005 mg/container to about 2.5 mg/container, about 0.00005 mg/container to about 1.0 mg/container, or about 0.00005 mg/container to about 0.5 mg/container of the one or more pesticides.

[0052] Containers described herein may comprise from about 1 mg/container to about 20 mg/container of the one or more surfactants. The amount of surfactant that may be used per container does not over-saturate a substrate described herein and, therefore, does not result in a sticky effect that traps pests to the substrate. In various implementations, exemplary containers may comprise surfactant at an amount of at least 1 mg/mL; at least 2.5 mg/mL; at least 5 mg/mL; at least 10 mg/mL; at least 15 mg/mL; or at least 20 mg/mL. [0053] A container may comprise from about 0.5 mb to about 1 .2 mb, about 0.8 mb to about 1.2 mb, about 1 mL to about 1.2 mb, about 0.5 mL to about 1 mb, or about 0.5 mb to about 0.8 mb of a composition described herein.

[0054] Exemplary methods may also include monitoring the first sample of pests and the second sample of pests for mortality within one or more predetermined periods of time.

[0055] In some embodiments, the one or more predetermined periods of time may be between one or more predetermined periods of time are between 5 minutes and 24 hours. The one or more predetermined periods of time may be one or more of 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes, 120 minutes, 135 minutes, 150 minutes, 165 minutes, 180 minutes, and 24 hours.

[0056] Exemplary methods may also include drying the acetone onto the inside of the first container and drying the composition onto the inside of the second container. In some embodiments, drying operations may comprise air drying and/or bottle rolling. When the composition is dried onto the inside of the second container, the bottle may be visually inspected to ensure no liquid remains inside the bottle.

[0057] Exemplary methods may also include determining a mortality ratio for each predetermined period of time for the first sample of pests and determining a mortality ratio for each predetermined period of time for the second sample of pests. The pests are susceptible to the pesticide when the mortality ratio is higher for the second sample of pests as compared to the first sample of pests.

B. Methods of Determining Resistance of Pests to a Pesticide

[0058] Described herein are methods of determining resistance of pests to an exemplary pesticide. Exemplary methods may include collecting a first sample of pests inside of a first container and collecting a second sample of pests inside of a second container. In some embodiments, the pests in the samples of pests may be mosquitoes.

[0059] In some embodiments, the inside of the first container may be coated with a composition comprising an exemplary pesticide, one or more exemplary surfactants, and acetone. In some embodiments, the inside of the second container may be coated with the same composition comprising an exemplary pesticide, one or more exemplary surfactants, and acetone.

[0060] In some embodiments, the first sample of pests may be susceptible to the pesticide. In some embodiments, the susceptibility of the second sample of pests to the pesticide may be unknown.

[0061] Exemplary methods may also include monitoring the first sample of pests and the second sample of pests for mortality within one or more predetermined periods of time. Exemplary methods may also include determining a mortality ratio for each predetermined period of time for the first sample of pests and determining a mortality ratio for each predetermined period of time for the second sample of pests. The second sample of pests are resistant to the pesticide when the mortality ratio is lower for the second sample of pests as compared to the first sample of pests.

[0062] In some embodiments, the one or more predetermined periods of time may be between one or more predetermined periods of time are between 5 minutes and 24 hours. The one or more predetermined periods of time may be one or more of 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes, 120 minutes, 135 minutes, 150 minutes, 165 minutes, 180 minutes, and 24 hours.

[0063] Exemplary methods may also include drying the composition onto the inside of the first container, the second container, or both the first container and the second container. In some embodiments, drying operations may comprise air drying and/or bottle rolling. When the composition is dried onto the inside of the first container, the second container, or a combination thereof, the bottle may be visually inspected to ensure no liquid remains inside the bottle.

IV. Examples

[0064] The foregoing may be better understood by reference to the following examples, which are presented for purposes of illustration and are not intended to limit the scope of the invention. The present disclosure has multiple aspects and embodiments, illustrated by the appended non-limiting examples. Example 1

Standard CDC Bottle Bioassay

[0065] Based on the CDC Bottle Bioassay protocol (CONUS Manual for Evaluating Insecticide Resistance in Mosquitoes Using the CDC Bottle Bioassay Kit, CDC 2020), four different doses of the insecticide imidacloprid were evaluated. For this evaluation, about 18-25 females of Ae. aegypti (3-6 days) per bottle were used and replicated two times per day over 3 days. Puerto Rico and Rockefeller strains were tested. The mosquitoes were exposed to 0.2 mg/mL, 1 mg/mL, 2 mg/mL, and 4 mg/mL of imidacloprid which are lOO-lOOOx more than the conventional dosage used for the CDC Bottle Bioassay. Each dose was dissolved in acetone, applied to the inside of a bottle, and allowed to dry onto the inside of the bottle in accordance with the CDC Bottle Bioassay protocol.

[0066] Clean, 250 mL (8oz) Boston Round bottles (Uline item #: S-23397 [with caps]) were used for each sample and replicate. Each bottle received 1 mL of acetone (control) or 1 mL of each of the acetone and imidacloprid compositions. The bottles were capped and swirled and inverted to coat the bottom, cap, and sides of the inside of the bottles with their respective composition. The bottle caps were removed to allow the compositions to dry for at least 1 hour. Once the compositions were dry, about 20 mosquitoes were added to each bottle and the bottles were sealed with their respective caps. Mosquito mortality was monitored at 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes, and 120 minutes. Mosquito mortality may be monitored every 15 minutes for up to 3 hours. After exposure in the bottles, all the mosquitoes from each of the bottles were transferred into cups that corresponded with each treatment and each cup opening was covered with a cotton pad that was moistened with a 20% sugar solution. Exposure time in the bottles may be 1, 2, or 3 hours. After 24 hours in the cups, the percent mortality for each time interval was calculated.

[0067] FIG. 1 shows that these high concentrations of imidacloprid do not reach 100% mortality within 2 hours of exposure to the mosquitoes. Increasing the concentration of imidacloprid does not yield an increase in mortality and therefore, saturation was achieved at about 90-95% mortality. Example 2

Selecting Surfactants

[0068] To mitigate the limitations of the CDC Bottle Bioassay with imidacloprid shown in Example 1, surfactants were utilized to help imidacloprid penetrate the mosquito cuticle during the exposure time in the bottle. A surfactant will help carry imidacloprid or other pesticides though the hydrophobic and hydrophilic layers of the mosquito cuticle. Not all surfactants are appropriate, however, for the bottle bioassay.

[0069] First, surfactants that are used in current formulations for a pesticide were tested for their compatibility with the bottle bioassay. If there is not a current formulation for a pesticide that comprises a surfactant, then new surfactants that are compatible with the pesticide and the bottle bioassay will be identified. It was found that many surfactants, including ethoxylated lauryl alcohols and alkoxylated alcohols, collect moisture from their environment during the drying period and form droplets inside of the bottles to which the mosquitoes stick (FIG. 2). In addition, mosquitoes exhibited lethargic behavior in response to the foregoing surfactants.

[0070] In contrast, surfactants such as polyoxyethylene - polyoxypropylene block copolymer surfactants, ethoxylated oleyl alcohol surfactants, and monobutyl ether copolymer surfactants did not collect moisture from their environment during drying and completely dried onto the inside of the bottles. In addition, these surfactants did not cause any detrimental effects on the mosquitoes or the integrity of the bottle bioassay. Therefore, these surfactants were found to be compatible with the bottle bioassay.

[0071] Exemplary compatible surfactants are nonionic. Exemplary compatible surfactants have a hydrophilic-lipophilic balance (HLB) of at least 5 on a scale of 0 to 18. Exemplary compatible surfactants are ethoxylated. Exemplary compatible surfactants typically have less than 0.01 weight percent (wt%) of moisture. Example 3

Modified CDC Bottle Bioassay

[0072] The assay used in these experiments was similar to the one used in Example 1, except a surfactant was added to some of the test compositions. Clean, 250 ml (8oz) Boston Round bottles (Uline item # S-23397 [with caps]) were used for each sample and replicate. Each bottle received 1 mb of acetone (control) or 1 mb of each test composition (i.e., acetone and imidacloprid, acetone and surfactant, or acetone, imidacloprid, and surfactant). The bottles were capped and swirled and inverted to coat the bottom, cap, and sides of the inside of the bottles with their respective composition. The bottle caps were removed to allow the compositions to dry for at least 1 hour. Once the compositions were dry, about 20 females of Ae. aegypti (3-6 days) were added to each bottle and the bottles were sealed with their respective caps. The experiments were replicated three times for each of the treatments. Mosquito mortality was monitored at 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes, and 120 minutes. Mosquito mortality may be monitored every 15 minutes for up to 3 hours. After exposure in the bottles, all the mosquitoes from each of the bottles were transferred into cups that corresponded with each treatment and each cup opening was covered with a cotton pad that was moistened with a 20% sugar solution. Exposure time in the bottles may be 1 , 2, or 3 hours. After 24 hours in the cups, the percent mortality for each time interval was calculated.

[0073] Tested compositions comprised 0.2 mg/mL imidacloprid alone, 1 mg/mL polyoxyethylene - polyoxypropylene block copolymer alone, or 0.2 mg/mL imidacloprid and 1 mg/mL polyoxyethylene - polyoxypropylene block copolymer, where each composition was dissolved in acetone. The data shown in FIG. 3 demonstrate that the use of the pesticide in combination with the surfactant decreased the time to mortality significantly as compared to the pesticide alone.

[0074] Compositions comprising 0.2 mg/mL imidacloprid alone dissolved in acetone or 0.2 mg/mL imidacloprid in combination with 1 mg/mL polyoxyethylene - polyoxypropylene block copolymer dissolved in acetone were also tested against Ae. aegypti Rockefeller (ROCK) and Puerto Rico (PR) strains. As shown in FIG. 4, a similar effect was visible against both strains. The diagnostic time (DT), which is the diagnostic dose (DD) of the insecticide dose that causes 100% mortality in a susceptible mosquito population within a certain time period, was reduced. In addition, imidacloprid worked on both a susceptible and a pyrethroid resistant strain.

[0075] The foregoing description of the specific aspects will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific aspects, without undue experimentation, without departing from the general concept of the present disclosure. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed aspects, based on the teaching and guidance presented here. It is to be understood that the phraseology or terminology is for the purpose of description and not of limitation such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0076] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary aspects, but should be defined only in accordance with the following claims and their equivalents.

[0077] For reasons of completeness, the following Clauses are provided.

Clause 1. A method of determining susceptibility of pests to a pesticide, the method comprising: collecting a first sample of pests inside of a first container, wherein the inside of the first container is coated with acetone; collecting a second sample of pests inside of a second container, wherein the inside of the second container is coated with a composition comprising the pesticide, one or more nonionic surfactants, and acetone; monitoring the first sample of pests and the second sample of pests for mortality within one or more predetermined periods of time; determining a mortality ratio for each predetermined period of time for the first sample of pests; determining a mortality ratio for each predetermined period of time for the second sample of pests; and when the mortality ratio is higher for the second sample of pests as compared to the first sample of pests, determining the pests are susceptible to the pesticide. Clause 2. The method of clause 1, wherein from about 0.8 mL to about 1 .2 mL of the composition is used to coat the inside of the second container.

Clause 3. The method of clause 1 or claim 2, wherein from about 0.8 mL to about 1.2 mL of the acetone is used to coat the inside of the first container.

Clause 4. The method of any one of clauses 1-3, further comprising: drying the acetone onto the inside of the first container; and drying the composition onto the inside of the second container.

Clause 5. The method of clause 4, wherein when the composition is dried onto the inside of the second container, the one or more nonionic surfactants have less than 0.01% of moisture and are non -toxic to the pests.

Clause 6. The method of any one of clauses 1-5, wherein the composition comprises from about 0.01-10 mg/mL of the one or more nonionic surfactants.

Clause 7. The method of any one of clauses 1-6, wherein from about 1-20 mg of the one or more nonionic surfactants are dried onto the inside of the second container.

Clause 8. The method of any one of clauses 1-7, wherein the one or more nonionic surfactants have a hydrophilic-lipophilic balance (HLB) of at least 5 on a scale of 0 to 18.

Clause 9. The method of any one of clauses 1-8, wherein the one or more nonionic surfactants are ethoxylated.

Clause 10. The method of any one of clauses 1-9, wherein the one or more predetermined periods of time are between 5 minutes and 24 hours.

Clause 11. The method of any one of clauses 1-10, wherein the pests in the first sample of pests and the second sample of pests are mosquitoes.

Clause 12. A method of determining resistance of pests to a pesticide, the method comprising: collecting a first sample of pests inside of a first container, wherein the inside of the first container is coated with a composition comprising the pesticide, one or more nonionic surfactants, and acetone, and wherein the first sample of pests are susceptible to the pesticide; collecting a second sample of pests inside of a second container, wherein the inside of the second container is coated with the composition comprising the pesticide, the one or more nonionic surfactants, and acetone, and wherein susceptibility of the second sample of pests to the pesticide is unknown; monitoring the first sample of pests and the second sample of pests for mortality within one or more predetermined periods of time; determining a mortality ratio for each predetermined period of time for the first sample of pests; determining a mortality ratio for each predetermined period of time for the second sample of pests; and when the mortality ratio is lower for the second sample of pests, determining the second sample of pests is resistant to the pesticide.

Clause 13. The method of clause 12, wherein from about 0.8 mL to about 1.2 mL of the composition is used to coat the inside of the first container, the second container, or combination thereof.

Clause 14. The method of clause 12 or claim 13, further comprising drying the composition onto the inside of the first container, the second container, or both the first container and the second container.

Clause 15. The method of clause 14, wherein when the composition is dried onto the inside of the first container, the second container, or a combination thereof, the one or more nonionic surfactants have less than 0.01% of moisture and are non-toxic to the pests.

Clause 16. The method of any one of clauses 12-15, wherein the composition comprises from about 0.01-10 mg/mL of the one or more nonionic surfactants.

Clause 17. The method of any one of clauses 12-16, wherein from about 1-20 mg of the one or more nonionic surfactants are dried onto the inside of the first container, the second container, or a combination thereof.

Clause 18. The method of any one of clauses 12-17, wherein the one or more nonionic surfactants have a hydrophilic-lipophilic balance (HLB) of at least 5 on a scale of 0 to 18.

Clause 19. The method of any one of clauses 12-18, wherein the one or more nonionic surfactants are ethoxylated. Clause 20. The method of any one of clauses 12-19, wherein the one or more predetermined periods of time are one or more of 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes, 120 minutes, 135 minutes, 150 minutes, 165 minutes, 180 minutes, and 24 hours.

Clause 21. The method of any one of clauses 12-20, wherein the pests in the first sample of pests and the second sample of pests are mosquitoes.

Clause 22. The method of any one of clauses 1-11, wherein the surfactant is an ethoxylated lauryl alcohol, a polyoxyethylene - polyoxypropylene block copolymer, an ethoxylated oleyl alcohol, a monobutyl ether copolymer, or an alkoxylated alcohol.

Clause 23. The method of any one of clauses 12-21, wherein the surfactant is an ethoxylated lauryl alcohol, a polyoxyethylene - polyoxypropylene block copolymer, an ethoxylated oleyl alcohol, a monobutyl ether copolymer, or an alkoxylated alcohol.

Claims

1. A method of determining susceptibility of pests to a pesticide, the method comprising: collecting a first sample of pests inside of a first container, wherein the inside of the first container is coated with acetone; collecting a second sample of pests inside of a second container, wherein the inside of the second container is coated with a composition comprising the pesticide, one or more nonionic surfactants, and acetone; monitoring the first sample of pests and the second sample of pests for mortality within one or more predetermined periods of time; determining a mortality ratio for each predetermined period of time for the first sample of pests; determining a mortality ratio for each predetermined period of time for the second sample of pests; and when the mortality ratio is higher for the second sample of pests as compared to the first sample of pests, determining the pests are susceptible to the pesticide.

2. The method according to claim 1, wherein from about 0.8 mb to about 1.2 m of the composition is used to coat the inside of the second container.

3. The method of claim 2, wherein from about 0.8 mb to about 1.2 mb of the acetone is used to coat the inside of the first container.

4. The method of claim 3, further comprising: drying the acetone onto the inside of the first container; and drying the composition onto the inside of the second container.

5. The method according to claim 4, wherein when the composition is dried onto the inside of the second container, the one or more nonionic surfactants have less than 0.01% of moisture and are non-toxic to the pests.

6. The method according to claim 5, wherein the composition comprises from about 0.01-10 mg/mL of the one or more nonionic surfactants.

7. The method according to claim 6, wherein from about 1-20 mg of the one or more nonionic surfactants are dried onto the inside of the second container.

8. The method according to claim 7, wherein the one or more nonionic surfactants have a hydrophilic-lipophilic balance (HLB) of at least 5 on a scale of 0 to 18.

9. The method according to claim 8, wherein the one or more nonionic surfactants are ethoxylated; and wherein the one or more predetermined periods of time are between 5 minutes and 24 hours.

10. The method according to claim 9, wherein the surfactant is an ethoxylated lauryl alcohol, a polyoxyethylene - polyoxypropylene block copolymer, an ethoxylated oleyl alcohol, a monobutyl ether copolymer, or an alkoxylated alcohol.

11. The method according to claim 10, wherein the pests in the first sample of pests and the second sample of pests are mosquitoes.

12. A method of determining resistance of pests to a pesticide, the method comprising: collecting a first sample of pests inside of a first container, wherein the inside of the first container is coated with a composition comprising the pesticide, one or more nonionic surfactants, and acetone, and wherein the first sample of pests are susceptible to the pesticide; collecting a second sample of pests inside of a second container, wherein the inside of the second container is coated with the composition comprising the pesticide, the one or more nonionic surfactants, and acetone, and wherein susceptibility of the second sample of pests to the pesticide is unknown; monitoring the first sample of pests and the second sample of pests for mortality within one or more predetermined periods of time; determining a mortality ratio for each predetermined period of time for the first sample of pests; determining a mortality ratio for each predetermined period of time for the second sample of pests; and when the mortality ratio is lower for the second sample of pests, determining the second sample of pests is resistant to the pesticide.

13. The method according to claim 12, wherein from about 0.8 mb to about 1.2 mb of the composition is used to coat the inside of the first container, the second container, or combination thereof.

14. The method according to claim 13, further comprising drying the composition onto the inside of the first container, the second container, or both the first container and the second container.

15. The method according to claim 14, wherein when the composition is dried onto the inside of the first container, the second container, or a combination thereof, the one or more nonionic surfactants have less than 0.01% of moisture and are non-toxic to the pests.

16. The method according to claim 15, wherein the composition comprises from about 0.01- 10 mg/mL of the one or more nonionic surfactants; and . wherein from about 1-20 mg of the one or more nonionic surfactants are dried onto the inside of the first container, the second container, or a combination thereof.

17. The method according to claim 16, wherein the one or more nonionic surfactants have a hydrophilic-lipophilic balance (HLB) of at least 5 on a scale of 0 to 18.

18. The method according to claim 17, wherein the surfactant is an ethoxylated lauryl alcohol, a polyoxyethylene - polyoxypropylene block copolymer, an ethoxylated oleyl alcohol, a monobutyl ether copolymer, or an alkoxylated alcohol.

19. The method according to claim 18, wherein the one or more predetermined periods of time are one or more of 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes, 120 minutes, 135 minutes, 150 minutes, 165 minutes, 180 minutes, and 24 hours.

20. The method according to claim 19, wherein the pests in the first sample of pests and the second sample of pests are mosquitoes.