CROPS"

The present invention relates to a formulation that promotes pollination of apple crops (Malus silvestris) by targeting collection preferences of honey bees (Apis mellifera) . The same comprises Citral, Benzaldehyde and Limonene. Particularly, the formulation comprises from about 35% to about 60% of Citral, from about 30% to about 50% of Benzaldehyde and from about 10 % to about 20% of Limonene. Preferably, the formulation of the invention comprises 44% of Citral, 40% of Benzaldehyde and 16% of Limonene .

Background of the invention

In melliferous bees, scents play an important role in the selection and discovery of food sources. It is known that bees target collection of sources not only according to innate search images but also according to previous experiences, whether in the field or within the social environment of the beehive (Ribbands CR (1955) The scent language of honey bees; Ann Rev Smithson Inst 368-377; von Frisch K (1967) The dance language and orientation of bees; Harvard University Press, Cambridge, MA) . In this sense, the scent memory established in the field from floral scents and nectar can be retrieved (recalled) when said floral scent appears in the beehive (Ribbands CR (1954) Communication between honeybees: the response of crop-attached bees to the scent of their crop; Proc R Entomol Soc Lond A 29:141-144; Johnson DL and Wenner AM (1966) A relationship between conditioning and communication in honeybees; Anim Behav 14:261-265; Jakobsen HB et al . , (1995) Can social bees be influenced to choose a specific feeding station by adding the scent of the station to the hive air? J Chem Ecol 21 (11) : 1635-1648; Reinhard J et al . , (2004) Floral scents induce recall of navigational and visual memories in honeybees , J Exp Biol 207:4371-4381) . Also, the ingestion of scented food that collector bees take to the hive can promote targeting of the collection preferences of congeners (von Frisch K (1923) liber die Sprache cler Bienen; Zool Jb Physiol 40:1-186; Wenner AM et al. (1969) Honey bee recruitment to food sources : olfaction or language? Science 164:84-86; Dornhaus A, Chittka L (1999) Evolutionary origins of bee dances; Nature 401:38). During collection from scented food sources, the members of the beehive are able to learn odors when these remain impregnated on the body of the collector bee (von Frisch κ

(1967) The dance language? and orientation of bees; Harvard University) and/or when small samples of scented food are transferred from mouth to mouth between hive members (trophallaxis)

(Farina, WM et al . (2007) Honeybees learn floral odors while receiving nectar from foragers within the hive; Naturwissenschaften, 94:55-60) .

Also, it has been shown that scented food introduced by collector bees that circulates within the hive may affect behavior of young bees that are not involved in collection yet (Gruter C et al . (2006) Propagation of olfactory information within the honeybee hive, Behav Ecol Socrobiol 60:707-71; Gruter C et al . (2009). Retention of long-term memories in different age-groups of honeybee (Apis mellifera) workers. Insectes Sociaux, DOI 10.1007/S00040-009-0034-0) .

Moreover, there are numerous crops that require to be pollinated by insects that upon visiting the different flowers of the same species benefit cross fertilization. Particularly, the bee Apis mellifera is the most numerous pollinator of monocrops worldwide and is considered of the highest importance to improve fruit tree and seed yields.

In Argentina apiculture is essentially conceived to obtain the products stocked in the hive (honey, propolis, royal jelly, etc.) and that is how the country is among the most important honey producers worldwide. However, the use of melliferous bee as a pollination agent is not very developed when compared to the importance it has in countries as the United States or France. However, with the advance of the monocrop, there is an increasing need to increment targeted and sustainable pollination of these crops .

Targeted pollination poses a primary problem related to the change in floral availability when beehives are taken from a location where collector bees are familiarized to a new one. In the new location bees do not have reliable and updated information that can be recalled during collection.

In practice, it is observed that after beehive migration, collector bees stay relatively inactive during the first days and do not visit immediately the flowers to be pollinated even when they offer a great reward. This is because the recently introduced bees do not have previous experiences related to the crop of interest, and therefore there are not previously established memories that lead to or make easing finding this floral type. During a variable time (that could last several days) bees establish new associations (memories) and update the information that allows them finding new floral types available in the environment .

Thus, the method of stimulating beehives with natural scents, particularly flower grinding, was used in the 30s by former URSS and Germany beekeepers (von Frisch 1967) .

The inventors of this invention have reported results indicating that bees can create scent memories within the hive during circulation of scented food that can be retrieved (recalled) days after being out of the hive, which is viewed at the targeting of collection preferences. Creation of these memories does not depend on recruiting mechanisms that are common to the hives or the presence of scented food stocks that make easier recovery of previously acquired memories. (Arenas A et al., (2007). Floral odor learning within the hive affects honeybees ^' foraging decisions. Naturwissenschaften, 94:218-222) .

Two products are commercially available, POLLINUS ^® and BEE SCENT ^® that represent the general profile of an orientation pheromone in bees, therefore its presence on flowers attracts new bees. They must be spayed on the crop flowering (whatever this is) that can be useful in small areas (orchards, nurseries) but not in large areas. This practice would require several applications of the product which would turn the operation costly and unpractical for extensive crops. Being products that directly function as bee attractors (though they might attract other insects) they have no specificity to a given floral type and could directly interfere with other behaviors of bees. None of the products is based on floral odors associated to the appetite context nor do they involve scent memory of bees.

Moreover, in the patent application JP2008212148 "Method for promoting pollination of plant including induction of flower bee to floral organ of specific plant by taking advantage of floral fragrance component of flower organ of this plant", there is no description of a low cost formulation that specifically simulates the apple flower fragrance that comprises only three aromatic substances.

In "Floral odor learning within the hive affects honeybees ^' foraging decisions" , published in Naturwissenschaften, 94:218-222, Arenas A et al . , it is also disclosed that the odor of food offered "inside the hive" generates long lasting odor memories that affect the selection behavior of food sources outside the hive. Also, it is mentioned that to obtain long lasting memories of a specific floral odor (pure), it must be offered dissolved in a sweetened solution (50% saccarose solution w/w), that can be offered in a feeder inside the hive (usual technique in beekeeping) . The pure odor must be dissolved in the sugar solution at minimum quantities (concentration: 50 μΐ of odor per liter of offered sweetened solution) .

In "Floral scents affect the distribution of hive bees around dancers" , Behavioral Ecology and Sociobiology (2007) 61:1589-1597, July 04, 2007, Diaz, Paula C. et al . , discloses that floral scents impregnated in the body of a bee returning from flowers attracts congeners at the time of following the "bees dance", a mechanism used by this species to recruit other bees to food sources inside the hive, besides incrementing the occurrence of transfers of mouth to mouth samples of food.

Finally in "Floral scents experienced within the colony affect long-term foraging preferences in honeybees" . Apidologie 39:714-722, 2008, Arenas A et al . , it is disclosed that it is the food odor circulating inside the hive and not the same odor exposed as volatile, the one that increases the number of landings to the source of food with that specific scent. Also, in "Passive volatile exposure within the honeybee hive and its effect on odor discrimination" , Journal of Comparative Physiology A, 195:759-768, 2009, Fernandez V et al., it is disclosed that the floral odor exposed as volatile "inside the hive" makes that odor learning difficult in later trainings. This shows that those odors exposed as volatile compounds inside the nest are not preferred if present in flowers of the environment even when these types of flowers offer plenty of nectar.

Brief description of the invention

Therefore it is an object of the present invention to provide a formulation that simulates the floral scent of the apple flower in order to create specific odor memories in bees of hives to be transferred to apple crops and used to favor pollination thereof. Memories established by stimulation with the formulation allow reducing times of inactivity of bees before starting collection. The formulation of the invention stimulates collection by promoting a quicker and sustained collection action on the crop. By using the invention, it is expected to reduce the residence time of hives at the crop as well as the number of hives to use. On the other hand, the cost of the inventive formulation is very low and does not require sophisticated techniques to administer the same.

Description of the drawings

Figure 1 : Response of proboscis extension (PER) of attached bees subjected to a classic conditioning wherein two scents are presented, four times each. One of the scents was compensated with a sweetened solution (CS+) and the other was not compensated (CS-) . Acquisition curves during training are shown on the right of the graphic and the scent evaluation after training to the left. A) The natural scent of apple flowers was the CS+ and the jasmine synthetic scent was CS- B) Jasmine synthetic scent was the CS+ and the natural scent of apple flowers was CS-. C) The natural scent of apple flowers was the CS+ and the apple synthetic formulation was the CS- D) Apple synthetic formulation was the CS+ and the natural scent of apple flowers the CS-. The number of conditioned bees is shown in the upper part of each figure. M: apple, J: jasmine, FS: synthetic formulation.

Figure 2 : Description of the progress of flower opening of apple trees through the week of recording (A) . The first variety of apples started to flower on September 29. Between the first day and the second of October was the peak of flowering of all varieties around the place of study. Before apple trees there were other fruit trees flowered in the area. The average collection activity of 5 beehives was measured as the total entry of bees in the hive per minute (B) . Treatment with the Apple synthetic formulation was positive, as the total entry of bees to beehives with this treatment was equal or higher than to untreated beehives through the days (Repeated measures ANOVA: treatment effect: F=6.95, gl=l, p=0.029, Figure 2B) . This difference increased to the last days of recording, a moment that matches with the flowering peak of apple trees. Bees of hives fed with a sweetened solution aromatized with the Apple synthetic formulation would be increasing their general activity and also collecting higher amounts of nectar when the predominant floral offer at that time is the apple tree.

Detailed description of the invention

In the last years pollination con beehives of melliferous bees en apple crops became very relevant due to the higher yield of fruits obtained.

The presence of beehives of melliferous bees near apple trees from the beginning of flowering improves the number of fertilized flowers, therefore a higher number of fruit set is obtained, which produces a higher number of fruits obtained. Also, resulting fruits are bigger and bear a higher number of seeds per fruit, parameters related to a higher quality of apples of the different varieties exported as premium products worldwide.

For these reasons apple producers hire a beekeeper pollination service devoted to beehive migration, whereby they hire during the flowering period a certain number of beehives depending on the type of crop to pollinate. In this context and considering beekeepers commonly stimulate hives with sweetened solutions, the application of a supplement containing the synthetic formulation of apple does not represent a significant change in the beekeeping practice. When beekeepers perform hive migration during this period, bees of those populations do not immediately start to visit new flowerings. That is why the power to establish "memories of a synthetic formulation that simulates the apple scent" inside beehives would allow to reduce delays to start collection of this crop besides turning pollination more efficient.

Therefore it is an object of the present invention to provide a formulation that simulates the natural scent of the apple flower so that bees start collection immediately in this crop. Said formulation comprises the compounds Citral, Benzaldehyde and Limonene.

Particularly, the formulation of the invention that simulates apple tree natural scent comprises from about 35% to about 60% of Citral, from about 30% to about 50% of Benzaldehyde and from about 10 % to about 20% of Limonene. Preferably, the formulation of the invention that simulates the floral odor of the apple tree comprises 44% of Citral, 40% of Benzalde yde and 16% of Limonene . In the present application, unless otherwise provided, concentrations are expressed as volume in volume that is the compound volume, expressed in ml by 100 ml of total volume. Also, according to the present application the terms "Citral", "Benzaldehyde" and "Limonene" comprise the compounds themselves and derivatives, polymorphs, hydrates, solvates, etc. thereof.

Surprisingly the present inventors discovered that bees cannot tell (confuse) between the odor of the apple flower natural fragrance and this formulation. Furthermore, field tests also show that beehives fed with the formulation of the invention start collection in apple crops earlier than control beehives and for a longer time.

As to the use procedure, the formulation of the invention can be added to the hive before or after installation of beehives in the apple crops using common methods known by beekeepers. The formulation of the invention can be introduced in the hive diluted in a 50%w/w sweetened solution, at a concentration of about 50μ1 of formulation per liter of sugar solution placed in an artificial feeder inside the hive or directly pouring from 500 to 1000ml of the sweetened solution on the hive frames with bees in two successive applications.

Adding the formulation of the invention, associated to sweetened syrup before or during installation of beehives in apple crops promotes a quick and sustained collection activity on the crop, as well as a higher collection activity in general. This simple method of stimulation makes pollination of the apple crop easier, takes a shorter time to pollinate and requires a lower number of beehives. On the other hand, it is noted that this formulation could be commercialized at a low cost.

Unlike POLLINUS ^® and BEE SCENT ^® , products that produce innate responses on the bee behavior, the formulation of the invention does not induce "rigid" responses on the bee behavior (which can be counterproductive in many cases) . On the contrary, it influences the decision making processes that are flexible and can be adjusted according to the needs of each beehive or the environment changing conditions. This is essential when it is desired to integrally manage beekeeping and agriculture without generating the benefit of one of the practices to the detriment of the other.

On the other hand, it is another object of the invention, a formulation that promotes targeted pollination of melliferous bees to apple crops that comprises Citral, Benzaldehyde, Limonene, sugar and water. Preferably, it comprises about 50 μΐ of a formulation comprising from about 35% to about 60% of Citral, from about 30% to about 50% of Benzaldehyde and from about 10 % to about 20% of Limonene, diluted in a sweetened solution.

More preferably, it comprises 50 μΐ of a formulation that comprises 44% of Citral, 40% of Benzaldehyde and 16% of Limonene, diluted in a 50% w/w sweetened solution.

Thus, the invention can be used in small amounts to stimulate the beehive. That added to the fact that the formulation is simple, as it consists of only three of the numerous volatiles that comprise the apple tree fragrance, makes it an economical product, easy to manufacture and use. Also, the formulation stimulates the immediate collection in apple crops. The formulation can be prepared before migrating the bees to the crop or even before starting flowering of such plant species. Also it would stimulate generalized pollen collection that is from different floral species, which is particularly important even if it is desired that bees have a rich and diverse diet .

Examples

Figure 1 : Response of proboscis extension (PER) of attached bees, subjected to a classic conditioning wherein two scents are presented, four times each. One of the scents was compensated con sweetened solution (CS+) and the other wasn't (CS-) . Through this procedure, bees can be trained to differentiate scents under lab conditions (Bitterman, M.E., Menzel, R., Fietz, A., and Schaefer, S. 1993. Classical conditioning of proboscis extension in honeybees. J. Comp. Psychol. 97, 107-119) . Acquisition curves during training are shown on the right of the graphic and the scent evaluation after training to the left.

A) Apple tree natural scent was the CS+ and the jasmine synthetic scent (Pfeiffer- Gerhards Keramik - Germany) was CS-: Bees learn to distinguish both scents during training (Repeated measures ANOVA: CS+ higher than the CS-: F=111.24; gl=l; N=32; p<0.001) and in the test phase (X ² =40.82; p<0.001) .

B) Jasmine synthetic scent was the CS+ and the natural scent of apple flowers was CS-: Bees learn to distinguish both scents during training (Repeated measures ANOVA: CS+ higher than CS-: F=15.366; gl=l; N=36; p<0.001) and in the test phase (X ² =32.956; p<0.001).

c) Apple natural scent was the CS+ and the Apple scent of the synthetic formulation of the invention was the CS-: Bees learn to distinguish both scents during training (Repeated measures ANOVA: significant interaction: F=9.008; gl=2; p<0.001; third training: F=6.75; gl=l; p=0.01, fourth training: F=29.79; gl=l; p<0.001) and in the test phase (X ² =28.22; p<0.01) .

D) Apple synthetic formulation of the invention was the CS+ and the apple natural fragrance was the CS-. With the training tests response increases (Repeated measures ANOVA: Tests: F=5.29; gl=2; p=0.0064) but does not separate the response to CS+ of CS- (Repeated measures ANOVA: CS+ vs CS- : F=0.678; gl=l; p= 0.414) . Bees are not able to distinguish between either scents during training or the test phase (X ² =2.915; p>0.05) .

***:p<0.001, **: p<0.01, n.s.: not significant. Number of conditioned bees is listed on the upper part of each figure .

The different graphics show examples indicating that bees learn to distinguish the scent of apple flowers from a synthetic scent as the jasmine scent (A and B) through different tests. When the Apple synthetic formulation is offered together with food as CS+ (a situation similar to a hive treated with apple synthetic formulation) , bee cannot differentiate the same from the apple flower natural scent (D) . However bee can do that when the synthetic formulation is offered not associated to food that is as CS- (C) .

Figure 2 : Description of the progress of flower opening of apple trees through the week of recording (A) . The first variety of apples started to flower on September 29. Between the first day and the second of October was the peak of flowering of all varieties around the place of study. Before apple trees there were other fruit trees flowered in the area. The average collection activity of 5 beehives was measured as the total entry of bees in the hive per minute (B) . Treatment with the Apple synthetic formulation was positive, as the total entry of bees to beehives with this treatment was equal or higher than to untreated beehives through the days (Repeated measures ANOVA: treatment effect: F=6.95, gl=l, p=0.029, Figure 2B) . This difference increased to the last days of recording, a moment that matches with the flowering peak of apple trees. Bees of hives fed a with sweetened solution aromatized with the Apple synthetic formulation would be increasing their general activity and also collecting higher amounts of nectar when the predominant floral offer at that time is the apple tree.