BACKGROUND OF THE INVENTION

Honey bees, Apis mellifera, are required for the effective pollination of crops and are therefore critical to world agriculture. Honey bees also produce economically important products, including honey and bees wax. Honey bees are susceptible to a number of pests including parasites and pathogens, such as the ectoparasitic mite, Varroa destructor. Varroa mites parasitize pupae and adult bees and reproduce in the pupal brood cells. The mites use their mouths to puncture the exoskeleton and feed on the bee's hemolymph. These wound sites in the exoskeleton harbor bacterial infections, such as Melissococcus pluton, which causes European foulbrood.

In addition, to their parasitic effects, Varroa mites are suspected to act as vectors for a number of honey bee pathogens, including deformed wing virus (DWV), Kashmir bee virus (KBV), acute bee paralysis virus (ABPV) and black queen cell virus (BQCV), and may weaken the immune systems of their hosts, leaving them vulnerable to infections.

If left untreated Varroa infestations typically result in colony-level mortality.

Maintaining a supply of strong honey bee colonies available for pollination is essential for the sustained production of farm crops worth more than $14 billion to U.S. agriculture. During the 2014-2015 (October-September) pollination year, an estimated 40% of the honey bee colonies in the U.S. were weakened or collapsed due to Varroa infestation (Seitz et al (2016). A National Survey of Managed Honey Bee 2014-2015 Colony Losses in the USA. Journal of Apiculture Research 54(4)). The Varroa mite is considered the most critical pest to honeybee health and consequently pollination services rendered by beekeepers. Migratory beekeepers assume that 30% of all hives will die during annual moves to pollinate crops across the US and consider the Varroa mite to be a significant cause of die off. The industry is desperate for new effective strategies to control the mite, primarily because mites develop resistance rapidly and beekeepers realize that bees also suffer from the effects of miticides.

Increasing the health of honey bees by controlling this parasite will result in many millions of dollars in savings to beekeepers alone. Additionally, pollination services will be increased because the numbers of colonies available to pollinate will be increased and healthy bees are more effective in pollination than weakened bees. Effectively controlling Varroa mite will therefore have significant impacts on crop production throughout the US and other countries.

Current methods of treating Varroa infestations are proving to be ineffective as the mites develop resistance to existing miticides. New compositions and methods for treating or preventing Varroa mite infestations are urgently required. Desirably, such compositions would include only natural ingredients that pose no risk to human health.

SUMMARY OF THE INVENTION

As described below, the present invention features methods and compositions for controlling a honey bee pests including parasitic mitea or for the treatment or prevention of honey been pests including parasitic mite infestation in a honey bee hive.

In accordance with the present invention there is provided a method for treating the object or area containing honey bee pests, comprising treating the object or area with a composition containing an effective amount of mono- and di-fatty acids comprising at least 99% C ₅ to C ₂ o, and mixtures thereof. It is understood that fatty acids are carboxylic acid with a long aliphatic chain, which is either saturated or unsaturated.

In another embodiment of the present invention, there is provided a method for treating the object or area containing honey bee pests, comprising treating the object or area with a composition containing an effective amount of mono- and di-fatty acids comprising at least 99% C ₅ to Co, and mixtures thereof such as C8910 fatty acid blend. As used herein, C8910 fatty acid blend means a mixture or blend of caprylic acid C8, pelargonic acid C9 and decanoic acid CIO carboxylic acids.

Other embodiments of the invention include a method for treating the object or area containing honey bee pests, comprising treating the object or area with a composition containing an effective amount of mono- and di-fatty acids comprising pelargonic acid C9, and C679 fatty acid blend, which is a mixture of from 24 to 30% by weight of C679 fatty acid blend of pelargonic acid C9, from 24 to 30% by weight of the C679 fatty acid blend of heptanoic acid C7, from 24 to 30% by weight of the C679 fatty acid blend of caproic acid C6, from 10 to 14% by weight of the C679 fatty acid blend of caprylic acid C8 and from 2 to 5% by weight of the C679 fatty acid blend of valeric acid C5. The C679 fatty acid blend is commercially available from Emery Oleochemicals LLC as EMERY™ 1210 FATTY ACID.

DEFINITIONS

By "controlling a parasitic mite" is meant inhibiting mite survival or reducing, slowing, or stabilizing the growth of a mite population.

By "effective amount of a miticide" is meant an amount effective to disrupt a mite biological function. By "hive" is meant a structure that contains a bee colony. A modern box hive typically includes a bottom board, cover, and one or more boxes, stacked one above the other. Inside, each box contains a series of movable frames of comb or foundation held in a vertical position a bee space apart.

By "honey bee" is meant a Hymenopteran insect of the genus Apis. The term "honey bee" is not limited to the adult form of the insect, but encompasses all honey bee developmental stages, including but not limited to egg, larva, and pupa. Exemplary honey bee species include Apis mellifera and Apis cerana.

By "honey bee pest" is meant any insect, acaraid, fungal or bacterial agent selected from the group consisting of Varroa mite, small hive beetle, bee louse, wax moth, lessor wax moth, tracheal mites, external mites, American foulbrood, European foulbrood, chalkbrood and nosema.

By "honey bee parasitic mite" is meant any acarid that parasitizes a honey bee.

Exemplary honey bee parasitic mite include Varroa mites and tracheal mites.

By "miticide" is meant an agent that inhibits a biological function of a mite.

By "miticidal activity" is meant any activity that inhibits the growth, reproduction, or survival of a mite or other acarid.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments described herein can be understood more readily by reference to the following detailed description and examples. Elements, apparatus, and methods described herein, however, are not limited to the specific embodiments presented in the detailed description, drawings, and examples. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Numerous modifications and adaptations will be readily apparent to those of skill in the art without departing from the spirit and scope of the invention.

In addition, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein, including the end points of the range, unless expressly stated otherwise. For example, a stated range of " 1.0 to 10.0" should be considered to include any and all subranges beginning with a minimum value of 1.0 or more and ending with a maximum value of 10.0 or less, e.g., 1.0 to 5.3, or 4.7 to 10.0, or 3.6 to 7.9.

The technology disclosed herein concerns a method for treating honey bee pests to an object or area, involving treating (or exposing) the object (e.g., insect trap) or area (e.g., field, orchard) with a composition containing a honey bee pests treating effective amount of a composition comprising saturated and unsaturated mono- and di-fatty acids comprising at least 99% C ₅ to C ₂₀ , or at least 99% C ₅ to C _i2 , C9, and mixtures thereof including C8910 and C679 fatty acid blends, and optionally a carrier material. The carrier or carrier material may be, for example, agronomically or physiologically acceptable carriers or carrier materials. The carrier material as used herein is defined as not including the body of an insect (e.g., honey bees) or the extract from which a composition is isolated. The temperature flexible polymer matrix may include polyethylene glycol (PEG).

The present invention is directed to methods and compositions to control acarids and other related species of the family Varroidae.

Parasitic mites that prey on honey bees include Varroa mites (e.g., Varroa destructor, Varroa jacobsoni) and tracheal mites (e.g., Acarapis woodi). Tracheal mites are microscopic mites that inhabit the respiratory tubes of bees. Varroa mites are ectoparasites that feed on bee hemolymph, and infest wild and domestic honey bee colonies. Varroa mite reproduction begins when the adult female mite enters a brood cell shortly before it is capped. Drone brood, which is reared in larger cells than worker brood, is preferentially targeted for mite infestation. The female mite feeds on the larval hemolymph prior to depositing her eggs. The Varroa eggs are enclose under the sealed cell, and the developing mites feed on the bee pupa. The first egg laid by the female Varroa develops into a male. Subsequent eggs develop into females that mate with their brother. The mated female mites along with their mother are released from the capped cell when the bee emerges. The female mites typically attach to adult bees between the abdominal segments or between body regions, where they feed on the bees' hemolymph. Adult bees serve as intermediate hosts and as a means of transport to new sites of infestation.

Desirably, miticides used in acarid control should address the following four needs: i) should disrupt a physiological function required for mite survival; ii) should cause no adult bee mortality; iii) should have no adverse effects on human bee keepers or honey intended for human consumption; and iv) should be capable of delivery into the hive.

The composition of the present invention may be applied either via aerosol or with a carrier component or with a patty. Aerosol is a flash treatment suitable for broodless periods. The aerosol may be used in the late fall, winter, or early spring. A patty treatment is a "time release" formulation that kills mites on mature or newly hatched adults. The patty treatment may be used in the spring, summer and/or fall. The carrier component can be a spray or a solid material. As is known in the art, the vehicle or carrier to be used refers to a substrate such as a membrane, hollow fiber, microcapsule, gel, polymers, or the like. All of these substrates have been used to release insecticide compositions in general and are well known in the art. In aerosol form, the fatty acids will come in contact with the mite when sprayed into the honey bee hive. In solid form, the hygienic action of the honey bees is the proposed mechanism by which the material will be spread around the hive. Additionally, the slow release of the fatty acids from the solid matrix may also have miticidal properties.

The acid phase used for this purpose comprises preferably a fatty acid, a dicarboxylic acid, a dimeric fatty acid, an oligomeric fatty acid, a hydroxy fatty acid or a mixture of at least two of these acids, fatty acids and hydroxy fatty acids being particularly preferred and fatty acids most preferred.

Fatty acid is in particular that of general formula R-COOH in which R represents a saturated or unsaturated, branched or linear alkyl or alkenyl radical with 5 to 20 C atoms, or from 5 to 15 C atoms, or from 5 to 12 C atoms. Particularly preferred in this connection are fatty acids selected from the group consisting of valeric acid, caproic acid, enanthic acid, caprylic acid, 2- ethylhexanoic acid, pelargonic acid, myristic acid, palmitic acid, palmoleic acid, stearic acid, isostearic acid, oleic acid, linoleic acid, and arachidic acid, and also mixtures thereof.

Temperature flexible polymer matrix is that the exact composition of the matrix can be altered to adjust the melting point of the material, and thus, the degree of solidness. One embodiment of the temperature flexible polymer matrix solid is an esterification reaction between valeric acid (pentanoic acid) (C ₅ ) and/or caproic acid (hexanoic acid) (C ₆ ) and/or enanthic acid (heptanoic acid) (C ₇ ) and glycerin. Another embodiment of the temperature flexible polymer matrix solid is an esterification reaction between azelaic acid (nonanedioic acid) (C ₉ ), stearic acid (octadecanoic acid) (C _i8 ), and glycerin (1,2,3-propanetriol) . Other

embodiments may include adipic acid (C ₆ ) and lauric acid (Ci ₂ ). Polyethylene glycol (PEG) may also be used as a solid matrix. The amount of composition used will be at least an effective amount. The term "effective amount," as used herein, means the minimum amount of composition needed to treat honey bee pests to a treated area or object when compared to the same area or object which is untreated. Effective concentrations of the composition in the compositions may vary between about 0.00001% to about 99.99% (preferably about 0.00001% to about 50%, more preferably about 0.00001%) to about 10%>, more preferably about 0.00001%) to about 1%, more preferably about 0.00001% to about 0.1%, more preferably about 0.00001% to about 0.01%). Of course, the precise amount needed will vary in accordance with the particular composition used; the type of area or object to be treated; the number of days of treatment needed; and the environment in which the area or object is located. The precise amount of composition can easily be determined by one skilled in the art given the teaching of this application. For example, one skilled in the art could follow the procedures utilized below; the composition would treat more than 50%> of the honey bee pests and would be statistically significant in comparison to a control. The

composition may or may not contain a control agent for honey bee pests, such as a biological control agent or an insecticide known in the art to kill honey bee pests. Other compounds may be added to the composition provided they do not substantially interfere with the intended activity of the composition; whether or not a compound interferes with composition activity can be determined, for example, by the procedure utilized below. Such other compounds may be present generally from about 0.0025%> to about 20% in the composition.

The following examples are intended only to further illustrate the invention and are not intended to limit the scope of the invention as defined by the claims.

Example 1 Laboratory vial bioassays were performed to compare the acute contact toxicity of C8910 fatty acid blend to honey bees and Varroa mites. Vial testing was performed by dissolving the blend in acetone and coating the sides of a 1.5-quartjar (honey bees) or a 20-mL scintillation vial {Varroa mites). The vials and jars were rotated on a hot dog roller to allow the solvent to evaporate and the fatty acid blend to form an even coat inside the container. Containers were prepared with varying concentrations of the C8910 fatty acid blend, plus a negative solvent control. Each container received 10 mites collected from adult bees using a "sugar-shake" (Macedo, P. A.; Wu, J.; and Ellis, Marion D. (2002). Using inert dusts to detect and assess Varroa infestations in honey bee colonies. Faculty Publications: Department of Entomology) or 20 three-day-old adult bees taken from managed apiaries (Johnson, R.M., Dahlgren, L.,

Siegfried., B.D. Ellis, M.D. (2013) Acaricide, Fungicide and Drug Interactions in Honey Bees (Apis mellifera). PLoS ONE 8(1): e54092.). Mites were maintained in the dark at 27°C and fed on a pre-pupa bee supplied 1 hour after initiation of the test. Bees were maintained in a dark humid incubator (34°C) and supplied 1 : 1 (w/w) sucrose water ad libitum. Mortality was scored at 1, 4 and 24 hour(s). At least three replicates were performed for each treatment. The LC ₅₀ was calculated using a log-probit analysis implemented in R. Preliminary experiments show that the C8 and C9 fatty acid components of the C8910 fatty acid blend were the most toxic to mites.

The toxicity of pure C8910 fatty acid blend (in a 1 : 1 : 1 : ratio) was found to be

significantly higher for Varroa mites than for honey bees. The LC ₅₀ for honey bees is

approximately 2-fold higher than for Varroa mites, which suggests that there is a sufficient margin of safety to use the existing C8910 fatty acid blend as a miticide.

Examples 2-8. A polyester polymer matrix was prepared through an esterification reaction of Emerox 1110 azelaic acid, Emersol 132 stearic acid, and Emery 916 glycerin at 220°C for 24 to 36 hours until the acid value (AV) was less than 5 mg KOH / gram of polymer. A low AV indicates that there are few free fatty acids in the sample; therefore, most of the product exists as esters. The melting point of the final polyester polymer was adjusted through alterations to the weight percent of each component. Optionally, Emersol 221 oleic acid or adipic acid was added to adjust the melting point. See Table 1.

Table 1.

In the preferred form, the starting materials were 21.3 % w/w of Emerox 1110 azelaic acid, 62.8 % w/w of Emersol 132 stearic acid, and 15.8 % w/w of Emery 916 glycerin.

Example 9

The polyester polyols from examples 6, 7, and 8 were evaluated in-hive to determine which the most promising delivery system for the C8910 fatty acid blend was. One 25g patty of each polyol was placed between two boxes in a strong full-sized colony. The patties were prepared by heating each polyol in a microwave and then pouring 25 grams into a weigh boat. The polyester polyol was then scooped out of the weigh boat and spread evenly onto a sheet of paper towel and allowed to solidify. Three polyester polyol patties and one control patty

(vegetable shortening) were placed in the same colony to allow direct comparisons between formulations. This was repeated in five colonies from the same apiary. Percent remaining of the patties was assessed visually and through digital photos every 3 days for 10 days. A two-way ANOVA test in SPSS was used to compare percent removal of polyol in the treatment groups.

The polyester polymer from example 6 was removed at the same rate as the control patty, which is a commonly used material by backyard and sideline beekeepers. The polyester polyols from examples 7 and 8 were removed at a slower rate than either example 6 or the control, but similar to each other.

Example 10-18

Table 2 shows the bee/mite LC ₅₀ ratio, upper and lower confidence intervals, and margin of safety. An asterisks * indicates that there is a significant difference between the bee and mite LC ₅₀ values using a ratio test. The LC ₅₀ values for Varroa mites for pure C5, C6, C7, and CI 1 acids were approximately the same (2.08 mg/cm ² ; 1.61 mg/cm ² ; 1.87 mg/cm ² ; 1.94 mg/cm ² ). Additionally, the LC ₅₀ for Varroa mites of the C8910 blend was about 2-fold higher than the LC ₅₀ for C5, C6, C7, and CI 1 acids, suggesting that the pure fatty acid chains (C5, C6, C7, CI 1) are more toxic to Varroa mites than the C8910 fatty acid blend. The LC ₅₀ for honey bees of pure C5 was the largest (>3000 mg/cm ² ), which suggests that C5 provides the largest margin of safety for honey bees (Robertson, J. L., & Robertson, J. L. (2007). Bioassays with arthropods. Boca Raton: CRC Press). Table 2

Example 19

The C8910 fatty acid blend was incorporated into polyester polyol example 6 to determine a colony-level dose and if there was a repellency effect of the formulated C8910 fatty acid blend. Two lOg patties of a 1% w/w C8190 fatty acid blend and a 4% w/w C8910 fatty acid blend treatment were placed in 5 full-sized bee colonies in the same apiary. The patties were prepared similarly to previous examples by heating the polyester polyol example 6 and pouring out 10 grams into a weigh boat. Either 1% (lOOmg of C8910 fatty acid blend) or 4% (400mg C8910 fatty acid blend) was added to the lOg patty and mixed thoroughly. Two patties with 4% C8910 fatty acid blend, two patties with 1% C8910 fatty acid blend, and two control patties (polyester polyol example 6 without C8910 fatty acid blend) were placed in the same colony. The patties were assessed visually through digital photos every 3-4 days for 7 days. A two-way ANOVA test was used to compare mean percent removal of the control, low dose, and high dose patties. After 7 days, the removal of the control patty was significantly greater than the high dose patty, but not significantly different from the removal of low dose patty. The removals of the high and low dose patties were not significantly different from each other. Example 20

The efficacy of the C8910 fatty acid blend was tested in 36 full-sized hives. Two colonies in each apiary received a 50g patty with 4% w/w C8910 fatty acid blend in polyester polyol example 6, Hopguard (a registered varroacide), or no treatment. The patties were prepared by heating the polyester polyol example 6 in a microwave and pouring 50g into a weigh boat. The patty was then removed and spread evenly across one sheet of paper towel and allowed to cool. Once the paper towel was hardened it was cut in half to make two 25g rectangular strips.

Treatments were replicated across 6 apiaries for a total of 12 colonies per treatment

All colonies were in standard 8-frame Langstroth hives with solid bottom boards.

Colonies were a mix of overwintered colonies and new colonies started from packages in spring. All colonies were maintained according to standard beekeeping practice, but had not been treated for Varroa mites previously in the season. Drop zone dead bee traps were placed at the entrance of the colonies at three apiaries to determine the number of dead bees before and after treatment. Dead bee traps were made with 2'x4'xl/2' wooden frames with ½" hardware cloth on top and window screen on the bottom. Colonies were visited on a weekly basis where the following measurements were taken: number of dead bees in the drop-zone trap, number of adult mites under 100 cappings, and number of phoretic mites on -300 adults bees using an alcohol wash. The 50g C8910 fatty acid blend patty was replaced each week and weighed to determine the rate of removal.

A two-way ANOVA was used to compare the effect of treatment on Varroa mites on adult bees and under cappings. Results showed that there was no significant difference in mites per 100 adult bees between the different treatment groups or the control. However, over half of the hives that started the experiment had a mite population of greater than 3%, which is the treatment threshold during the late summer (Honey bee heath coalition (2015). The Keystone Policy Center. Tools for Varroa Management: A guide to effective Varroa sampling and control). Nearly 20% of the hives had mite populations greater than 10%.

Example 21

Using caged adult bees, the safety and efficacy of 15% C8910 fatty acid blend in food grade mineral oil as an aerosol application was tested. Approximately 300 bees (by weight) were collected from brood frames of a colony and placed in a cylindrical cage (10 cm x 20 cm) constructed of #8 hardware cloth and inserted into a plastic petri dish to collect dead Varroa mites falling from the bees. The cage was placed inside an deep hive body with empty frames to fill out the internal space. A cold-fogger used for structural pest control (Actisol Compact Portable Aerosol Unit) was filled with either 15% C8910 fatty acid blend in mineral oil or straight mineral oil, set to 15-20 psi and the aerosol was applied through the entrance for 30 sec while moving the nozzle around inside the hive. After application, the cage was removed, the bees fed with sugar water and maintained in an incubator for 24 hours to assess total mite fall.

Three replicate cages were treated with 15% C8910 fatty acid blend for 30 sec. The observed bee mortality was low (1-2 bees) and comparable to the single control cage. A mean of 75%) of mites present on bees treated with 15%> C8910 fatty acid blend fell over the 24 hours following treatment compared to 19% mite fall in the mineral oil control cage. Longer applications of the mineral oil blend with several cages of bees found that there was no increased bee mortality even after 2 min of aerosol application.

Example 22

A field experiment was conducted in which 15%> C8910 fatty acid blend aerosol was applied to a single full-sized colony in August to test the application method and further assess any negative effect on the colony. The C8910 fatty acid blend was applied with the Actisol as in the preliminary caged bee experiment, but the duration of treatment was extended to 2 min and 3 repeat applications were made over 3 weeks. Mite fall was measured using a "sticky board" made of a manilla file folder smeared with petroleum jelly that was placed on the bottom board of the hive. Samples of bees were taken for standardized alcohol washes. A drop zone dead bee trap was placed in front of the colony to collect dead bees. This single-colony experiment was considered a failure because, despite seeing hundreds of mites fall after each C8910 fatty acid blend application and no accumulation of dead bees in dead bee traps, the numbers of mites present in the alcohol washes continued to climb over the 3 weeks of the experiment. It was determined that the C8910 fatty acid blend aerosol had no effect on developing mites under cappings in the brood and that the population of phoretic mites on adult bees was being rapidly replenished by this protected population of mites. We decided that, while it would theoretically be possible to control mites in the summer with C8910 fatty acid blend aerosol, the amount of labor and the number of visits to the apiary that would be required would be unacceptable to nearly all beekeepers.

Example 23

A second field experiment was conducted with two apiaries and 16 colonies during November on a day when temperatures were over 40°F and bees were not so tightly clustered such that the vapor could not penetrate the cluster. After each treatment a manilla file folder covered with petroleum jelly was placed on the bottom board to catch falling mites. The folder was retrieved after 24 hours to count the number of mites killed in each application.

C8910 Fatty Acid Blend Application: Pure C8910 fatty acid blend (1 : 1 : 1) was mixed in USP mineral oil to a concentration of 15% and poured into the tank of the Actisol compact unit. To apply the 15% C8910 fatty acid blend, the Actisol was plugged in to a car inverter, powered on and the pressure adjusted to 15-20 psi. The handle was pressed outside a colony to insure material was flowing and ready for application. The wand was inserted into the open entrance of an 8-frame colony and paper towel was used to plug up the remainder of the entrance. The tip of the wand as turned to a 90-degree angle to avoid direct application to bees and held on the side of the bottom board. The handle was pressed and held for 2 minutes. The wand was removed and the entrance completely plugged with paper towel for an additional 10 minutes.

Oxalic Acid Application: A Varrox vaporizer was filled with 2 g oxalic acid (Brushy

Mountain) for each colony to be treated. The vaporizer was inserted into the middle of the colony and the entrance blocked with toweling. The vaporizer was energized by connecting to a 12V battery for 4 minutes. Electricity was disconnected and the vaporizer and towels were allowed to sit for 10 minutes prior to removal.

The standard oxalic acid treatment was much more effective in the 8 colonies receiving this treatment than the 8 colonies receiving C8910 fatty acid blend application (p<0.05, T-test).

In the oxalic acid treated colonies 635 (215 SE) fell in the 24 h following application. In the C8910 fatty acid blend treated colonies 75 (16 SE) mites fell over the same period. While dead bee traps were not used for this trial, there were no additional dead bees noted stuck to the sticky card or in front of colonies subjected to either treatment.

It appears that an aerosolized 15% solution of C8910 fatty acid blend in mineral oil is safe for bees, but does not have sufficient toxicity to Varroa to serve as an mite control when applied as an aerosol in either summer or winter applications. While multiple applications may achieve acceptable mite control, this would require more effort on the part of the beekeeper than the current standard wintertime treatment, oxalic acid. It is to be understood that the various components of a method for treating honey bee pests to an object described herein can be combined in any manner or combination not inconsistent with the objectives of the present invention. For example, a method for treating honey bee pests to an object can comprise any step described herein in combination with any mono- and di -fatty acids comprising C ₅ to C ₂ o, and mixtures thereof herein in combination with any carrier material described herein.

Various embodiments of the carrier material have been described in fulfillment of the various objectives of the invention. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Numerous modifications and adaptations thereof will be readily apparent to those skilled in the art without departing from the spirit and scope of the invention.