Flavonoid compositions and methods of use thereof for cognitive protection of insect pollinators

Abstract

Compositions and methods for forming dilute compositions suitable for administration to or ingestion by insect pollinators such as honeybees. Provided herein are concentrated formulations containing one or more flavonoids e.g., rutin, and a solvent e.g., sodium bicarbonate. The flavonoids are present in the composition in an effective amount to protect against impairment of a cognitive and / or immune function of the insect pollinator, and are more soluble in the solvent than in a 1M aqueous sucrose solution. Also provided are dilute compositions formed from the concentrated formulations that are suitable for administration to or ingestion by insect pollinators to protect them against the neurocognitive effects of pesticides. Also provided are methods of protecting the cognitive function of an insect pollinator from a pesticide. The methods generally include applying or placing a dilute composition in a hive located in an area that is occupied by one or more insect pollinators.

Description

[0001] ATTORNEY DOCKET NO. UA 23-279 PCT

[0002] FLAVONOID COMPOSITIONS AND METHODS OF USE THEREOF FOR COGNITIVE PROTECTION OF INSECT POLLINATORS CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims benefit of and priority to U.S. Provisional Application No. 63 / 729,883, filed December 9, 2024, which is specifically incorporated by reference herein in its entirety.

[0004] FIELD OF THE INVENTION

[0005] The disclosed invention is generally in the field of insect pollinators.

[0006] BACKGROUND OF THE INVENTION

[0007] Honeybee hive mortality is a global concern, driven by multiple factors impacting bee health both inside and outside the hive (Brodschneider et al., 2018; Hristov et al., 2020; Kulhanek et al., 2017; Moritz & Erlcr, 2016; Potts et al., 2010; Rcquicr et al., 2018). External threats, such as exposure to agrochemicals, cause lethal and sublethal effects, while internal stressors, including pathogens and Varroa mites, weaken individual bees and compromise hive survival (Dively et al., 2015; Quinlan et al., 2023; Tashakkori & Ghadiri, 2015; de Mattos et al., 2017; Pankiw et al., 2018; Ramsey et al., 2019). The combined effects of pesticides and Varroa infestation reduce foraging activity, larval nutrition, and worker bee production, significantly impacting colony health (Avni et al., 2014; Horn et al., 2016; Tsvetkov et al., 2017; van Dooremalen et al., 2013).

[0008] Nutrition is important for mitigating these stress factors. Beekeeping practices often use supplements, such as sucrose syrups, to improve bee health and productivity, though these lack the nutritional complexity of natural nectar and pollen, which contain important phytochemicals (Brodschneider & Crailsheim, 2010; Di Pasquale et al., 2016; Gong & Diao, 2017; Pudasaini et al., 2020). The protective effects of phytochemicals like flavonoids, phenolic acids, and alkaloids, which improve motor abilities, immune responses, survival, and metabolic activity under pesticide stress have been explored (Ardalani et al., 2021; Balieira et al., 2018; Liao et al., 2017, 2019; Mitton et al., 2020; Wong et al., 2018). However, some phytochemicals pose risks, as many act as plant defense mechanisms with toxic effects on bees, such as reduced longevity, survivability, and altered reproductive behavior (Bourgaud et al., 2001; Gao et al., 2010; Kevan & Ebert, 2005; London-Shafir et al., 2003; Lucchetti et al., 2018; Stevenson et al., 2017; Wink, 2003).

[0009] Flavonoids are nutraceutical molecules derived from plants and studied for more than a century in diverse contexts, from cell culture to clinical trials. However, constraints in flavonoid solubility in water have been a challenge for research, subject administration, and storage.

[0010] 1

[0011] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT

[0012] Dimethyl sulfoxide (DMSO) is the most common solvent used to dissolve flavonoids as well as other polar and nonpolar molecules. DMSO has been widely recognized for its presumed innocuity and is approved for tests in toxicology. However, increasing evidence suggests that the DMSO may have toxic effects even at exceptionally low concentrations (e.g., Galvao et al., 2013: Milchreit et al., 2016; Verheijen et al., 2019; Kim & Lee, 2021). For example, administration of DMSO to cell cultures of retina induces apoptosis even at concentrations as low as 1% (Galvao et al., 2013) while administration of 0.1% alters epigenetic regulations in cardiac cells (Verheijen et al., 2019). Also, in honeybees brood production is reduced after the exposure to DMSO at concentrations as low as 0.5% (Milchreit et al., 2016).

[0013] Research has demonstrated that low concentrations (~1 mM) of rutin protect honeybees and bumble bees against sensory, cognitive and motor impairments by imidacloprid, dcltamcthrin and fipronil (Riveros ct al. 2020, Riveros & Groncnbcrg, 2022, Garcia ct al. 2024). At such low concentrations, the low solubility of flavonoids in water is not an issue. However, mass production, transportation, and administration in large apiaries or otherwise, demand preparation of concentrated solutions. For example, it is estimated that a colony of 30,000 bees may require two liters of solution per week, making it impractical for the transportation of sufficient volumes. Thus, improved concentrated formulations that increase the dissolution of flavonoids or minimize their precipitation are needed. Also needed are more effective methods for delivering these flavonoids in a diluted form, suitable for large-scale use in apiaries. Improved compositions containing flavonoids, derived from the concentrated formulations, which do not negatively affect the physiology of bees are also needed.

[0014] Thus, the object of the present invention is to provide concentrated formulations of flavonoids.

[0015] It is also an object of the present invention to provide compositions for protecting insect pollinators from pesticides.

[0016] It is a further object of the invention to provide a method for delivering improved compositions to protect insect pollinators.

[0017] BRIEF SUMMARY OF THE INVENTION

[0018] Compositions and methods for forming dilute compositions suitable for administration to or ingestion by insect pollinators are provided. Provided herein are concentrated formulations containing one or more flavonoids and a solvent. Generally, the one or more flavonoids are (i) present in the composition in an effective amount to protect against impairment of a cognitive function of the insect pollinator, and (ii) more soluble in the solvent than in a control solvent e.g., aqueous sucrose solution. For example, the one or more flavonoids are more soluble in the 2

[0019] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT solvent than in IM aqueous sucrose solution. The concentrated formulation has a pH ranging from about 7.0 to about 9.0.

[0020] The flavonoid in the concentrated formulation can be quercetin, rutin, myricetin, kaempferol, fisetin, or apigenin, or a combination thereof. In one exemplary form, the flavonoid in the concentrated formulation is rutin. Generally, the one or more flavonoids are present in the concentrated formulation in a range from about 30 mg / E to about 500 mg / E.

[0021] Any solvent that is capable of solubilizing high concentrations of the flavonoid (e.g., rutin) while remaining non-toxic and tolerable to bees may be used. Tor example, the solvent can be sodium bicarbonate, methanol, ethanol, 1 -propanol, 2-propanol, 1 -butanol, acetone, ethyl acetate, glycerin, or a combination thereof. In one exemplary form, the solvent is sodium bicarbonate. Generally, when the solvent is aqueous sodium bicarbonate, the sodium bicarbonate is present in the concentrated formulation at a concentration ranging from about 40 mg / L to about 500 mg / L. The solvent can optionally include one or more pH modifiers to achieve the desired pH range. Optionally, the solvent is not Dimethyl sulfoxide (DMSO).

[0022] Optionally, the concentrated formulation contains one or more additional nutrients and / or ingredients selected from the group consisting of carbohydrates, proteins, lipids, vitamins, minerals, and water, or a combination thereof. Exemplary additional nutrients and / or ingredients are selected from the group consisting of natural or artificial nectar, honey, sugar, sugar syrup, pollen or pollen substitute, soy flour, soy meal, gluten, skim milk, yeast, pollard, and oil, or combinations thereof.

[0023] Generally, the insect pollinator can be a butterfly, a moth, a fly, a beetle, a wasp or a bee. In one exemplary form, the insect pollinator is a bee, such as honeybee, bumblebee, carpenter bee, leafcutter bee, blueberry bee, squash bee, mason bee, orchid bee, stingless bee, or sweat bee.

[0024] Also provided are dilute compositions formed from the concentrated formulations. Generally, the disclosed dilute compositions are safe for use as a food, feed additive or supplement, and / or nutraceutical. The dilute compositions are suitable for administration to or ingestion by insect pollinators to protect them against the harmful neurocognitive effects of pesticides.

[0025] Also provided are methods of protecting the cognitive function of an insect pollinator (e.g., a honeybee) from a pesticide (e.g., fipronil). The method generally includes (i) applying or placing a dilute composition in a hive located in an area that is occupied by one or more insect pollinators. The dilute composition includes a dilute form of the concentrated formulation described above.

[0026] 3

[0027] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT

[0028] The dilute composition contains an effective amount of one or more flavonoids to prevent or reduce impairment of a cognitive function and / or increase a cognitive function in the insect pollinator following exposure to a pesticide. In some forms, the one or more flavonoids is present in the dilute composition in an amount from about 0.01 pM to about 100 mM.

[0029] In some forms, the method further includes prior to step (i), diluting the concentrated formulation to form a diluted composition comprising about 1 pM of the one or more flavonoids, wherein the diluent is or comprises an aqueous sucrose solution, such as a IM sucrose solution. In some forms, the method further includes incorporating the dilute composition in a solid food source prior to step (i). In some forms, step (i) of the method includes the dilute composition is placed in the hive via any form of internal or external feeder such as a deep division board feeder.

[0030] BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings illustrate several embodiments of the disclosed method and compositions and together with the description, serve to explain the principles of the disclosed method and compositions.

[0032] Figure 1 is a schematic showing the impact of pesticide exposure and Varroa sp. infestation in honey bee health indicators.

[0033] Figure 2 is a schematic of an experimental set-up for the evaluation of colony and individual health for Study Site 1 (HPG’s = Hypopharyngeal glands).

[0034] Figures 3A-3D are results showing hive health indicators evaluated at Site 1 apiary by treatment, by hive, by replicate through the evaluation period. Figures 3A and 3B show the number of brood and food storage frames (in grey and white bars), the amount of collected pollen (gray lines), the amount of dead bees collected (red and blue lines) and the percentage of infestation Varroa sp. levels evaluated at the beginning and the end of the feeding period for Replicate 1 (Figure 3A) and Replicate 2 (Figure 3B). In both replicates, there was a pesticide aspersion event on week 3 (not intended in the methodology and represented with a skull illustration on the graph). Figures 3C and 3D show the amount of pollen collected for Replicate 1 (Figure 3C) and Replicate 2 (Figure 3D).

[0035] Figure 4 is a bar graph showing Varroa sp. infestation relative change percentage (infestation levels from week 1 compared to levels of week 4) for each test colony.

[0036] Figures 5A-5F show total, head and thorax, respectively, mean dry mass from worker bees from Rut and Control group hives at Replicate 1 (Figures 5A-5C) and Replicate 2 (Figures 5D-5F). **p < 0.01.

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[0038] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT

[0039] Figures 6A-6C show the effects of rutin supplementation on total head and thorax dry mass of bees on Site 3. Data from bees was collected in Week 4. Only significant differences are indicated to facilitate visualization. ***p < 0.001.

[0040] Figures 7A and 7B show the area of hypopharyngeal glands acini from nurse bees from Rut and Control group colonies. Differences in acini area measurements were detected in the same hive between replicates. ***p < 0.001.

[0041] Figures 8A-8C are line graphs showing variation in absorbance and pH for solutions of rutin in different concentrations of Nal ICO;. Shown are absorbance at 256nm (Figure 8A), Absorbance at 352nm (Figure 8B), and pH under different concentrations of Nal ICO; (Figure 8C).

[0042] Figures 9A-9C are results from studies testing the effects of an exemplary formulation of rutin inducing protection of learning in honeybees treated with the pesticide fipronil compared with honeybees receiving only sucrose (Control) and bees receiving only fipronil.

[0043] Figures 10A and 10B are results from studies testing the effects of an exemplary formulation of rutin inducing protection of learning in honeybees treated with the pesticide deltamethrin compared with honeybees receiving only sucrose (Control) and bees receiving only deltamethrin.

[0044] DETAILED DESCRIPTION OF THE INVENTION

[0045] The disclosed method and compositions can be understood more readily by reference to the following detailed description of particular embodiments and the Example included therein and to the Figures and their previous and following description.

[0046] I. Definitions

[0047] As used herein, “bee” refers to members of the Family Apidae, Order Hymenoptera. A “honeybee” refers to members of the genus, Apis. For example, the domesticated honeybee is Apis mellifera. A “bumblebee” refers to the genus ‘Bombas’ . For example, a domesticated species of bumblebee include the buff-tailed bumblebee, Bombus terrestris and the bumblebee Bombus impatiens.

[0048] As used herein, “pollinator” means an animal that moves pollen from the male anther of a flower to the female stigma of a flower, which helps bring about fertilization of the ovules of the flower by the male gametes in the pollen grains. “Insect pollinators” are insects whose behavior results in pollination of one of more species of plant. The term does not denote a particular age or sex.

[0049] As used herein, “pest” and “insect pest” (used interchangeably) mean an insect or arthropod that damages agricultural products or reduces agricultural yield of agricultural

[0050] 5

[0051] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT products that are economically useful or that find desirable utility in human or animal consumption. “Pest” and “insect pest” are also understood as any arthropod or insect that is destructive by infesting and damaging pollinating insects, bee hives, or reducing honey bee populations, or by causing a reduction in honey production.

[0052] As used herein, “exposure” when used in the context of a pesticide describes the state of having contact with the pesticide. Exposure can be obtained through a variety of mechanisms. For example, an insect pollinator can be exposed to a pesticide through food or water ingestion, nesting material (e.g., resin, wax etc.), contact with spray drift and dust drift generated by pesticide application, contact with contaminated plants, soil, water, and inhalation.

[0053] As used herein, “sublethal” describes the amount or concentration of pesticide that does not give rise to acute mortality. Sublethal effects on an insect may involve modifications of insect (e.g., honeybee) behavior and physiology (e.g., immune system). They do not directly cause the death of the individual or the collapse of a colony but may become lethal in time and / or may make the colony more sensitive (e.g., more prone to diseases), which may contribute to its collapse. For instance, an individual with memory, orientation or physiological impairments might fail to return to its hive, dying from hunger or cold. In the context of neonicotinoids, a sublethal dose can be a field dose, which is an amount or concentration of the pesticide that an insect pollinator may be exposed to during normal foraging. In some forms, a field dose of imidacloprid is about 1 pg / L.

[0054] As used herein, “cognitive function” when used in the context of an insect pollinator, encompasses acquisition, storage, processing, retrieval and use of information, and hence include perception, attention, memory, learning, motor skills, and navigation.

[0055] As used herein, “impair” in the context of a biological function or parameter means to weaken, reduce or otherwise adversely alter that function or parameter.

[0056] As used herein, the term “neurotoxic” means poisonous or otherwise harmful to the nervous system.

[0057] As used herein, “effective amount” means that the amount of the composition used is of sufficient quantity / concentration to affect an intended response. For example, the amount of the neuroprotective compositions disclosed herein can be effective to ameliorate one or more symptoms or effects of a pesticide on an insect pollinator. Non-limiting examples of symptoms or effects of pesticides include reduction in information processing including, for example, learning, memory or navigation. Such amelioration only requires a reduction or alteration, not necessarily elimination. The precise dosage will vary according to a variety of factors such as species, subject-dependent variables (e.g., organism size, age, immune system health, etc.), the 6

[0058] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT pesticide being protected against, as well as the route of administration and the pharmacokinetics of the agent being administered. Alternatively, the amount of pesticide can be effective to reduce the number of insect pests in a treated area. In some examples, the amount of pesticide is effective to kill (e.g., be lethal to) insect pests.

[0059] As used herein, “reduce” means to decrease an activity, function, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete ablation of the activity, function, response, condition, or disease. This may also include, for example, at least a 5% decrease in the activity, function, response, condition, or disease, or other biological parameter as compared to a native or control level (e.g., before or after administration of a disclosed composition or levels in an insect pollinator not administered a disclosed composition). Thus, the reduction can be a 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of decrease in between as compared to native or control levels.

[0060] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[0061] Use of the term “about” is intended to describe values either above or below the stated value in a range of approx. + / - 10%; in other embodiments the values may range in value either above or below the stated value in a range of approx. + / - 5%; in other embodiments the values may range in value either above or below the stated value in a range of approx. + / - 2%; in other embodiments the values may range in value either above or below the stated value in a range of approx. + / - 1%. The preceding ranges are intended to be made clear by context, and no further limitation is implied.

[0062] All methods described herein can be performed in any suitable order unless otherwise indicated or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0063] II. Formulations

[0064] Disclosed are concentrated formulations for forming dilute compositions suitable for administration to or ingestion by an insect pollinator (e.g., honeybees). Generally, the concentration of the flavonoid in solution in the concentrated formulation is at least 100 mM. For example, the concentration of the flavonoid in solution in the concentrated formulation is about 7

[0065] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT

[0066] 102.4 mM, about 150 mM, about 200 mM, about 204.7 mM, about 250 mM, about 300 mM, about 350 mM, about 380 mM, about 409.5 mM, about 500 mM, about 600 mM, about 700 mM, or about 750 mM.

[0067] Generally, the concentrated formulations include one or more flavonoids, such as rutin, and a solvent that increases solubility of the one or more flavonoids compared to the same flavonoid in an aqueous sucrose formulation. Typically, the one or more flavonoids are present in the formulation in an effective amount to protect against impairment of cognitive function of the insect pollinator. Also, the one or more flavonoids are more soluble in the solvent than in a control solvent e.g., aqueous sucrose solution. For example, the one or more flavonoids are more soluble in the solvent than in 1 M aqueous sucrose solution. As demonstrated in non- limiting Example 2, 400 mg of sodium bicarbonate is effective to dissolve up to about 250 mg of rutin without precipitation. In other forms, solvents other than sodium bicarbonate can dissolve more than 250 mg of rutin without precipitation. The concentrated formulation is typically maintained at an alkaline pH to ensure the complete solution of one or more flavonoids. Generally, the concentrated formulation has a pH ranging from about pH 7.0 to about pH 9.0. For example, the concentrated formulation can have a pH ranging from about pH 7.0 to about pH 8.8, from about pH 7.0 to about pH 8.6, from about pH 7.0 to about pH 8.4, from about pH 7.0 to about pH 8.2, from about pH 7.0 to about pH 8.0, from about pH 7.0 to about pH 7.8, from about pH 7.0 to about pH 7.6, from about pH 7.0 to about pH 7.4, from about pH 7.0 to about pH 7.2, such as about pH 7.5.

[0068] The concentrated formulations can be diluted to produce dilute compositions suitable for administration to or ingestion by insect pollinators. The dilute compositions contain an effective amount of the one or more flavonoids to reduce or prevent one or more of the harmful effects of neonicotinoids and other neuro-pesticides on insect pollinators such as honeybees. The dilute compositions can be in the form of a solid or a liquid composition.

[0069] The dilute compositions can further contain a source of carbohydrates, proteins, lipids, vitamins, minerals, water or combinations thereof, such as, for example, natural or artificial nectar, honey, sugar, sugar syrup, pollen or pollen substitute, soy flour, soy meal, gluten (e.g., soy or corn), skim milk, yeast, pollard, oil, and combinations thereof. In some forms, the source of carbohydrates, proteins, lipids, vitamins, minerals, or water can be derived from a commercially available feed such as AP23®, BEE-PRO®, FEEDBEE, MEGABEE and ULTRA BEE.

[0070] The dilute compositions containing the one or more flavonoids for administration to the insect pollinators generally have a pH ranging between about pH 6.0 and about pH 9.0, similar to 8

[0071] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT the pH of the digestive tract, of the insect pollinators and / or the pH of nectar. The pH of the of the digestive tract of honeybees ranges from about pH 5.2 to about pH 7 (Zheng et al., Proc Natl Acad Sci USA, 2017). The pH of nectar ranges from about 4.2 to about 8.5 (Baker et al., Apidologie 8 (4), 1977). For example, the pH of the dilute compositions containing the one or more flavonoids for administration to the insect pollinators can have a pH ranging from about pH 6.0 to about pH 8.8, from about pH 6.0 to about pH 8.6, from about pH 6.0 to about pH 8.4, from about pH 6.0 to about pH 8.2, from about pH 6.0 to about pH 8.0, from about pH 6.0 to about pH 7.8, from about pH 6.0 to about pH 7.6, from about pH 6.0 to about pH 7.4, or from about pH 6.0 to about pH 7.2. In one exemplary form, the pH of the dilute compositions containing the one or more flavonoids have a pH of about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5.

[0072] A. Flavonoids

[0073] The concentrated formulation as described herein contains one or more flavonoids. Flavonoids are secondary metabolites and are found in several parts of the plant (e.g., fruits, vegetables, grains, bark, roots, stems, flowers). Flavonoids are used by vegetables for their growth and defense against plaques. Flavonoids protect plants from different biotic and abiotic stresses and act as unique UV filters, function as signal molecules, allopathic compounds, phytoalexins, detoxifying agents and antimicrobial defensive compounds (Pance, et al., J. Nutr. Sci., 5: e47, 2016). Flavonoids are considered an indispensable component in a variety of nutraceutical, pharmaceutical, medicinal and cosmetic applications. This is attributed to their anti-oxidative, anti-inflammatory, anti-mutagenic and anti-carcinogenic properties coupled with their capacity to modulate key cellular enzyme function.

[0074] Flavonoids possess potent antioxidant properties and can protect against damage caused by free radicals through multiple mechanisms. One mechanism involves the direct scavenging of free radicals, during which flavonoids are oxidized, resulting in the formation of more stable and less reactive radicals. This process stabilizes reactive oxygen species by permitting flavonoids to react with and neutralize the reactive components of the radicals. The high reactivity of the hydroxyl groups in flavonoids plays an important role in rendering radicals inactive. Additionally, in certain forms, flavonoids enhance or increase the expression and / or activity of components within antioxidant pathways, such as the Nrf2 or AP-1 pathways (Farooqui, T. Front. Genet., 5:60, 2014). Furthermore, the disclosed formulations, compositions, and methods leverage the diverse biological activities of flavonoids, including their antioxidant, antiinflammatory, anti-mutagenic, and anti-carcinogenic properties, as well as their ability to modulate key cellular enzyme functions.

[0075] 9

[0076] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT

[0077] Exemplary flavonoids that can be used in the composition include but are not limited to rutin, quercetin, fisetin, myricetin, kaempferol, and apigenin. The structures of these exemplary flavonoids are provided in Table 1. In one particular form, the flavonoid included in the concentrated formulation is rutin. In some forms, the flavonoid included in the concentrated formulation is quercetin. In some forms, the flavonoid included in the concentrated formulation is fisetin. In some forms, the flavonoid included in the concentrated formulation is myricetin. In some forms, the flavonoid included in the concentrated formulation is kaempferol. In some forms, the flavonoid included in the concentrated formulation is apigenin.

[0078] Table 1: Exemplary flavonoids

[0079] 10

[0080] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT

[0081] The concentrated formulation can contain only one flavonoid. Alternatively, the concentrated formulation can contain two, three, four, or more different flavonoids. One of ordinary skill in the art can readily determine the appropriate ratios (e.g., molar or mass) of the flavonoids in such formulations. It is to be understood that in some forms, having more than one type of flavonoid may confer a synergistic effect (e.g., the effect of the combination is higher than the sum of the effect of each flavonoid alone). For example, when the concentrated formulation contains two flavonoids, e.g., quercetin and rutin, the quercetin and rutin are present in the concentrated formulation at a quercetin: rutin molar ratio of 1 : 1, 0.75:25, or 0.25:0.75. In a second exemplary form, the concentrated formulation contains kaempferol and rutin at a kaempferol: rutin molar ratio of about 100:1, about 131.6: 1, or about 200:1. For example, a concentrated formulation containing kaempferol and rutin can contain from about 100 pM to about 200 pM kaempferol and from about 0.5 pM to about 2.0 pM rutin, such as about 131.6 pM kaempferol and about 1 pM. Although not described, concentrated formulations containing other combinations of flavonoids are also contemplated e.g., myricetin and rutin; apigenin and rutin, or fisetin and rutin.

[0082] The one or more flavonoids can be present in the concentrated formulation in a range from about 30 mg / L to about 500 mg / L. The one or more flavonoids can be present in the concentrated formulation in concentration ranging from about 30 mg / L to about 480 mg / L, from about 30 mg / L to about 460 mg / L, from about 30 mg / L to about 440 mg / L, from about 30 mg / L to about 420 mg / L, from about 30 mg / L to about 400 mg / L, from about 30 mg / L to about 380 mg / L, from about 30 mg / L to about 360 mg / L, from about 30 mg / L to about 340 mg / L, from

[0083] 11

[0084] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT about 30 mg / L to about 320 mg / L, from about 30 mg / L to about 300 mg / L, from about 30 mg / L to about 280 mg / L, from about 30 mg / L to about 260 mg / L, from about 30 mg / L to about 240 mg / L, from about 30 mg / L to about 220 mg / L, from about 30 mg / L to about 200 mg / L, from about 30 mg / L to about 180 mg / L, from about 30 mg / L to about 160 mg / L, from about 30 mg / L to about 140 mg / L, from about 30 mg / L to about 120 mg / L, from about 30 mg / L to about 100 mg / L, from about 30 mg / L to about 90 mg / L, from about 30 mg / L to about 80 mg / L, from about 30 mg / L to about 70 mg / L, from about 30 mg / L to about 60 mg / L, or from about 30 mg / L to about 50 mg / L. Eor example, the one or more flavonoids is present in the concentrated formulation at a concentration of about 62.5 mg / L, about 125 mg / L, about 250 mg / L, or about 500 mg / L. Concentrations of flavonoids of up to and including 1000 mg / L are also contemplated.

[0085] B. Solubility-Enhancing Solvents

[0086] The concentrated formulation also contains a suitable solubility-enhancing solvent (also referred to herein as “solvent”), where the solubility of the one or more flavonoids in the solvent is greater than its solubility in a control solvent e.g., aqueous sucrose solution. For example, the solvent is capable of increasing the solubility of the one or more flavonoids by about 10%, about 15%, about 20%, about 25%, about 30%, or more, up to about 100%, relative to their solubility in a control solvent such as 1 M aqueous sucrose solution.

[0087] Flavonoids, including rutin, are generally difficult to dissolve in water or aqueous sucrose potentially because they contain both hydrophobic aromatic rings and multiple hydroxyl groups that form strong internal hydrogen bonds. These structural features reduce their ability to interact with water, causing flavonoids to aggregate and crystallize when present at higher concentrations. Mildly alkaline solvents can improve flavonoid (e.g., rutin) solubility in part by deprotonating some of their phenolic hydroxyl groups. This in part, increases the ionic character of the molecules, reduces aggregation, and allows the flavonoid to remain dissolved when higher concentrations of the flavonoid (e.g., rutin) is used. Therefore, any solvent that can solubilize high concentrations of the flavonoid (e.g., rutin) compared to a control solvent e.g., an aqueous sucrose solution, while remaining non-toxic and tolerable to bees, can be used as the solvent. For example, as demonstrated in Example 2, the solubility of the flavonoid, rutin, in an aqueous solution of sodium bicarbonate is greater than its solubility in a control solvent e.g., an aqueous sucrose solution, such as a IM aqueous sucrose solution.

[0088] Therefore, the solvent can be any mildly alkaline aqueous medium that increases the solubility of flavonoids without exhibiting toxicity to insect pollinators such as honeybees. For example, the solvent is any alkaline solution that increases the solubility of the selected

[0089] 12

[0090] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT flavonoid(s) relative to their solubility in neutral or slightly acidic aqueous solutions (e.g., aqueous sucrose solution). Suitable solvents include aqueous solutions containing solubilizing agents that raise the pH sufficiently to disrupt or prevent flavonoid aggregation or crystallization in aqueous media, such as by modulating ionic strength, altering hydrogen-bonding interactions, or partially deprotonating phenolic hydroxyl groups of the flavonoids. For example, the solvent increases flavonoid dissolution by reducing intramolecular hydrogen bonding, and by increasing partial deprotonation of the phenolic hydroxyl groups present on flavonoid molecules (e.g., rutin).

[0091] The solubilizing agent (e.g., sodium bicarbonate) generally has a pKa sufficient to produce a mild alkaline solvent capable of increasing solubility of the one or more flavonoids. For example, the solubilizing agent (e.g., sodium bicarbonate) has a pKa between about 6.3 and about 12.3, such as between about 6.3 and about 10.3, between about 6.3 and about 8.2, or between about 6.3 and about 7.2. Examples of such solubilizing agents include but are not limited to alkaline salts such as bicarbonate salts (e.g., sodium bicarbonate, potassium bicarbonate), or carbonate salts (e.g., sodium carbonate, potassium carbonate).

[0092] Optionally the pH of the concentrated formulation is in the range of about pH 6.0 to about pH 9.0. For example, the pH of the concentrated formulation ranges from about pH 7.0 to about pH 8.8, from about pH 7.0 to about pH 8.6, from about pH 7.0 to about pH 8.4, from about pH 7.0 to about pH 8.2, from about pH 7.0 to about pH 8.0, from about pH 7.0 to about pH 7.8, from about pH 7.0 to about pH 7.6, from about pH 7.0 to about pH 7.4, from about pH 7.0 to about pH 7.2, such as about pH 7.5. In certain forms, the pH of the concentrated formulation is maintained at or below pH 9.0. For example, when rutin is the flavonoid, or one of the flavonoids, included in the formulation, the pH is kept at or below pH 9.0 to prevent conditions that favor the conversion of rutin into quercetin, which would reduce the concentration of rutin in the formulation.

[0093] As demonstrated in Example 2, the administration to multiple hives of an exemplary composition containing rutin and sodium bicarbonate dissolved in sucrose solution is effective to protect honeybees against cognitive impairments induced by the exemplary pesticides, fipronil and deltamethrin (Example 2).

[0094] In some instances, the solvent contains sodium bicarbonate, for example, the solvent is an aqueous solution of sodium bicarbonate, such as a 1 mM sodium bicarbonate solution. In some forms, the molar concentration of sodium bicarbonate in the aqueous solution can range from about 0.4 mM to about 5 mM, such as about 0.6 mM, about 1.19 mM, or about 4.76 mM. For example, the molar concentrate of sodium bicarbonate in the aqueous solution can range from

[0095] 13

[0096] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT about 0.40 mM to about 4.80 niM, from about 0.60 mM to about 4.50 mM, from about 0.40 mM to about 4.25 mM, from about 0.40 mM to about 4.00 mM, from about 0.40 mM to about 3.50 mM, from about 0.40 mM to about 3.00 mM, from about 0.40 mM to about 2.50 mM, from about 0.40 mM to about 2.00 mM, from about 0.40 mM to about 1.50 mM, from about 0.40 mM to about 1.20 mM, from about 0.4 mM to about 1.0 mM, or from about 0.4 mM to about 0.70 mM. Alternatively, solvents that produce an alkaline solution can be used. Optionally, the solvent includes one or more nontoxic pH modifiers to achieve the desired pH range. Exemplary acidifier compounds are the organic acids such as citric acid, acetic acid, lactic acid, malic acid, fumaric acid, or succinic acid and combinations of organic acids such as malic acid, fumaric acid, and pyruvic acid.

[0097] Additional solvents that can be used include but are not limited to methanol, ethanol, 1 - propanol, 2-propanol, 1 -butanol, acetone, glycerin, sodium carbonate, potassium bicarbonate, and potassium carbonate. For example, the solvent is an aqueous solution of sodium carbonate, potassium bicarbonate, and potassium carbonate. In these forms, the molar concentration of sodium carbonate, potassium bicarbonate, or potassium carbonate in the aqueous solution can range from about 0.4 mM to about 5 mM, such as about 0.6 mM, about 1.19 mM, or about 4.76 mM. The solvent is not and does not contain DMSO.

[0098] The solvent can be included at a concentration sufficient to reduce or prevent the formation of flavonoid crystals, such as rutin crystals, in the concentrated formulation. When the solvent is an aqueous sodium bicarbonate solution, the sodium bicarbonate may be present in the concentrated formulation at a concentration ranging from about 40 mg / L to about 500 mg / L. For example, sodium bicarbonate can be present in the concentrated formulation in concentration ranging from about 40 mg / L to about 450 mg / L, from about 40 mg / L to about 460 mg / L, from about 30 mg / L to about 440 mg / L, from about 40 mg / L to about 420 mg / L, from about 40 mg / L to about 400 mg / L, from about 40 mg / L to about 380 mg / L, from about 40 mg / L to about 360 mg / L, from about 40 mg / L to about 340 mg / L, from about 40 mg / L to about 320 mg / L, from about 40 mg / L to about 300 mg / L, from about 40 mg / L to about 280 mg / L, from about 40 mg / L to about 260 mg / L, from about 40 mg / L to about 240 mg / L, from about 40 mg / L to about 220 mg / L, from about 40 mg / L to about 200 mg / L, from about 40 mg / L to about 180 mg / L, from about 40 mg / L to about 160 mg / L, from about 40 mg / L to about 140 mg / L, from about 40 mg / L to about 120 mg / L, from about 40 mg / L to about 100 mg / L, from about 40 mg / L to about 90 mg / L, from about 40 mg / L to about 40 mg / L, from about 40 mg / L to about 70 mg / L, from about 40 mg / L to about 60 mg / L, or from about 40 mg / L to about 50 mg / L. In one exemplary form, the

[0099] 14

[0100] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT sodium bicarbonate may be present in the concentrated formulation at a concentration of about 400 mg / L.

[0101] In certain forms, the solvent used in the concentrated formulation is selected from methanol, ethanol, 1 -propanol, 2-propanol, 1 -butanol, acetone, ethyl acetate, glycerin, or hexane. In these cases, the solvent is incorporated at a concentration that is non-toxic to insect pollinators. This means the concentration is low enough to avoid harmful effects on the health, behavior, or survival of the insect pollinators, such as honeybees, while still effectively dissolving the flavonoid. The selection of a suitable solvent concentration may involve consideration of factors such as the solvent's toxicity profile, bioaccumulation potential, i.e., the ability of the solvent to build up, or accumulate, in the tissues of the insect pollinators over time.

[0102] C. Diluted Compositions for Administration to Insect Pollinators

[0103] The disclosed concentrated formulation can be diluted to form diluted compositions such as animal feed or feed supplements for administration to insect pollinators.

[0104] In some forms, when the solvent used is aqueous sodium bicarbonate, the amount of sodium bicarbonate delivered to each bee should not exceed about 24 pg per bee. For instance, if a bee consumes 60 pL of the solution per day, the molar concentration of sodium bicarbonate in the solution should be limited to no more than between about 4.60 mM to about 4.80 mM, such as about 4.76 mM.

[0105] In some forms, the dilute compositions can contain a flavonoids in the concentration range of about 0. 1 pM to about 100 mM, about 0.1 pM to about 10 mM , or about 0.1 pM to about 10 mM, or about 1 pM to about 10 mM, or 0.5 pM to about 5 mM, or about 0.1 pM to about 100 pM, or about 0.1 pM to about 10 pM, or about 1 pM to about 1 mM, or about 1 pM to about 100 pM, or any subrange between any of the foregoing, such as about 1 pM.

[0106] The diluted compositions typically have a low viscosity at mean hive temperature (i.e., about 30°C). The viscosity of the dilute compositions can range from about 1x10'3Pa-s to about 0.01 Pa-s, such as about 3.27xl0’3Pa-s. Viscosity can be determined based on Hidayanto, et al. “Measurement of viscosity and sucrose concentration in aqueous solution using portable brix meter”, Berkala Fisika, vol. 13, issue 2, pages A23-A28, 2010.

[0107] An animal feed or feed supplement for use in the methods described herein may include the composition containing one or more flavonoids and the solubility enhancing solvent, and one or more nutrients or ingredients such as one or more proteins, lipids, carbohydrates, minerals, water, or combinations thereof. The one or more nutrients and / or ingredients such as sucrose can facilitate the consumption of the diluted composition and increase the caloric value of the diluted composition for the insect pollinators.

[0108] 15

[0109] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT

[0110] Protein and lipid sources can be corn gluten sources in combination with soy concentrate, barley flour, yeast, and / or com distillers’ dry grains, soy sources and egg or egg product sources. The egg source is also a good source of cholesterol. Soy contributes one of the most complete profiles of essential amino acids of any plant material, and it is also a source of lipoproteins, which help deliver sterols and polyunsaturated fatty acids. It is a naturally lipid-rich food material. Examples of soy sources include soy flour (e.g., expeller pressed, solvent extracted), soy meal, soy milk, and suspended soy extract. Eggs, especially egg yolks, are sources of extremely high amounts of proteins that are a standard of nutritional completeness. Eggs are also a rich source of lipids, including cholesterol which, as a sterol, is an essential nutrient for honeybees. The lipids in egg yolk are also rich in polar components such as lecithin, which is highly digestible, nutritious, and a natural emulsifier. Eggs also contribute to texture by increasing viscosity. Eggs arc a complete source of B vitamins, vitamin A (in a complex of carotenoids), and vitamin E, and are also a fairly complete source of minerals. Also, vegetable oils can be used as a lipid source. Examples include soy oil, safflower oil, corn oil, peanut oil, sunflower oil, canola oil, rapeseed oil, cottonseed oil, and flax oil.

[0111] The carbohydrate (e.g., sugar) source in the formulations can serve as a feeding stimulant, a source of carbon for building blocks for growth, a source of energy, a viscosity increasing agent (texturizer), and a humectant (water retaining agent), which lowers water activity that reduces microbial growth and inhibits chemical reactions that help deteriorate diets. Examples of sugar sources include sucrose, e.g., crystalline or granulated sugar; other crystalline or granulated sugars, e.g., fructose, glucose (also denoted as dextrose) or maltose; high fructose corn syrup, e.g., HFCS55, or other sugar syrup. They can be used in the solid form or as a syrup.

[0112] Anti-fungal and / or anti-microbial agents are optionally added to the formulations to prevent premature deterioration of the formulations. These are desirable because at typical hive temperatures, microbes can proliferate rapidly and spoil the feeds and serve as potential pathogens to the bees. Exemplary antimicrobial agents are sorbic acid and its salts, propionic acid and its salts, the series of parabens (methyl, ethyl, propyl, and butyl form), and benzoic acid and its salts. Potassium sorbate, a fatty acid, has well-demonstrated anti-fungal and antimicrobial properties. Sodium propionate is an effective anti-microbial agent and approved preservative. Other anti-fungal and / or antimicrobial agents are known in the art, including for example calcium propionate.

[0113] The compositions can be formulated in any form suitable for administration (e.g., ingestion) to the insect pollinator. In some forms, the compositions can be in the form of a liquid (e.g., sugar syrup, high-fructose com syrup). A liquid formulation can be a solution, suspension 16

[0114] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT or emulsion. In some forms, liquid formulations provide an evenly mixed, water-dispersible, substantially homogeneous, substantially non-clumping, pourable, or flowable liquid wherein nutrients are dissolved, suspended, and / or emulsified therein.

[0115] In some embodiments, the compositions are formulated as a solid (e.g., a powder, patty, candy). The dry formulations may be directly administered to insect pollinators such as bees without liquid being added. Tn some forms, the compositions is formulated in dry form, which may subsequently be mixed with water or a sugar syrup prior to use to form a liquid. A patty or a semi -dry formulation may be formed by using less syrup or a solid carbohydrate source and bringing the product to a dough-like or powdery consistency, e.g., that can be placed in or near a colony for feeding. For example, an effective amount of the concentrated formulation can be processed into a solid formulation, such as a dietary supplement, suitable to deliver about 12 ng of the one or more flavonoids per insect pollinator e.g., honeybee.

[0116] Formulations of the various compositions can be pH balanced, e.g., to mimic the pH of natural pollen and promote a healthy gut environment in the pollinator. Accordingly, the formulation can include one or more organic acids or phosphoric acid as acidifier sources to achieve the desired pH (e.g., in the range of 6.5 to 9.0), such as between about pH 7.0 to about 7.5. Exemplary acidifier compounds are organic acids such as citric acid, acetic acid, lactic acid, malic acid, fumaric acid, or succinic acid and combinations of organic acids such as malic acid, fumaric acid, and pyruvic acid.

[0117] In some forms, the particle size of the composition that is applied at the pollinator’s site is controlled to mimic the particle size of natural pollen. For example, the particle size of the formulation is about 35 microns or smaller. Optionally, anti-microbial agents may be included to prevent pre-mature deterioration of the formulations.

[0118] D. Kits

[0119] Kits containing the disclosed concentrated formulations are also disclosed. The kit can include a separate container containing a suitable carrier, diluent or excipient. Additionally, the kit can include instructions for mixing or combining ingredients and / or administration.

[0120] An exemplary kit includes the concentrated formulation of one or more flavonoids and a solvent. For example, the kit can include a concentrated formulation of rutin and sodium bicarbonate.

[0121] Optionally, the kit can include one, two, three, four, or more dosages of the concentrated formulation per hive. It can also contain other materials useful to the end-user, including other

[0122] 17

[0123] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT pharmaceutically acceptable formulating solutions such as buffers, diluents, or other delivery device(s).

[0124] The kit can also include a package insert containing written instructions for methods of diluting the concentrated formulation and / or administration to hives of insect pollinators.

[0125] IL Methods of use

[0126] Methods of using the disclosed concentrated formulations are provided. The concentrated formulations can be diluted to form diluted compositions suitable for large scale administration to insect pollinators.

[0127] A. Methods of Preparing Dilute Compositions

[0128] The concentrated formulation containing one or more flavonoids and a solubility enhancing solvent can be diluted to form dilute compositions for administration to apiaries using any suitable method. In some forms, an amount of the concentrated formulation can be diluted in an aqueous sucrose solution prior to use for administration to bee hives.

[0129] Optionally, the concentrated formulation is diluted to form a dilute composition having about a 1 pM of the one or more flavonoids. Optionally, the diluent is or comprises an aqueous sucrose solution, such as a IM sucrose solution.

[0130] In one exemplary form, to achieve a final ratin concentration of 1 p M, 5 mL of the concentrated solution, which contains 400 mg / L of sodium bicarbonate and 250 mg / L of rutin, can be diluted with sucrose- water to a total volume of 2 L. The solution can be mixed thoroughly to facilitate homogeneity. For applications to a bee colony housing about 40,000 individuals, it is estimated that each bee ingests about 50 pl. of the diluted solution, providing a guideline for the volume needed to meet dietary or experimental requirements. These exemplary amounts can be extrapolated to prepare dilute compositions for administration to multiple bee colonies.

[0131] Flavonoids, e.g., rutin, are sensitive to ultraviolet radiation, and should be packaged properly to maintain the formulation's stability and efficacy. Optionally, the dilute compositions can be stored in amber containers to block UV light and protect the flavonoid compounds. The containers can optionally be made of UV-resistant, chemically inert materials, such as amber glass or plastic, and may be airtight to prevent contamination and degradation caused by environmental factors.

[0132] The stability of the concentrated and diluted formulations can also be influenced by storage conditions. In some forms, the concentrated formulations and compositions thereof can be refrigerated at about 2°C to about 8°C or at room temperature (i.e., from about 20°C to about 25 °C), depending on the available storage facilities and the anticipated duration of storage. In

[0133] 18

[0134] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT some forms, containers can optionally be kept in a dark or low-light environment to further mitigate UV exposure.

[0135] The diluted compositions provided herein can be used as a food, feed additive or supplement, or nutraceutical. Such foods, feed additives or supplements, or nutraceuticals are useful for prophylactically protecting insect pollinators against the negative effects of neurotoxic pesticides (e.g., by administration before exposure to a pesticide). Tn some forms, the disclosed compositions including foods, feed additives, feed supplements, or nutraceuticals are useful for treating insect pollinators against the negative effects of neurotoxic pesticides (e.g., by administration during and / or after exposure to a pesticide).

[0136] B. Methods For Protecting Cognitive Function of Insect Pollinators

[0137] Methods of protecting the cognitive function of an insect pollinator by administering an effective amount of any of the disclosed compositions to an insect pollinator in need thereof arc provided. For example, in some forms, the methods include protecting the learning and / or memory capabilities of an insect pollinator by administering an effective amount any of the disclosed compositions to the insect pollinator.

[0138] Methods for preventing or reducing impairment of cognitive function in an insect pollinator by administering to the insect pollinator a diluted composition (e.g., a food, feed additive or supplement, or nutraceutical) containing an effective amount of one or more flavonoids to prevent or reduce impairment of a cognitive function in the insect pollinator are also provided. Preferably, the cognitive function includes but is not limited to, learning, memory, attention, decision-making and other forms of information acquisition, storage, processing, retrieval and use, navigation, motor activity, sucrose sensitivity, and combinations thereof.

[0139] Thus, in some forms, the methods reduce, diminish, or prevent a reduction or loss in memory, learning, navigation skills, motor activity, or combination thereof in an insect pollinator upon exposure to a pesticide, by administering diluted compositions formed from the concentrated formulations.

[0140] Methods of controlling insect pests are also provided. In some forms, a method of controlling insect pests involves treating an area with a pesticide and administering to the insect pollinator a composition including an effective amount of the diluted compositions to prevent or reduce impairment of a cognitive function and / or increase a cognitive function in the insect pollinator. The area can be a geographical space (e.g., a field) or part of an organism (e.g., a plant). In some forms, the area is occupied by insect pests and insect pollinators. The area can be treated with the pesticide at any suitable time, e.g., before or after the diluted composition is administered to the insect pollinators. In some forms, the area is treated with the pesticide by

[0141] 19

[0142] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT foliar application, soil injection, tree injection, ground application as a granular or liquid formulation, or as a pesticide-coated seed treatment.

[0143] The area can be treated with an effective amount of pesticide to reduce the number of insect pests in the area. For example, the pesticide can be lethal to insect pests and / or in an amount that is sublethal to insect pollinators. Exemplary insect pests that can be targeted include aphids, thrips, whiteflies, mites, leafhoppers, mealybugs, spittlebugs, fleas, termites, scales (e.g., armored scales, soft scales), and beetles.

[0144] Typically, the disclosed methods prevent, reduce, decrease, or inhibit one or more adverse effects associated with pesticide exposure. For example, in some forms, the disclosed methods reduce, decrease, or inhibit one or more mechanisms of mitochondrial dysfunction (e.g., reduced electron transport, reduced ATP production, altered mitochondrial membrane potential), e.g., in the brain of an insect pollinator by administering any of the disclosed compositions. In some forms, the disclosed methods induce or enhance expression and / or activity of one or more components of antioxidant and overall detox pathways (e.g., Nrf2 pathway, AP-1 pathway, CYP450 proteins) in the brain or other organs of the insect pollinator by administering any of the disclosed compositions to the insect pollinator.

[0145] In some forms, the methods enhance cognitive function. For example, methods for enhancing cognitive function (e.g., learning, memory, speed and accuracy of decisions) in an insect pollinator by administering to the pollinator a disclosed composition containing an effective amount of one or more phenolic compounds are provided.

[0146] C. Methods For Protecting Insect Pollinators from Impairment of the Immune System and / or Parasites

[0147] Honey bees have an immune system that only includes innate immunity. In these bees, cellular responses such as phagocytosis are mediated by hemocytes (analogous to white blood cells in humans). Humoral responses include unique processes in invertebrates, such as the encapsulation response (ER), which is an important immune process for bees. In this response, reactive oxygen species (ROS) are produced, and cells are recruited to neutralize different pathogens (such as parasites and fungi) by forming a capsule around them. To achieve this, bee hemocytes use different soluble molecules to catalyze melanin biosynthesis. Melanin acts both as a substrate for ROS production and a recruitment signal for hemocytes (Wilson et al. 2019). Also, in honey bees, ER correlates positively with resistance to bacterial and viral infections (Mao et al. 2013;).

[0148] The methods described herein can protect insect pollinators from impairment of the immune system. The methods can prevent the suppression of the immune system caused by pesticides

[0149] 20

[0150] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT e.g., imidacloprid as well as improve overall immune responses. Cor instance, the methods can improve both cellular and humoral immune functions, such as the encapsulation response, which is important for bees to fight off infections. The disclosed dilute compositions and methods can therefore mitigate the adverse effects of pesticide exposure on honey bees, thereby supporting their health. This in turn can support the broader ecosystem bees sustain through pollination.

[0151] For example, a method of protecting an insect pollinator of impairment of the immune system from a pesticide and / or parasite, comprises:

[0152] (i) applying or placing a dilute composition in a hive located in an area that is occupied by one or more insect pollinators, wherein the dilute composition comprises a dilute form of the concentrated formulation of any one of claims 1 to 15. Optionally, the method further includes prior to step (i) diluting the concentrated formulation to form the dilute composition, wherein the dilute composition comprises about 1 p M of the one or more flavonoids, optionally wherein the diluent is or comprises an aqueous sucrose solution, such as a IM sucrose solution.

[0153] Inside the hives, pathogens and parasites, such as Varroa sp., lead to weakening the health of bees, such as nurse bees, causing diseases (Ramsey et al., 2019). For example, Varroa mites contribute to poor nutrition in the brood by altering the protein levels of nurse bees, which impairs the function of their hypopharyngeal glands (hereafter HPG’s) and affects the quality of their food secretions (van Dooremalen et al., 2013). The low nutritional content for the brood, combined with the effects of Varroa mites and pesticide exposure, impacts worker bee production, survivability, and size resulting in lower body mass (Figure 1). The methods described herein can increase the resistance of insect pollinators e.g., honeybees to the effects of one or more parasites, such as for example, Varroa sp., Nosema sp., and / or Deformed wing virus.

[0154] For example, a method of increasing the resistance of an insect pollinator the effects of one or more parasites, comprises:

[0155] (i) applying or placing a dilute composition in a hive located in an area that is occupied by one or more insect pollinators, wherein the dilute composition comprises a dilute form of the concentrated formulation described above. Optionally, the method further includes prior to step (i) diluting the concentrated formulation to form the dilute composition, wherein the dilute composition comprises about 1 pM of the one or more flavonoids, optionally wherein the diluent is or comprises an aqueous sucrose solution, such as a IM sucrose solution.

[0156] 21

[0157] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT

[0158] D. Methods of Administration of Dilute Compositions

[0159] The dilute compositions can be administered to insect pollinators by a variety of suitable means. For example, the compositions and formulations can be ingested by the recipient insect pollinator.

[0160] Typically, the methods provide for administration of the disclosed compositions and formulations via ingestion through the normal feeds and feeding schedule of the insect pollinator. The provided compositions and formulations can be provided as food, feed additive or supplement, or nutraceutical. The compositions and formulations can be placed in an area where bees are located or within feeding vicinity of bees, such as in or adjacent to a bee hive or bee cage, or also inside the hive as a patty or as a liquid. Optionally, the feeder is made of amber colored material or otherwise darkened to protect the one or more flavonoids in the compositions from direct sun light. By “patty” is meant a mixture of sugar syrup and a bee diet formulation to form soft pliable dough-like consistency that is pressed into a thin patty. Patties are typically provided to honey bee colonies to support the protein and nutritional need of the colony.

[0161] The dilute compositions can be placed in a feeder suitable for the insect pollinator e.g., honeybees or bumble bees. The feeder can be an internal feeder or an external feeder. Internal feeders are placed inside the hive or colony, providing convenient access to the insect pollinator while minimizing exposure to external factors. External feeders, on the other hand, are positioned outside the hive, allowing for easier refilling and monitoring. The type of feeder often depends on the species and needs of the colony; for instance, feeders designed for bumble bee colonies differ from those used for honeybee hives. Exemplary feeders include deep division board feeders, frame feeders, entrance feeders, top feeders, gravity feeders, bucket or pail feeders, or open feeders.

[0162] A deep division board feeder is an internal feeder that fits inside a hive like a standard frame. It is configured to hold the dilute composition in a compartment, and includes a ladder or textured surface to help bees access the feed safely without drowning. Its enclosed design minimizes robbing, reduces evaporation, and provides easy access for bees within the hive, making it suitable for supplemental feeding during nectar shortages or colder months. In addition to deep division board feeders, there are several other types of feeders commonly used for bees. Frame feeders, for example, fit inside the hive like a standard frame and provide an enclosed reservoir for liquid feed, reducing the risk of robbing. Entrance feeders are external devices that attach to the hive entrance, making them easy to refill but potentially increasing the risk of attracting pests. Top feeders sit on top of the hive, usually under the cover, and are designed to hold larger quantities of feed, ideal for times when extended feeding is required. Gravity feeders, 22

[0163] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT similar to poultry feeders, allow syrup to flow into a feeding trough as bees consume it, ensuring a steady supply. Bucket feeders or pail feeders involve an inverted container with small holes in the lid that bees access; they are often placed directly on the hive cover. Open feeders are communal feeding stations set up away from the hives, useful for feeding multiple colonies at once but requiring careful management to prevent robbing or competition with other insects.

[0164] By way of example, mechanisms for providing the disclosed compositions and formulations to bees are summarized below. However, these are not intended to be limiting. One of skill in the art can readily determine appropriate methods for administration of the composition to any insect pollinator of interest. Appropriates amounts and timing of feeding for various organisms are known to those skilled in the art and are readily ascertainable.

[0165] The compositions and formulations may be fed to bees or a colony of bees in a variety of ways. For example, the compositions may be formulated as a liquid and fed within a hive in a horizontal feeder in place of a comb. Alternatively, or in addition, the composition may be placed in a vertical feeder, which is in turn placed on top of a comb within the hive.

[0166] The compositions can be formulated as a liquid, a patty, or a biscuit. In certain forms, the composition is provided adjacent to a comb in the hive, e.g., on top of the comb. The compositions can be provided on a mesh through which the bees can pass. In certain forms, the compositions formulated as a liquid is provided in an inverted jar inserted into a hole in the roof of a hive. In certain forms, the compositions are provided in an area surrounding a hive (e.g., within an apiary). Thus, the bees can eat the composition as part of their normal foraging. In such forms, the composition can be formulated as a liquid or a powder.

[0167] In some forms, the composition are for example provided via a frame feeder, or is poured or sprayed.

[0168] In any of the foregoing, the compositions and formulations can be provided to the insect pollinator before or after exposure to one or more pesticides. This can be over any period of time, for example, minutes, hours, days, or weeks. In some forms, the compositions and formulations are provided to the insect pollinator one or more times between about 1 hour and about 10 days after pesticide exposure.

[0169] Additionally, or alternatively, in preferred forms, the compositions and formulations can be provided to the insect pollinator before exposure to one or more pesticides. For example, the compositions and formulations can be provided in the range of between about 1 hour and about 10 days before exposure to one or more pesticides.

[0170] Typically, the compositions and formulations are provided 1-3 days (e.g., 1, 2, or 3 days) before exposure to one or more pesticides. For example, migratory bee keepers who use their

[0171] 23

[0172] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT hives to pollinate almond, apple, plum and other orchards can provide the compositions and formulations to their colonies before (e.g., 1, 2, or 3 days) releasing them to the open for foraging.

[0173] When administration is via ingestion, the compositions and formulations are typically provided to the insect pollinators ad lib, i.e., the pollinator feeds freely as desired. For example, the compositions containing an effective amount of the compositions placed in a bag or jar in, on, or near the hive. The bees can access and ingest the composition as desired over the period of time before exposure to a pesticide. For example, the bees can be fed with the composition ad lib over a period of 1-10 days before exposure to one or more pesticides.

[0174] The pollinator may ingest an effective amount of the compositions on a single, repeated, or regular basis. For example, the pollinator may ingest an effective amount of the compositions one, two, three, or more times weekly, every other day, every day, or more than once every day (e.g., once, twice, three, or more times every day) during the performance of the disclosed methods or uses. In some forms, diluted compositions of the concentrated formulation may be administered to the honeybees once weekly. In some forms, the insect pollinator may ingest an effective amount of the dilute compositions once per week. In some forms, the compositions are included in a feed, a feed supplement, and / or in drinking water and the pollinator ingests the compositions when they eat and / or drink, and optionally every time they eat and / or drink. This ingestion of an effective amount of the compositions may continue through a period of time of the animal’s lifespan that may correspond to a period of time that is, is up to, or is at least, 5%, 10%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or substantially 100% of the life of the animal from birth to death. The ingestion of an effective amount of the compositions may start on the day of the animal’s birth, or at the age of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 days, or more. After the pollinator starts to ingest or absorb the one or compounds, it may continue to do so on a regular and repeated basis for a period of time that can be, or be up to. or at least, 1. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 days, or more. Ingestion can also start while in larval stages, where adults can provide a brood with a single (mass provisioning species) or multiple (sequential provisioning) loads of supplemented food.

[0175] 1. Effective amounts

[0176] Because the administration of the compositions elicits a beneficial effect on one or more cognitive functions of the insect pollinator, the amount of the composition administered can be 24

[0177] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT expressed as the amount effective to achieve a desired effect in the recipient pollinator. Tor example, in some forms, the amount of the compositions is effective to enhance cognitive function (e.g., learning, memory, navigation) in an insect pollinator.

[0178] In some forms, the amount of the compositions is effective to protect insect pollinators against the harmful cognitive effects of pesticides (e.g., neurotoxic pesticides). The amount of the one or more flavonoids in the compositions can be effective to prevent reduction in memory, learning, navigation skills, motor activity, or combinations thereof in an insect pollinator upon exposure to a pesticide. The amount of one or more flavonoids in the compositions can be effective to reduce or diminish loss of memory, learning, navigation skills, motor activity, or combinations thereof in the insect pollinator upon exposure to a pesticide.

[0179] The amount of a composition administered to an insect pollinator is typically enough to prevent, reduce, decrease, or inhibit one or more adverse effects associated with pesticide exposure. For example, in some forms, the amount of one or more flavonoids in the compositions is effective to prevent, reduce, decrease, or inhibit one or more mechanisms of mitochondrial dysfunction (e.g., reduced electron transport, reduced ATP production, altered mitochondrial membrane potential). In other forms, the amount of one or more flavonoids in the compositions is effective to induce or enhance expression and / or activity of one or more components of antioxidant pathways (e.g., Nrf2 pathway, AP-1 pathway) in the brain or other organs of the insect pollinator.

[0180] Effective amounts can be expressed as total mass (e.g., mg), an amount per unit body weight of the recipient (e.g., mg / kg), as body surface-area based dosing e.g., mg / m2) and the like. In some forms, the disclosed compositions can contain a flavonoids in the concentration range of about 0. 1 pM to about 100 mM, about 0.1 M to about 10 mM , or about 0.1 M to about 10 mM, or about 1 pM to about 10 mM, or 0.5 pM to about 5 mM, or about 0. 1 pM to about 100 pM, or about 0.1 pM to about 10 pM, or about 1 pM to about 1 mM, or about 1 pM to about 100 pM, or any subrange between any of the foregoing. In some forms, the dosage is a specific dose between the foregoing ranges and subranges, inclusive of the end points. For example, in the experiments below, honeybees received 1 mM Rutin and 6 mM Quercetin. Bumblebees received IpM Rutin.

[0181] When the solvent used is aqueous sodium bicarbonate, the amount of sodium bicarbonate delivered to each bee should not exceed about 24 pg per bee. For instance, if a bee consumes 60 pL of the solution per day, the molar concentration of sodium bicarbonate in the solution should be limited to no more than between about 4.60 mM to about 4.80 mM, such as about 4.76 mM. The actual effective amounts of an active agent (e.g., flavonoids) can vary according to factors 25

[0182] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT including the specific flavonoid, the particular composition formulated, the mode of administration, and the weight or condition of the subject (e.g., insect pollinator) being administered, as well as the biological parameter (e.g., learning, memory, navigation) being evaluated or impacted.

[0183] Flavonoids exhibit a hormetic effect (i.e. low concentrations can often be better) and high concentrations may be toxic. Thus, typically a lower, but still effective amount, is preferred over of a higher amount. Preferably, the amount is nontoxic or at least sublethal for pollinators, particular honeybees and / or bumblebees.

[0184] 2. Insect pollinators

[0185] The disclosed compositions can be used to protect the cognitive function of a pollinator (e.g., an insect pollinator). For example, the compositions can be used for preventing or reducing impairment of cognitive function (e.g., learning, memory, navigation) in an insect pollinator. Pollination is the process by which a pollen grain moves from the anther (male part) of a flower to the stigma (female part). This is the first step in a process that produces seeds, fruits, and the next generation of plants. This can happen through self-pollination, wind and water pollination, or through the work of vectors (e.g., animals) that move pollen within the flower. Notably, it is estimated that between 75% and 95% of all flowering plants on the earth need help with pollination, i.e., they need pollinators (Oilerton J„ et al., Oikos, 120:321-326. (2011)).

[0186] Many crops benefit from pollination by animals. Birds, bats, butterflies, moths, flies, beetles, wasps, small mammals, and bees are known pollinators. They visit flowers to drink nectar or feed off of pollen and transport pollen grains as they move from spot to spot. Crops that benefit from such pollinators include almond, apple, avocado, blueberry, canola, cantaloupe, cherry, blueberry, cranberry, cucumber, kiwifruit, nectarine, peach, pear, pepper, plum, prune, raspberry, squash (including pumpkin and gourd), strawberry, sunflower, and tomato; and also crops for seed production, such as alfalfa, asparagus, beet, cabbage and other crucifers, carrot, clover and onion.

[0187] In preferred forms, the pollinator is a managed species such as bees (for example bumblebees, honeybees, squash bees, mason bees, stingless bees, carpenter bees, etc.) but other wild bees (orchid bees, sweat bees). Additionally, or alternatively, the pollinator includes or consists of one or more other insect pollinators (e.g. butterflies, moths, flies, beetles, wasps). i. Honeybees and Bumblebees

[0188] In some forms, the insect pollinator is a member of the Apidae (which includes honeybees and bumblebees), Halictidae, Andrenidae, Megachilidae, or Colletidae families. In preferred forms, the insect pollinator is a member of the Apis genus within the Apidae family,

[0189] 26

[0190] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT such as but not limited to, A. mellifera, A. mellifera liguistica, A. mellifera camica, A. mellifera caucasica, A. mellifera caucasica, A. mellifera iberiensis, A. mellifera scutellata, A. cerana, and A. dorsata.

[0191] In some forms, the insect pollinator is a bumblebee such as, but not limited to, buff-tailed bumblebee, cuckoo bumblebee, tree bumblebee, garden bumblebee, heath bumblebee, ruderal bumblebee, broken-belted bumblebee, white-tailed bumblebee, red-tailed bumblebee, early bumblebee, red-shanked bumblebee, and bilberry bumblebee. In some forms, the insect pollinator is a member of the Bombus genus within the Apidae family, such as but not limited to, Bombus terrestris, Bombus impatiens, Bombus occidentalis, Bombus atratus, Bombus vestalis, Bombus lucorum, Bombus bohemicus, Bombus hypnorum, Bombus sylvestris, Bombus hortorum, Bombus barbutellus, Bombus jonellus, Bombus ruderatus, Bombus soroeensis, Bombus lapidarius, Bombus rupestris, Bombus pratorum, Bombus ruderarius, Bombus monticola, Bombus pascuorum, Bombus campestris, Bombus humilis, Bombus muscorum, Bombus distinguendus, and Bombus sylvarum.

[0192] The disclosed compositions and methods can be used to limit the harmful effects of neurotoxic pesticides on any of the foregoing pollinators.

[0193] 3. Pesticides

[0194] Typically, impairment of a cognitive function is induced by exposure to a pesticide, for example, a neurotoxic pesticide. Accordingly, the disclosed compositions may be useful to protect against pesticides that are neurotoxic. The disclosed compositions may be useful to protect against pesticides that can adversely affect cholinergic, GABAergic or glutamatergic neurotransmission. In some forms, the disclosed compositions may be useful to protect against pesticides that are toxic to mushroom bodies (main centers of learning and memory and integration in the insect brain) and the antennal lobes (major centers for primary integration of olfactory information). In some forms, the disclosed compositions are useful to protect against pesticides that negatively affect mitochondrial function (e.g., electron transport, ATP production, mitochondrial membrane potential), increase apoptosis, increase oxidative stress, or combinations thereof in the brain, for example, the mushroom bodies of the insect pollinator.

[0195] Insecticides play an important role in the control of insect pests. Most of the chemical insecticides in use today are neurotoxic pesticides, and act by poisoning the nervous systems of the target organisms. The target sites for insecticides in insects are also found in mammals, hence insecticides vary in their levels of selectivity with regard to targets of toxicity, and mammals, including humans, may be sensitive to their toxicity. Insecticides have higher acute toxicity toward non-target species compared to other pesticides. There are several classes of

[0196] 27

[0197] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT insecticides that affect the pre- or post- synaptic terminals (e.g. pyrethroids, neonicotinoids, sulfomine derivatives, fipronil) as well as the dynamics occurring in the synaptic cleft (e.g. organophosphates and carbamates). a. Neonicotinoids

[0198] In preferred forms, impairment of a cognitive function is induced by exposure to one or more pesticides that target (e.g., agonists of) the nicotinic acetylcholine receptor (nAChR). Tn preferred forms, impairment of a cognitive function is induced by exposure to one or more neonicotinoids. In preferred forms, methods of controlling insect pests involve treating an area with a pesticide, such as one or more neonicotinoids.

[0199] The introduction to the market in the early 1990s of imidacloprid and thiacloprid opened the neonicotinoid era of insect pest control. Neonicotinoids are now the most widely used pesticides in the world with a global market share of at least 26% of the insecticide market. Two of them — clothianidin and thiamethoxam — dominate the global market for insecticidal seed treatments and are used to coat the seeds of most of the annual crops planted around the world. In fact, more than 94% of the com and more than 30% of the soy planted in the United States is pretreated with neonicotinoids. Acting systemically, this class of neurotoxic insecticides is taken up by plants, primarily through the roots, and translocates to all parts of the plant through xylemic and phloemic transport. Therefore, unlike older pesticides that evaporate or disperse shortly after application, neonicotinoid insecticides incorporate themselves into plant tissues, turning the plant itself into a tiny poison factory emitting toxin from its roots, leaves, stems, pollen, and nectar.

[0200] As the name suggests, neonicotinoids are similar in structure to nicotine and interact with the nicotinic acetylcholine receptors (nAChRs) of the insect central nervous system. Their capacity to cross the ion-impermeable barrier surrounding the central nervous system (BBB, blood-brain barrier) and their strong binding to nAChR in the bee's central nervous system are responsible for a unique chronic and sublethal toxicity profile.

[0201] Neonicotinoids mainly act agonistically on nAChRs on the post-synaptic membrane, mimicking the natural neurotransmitter acetylcholine by binding with high affinity (Van der Sluijs, JP., et al., Current Opinion in Environmental Sustainability, 5(3-4), 293-305. 2013). This induces a neuronal hyper-excitation, which can lead to the insect’s death within minutes. Some of the major metabolites of neonicotinoids are equally neurotoxic, acting on the same receptors thereby prolonging the effectiveness as systemic insecticide. The nAChR binding sites in the vertebrate nervous system are different from those in insects, and in general they have lower numbers of nicotinic receptors with high affinity to neonicotinoids, which are the reasons that

[0202] 28

[0203] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT neonicotinoids show selective toxicity for insects over vertebrates (Van der Sluijs, IP., et al. 2013).

[0204] At their introduction, neonicotinoids were assumed to be more efficient than the organophosphates and carbamates that they replaced. As a seed treatment, they could be used in much lower quantities and they promised to be less polluting to the environment. It is however not the quantity that is relevant but the potency to cause harm, which results from toxicity, persistence and bioavailability to non-target species. Soon after the introduction of neonicotinoids, exposure to its residues in pollen, nectar, sowing dust etc., of non-target pollinating insects became clear.

[0205] Exemplary neonicotinoids include imidacloprid, thiacloprid, clothianidin, thiamethoxam, acetamiprid, nitenpyram, dinotefuran, and nithiazine. Many neonicotinoids are commercially available, for example as various insecticide formulations. Table 2 provides an exemplary list of neonicotinoids and their trade names.

[0206] Table 2. Exemplary list of commercial neonicotinoid containing products.

[0207] Pollinators’ exposure to neonicotinoids can occur through multiple pathways including ingestion, contact (e.g., contaminated nesting material, plants, soil, water), and inhalation (aerosols). At low concentrations of neonicotinoids, sublethal effects can occur. Sublethal effects involve modifications of honeybee behavior and physiology (e.g., immune system). They do not directly cause the death of the individual or the collapse of the colony but may become lethal in time and / or may make the colony more sensitive (e.g., more prone to diseases), which may contribute to its collapse. For instance, an individual with memory, orientation or physiological impairments might fail to return to its hive, dying

[0208] 29

[0209] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT from hunger or cold. It has been reported that sub-lethal effects of neonicotinoids exist on neurophysiology, larval development, moulting, adult longevity, immunology, fecundity, sex ratio, mobility, navigation and orientation, feeding behavior, oviposition behavior, and learning.

[0210] In some forms, there can be a synergism among stressors that negatively impact insect pollinators. A synergistic effect occurs when the effect of a combination of stressors is higher than the sum of the effect of each stressor alone. When neonicotinoids are combined with certain fungicides (e.g., azoles, such as prochloraz, or anilides, such as metalaxyl) or other agrochemicals that block cytochrome p450 detoxification enzymes, their toxicity increases by a factor from 1.52 to 1141 depending on the combination. Synergy has also been demonstrated for neonicotinoids and infectious agents. Prolonged exposure to a non-lethal dose of neonicotinoids renders beehives more susceptible to parasites such as Nosema ceranae. Therefore, in some forms, the disclosed compositions can be useful to protect against synergistic effects of pesticides and other pollinator stressors (e.g., parasites). b. Carbamates and organophosphates

[0211] Impairment of a cognitive function can be induced by exposure to a carbamate or organophosphate. Thus, the disclosed compositions can also be useful to protect against harmful cognitive effects of one or more carbamates or organophosphates. In some forms, the pesticide is a carbamate or organophosphate.

[0212] Organophosphates are phosphoric acid esters or thiophosphoric acid esters. When developed in the 1930s and 1940s, their original compounds were highly toxic to mammals. Organophosphates manufactured since then are less toxic to mammals but toxic to target organisms, such as insects. Malathion, dibrom, chlorpyrifos, temephos, diazinon and terbufos are exemplary organophosphates.

[0213] Carbamates are esters of N-methyl carbamic acid. Aldicarb, carbaryl, propoxur, oxamyl and terbucarb are exemplary carbamates. Carbamates are slruclurally and mechanistically similar to organophosphate insecticides but differ in action from the organophosphates in that the inhibitory effect on cholinesterase is generally brief.

[0214] Although carbamate and organophosphate pesticides differ chemically, they act similarly. When applied to crops or directly to the soil as systemic insecticides, organophosphates and carbamates generally persist from only a few hours to several months. Organophosphates and carbamates are potent inhibitors of acetylcholinesterase, thereby inhibiting the normal breakdown of Acetylcholine (ACh). ACh is a neurotransmitter, a chemical produced by a neuron that transmits signals from that neuron to another neuron, an exocrine gland, or a muscle. Importantly, whereas ACh prevails as a neurotransmitter at the neuromuscular junction in

[0215] 30

[0216] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT vertebrates with less prevalence in the central nervous system, it is the main neurotransmitter in the nervous system of invertebrates. ACh is released in the junction (synapse) between two cells (a neuron and a muscle or two neurons) where it binds to its receptor on the target cell (the postsynaptic terminal), inducing its activation and relaying the signal. Acetylcholinesterase (AChE) is an enzyme located in the intercellular space that is responsible for ACh degradation. Organophosphates and carbamates block the site where the neurotransmitter attaches to the Acetylcholinesterase leading to the buildup of ACh and continuous stimulation of the receptors on the target cells. In vertebrates, this results in parasympathetic and sympathetic overstimulation and eventual muscle paralysis. Overstimulation also underlies effects in invertebrates. c. Fipronil

[0217] In some forms, the pesticide is fipronil. Fipronil targets two main receptors in the insect brain. Fipronil can bind to GABA receptors or to Glutamate ionotropic receptors (chloride channels). GABA is a neurotransmitter that activates chloride channels, thus inducing a hyperpolarization (i.e. making more negative the neuron membrane) and depressing the neural activity. On the other hand, a population of glutamate ionotropic receptors in the insect brain can allow the movement of chloride, also leading to hyperpolarization. Thus, these networks act as modulatory of the activity during information processing. As fipronil targets GABAergic and glutamatergic receptors it acts, unlike other pesticides, on inhibitory, not excitatory, networks. Fipronil may affect the dopaminergic network and thus motor control and the value assigned to a reward. Fipronil effects include mitochondrial impairment, disrupting processing of information. d. Sulfoximine derivatives

[0218] In some forms, the pesticides is a derivative of sulfloxamines, such as Sulfoxaflor. Similar to neonicotinoids, Sulfoxaflor targets nicotinic cholinergic receptors (nAChR) being a competitor agonist of ACh. Major impairments at sublethal levels include motor control and reproduction.

[0219] B. Controls

[0220] The methods disclosed herein typically include comparing the level of a biological parameter in the subject pollinator to a control. Suitable controls will be known to one of skill in the art. Controls can include, for example, standards obtained from subjects not exposed to compositions containing one or more flavonoids. Controls can also include the level of a biological parameter at a specific reference point, for example, before administration of compositions containing one or more phenolic compounds. A control can be a single or pooled or averaged values assessed / assayed equivalent to the experimental subject. Reference indices 31

[0221] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT can be established by using pollinators that have been exposed to one or more pesticides and that exhibit one or more cognitive impairments with different known severities or prognoses.

[0222] C. Methods of Making

[0223] Methods for the production of the disclosed compositions including food, feed additive or supplements, and nutraceuticals are provided. Exemplary types of feed include feed for honeybees (e.g., sugar syrup, patties). The methods can include the steps of incorporating one or more of the flavonoids into the feed product or feed supplement product during the preparation of the feed or supplement. An animal feed or feed supplement for use in the methods described herein can include one or more flavonoids, proteins, lipids, carbohydrates, minerals, water, other nutrients or ingredients, or combinations thereof.

[0224] The one or more flavonoids can be incorporated into the feed product at any stage during the production process including before one or more heating steps or mixing steps.

[0225] The following processes can be used alone or in combination, as needed to provide the disclosed compositions and formulations: stirring, mixing, size reduction, and heating. Dry ingredients can be mixed and blended in a high-speed mixer / blender to achieve complete mixing and size reduction of the particles. The mixing is carried out sufficient to render the components into a well-dispersed form that is available in a substantially homogeneous manner. If desired, size reduction is carried out sufficient to render the components to be of a size and form so as to remain suspended in the final formulation and be of size acceptable to the mouthparts of an insect, e.g., a bee. In cases where the source ingredients are not greater than about 35 microns, size reduction may not be required. Heating can serve to increase the digestibility and absorption potential for components such as proteins and to destroy microbes, especially those in vegetative phases of their life cycle. Preferably heating is carried out sufficient to accomplish the foregoing but insufficient to cause excessive destruction or breakdown of the nutrients. Mixing and heating parameters for a particular set of circumstances can be readily determined by routine experimentation. In some forms, dry and / or solid formulations are mixed with water or liquid formulations to provide the final feed product.

[0226] It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0227] The disclosed concentrated formulations, diluted compositions made therefrom, and methods of use thereof, can be further understood by the following numbered paragraphs:

[0228] 32

[0229] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT

[0230] Paragraph 1. A concentrated formulation for forming a dilute composition suitable for administration to or ingestion by an insect pollinator, wherein the concentrated formulation comprises:

[0231] (i) one or more flavonoids, and

[0232] (ii) a solvent, wherein the one or more flavonoids are present in the concentrated formulation in an amount that, upon dilution of the concentrated formulation to form the dilute composition, is effective to protect against impairment of a cognitive function of the insect pollinator. Paragraph 2. The formulation of paragraph 1, wherein the one or more flavonoids is quercetin, rutin, myricetin, kaempferol, fisetin, or apigenin, or a combination thereof.

[0233] Paragraph 3. The formulation of paragraph 1, wherein the one or more flavonoids is rutin. Paragraph 4. The formulation of any of paragraphs 1-3, wherein the one or more flavonoids arc present in the concentrated formulation at a concentration ranging from about 30 mg / L to about 500 mg / L.

[0234] Paragraph 5. The formulation of any of paragraphs 1-4, wherein the solvent is selected from the group consisting of sodium bicarbonate, methanol, ethanol, 1 -propanol, 2-propanol, 1- butanol, acetone, ethyl acetate, glycerin, potassium bicarbonate, sodium carbonate, potassium carbonate, or a combination thereof.

[0235] Paragraph 6. The formulation of any of paragraphs 1-5, wherein the solvent is sodium bicarbonate.

[0236] Paragraph 7. The formulation of paragraph 6, wherein the sodium bicarbonate is present in the concentrated formulation at a concentration ranging from about 40 mg / L to about 500 mg / L. Paragraph 8. The formulation of any of paragraphs 1-7, wherein the concentrated formulation has a pH ranging from about 6.0 to about 9.0.

[0237] Paragraph 9. The formulation of any one of paragraphs 1-8, wherein the solvent comprises one or more pH modifiers.

[0238] Paragraph 10. The formulation of any one of paragraphs 1-9, wherein the solvent is not or does not contain DMSO.

[0239] Paragraph 11. The formulation of any one of paragraphs 1-10, further comprising one or more additional nutrients and / or ingredients selected from the group consisting of carbohydrates, proteins, lipids, vitamins, minerals, and water, or a combination thereof.

[0240] Paragraph 12. The formulation of paragraph 11, wherein the one or more additional nutrients and / or ingredients are selected from the group consisting of natural or artificial nectar, honey,

[0241] 33

[0242] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT sugar, sugar syrup, pollen or pollen substitute, soy flour, soy meal, gluten, skim milk, yeast, pollard, and oil, or combinations thereof.

[0243] Paragraph 13. The formulation of any one of paragraphs 1-11, wherein the flavonoid is rutin and the solvent is sodium bicarbonate.

[0244] Paragraph 14. The formulation of paragraph 13, wherein rutin is present in the formulation at a concentration ranging from about 30 mg / E to about 500 mg / L, and sodium bicarbonate is present in the formulation at a concentration ranging from about 40 mg / L to about 500 mg / L.

[0245] Paragraph 15. The formulation of paragraph 14, wherein rutin is present in the formulation at a concentration of 30 mg / L to 260 mg / L, and sodium bicarbonate is present in the formulation at a concentration ranging from about

[0246] 40 mg / L to about 420 mg / L.

[0247] Paragraph 16. The formulation of paragraph 14 or 15, wherein rutin is present in the formulation at a concentration of 62.5 mg / L, and sodium bicarbonate is present in the formulation at a concentration of 400 mg / L.

[0248] Paragraph 17. The formulation of paragraph 14 or 15, wherein rutin is present in the formulation at a concentration of 250 mg / L, and sodium bicarbonate is present in the formulation at a concentration of 400 mg / L.

[0249] Paragraph 18. The formulation of any one of paragraphs 1-17, wherein when the formulation is diluted to form a dilute composition comprising at least about 1 pM of the one or more flavonoids, the dilute composition is safe for use as a food, feed additive or supplement, or nutraceutical.

[0250] Paragraph 19. The formulation of any one of paragraphs 1-18, wherein the insect pollinator is a butterfly, moth, fly, beetle, wasp or bee.

[0251] Paragraph 20. The formulation of any one of paragraphs 1-18, wherein the insect pollinator is a bee, optionally wherein the bee is selected from honeybee, bumblebee, carpenter bee, leafcutter bee, blueberry bee, squash bee, mason bee, orchid bee, stingless bee, or sweat bee.

[0252] Paragraph 21. The formulation of any one of paragraphs 1-20, wherein the one or more flavonoids are more soluble in the solvent than in a control solvent consisting of 1 M aqueous sucrose solution.

[0253] Paragraph 22. A method of protecting the cognitive function of an insect pollinator from a pesticide, the method comprising:

[0254] 34

[0255] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT

[0256] (i) applying or placing a dilute composition in a hive located in an area that is occupied by one or more insect pollinators, wherein the dilute composition comprises a dilute form of a concentrated formulation comprising (i) a flavonoid and (ii) a solvent, wherein the flavonoid is rutin, wherein rutin is present in the concentrated formulation at a concentration ranging from 30 mg / L to about 500 mg / L, wherein the solvent is sodium bicarbonate, and wherein sodium bicarbonate is present in the concentrated formulation at a concentration ranging from about 40 mg / L to about 500 mg / L.

[0257] Paragraph 23. A method of protecting the cognitive function of an insect pollinator from a pesticide, the method comprising:

[0258] (i) applying or placing a dilute composition in a hive located in an area that is occupied by one or more insect pollinators, wherein the dilute composition comprises a dilute form of a concentrated formulation comprising (i) a flavonoid and (ii) a solvent, wherein the flavonoid is rutin, wherein rutin is present in the concentrated formulation at a concentration ranging from

[0259] 30 mg / L to 260 mg / L, and wherein the solvent is sodium bicarbonate, wherein sodium bicarbonate is present in the formulation at a concentration ranging from about 40 mg / L to about 420 mg / L.

[0260] Paragraph 24. A method of protecting the cognitive function of an insect pollinator from a pesticide, the method comprising:

[0261] (i) applying or placing a dilute composition in a hive located in an area that is occupied by one or more insect pollinators, wherein the dilute composition comprises a dilute form of a concentrated formulation comprising (i) a flavonoid and (ii) a solvent, wherein the flavonoid is rutin, wherein rutin is present in the concentrated formulation at a concentration of 62.5 mg / L, and wherein the solvent is sodium bicarbonate, wherein sodium bicarbonate is present in the formulation at a concentration of 400 mg / L.

[0262] 35

[0263] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT

[0264] Paragraph 25. A method of protecting the cognitive function of an insect pollinator from a pesticide, the method comprising:

[0265] (i) applying or placing a dilute composition in a hive located in an area that is occupied by one or more insect pollinators, wherein the dilute composition comprises a dilute fomr of a concentrated formulation comprising (i) one or more flavonoids and (ii) a solvent, wherein the one or more flavonoids are selected from the group consisting of quercetin, rutin, myricetin, kaempferol, fisetin, or apigenin, or a combination thereof, wherein the one or more flavonoids are present in the concentrated formulation at a concentration ranging from 30 mg / L to 500 mg / L, and wherein the solvent is selected from the group consisting of methanol, ethanol, 1- propanol, 2-propanol, 1 -butanol, acetone, ethyl acetate, glycerin, potassium bicarbonate, sodium carbonate, potassium carbonate, or a combination thereof, wherein the solvent is present in the concentrated formulation at a concentration ranging from 40 mg / L to 500 mg / L.

[0266] Paragraph 26. The method of any one of paragraphs 22-25, wherein the flavonoid(s) is more soluble in the solvent than in a control solvent consisting of 1 M aqueous sucrose solution. Paragraph 27. The method of any one of paragraphs 22-25, comprising prior to step (i) diluting the concentrated formulation to form the dilute composition, wherein the dilute composition comprises at least about 1 pM of the one or more flavonoids, optionally wherein the diluent is or comprises an aqueous sucrose solution, such as a IM sucrose solution.

[0267] Paragraph 28. The method of paragraph 27, wherein the dilute composition is incorporated in a solid food source prior to step (i).

[0268] Paragraph 29. The method of any one of paragraphs 22-25, wherein in step (i) the dilute composition is placed in the hive via a form of internal or external feeder such as a deep division board feeder.

[0269] Paragraph 30. The method of any one of paragraphs 22-25, wherein the dilute composition comprises an effective amount of the one or more flavonoids to prevent or reduce impairment of a cognitive function and / or increase a cognitive function in the insect pollinator following exposure to a pesticide, compared to an insect pollinator not administered the dilute composition comprising the one or more flavonoids.

[0270] Paragraph 31 . The method of paragraph 27, wherein the one or more flavonoids are present in the dilute composition in an amount from about 0.01 pM to about 100 mM.

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[0272] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT

[0273] Paragraph 32. The method of any one of paragraphs 22-25, wherein the cognitive function is selected from learning, memory, attention, decision accuracy, decision speed, navigation, motor activity, sucrose sensitivity, and combinations thereof.

[0274] Paragraph 33. The method of any one of paragraphs 22-25, wherein exposure to the pesticide induces cognitive impairment.

[0275] Paragraph 34. The method of any one of paragraphs 22-25, wherein the pesticide is neurotoxic. Paragraph 35. The method of any one of paragraphs 22-25, wherein the pesticide adversely affects GABAergic or glutamatergic neurotransmission, the mushroom bodies, the antennal lobes, the optic lobes or mitochondrial function; increases apoptosis; increases oxidative stress; or combinations thereof in the pollinator.

[0276] Paragraph 36. The method of any one of paragraphs 22-25, wherein the insect pollinator is a butterfly, moth, fly, beetle, wasp or bee.

[0277] Paragraph 37. The method of any one of paragraphs 22-25, wherein the insect pollinator is a bee, and wherein the bee is selected from honeybee, bumblebee, carpenter bee, leafcutter bee, blueberry bee, squash bee, mason bee, orchid bee, stingless bee, or sweat bee.The present invention will be further understood by reference to the following non-limiting examples.

[0278] Examples

[0279] Example 1: Innocuous Effects of the Supplementation with the Flavonol Rutin on HoneyBee Health Indicators

[0280] Materials and Methods

[0281] Study sites

[0282] The experiments were conducted at three study sites: Bogota (Study site 1), Viracacha (Study site 2), and Facatativa (Study site 3).

[0283] The Study site 1 (hereafter SI), located in Bogota (Universidad del Rosario: 4°39’ 17.154” N, 74°4’25.515”W; 2640 asl; Figure 1) had five Africanized honeybee hives. The apiary is situated in a high-density urban area where the main resource for the colonies conies from flowers within the university campus. Two hives were selected (Hive 1, Hive 2) with similar colony sizes (approximately 35,000 to 40,000 individuals), strength, pollen and honey reserves, and Varroa sp. levels. Each colony was maintained in a standard Langstroth hive consisting of a 10-frame deep super (i.e., the height of a basic Langstroth hive) and a 10-frame medium super (i.e., the height extensions as the colony grows). The location of this apiary within the campus allowed more detailed and extended analyses than with hives from the other study sites (see below Study design).

[0284] 37

[0285] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT

[0286] The Study site 2 (hereafter S2) was located in the rural area of Viracacha (Apicultura Los Cerezos: 5°26’48” N, 73°17’41.8”W. 2.520 asl; 155km north from Bogota; Figure 2) and had 90 Africanized honeybee hives with heterogeneous colony sizes and primarily used for intense pollen harvesting (estimated in five tons / year according to the owner). Ten hives (Hivel-10) were randomly selected, facilitating between 8 to 10 frames and the presence of the queen and all-stages brood. According to the beekeeper, these hives were healthy, with suspicion of low Varroa sp. presence. The colony resources were obtained from the native flora of the area and eucalyptus flowers.

[0287] Study site 3 was located in a rural area of Facatativa (Aromas & Sabores: 4°46’44.7” N, 74°20’36.9”W. 2.586 asl). When the study was planned, the apiary had more than 30 strong hives. However, before the onset of the experiments, the apiary experienced massive losses, presumably due to the pesticides used in a near blue cryngo (Eryngium planum) crop. Thus, the study was initiated with 10 Langstroth nucleus hives (with a population of around 5.000 individuals) but was reduced due to problems in meeting the criteria for data collection.

[0288] Study design

[0289] For SI, hive and individual health indicators (as described in Figure 2) were recorded for seven weeks. The data collected during week 0 was used as the baseline. Then, the colonies were randomly assigned to receive one of two treatments: i. supplementation with sucrose syrup (Control), ii. supplementation with rutin (Rut). The assigned supplement was provided for four weeks (weeks 1 -4) followed by two weeks where no supplement was provided to the site (weeks 5-6) to evaluate any potential changes occurring after the final administration. To balance the design and control for genetic variation, the treatments were reversed and repeated between colonies. The second phase of the experiments had a duration of six extra weeks to ensure a new cohort of worker bees within the colony.

[0290] For S2 and S3, the evaluation period spanned four weeks where the selected colonies were randomly assigned to the Control and Rut treatments. The treatments were administrated through all the evaluation periods following the feeding protocol (see below). For S2, only the Varroa sp. infestation levels (see below) were evaluated at the beginning (week 1) and the end (week 4) of the evaluation period. For S3, only the dry mass was assessed in this evaluation as an individual health parameter at the beginning and the end of the evaluation period.

[0291] Dosages and supplementation schedule

[0292] During weeks 1-4, hives from SI and S2 were fed with 2L of a IM sucrose solution (Control) using a deep division board feeder. Due to the reduced population of bees on S3, hives were fed with 1 L of the sucrose solution (Control). It is a common beekeeping practice to use a 38

[0293] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT

[0294] 1: 1 proportion of sugar and water to feed colonies (2.92M sucrose concentration; Eaucon et al., 2005; Overmyer et al., 2018). However, the high viscosity of this solution at the mean temperature of a beehive (0.01 Pa / s at 30°C) decreases its palatability and consumption by bees (Hidayanto et aL, 2010). A concentration of IM of sucrose solution matches with the concentrations that are found in multiple flower nectar that are visited by honeybees (Kim et al., 2011 ). Also, this concentration improves the sucrose solution feeding in honeybees under laboratory conditions (Shi et al., 2020). Because of its low viscosity even at a mean hive temperature (3.27xlO-3Pa / s at 30°C), IM sucrose concentration facilitates its consumption and increases the caloric value of the solution for bees (Hidayanto et aL, 2010; Kim et aL, 2011).

[0295] The Rut hive was fed with 2L of a supplemented solution of IpM rutin (Sigma- Aldrich R5143)+29.9 pM sodium bicarbonate+lM sucrose using a deep division board feeder every week of the feeding period. The volume of syrup per colony is based on an assumed ingestion of approximately 50.5 pl per day (for 40,000 individuals). This dosage of rutin is based on controlled laboratory experiments demonstrating positive effects in Africanized Apis meJlifera and Bombus impatiens (Garcia et aL, 2024; Riveros & Gronenberg, 2022). Similar results are found when supplementing hives with protein patties (Velandia, 2021).

[0296] Proxies of colony health

[0297] Three variables were assessed: allocation of frames to brood or resource storage, mortality, pollen productivity, and levels of Varroa sp. infestation during the evaluation time on Site 1. First, allocation of frames was determined by monitoring the number of frames used for brood and food storage present in the deep super (a total of 9 frames per hive after the introduction of the deep division board feeder) every two weeks. A frame was considered a brood frame if it contained eggs and uncapped or capped brood of any developmental stage. Second, mortality was determined by installing a plastic recipient at the front of the hive entrance at the bottom of the deep super to collect the dead bees removed from the colony by workers. This recipient was monitored weekly and dead individuals (of any caste or stage) were counted.

[0298] Third, colony productivity was assessed by installing pollen traps to measure the amount of pollen collected weekly (Replicate 1 : weeks 1-6; Replicate 2: weeks 0-6). Fourth, the levels of Varroa sp were assessed during two times: week 0 (baseline) and week 4, (one month between evaluations being the interval recommended by beekeeping practices). For this assessment, 100 individuals (including adult drones and workers) were collected randomly from frames with uncapped brood. Varroa sp. mites were separated and counted to obtain the number of parasites

[0299] 39

[0300] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT by the number of captured bees expressed in an infestation percentage (Ramsey et al., 2019). This method was also used on the Varroa sp. infestation levels evaluated on S2 at weeks 1 and 4.

[0301] Worker bees health proxies

[0302] For workers from the hives at SI, the dry mass and the size of the hypopharyngeal glands in nurse bees collected on week 4 were measured. For this, worker bees of multiple ages were collected from an inner brood frame with an uncapped brood (100 individuals from each colony). Then bees were cold-sacrificed placed in a drying incubator at 60°C for seven days (Henderson, 1992). Then, using a digital scale, the dry mass of the head and the thorax of each bee were individually weighed. The dry mass of the abdomen was not measured due to variations derived from crop content. On S3, from ten test hives, only one hive per treatment met the requirement of hive size and population to extract individuals for the dry mass evaluation. (Week 1 : Control Hive 1 N = 24. Rut Hive 1 N = 21 ; week 4: Control Hive 1 N = 42. Rut Hive 1 A = 44).

[0303] For measures of hypopharyngeal glands, ten nurse bees were collected from each hive. Nurse bees were identified as bees that keep their heads in larval cells for more than three seconds (after Vannette et al., 2015). Hypopharyngeal glands (hereafter HPG’s) were extracted and photographed using a Eeica MCI 70 HD microscope camera. Then, from each bee, ten acini were randomly selected for area measurement using the ImageJ version 1.53t software. HPG were only measured on S 1 due to the proximity of the hives to the laboratory, which facilitated the study of freshly collected individuals, required for the study of these glands.

[0304] Data analyses

[0305] Analysis for Site 1 were separated by replica due to unexpected events occurring in each moment. To analyze the indicators of hive health (number of brood and resources frames, collected dead bees, and Varroa sp. infestation levels), a Paired sample T Test was used to examine the difference of the data between weeks by treatment by weeks (from week 0 to week 6). Due to the low number of sampling units collected for the hive health indicators on SI, it was not possible to execute statistical analysis to test the differences between treatments by week. To test the Varroa sp. infestation levels observed on Site 2, and to compare differences between treatments and weeks, a General Mixed Model (GEMM) analysis was performed. For the individual health indicators, normality and uniformity of distribution of the data were evaluated using the Shapiro-Wilk W test and the Eevene’s test for Equality of Variance. Total, head, and thorax mass were analyzed independently by replicate by using a Wilcoxon signed- rank test to test the differences between Rut and Control treatments. For Site 3 dry mass, a Oneway MANOVA was used followed by an ES Means Student’s t post hoc test. A General Lineal 40

[0306] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT model (GLM) was used to analyze the difference between HPG’s area by treatment for each replicate independently. All the analyses were done using JMP 17.2.0 (SAS Institute).

[0307] Results

[0308] Proxies of colony health

[0309] Site 1

[0310] Honeybee hives treated with rutin supplementation showed a different performance of the hive health indicators compared to Control hives through the evaluation time in both replicates (Eigures 3A-3D). At Replicate 1 (Eigure 3A), the Control hive exhibited a variability in the number of brood frames by increasing the number of frames from week 2 to week 6 yet no significant (ti= -1.942, p = 0.192) and a low reduction of Varroa sp. infestation from 3.33% on week 1 to 3.15% on week 4 (a decrease of 5.4%). Control hive collected a total amount of pollen of 6.8kg and a total of 66 dead bees collected through the evaluation period. In week 3, there was an event of pesticide aspersion (not intended or planned in this study) on the vegetation near the apiary that coincided with first, a reduction of pollen collected 51.11% compared to the amount of week 4 (356.80 g collected pollen at week 3 and 730.94 g at week 4; yet no significant through time; ti= -1.583, p = 0.36; Figure 3C), and second, a maximum number of dead bees 43 individuals at week 4 but does not represent a significant change in dead bees collected ti= -2.17, p = 0.163).

[0311] Conversely, the Rut hive exhibited a stable number of brood and resource frames through the evaluation period, a great reduction in the percentage of infestation from 2.97% at week 1 to 0.66% at week 4 (a reduction of 77.77%), a total amount of 8.19 kg of collected pollen and a total of 26 dead bees collected through the evaluation period. In week 3, where the aspersion event occurs, the hive exhibited a 26.27% reduction of the collected pollen compared to week 4 (737.49 g collected pollen at week 3 and 999.7 g at week 4; Figure 3A and 3C), nevertheless, the changes of pollen collection over time were not significant (0= -1.735, p = 0.333). Also, the number of dead bees in week 4 was 12, representing a significant change over time (tz = -4.914, p < 0.05).

[0312] At Replicate 2 (Figure 3B and 3D), the Control hive (Rut hive at Replicate 1) showed an increase in the number of brood frames and a reduction of resource frames, yet not significant (Brood frames: fo = -2, p - 0.184; Resource frames: t ~ -2, p - 0.184). Also, the control hive exhibited an increase in the percentage of infestation Varroa sp. infestation from 2.25% to 4.4% (representing an increase of 76%). As in Replicate 1, in Replicate 2 a new spraying event took place but did not affect the pollen collection in week 3, nevertheless, the Control hive did not show a reduction in pollen collection with a total amount of 9.08 kg collected that varied

[0313] 41

[0314] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT throughout time (?2 = -9.581, p < 0.05). Also, even with the presence of aspersion, the Control hive did not show an increase in the number of dead bees at week 4; and the colony exhibited 43 dead bees present in the recipient of collection but did not vary through the evaluation period (fe = 1.625, / ? = 0.246).

[0315] In contrast, the Rut hive (Control hive at Replicate 1) exhibited a high but stable number of brood and resource frames through the feeding period, a slight rise of Varroa sp. infestation levels from 6.15% to 7.04% at week 4 (representing a rise of 14.47%). Likewise, Control hive in Replicate 2, the Rut hive did not show a reduction in pollen collection or an increase in dead bees during the aspersion event; and a total number of 35 dead bees collected which pattern was stable through time (fo = -2.46, p = 0.133). Also, the Rut hive exhibited a total amount of 8.78 kg of pollen collected that varied significantly during the tested weeks (t = -6.785, p < 0.05).

[0316] Site 2

[0317] Of the ten hives tested at the start of the evaluation period, only eight remained by week 4; where two hives from the Rutin treatment were excluded from the study presented due to the absence of the queen. All the colonies from the Control treatment remained by the end of the experimentation. In the evaluation of levels of Varroa sp. infestation, overall, both treatment hives presented a decrease in the percentage of infestation from week 1 to week 4 (Figure 4). All Rut hives exhibited a reduction of 64.62% of the mean percentage of infestation (mean ± s.d.m. Week 1 : 3.18+1.8%. Week 4: 1.13+0.91%) where, within colonies, Hive 8 showed a notorious reduction of 98.46% by week 4 (Figure 4). Control hives showed a mean reduction of the infestation percentage of 41.71% (Week 1: 2.11±2%. Week 4: 1 ,23±1.16%); where Ilive 9 exhibited an important reduction of 87.2% (Figure 4). However, two colonies 2 Control treatment showed an increase in the percentage of infestation, where the highest percentage was exhibited by Hive 2 with 1340%. Despite the observation of the overall reduction of the percentage of Varroa sp. infestation, no significant differences were detected in either treatment between week 1 or week 4 (GLMM: Treatment * Week: F = 0.21, p = 0.655).

[0318] Site 3

[0319] Ten nucleus hives were tested to evaluate the effects of supplementation against pesticide exposure. All colonies survived until the end of the experimental period; however, only two colonies met the criteria for extracting individuals to assess the dry mass of the worker bees. By week 4, eight colonies (four from each treatment) were classified as weak nucleus hives due to having two or fewer populated frames. It was not possible to evaluate the levels of Varroa sp. in any of the nucleus hives due to the low population of worker bees.

[0320] 42

[0321] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT

[0322] Proxies of individual health

[0323] Worker bees dry mass evaluation

[0324] To determine the effects of the supplementation on the worker bees from the test hives, the dry mass of the head and the thorax body segments were evaluated. The test was conducted with 400 bees and distributed equally in four groups: Replicate 1 Control (N = 100) and Rut (N = 100) and Replicate 2 Control (N = 100) and Rut (TV = 100). The rutin administration had a significant positive impact on total dry mass on worker bees by resulting on 8.64% more total mass (head and thorax mass; Eigures 5A-5F) compared to the Control worker bees (mean ± SDM; Replicate 1 : Control: 16.292±3.9mg; Rut 17.698±3.65mg; Wilcoxon signed-rank test: W = 11372.0, z = 3.29, p < 0.01) at Replicate 1 and a 16.13% more total mass at Replicate 2 ( mean ± SDM; Replicate 2: Control: 15.162±4.327mg; Rut 17.607+6.191 mg; Wilcoxon signed-rank test: W = 11328, z = 3.122, p < 0.01). This is confirmed by a significant increase of 10.6% of the head dry mass of Rut worker bees ( mean ± SDM; Control: 4.516 ± 1.606 mg; Rut 4.995 ± 1.201mg; Wilcoxon signed-rank test: W = 11305.5, z = 3.068, p < 0.01; Fig 5B) and 7.87% significant more of thorax mass for Rut worker bees (mean ± SDM; Control: 11 .776±3.9mg; Rut 12.703+3.0.12 mg; Wilcoxon signed-rank test: W = 11305.5, z = 3.068, p < 0.01; Figures 5A- 5F) both in Replicate 1. As for Replicate 2, as well as Replicate 1, rutin administration increases the head dry mass by 14.97% (mean + SDM; Control: 4.185 + 1.468 mg; Rut 4.812 + 1.705 mg; Wilcoxon signed-rank test: W = 11120.5, z = 2.616, p < 0.01; Figures 5A-5F) as for the thorax dry mass by a 16.56% (mean + SDM; Control: 10.977 + 3.198 mg; Rut 12.795 + 4.713 mg; Wilcoxon signed-rank test: W = 11380, z = 3.249, p < 0.01; Figures 5A-5F).

[0325] The dry mass evaluation for Site 3 was conducted with 187 individuals and distributed in four groups: Week 1 evaluation 46 worker bees (Control: N = 25 and Rut N = 21) and 141 worker bees at Week 4 (Control: N = 66 and Rut N = 75). The supplementation with rutin causes a positive effect on the total dry mass of the head and thorax segments of the worker bees through the evaluation period (MANOVA: Total: Treatment * Week: Total Fi, 44=0.730, p < 0.001; Head: Treatment * Week: FI.44=0.742, p < 0.001. Thorax: FI,44= 0.41, Treatment * Week: p < 0.001 ; Figure 6).

[0326] At week 1, bees from Control hive exhibited a significantly large total dry mass (19.06% more) compared to the Rut bees (mean ± SDM. Control: 15.212±0.794 mg. Rut: 12.777±1.509 mg. p < 0.05; Figure 6). Also, bees from the Control hive exhibited a significantly higher head dry mass of 30.53% compared to Rut hive bees (mean + SDM. Control: 4.52+0.8 mg. Rut: 3.14+0.57 mg. 30.53% more mass. GLM: F = 1.517, p < 0.01 ; Figure 6) but the thorax dry mass

[0327] 43

[0328] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT did not show significant differences between treatments (Control: 10.69+0.80 mg. Rut: 9.64+1.30 mg. Difference of 9.8%. GLM: F = 1.685, p = Figure 6).

[0329] Interestingly, at the end of the feeding period (Week 4), Rut bees exhibited a significant greater increase of the total dry mass by 81.920% compared to Week 1 (Rut: 23.250±4.670mg, p < 0.0001). This was also observed on a higher head dry mass of a 94.27% more mass (Rut: 6.10+1 .38mg, p < 0.0001 ; Figure 6) and a 56.2% more thorax dry mass (Rut: 15.05+2.42mg, p < 0.0001; Figure 6). There was also significant differences by comparing the total (p < 0.0001), head (p < 0.0001) and thorax dry mass (p < 0.0001) between treatments at Week 4. In contrast, Control bees at Week 4 did not exhibit a significant change of the total (Control: 16.657±4.568 mg, p = 0.137), head (Control: 4.46+1 .17mg, p = 0.663) nor thorax dry mass (Control: 11.43+2.28mg,p = 0.117).

[0330] Nurse bee hypopharyngeal glands measurement

[0331] The test was conducted with 400 measurements and distributed equally in four groups: Replicate 1 Control (A = 100) and Rut (N = 100) and Replicate 2 Control (N = 100) and Rut (N = 100) on Site 1 . The area from the acini of the hypopharyngeal glands by the individual nurse bees did not significantly differ between treatments at Replicate 1 (mean+s.d.m. Replicate 1. Control: 2.39xl04± 1.8 x 104pm2; Rut: 2.7 x 104± 1.306 x 104pm2; GLM: F= 753.784, / ? = 0.495; Figure 7) Nevertheless, at Replicate 2, Rut hive nurse bees exhibited a 39.31% significant higher area compared to the Control nurse bees (mean + s.d.m. Replicate 1. Control: 1.3 x 104+ 7.032 x 103pm2; Rut: 1.804 x 104+ 8.729 x 103pm2; GLM: F= 753.784, p < 0.001). The individual from whom the gland came from has no effect in the area of the IIPG’s (GLM: Treatment x Id: Replicate 1: F = 5120.138, p = 0.15; Replicate 2: F= 553.231 , p = 0.759).

[0332] Discussion

[0333] Honeybees play an important global role due to their function as pollinators. Unfortunately, honeybee health is affected by multiple factors occurring inside and outside the hive. Supplementation with secondary metabolites has surged as a strategy to improve honeybee health; yet these molecules might also have toxic effects. Thus, here, the goal was to determine whether the intermittent long-term supplementation of entire hives with the flavanol rutin, previously shown to protect bees against pesticides with different toxicodynamic, has an innocuous impact on the health of the hive and individuals. The results demonstrate that supplementation with rutin does not negatively affect the health of hive or the individuals; rather, the supplementation had a positive effect against pesticide exposure and Varroa sp. infection. Together, the results highlight three key aspects of the effects of rutin supplementation: (i) the

[0334] 44

[0335] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT innocuous effect of rutin, (ii) the positive effects on individuals against pesticide exposure, and (iii) the potential effects on the immune systems favoring the protection against Varma sp.

[0336] First, supplementation did not appear to impair the health of the individuals or the hive under field or semi-field conditions. At the individual level, the mass and size of HPG’s in individuals from Rut hives was not significantly different from bees in the Control group. Similarly, at the hive level, no significant change was observed in the proportion of frames devoted to resources or brood in the hive, a proxy of growth, or the number of dead bees, a proxy of resilience against environmental and internal stressors, were detected on Rut hives (see Figures 3A- 3D). Importantly, supplementation with rutin did not interfere with the effects intrinsic to the administration of the sucrose syrup. As expected, the sole administration with sucrose led to a change in the rate of pollen collection presumably as an attempt to equilibrate the stored resources (Free, 1965; Goodwin & Houten, 1991; sec Figure 3C and 3D).

[0337] These innocuous results were predicted due to the common presence of the flavonol rutin in natural sources of food (primarily pollen and nectar) and, not surprisingly, in byproducts inside the hive (propolis, beebread and honey; Bakchiche, 2017; Kaskoniene et al., 2009). Flavonoids are commonly found in nectar from multiple floral sources and plant families, in extrafloral nectar (honeydew honey), and in resins collected by honeybees (Guffa et al., 2017; Bakchiche, 2017: Harun Kurtagic et al., 2015; Kaskoniene et al., 2009; Silici et al., 2013).

[0338] Besides innocuity, the concentration and doses of IpM-1rutin consumed rendered positive results against stress factors of the hive and individual health parameters. Such positive effects with an exceptionally low concentration suggest a hormetic effect on the induction of protection (Chattopadhyay et al., 2017; reviewed by Martel et al., 2019). Although secondary metabolites produced by plants possess insecticide and antifeeding properties, the presence of low concentrations of phytochemicals on nectar functions as attractants of pollinators (associated with petal color and floral scents; reviewed by Wink, 2003) and generates positive effects in their system-tested on different models (reviewed by Stevenson et al., 2017). For example, in Drosophila melanogaster, the administration of rutin between 100 and 200 pM for 15 days increased survival, development, and resistance to oxidative and temperature stress; in contrast, concentrations above 400p M reduced fecundity and food intake (Chattopadhyay & Thirumurugan, 2020). Also, the daily supplementation of colonies with high concentrations of the flavonoid quercetin (0.33M) for 15 days induced the development of ovaries in worker bees, compromising the stability of the entire hive (Gao et al., 2010). On the other hand, the daily administration of rutin in concentrations as low as 1 p M for three days (12 ng / bee*day) in honeybees induced a protective effect in learning and motor impairment against the pesticide

[0339] 45

[0340] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT fipronil (Garcia et al., 2024). Similarly, in bumble bees, low dosages (24ng / bee*day) induced protection against fipronil and imidacloprid (Riveros & Gronenberg, 2022).

[0341] In the present study, the dosages are not directly comparable given the intermittent and ad libitum administration. Yet, the dosages are most likely exceptionally low. Considering an approximate population of 40,000 bees, a single adult would invest approximately 30 ng / week (assuming only the supplemented amount). Moreover, honeybees, during their development, could be exposed to even lower doses of rutin due to the conversion of the syrup into beebread and the metabolism of the nurse bees (Crailsheim et al., 1992; Lucchetti et al., 2018; Vidkjaer et al., 2024); yet still exhibiting positive effects on individual health proxies. Also, an innocuous effect of the feeding of supplemented pollen patties with 1 p M for one month using bees from the same apiary of SI was observed (Velandia and Riveros unpublished). Altogether, if the hermetic effect underlies the induced protection, the bees presumably relied on mechanisms to respond to the negative effects by activating pathways that result in positive effects against health stress factors (reviewed by Berenbaum & Johnson, 2015; reviewed by Gong & Diao, 2017).

[0342] Second, On SI and S3, rutin supplementation reduces the negative impact of worker bees from hives exposed to pesticides on hive and individual health parameters. On SI and Replicate 1 , both treatment colonies experienced an aspersion event in flower plants in week 3 (presumably neonicotinoids, an event not planned or intended in the present methodology). This event coincides with a high number of dead bees in the plastic recipients on Control Hive and a reduction of pollen collection (see Figures 3A-3D), which can be translated into the necessity of producing more brood seen in an increase in the number of brood frames to satisfy the lack of hive members (Castle et al., 2022; Dively et al., 2015). Also, on SI (Replicate 1) and S3, the exposure to pesticides could affect the dry mass of the tested individual of Control hive worker bees (see Figures 5A-5F and Figures 6A-6C). These chemicals (in doses commonly found in the field) cause negative effects on the timing, metabolism, and nutrition during development causing underweight in newly emerged bees (Bohme et al., 2017; Cook, 2019). On the other hand, on SJ (Replicate 1), the Rut hive exhibited a better response to the pesticide aspersion event of week 3 seen on the number of dead bees, a greater amount of pollen collected compared to Control hive and the constant number of brood frames (see Figures 3A-3D). Also, on SI and S3 (week 4), worker bees exhibited a significantly higher total, head and thorax dry mass after four weeks of rutin supplementation (see Figures 5A-5F and Figures 6A-6C).

[0343] According to findings of the metabolization of rutin in insects, at the gut, the rutinose structure of rutin is excreted and the main structure (3-O-glucoside of quercetin) is metabolized (Bernays & Chapman, 2000). Quercetin is a flavonoid that has beneficial effects on insects such 46

[0344] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT as bees (Ardalani et al., 2021; Liao et al., 2017, 2019; Wong et al., 2018). Therefore, the positive effects seen in the present study against pesticide exposure could originate from the presence of quercetin in the worker bees resulting from the metabolism of rutin. The positive mechanism of quercetin consumption lies in its ability to enhance the activity of the enzymatic complex of cytochrome P450 monooxygenases (Ardalani et al., 2021; Mao et al., 2011). Also, quercetin can increase the expression of the enzymes of the family CYP9 and CYP6AS which allows for efficient metabolization of pesticides causing them to be evacuated more quickly from the system (Johnson et al., 2012; Mao et al., 2011, 2017b). The consumption of quercetin facilitates a reduction in the concentration of pesticides in the system and in the lethal and sublethal effects (Ardalani et al., 2021; Liao et al., 2017, 2019; Mao et al., 2017; Wong et al., 2018). Also, the consumption of quercetin during the development of honeybees (in the larvae stage) ameliorates the flying activity and the ATP production affected by a fungicide (Liao et al., 2019). From these findings, it is likely that the supplementation of rutin could protect bees the lethal effects of the pesticide event on week 3 on 51 by enhancing the metabolization pathways reported on the literature, which can explain the differences of bee mortality (see Figure 3A-3D). This hypothesis can be demonstrated by a constant number of brood frames by a low loss of individuals and a greater pollen collection on .S' I (Replicate 1; see Figure 3 A and 3C). Also, it is possible that the consumption of rutin during the development of Rut hive worker bees (possibly by the secretion of nurse bees that consume beebread prepared with sugar syrup at week 1 on both SI and S3), also generates a protector effect against the low weight effect from pesticide exposure.

[0345] Third, on SI (Replicate 2; see Figure 3) and S2 (see Figure 4) at both Replicates, the hives were exposed at low and medium levels of Varroa sp. infection (Coopera on Colombiana de Investigation Agropecuaria (AGROSAVIA, n.d.), but both Rut group colonies exhibited a higher resistance of mites presence by reducing the percentage of infection or by minimizing the negative effects of Varroa sp. Taking account that Varroa sp. mites are considered a high-risk factor in beekeeping practices due to the impact on the young disrupting the mechanisms of xenobiotics detoxifying, protein synthesis, and immune process that occurs in the fat body, affecting the quality of the future foragers (Kovac & Crailsheim, 1988; Ramsey et al., 2019). In the nurse bees, the mites affected the size of the hypopharyngeal glands (HPG’ s) and, therefore, a decrease in the quantity, the protein content, and the vitellogenin protein concentration on the brood food, which negatively affects immunocompetence and the survivability in young bees (Alaux et al., 2011; Bruckner et al., 2023; van Dooremalen et al., 2013). The negative effect of Varroa sp. infestation was exhibited on a variable number of brood frames through

[0346] 47

[0347] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT experimentation time on both hives on SI (Replicate 2; see Eigure 3A-3D), a reduction of the dry mass of worker bees and in size of the HPG’ s of nurse bees from Control hive compared to Rut hive worker bees (see Figures 5A-5F and Figure 7A-7B). On the other hand, the consumption during the development by Rut hive could have prevented an important reduction of dry mass and size of the HPG’s and increase the activity of the immune system of the members of the hive (see Figure 5A-5F and Figure 7A-7B). The resistance of pathogens in bees depends, in part, on the levels of expression of the genes encoding antimicrobial peptides such as defensin - 1 and apisimin produced at the HPG's in both nurse and forager bees (Alaux et al., 2011). Although no evidence of the effect of flavonoids in the immune system in bees is known to date, the consumption of low concentrations of quercetin during the development of Bombyx rnori results in an increase in the body mass, the expression of antimicrobial peptides such as defensin-1, multiple immune pathways and various antioxidant enzymes such as supcroxidasc dismutase and catalase which is also part of bees (Korayem et al., 2012; G. Shi et al., 2020). Also, the consumption of the phenolic acid p-coumaric acid in newly emerged bees causes an upregulation of the apisimin antimicrobial peptide and positively affects the insulin / insulin-like pathway that is responsible for the size of the bees (Ihle et al., 2014; Lin & Smagghe, 2019). Also, the consumption of this phenolic acid increases the size of the HPG’ s compared to other phytochemicals in younger bees (Nino et al., 2022). In addition, the reduction of the levels of Varroa sp. on both Rut group hive could be explained by the evidence of sensitivity of the mites to the contact to hive materials (such as collected resins or propolis) with high content of flavonoids (Damiani et al., 2010; Pusceddu et al., 2019). Considering that the sugar syrup could be also stored as honey yield, the contact of the Varroa sp. mites with this hive structure could result in a reduction of this parasite.

[0348] Example 2: A Method to Increase the Solubility of the flavonoid rutin for biological assays and administration for protective effects

[0349] Materials and Methods

[0350] Chemicals

[0351] Rutin hydrate was acquired from Tokyo Chemical Industry (R0035), Sodium bicarbonate was acquired from AMRESCO (0865).

[0352] Solutions

[0353] Five concentrations of rutin solutions were prepared based on the reported solubility of 125mg / L (DrugBank Online): 62.5mg / L (102.4mM), 125mg / L (204.7mM), 250mg / L (409.5mM), 500mg / L (819mM). These concentrations include the range most often used in bioassays and their biological effectiveness. Also, some of these concentrations exceed the

[0354] 48

[0355] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT values of solubility reported for rutin. Previous attempts indicated that 125mg / L of rutin would lead to precipitation and that the solubility in water might be lower, at around 30 mg / L.

[0356] To test the effectiveness of sodium bicarbonate as solvent, four concentrations were used: 4.76 mM (400 mg / L), 1.19mM (100 mg / L), 0.6mM (50 mg / L). These concentrations were selected because the LCso and the NOEC (No Observed Effect Concentration) for sodium bicarbonate in honeybees is 24 mg / bee (4.76 mM assuming an ingested volume of 60 ml. per bee; OECD SIDS, 2002).

[0357] Behavioral assays

[0358] Experimental subjects

[0359] Four hives from the Apiary at Universidad del Rosario (Bogota, Colombia). Two hives were designated as ‘Controls’ and received only a solution with the vehicle (sucrose + sodium bicarbonate) whereas the other two, designated as ‘Supplemented’, received the full composition (sucrose + sodium bicarbonate + rutin). For experiments, only foragers were collected at the entrance of the hive (see below). The hives were matched into two groups according to their approximate size.

[0360] Feeding solutions and schedule

[0361] The four hives received 2L of the designated solution every seven days. Thus, the Control hives received 2L of a IM sucrose / 29 mM sodium bicarbonate: the supplemented colonies received 2L of a 1 mM rutin / 1 M sucrose / 29 mM sodium bicarbonate.

[0362] Bee collection and administration of pesticides

[0363] Foragers leaving the hive were collected on the third and fourth days following the administration of the solution to each colony. Upon collection, bees were transferred to the laboratory, briefly anesthetized in an ice bath, and harnessed in plastic tubes. After recovery, the bees were fed to satiation with a sucrose solution and maintained in a plastic container with wet cotton.

[0364] The following day, the bees were tested in their motivation by gently touching their antenna with a sucrose solution. Bees not responding with the extension of their proboscis were discarded. The rest of the bees were randomly assigned to one of two treatments: 10 mL of a IM sucrose solution or 10 mL of a sucrose solution containing one of two insecticides: fipronil (1 ng / bee) or Deltamethrin (45 ng / bee). For the oral administration of the pesticides, the antennae of the bees were gently touched with a clean sucrose solution and provided the contaminated solution directly to the proboscis. Thus, the antennae were not contaminated with pesticide. Following this protocol, bees belong to one of four treatments (for each pesticide):

[0365] 1. Control- fed with the sucrose + bicarbonate solution and then with sucrose solution.

[0366] 49

[0367] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT

[0368] 2. Pesticide- fed with the sucrose + bicarbonate solution and then with either pesticide.

[0369] 3. Rutin- fed with the sucrose + bicarbonate + rutin solution and then with the sucrose solution.

[0370] 4. Rutin + Pesticide-fed with the sucrose + bicarbonate + rutin solution and then with either pesticide.

[0371] Learning and memory tests

[0372] The appetitive olfactory conditioning of the proboscis extension reflex (PER) was used, a standard method used to evaluate the impact of pesticide administration in bees. During conditioning, the bees were trained to learn the association between an odor (1 -hexanol; Sigma- Aldrich 471402) and a reward (approx. 1 mL of a 1.5M sucrose solution). Each pairing is known as a training trial, and each bee was presented with six training trials separated in time by 10 minutes. A bee was said to have a conditioned response if it exhibits the PER upon the initial presentation of the odor. Each bee was assigned a learning score (0-5) that represents the number of conditioned responses that follow the initial training trial (during which the bee is not expected to exhibit a response). Memory retention was tested after 24 hours. By presenting only the odor and recording whether a conditioned PER is exhibited.

[0373] The training tests started at different times after administration of the insecticide depending on the known time of activity: Fipronil (3 hours), Deltamethrin (5 hours), Imidacloprid (2 hours).

[0374] Results

[0375] Measure of solubility and pH

[0376] The concentrations of each solution were compared by relying on spectrophotometric readings (NanoDrop™ 2000 / 2000c Spectrophotometers) and direct observations of precipitate were made. The pH of each solution was measured as a parameter relevant for its use in biological assays.

[0377] It was observed that some amount of rutin always precipitated when dissolved in water (Figure 8A, blue curve). When NaHCCE was added, the absorbance increased, indicating a larger portion of rutin in solution. At 62.5 mg / L of rutin, all concentrations of NaHCCh were sufficient to produce a solution without rutin precipitation. Also, 400 mg / L of NaHCCL allowed up to 250 mg / L of rutin to dissolve without precipitating (Figure 8A, 8B). This variation observed in absorbance was also present in pH; the addition of rutin acidified the solutions (Figure 8C). The lowest pH was measured in a solution of 250 mg / L of rutin in water and the highest was measured in a solution of 62.5 mg / L of rutin in 400 mg / L of Nal lCOi.

[0378] 50

[0379] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT

[0380] After excluding the concentrations that led to precipitation and pH > 8.0, it was concluded that the most effective composition had 102.4 mM (62.5mg / L) of rutin and 1.19 mM (100 mg / L) of NaHCOs. The targeted concentration to feed a hive of 30 000 bees is 2L of ImM rutin. Thus, lOOmL of the concentrated composition as enough to feed five colonies after dilution. Also, given the initial concentration of NaHCCE (1.19 mM), its final concentration would be 29.9 mM after dilution. Importantly, 1 L of a concentrated solution of 250 mg / E of rutin (409.49 mM) and 400 mg / L of NaHCOs (4.76 mM) would be enough after dilution to feed 200 colonies (5mL / 30 000 beehive). Such a solution (5 mL) had a pH of 7.54 at the final volume to be delivered to the bees (2 L).

[0381] Cognitive Protection and Protection against impairment by Fipronil

[0382] Next, the innocuity of this composition was tested and its effectiveness in inducing cognitive protection against impairments produced by two pesticides with different toxicodynamic properties were assessed.

[0383] In total, 290 bees were collected and prepared from the Control and Supplemented colonies. For final analysis, 18 bees that stopped responding during training were excluded. Thus, 272 bees were distributed across four treatments: Control (N = 68), Rut (N = 70), Rut + Fip (N = 69), Fip (N = 65).

[0384] It was found that the administration of fipronil significantly decreased the learning score of bees relative to bees fed with sucrose and bicarbonate (Figure 9A; Wilcoxon Method: Control vs Fip: Z=3.34, P < 0.001). Interestingly, the administration of rutin led to a significant improvement in the learning score relative to Control bees (Figure 9A; Wilcoxon Method: Control vs. Rut: Z=-5.78, P < 0.001). Most importantly, bees fed with Rutin + bicarbonate and then exposed to fipronil (Rut + Fip treatment) exhibited learning scores significantly higher than the ‘unprotected bees’ only exposed to fipronil (Figure 9A; Wilcoxon Method: Rut + Fip vs. Fip: Z=6.40, P < 0.001). Nevertheless, the learning score of the ‘protected’ bees (Rut + Fip) was significantly lower than the bees receiving only Rutin + bicarbonate, indicating partial protection (Figure 9A; Wilcoxon Method: Rut+Fip vs. Rut: Z = -4.73, P = 0.001). However, the learning score of the ‘protected’ bees (Rut+Fip) was significantly higher than Control bees (Figure 9A; Wilcoxon Method: Control vs. Rut+Fip: Z=-2.28, P=0.022).

[0385] It was found that the probability of the bees exhibiting a conditioned PER was significantly affected by training across trials and by feeding treatment (Figure 9B; GLMM: Trial: F=284.39, P<0.0001; Treatment: F=30.6, P<0.0001). However, the effect of Trial did not vary across treatments (GLMM: Treatment*Trial: F=0.38, P=0.77). Therefore, the following analyses were conducted using a reduced model excluding the interaction. The reduced model 51

[0386] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT confirmed the pattern described above (GEMM: Trial: F=311.41, PcO.OOOl; Treatment: F=32.09, PcO.OOOl). Post hoc comparisons indicated that the administration of fipronil significantly impaired the performance of bees across trials (Dunnett adjusted for Fip vs. Control: t=4.27, PcO.OOOl) whereas the administration of rutin enhanced the performance relative to Control bees (Dunnett adjusted for Rut vs. Control: t=-5.58, PcO.OOOl). Most importantly, the bees fed with ratin and then administered with fipronil exhibited a performance across trials that was significantly higher than Fip bees (Dunnett adjusted for Rut+Fip vs. Fip: t=6.85, PcO.OOOl). However, the bees fed with ratin and then administered with fipronil exhibited a performance across trials that was significantly lower than bees fed only with rutin (Dunnett adjusted for Rut+Fip vs. Rut: t=-2.93, P=0.011).

[0387] After 24h, it was observed that memory retention was not negatively impacted. Rather, bees in some groups even exhibited moderate or significant increase in responsiveness (Figure 9C; Control: Likelihood ratio-test: Chi Squarei.es: 8.58, P=0.0034; Rut: Likelihood ratio-test: Chi Squarei,7o: 3.78, P=0.05; Fip: Likelihood ratio-test: Chi Squarei.65: 43.6, PcO.OOOl; Rut+Fip: Likelihood ratio-test: Chi Squarei,69: 0.64, P=0.42).

[0388] Protection against impairment by Deltamethrin

[0389] In total, 336 bees were collected and prepared from the Control and Supplemented colonies. For final analysis, 69 bees were excluded that stopped responding during training (Control=6; Rut=2; Rut+Delta=22; Delta=40). Thus, 267 bees were distributed across four treatments: Control (N =78), Rut (N=86), Rut+Delta (N=61), Delta (N=42).

[0390] It was found that the administration of deltamethrin significantly decreased the learning score of bees relative to bees fed with sucrose and bicarbonate (Figure 10A; Wilcoxon Method: Control vs Delta: Z=4.98, P=0.0005). In contrast, the administration of rutin was innocuous and did not significantly impact on the learning score relative to Control bees (Figure 10A; Wilcoxon Method: Control vs. Rut: Z=-1.53, P=0.13). Most importantly, bees fed with Rutin+bicarbonate and then exposed to deltamethrin (Rut+Delta treatment) exhibited learning scores significantly higher than the ‘unprotected bees’ only exposed to deltamethrin (Figure 10A; Wilcoxon Method: Rut+Delta vs. Delta: Z=3.58, P=0.0005). Nevertheless, the learning score of the ‘protected’ bees (Rut+Delta) was significantly lower than the bees receiving only Rutin+bicarbonate (Figure 10A; Wilcoxon Method: Rut+Delta vs. Rut: Z=-3.74, P=0.0005), or than Control bees (Figure 10A; Wilcoxon Method: Control vs. Rut+Delta: Z=2.1, P=0.046).

[0391] It was observed that the probability of the bees exhibiting a conditioned PER was significantly affected by training across trials and by feeding treatment (Figure 10B; GLMM: Trial: F=163.9, PcO.OOOl; Treatment: F=12.89, PcO.OOOl). However, the effect of Trial did not 52

[0392] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT vary across treatments (GLMM: Treatment*Trial: F=1.56, P=0.20). Therefore, the following analyses were conducted using a reduced model excluding the interaction. The reduced model confirmed the pattern described above (GLMM: Trial: F=349.83, PcO.OOOl; Treatment: F=16.13, P<0.0001). Post hoc comparisons indicated that the administration of deltamethrin significantly impaired the performance of bees across trials (Dunnett adjusted for Delta vs. Control: t=-5.34, P<0.0001) whereas the administration of ratin led to performance that did not significantly differ from Control bees (Dunnett adjusted for Rut vs. Control: t=l .78, P=0.20). Most importantly, the bees fed with rutin and then administered with deltamethrin exhibited a performance across trials that was significantly higher than Delta bees (Dunnett adjusted for Rut+Delta vs. Delta: t=3.68, P=0.0007). However, the bees fed with rutin and then administered with deltamethrin exhibited a performance across trials that was significantly lower than bees fed only with rutin (Dunnett adjusted for Rut+Delta vs. Rut: t=-3.81, P=0.0005).

[0393] Discussion and Conclusion

[0394] Flavonoids are secondary metabolites derived from plants that are broadly used in nutraceutical research; however, their solubility in water remains a constraint in biological assays and administration to subjects. The flavonol rutin, for example, has proved to be effective against multiple physiological impairments; yet, due to its low water solubility, rutin is typically dissolved using Dimethyl sulfoxide (DMSO). However, although DMSO is broadly used as a major solvent, recent accounts have shown cell toxicity even at very low concentrations. Here, whether sodium bicarbonate at very low concentrations is sufficient to allow dissolution of the flavonol rutin was investigated. Also tested was the efficacy of the solution to protect honeybees against cognitive impairment induced by the oral administration of three insecticides with different modes of action. It was found that a composition of 62.5mg / L (102.4 of rutin and 100 mg / L of Nal lCOi achieved a fully dissolved solution and a pH that resembles physiological levels in bees. It was also demonstrated that the prophylactic administration of the composition to honeybee hives induces protection against the three pesticides tested.

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[0492] It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these can vary. It is also to be

[0493] 60

[0494] 45807824.1 ATTORNEY DOCKET NO. UA 23-279 PCT understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0495] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

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[0497] 45807824.1

Claims

ATTORNEY DOCKET NO. UA 23-279 PCTCLAIMSWe claim:

1. A concentrated formulation for forming a dilute composition suitable for administration to or ingestion by an insect pollinator, wherein the concentrated formulation comprises:(i) one or more flavonoids, and(ii) a solvent, wherein the one or more flavonoids are present in the concentrated formulation in an amount that, upon dilution of the concentrated formulation to form the dilute composition, is effective to protect against impairment of a cognitive function of the insect pollinator.

2. The formulation of claim 1 , wherein the one or more flavonoids is quercetin, rutin, myricetin, kaempferol, fisetin, or apigenin, or a combination thereof.

3. The formulation of claim 1, wherein the one or more flavonoids is rutin.

4. The formulation of claim 1, wherein the one or more flavonoids are present in the concentrated formulation at a concentration ranging from about 30 mg / L to about 500 mg / L.

5. The formulation of claim 1, wherein the solvent is selected from the group consisting of sodium bicarbonate, methanol, ethanol, 1 -propanol, 2-propanol, 1 -butanol, acetone, ethyl acetate, glycerin, potassium bicarbonate, sodium carbonate, potassium carbonate, or a combination thereof.

6. The formulation of claim 1 , wherein the solvent is sodium bicarbonate.

7. The formulation of claim 6, wherein the sodium bicarbonate is present in the concentrated formulation at a concentration ranging from about 40 mg / L to about 500 mg / L.

8. The formulation of claim 1, wherein the concentrated formulation has a pH ranging from about 6.0 to about 9.0.

9. The formulation of claim 1, wherein the solvent comprises one or more pH modifiers.

10. The formulation of claim 1, wherein the solvent is not or does not contain DMSO.

11. The formulation of claim 1, further comprising one or more additional nutrients and / or ingredients selected from the group consisting of carbohydrates, proteins, lipids, vitamins, minerals, and water, or a combination thereof.

12. The formulation of claim 11, wherein the one or more additional nutrients and / or ingredients are selected from the group consisting of natural or artificial nectar, honey, sugar, sugar syrup, pollen or pollen substitute, soy flour, soy meal, gluten, skim milk, yeast, pollard, and oil, or combinations thereof.

13. The formulation of claim 1, wherein the flavonoid is rutin and the solvent is sodium bicarbonate.6245807824.1ATTORNEY DOCKET NO. UA 23-279 PCT14. The formulation of claim 13, wherein rutin is present in the formulation at a concentration ranging from about 30 mg / L to about 500 mg / L, and sodium bicarbonate is present in the formulation at a concentration ranging from about 40 mg / L to about 500 mg / L.

15. The formulation of claim 14, wherein rutin is present in the formulation at a concentration of 30 mg / L to 260 mg / L, and sodium bicarbonate is present in the formulation at a concentration ranging from about 40 mg / L to about 420 mg / L.

16. The formulation of claim 14 or 15, wherein rutin is present in the formulation at a concentration of 62.5 mg / L, and sodium bicarbonate is present in the formulation at a concentration of 400 mg / L.

17. The formulation of claim 14 or 15, wherein rutin is present in the formulation at a concentration of 250 mg / L, and sodium bicarbonate is present in the formulation at a concentration of 400 mg / L.

18. The formulation of claim 1 , wherein when the formulation is diluted to form a dilute composition comprising at least about 1 pM of the one or more flavonoids, the dilute composition is safe for use as a food, feed additive or supplement, or nutraceutical.

19. The formulation of claim 1, wherein the insect pollinator is a butterfly, moth, fly, beetle, wasp or bee.

20. The formulation of claim 1, wherein the insect pollinator is a bee, optionally wherein the bee is selected from honeybee, bumblebee, carpenter bee, leafcutter bee, blueberry bee, squash bee, mason bee, orchid bee, stingless bee, or sweat bee.

21. The formulation of claim 1, wherein the one or more flavonoids are more soluble in the solvent than in a control solvent consisting of 1 M aqueous sucrose solution.

22. A method of protecting the cognitive function of an insect pollinator from a pesticide, the method comprising:(i) applying or placing a dilute composition in a hive located in an area that is occupied by one or more insect pollinators, wherein the dilute composition comprises a dilute form of a concentrated formulation comprising (i) a flavonoid and (ii) a solvent, wherein the flavonoid is rutin, wherein rutin is present in the concentrated formulation at a concentration ranging from 30 mg / L to about 500 mg / L,6345807824.1ATTORNEY DOCKET NO. UA 23-279 PCT wherein the solvent is sodium bicarbonate, and wherein sodium bicarbonate is present in the concentrated formulation at a concentration ranging from about 40 mg / L to about 500 mg / L.

23. A method of protecting the cognitive function of an insect pollinator from a pesticide, the method comprising:(i) applying or placing a dilute composition in a hive located in an area that is occupied by one or more insect pollinators, wherein the dilute composition comprises a dilute form of a concentrated formulation comprising (i) a flavonoid and (ii) a solvent, wherein the flavonoid is rutin, wherein rutin is present in the concentrated formulation at a concentration ranging from 30 mg / L to 260 mg / L, and wherein the solvent is sodium bicarbonate, wherein sodium bicarbonate is present in the formulation at a concentration ranging from about 40 mg / L to about 420 mg / L.

24. A method of protecting the cognitive function of an insect pollinator from a pesticide, the method comprising:(i) applying or placing a dilute composition in a hive located in an area that is occupied by one or more insect pollinators, wherein the dilute composition comprises a dilute form of a concentrated formulation comprising (i) a flavonoid and (ii) a solvent, wherein the flavonoid is rutin, wherein rutin is present in the concentrated formulation at a concentration of 62.5 mg / L, and wherein the solvent is sodium bicarbonate, wherein sodium bicarbonate is present in the formulation at a concentration of 400 mg / L.

25. A method of protecting the cognitive function of an insect pollinator from a pesticide, the method comprising:(i) applying or placing a dilute composition in a hive located in an area that is occupied by one or more insect pollinators, wherein the dilute composition comprises a dilute form of a concentrated formulation comprising (i) one or more flavonoids and (ii) a solvent, wherein the one or more flavonoids are selected from the group consisting of quercetin, rutin, myricetin, kaempferol, fisetin, or apigenin, or a combination thereof,6445807824.1ATTORNEY DOCKET NO. UA 23-279 PCT wherein the one or more flavonoids are present in the concentrated formulation at a concentration ranging from 30 mg / L to 500 mg / L, and wherein the solvent is selected from the group consisting of methanol, ethanol, 1- propanol, 2-propanol, 1 -butanol, acetone, ethyl acetate, glycerin, potassium bicarbonate, sodium carbonate, potassium carbonate, or a combination thereof, wherein the solvent is present in the concentrated formulation at a concentration ranging from 40 mg / L to 500 mg / L.

26. The method of any one of claims 22-25, wherein the flavonoid(s) is more soluble in the solvent than in a control solvent consisting of 1 M aqueous sucrose solution.

27. The method of any one of claims 22-25, comprising prior to step (i) diluting the concentrated formulation to form the dilute composition, wherein the dilute composition comprises at least about 1 pM of the one or more flavonoids, optionally wherein the diluent is or comprises an aqueous sucrose solution, such as a IM sucrose solution.

28. The method of claim 27, wherein the dilute composition is incorporated in a solid food source prior to step (i).

29. The method of any one of claims 22-25, wherein in step (i) the dilute composition is placed in the hive via a form of internal or external feeder such as a deep division board feeder.

30. The method of any one of claims 22-25, wherein the dilute composition comprises an effective amount of the one or more flavonoids to prevent or reduce impairment of a cognitive function and / or increase a cognitive function in the insect pollinator following exposure to a pesticide, compared to an insect pollinator not administered the dilute composition comprising the one or more flavonoids.

31. The method of claim 27, wherein the one or more flavonoids are present in the dilute composition in an amount from about 0.01 pM to about 100 mM.

32. The method of any one of claims 22-25, wherein the cognitive function is selected from learning, memory, attention, decision accuracy, decision speed, navigation, motor activity, sucrose sensitivity, and combinations thereof.

33. The method of any one of claims 22-25, wherein exposure to the pesticide induces cognitive impairment.

34. The method of any one of claims 22-25, wherein the pesticide is neurotoxic.

35. The method of any one of claims 22-25, wherein the pesticide adversely affects GABAergic or glutamatergic neurotransmission, the mushroom bodies, the antennal lobes, the optic lobes or mitochondrial function; increases apoptosis; increases oxidative stress; or combinations thereof in the pollinator.6545807824.1ATTORNEY DOCKET NO. UA 23-279 PCT36. The method of any one of claims 22-25, wherein the insect pollinator is a butterfly, moth, fly, beetle, wasp or bee.

37. The method of any one of claims 22-25, wherein the insect pollinator is a bee, and wherein the bee is selected from honeybee, bumblebee, carpenter bee, leafcutter bee, blueberry bee, squash bee, mason bee, orchid bee, stingless bee, or sweat bee.6645807824.1

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