# Patent

## Title
Stabilized synthetic brood pheromone and race-specific ratios of components for manipulating the behavior and physiology of honey bees

## Abstract

This invention relates to a 10-component stabilized synthetic honey bee brood pheromone and methods of stabilizing said pheromone by adding one or more antioxidants, thereby enabling the production and sustained use of commercial products based on that pheromone. The 11-component stabilized pheromone composition formed by adding the antioxidant tertiary-butyl hydroquinone to a synthetic blend of ethyl linoleate, ethyl linolenate, ethyl oleate, ethyl palmitate, ethyl stearate, methyl linoleate, methyl linolenate, methyl oleate, methyl palmitate and methyl stearate can be used in generic or race-specific ratios to manipulate the behavior and improve the performance of worker honey bees, resulting in overall increased vigor of the hive.

## Background
REFERENCE TO RELATED APPLICATION
This application is a continuation-in-part of application Ser. No. 11/470,762, filed Sep. 7, 2006.


FIELD OF THE INVENTION
This invention relates to a stabilized 11-component synthetic honey bee brood pheromone composition comprising a 10-component synthetic honey bee brood pheromone composition and an effective amount of an antioxidant and a method of stabilizing said 10-component composition by adding an antioxidant to form an 11-component composition, thereby enabling the production and sustained use of commercial products based on said pheromone composition. The stabilized synthetic pheromone composition can be used to manipulate the behavior and improve the performance of worker honey bees, resulting in overall increased vigor of the hive. Larvae of different races of honey bees produce the components of the natural pheromone composition in race-specific ratios. Worker honey bees respond best to said synthetic composition formulated in a ratio specific to their own race. Specific behaviors influenced by the 11-component stabilized synthetic pheromone composition applied on an inert substrate include: age of first foraging, proportion of workers that are foragers, ration of pollen to nectar foragers, pollen loading by individual bees, efficiency of pollination of target crops, consumption of supplementary diet components and antibodies, rearing of workers, and propensity to swarm. The 11-component stabilized synthetic pheromone composition can also be used to influence the development of the hypopharyngeal food gland and the production of protein by the gland. The 11-component stabilized synthetic pheromone composition can further be used in conjunction with the honey bee queen mandibular gland pheromone, particularly in influencing pollination of target crop plants.
BACKGROUND OF THE INVENTION
A colony of honey bees, Apis mellifera, 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 (Robinson 1992; Anderson and Franks 2001). When worker honey bees emerge from their cells as adults their first “job” is normally to clean cells. As the workers age they take on a succession of other jobs: feeding larvae, processing and storing food, secreting wax and constructing comb, and guarding the entrance to the hive. The most obvious change in behavior occurs when worker bees are about three weeks old, and they begin foraging (Lindauer 1952; Seeley and Kolmes 1991). Foraging labor is also divided, whereby some workers return to the colony carrying only nectar, some carry only pollen, and some return carrying both nectar and pollen. Pollen and nectar are sources of protein and carbohydrate, respectively.
Honey bee foragers collect pollen from available plant sources. They then return to the nest and deposit their loads of pollen directly into cells. Stored pollen is consumed by young nurse bees that use the proteins derived from the pollen to produce proteinaceous hypopharyngeal gland secretions that are fed to developing larvae (Crailsheim et al. 1992). By consuming pollen in this manner, nurse bees reduce the quantities of stored pollen. The presence of young larvae affects the proportion of foragers collecting pollen. The more larvae in a colony, the more pollen collected by foraging workers (Al-Tikrity et al. 1972; Barker 1971; Free 1967, 1979; Jaycox 1970; Todd and Reed 1970).
The hypothesis that larval honey bees produce a pheromone that stimulates foraging (Filmer 1932: Free 1967) was tested and verified by Pankiw et al. (1998), who found a 2.5 fold increase in the number of pollen foragers when colonies with 1,000 larvae were provided with hexane extracts of 2,000 honey bee larvae. Pankiw et al. (1998) also found that the same hexane larval extract stimulated pollen foraging in colonies with no larvae, i.e. the extract replaced the larvae as a source of pheromone. Thus, bees apparently determine the number of larvae in a colony by sensing the concentration of chemicals in brood pheromone produced by the larvae.
Pankiw and Page (2001) and Page and Pankiw (2002) tested a synthetic pheromone blend that comprised ten non-volatile esters of fatty acids (LeConte et al. 1990) in the following percentages: 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 stearate 17%. This blend was found to lower the neurosensory response threshold to sucrose in worker bees, which is a measure of propensity to forage for pollen. Worker honey bees with low sucrose response thresholds are most likely to forage for pollen, and those with high response thresholds are most likely to forage for nectar (Pankiw and Page 2000; Pankiw 2003; Pankiw et al. 2004a). Within one hour of being placed in the center of a colony, larval extract or the synthetic brood pheromone blend increased the ratio of pollen to non-pollen foragers of colonies in an almond orchard. Based on their results, Pankiw and Page (2001) suggest that “brood pheromone modulates response threshold in pre-foragers, primes them to be pollen foragers, and releases pollen foraging behavior.” It is not known whether different races of honey bees produce pheromone with different ratios of the ten components, or whether worker bees respond preferentially to the blend of pheromone components produced by larvae of their own race.
In a subsequent study, Pankiw et al. (2004b) exposed honey bee colonies to 2,000 larval equivalents (1.12 mg) of synthetic brood pheromone daily for 28 days. As well as increasing the number of pollen foragers, as predicted by earlier studies, brood pheromone had several other important effects. Compared to untreated control colonies, the weights of pollen loads returned to the hive were significantly greater in colonies treated with brood pheromone. Pheromone-treated colonies reared significantly more brood, as measured by the area of comb housing eggs, larvae and pupae. The number of adult honey bees that emerged from this brood was significantly greater in colonies treated with brood pheromone than in untreated control colonies. Thus the overall size of colonies was increased.
Brood pheromone can have dose-dependent effects (Pankiw 2004a; LeConte et al. 2000). At a low, biologically realistic dose of 2,000 larval equivalents, brood pheromone recruited honey bee workers to become foragers at an earlier age than in control colonies (Pankiw et al. 2004), whereas at a much higher dose of 6,200 larval equivalents, brood pheromone delayed the recruitment of workers to become foragers (LeConte et al. 2000).
Pankiw et al. (2004) found that although exposure to brood pheromone generally lowered the age of first foraging, the onset of foraging was delayed in a specialized cohort of worker bees that had a very high content of protein in their hypopharyngeal glands. This high protein level is an indication of increased probability of brood rearing behavior and a correspondingly higher quality of protein fed to larvae compared to that in control colonies.
Pankiw (2004b) found that the ratio of pollen foragers to non-pollen foragers increased within one hour of placement of 2,000 larval equivalents of synthetic brood pheromone into a honey bee colony. Foragers from pheromone-treated colonies returned with heavier pollen loads than foragers from control colonies, and this pollen was 43% more likely to originate from the target crop within which colonies were placed to ensure pollination. Non-pollen foragers, that may visit and pollinate more flowers than pollen foragers while searching for the best nectar sources, had more pollen grains on their bodies than non-pollen foragers from untreated control colonies. Similar to pollen foragers, these non-pollen foragers bore pollen that was 54% more likely to originate from the target crop than pollen borne by non-pollen foragers from control colonies. By increasing the activity of both pollen foragers and non-pollen foragers, brood pheromone has the potential to greatly increase the effectiveness of colonies used in custom pollination of target crops.
Another potential use for brood pheromone is stimulating the consumption of dietary protein supplements. Beekeepers commonly provide a protein supplement in winter or early spring to promote colony growth (Herbert 1992). Often an antibiotic is blended with the protein supplement as a prophylactic against larval bacterial diseases. However, because larvae are absent or at their lowest levels in late winter and early spring, there is very little, if any, brood pheromone being produced within the colony that might stimulate the consumption of protein and antibiotic dietary supplements, as well as hypopharyngeal gland development and protein biosynthesis. Administration of synthetic brood pheromone within a colony could provide the necessary stimulus.
Despite the practical promise of brood pheromone (Pankiw 2004b), no commercial products have been developed that incorporate its ten components, apparently because of their instability at room temperature. 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 −70° C., if it will be stored for any long period of time.” Even freezing at −70° C. may not stabilize the pheromone. For example, crude honey bee larval extract lost all pheromonal activity after only three months in a laboratory freezer (T. Pankiw, Texas A&M University, unpublished observation). Thus any commercial product that incorporated either the natural or synthetic pheromone would have almost no shelf life, and would potentially lose all potency during storage and shipping. Freezing at would be prohibitively expensive, and would probably not prolong the shelf life for an acceptable duration. Recognizing the instability of the synthetic pheromone, researchers have invariably provided their experimental bees with fresh pheromone each day, usually on glass plates (Pankiw et al. 1998; Pankiw and Page 2001; Page and Pankiw 2002; Pankiw and Rubink 2002; Pankiw et al. 2004b). In commercial operational use, daily application of pheromone would be impractical and expensive, and would not eliminate the need to freeze the pheromone during storage.
Two main hypotheses could potentially explain the short duration of bioactivity of the synthetic and natural pheromone: 1) minute amounts of breakdown products have a positive effect at first, but over time rise to inhibitory levels, or 2) the fresh pheromone is biologically active, but some or all of its constituents break down rapidly to sub-threshold levels. If the synthetic pheromone composition could be stabilized, and if it were then inactive when experimentally tested, the results would support the first hypothesis. If the synthetic pheromone composition were stabilized and remained active in an experimental test, the results would uphold the second hypothesis. The first hypothesis appears most likely, as it is supported by the fact that freshly prepared synthetic brood pheromone can have dose-dependent effects, with high doses negating the positive effect seen with low doses (Pankiw 2004a; LeConte et al. 2000). In this case, there would be no practical purpose in stabilizing the pheromone. On the other hand, if the second hypothesis were upheld, the stabilized synthetic pheromone would have practical utility.
However, if the stabilization process involved adding a chemical stabilizer, such as an antioxidant, the stabilizer could potentially cause the synthetic pheromone composition to become inactive. This could occur through denaturing one or more of the essential components in a complex pheromone blend. It could also inhibit the pheromone receptor membrane on the antennal or palpal sensilla, causing the insect not to perceive the pheromonal message. Finally, employing an altered composition could cause an insect to perceive the “wrong” pheromone blend and thus not to respond.
Antioxidants are not naturally occurring in honey bee colonies. The long-term storage of honey bee food does not involve antioxidants, but relies instead on other methods of preservation. For example, the enzymes diastase, invertase, and glucose oxidase are added by the bees to nectar soon after it is deposited into cells. Glucose oxidase breaks down glucose and releases hydrogen peroxide, an antibacterial agent of anaerobic bacteria. The water content of honey is reduced to less than 18%. This supersaturated solution of sucrose inhibits fungal growth. Fermentation is negated by preventing re-hydration by sealing honey with a thin sheet of wax. Stored pollen is preserved using honey. The enzymes in the honey prevent pollen germination and microbial growth.
Even if it were conceivable to add an antioxidant to a fatty acid pheromone, it is not evident to a person skilled in the pheromone art that the bioactivity of the pheromones would be retained with the addition of an antioxidant. The bioactivity of pheromones is very sensitive and unpredictable. The addition of a foreign substance to a complex pheromone blend is counterintuitive to a person skilled in the art of pheromone biology. To such a person, the addition of an antioxidant might just as easily have negative rather than positive consequences on honey bee foraging behaviors and physiological effects. For example, an antioxidant could prove to be repellent or toxic to honey bees, negating any positive effect of the formulated pheromone. In addition, bioactivity could reside in part in minute amounts of breakdown products, that later accumulate to inhibitory levels, and that would not be produced in the presence of an antioxidant. The relevance of the antioxidant is not centered on chemical stability alone, but rests also on maintaining the behavioral and physiological responses to the pheromone in the presence of an antioxidant that is not naturally occurring with the pheromone or a honey bee colony.
Doisaki et al. (2006), Quimica Nova (2006), and Critical Reviews in Food Science and Nutrition (1995) disclose antioxidants but do not deal with pheromones. The disclosed antioxidants can be used as additives for fats and oils to enhance their oxidative stability. There is no teaching in any of these references that the disclosed antioxidants can be used with pheromones. The respective arts of pheromones and stabilizing fats and oils with antioxidants are distinct and not related. A person skilled in the art of pheromones is not likely to look to the other art for inspiration.
SUMMARY OF THE INVENTION
Throughout the following description, specific details are set forth in order to provide a more thorough understanding of the invention. However, the invention may be practiced without these particulars. In other instances, well known elements have not been shown or described in detail to avoid unnecessarily obscuring the invention. Accordingly, the specification is to be regarded in an illustrative, rather than a restrictive, sense.
The invention is directed to a method of stabilizing synthetic honey bee brood pheromone comprised of ten non-volatile esters of long-chain carboxylic acids, specifically ethyl linoleate, ethyl linolenate, ethyl oleate, ethyl palmitate, ethyl stearate, methyl linoleate, methyl linolenate, methyl oleate, methyl palmitate and methyl stearate by incorporating an effective amount of a food-grade antioxidant into the pheromone. The invention is further directed toward demonstration that the stability imparted by adding said food-grade antioxidant is sustainable over a duration long enough to ensure adequate shelf life of a commercial product derived from the stabilized composition.
The antioxidant can be a food-grade antioxidant. In particular, the food-grade antioxidant can be selected from among the group comprised of (but not limited to) butylated hydroxyanisole, butylated hydroxytolulene, tertiary-butyl hydroquinone, polypropylene glycol, citric acid and vitamin E and its derivatives (tocopherols) or any combination of two or more of said antioxidants.
In the method, the antioxidant acts by inhibiting the breakdown of methyl linoleate, methyl linolenate, ethyl linoleate, ethyl linolenate, methyl oleate and ethyl oleate.
In another aspect, the invention is directed to an 11-component synthetic pheromone composition comprised of ethyl linoleate, ethyl linolenate, ethyl oleate, ethyl palmitate, ethyl stearate, methyl linoleate, methyl linolenate, methyl oleate, methyl palmitate, methyl stearate and an effective amount of a food-grade antioxidant selected from among the group comprised of (but not limited to) butylated hydroxyanisole, butylated hydroxytolulene, tertiary-butyl hydroquinone, polypropylene glycol, citric acid and vitamin E and its derivatives (tocopherols) for administration to honey bees or honey bee colonies.
In a further aspect of the invention, the effect of the 11-component stabilized synthetic pheromone composition on workers of a given race of honey bees can be optimized by treating them or their colonies with said synthetic pheromone composition formulated so that the ratio of fatty acid ester components mimics the ratio of components in the natural pheromone composition produced by larvae of that specific race of honey bees.
The proportions by weight of the 11 components of the composition can be ethyl linoleate (0.1-50%), ethyl linolenate (0.9-50%), ethyl oleate (0.8-50%), ethyl palmitate (0.3-50%), ethyl stearate (0.6-50%), methyl linoleate (0.2-50%), methyl linolenate (0.2-50%), methyl oleate (0.3-50%), methyl palmitate (0.3-50%), methyl stearate (0.7-50%), and antioxidant (0.005-5.0%).
The stabilized 11-component composition as described can be administered to honey bees, honey bee colonies or pollination units in an inert substrate selected from among the group comprised of paraffin, vegetable wax, beeswax, silica, alumina, cellulose, modified cellulose (paper and paper products), dried plant tissue, wood, or a synthetic polymer.
The method and the stabilized 11-component composition can be used to increase the ratio of pollen to non-pollen foragers among worker honey bees, or used to lower or delay the age of first foraging by worker honey bees, or can be used to increase the total number of foraging worker honey bees, or can be used to increase the load weight of pollen returned to the colony of pollen-foraging worker honey bees, or used to increase the number of pollen grains carried by the bodies of non-pollen-foraging worker honey bees, or used to increase the pollination of the target crop near or within which honey bee colonies or pollination units are placed, or used to stimulate hypopharyngeal brood food gland development and gland protein production in honey bee workers, or used to stimulate the consumption of dietary protein or other diet components and antibiotic supplements by honey bees.
In the method, continual administration of the stabilized 11-component composition to a honey bee colony for a period of about two weeks or longer can be used to increase the number of bees reared within the colony, or used to inhibit absconding (swarming) of bees from the colony.
In the method, the stabilized 11-component composition can be administered to honey bees, honey bee colonies or pollination units in combination with synthetic honey bee queen mandibular gland pheromone administered to honey bees, honey bee colonies or pollination units or to 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.
FIG. 1 illustrates gas chromatograph tracings of blends of methyl oleate (1), methyl linoleate (2) and methyl linolenate (3) formulated with the antioxidant stabilizer tertiary-butylhydroquinone at time zero and at two weeks after exposure to air at room temperature (approximately 20° C.), in comparison to an otherwise identical unstabilized formulation after two weeks exposure at 20° C.
FIG. 2 illustrates gas chromatograph tracings of blends of ethyl oleate (1), ethyl linoleate (2) and ethyl linolenate (3) formulated with the antioxidant stabilizer tertiary-butylhydroquinone at time zero and at two weeks after exposure to air at room temperature (approximately 20° C.), in comparison to an otherwise identical unstabilized formulation after two weeks exposure at 20° C.
FIG. 3 illustrates gas chromatograph tracing of blend of ethyl oleate (1), ethyl linoleate (2) and ethyl linolenate (3) formulated with the antioxidant stabilizer tertiary-butylhydroquinone after 20 weeks exposure to air at room temperature (approximately 20° C.), in comparison to tracing of an otherwise identical unstabilized formulation after 20 weeks exposure at 20° C.
FIG. 4 illustrates gas chromatograph tracing of blend of ethyl oleate (1), ethyl linoleate (2), ethyl linolenate (3), ethyl palmitate (4) and ethyl stearate (5) formulated with the antioxidant stabilizer tertiary-butylhydroquinone after 72 weeks exposure to air at room temperature (approximately 20° C.), in comparison to tracing of an otherwise identical unstabilized formulation after 72 weeks exposure at 20° C.



DETAILED DESCRIPTION OF THE INVENTION
Honey bee foraging pheromone is easily oxidizable and unstable at room temperature, loses bioactivity rapidly, and must be held under low oxygen conditions and preferably frozen at very low temperatures, e.g. −70° C., if it to be stored for long durations. Under normal conditions, any product based on a synthetic 10-component pheromone composition would have almost no shelf life, and would not be commercially viable. Thus, apart from use of the pheromone in research, there has been no commercial application of the pheromone.
We have discovered that the synthetic 10-component honey bee brood pheromone composition can be stabilized for a substantial period of time by incorporating an antioxidant, particularly a food-grade antioxidant, into the synthetic pheromone to form an 11-component composition. Stabilized synthetic brood pheromone compositions can be formulated in race-specific ratios, and delivered to the hive on an inert substrate. Used alone, or with the honey bee queen mandibular gland pheromone, the 11-component synthetic brood pheromone compositions can improve the performance of worker honey bees and enhance the overall vigor of the hive.
In general terms, the invention is directed to a method of stabilizing the 10-component honey bee brood pheromone using one or more acceptable antioxidants, especially one or more food-grade antioxidant, including (but no limited to) butylated hydroxyanisole, butylated hydroxytolulene, tertiary-butyl hydroquinone, polypropylene glycol, citric acid and vitamin E and its derivatives. 11-Component formulations of the synthetic brood pheromone that include an antioxidant as one of the components can be delivered in a sustained manner to a hive on, or incorporated into, an inert substrate or device.
By using the antioxidant stabilizer tertiary-butylhydroquinone, we have discovered that certain unstable pheromone components, particularly methyl linoleate, methyl linolenate, ethyl linoleate and ethyl linolenate, but also including methyl oleate and ethyl oleate, remained intact for 72 weeks. We have also discovered that a formulation stabilized with tertiary-butylhydroquinone retained bioactivity for 231 days, while an unstabilized formulation lost almost all bioactivity after 123 days. We have further discovered that the stabilized 11-component synthetic brood pheromone composition increases pollen foraging among worker honey bees, and stimulates pollen diet consumption.
In another aspect, we have discovered that an 11-component stabilized synthetic brood pheromone composition formulated using technical-grade components that are less pure than reagent-grade components, and with a proportional composition that varies slightly from that achieved with reagent-grade components, has the same biological effect as a reagent-grade composition. Such durability and robustness enables the development and sustained use of commercial products based on the brood pheromone stabilized with food-grade antioxidants.
Lastly, we have discovered that worker honey bees of different races respond to said generic reagent-grade 11-component composition, but that for a given race they respond at significantly greater levels to a synthetic pheromone composition in which the components are formulated in the precise ratio produced by larvae of that race.
This invention also pertains to compositions in which the 10 pheromone components, comprised of methyl palmitate, ethyl palmitate, methyl stearate, ethyl stearate, methyl oleate, ethyl oleate, methyl linoleate, ethyl linoleate, methyl linolenate and ethyl linolenate, are formulated in effective amounts with a food-grade antioxidant, including (but not limited to) butylated hydroxyanisole, butylated hydroxytolulene, tertiary-butyl hydroquinone, polypropylene glycol, citric acid and vitamin E and its derivatives to form 11-component compositions. These compositions can be delivered to honey bees within a hive on an inert substrate, including (but not limited to) paraffin, vegetable wax, beeswax, silica, alumina, cellulose, modified cellulose (paper and paper products), dried plant tissue, wood, a synthetic polymer, or a device containing liquid pheromone. These compositions can be used to manipulate the behavior of honey bee workers, including (but not limited to): increasing the ratio of pollen to non-pollen foragers, lowering the age of first foraging, inducing foraging behavior, stimulating pollen collection and transport of pollen back to the hive, stimulating consumption of supplementary diet components and antibiotics, inhibiting swarming and improving pollination of target crops. The stabilized pheromone compositions can also be used to stimulate the development and production of protein by the hypopharyngeal food gland. Combined application of stabilized brood pheromone compositions with the honey bee queen mandibular gland pheromone can be used to improve conditions within the hive and enhance the pollination of target crops.
Example 1
Formulation
Because of the possibility of tainting honey, only food-grade antioxidants were considered as potential stabilizers. Brood pheromone was formulated with tertiary-butylhydroquinone as a test stabilizer in the following proportions (percentages weight/weight) and amounts as follows: methyl palmitate 3% (0.0225 g), ethyl palmitate 3% (0.0225 g), methyl stearate 17% (0.1275 g), methyl oleate 24.95% (0.1871 g), ethyl stearate 7% (0.0525 g), ethyl oleate 8% (0.0600 g), methyl linoleate 2% (0.0150 g), ethyl linoleate 1% (0.0075 g), methyl linolenate 21% (0.1575 g), ethyl linolenate 13% (0.0975 g), and tertiary-butylhydroquinone 0.05% (0.0004 g). Components were mixed at approximately 20° C. in the above order using a magnetic stirrer and were blended until homogenous, resulting in 0.75 g of product.
Example 2
Chemical Stability
Short-term and long-term stability studies were conducted, with simplified blends of potentially unstable brood pheromone components mixed in approximately the same proportions as in Example 1. Short-term evaluation for a two-week duration was done on blends of methyl oleate, methyl linoleate and methyl linolenate, and ethyl oleate, ethyl linoleate and ethyl linolenate, each with and without tertiary-butylhydroquinone. Twenty-week tests were done with identical blends as for the two-week experiments, but 72-week tests had methyl and ethyl palmitate and stearate added in the appropriate proportions to their respective blends. Approximately 250 mg of a blend was placed in a 4 mL (15×45 mm) plastic vial (Chromatographic Specialties) fitted with a diffusion insert screw cap (Agilent Technologies) with a 1 mm diameter hole to allow air circulation over the sample. All blends were continuously held at a constant temperature of 30° C.
For analysis by capillary gas chromatography, approximately 1 mg of a blend was dissolved in 1 mL of diethyl ether, and a 1 μL aliquot of each extract was analyzed. Analyses were done at time zero and after the given duration of exposure to air at 30° C., 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 80° C. rising at 5° C. per min for 24 min to 200° C., and then rising at 20° C. per min for 3 min to 260° C.
Two-week results are given for methyl and ethyl esters (FIGS. 1 and 2), while long-term 20-week and 72-week results are given for only ethyl esters (FIGS. 3 and 4). Long-term results for methyl esters were virtually identical to those for the ethyl esters.
After two weeks, the proportional composition of methyl esters in the blend that was stabilized with tertiary-butylhydroquinone remained the same as in the unstabilized blend at time zero (FIG. 1). However, in the unstabilized blend, after two weeks, the amount of methyl linoleate had fallen by 85%, and the amount of methyl linolenate had decreased by 90%. Because of breakdown of the other components, the proportional composition of methyl oleate had risen from 48% of the total blend to 82%. Similar results were obtained with the ethyl esters after two weeks. The amount of ethyl linoleate fell by 74%, and the amount of ethyl linolenate decreased by 94%, while ethyl oleate rose from 40% of the total blend to 66% (FIG. 2). These results indicate that breakdown of methyl and ethyl linoleate and linolenate occurs rapidly at room temperature, and would probably result in complete loss of bioactivity of the full pheromone blend after 2-4 weeks. The results also indicate that without stabilization, the shelf life of any brood pheromone product would be unacceptably short.
After 20 and 72 weeks at 20° C., the proportional composition of ethyl oleate, ethyl linoleate and ethyl linolenate in the stabilized blends (FIGS. 3 and 4, respectively) remained essentially the same as in the stabilized blend after two weeks exposure (FIG. 2). In contrast, after 20 weeks, no ethyl linoleate and no ethyl linolenate remained in the unstabilized blend, and the amount of ethyl oleate had risen only slightly from 37% to 43% of the total blend, despite the complete absence of the other two major components (FIG. 3). This indicates complete breakdown of ethyl linoleate and ethyl linolenate after 20 weeks exposure, and also suggests partial breakdown of ethyl oleate.
As expected from the results in FIG. 3, no ethyl linoleate or ethyl linolenate remained after 72 weeks of exposure to room temperature, indicating complete breakdown of these two pheromone components (FIG. 4). The percentage compositions of ethyl palmitate and ethyl stearate rose from 8% and 23% to 20% and 57%, respectively, suggesting no breakdown even after 72 weeks. However, the percentage of ethyl oleate fell from 24% to 10%. Together with the results after 20 weeks, this decline confirms a slow breakdown of ethyl oleate in the unstabilized formulations.
These results indicate that stabilization of honey bee brood pheromone with tertiary-butylhydroquinone will result in no breakdown of any unstable pheromone component for well over a year. The lack of even a slight indication of breakdown after 72 weeks (FIG. 4) suggests that the shelf life of a stabilized pheromone formulation will be at least two years. This is a sufficiently long duration to justify development of a commercial product.
Example 3
Longevity of Stabilized Brood Pheromone Measured by Sucrose Response Threshold
Honey bees respond reflexively by extending the proboscis when a drop of sucrose solution is applied to the antennae. This is called the proboscis extension response threshold (PER-RT) assay to sucrose. The assay consists of a sucrose concentration course of 0.1, 0.3, 1, 3, 10 and 30% (wt/vol), corresponding to a logarithmic series of −1, −0.5, 0, 0.5, 1 and 1.5. The antenna of each bee is touched once with a droplet of sucrose. The lowest concentration that causes proboscis extension is interpreted as an estimate of individual sucrose response threshold (Page et al. 1998; Pankiw and Page 1999). The results from the PER assay with ascending concentrations of sucrose are transformed to sucrose scores. A PER score is directly related to the response threshold of the individual bee, because most bees continue to respond to all increasing concentrations of sucrose following initial response. A PER score of 6, wherein an individual bee responds to all 6 sucrose concentrations, beginning with 0.1% sucrose in the PER assay, represents a response threshold 300 times lower than a score of 1, wherein an individual responds only to 30% sucrose. The response threshold (RT) of an individual bee is a “window” into the neurosensory system that is correlated with foraging behavior (Pankiw and Page 2000), and age (Pankiw and Page 1999; Pankiw 2003), and is modulated by brood pheromone (Pankiw and Page 2001, 2003). We used the PER-RT assay to sucrose as one method of assessing the bioactivity of stabilized brood pheromone stored for various durations at room temperature.
Honey bees from at least six unrelated “wild-type” sources were emerged for 3 h from their combs in an incubator maintained at 33° C. and 55% RH. Three hundred newly-emerged bees were randomly placed into plexiglass wire mesh cages (14.6×10×7.7 cm). Cages were provided with 30% sucrose solution, water ad libitum, and reared in the above incubator conditions. Individual cages were randomly selected for the following treatments: 1) unstabilized brood pheromone stored at room temperature, 2) brood pheromone stabilized with tertirary-butyl hydroquinone and stored at room temperature, 3) freshly prepared unstabilized brood pheromone, and 4) a control with no pheromone. The composition of the prepared pheromone was: 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 stearate 17%. Pheromone-treated cages received 300 larval equivalents (LEq) per day (1:1 ratio of brood pheromone to bees). The pheromone was formulated in isopropanol such that 300 LEq was delivered in approximately 15 μL of solution. The control cages received an equal volume of isopropanol daily. Respective treatments were delivered daily on a glass plate (7×8 cm) after the isopropanol was evaporated. Bees were reared as above for six days, and then tested for their sucrose response threshold as described above. This protocol was followed on days 48, 123 and 231 after the preparation of unstabilized and stabilized formulations of brood pheromone. Between treatment scores were compared using Kruskal-Wallis and Mann-Whitney U tests.
Stabilized brood pheromone unexpectedly showed bioactivity that was no different from freshly formulated brood pheromone (Table 1). Honey bees exposed to stabilized brood pheromone consistently showed a sucrose response threshold that was at least 1.75 times higher than the response of unexposed bees in control cages. Additionally, sucrose responsiveness of honey bees exposed to unstabilized brood pheromone stored at room temperature for 48, 123 and 231 days was not significantly different from that of control bees exposed to no pheromone.
These results demonstrate that stabilized brood pheromone shows long-term bioactivity when stored at room temperature. This bioactivity is statistically the same as that induced by freshly-formulated brood pheromone. The results do not support the hypothesis that bioactivity is due to breakdown products of the unstable brood pheromone components that build up to inhibitory levels over time. In contrast, they support the alternative hypothesis that bioactivity depends on the presence in the pheromone blend of sufficient amounts of the unstable fatty acid esters. The results also unexpectedly indicate that addition of tertiary-butylhydroquinone to the ten pheromone components had no effect on bioactivity of the pheromone. Thus it does not adversely affect the chemical integrity of any of the ten pheromone components, does not inhibit pheromone receptor membranes in the sensilla, is not repellent and does not contribute to a false message that would have prevented a physiological and behavioral response to the pheromone. Therefore, an 11-component stabilized synthetic pheromone composition has probable practical utility in apiculture.
Table 1. Bioactivity of unstable and stabilized brood pheromone stored at room temperature versus freshly formulated brood pheromone as a percent of sucrose responsiveness in honey bees held in a no-pheromone control environment. Probabilities below each value indicate likelihood that sucrose responsiveness was similar to that of bees in the no-pheromone control environment.
















Freshly-



Unstable brood
Stabilized brood
formulated


Days at room
pheromone
pheromone
unstable brood


temperature
%
%
Pheromone


















48
17.0 
88.1
78.7



(P > 0.05)
(P < 0.01) 
(P < 0.001)


123
6.8
75.6
68.5



(P > 0.05)
(P < 0.001) 
(P < 0.001)


231
3.7
89.7
91.0



(P > 0.05)
(P < 0.0001)
 (P < 0.0001)









Example 4
Brood Pheromone Formulated from Reagent-Grade and Technical-Grade Fatty Acid Esters Releases Honey Bee Pollen Foraging
All previous formulations of brood pheromone were comprised of reagent grade compounds of greater than 99% purity supplied by Sigma-Aldrich (St. Louis, Mo.). Reagent grade compounds are most commonly used as standards in chemical identification, for chemical synthesis, or in any other application where purity is essential. However, amount of purity is directly proportional to the cost of a compound. One way to reduce the cost of brood pheromone is to formulate it using less pure, technical grade components. Although cost is significantly reduced with technical grade components there is a trade-off. Technical grade components are derived from natural sources containing blends of fatty acids not in the exact proportions of the natural brood pheromone. We hypothesized that bees exposed to brood pheromone formulated from either reagent-grade or technical-grade components would forage for pollen at a higher level than bees exposed to no pheromone.
This experiment was replicated six times on Jun. 7, 2005. Typical honey bee colonies were randomly selected to receive one of the following treatments; 1) control, blank glass plate, 2) reagent-grade brood pheromone stabilized with tertiary-butyl hydroquinone, or 3) similarly stabilized technical-grade brood pheromone. Pheromone blends were formulated with tertiary-butyl hydroquinone according to the proportions given in Table 2. A total of 1.12 mg of total esters was applied to a glass plate (20 cm×10 cm), and the plate was placed in the center of the brood nest area of the colony. One hour later, we conducted five minute entrance counts of returning pollen and non-pollen foragers. Pollen foragers are visibly apparent because pollen is carried outside the body on specialized areas of the hind legs called the corbiculae. Liquid loads such as nectar and water are carried internally in the crop and are not visibly apparent. When type, presence or absence of load is not visibly apparent, such returning bees are classified as non-pollen foragers. Observers use hand-held tally counters, one hand for pollen and one hand for non-pollen foragers. Each colony was measured two times per hour for two hours by two different people for a total of four five-minute counts per colony. The people performing the counts were blind to the treatments.
Count data were analyzed using Contingency Table Analysis. Compared to the no pheromone blank control, both the technical-grade (χ2=7.0, df=1, P<0.01) and the reagent-grade (χ2=15.0, df=1, P<0.0001) formulations induced significantly greater ratios of pollen to non-pollen foragers entering colonies (Table 3). The reagent-grade formulation released a slightly higher proportion of pollen foragers than the technical grade formulation (χ2=3.9, df=1, P=0.048).
Two pertinent findings arise from this study. First, stabilized brood pheromone of either grade is capable of inducing enhanced pollen foraging. Second, technical-grade formulated brood pheromone increased the proportion of pollen foragers by approximately 116% over the no pheromone control treatment, only 3.5% less than that induced by the reagent-grade formulation. Thus, at a cost of only 7% of the reagent-grade formulation, technical-grade formulated brood pheromone stabilized with tertiary-butyl hydroquinone appears to be an efficacious and economically viable product.







TABLE 2







Formulation of reagent-grade and


technical-grade brood pheromone.










Reagent-grade fatty
Technical-grade fatty



acid esters + 0.05%
acid esters + 0.05%



tertiary-butyl
tertiary-butyl



hydroquinone
hydroquinone


Compound
(% of total esters)
(% of total esters)












Methyl Palmitate
3.00
2.03


Ethyl Palmitate
3.00
4.23


Methyl Stearate
17.00
15.17


Methyl Oleate
25.00
25.04


Ethyl Stearate
7.00
8.88


Ethyl Oleate
8.00
6.72


Methyl Linoleate
2.00
8.63


Ethyl Linoleate
1.00
3.5


Methyl Linolenate
21.00
17.07


Ethyl Linolenate
13.00
8.73
















TABLE 3







Ratio of pollen to non-pollen foragers


entering colonies in a 5 min period.










Ratio of pollen/



Treatment
non-pollen foragers
Probability1












Control
0.38



Technical-grade fatty acid esters +
0.82
<0.01


Tertiary-butyl hydroquinone)




Reagent-grade fatty acid esters +
0.85
<0.0001


Tertiary-butyl hydroquinone)





1Contingency Table Analysis probabilities where the null hypothesis is that control and treatment ratios are the equal.






Example 5
Racial Specificity in Pheromone Composition
Until 1990, honey bees in the USA were considered to be a mixture of seven subspecies introduced since the 1600s from Europe (Sheppard 1989). In 1990, Africanized honey bees (produced by cross mating of European honey bees with the South African sub species, Apis mellifera scutellata) were first found in the USA (Pinto et al. 2005). Thus, both European and Africanized honey bees now occur in the USA. The inventors hypothesized that different races of honey bees, with race-specific genetic make up, will produce brood pheromone with distinctly different ratios of components.
Honey bees from three populations were procured and maintained in an apiary at Texas A&M University, College Station, Tex., USA. European queens and package bees, with European DNA (Georgia-European race) were purchased from a breeder in Moultrie, Ga., USA. Africanized colonies, with African DNA (Texas-Africanized race) were collected from Mission, Tex., USA. Texas-European honey bees, with mixed African and European DNA (Texas-European race) were from Navasota, Tex., USA. European and African lineage was confirmed by analysis of mitochondrial DNA using the method of Pinto et al. (2003). Colonies of Apis mellifera scutellata (South African “race”) were maintained in an apiary at the University of Pretoria, Pretoria, South Africa.
For extraction of brood pheromone, larvae were collected from eight Texas-Africanized, eight Georgia-European, 11 Texas-European and seven South African colonies. Larvae were removed from cells, washed gently in water and shaken dry. Ten fifth instars from each race were pooled in a glass beaker and soaked for 1 min in 2 mL of 95% n-hexane containing 1 μg methyl myristate as an internal standard and 0.05% tertiary-butyl hydroquinone as an antioxidant. Extracts were filtered through a Buchner funnel, and the larvae and glassware were washed further with 2 mL of 95% n-hexane, which was added to the extract.
Extracts were concentrated to 1 mL by using a nitrogen stream in a water bath at 55° C., and then fractionated by liquid chromatography using a silica gel column (0.5 g, 70-230 mesh/60 Å; Sigma-Aldrich, St. Louis, Mo., USA) in a Pasteur pipette plugged with a piece of dust-free paper cloth. Extracts were placed on freshly prepared and rinsed columns, and two fractions were eluted: the first, containing long and short-chain hydrocarbons, but no esters, with 10 mL of 95% n-hexane, and the second, containing the esters, with 10 mL of 99% dichloromethane. The dichloromethane fraction was evaporated to apparent dryness using a nitrogen stream in a water bath at 50° C., and then immediately reconstituted in 1 mL of 95% n-hexane and vortexed for 30 sec. This was concentrated to ca. 100 μL and transferred to an autosampler vial containing a 250 μL glass insert; the vial previously containing the fraction was rinsed three times with 50 μL hexane and the rinse added to the insert. Solvent was removed from the fraction by heating to 50° C., after which the vial was cooled and 20 μL hexane containing 2 μg of n-octadecane (Sigma-Aldrich, St. Louis, Mo., USA) was added to the fraction as a reference.
Extracts were analyzed with a Hewlet-Packard 6890 gas chromatograph (GC) (Hewlet-Packard, Palo Alto, Calif., USA) equipped with splitless injection, flame ionization detection, a 60 m×0.25 mm ID HP-88 ([88%-cyanopropyl]-methylarylpolysiloxane, Agilent, Santa Clara, Calif., USA) column. The inlet temperature was held at 60° C. for 0.1 min and then increased to 250° C. at 50° C. per min. The column oven temperature was held at 50° C. for 2 min, then increased to 170° C. at 20° C. per min, held for 3 min, then increased to 230° C. at 30° C. per min. The carrier gas was hydrogen at a constant flow of 2 mL per min. Individual fatty acid ester retention times were identified by comparison with >99% pure standards (Sigma-Aldrich, St. Louis, Mo., USA). For quantification, standard curves of peak area for each fatty acid ester were constructed. Extract ester quantities were corrected for methodological error by reference to the methyl myristate internal standard.
GC-mass spectrographic (MS) analyses were performed on a 6890 GC coupled with a 5957C Inert XL quadruoole MS (Agilent, Santa Clara, Calif., USA). The GC was equipped with a 30×0.25 mm HP-5MS [(5%-phenyl)-methylpolysilozane], fused silica capillary column (Agilent, Santa Clara, Calif., USA), and a splitless injector using helium as carrier gas at a constant flow of 1.0 mL per min and an inlet temperature of 300° C. The column temperature was programmed from 150° C. to 320° C. at 20° C. per min. The MS was operated in electron ionization mode (EI) at 70-eV electron energy, using selected ion monitoring (SIM) mode for the following m/z (indicative of): 74, 242 (methyl myristate), 74, 270 (methyl palmitate), 88, 284 (ethyl palmitate), 81, 294 (methyllinoleate), 84, 264 (methyl oleate), 79, 292 (methyl linolenate), 74, 298 (methyl stearate), 81, 308 (ethyl linoleate), 98, 310 (ethyl oleate), 79, 306 (ethyl linolenate), 88, 312 (ethyl stearate), and 85, 254 (octadecane).
GC and GC-MS analyses revealed surprising differences in mean percentage composition of fatty acid ester components of brood pheromone among the extracts from the four races of honey bees analyzed, and the composition of the brood pheromone from a French race (personal communication, Y. LeConte, INRA, Avignon, France) (Table 4). The smallest range between lowest and highest percentage composition was 1.6 times for ethyl linolenate, and the largest range between lowest and highest percentage composition was 12.1 times for ethyl linoleate.







TABLE 4







Percentage composition of brood pheromone components


among five races of honey bees, three with European


DNA, and two with African DNA.
















Texas-





Texas-
Georgia-
Afri-
South



French
European
European
canized
African


Fatty acid ester
race
race
race
race
race















Methyl palmitate
3.0
6.9
2.0
4.1
6.0


Ethyl palmitate
3.0
3.9
3.9
4.7
8.8


Methyl stearate
17.0
15.6
6.5
14.9
25.6


Ethyl stearate
7.0
6.4
8.1
10.3
14.5


Methyl oleate
25.0
16.8
20.6
9.0
11.5


Ethyl oleate
8.0
15.4
13.8
15.9
12.0


Methyl linoleate
2.0
2.9
4.2
9.3
4.5


Ethyl linoleate
1.0
5.4
12.1
6.4
4.9


Methyl linolenate
21.0
13.8
16.9
11.7
2.8


Ethyl linolenate
13.0
12.9
11.0
13.7
8.5









There were also substantial differences in ranking for all of the 10 components among races. The brood pheromone from the French race ranked highest in composition of methyl oleate and methyl linolenate and lowest in composition of ethyl palmitate, ethyl oleate, methyl linoleate and ethyl linoleate. The pheromone from the Texas-European race ranked highest in composition of methyl palmitate and lowest in composition of ethyl stearate. The pheromone from the Georgia-European race ranked highest in composition of ethyl linoleate and lowest in composition of methyl palmitate and methyl stearate. The pheromone from the Texas-Africanized race ranked highest in composition of ethyl oleate, methyl linoleate and ethyl linolenate, and lowest in composition of methyl oleate. Finally, the pheromone from the South African race ranked highest in composition of ethyl palmitate, methyl stearate and ethyl stearate, and lowest in composition of methyl linolenate and ethyl linolenate.
Example 6
Racial Specificity in Pheromone Response
The large differences in percentage composition in brood pheromone components among honey bee races, led the inventors to hypothesize that bees of each race would respond optimally to synthetic brood pheromone blends that reproduced the ratio of components produced by larvae of that race. This hypothesis was tested by conducting three experiments in which the ratio of pollen foragers to non-pollen foragers was determined for honey bee races exposed to the pheromone blend of their own race, the pheromone blend of another race, or to a solvent control treatment.
The first experiment compared the response of bees of the Texas-European race to their own pheromone blend, the French pheromone blend, and a solvent control. Twelve similarly-sized Texas-European colonies were established, each comprising approximately 1 kg of bees, one queen and a few very young larvae. Colonies received 0.6 mg of the Texas-European or French pheromone blend (Table 4). This dose was selected through a series of dose-response trials following the method of Pankiw et al. (1998). Pheromone was applied on glass plates measuring 14×7 cm. Solvent was allowed to evaporate prior to placement in colonies. A single plate was then hung on metal wire between two frames in the middle of the colony. One hour after placement of glass plates, the number of pollen and non-pollen foragers entering colonies was counted for a 5 min period according to the method of Pankiw et al. (1998). Pollen foragers were distinguishable from non-pollen foragers by the pollen pellets on their hind legs. Counts were performed between 0800 and 1000 h. All colonies received all treatments on subsequent days in a random, non-repeating manner.
The pollen foraging bioassay was performed as above in two additional experiments, one with six Texas-Africanized colonies and the other with six Georgia-European colonies, each treated with 1.0 mg of the synthetic blend of their own race, the other race or a solvent control.
For each experiment, counts were analyzed by 3×2 Chi-square contingency table analysis for the effect of pheromone treatment on pollen to non-pollen forager ratio. Pairwise comparisons were performed using 2×2 Chi-square contingency table analysis with the Dunn-Sidak method of correcting for possible Type 1 errors.
Surprisingly, in each experiment, bees in the test race had the highest ratio of pollen to non-pollen foragers when exposed to the brood pheromone blend formulated in the percentage composition characteristic of their own race (Table 5). In the first experiment with the Texas-European race, statistical analysis indicated that the ratios differed among treatments (χ2=64.0, df=2, P<0.001). Pairwise comparisons indicated that all treatments were different from each other, with the ratio in response to the Texas-European blend being greater than the response to the French blend (χ2=15.1, df=1, P<0.001), which in turn was greater than to the solvent control (χ2=22.1, df=1, P<0.001).
Similarly, in the experiment with the Georgia-European race, statistical analysis indicated that the ratios differed among treatments (χ2=121.4, df=2, P<0.001) (Table 5). Pairwise comparisons indicated that all treatments were different from each other, with the ratio in response to the Georgia-European blend being greater than the response to the Texas-Africanized blend (χ2=3.3, df=1, P=0.038), which in turn was greater than to the solvent control (χ2=72.6, df=1, P<0.001).
Table 5. Ranked ratios of pollen to non-pollen foragers in three experiments in which honey bee forgers were exposed to brood pheromone blends characteristic of their own race, another race or a solvent control. For each experiment, ratios followed by different letters are significantly different, Chi-square test, P<0.05.














Mean ratio of pollen to


Race of test bees
Pheromone blend tested
non-pollen foragers ± SE







Texas-European
Texas-European
1.27 ± 0.34 a



French
0.61 ± 0.13 b



Solvent control
0.19 ± 0.03 c


Georgia-European
Georgia-European
0.97 ± 0.05 a



Texas-Africanized
0.86 ± 0.09 b



Solvent control
0.35 ± 0.03 c


Texas-Africanized
Texas-Africanized
2.44 ± 0.30 a



Georgia-European
1.78 ± 0.52 b



Solvent control
1.69 ± 0.22 c









Finally, in the experiment with the Texas-Africanized race, statistical analysis indicated that the ratios differed among treatments (χ2=59.8, df=2, P<0.001) (Table 5). Pairwise comparisons indicated that all treatments were different from each other, with the ratio in response to the Texas-Africanized blend being greater than the response to the Georgia-European blend (χ2=58.8, df=1, P<0.001), which in turn was greater than to the solvent control (χ2=7.1, df=1, P=0.004).
These results unexpectedly indicate that the practical efficacy of products utilizing a synthetic honey bee brood pheromone composition can be optimized by using a pheromone blend that is formulated in the precise ratio of components found in the natural pheromone produced by larvae of the target race or sub-species.
Example 7
Stimulation of Pollen Diet Consumption
This experiment was conducted from 1-15 Mar. 2004. Twelve typical colonies from Texas A&M University apiaries were randomly selected. Colony measures were estimated by the use a grid divided into square sections, 6.45 cm2. The area covered by bees was converted to bee numbers; approximately 1.5 bees per cm2 (Pankiw and Page 2001; Pankiw et al. 2002). Comb area occupied by eggs, larvae, and pupae collectively known as brood were measured. Six colonies were randomly selected to receive daily treatment of brood pheromone at three larval equivalents/bee/day (1.55 μg/bee/day), and six colonies received a blank glass plate. A dose of three larval equivalents/bee/day is known to increase amount of pollen foraging in large colonies (Pankiw and Page 2001). Each colony was provisioned with a commercially available pollen diet supplement: 450 g of Brood BuilderTM (Dadant & Sons, Hamilton, Ill.) shaped like a hamburger patty between two pieces of scored wax paper. Fresh patties were provided upon consumption of the previous patty. Every three days for fifteen days colonies were inspected for patty consumption. A new patty was provided to colonies that fully consumed the previous patty. Colony measures were conducted on days 7 and 15. All colonies received a fresh pollen supplement patty regardless of amount used on day 7. Unconsumed old patties were removed and weighed to estimate amount consumed.
Analysis of variance was used to compare pheromone treated and control colonies. It was not necessary to perform analysis of co-variance because at the outset of the experiment number of bees and amounts of comb space used by brood, pollen, honey, and empty cells was not significantly different (P>0.05) between treatment groups.
Colonies treated with brood pheromone consumed significantly more pollen supplement than did control colonies in weeks 1 and 2 (Table 4). Brood area was not different between treatments in week 1 (F1,11=0.3, P>0.05). In week 2 brood area was significantly greater in the brood pheromone treated group (F1,11=10.6, P<0.01; Table 2).
These results indicate that stabilized brood pheromone can be used to stimulate consumption of food, resulting in increased production of brood, which in turn can lead to greater colony vigor. Increased consumption of food can also be exploited by incorporating dietary supplements or antibiotics into a pollen patty or other food source.
Table 6. Comparison of pollen consumption and brood area between honey bee colonies exposed to brood pheromone and unexposed control colonies.












Treatment/
Consumption (g)
Brood area (cm2)*











statistics
wk 1
wk 2
wk 1
wk 2





Brood
406.6 ± 9.5 
422.5 ± 17.0
3928 ± 586
2975 ± 330


pheromone






Control
185.0 ± 21.4
178.3 ± 27.2
3472 ± 668
1800 ± 142


ANOVA F1, 11
89.7
57.2
0.3
10.7


Probability
.0001
.0001
0.6
0.008









While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and sub-combinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are within their true spirit and scope.
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## Claims

1. An 11-component stabilized synthetic honey bee brood pheromone composition comprised of ethyl linoleate, ethyl linolenate, ethyl oleate, ethyl palmitate, ethyl stearate, methyl linoleate, methyl linolenate, methyl oleate, methyl palmitate and methyl stearate and an effective amount of a food-grade tertiary-butyl hydroquinone, wherein the proportions by weight of the components are: ethyl linoleate (0.1-50%), ethyl linolenate (0.9-50%), ethyl oleate (0.8-50%), ethyl palmitate (0.3-50%), ethyl stearate (0.6-50%), methyl linoleate (0.2-50%), methyl linolenate (0.2-50%), methyl oleate (0.3-50%), methyl palmitate (0.3-50%), methyl stearate (0.7-50%) and tertiary-butyl hydroquinone (0.005-5.0%).