SLOW-RELEASE DEVICE FOR DELIVERING STABILIZED HONEY BEE BROOD PHEROMONE WITHIN THE HIVE

FIELD OF THE INVENTION This invention relates to a novel device and method of introducing a stabilized formulation of the 10-component honey bee brood pheromone into a hive in such a manner that worker honey bees are exposed for several weeks to the pheromone exuding though a plastic membrane on one side of a heat-sealed bag reservoir held in a solid casing. The invention further relates to a method of affixing said casing to a T-shaped holder, or incorporating said casing into the holder, and suspending the holder between the comb frames in a hive unit or Super. Lastly the invention relates to methods of using said holder, with the pheromone-containing bag and casing attached or incorporated, to enhance the vigor of the honey bee colony and to induce increased collection of pollen and nectar.

BACKGROUND OF THE INVENTION

A honey bee colony consists of a single queen, who is usually the mother of all other colony members, 10,000-30,000 semi-sterile female workers, and from zero to a few thousand males (drones), depending on the time of year. Eggs, larvae, and pupae are collectively referred to as brood. Female adult workers perform all of the behavioral tasks associated with colony growth and maintenance. Worker honey bees perform different tasks as they age, a phenomenon referred to as division of labor or temporal polyethism. When bees emerge from their cells as adults they normally clean cells. As they age, they take on other tasks, passing through sequential stages in which they feed larvae, process and store food, secrete wax and construct comb, and guard the entrance. The most obvious, and final, change in behavior occurs when bees are about three weeks old, when they begin foraging (Lindauer 1952; Seeley and Kolmes 1991). In this way, the adult work force may be viewed as divided between non-foraging bees, that rear brood and maintain the hive, and foragers that collect food outside the hive. Foraging labor is also divided. Some foragers return to the nest carrying nectar, some return with

loads of pollen carried on their rear legs, some return carrying both nectar and pollen, and a small proportion of foragers collect water and plant resin called propolis.

Pollen foragers collect pollen from available plant sources. They then return to the nest and deposit their loads of pollen directly into cells of wax comb typically located on the perimeter of the brood nest area. The brood nest area is in the center of the colony where each immature bee is cradled in her own cell. Stored pollen is consumed by nurse bees that use the proteins derived from the pollen to produce proteinaceous hypopharyngeal glandular secretions that are fed to developing larvae. This process reduces the quantity of stored pollen in the hive .

Since the early 1930s apicultural researchers have hypothesized that larval honey bees produce a pheromone that stimulates worker bees to leave the hive and forage for pollen that they will bring back to the hive to replenish and increase the amount of stored pollen in the hive, so that the larvae can be fed (Filmer 1932: Free 1967). More recently, evidence has accumulated in support of this hypothesis (Al-Tikrity et al. 1972; Free 1979; Eckert et al. 1994; Dreller et al. 1999).

As evidence accrued, the huge implications of the potential availability of a synthetic pheromone became apparent. A synthetic honey bee brood foraging pheromone introduced to a hive could induce enhanced pollination of essential crops by honey bees, an enterprise valued at an estimated $15 billion USD annually (Morse and Calderone 2000). Additional pollen brought back to a pheromone-treated hive could be used as a resource to stimulate and support colony growth (Fewell and Winston 1993; Pankiw et al. 2004a). Such an outcome would be particularly advantageous in current times, when the demand for honey bees as pollinators remains high or is increasing, at the same time that honey bee colonies have been depleted by natural enemies like the varroa mite (Sammantaro et al. 2000), and by new afflictions like colony collapse disorder (Stokstad 2007). Beekeepers routinely invest in labor and inputs such as protein supplements to stimulate colony growth so that maximum colony size is synchronized with blossoming of the crop for optimizing pollination, and major nectar flows for optimizing honey

production (Ambrose 1992). Larger colonies field more foragers for pollination and nectar collection (Free 1967), and are better able to withstand parasites and pathogen infections (Sammantaro et al. 2000).

Colony growth also leads to colony-level reproduction. Honey bee colonies reproduce through a process of budding, commonly referred to as swarming (Winston 1987). Colony growth rates and trajectories are critical to colony reproductive rate, the size of swarms, and the timing of swarming (Lee and Winston 1985a,b, 1987). Survival of swarms is likewise dependent on parental colony size and the timing of issue (Seeley 1985; Lee and Winston 1985a,b, 1987). Lee and Winston (1985b) found a positive correlation between swarm size and both brood production and emergent worker weight in newly founded colonies. Larger colonies invest more workers in swarms, which confers an increased probability of swarm survival (Lee and Winston 1987). Larger swarms also produce more total brood comb, the area in which brood are reared (Lee and Winston 1985a). The number of swarms that a colony produces is positively correlated with the amount of pupae at the time the first swarm issues (Winston 1987).

Beekeepers take advantage of honey bee colony growth leading to reproduction as the only means by which they can increase colony numbers or replace dead colonies. Instead of allowing colonies swarm naturally, beekeepers choose the timing of swarming by dividing a large colony into two or three additional units, depending on parental colony size. To each divide, a mated queen is introduced, resulting in a new colony. Frequency of colony division and number of divisions is dependent on rate of colony growth and size of the parental colony. The new colonies may be added to the beekeeper's apiary or sold to other beekeepers as: 1) packaged bees in a screened cage, 2) a five-frame nucleus colony, or 3) a standard 10-frame colony. If a synthetic honey bee brood pheromone could be used to promote colony growth it would provide beekeepers with a tool to increase the numbers of colonies in the apiary and to replace dead colonies, and in addition it would provide for increased profits through the sale of packaged bees, nuclei or colonies to other beekeepers.

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The path to a synthetic pheromone opened up when LeConte et al. (1990) identified 10 fatty acid ester components of the putative foraging pheromone. Pankiw and Page (2001) and Page and Pankiw (2002) subsequently found that a synthetic blend of the pheromone (ethyl linoleate 1%, ethyl linolenate 13%, ethyl oleate 8%, ethyl palmitate 3%, ethyl stearate 7%, methyl linoleate 2%, methyl linolenate 21%, methyl oleate 25%, methyl palmitate 3%, and methyl stearte 17%) lowered the foraging age of worker bees, and also lowered the neurosensory response threshold to sucrose, a measure of increased propensity to forage for pollen rather than nectar (Pankiw and Page 2000; Pankiw 2003; Pankiw et al. 2004b).

Many studies have been done with the 10-component synthetic pheromone, offering minute amounts of the composition to honey bees within the hive by dispensing it daily on glass plates. Using this methodology, a remarkable list of phenomena induced by the synthetic brood pheromone has been compiled. These phenomena and the accompanying benefits include:

• increasing the ratio of pollen to non-pollen foragers (Pankiw and Page 2001),

• increasing the number of pollen foragers by up to 150% (Pankiw 2004a,b; Pankiw and Page 2001; Pankiw and Rubin 2002; Pankiw et al. 1998; 2004a; Sagili 2007; Schulz et al. 2002), • increasing the number of pollen forager trips per unit time (Pankiw 2007),

• raising the weight of pollen returned to the hive (Pankiw 2004a; Pankiw et al. 2004a),

• increasing colony growth rate by inducing the colony to construct more brood cells, raise more larvae and increase the population of adult worker bees in the colony in summer and winter (Pankiw et al. 2004a; 2006),

• lowering the age at which worker bees become foragers (LeConte et al. 2001 ; Pankiw 2004; Pankiw et al. 2004a,b; Sagili 2007),

• increasing the protein content in the hypopharyngeal glands of nurse bees, thus increasing the quality of the diet fed to larvae (Mohammedi et al. 1996; Pankiw et al. 2004a,b; Sagili 2007),

• raising the number and activity level of nectar foragers, thereby further increasing pollination of flowers that are visited (Pankiw 2004a; Pankiw et al. 2004a), and

• stimulation of consumption of dietary pollen in the winter, leading to increased colony vigor (Pankiw et al. 2006).

Despite the above benefits, and a high demand for a commercial product based on the synthetic brood pheromone, no such product has been developed since the 5 December 2002 issue of the patent describing the pheromone and its uses (Page and Pankiw 2002). This was largely because four of the 10 components of the synthetic pheromone are unstable, and break down at room temperature, giving any product based on the unstabilized pheromone an unacceptably short shelf life (Pankiw et al. 2006). In fact, Page and Pankiw (2002) teach that the "synthetic brood pheromone is easily oxidizable, and must be stored in low-oxygen conditions, preferably at -20°C, and most preferably at -7O ⁰ C if it will be stored for any long period of time. "

It was not clear, whether the short duration of bioactivity of the synthetic and natural pheromone was based on biologically active levels of its constituents, which then fell to sub-threshold levels, or to minute amounts of breakdown products that over time rose to inhibitory levels. This question was answered by Pankiw et al. (2006), who added an antioxidant to the synthetic pheromone and extended the lifetime of the stabilized synthetic pheromone to well over a year. Moreover, the antioxidant had no effect on either the bioactivity of the synthetic pheromone or the health of bees to which it was exposed.

There is abundant well-developed technology for the controlled release of volatile pheromones. However, the honey bee brood pheromone is comprised of 10 non- volatile components. After being produced by larval bees, it becomes impregnated into the wax comb, and worker bees are exposed by contact to minute amounts of the pheromone when they perform tasks within the hive. Therefore, despite the availability of stabilized brood pheromone, there remained a problem of delivery of the non- volatile pheromone

within the hive at biologically realistic minute doses over a sustained period of time. Although Page and Pankiw (2002) and Pankiw et al. (2006) mention a number of substrates that could be used to incorporate the pheromone and serve as a slow-release device, none had been tested.

SUMMARY OF THE INVENTION

This invention is directed to a method of releasing the stabilized synthetic 10-component honey bee brood pheromone at a constant rate for an extended duration when it is contained in a device that is inserted into a honey bee hive.

Conceptually the method for releasing the honey bee brood pheromone involves placing a formulated composition in a reservoir that is designed to release the pheromone in a sustained manner at a controlled rate. However, with the specific non-volatile honey bee brood pheromone formulation, we show that practical achievement of this method is uncertain. In the following examples, we document unsatisfactory performance with respect to release rate, longevity or modification of honey bee behavior, with many pheromone-laden devices, including the following: wooden tongue depressors, perforated culture tubes with a sponge insert, water-based gels, transfer pipette bulb ends, beeswax discs, bubble caps with a sponge insert, and plastic pouches with a filter paper insert. We also note other unsuccessful devices and formulation ingredients, including: cardboard, lard, hard sugar candy, felt and cigarette filters.

In the successful method, the stabilized formulation is placed in a small flat heat-sealed pouch with one or both sides permeable to the pheromone composition, which slowly exudes through one or both walls of the pouch. The pouch can be mounted in a firm casing, which in turn can be placed in a holder that is suspended between the frames in a hive unit, or super.

Be example, we show that when 200 μL of a pheromone composition comprising methyl palmitate 2.11%, ethyl palmitate 4.19%; methyl stearate 17.30%, ethyl stearate 8.04%, methyl oleate 21.66%, ethyl oleate 7.50%, methyl linoleate 8.09%, ethyl linoleate 3.82%,

methyl linolenate 16.53%, ethyl linoenate 10.77% and tertiary-butylhydroquinone 0.05% was loaded into small plastic pouches, measuring 38.0 x 35.0 mm, made of pheromone- impermeable 4.0 mil Mylar on one side and pheromone-permeable 6.0 mil brown low- density polyethylene (LDPE) on the other, a constant release rate of approximately 0.3 mg per day was obtained. By further example, we show that when said pheromone-laden pouch was mounted in a 35 mm slide frame casing and suspended by wire between the frames of a hive unit, or super, in an active hive, the ratio of pollen forager worker honey bees to non-pollen foragers was unexpectedly and significantly increased for 36 days, the weight of pollen brought back to the hive by returning foragers was significantly increased, and after 31 days the number of adults bees and the area of comb in which brood are raised also significantly increased by 40.7% and 38.5%, respectively.

In another aspect, this invention is directed to a family of devices: one that contains the stabilized, synthetic 10-component honey bee brood pheromone composition in a reservoir and releases it at a constant rate through a membrane bounding the reservoir; a second device in which the reservoir is mounted in a firm casing; and a third device comprising a holder in which the casing holding the reservoir can be suspended between the frames in a honey bee hive unit, or super. In a modification of this aspect, any two devices, or all three of the devices, can be combined.

The pheromone-releasing device can be a heat-sealed plastic pouch containing synthetic stabilized 10-component honey bee brood pheromone, comprising methyl palmitate (0.3- 50%), ethyl palmitate (0.3-50%), methyl stearate (1.7-50%), ethyl stearate (0.7-50%), methyl oleate (0.3-50%), ethyl oleate (0.8-50%), methyl linoleate (0.2-50%), ethyl linoleate (0.1 -50%), methyl linolenate (0.2-50%), ethyl linolenate (1.3-50%) and a food- grade antioxidant selected from among the group comprised of butylated hydroxyanisole, butylated hydroxytolulene, tertiary-butyl hydroquinone, citric acid, vitamin E or its derivatives, or any combination of two or more of said antioxidants (0.001-50%), with one or both faces of the pouch device permeable to the pheromone.

The pheromone-containing plastic pouch device can have one pheromone-impermeable face, firm or with varying degrees of flexibility, composed of a substance such as Mylar, and a pheromone-permeable face composed of a material, such as polyethylene.

Said pheromone-containing plastic pouch, if flexible, can be mounted in a firm casing, such as a 35 mm slide frame, or if the pheromone-impermeable face is firm, the need for a firm mounting can be eliminated.

Said mounted pouch in a casing, or unmounted pouch with a firm backing, can be placed in a holder made of cord, metal, wood or plastic and suspended between the frames of a hive unit, or super, in an active hive.

Said holder can be flexible flat plastic, with a hinged portion that bends to a 90° angle from the vertical portion to form a T-shaped structure, and has slots in which the mounted pouch in a casing, or unmounted pouch with a firm backing can be inserted.

Said holder can them be suspended by the hinged portion bent to a 90° angle between two frames in a hive unit, or super, so that the pheromone-permeable side of the pouch is in juxtaposition to honey bee brood comb.

Said holder can be modified so that the end nearest the brood comb, becomes the pheromone-impermeable backing to the pheromone-containing pouch, combining all three said devices into one device.

In a third aspect, the invention is directed to methods wherein the pheromone-containing pouch mounted in a firm casing placed in a holder, or any combination of said three devices that results in two devices or one composite device, can be suspended between the frames in a hive unit, or super, and over a period of two weeks or longer can be used to: to increase the ratio of pollen to non-pollen foragers among worker honey bees; lower or delay the age of first foraging by worker honey bees; increase the total number of foraging worker honey bees; increase the load weight of pollen returned to the colony by

pollen-foraging worker honey bees; increase the number of pollen grains carried by the bodies of non-pollen-foraging worker honey bees; increase pollination of the target crop near or within which honey bee colonies or pollination units are placed; stimulate hypopharyngeal brood food gland development and gland protein production in honey bee workers; stimulate the consumption of dietary protein or other diet components and antibiotic supplements by honey bees; increase the number of bees reared within the colony; or inhibit absconding (swarming) of bees from the colony.

In a fourth aspect, the invention is directed to methods wherein the pheromone- containing pouch mounted in a firm casing placed in a holder, or any combination of said three devices that results in two devices or one composite device, can be suspended between the frames in a hive unit, or super, in combination with synthetic honey bee queen mandibular gland pheromone.

In a final aspect, the invention is directed to methods wherein the pheromone-containing pouch mounted in a firm casing placed in a holder, or any combination of said three devices that results in two devices or one composite device, can be suspended between the frames in a hive unit, or super, and synthetic honey bee queen mandibular gland pheromone is administered at the same time to nearby flowering plants on which enhanced pollination is desired.

DRAWINGS

Exemplary embodiments are illustrated in referenced figures of the drawings. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

Figure 1 illustrates an exploded isometric view of a pouch comprising a base with reservoir, a pheromone permeable membrane and membrane holding frame.

Figure 2 illustrates a pheromone permeable membrane.

Figure 3 illustrates a composite isometric view of a pouch.

Figure 4 illustrates a front view of a holder.

Figure 5 illustrates an isometric view of a holder.

Figure 6 illustrates an isometric view of a holder with a top hinged portion bent at a 90° angle.

Figure 7 illustrates an isometric view of a holder with pouch ready for insertion in the opening in the holder.

Figure 8 illustrates an isometric view of a holder with the pouch in place in the opening in the holder.

Figure 9 illustrates a front view of the holder with pouch in place.

Figure 10 illustrates a section view of the holder and pouch taken along section line A-A of Figure 9.

Figure 11 illustrates an isometric view of a holder with part of the holder and pouch cut away.

Figure 12 illustrates an enlarged detail of the portion indicated by circle B of Figure 11.

DETAILED DESCRIPTION QF THE INVENTION

This application contains certain terminology in common usage in the apicultural world.

Although the term hive technically refers to the physical structure in which honey bees reside, in common usage it often describes the inhabited structure. This is also referred to as a colony. Therefore, throughout this application the terms hive and colony are used interchangeably. In much of the world, supers, or hive units, are the box-like structures

that are super-imposed in a stack to expand the physical structure of the hive. Hung inside each super are numerous removable rectangular frames, on which honey bees build wax comb comprised of many hexagonal wax cells, in which they raise their brood and in which they store pollen or honey.

We set out to develop an operational slow-release device for the stabilized synthetic 10- component honey bee brood pheromone. To avoid excessive costs to beekeepers in handling hundreds to thousands of hives, we set a minimum target for longevity of the device at 14 days, and a target constant release rate over that time of 0.1-1.0 mg per day. This application describes numerous failures to meet these targets. After two years of intensive effort, we constructed and successfully field tested an operationally-feasible device.

As will be apparent from the following examples, many potential release devices were developed, tested, and found to be unsatisfactory. Not included in the detailed examples are the following unsatisfactory potential pheromone delivery substrates and devices: pheromone-impregnated cardboard hung between the frames (shredded and destroyed by the bees in short order); lard (would be rapidly consumed as food); hard sugar candy (would probably last a fairly long time, but would require heat to make, causing breakdown of pheromone components); felt (became tangled in the bees tarsi); and cigarette filters (rapidly destroyed by the bees).

As will further be apparent from the following examples we have followed a path that led to the unexpected discovery that the stabilized synthetic 10-component honey bee brood pheromone can be dispensed from a thin heat-sealed plastic pouch with one face made of impermeable plastic, and the other made of permeable plastic, through which the pheromone exudes. We have also discovered that the plastic pouch can be firmly fixed into a rigid plastic casing, which in turn can be offered to honey bees by suspending it between the frames within a hive unit or super. Finally we have found that the slow- release device suspended between the frames mimics the effects of dispensing minute amounts of the pheromone daily on glass plates within the hive. Specific statistically-

significant effects measured were an increase in the ratio of pollen to non-pollen foragers, an increase in the weight of the pollen load carried back to the hive by returning worker honey bees, an increase in the area of brood comb constructed for rearing larvae, and a significantly larger population of adult worker honey bees than in untreated control colonies. Unexpectedly a single device placed with the hive was effective for up to 36 days, well beyond the minimum target longevity that we set at 14 days for an operational device.

The drawings generally illustrate a holder made of rigid to semi-flexible material (most likely plastic), fitted with slots that can hold a slow-release pheromone device (in this case a plastic pouch with one side pheromone impermeable, and the other side permeable to the non-volatile stabilized synthetic 10-component honey bee brood pheromone), fixed in a rigid to semi-flexible casing (in this case a 35 mm plastic side frame mount). The holder can be bent 90 degrees at the top, taking a T configuration that allows it to be suspended between two frames in a honey bee hive unit, or super, such that the pheromone release device hangs adjacent to the comb area of two frames.

Figure 1 illustrates an exploded isometric view of a pouch comprising a base with reservoir, a pheromone permeable membrane and membrane holding frame. Figure 2 illustrates a pheromone permeable membrane. Figure 3 illustrates a composite isometric view of a pouch. Figure 4 illustrates a front view of a holder. Figure 5 illustrates an isometric view of a holder. Figure 6 illustrates an isometric view of a holder with a top hinged portion bent at a 90° angle. Figure 7 illustrates an isometric view of a holder with pouch ready for insertion in the opening in the holder. Figure 8 illustrates an isometric view of a holder with the pouch in place in the opening in the holder. Figure 9 illustrates a front view of the holder with pouch in place. Figure 10 illustrates a section view of the holder and pouch taken along section line A-A of Figure 9. Figure 11 illustrates an isometric view of a holder with part of the holder and pouch cut away. Figure 12 illustrates an enlarged detail of the portion indicated by circle B of Figure 11.

Example 1

Wooden Tongue Depressor Device

Honey bees routinely destroy and remove foreign organisms and objects that enter (or are put into) their hive. We tried to use or construct devices that were resistant to the honey bees' best defensive and destructive efforts, to ensure they met our longevity target of 14 days. One such device was a wooden tongue depressor. We tested infant and regular size wooden tongue depressors (Puritan No.700, Hardwood Products Co., Guildford, Maine USA). Ten tongue depressors of each size were cut in half, and a small hole drilled in each cut end for hanging. Each end was then treated with 40 μL of stabilized synthetic brood pheromone delivered from a 200 μL pipette in a 3-5 cm long swath.

Treated devices were allowed to dry at room temperature for three days. They were then stacked, wrapped in aluminum foil, bagged in Mylar and shipped to Texas A&M University.

The devices were introduced for 26 days to 20 colonies, 10 per treatment. Colonies were initiated as 2 pound packages of bees. Factors measured included: 1) ratio of pollen foragers to non-pollen foragers entering colonies during a 5 minute interval measured two times per week for four weeks; 2) estimate of adult bee numbers; 3) brood comb area; 4) pollen area; 5) honey area; and 6) empty comb area. Proportional data for ratios of pollen to non-pollen foragers were analyzed using Chi square contingency tables. Data for adult bee numbers, brood comb area, pollen area, honey area and empty comb area were analyzed by one-way analysis of variance (ANOVA). In all cases α = 0.05.

Although the tongue depressors were not chewed to pieces, and therefore were resistant to destruction, there was no significant effect on the ratio of pollen to non-pollen foragers, as measured by 5-minute entrance counts (Table 1), nor was there any effect on colony growth (Table 2). We concluded that the brood pheromone readily impregnates the wooden substrate offered by the tongue depressors, but that in doing so it becomes unavailable to the bees.

Table 1. Daily pollen to non-pollen forager ratios for honey bee workers returning to hives in which pheromone-treated or non-treated control tongue depressor devices were placed.

aAsterisks indicate higher ratios in pheromone-treated than control colonies.

Table 2. Effect on colony growth of 26 days ' exposure to tongue depressor halves treated with brood pheromone or untreated controls.

Example 2

Perforated Culture Tube and Sponge Device Another potential delivery device was constructed from plastic culture tubes (Grenier Bio-One Ltd., Stonehouse, UK) with holes drilled into the sides and a pheromone-laden sponge inside. We hypothesized that this device would allow honey bees to come in contact with the pheromone, which would be released in a controlled fashion through the holes of the sponge-filled reservoir. Twenty small holes less than 1 mm in diameter were drilled at regular intervals (5 per quadrant) through the wall of each culture tube. The tubes were then tightly filled with industrial absorbent sponge (Pig Mat, New Pig Corp., Tipton, PA, USA). We then prepared a water-based micro-emulsion containing 1% by weight of the stabilized synthetic 10-component brood pheromone. The exact emulsion was determined by adding varying amounts of isopropyl alcohol and isopropyl amine branched dodecylbenzene sulfonate (IPAM) until stability was achieved, i.e. the solution

remained clear and colorless. The final emulsion used was created with a pre-mix of brood pheromone, isopropyl alcohol and IPAM in a 20:40:40% w/w ratio, which was added to water in a 5:95% weight to weight ratio.

Four culture tubes packed with sponges were sent with a bottle of the brood pheromone emulsion to Texas A&M University, where sponges in two of them were each soaked with 1,000 μL of the emulsion, and two were held as untreated controls. The devices were placed in hives, and entrance counts of pollen foragers and non-pollen foragers were done for all four hives at two times during the following week. The mean ratio of pollen to non-pollen foragers was 0.64 for hives that received the pheromone-loaded devices, and 0.80 for control hives, with no significant difference between them. We concluded that the lack of efficacy was because insufficient brood pheromone reached the surface of the sponges inside the holes on the walls of the tubes.

Example 3

Gel Device

The next attempt to create a delivery device for the stabilized synthetic 10-component honey bee brood pheromone consisted of water-based gel formulations, with varying degrees of firmness. Gels were made from modified polysaccharides including Noveon C865 & G888 (Lubrizol Advanced Materials, Inc., Cleveland, OH, USA), and Genugel CI- 102 (CP Kelco, Atlanta, GA, USA), calcium acetate, potassium chloride, sodium hydrosulfate and water. Gels included a biocide (Kathon CG, Rohm & Haas Co., Calgary, AB, Canada) to stop fungal or bacterial growth. The brood pheromone mixture was pre-mixed with Kathon CG, isopropanol, and isopropylamine branched dodecyl benzene sulfonate.

To make the gel, all the solids including the thickeners were dry-mixed using a metal spatula. Water was charged into a 600 ml beaker equipped with an overhead stirrer and heated. At 53 ⁰ C, the solids were trickled into the stirring hot water. Heating and mixing was continued until 73 ⁰ C and held at this temperature for 15 minutes. Heat was removed and the mixture allowed to continue mixing as it cooled down. Once it had cooled down

to 53 ⁰ C, the organic pre-mix which consisted of Kathon CG, Isopropanol, IPAM and the stabilized brood pheromone mixture was quickly added. Mixing was continued for a further 3 minutes after which the mixture was poured into an aluminum cookie pan measuring 28.4 x 18.6 x 1.6 cm (Safeway, Vancouver, BC, Canada and allowed to cool further until the mixture gelled.

Different firmness of gels was created by varying the ratios of the modified polysaccharides. Gels treated with brood pheromone contained the water-based micro emulsion as described in Example 2.

Six replicates of treated and control gels were sent to Texas A&M University. Gels were placed in the hives and hives were monitored daily. Control gels were eaten by worker honey bees within 4.3 days and pheromone-laden gels were consumed within 3.6 days. When bees ingest the brood pheromone, it is removed from circulation, and no longer has the desired effects. These results indicate that gel formulations are unlikely to meet the targets for either a sustained daily dose of 1 mg per hive or a minimum longevity of 14 days.

Example 4 Transfer Pipette Bulb End

A fourth potential delivery device was constructed from a polyethylene transfer pipette bulb with a 0.9 mL bulb capacity (DiaMed Lab Supplies Inc., Mississauga, ON, Canada). The tapered end was cut off at the halfway mark, the bulb end was filled with 300 μL of neat stabilized synthetic 10-component honey bee brood pheromone, and the pheromone- containing reservoir was thermally sealed. Three replicates of three treatments were crafted, empty control bulbs, bulbs filled with the neat pheromone formulation, and bulbs filled with the neat pheromone formulation and enough imbiber beads to immobilize the pheromone.

Each device was placed in a 2O ⁰ C or 3O ⁰ C chamber and swabbed with a 2 cm x 1 cm piece of paper towel after 1 day and after 3 days to remove any brood pheromone that

migrated across the pipette bulb plastic membrane. The paper towel was placed in a small glass vial to which 1.0 mL of a solution of C14 in hexane (10.75% C14) was added as an internal standard for gas-chromatographic analysis, and allowed sit for 20 minutes or longer. Samples were then agitated using a vibrating mixer for 5 seconds and prepared for analysis by capillary gas chromatography.

For analysis by capillary gas chromatography, approximately 1 mg of a blend was dissolved in 1 mL of hexane, and 1 μL of this solution was analyzed. Analyses were done using a Hewlett Packard HP5890 gas chromatograph equipped with a flame ionization detector and a 30 m long BPX-70 capillary column (J&W Scientific). The injector and detector temperatures were 260°C and 285°C, respectively. The temperature program was 8O ⁰ C rising at 5°C per min for 24 min to 200°C, and then rising at 2O ⁰ C per min for 3 min to 26O ⁰ C.

An acceptable amount of BP released from devices would be 1.0 mg perday. However, the amounts of brood pheromone released from these devices were very small and potentially not high enough to elicit a response in a bee hive (Table 1). These devices were considered unacceptable, and were not placed in hives for further testing.

Table 3. Mean amounts of stabilized synthetic 10-component honey bee brood pheromone released from polyethylene pipette bulb devices after 1 and 3 days.

Example 5

Wax Discs

Refined beeswax obtained from Sigma- Aldrich was cooled with dry ice, and then ground to a fine powder in a blade mill. To remove moisture, the powder was placed in a desiccator overnight under an aspirator vacuum, and then for 30 minutes under a mechanical pump vacuum. Each device was made separately by adding 50 uL of stabilized synthetic 10-component honey bee brood pheromone to 3.0 g of beeswax powder. To aid in assuring complete mixing of the brood pheromone, a small amount of Solvent Blue dye was added. The mixture was stirred until homogenous, and then pressed into 1.5 inch diameter discs using a wooden mold and a 4 ton hydraulic press. Control discs did not contain brood pheromone or Solvent Blue dye. The discs were sealed in Mylar bags and shipped to Texas A&M University for further study.

Discs were placed in hives, which were monitored periodically for 26 days. There was a highly significant difference in the ratio of pollen to non-pollen foragers for the first two observations, but the ratio favored colonies exposed to control discs over those exposed to pheromone-laden discs (Table 4). The bees chewed and destroyed the pheromone- treated discs and possibly recycled the pheromone-laden wax. They did not touch the untreated control discs.

Table 4. Pollen to non-pollen forager ratios for honey bee workers returning to hives in which pheromone-laden or untreated beeswax discs were placed for 26 days.

Example 6

Bubble Cap Device with Sponge Insert

A bubble cap device was constructed from two plastic membranes; the impermeable bubble membrane was 4 mil Mylar and the release membrane was made from 10 mil clear low-density polyethylene (LDPE). To aid in retention of the pheromone, and its controlled release over time, a 2.5 cm diameter disc cut from industrial absorbent sponge (Pig Mat, New Pig Corp., Tipton, PA, USA) was placed in the bubble, the sponge was then loaded with a 30% formulation of stabilized synthetic 10-component honey bee

brood pheromone in a 3:2 blend of methanol and ethanol and the bubble cap was heat sealed.

Gas chromatographic analysis of extracted swabs of the both membrane surfaces, as in Example 4, indicated that the brood pheromone was not passing through either wall of the bubble cap devices. From Example 4 we knew that the brood pheromone can travel through a plastic membrane, and we surmised that the sponge withheld the brood pheromone. We therefore sought a reservoir insert material with potentially weaker retention properties.

Example 7

Plastic Pouch Device with Filter Paper Insert

Small plastic pouches were constructed, from two different materials, one impermeable and the other impermeable to the stabilized synthetic 10-component honey bee brood pheromone. The impermeable side was 4.0 mil Mylar measuring 29.0 x 57.0 mm, and the pheromone-permeable side, measuring 29.0 x 29.0 mm, was made from either 6.0 mil brown low-density polyethylene (LDPE), 1.75 mil brown LDPE, 1.75 mil white LDPE, 3.0 mil white LDPE, 10 mil polyvinylchloride (PVC), 40 micron clear LDPE, or 40 micron smoky gray LDPE. The pouch was formed by heat-sealing the pheromone permeable membrane to the Mylar on three edges, leaving one edge open. To aid in retention of the pheromone, a 25 x 25 mm piece of filter paper (Whatman No.l, Sanford, MA, USA) was inserted inside the pouch. Using a hypodermic syringe, the filter paper was then loaded with 100 μL of brood pheromone, and the open edge was sealed.

One device of each type was placed in a 3O ⁰ C chamber and swabbed with a 1 x 2 cm piece of paper towel every 24 hours to remove any BP that migrated across the plastic membrane. The paper towel was placed in a small glass vial to which 1.0 mL of a solution of C 14 in hexane (0.1075 mg C14 per mL) was added as an internal standard for gas-chromatographic analysis, and allowed sit for 20 minutes or longer. Samples were then agitated using a vibrating mixer for 5 seconds.

For analysis by capillary gas chromatography, approximately 1 mg of a blend was dissolved in 1 mL of hexane, and 1 μL of this solution was analyzed. Analyses were done using a Hewlett Packard HP5890 gas chromatograph equipped with a flame ionization detector and a 30 m long BPX-70 capillary column (J&W Scientific). The injector and detector temperatures were 26O ⁰ C and 285 ⁰ C, respectively. The temperature program was 8O ⁰ C rising at 5°C per min for 24 min to 200 ⁰ C, and then rising at 20 ⁰ C per min for 3 min to 26O ⁰ C.

Released amounts of brood pheromone ranged widely from these devices, and were potentially either too high or not high enough to elicit a response in a bee hive (Table 2). However, two of the devices were chosen to be tested in the hive, the brown 6 mil LDPE and the white 1.75 mil LDPE, because they released the greatest and the least amounts of brood pheromone, without releasing more than 1.0 mg.

Table 5. Mean amounts of stabilized synthetic 10-component honey bee brood pheromone released daily at 30°C from plastic pouch devices with filter paper inserts over 7 days. Each device had an impermeable Mylar side heat sealed to apheromone- permeable low density polyethylene (LDPE) side.

Twenty-four devices were placed in hives at Texas A&M University, six pheromone- loaded 6 mil LDPE brown devices, six control brown devices with filter paper inserts but no pheromone, six pheromone-loaded 1.75 mil LDPE white devices, and 6 control white devices with filter paper inserts but no pheromone. The six criteria described in Example 1 were used to evaluate the performance of each device. The proportional data for ratios of pollen to non-pollen foragers were analyzed using Chi square contingency tables. Data for adult bee numbers, brood comb area, pollen area, honey area and empty comb area were analyzed by one-way analysis of variance (ANOVA). In all cases α = 0.05.

The results indicated that overall the mean ratio of pollen to non-pollen foragers was not significantly different between the brown and white device and control devices (Table 6).

The results for daily pollen to non-pollen forager ratios for both devices were inconsistent, with the ratios for pheromone-loaded brown membrane devices being significantly higher than for control devices in three instances, and control devices being significantly higher in two instances (Table 7), and the ratios for pheromone-loaded white membrane and control devices being significantly higher in one instance each (Table 8).

The total brood area (mean + SE) was significantly greater in hives where empty control brown membrane devices were inserted (3,729.33 cm ² ± 174.15) than in hives with pheromone-loaded brown membrane devices (2,881.33 cm ² ± 289.43) (One-way ANOVA, F ₁₎₁₀ = 6.302, P < 0.05). All other measures showed no significant difference

Table 6. Ratio of pollen to non-pollen foragers among worker honey bees exposed for 17 days to pheromone-laden or empty control plastic pouch devices with filter paper inserts and two different kinds ofpheromone-permeable membranes.

Table 7. Daily pollen to non-pollen forager ratios for honey bee workers returning to hives in which devices with 6.0 mil brown LDPE pheromone-permeable membranes were placed.

a Asterisks indicate higher ratios in pheromone-treated than control colonies.

Table 8. Daily pollen to non-pollen forager ratios for honey bee workers returning to hives in which devices with 1.75 mil white LDPE pheromone-permeable membranes were placed.

aAsterisk indicates higher ratio in pheromone-treated than control colony, between brood pheromone and control treatments (One-way ANOVA, P > 0.05).

We concluded that devices with a 6 mil brown LDPE membrane showed some promise, but that the results were not nearly consistent enough to justify using these devices for development of an operational commercial product. Moreover, the devices tended to curl up, leaving the permeable membrane inaccessible to bees on the inside of the curl.

Example 8

Prototype Operational Device

We hypothesized that the pouch used in Example 7 might be converted into an acceptable device if the filter paper insert were removed, allowing the liquid pheromone composition to make contact at all times with the permeable membrane, and if the pouch were mounted in a rigid casing to eliminate curling.

We constructed small plastic pouches, measuring 38.0 x 35.0 mm, made of two different materials. An impermeable side was 4.0 mil Mylar, and the other pheromone-permeable side was made from 6.0 mil brown low-density polyethylene (LDPE). The pouch was formed by heat-sealing on three edges, leaving one edge open. Using a hypodermic syringe, the pouch was filled with 200 μL of synthetic pheromone formulation made up using technical-grade chemicals with the following composition: methyl palmitate 2.11%, ethyl palmitate 4.19%; methyl stearate 17.30%, ethyl stearate 8.04%, methyl oleate 21.66%, ethyl oleate 7.50%, methyl linoleate 8.09%, ethyl linoleate 3.82%, methyl linolenate 16.53%, ethyl linoenate 10.77% and tertiary-butylhydroquinone 0.05%. The filled pouches were immediately heat sealed, and mounted in 35 mm plastic slide holders (Gepe slide mount), so that the permeable membrane was centered in the window on the

white side of the casing, while the impermeable Mylar backing was exposed in the window of the dark side of the casing. A 2 mm diameter hole was drilled in the midpoint of one margin of the casing to facilitate hanging the device with a wire or string.

To test for longevity and durability, as well as the presence of air bubbles as an outcome of manufacturing, we manufactured nine prototype devices Six of the prototypes had a minimal air bubble, and were packaged in stacks of three and sealed in separate Mylar bags. One Mylar bag was held for five days at room temperature, the other was placed in a freezer for two days then allowed sit for an additional three days at room temperature. The remaining three prototypes were manufactured with a large air bubble. They were also sealed in Mylar bags and held for five days at room temperature. The outer surface of the permeable membrane was then wiped clean and all nine bags were placed in a 30°C chamber. Pheromone release was then determined as in Example 7 for seven 24- hour periods over a 10 day duration. The daily mean release rates for each type of device (N = 3) were analyzed by ANOVA, α = 0.05.

Released amounts of brood pheromone were consistent from all devices. Neither air bubbles nor freezing had a significant effect on the amount of pheromone released from the permeable LDPE membrane (Table 9) (F = 1.5508; df = 2,6, P = 0.2865). The release rates were well within the target range of 0.1 -1.0 mg per day, justifying testing of this prototype for efficacy in the field.

Table 9. Mean amounts of stabilized synthetic 10-component brood pheromone released daily at 30 ⁰ C from plastic pouch devices manufactured with or without air bubbles and held frozen or unfrozen prior to determination of daily release rate. Each device had an impermeable Mylar side heat sealed to a 6 mil LDPE pheromone-permeable side.

Example 9

Efficacy of Prototype Operational Device: Effect on Pollen Foraging

Twenty plastic pouch devices with 6 mil brown LDPE permeable membranes, mounted in 35 mm plastic slide frames, as described in Example 8 were shipped to Texas A&M University. Ten of the devices were loaded with 200 μL of the stabilized synthetic 10- component honey bee brood pheromone, and 10 were empty control devices. Twenty plastic pouch devices with 1.75 mil white LDPE permeable membranes were also sent to Texas A&M University, 10 loaded with 200 μL of stabilized synthetic brood pheromone, and 10 as empty control devices.

A total of 24 colonies (with approximately 10,000 to 12,000 bees each) were randomly selected for pheromone (N=6) and control (N=6) treatments with both the brown and white LDPE permeable membranes. The experiments ran from 28 September to 5

October 2007. Five-minute entrance counts were conducted prior to 12 noon, at least 2 times per week or as weather permitted. An observer sat by the colony entrance and recorded the type of load for every returning forager, pollen or non-pollen. Because pollen is carried on the outside a forager's body, pollen foraging is easily distinguished. Returning non-pollen foragers may be unsuccessful foragers, or may be successful foragers returning with a liquid load such as nectar or water carried internally in the crop. The rate of colony-level foraging activity was determined, as well as the ratio of pollen to non-pollen foragers. Colonies exposed to white devices were followed for 29 days, and those with brown devices for 43 days. The proportional data for ratios of pollen to non- pollen foragers were analyzed using Chi square contingency tables (α = 0.05). A greater ratio signifies a greater amount of pollen foraging.

On days 8 and 27 pollen load weight of returning pollen foragers was measured for colonies exposed to devices with 6.0 mil brown LDPE permeable membranes. Following the five-minute entrance counts, colony entrances were blocked with wire mesh for 15 minutes, and returning pollen foragers were randomly collected as they returned. A total of 10 foragers per colony were collected each day. Individual foragers were placed in 1.5 ml plastic snap-cap tubes (Eppendorph ^® ) and stored on dry ice for transport to the lab for processing. One pollen pellet was removed and weighed from each individual. Because bees carry a balanced pollen load (Pankiw et al. 2002), the weight of a single pellet was doubled to determine total pollen load weight. We predicted that pheromone treatment would cause an increase in the weight of pollen brought back to the hive. Therefore, oneway ANOVA (α = 0.05) was used to analyze the difference between means for pheromone-treated and control colonies.

For colonies treated with devices having 6 mil brown LDPE pheromone-permeable plastic membranes, the ratio of pollen to non-pollen foragers was unexpectedly and significantly greater from 8 to 36 days in pheromone-treated than in control colonies (Table 9). Similar results (not shown) were achieved with devices having 1.75 mil white LDPE pheromone-permeable membranes, but the effect disappeared after 27 days.

Table 9. Daily pollen to non-pollen forager ratios for honey bee workers returning to hives in which devices with 6 mil brown LDPE pheromone-permeable membranes were placed.

^a Asterisks indicate higher ratios in pheromone-treated than control colonies.

Surprisingly, in the second and fourth week of treatment, pollen foragers from colonies treated with devices having 6 mil brown LDPE pheromone-permeable membranes returned to the hive with significantly heavier pollen loads than foragers from control colonies (Table 10).

The longevity of 36 days is far longer than the minimum target of 14 days for an operational device. This finding, along with the change in ratio in favor of pollen foragers over non-pollen foragers, and the increase in pollen load by foragers returning to pheromone-treated colonies, indicate that we have discovered an operational device with extraordinary potential for increasing pollination of essential agricultural crops as well as the size and quality of the harvested product.

Table 10. Mean weight of pollen carried by worker honey bee foragers returning to the hive at 8 and 27 days after hive had received either brood pheromone-loaded devices with 6 mil brown LDPE pheromone-permeable membranes, or empty control devices.

Example 10

Efficacy of Prototype Operational Device: Effect of Long-Term Exp on Colony Growth

Providing beekeepers with a tool to stimulate colony growth is important both to apiculture and to farmers dependent on honey bees for crop pollination. Therefore, an experiment to evaluate colony growth was initiated 21 August 2007 at Texas A&M University.

At the start of the experiment, 12 colonies were measured for amount of adult bees and brood area. Colony size was measured using a grid the size of a Langstroth-deep frame divided into 6.45 cm ² sections. The area covered by bees was converted to bee numbers, approximately 1.5 bees per cm ² (Pankiw et al. 2004a). Comb area occupied by eggs, larvae, and pupae collectively named brood was also measured. Six colonies were randomly selected to receive a slow release identical to the device with a pheromone- permeable membrane made from 6.0 mil brown LDPE plastic. Each of these six devices was loaded with 200 μL of the stabilized synthetic 10-component honey bee brood pheromone. The remaining six colonies received a control device with no brood pheromone. Thirty one days later, on 21 September 2007, colonies were again measured for amount of bees and brood area. Colony size data and brood comb area at the start of the experiment were statistically analyzed using two-way ANOVA. At the end of the experiment we used covariate ANOVA, with the pre-treatment measure as the covariate.

At the start of the experiment, numbers of adult bees estimated and brood area were not significantly different between control and treatment hives. After 31 days, numbers of adult bees in hives that received devices laden with brood pheromone had increased by 38.5%, while numbers in control colonies had decreased by 10.7% (Table 11), for a highly significant summed difference of almost 50%.

Table 11. Effect of 31 days exposure to devices with 6 mil LDPE pheromone-permeable membranes loaded with stabilized synthetic brood pheromone or empty control devices on growth in numbers of adult worker honey bees in the colony.

The amount of brood comb area also increased significantly greater in hives treated with devices loaded with brood pheromone (Table 12). Brood area in pheromone-treated colonies increased by 40.7%, while it decreased by 24.2% in control colonies, for a highly significant summed difference of almost 65%.

Table 12. Effect of 31 days exposure to devices with 6 mil LDPE pheromone-permeable membranes loaded with stabilized synthetic brood pheromone or empty control devise on the amount of brood comb area in the colony.

The reduction in number of adults and brood area observed in control colonies is a natural phenomenon occurring in the autumn as colonies prepare for winter. During this period, the mortality rate among the oldest bees increases and there is less brood rearing in response to decrease pollen intake (Seeley 1985; Winston 1987). The reversal of this negative seasonal trend in favor of pronounced growth in both brood comb area and adult bee numbers is particularly striking and unexpected. Because the brood comb area would likely contain many developing larvae, one should expect sustained growth in numbers of adult worker honey bees for some time following depletion of the pheromone reservoir in the devices.

We conclude that devices that provide sustained and controlled release of the stabilized synthetic 10-component honey brood pheromone would yield highly positive benefits at several times of the year, including, but not limited to: 1) in the spring and early summer to enhance colony growth and vigor, to improve pollination, and potentially to increase crop yield, and in some cases to improve crop quality, 2) at the end of the summer to counteract decline in colony size and to improve the health of a colony as winter approached, and 3) at the end of the winter to stimulate feeding on pollen patties and uptake of antibiotics within the hive at a critical period for the maintenance of colony health.

Example 11

Holder for Placing Operational Device in Hive

The holder (Figure 1) consists of a single part of plastic material such as printable polyethylene (PE), polyethylene terephthalate (PET) or polycarbonate (PC). The parts

can be manufactured by die cutting them out of sheet plastic material ranging in thickness from 20-60 mil (0.5-1.5 mm) with the shapes closely arranged or ganged in a pattern to minimize wasted material.

Details are incorporated in the part to provide functional benefits. Slotted holes allow specific portions to flex away from the flat shape without damaging the part, enabling the casing to be inserted and held in place. The retention of the casing is achieved with integral shoulders in the flat part which rest against the edge of the casing. Other slots and a crease, essentially a compressed linear section, allow a portion of the holder to be bent at a 90° angle so that the resulting T-shaped structure can suspend between two frames in a hive unit.

A specific shape is cut out of the area where the casing is held, consisting of two juxtaposed rotated rectangular holes. They allow the pheromone-permeable flexible side of the pouch to be exposed on either side of the holder, regardless of the orientation of the casing, as shown in Figure 1.

It is possible that injection molding can be considered as an alternative manufacturing process to die cutting sheet material.

As will be apparent to those skilled in the art in the light of the foregoing disclosure, many alterations and modifications are possible in the practice of this invention without departing from the spirit or scope thereof. Accordingly, the scope of the invention is to be construed in accordance with the substance defined by the following claims.

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