Adjuvants to improve efficacy of varroa control active ingredients in managed honey bee colonies

Abstract

Provided is a composition comprising a miticidal ingredient and an adjuvant and methods of using the composition for mite control in honey bee colonies.

Description

PCT / US24 / 56156 15 November 2024 (15.11.2024)Attorney Docket No. 103361-545WO1ADJUVANTS TO IMPROVE EFFICACY OF VARROA CONTROL ACTIVE INGREDIENTS IN MANAGED HONEY BEE COLONIESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 599,164, filed November 15, 2023, which incorporated by reference herein in its entirety.BACKGROUND

[0002] The Varroa mite (Varroa destructor) is the most damaging pest to honey bees and is responsible for widespread honey bee colony losses1. In 2021, commercial beekeepers selfreported that 6 in 10 colony losses were attributable to Varroa2. Varroa mites can be a problem not only for the individual colony affected but can spread to other managed and feral honey bee colonies within foraging distance through robbing and drifting3,4’5.

[0003] Control of Varroa is essential to maintaining healthy colonies. Unfortunately, many of the approved miticides have limited efficacy6,7. Varroa mites have exhibited resistance to many available miticide active ingredients, including pyrethroids such as fluvalinate and flumethrin8,9’10’11, organophosphates such as coumaphos11,12, and formamidines such as amitraz13,14Miticide applications can be expensive and labor-intensive to apply, which can limit their use for mite control. Increasing the efficacy of currently available active ingredients and finding better formulations for miticides, with a focus on reducing cost and ease of application, can give beekeepers better tools to control mites in colonies.

[0004] As such, there exists a need for improved miticides with greater efficacy, lower resistance, and minimal toxicity to honey bees. These needs and others are at least partially satisfied by the present disclosure.SUMMARY

[0005] Disclosed herein is the addition of pesticide adjuvants, commonly used elsewhere in the pesticide industry, to improve the efficacy of Varroa control. This problem is approached by combining a range of adjuvant components with oxalic acid in glycerin and testing efficacy in cage trials using 300 honey bees infested with mites. Alcohol ethoxylate, e.g., Eco BC-12, is one such candidate. A miticide such as, for example, oxalic acid in glycerin with 1% Eco BC-12 in whole honey bee colonies, can improve the efficacy of oxalic acid and other traditional miticides for Varroa mite control.PCT / US24 / 56156 15 November 2024 (15.11.2024)Attorney Docket No. 103361-545WO1

[0006] In an aspect, provided is a composition, comprising a miticidal ingredient and an adjuvant.

[0007] In another aspect, provided is a method of mite control in honey bee colonies, the method comprising introducing any of the disclosed compositions into a honey bee colony.

[0008] In another aspect, provided is an article comprising an absorbent and any of the disclosed compositions, wherein the composition is absorbed into the absorbent.

[0009] In another aspect, provided is a method of mite control in honey bee colonies, the method comprising introducing any of the disclosed articles into a honey bee colony.

[0010] Other systems, methods, features and / or advantages will be or may become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features and / or advantages be included within this description and be protected by the accompanying claims.BRIEF DESCRIPTION OF DRAWINGS

[0011] FIGURE 1 depicts efficacy of solvent control, oxalic acid alone, and oxalic acid plus adjuvant treatment in apiarium cage trials. 24-hour efficacy is the number of Varroa that fell through the cage divided by the total number of mites, expressed as a percentage. All treatments were significantly different from the glycerin solvent (P < 0.05), but there were no statistically significant differences between the adjuvant treatments and the oxalic acid alone.

[0012] FIGURE 2 depicts a pre- and post-treatment mite wash using oxalic acid.

[0013] FIGURE 3 depicts dose-response curves for the two adjuvants that were determined statistically to have no relationship between dose and mortality at the concentrations tested (up to 20% concentration).

[0014] FIGURE 4 depicts dose-response curves for the three adjuvants that demonstrate low risk to honey bee toxicity. In all cases, the LCso was determined to be above the maximum concentration tested (20% concentration).

[0015] FIGURES 5A-5B depict preliminary field trial data.

[0016] FIGURE 6 depicts additional preliminary field trial data.

[0017] FIGURES 7A-7B depict Varroa levels from pre- and post-treatment alcohol washes in the year 1 (FIG. 7A) and year 2 (FIG. 7B) field trials. Points indicate mites per 100 bees for each colony, and lines connect paired pre- and post-treatment data for each individual colony. Significant difference within treatments, indicated by an asterisk, was determined for the Oxalic Acid Plus Adjuvant Treatment in Year 1 (P = 0.0122, n=6, t= -3.189).PCT / US24 / 56156 15 November 2024 (15.11.2024)Attorney Docket No. 103361-545WO1

[0018] FIGURES 8A-8B depict the change in Varroa levels from alcohol washes for year 1 (FIG. 8A) and year 2 (FIG. 8B) field trials. Points indicate the post-treatment minus the pretreatment Varroa per 100 bees for individual colonies. Significant difference between treatments, indicated by an asterisk, was determined between the Solvent Control and Oxalic Acid Plus Adjuvant Treatment in Year 2 (P = 0.005).

[0019] FIGURE 9 depicts mite drop data collected over 48-hour periods, where the treatment was applied at the start of day 0.DETAILED DESCRIPTION

[0020] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, can also be provided in combination with a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, can also be provided separately or in any suitable subcombination. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure.

[0021] In an aspect, provided is a composition, comprising a miticidal ingredient and an adjuvant.

[0022] In some aspects, the miticidal ingredient is oxalic acid, thymol, fenazaquin, or a derivative or combination thereof.

[0023] In some aspects, the adjuvant is an oil. In some aspects, the adjuvant comprises Stepan™ C-65, Stepan™ 108, Steposol™ C-65, ECOSTEP™ CE-13, Tall Oil Fatty Acids, or any combination thereof.

[0024] In some aspects, the adjuvant is a traditional surfactant, an organo-silicone surfactant, or a combination thereof. In some aspects, the adjuvant comprises Ninate™ 60E, Toximul™ 8240, Toximul™ 8320, ECOSTEP™ SE-11, ECOSTEP™ AE-13, ECOSTEP™ BC-12, Biosoft™ Nl-7, Makon™ 10, Makon™ P104, Step-Flow™ 26, Ninex™ MT-615, Toximul™ TA-8, Steol™ TSP-16N, Silwet™ Eco, Sylgard™ 309, Silwet™ L-7500, or any combination thereof.

[0025] In some aspects, the adjuvant is an emulsifier. In some aspects, the adjuvant comprises pinene monomers, polyterpenes, or a combination thereof.

[0026] In some aspects, the composition further comprises a solvent, wherein the miticidal ingredient and the adjuvant are dissolved or suspended in the solvent. In some aspects, the solvent comprises glycerol, glycerin, or any combination thereof.PCT / US24 / 56156 15 November 2024 (15.11.2024)Attorney Docket No. 103361-545WO1

[0027] In some aspects, the miticidal agent is present in a concentration of from about 0.1% to about 50%, or about 0.5% to about 45%, or about 1% to about 40%, or about 1.5% to about 35%, or about 2% to about 30%, or about 4% to about 25%, or about 6% to about 20%, or about 8% to about 10%, or from about 0.1% to about 10%, or from about 0.5% to about 8%, or from about 1% to about 6%, or from about 1.5% to about 4%, or from about 10% to about 50%, or from about 15% to about 45%, or from about 20% to about 40%, or from about 25% to about 35% relative to the total amount of the composition.

[0028] In some aspects, the adjuvant is present in a concentration of from about 0.1% to about 3%, or from about 0.2% to about 2.8%, or from about 0.4% to about 2.6%, or from about 0.6% to about 2.4%, or from about 0.8% to about 2.2%, or from about 1% to about 2%, or from about 1.2% to about 1.8%, or from about 1.4% to about 1.6%, or from about 0.1% to about 1.5%, or from about 0.2% to about 1.4%, or from about 0.4% to about 1.2%, or from about 0.6% to about 1%, or from about 1.5% to about 3%, or from about 1.6% to about 2.8%, or from about 1.8% to about 2.6%, or from about 2% to about 2.4% relative to the total amount of the composition.

[0029] In another aspect, provided is a method of mite control in honey bee colonies, the method comprising introducing any of the disclosed compositions into a honey bee colony.

[0030] In some aspects, the composition is sprayed into or coated on a portion of the honey bee colony.

[0031] In some aspects, the honey bee colony comprises a mite population, and wherein the composition reduces the mite population by from about 1% to about 100%, or from about 5% to about 95%, or from about 10% to about 90%, or from about 15% to about 85%, or from about 20% to about 80%, or from about 25% to about 75%, or from about 30% to about 70%, or from about 35% to about 65%, or from about 40% to about 50%, or from about 1% to about 40%, or from about 5% to about 35%, or from about 10% to about 30%, or from about 15% to about 25%, or from about 50% to about 100%, or from about 55% to about 95%, or from about 60% to about 90%, or from about 65% to about 85%, or from about 70% to about 80%.

[0032] In some aspects, the composition is introduced into the honey bee colony preventatively. In some aspects, the composition is used to treat, relieve, or mitigate a mite outbreak.

[0033] In some aspects, the composition reduces a population of the honey bee colony by less than 10%, or less than 9%, or less than 8%, or less than 7%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1%, or less than 0.5%.PCT / US24 / 56156 15 November 2024 (15.11.2024)Attorney Docket No. 103361-545WO1

[0034] In another aspect, provided is an article comprising an absorbent and any of the disclosed compositions, wherein the composition is absorbed into the absorbent.

[0035] In some aspects, the absorbent comprises felt, fabric, cellulose, or any combination thereof.

[0036] In some aspects, the composition is present in the article in an amount of from about 0.5 mL to about 5 mL, or from about 0.75 mL to about 4.75 mL, or from about 1 mL to about 4.5 mL, or from about 1.25 mL to about 4.25 mL, or from about 1.5 mL to about 4 mL, or from about 1.75 mL to about 3.75 mL, or from about 2 mL to about 3.5 mL, or from about 2.25 mL to about 3.25 mL, or from about 2.5 mL to about 3 mL, or from about 0.5 mL to about 2.5 mL, or from about 0.75 mL to about 2.25 mL, or from about 1 mL to about 2 mL, or from about 1.25 mL to about 1.75 mL, or from about 3 mL to about 5 mL, or from about 3.25 mL to about 4.75 mL, or from about 3.5 mL to about 4.5 mL, or from about 3.75 mL to about 4.25 mL.

[0037] In some aspects, the composition is present in the article in an amount of from about 60 mL to about 300 mL, or from about 80 mL to about 280 mL, or from about 100 mL to about 260 mL, or from about 120 mL to about 240 mL, or from about 140 mL to about 220 mL, or from about 160 mL to about 200 mL, or from about 60 mL to about 180 mL, or from about 80 mL to about 160 mL, or from about 100 mL to about 140 mL, or from about 180 mL to about 300 mL, or from about 200 mL to about 280 mL, or from about 220 mL to about 260 mL.

[0038] In another aspect, provided is a method of mite control in honey bee colonies, the method comprising introducing any of the disclosed articles into a honey bee colony.

[0039] In some aspects, the honey bee colony comprises a mite population, and wherein the article reduces the mite population by from about 1% to about 100%, or from about 5% to about 95%, or from about 10% to about 90%, or from about 15% to about 85%, or from about 20% to about 80%, or from about 25% to about 75%, or from about 30% to about 70%, or from about 35% to about 65%, or from about 40% to about 50%, or from about 1% to about 40%, or from about 5% to about 35%, or from about 10% to about 30%, or from about 15% to about 25%, or from about 50% to about 100%, or from about 55% to about 95%, or from about 60% to about 90%, or from about 65% to about 85%, or from about 70% to about 80%.

[0040] In some aspects, the article is introduced into the honey bee colony preventatively. In some aspects, the article is used to treat, relieve, or mitigate a mite outbreak.

[0041] In some aspects, the article reduces a population of the honey bee colony by less than 10%, or less than 9%, or less than 8%, or less than 7%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1%, or less than 0.5%.PCT / US24 / 56156 15 November 2024 (15.11.2024)Attorney Docket No. 103361-545WO1EXAMPLESExample 1

[0042] Lab Trial: The cage design for laboratory testing is a modification of the design of Bahreini et al.32. A 32 oz. deli cup (5.5” height x 4.5” diameter) with 2-mm holes added in the side can be used for airflow. 1 cm3sugar cubes can be hot glued to the top of the inverted cup. An 8 oz. deli cup (1.75” height x 4.5” diameter) can have the base removed and replaced with plastic mesh, held in place with hot glue, to allow dead mites to fall through. A lid can be placed on the 8 oz. cup, which can be placed inside the 32 oz. cup, taped in place, and inverted so that the lid is on the bottom to catch the fallen mites. A two-inch slit can be cut in the top of the 32 oz. deli cup, where the 4-cm x 10-cm rectangular felt strip can be inserted and held in place with a binder clip.

[0043] The treatment applied to the felt strip can include a 2-mL mixture of glycerol solvent, an adjuvant principal functioning agent at a concentration shown to cause minimal bee toxicity, and a miticidal active ingredient. The concentration of the active ingredient can vary based on control efficacy of each specific active ingredient without an adjuvant present. The solvent control can include glycerin solvent alone, with no adjuvant or active ingredient treatment. The active ingredient control can be dissolved in glycerin at the same concentration as the treatments, but with no adjuvant added.

[0044] Bees can be collected from hives with between 5 and 15 Varroa mites per 100 bees. Hive bees can be collected by shaking four brood frames into a plastic wash tub. Bees can be sprayed with water to discourage flying, and 1 / 3- 1 / 2 cup of bees (about 200 bees) can be scooped into each 32 oz. deli cup, prepared with the treatment strip, in random order. All adjuvant treatments and controls can be performed for a single trial with the same group of collected bees. Bees can be stored in a dark incubator at hive conditions (34° C, 60% humidity). Dead bees and dead Varroa mites can be counted after 1 hour, 24 hours, 48 hours, and 72 hours. After 72 hours, bees can be frozen at -4° C and dead bees can then counted followed by a standard alcohol wash to count the number of mites that were not killed by the treatment.

[0045] A minimum of three trials can be conducted using all nine adjuvant-active ingredient treatments, the active ingredient control, and the solvent control for each of the four active ingredients. The efficacy of Varroa mite control for adjuvant-active ingredient combinations can be compared to those of the active ingredient alone to determine if there is a significant increase in efficacy or bee mortality when an adjuvant is added. The efficacy of honey bee mortality can be compared to solvent control to determine if there was a significant increase in Varroa or honey bee mortality. Preliminary data from trials with oxalic acid is shown in FIG. 1 and FIG. 2.PCT / US24 / 56156 15 November 2024 (15.11.2024)Attorney Docket No. 103361-545WO1 Toxicity data for a selection of adjuvants is further shown in FIG. 3 and FIG. 4. While preliminary results demonstrated efficacy, honey bee mortality was significantly greater than miticide alone for many adjuvants tested, highlighting a need to focus on oils and non-ionic surfactants that do not contain ethoxylates or organosilicones for future testing.

[0046] Field Trial: The efficacy of the two most promising adjuvant-active ingredient strip formulations, based on the laboratory trial, can be tested in 32 full-sized hives managed by Ohio State University in central and northeast Ohio during the months of September and October 2023. Two colonies in each apiary can receive two strips per brood box of one of the two strip formulations, Apivar (amitraz) as a positive control, or felt strip with only glycerol solvent, for a total of six colonies tested per apiary. Treatment strip formulations can be prepared by placing a 10 x 24 cm felt strip in 12 mL adjuvant-active ingredient formulation in glycerol in a closed mason jar, warmed to hive temperature (34° C), for 24 hours.

[0047] Treatments can be replicated across four apiaries for a total of 8 colonies per treatment. Colonies can be randomly assigned to treatments and can be a mix of overwintered colonies and new colonies started in Spring 2023 using a randomized complete block design, using apiaries as blocks. All colonies can be managed using standard beekeeping practice but not be treated for Varroa in 2023 prior to tests in August. Drop zone bee traps, made with 2’ x 4’ x 0.5’ wooden frames with half inch hardware cloth on top and window screen on bottom33, can be placed at the entrance of the colonies to determine the number of dead bees before, during, and after treatment.

[0048] Colonies can be observed weekly for six weeks, and measurements can be taken for number of dead bees in the drop-zone trap and number of phoretic mites on about 300 adult bees, taken from an uncapped brood frame, using an ethanol wash. After three weeks, the treatment and positive control strips can be removed from hives and treatment strips analyzed using LCMS (available in the Ranger laboratory, USDA-ARS on OSU’s Wooster campus) to determine the concentration of active ingredient and adjuvant remaining on the felt strip. To compare the effect of treatment to the positive and negative controls on Varroa count and dead bees, an ANOVA test and Tukey’s Post-Hoc test can be performed. In addition, a chi-squared test can be performed to determine if there was a significant effect of treatment on the number of colonies that survived the winter.

[0049] Preliminary field trial results are given in FIGS. 5A-5B and FIG. 6.

[0050] Discussion: Adjuvants are often used in agricultural pesticide spray applications with the intention of improving the efficacy of active ingredients against the target pest and allowing reduced pesticide use while maintaining crop protectioms. Adjuvant products available on thePCT / US24 / 56156 15 November 2024 (15.11.2024)Attorney Docket No. 103361-545WO1 market contain one or more “principal functioning agents”, which are equivalent to “active ingredients” in regular pesticides. Testing the bee toxicity of both adjuvants and their constituent principal functioning agents and has led to the identification of materials showing high toxicity to bees. Adjuvants containing non-ionic surfactant ethoxylates and organosilicones as principal functioning agents generally demonstrate high bee toxicity, but adjuvants containing oils and fatty acids appear to be minimally bee toxic. These principal functioning agents serve to improve the spreading, and penetration of a pesticide through the waxy cuticle on crop leaves16,17’18. Similar waxes are found in the cuticle of Varroa mites, so it can be hypothesized that bee-safe adjuvants may improve the spreading and penetration of miticides through Varroa mite cuticle.

[0051] The active ingredients which can be studied are oxalic acid, thymol, and fenazaquin. Oxalic acid is currently commercially available as API-Bioxal19which is approved for application using vaporization methods, which can be restrictive for hobbyist beekeepers and have human health risks if not applied using proper PPE, or dribble methods, which is not effective through brood cappings21. Oxalic acid has been applied in experimental use through strip formulations with glycerin22. The mode of action of oxalic acid in Varroa mites is not well understood but it is thought to cause mite mortality through its acidity23. Thymol, an essential oil derived from Thymus vulgaris L.24, with multiples modes of action as a tyramine receptor agonist and GABA-gated anion channel agonist25,26’27, is available as Apiguard and Api Life VAR, but longevity of the current products within a hive may not last for a full brood cycle28and the gel products can be a repellant to bees29.

[0052] The adjuvants proposed for testing are oils, Stepan™ C-65, Stepan™ 108, and ECOSTEP™ CE-13, and nonionic surfactants, Ninate™ 60E, Toximul™ 8240, Toximul™ 8320, ECOSTEP™ SE-11, ECOSTEP™ AE-13, and ECOSTEP™ BC-12. These adjuvants are all commercially available through the Stepan Company, a major provider of adjuvant and inert ingredients to the pesticide industry, and are listed in TABLE 1. All adjuvants proposed for testing are listed on the EPA inert ingredients list and are approved for both food and nonfood use and four are OMRI listed for use in organic agriculture.PCT / US24 / 56156 15 November 2024 (15.11.2024)Attorney Docket No. 103361-545WO1TABLE 1. Adjuvants proposed for testing to improve efficacy of Varroa control active ingredients. Listed are the product trade name, the chemical makeup of each adjuvant, the type of adjuvant, and whether it is approved for use in organic agriculture. All adjuvants listed are approved on the EPA inert ingredients list for both food and nonfood use.PCT / US24 / 56156 15 November 2024 (15.11.2024)Attorney Docket No. 103361-545WO1

[0053] Beekeepers will benefit from improved tools for Varroa control. Strip formulations will be developed which are easy to apply and last over a full brood cycle period to effectively reduce colony Varroa levels. Currently available oxalic acid and thymol treatments demonstrate variable efficacy if used when brood is present in colonies and improvements in their formulation could increase efficacy during this period. Combining active ingredients with an adjuvant to create an effective strip formulation would expand a beekeeper’s miticide toolbox to control Varroa and manage resistance. Work to improve the delivery and increase the efficacy of these active ingredients through combination with adjuvants could improve the formulation for these and other Varroa control chemistries.Example 2

[0054] Potter Tower Toxicity Tests'. The LCso of all adjuvants used in this study was greater than the maximum concentration tested (TABLE 2). The LCso for the positive control was estimated to be 0.043%, (95% CI = 0.038 - 0.047).PCT / US24 / 56156 15 November 2024 (15.11.2024)Attorney Docket No. 103361-545WO1 TABLE 2. 48-hour LCso estimates of each adjuvant applied to adult bees using a Potter Spray Tower. All adjuvant LCso estimates were above the maximum rate tested. Values in parenthesis indicate 95% confidence intervals.

[0055] Apiarium Trials'.

[0056] Cage Design: Cage design was modified from (Rinkevich, 2020). The 1.25 x 8.5 cm treatment strip was inserted in a 15 mm x 3 mm slit cut in the top of the cage. Four equally spaced 3 -mm holes were added for airflow on the sides of the cage, 1 cm from the top of the cage. Two 1 cm3sugar cubes were fixed to the top of the cage using hot melt adhesive to allow bees to feed.

[0057] Honey Bees: Each group of honey bees were shaken from two to three deep brood frames of a single colony, chosen from a minimum of six colonies, between the months of July and October. Colonies were managed at The Ohio State University - Wooster campus apiaries and were not treated for mites for at least 6 months prior to collection. Queens were caged for at least 21 days prior to collection to increase the number of phoretic mites in the colony. All colonies were requeened with New World Carniolans the previous Spring. Bees were collected in an empty 4-frame deep nucleus box and stored in dark at ambient temperature for no longer than one hour. Collected bees were sprayed with DI water to discourage flying. Approximately 300 bees were scooped from the nucleus box and placed into each apiarium containing a treatment.

[0058] Experimental Design: Each treatment series included a negative control, an oxalic acid control, and four different oxalic acid-adjuvant combination treatments. Treatments were assigned in random order. Cages were placed inside an incubator (Humidaire Model No. 2048; The Humidaire Incubator Company, New Madison, OH, USA) and stored at hive conditionsPCT / US24 / 56156 15 November 2024 (15.11.2024)Attorney Docket No. 103361-545WO1 (34°C, 60% humidity, darkness). Bees were misted with water immediately after being placed in the incubator and 2 hours after being placed in the incubator.

[0059] Dead bees at the bottom of the cage and dead Varroa on the collection tray were counted after twenty-four hours. After bees and Varroa were counted, the plastic tray was discarded and cups were inverted and frozen at -20°C.

[0060] Bees were weighed (scale make / model) and the number of grams was multiplied by 11.34 to determine number of bees, the number of bees per gram that was determined from these colonies in preliminary trials. An ethanol wash was performed and the Varroa remaining on the bees were counted. Treatment efficacy was determined by dividing the number of Varroa fallen during treatment by the number of Varroa fallen during treatment plus the number of Varroa remaining on the bees.

[0061] A Kruskal-Wallace test was used to determine significant differences in bee mortality and Varroa control efficacy. This was followed with a pairwise Wilcoxan Rank-Sum Test using Benjamini -Hochberg post-hoc correction.

[0062] Oxalic acid. Bee mortality and Varroa mite drop efficacy results from oxalic acid are presented in TABLE 3. There was no significant difference in 24-hour bee mortality (Kruskal- Wallace; P > 0.05). The solvent control demonstrated 0.4 (c = 0.4) % bee mortality after 24 hours, while 20% oxalic acid without adjuvant demonstrated 0.1 (o = 0.2) % bee mortality. Though not statistically significant, the highest 24-hour bee mortality recorded was from the combination of oxalic acid and Ecostep SE-11 which demonstrated 0.7 (c = 0.4) % mortality.PCT / US24 / 56156 15 November 2024 (15.11.2024)Attorney Docket No. 103361-545WO1 TABLE 3. 24-hour bee mortality and Varroa mite drop efficacy for oxalic acid active ingredient, expressed as a percentage, with range of standard error listed in parenthesis. Efficacy is defined as the Varroa that were knocked down from each treatment divided by the total Varroa, expressed as a percentage. P-value of each treatment compared to the solvent control and active ingredient control were determined via a pairwise Wilcoxan Rank-Sum Test using Benjamini-Hochberg post-hoc correction.

[0063] There was a significant difference in 24-hour Varroa efficacy (Kruskal-Wallace; P = 0.0062). Pairwise Wilcox Test using Benjamini -Hochberg post-hoc correction determined significant differences between negative control with active ingredient control and all active ingredient-adjuvant combinations (P < 0.05), but no significant difference between the active ingredient control and any active ingredient - adjuvant combination (P > 0.05), though there was a trend for increased efficacy in all four adjuvant combinations. The solvent control demonstrated 27.5 (G = 10.5) % efficacy after 24 hours, while 20% oxalic acid without adjuvant demonstrated 75.9% (o = 17.1) % efficacy (Pcontroi = 0.0035). In order increasing mean efficacy, 20% oxalic acid in combination with Silwet L-7500 Copolymer™ demonstrated 84.6 (G = 11.4) % efficacy (Pcontroi = 0.0035), in combination with Ecosteop CE-13™ demonstrated 86.5 (G = 14.1) % efficacy (Pcontroi = 0.0035), in combination with Ecostep SE-11™ demonstrated 88.3 (G = 5.3) %PCT / US24 / 56156 15 November 2024 (15.11.2024)Attorney Docket No. 103361-545WO1 efficacy (Pcontroi = 0.0035), and in combination with Ecostep BC-12™ demonstrated 96.4 (c = 5.0) % efficacy (Pcontroi = 0.0035).

[0064] Year 1 Field Trials:

[0065] Honey bee colonies: Three apiaries, separated by a minimum of 5 km, located at The Ohio State University - Wooster campus, were used to conduct the trial. Nine hives from apiary 1, and six hives each from apiaries 2 and 3 were randomly assigned treatments so that each apiary had an equal number of replicates of each treatment. Each colony included a minimum of two deep 8-frame Langstroth boxes at the start of the experiment. Each colony had a screened bottom board for ease in collecting mite drop.

[0066] Treatments: For each uncut Swedish sponge, 70 g of solution, the amount needed for complete saturation of one Swedish sponge, was prepared and heated at less than 67° C for at least 1 hour with sonication (Kendal Digital Ultrasonic Heated Cleaner model HB-S-49DHT) and stirring, if necessary. The adjuvant combination treatment included 1% Ecostep BC-12™ adjuvant and 40% oxalic acid dihydrate dissolved in glycerin, the oxalic acid control included 40% oxalic acid dihydrate dissolved in glycerin, and the solvent control inlcuded glycerin only. Each Swedish sponge was saturated in the specified treatment by soaking in the treatment solution at 67° C for at least 1 hour.

[0067] Experimental Design: Before and after treatment, colonies were assessed by concurrently performing Varroa alcohol washes and seam counts. Seam counts are estimates of adult bees in a colony and include visually counting the “seams” of bees that are present between each frame for each box of a colony, where medium seams are multiplied by the height of a medium Langstroth box divided by the height of a deep Langstroth box (6.625 / 9.625). Any colonies with less than 9 seams prior to the treatment or less than 2 seams after the treatment were excluded from analysis. Alcohol washes involved collecting approximately 300 bees from 3 different worker brood frames, soaking in 70% ethyl alcohol, shaking for a minimum of 30 minutes, and counting the bees and Varroa that were strained from the wash.

[0068] One treatment strip, including a full Swedish sponge, saturated in treatment solution, was applied for every 9 seams of bees, rounded up, so that each colony had either 2 or 3 treatment strips. Treatment strips were placed between boxes of brood and colonies were left undisturbed during the treatment period. Treatments were applied starting on Sept 21st for apiary 1, Oct 9th for apiary 2, and Oct 14th for apiary 3. Treatment strips were removed after 23 days.

[0069] Efficacy within treatments was compared by performing a paired t-test on the difference between the pre- and post-treatment Varroa levels from alcohol washes using the stats package in R (R Core Team, 2023). The change in Varroa levels between treatments werePCT / US24 / 56156 15 November 2024 (15.11.2024)Attorney Docket No. 103361-545WO1 compared by performing an ANOVA on the difference between pre- and post-treatment mite wash levels using the stats package in R (R Core Team, 2023).

[0070] Year 2 Field Trials:

[0071] Honey bee colonies: The same three apiaries as used in year 1 were used in the year 2 trial. Nine hives each from 3 apiaries (27 total) were assigned treatments, so that each apiary had an equal number of replicates of each treatment. Treatments were stratified based on pretreatment Varroa levels. Each colony included a minimum of two deep 8-frame Langstroth boxes at the start of the experiment.

[0072] Treatments: Solutions were prepared in the same manner as in year 1 trials, except for the following changes: a 0.5% adjuvant concentration was used instead of the 1% concentration in the oxalic acid plus adjuvant treatment; and Swedish Sponges were cut in half to increase contact area with bees.

[0073] Experimental Design: Before and after treatment, colonies were assessed by concurrently performing Varroa alcohol washes and seam counts, in the same manner as in year 1. Treatment strips were applied in the same manner as year 1, except that strips were cut in half to increase contact area with bees. A saturated half-Swedish sponge treatment strip was applied for every 5 seams of bees, rounded up, so that each colony had at least 3 treatment strips. Treatments were applied starting on July 30 for all three apiaries for a 22-day period, which is earlier in the season and a decrease from the 23 -day treatment period in year 1. Data was analyzed independently of year 1, using the same statistical analysis methods for alcohol washes as year 1.

[0074] Pre- and Post-Treatment Ethanol Washes'. The pre- and post-treatment mite wash data summary for year 1 are listed in TABLE 4. One-sided paired t-tests are presented graphically in FIG. 7 and FIG. 8. A one-sided t-test on the difference between the pre- and posttreatment Varroa in ethanol washes determined an average increase of 8.4 (SE: 1.7 - 15.1) Varroa per 100 bees for the solvent control, which was not statistically significant (P > 0.05; n = 5, t = 1.260); an average decrease of 4.3 (SE: -8.7 - 0.1) for the oxalic acid control, which was not statistically significant (P > 0.05; n = 7, t = -0.975); and an average decrease of 3.1 (SE: -4.1 - -2.1) for the oxalic acid plus adjuvant treatment, which was determined to be a statistically significant decrease (P = 0.0122; n = 6, t = -3.189). An ANOVA between change in the initial and final Varroa from alcohol washes of treatments determined no significant differences between treatments (P > 0.05).PCT / US24 / 56156 15 November 2024 (15.11.2024)Attorney Docket No. 103361-545WO1 TABLE 4. Year 1 and 2 ethanol wash data for each treatment, where rate is defined as Varroa infestation rate per 100 bees. The value listed in parenthesis for the initial and final rate mean indicates standard deviation; the range listed in parenthesis for the mean difference indicates standard error. The column “n” indicates the number of colonies used for analysis for each treatment. P-value within treatments was determined using a one-sided t-test on the difference within treatments and P-value between treatments and each of the controls was determined with an ANOVA with Tukey’s Post-Hoc test of the change in mite levels, where an asterisk (*) indicates a statistically significant test (P < 0.05).

[0075] The pre- and post-treatment mite wash data summary for year 2 are also listed in TABLE 4. A one-sided t-test on the difference between the pre- and post-treatment Varroa in ethanol washes determined an average increase of 1.3 (SE: 0.9 - 1.7) Varroa per 100 bees forPCT / US24 / 56156 15 November 2024 (15.11.2024)Attorney Docket No. 103361-545WO1 the solvent control, which was not statistically significant (P > 0.05; n = 9, t = 3.409); an average increase of 0.7 (SE: 0.5 - 0.8) for the oxalic acid control, which was not statistically significant (P > 0.05; n = 9, t = 4.986); and an average decrease of 0.2 (SE: -0.5 - 0.2) for the oxalic acid plus adjuvant treatment, which was also not statistically significant (P > 0.05; n = 9, t = -0.521). An ANOVA between change in the initial and final Varroa from alcohol washes of treatments determined a significant difference between treatments (P = 0.007; df = 2, F = 6.143). A Tukey’s Post-Hoc test determined that there was a significant difference between the solvent control and the oxalic acid plus adjuvant treatment (P = 0.005), but no significant difference between the solvent control and oxalic acid control (P > 0.05) or between the oxalic acid control and the oxalic acid plus adjuvant treatment (P > 0.05).

[0076] Mite drop data is shown in TABLE 5 and FIG. 9.TABLE 5. Average daily Varroa mite drop from sticky traps below colonies over the treatment period, where sticky traps monitored for 48-hour periods. Day 0 is the initial time of treatment application. Baseline indicates daily mite drop for the 48-hour period prior to treatment application. Values indicate means for each treatment with standard deviation listed in parenthesis.

[0077] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:

[0078] Throughout the description and claims of this specification, the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and are not intended to exclude, for example, other additives, segments, integers, or steps. Furthermore, it is to be understood that the terms comprise, comprising, and comprises as they relate to various aspects, elements, and features of the disclosed invention also include the more limited aspects of “consisting essentially of’ and “consisting of.”PCT / US24 / 56156 15 November 2024 (15.11.2024)Attorney Docket No. 103361-545WO1

[0079] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an “adjuvant” includes aspects having two or more such adjuvants unless the context clearly indicates otherwise.

[0080] Ranges can be expressed herein as from “about” one particular value and / or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It should be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0081] As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0082] For the terms “for example” and “such as,” and grammatical equivalences thereof, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise.

[0083] The following patents, applications and publications as listed below and throughout this document are hereby incorporated by reference in their entirety herein.Reference List1. Ellis, J., D., & Nalen, C. M. Z. (n.d.). Varroa Mite — Varroa destructor Anderson and Trueman. Retrieved October 28, 2022.2. Steinhauer, N., et al. (2021). United States Honey Bee Colony Losses 2020-2021 : Preliminary Results. Bee Informed Partnership.3. Schmid-Hempel, P. (1998). Parasites in Social Insects. Princeton University Press.4. Peck, D. T., et al. (2016). Varroa destructor Mites Can Nimbly Climb from Flowers onto Foraging Honey Bees. PLOS ONE, 11(12), e0167798.5. Burnham, T. (2016). Varroa Bombs Are Real | Bee Culture.6. Gracia, M. J., et al. (2017). Field efficacy of acaricides against Varroa destructor. PLOS ONE, 12(2), e0171633.7. Vandervalk, L. P., et al. (2014). New Miticides for Integrated Pest Management of Varroa destructor (Acari: Varroidae) in Honey Bee Colonies on the Canadian Prairies. Journal of Economic Entomology, 107(6), 2030-2036.PCT / US24 / 56156 15 November 2024 (15.11.2024)Attorney Docket No. 103361-545WO18. Rodriguez-Dehaibes, S. R., et al. (2011). Susceptibility of Varroa destructor (Gamasida: Varroidae) to four pesticides used in three Mexican apicultural regions under two different management systems. International Journal of Acarology, 37(5), 441-447.9. Martin, S. J. (2004). Acaricide (pyrethroid) resistance in Varroa destructor. Bee World, 85(4), 67-69.10. Thompson, H., et al. (2003). Varroa destructor resistance to pyrethroid treatments in the United Kingdom. Bulletin of Insectology, 56, 175-184.11. Mozes-Koch, R., et al. (2000). First detection in Israel of fluvalinate resistance in the Varroa mite using bioassay and biochemical methods. Experimental & Applied Acarology, 24(1), 35- 43.12. Elzen, P. J., et al. (2000). Control of Varroa jacobsoni Oud. Resistant to fluvalinate and amitraz using coumaphos. Apidologie, 31(3), 437-441.13. Spreafico, M., et al. (2001). First detection of strains of Varroa destructor resistant to coumaphos. Results of laboratory tests and field trials. Apidologie, 32(1), 49-55.14. Rodriguez-Dehaibes, S. R., et al. (2005). Resistance to amitraz and flumethrin in Varroa destructor populations from Veracruz, Mexico. Journal of Apicultural Research, 44(3), 124-125.15. Maggi, M. D., et al. (2010). Resistance phenomena to amitraz from populations of the ectoparasitic mite Varroa destructor of Argentina. Parasitology Research, 107(5), 1189-1192.16. Castro, M. J. L., et al. (2014). Advances in surfactants for agrochemicals. Environmental Chemistry Letters, 12(1), 85-95.17. Knowles, A. (2001). Trends in pesticide formulation. AGROW Report DS-215. PJB Publications, Surrey.18. Hazen, J. L. (2000). Adjuvants — Terminology, Classification, and Chemistry. Weed Technology, 14(4), 773-784.19. Krogh, K. A., et al. (2003). Environmental properties and effects of nonionic surfactant adjuvants in pesticides: A review. Chemosphere, 50(7), 871-901.20. U.S. Department of Agriculture, (n.d.). Oxalic Acid FAQ’s: USDA ARS. Retrieved October 28, 2022.PCT / US24 / 56156 15 November 2024 (15.11.2024)Attorney Docket No. 103361-545WO121. Rademacher, E., & Harz, M. (2006). Oxalic acid for the control of varroosis in honey bee colonies - a review. Apidologie, 37(1), 98-120.22. Oliver, R. (2022, April 22). 2022 Extended-Release Oxalic Update: Part 1. Scientific Beekeeping.23. Heuvel, C. (n.d.). Oxalic Acid & Varroa | Bee Culture. Retrieved October 28, 2022.24. Escobar, A., et al. (2020). Thymol bioactivity: A review focusing on practical applications. Arabian Journal of Chemistry, 13(12), 9243-9269.25. Blenau, W., et al. (2012). Plant essential oils and formamidines as insecticides / acaricides: What are the molecular targets? Apidologie, 43(3), 334-347.26. Enan, E. E. (2005). Molecular response of Drosophila melanogaster tyramine receptor cascade to plant essential oils. Insect Biochemistry and Molecular Biology, 35(4), 309-321.27. Priestley, C. M., et al. (2003). Thymol, a constituent of thyme essential oil, is a positive allosteric modulator of human GABAA receptors and a homo-oligomeric GABA receptor from Drosophila melanogaster. British Journal of Pharmacology, 140(8), 1363-1372.28. Floris, I., et al. (2004). Comparison Between Two Thymol Formulations in the Control of Varroa destructor '. Effectiveness, Persistence, and Residues. Journal of Economic Entomology, 97(2), 187-191.29. Mondet, F., et al. (2011). Age-related changes in the behavioural response of honeybees to Apiguard™, a thymol-based treatment used to control the mite Varroa destructor. Journal of Comparative Physiology A, 197(11), 1055.30. Bahreini, R., et al. (2022). Miticidal activity of fenazaquin and fenpyroximate against Varroa destructor, an ectoparasite of Apis mellifera. Pest Management Science, 78(4), 1686-1697.31. Li, L., et al. (2017). The effects of clove oil on the enzyme activity of Varroa destructor Anderson and Trueman (Arachnida: Acari: Varroidae). Saudi Journal of Biological Sciences, 24(5), 996-1000.32. Bahreini, R., et al. (2021). New bioassay cage methodology for in vitro studies on Varroa destructor and Apis mellifera. PLOS ONE, 16(4), e0250594.PCT / US24 / 56156 15 November 2024 (15.11.2024)Attorney Docket No. 103361-545WO1 33. Riusech, N. S. (2017). Varroa mite control in honey bee colonies: The use of a fatty acid blend (C8910) for Varroa mite control and exploring management practices used by beekeepers in full-sized colonies [The Ohio State University],

Claims

PCT / US24 / 56156 15 November 2024 (15.11.2024)Attorney Docket No. 103361-545WO1 What is claimed is:

1. A composition, comprising a miticidal ingredient and an adjuvant.

2. The composition of claim 1, wherein the miticidal ingredient is oxalic acid, thymol, fenazaquin, or a derivative or combination thereof.

3. The composition of any one of claims 1-2, wherein the adjuvant is an oil.

4. The composition of claim 3, wherein the adjuvant comprises Stepan™ C-65, Stepan™ 108, Steposol™ C-65, ECOSTEP™ CE-13, Tall Oil Fatty Acids, or any combination thereof.

5. The composition of any one of claims 1-2, wherein the adjuvant is a traditional surfactant, an organo-silicone surfactant, or a combination thereof.

6. The composition of claim 5, wherein the adjuvant comprises Ninate™ 60E, Toximul™ 8240, Toximul™ 8320, ECOSTEP™ SE-11, ECOSTEP™ AE-13, ECOSTEP™ BC-12, Biosoft™ Nl-7, Makon™ 10, Makon™ P104, Step-Flow™ 26, Ninex™ MT-615, Toximul™ TA-8, Steol™ TSP-16N, Silwet™ Eco, Sylgard™ 309, Silwet™ L-7500, or any combination thereof.

7. The composition of any one of claims 1-2, wherein the adjuvant is an emulsifier.

8. The composition of claim 7, wherein the adjuvant comprises pinene monomers, polyterpenes, or a combination thereof.

9. The composition of any one of claims 1-8, further comprising a solvent, wherein the miticidal ingredient and the adjuvant are dissolved or suspended in the solvent.

10. The composition of claim 9, wherein the solvent comprises glycerol, glycerin, or any combination thereof.

11. The composition of any one of claims 9-10, wherein the miticidal agent is present in a concentration of from about 0.1% to about 50%.PCT / US24 / 56156 15 November 2024 (15.11.2024)Attorney Docket No. 103361-545WO112. The composition of any one of claims 9-11, wherein the adjuvant is present in a concentration of from about 0.1% to about 3%13. A method of mite control in honey bee colonies, the method comprising introducing the composition of any one of claims 1-12 into a honey bee colony.

14. The method of claim 13, wherein the composition is sprayed into or coated on a portion of the honey bee colony.

15. The method of any one of claims 13-14, wherein the honey bee colony comprises a mite population, and wherein the composition reduces the mite population by from about 1% to about 100%.

16. The method of any one of claims 13-15, wherein the composition is introduced into the honey bee colony preventatively.

17. The method of any one of claims 13-15, wherein the composition is used to treat, relieve, or mitigate a mite outbreak.

18. The method of any one of claims 13-17, wherein the composition reduces a population of the honey bee colony by less than 10%.

19. An article comprising an absorbent and the composition of any one of claims 1-12, wherein the composition is absorbed into the absorbent.

20. The article of claim 19, wherein the absorbent comprises felt, fabric, cellulose, or any combination thereof.

21. The article of any one of claims 19-20, wherein the composition is present in an amount of from about 60 mL to about 300 mL.

22. A method of mite control in honey bee colonies, the method comprising introducing the article of any one of claims 19-21 into a honey bee colony.PCT / US24 / 56156 15 November 2024 (15.11.2024)Attorney Docket No. 103361-545WO123. The method of claim 22, wherein the honey bee colony comprises a mite population, and wherein the article reduces the mite population by from about 1% to about 100%.

24. The method of any one of claims 22-23, wherein the article is introduced into the honey bee colony preventatively.

25. The method of any one of claims 22-23, wherein the article is used to treat, relieve, or mitigate a mite outbreak.

26. The method of any one of claims 22-25, wherein the article reduces a population of the honey bee colony by less than 10%.

Images