GB2095998A - Synthetic bee pheromone and its use in attracting a colony of bees to a hive or trap - Google Patents

Abstract

A synthetic lure which can coax a swarm of bees into making its home in an empty hive has as its essential components (1) E-citral or a mixture of (E)- and (Z)-citrals, (2) geraniol and (3) nerolic acid, preferably in the weight ratio of 1 : 0.1 : 0.1 to 1 : 10: 10. The invention includes (a) the method of luring a colony of bees to a hive or trap which comprises positioning the lure at or near the entrance to the hive or trap, (b) the lure composition itself and (c) a beekeeping kit in which a hive and the lure composition are sold together.

Description

SPECIFICATION Synthetic bee pheromone and its use in attracting a colony of bees to a hive or trap This invention relates to a synthetic bee pheromone and its use in attracting a colony of bees to a hive or trap.

When a queen honey bee ages, her colony is stimulated to rear a new queen. Frequently part of the colony then leaves the hive with the old queen to find a new nest. This phenomenon is known as swarming. The swarming bees are often lost to the beekeeper even when he has an empty hive available to accommodate a new colony. The present invention is concerned primarily with attracting swarms or migrating colonies of honey bees to an empty hive.

Honey bees secrete substances which they can release into the atmosphere to provide an attractive scent known as a pheromone. There are several different kinds of secretion, giving different pheromones. The presence or absence of a particular pheromone serves as a stimulus to fellow bees to perform or desist from performing a particular function. Knowledge and understanding of bee pheromones is at present relatively small. There are some honey bee pheromones which have been identified chemically, some which remain unidentified and probably some yet to be discovered. Understanding of the behavioural influences of pheromones on honey bees is only in its infancy, even for pheromones which have been identified chemically.

One pheromone which has been identified chemically is the Nasonov pheromone. The Nasonov scent gland of the worker honey bee secretes into a groove between the sixth and seventh dorsal abdominal tergites. The secretion is exposed by flexing the tip of the abdomen, thereby releasing Nasonov pheromone.

Worker bees which have had difficulty in finding the entrance to their hive release the pheromone and disperse it by fanning their wings. This behaviour helps their fellow bees to find the entrance. They also expose the Nasonov gland during swarming, when flying or clustering. Honey bees have been seen to release the Nasonov pheromone when foraging at a source of water or at an artificial food such as a dish of sugar syrup. This behaviour of bees when foraging is very different from the behaviour of swarming bees.

The bees will expose the gland only when the sugar syrup is highly concentrated and then only after 3 or 4 foraging trips to the syrup.

Some components of the Nasonov pheromone were identified chemically in the 1960s. These were citral (a mixture of isomers), geraniol and geranic and nerolic acids. However, the published results of the different researchers were not in agreement and some uncertainty remained. During the 1960s field trials were carried out on attracting foraging honey bees by use of some of the identified chemicals. They were carried out on bees foraging for sugar syrup. Again, the results of different workers were not in agreement. Probably the most definitive paper was that of Butler and Calam, J. Insect. Physiol 15,237-244(1969), which concluded that citral or a mixture of citral with geraniol was nearly as attractive to the bees as the natural Nasonov pheromone (obtained by wiping their gland surfaces). Geranic and nerolic acids were rated as of little consequence.In 1970, Waller, J. Ag ricultural Research, 9, 9-12 (1970) published results on attempts to attract honey bees to flowering plants. He tried various combinations of citral and geraniol (components of the Nasonov pheromone) and anise oil (anise oil is mainly 1-methoxy-4-propenylbenzene) and found that each was ineffective unless applied in solution in sucrose. Further, combinations of citral plus geraniol appeared to be less attractive than citral plus anise oil or geraniol plus anise oil.

Recently Pickett etal., J. Chemical Ecology 6,425-434(1980), have determined the full composition of the secretion giving rise to the Nasonov pheromone. Two further components were revealed and approximate proportions determined. The following approximate composition, relative to 100 parts of geraniol, was determined: Component Proportion by weight Geraniol 100 Nerolic acid 78 (E,E)-Farnesol 44 Geranic acid 11 (E)-Citral 1.1 (Z)-Citral 0.6 Nerol 0.4 It has now surprisingly been found that honey bees can be lured to a hive or trap by applying a synthetic pheromone at or near the entrance to the hive or trap, this synthetic pheromone lure having a different composition from the Nasonov pheromone secretion. The (E,E)-farnesol or nerol component or both are preferably omitted from the lure.If present in substantial amounts they diminish the attractiveness of the lure. It is also possible, although not necessarily always convenient, to omit geranic acid. The essential components are (1) (E)-citral or a mixture of (E)- and (Z)-citrals, (2) geraniol and (3) nerolic acid. The proportions of these three components can be varied considerably from those found in the Nasonov pheromone. In the Nasonov pheromone the proportions of these components (total citrals : geraniol nerolic acid) are about 1: 60 : 45.We have found that the proportion of total citrals relative to either of the other two essential components can be increased greatly over that present in the Nasonov pheromone secretion, desirably to within the range 1: 0.1: 0.1 to 1:10:10. Preferably the proportions of the essential components are within the range 1: 0.25: 0.3 to 1: : 2. Our experiments indicate that such proportions are usually more attractive to honey bees looking for a hive than other proportions. All proportions (including percentages and ratios) in this specification are by weight.

It is surprising that (E,E)-farnesol is an inessential component in a synthetic pheromone for the purposes described above. It is the third largest component, on a weight basis, in the Nasonov pheromone secretion. It is also surprising that the proportion of citral relative to the others can be so large. However, we have discovered that during release of the pheromone the honey bees convert geraniol into (E)-citral, thereby maintaining a higher proportion of (E)-citral in the air than in the gland.

A possible explanation for the present results compared with those obtained in the sugar syrup trials in the 1 960s is that different components or mixtures of components in Nasonov pheromone stimulate the bees more strongly than others in different behavioural contexts.

A feature of the invention is a method of luring a colony of honey bees to a hive or trap which comprises positioning at or near the entrance to the hive or trap a synthetic pheromone lure comprising (1) (E)-citral or a mixture of (E)- and (Z)-citrals, (2) geraniol and (3) nerolic acid and in which nerol and (E,E)-farnesol are substantially absent. Although the presence of small amounts of nerol and (E,E)-farnesol is not objectionable, our tests show that the bees prefer a lure in which they are absent. It is ordinarily convenient to omit them in order to reduce manufacturing costs. If they are included it would be desirable to keep their proportion to less than half that of the total citrals.

The most preferred proportions of the essential components (1), (2) and (3) are 1: 0.5: 0.5 to 1: 2 : 2, most especially about 1:1:1. Component (1) is reckoned as total citrals if (Z) and (E) isomers are both present.

However, some of our tests have indicated that a composition containing as little as one-tenth of a part of geraniol or one-tenth of a part of nerolic acid relative to total citrals is sometimes attractive. Also, a composition containing as much as 10 parts of geraniol or of nerolic acid is sometimes attractive.

Another feature of the invention is a composition useful for luring a colony of honey bees to a hive or trap, the composition comprising any composition as defined above in connection with the invention except a composition similar to the Nasonov pheromone secretion itself and except a composition consisting of geraniol, citrals and nerolic acid in a ratio of 1: 2 : 4, described in a test reported by Shearer and Boch. J.

Insect Physiol., 12, 1513-1521(1966). This This mixture, re-expressed in the ratio citrals : geraniol : nerolic acid 1: 0.5 : 2, was one of many which was the subject of experiments in the 1960s on bees foraging for sugar syrup.

The later paper by Butler and Calam, mentioned above, points away from the inclusion of nerolic acid but, more importantly, experiments on foraging bees are not a basis for predictions in relation to swarming bees, because of their different behaviour patterns in these different contexts.

The components of the lure can be used in unpurified form. The most convenient source of nerolic acid will usually be the product of oxidation of a mixture of citrals. The oxidation will ordinarily lead to a mixture of acids predominant in geranic acid, e.g. in a ratio of from 3:1 to 4:1 to nerolic acid. However, the geranic acid is not detrimental. Preferably the ratio of geranic: nerolic acid will not exceed 4:1.

The composition can contain excipient(s), i.e. other component(s) compatible with its utility as a lure.

Thus, it can contain an inert solid or liquid carrier or diluent, a component of another kind of honey bee pheromone than the Nasonov pheromone, or other attractive scent or honey bee food, e.g. sugar. It is particularly preferred to include queen honey bee pheromone component "902", i.e.9-oxo-2-decenoic acid.

Since the active ingredients are volatile, the composition must be sold in some form in which their escape is prevented until the required use or only slow escape permitted. Preferably the composition is put in a container constructed to provide a slow release of the vapour in amounts attractive to bees for at least one month. It is preferably sealed or otherwise enclosed in a plastics vial, e.g. of polyethylene, having walls of low permeability to the vapour of the composition, or microencapsulated, but it could be formulated as granules, impregnated foam, or a coated block or tablet, to give a slow or delayed release of vapour of the active ingredients. It could be sealed in micro-tubes (ultra-fine capillary tubing).The polyethylene vials are preferred because they have been found to reduce the rate of release of (E)-citral, which is much more volatile than the other components.

Each container or other carrier means can contain any desired amount of the lure but ordinarily at least one month's supply, for example typically from 10 mg to 10 g of citrals and the other essential components in proportion thereto.

The lure can be placed in an empty hive orearthe entrance on the outside of the hive. Another aspect of the invention is a beekeeping kit comprising a hive and a composition as defined above in connection with the method of the invention. It is contemplated that such a kit will be sold to beekeepers who want to increase their number of hives without going to the expense of buying another swarm or facing the hazards involved in trying to transfer swarming bees from a nearby hive. One or more containers of lure could be sold with each hive. Means for fixing the container to the hive, e.g. a hook co-operating with a flange on the container perforated to fit on the hook or an adhesive mounting on the container, are preferably also provided as part of the kit. Other beekeeping accessories of the conventional kind could be included as part ofthe kit.

The invention is also applicable to trapping bees. Some colonies of bees behave less benevolently to humans than others and it might be desirable on occasion to remove a swarm of relatively malevolent bees.

For this purpose, the lure can be positioned near the entrance to a trap, e.g. a trap of the known funnel type. It is contemplated also that the method of the invention can be used to trap the Africanised bees. These are a particularly malevolent strain of bee which has become established in parts of Brazil and it is feared that these bees might eventually migrate to the Central American isthmus and even into the Southern parts of the United States.

The following Examples illustrate the invention.

Example 1 Experiments 1 a to if were done, following the technique of Ferguson et al., Physiological Entomology 4, 339-344 (1979), in a bee-proof cage of nylon mesh (3 x 2 x 2m high) kept in a large glasshouse maintained at about 20"C. A roundabout that rotated at one revolution per minute was suspended from the centre of the roof of the cage; it was so constructed that the number of arms (50 cm long) projecting from it could be varied from two to twelve but remain equidistantly spaced. Cylindrical double-walled cages of perforated zinc (30 mm diameter and 55 mm long) were suspended by thin wire from the end of each arm of the roundabout, so the cages were about 1 m above the ground. A porous polyethylene block was held in the centre of each cylindrical cage where it could not be reached by the bees outside.

The Nasonov components tested were: (E)- and (Z)-citral, nerol, geraniol, nerolic and geranic acid, and (E,E)farnesol. Immediately before each trial, one or more of the Nasonov components in pentane (100 plug (E)-9-oxo-2-decenoic acid (1001lug) in ethanol (100F1) was applied to each polyethylene block.

Components were presented either in equal proportions with 100 worker bee equivalents of geraniol (200fug; 1 worker bee equivalent = 2.0Fg) or in proportions approximating those in the Nasonov secretion, i.e.

(Z)-citral (1 part), (E)-citral (1 part), nerol (1 part), geraniol (100 parts), nerolic acid (75 parts), geranic acid (12.5 parts) and (E,E)-farnesol (50 parts), each at 100 worker bee equivalents. The components were about 97-99% pure as determined by gas chromatography.

The positions of the samples on the roundabout were randomised for each trial.

About 1,500 worker bees were brushed from the combs of their colony into a plastic bucket with a lid about 30 minutes before a trial. No queen bee was present. When the trial began the bees were emptied onto the floor of the cage beneath the roundabout. Usually they began to cluster within 10 minutes on one of the cylindrical cages. When about twenty bees were present on the same cage the numbers increased rapidly, and the cluster soon contained 400-500 bees (called hereafter the primary cluster); at this stage additional clusters sometimes formed on one or two other cages (called hereafter secondary and tertiary clusters).

Experiment la In the experiment (Table 1), each cylindrical cage contained a different Nasonov component; the relative proportion of each component was similar to that occurring in the Nasonov secretion. To determine their relative potency in inducing clustering, the component that attracted most clusters was omitted in each succeeding test of a progressive series (tests A-H), the remaining components being left in the same proportions. Thus, for example, nerolic acid, which was chosen first by clusters in eleven of the twenty-three trials in test A, was omitted from test B and so on down the table.

Nerolic acid and the citrals were preferred. The bees were reluctant to cluster when only nerol or (E,E)-farnesol were present. Bees discriminated little, if at all, between the commercial preparations of (E,E)-farnesol and nerol and their purified forms. But purified nerolic acid was preferred to the preparation containing geranic acid, and the commerical preparation of (E)-citral together with (Z)-citral was preferred to either component in a purified state.

TABLE 1 Number of clusters formed (primary, secondary, tertiary) per n trials, components of Nasonov pheromone presented as choices, at approximately naturally occurring ratios Compounds present on the arms of the roundabout Geranic No. arms and of round- n (no. Nerolic (E)- and nerolic Geranic Test about used trials) acid (Z)-citral* acid acid A 11 23 11,0,0 3,4,0 3,2,0 4,1,0 B 10 24 - 7,4,0 6,5,0 6,3,0 C 9 27 - - 12,5,0 6,3,0 D 8 25 - - - 10,5,0 E 7 25 - - - - F 6 21 - - - - G 5 19 - - - H 4 12 - - - TABLE 1 (cont'd) Compounds present on the arms of the roundabout (E,E,) (E)- (Z)- Nerol farnesol (E,E) Test citral citral Geraniol (impure)* Nerol (impure)* farnesol A 2,0,0 0,2,0 0,1,0 0,0,1 0,0,1 0,0,0 0,0,0 B 2,0,0 1,2,0 2,2,1 0,0,1 0,0,1 0,0,0 0,0,0 C 4,0,0 2,5,0 2,2,1 0,0,1 1,0,1 0,0,0 0,0,0 D 5,0,0 5,5,0 2,2,1 0,0,1 3,1,1 0,0,0 0,0,0 E 8,0,0 6,6,1 4,2,1 2,1,0 3,1,1 1,1,0 1,0,0 F - 9,6,1 5,2,1 2,2,0 3,1,2 1,1,0 1,0,0 G - - 8,3,1 5,4,1 4,2,3 1,1,0 1,0,0 H - - - 6,4,1 4,2,3 1,1,0 1,0,0 The three figures separated by commas in each entry indicate the total numbers of, respectively, the primary, secondary and tertiary clusters; dashes indicate absence of component from the roundabout; zero signifies no cluster. n = the total number of trials of each set of components on the roundabout.

*commercial preparation.

Experiment 1b In the next experiment a roundabout with 8 arms was used. The eight mixtures compared (Table 2) consisted of all seven known components of the Nasonov pheromone in the proportions occurring naturally, or with one of the components missing. The absence of nerolic acid, geraniol or (E)-citral diminished attractiveness but the absence of other components had little effect.

TABLE 2 Numbers of choices made by clustering bees for 8 samples of synthetic Nasonov pheromone (natural proportions) from 7 of which one component was missing.

Components present No. clusters formed (primary, secondary, on roundabout* tertiary, quaternary) per 20 trails All components present 4, 4, 3, 0 All components less: Geranic acid 5, 2, 0, 0 Nerol 4, 4, 0, 0 (Z)-citral 4, 3, 0, 0 (E,E)-farnesol 3, 2, 3, 0 (E)-citral 0, 2, 2, 0 Geraniol 0, 0, 0, 1 Nerolic acid 0, 0, 0, 1 *Roundabout with eight test arms (one for each row of table) n = 20 trials; fourth clusters also recorded; other details as for Table 1.

Experiment ic A mixture of all the seven known components, in either the natural or equal proportions, and combinations of (E)-citral, (Z)-citral, nerolic and geranic acids in equal proportions were compared next (Table 3). Mixtures with all components present were preferred, especially when in equal proportions (Nos. 1 and 2). In the absence of mixtures containing all components (test C) those comprising (E)-citral plus nerolic acid, or (E)- and (Z)-citral plus nerolic acid were the most attractive (Nos. 3 and 4). Geranic acid combined with either of these two mixtures reduced their attractiveness (5 v. 3 and 6 v.4).The presence or absence of (Z)-citral in this combination made little difference (5 v. 6), as was confirmed in a further test of combinations 5.and 6 alone, when in twenty trials there were eleven primary clusters on No.6 (i.e. with (Z)-citral) and nine on No. 5 (without (Z)-citral). However, because a mixture of (E)- and (Z)-citrals was preferred to (E)-ctiral alone (Table 1) both isomers were used in subsequent tests.

TABLE 3 Number of choices made by clustering bees for combination of Nasonov components No. clusters formed Components present on roundabout* (primary, secondary, tertiary) A B C 1. All components present (equal proportions) 6,2,0 - 2. All components present (natural proportions) 2,2,1 6,2,2 3. (E)-citral + nerolic acid 3,0,0 3,2,0 8.4,0 4. (E)-citral + (Z)-citral + nerolic acid 1,0,0 3,1,1 6,2,3 5. (E)-citral + nerolic acid + geranic acid 1,2,0 1,3,2 3,4,3 6. (E)-citral + (Z)-citral + nerolic + geranic acid 1,2,1 1,3,1 2,7,2 7. (E)-citral + (Z)-citral + geranic acid 0,0,0 1,0,1 2,3,4 8. (E)-citral + geranic acid 0,0,1 0,0,1 2,0,2 9. (Z)-citral + nerolicacid 1,0,0 1,0,0 1,0,0 10. Nerolic acid 0,0,1 0,1,1 0,1,1 11. (Z)-citral + nerolic acid + geranic acid 0,0,0 0,0,0 0,0,0 12. (Z)-citral + geranic acid + 0,0,0 0,0,0 0,0,0 * Roundabout with twelve arms in test A, eleven in B, and ten in C. n was 15 in Test A, 16 in B, 24 in C.

Experiment id The addition of the other three Nasonov components to a mixture of equal proportions of (E)- and (Z)-citral + nerolic acid + geraniol if anything decreased its attractiveness (Table 4). Omission of geraniol substantially decreased it.

TABLE 4 Choices made by clustering bees for different proportions of various Nasonov components Amounts (llg/lll) on each roundabout arm* Test (E)- and (Z)- Nerolic citrals acid Geraniol A B 2.00 2.00 2.00 6,6,1 2.00 2.00 2.00 (plus)0 4,3,0 6,3,0 2.00 2.00 0 2,0,2 2,0,2 2.66 1.33 0 1,1,2 1,1,2 1.33 2.66 0 1,1,0 1,1,0 3.33 0.66 0 0,0,0 0,0,0 0.66 3.33 0 0,2,2 0,2,2 4.00 0 0 0,1,2 0,1,2 0. 4.00 0 0,0,0 0,0,0 0 0 4.00 0,0,0 0,0,0 * Roundabout with ten arms in A, nine in B, n was 14 in Test A, in B.

Id The three main components plus an equal amount (2.00CLg/CLl) of each of the other three components. Other details as in Table 1.

Experiment le When only the citrals and nerolic acid were present, the two mixtures in which the components approached equal proportions were preferred. A 2:1 excess of citrals over nerolic acid was preferred to a 1 2 deficiency (Table 5).

TABLE 5 Attractiveness for clusters of various ratios of (E)- and (Z)-citrals: nerolic acid, and of geraniol Amount present fR911l/) on each roundabout arm* (E)- and (Z)-citrals Nerolic acid Clusters 2.00 2.00 5,4,2 2.66 1.33 5,4,2 1.33 2.66 2,3,0 3.33 0.66 0,2,2 0.66 3.33 1,4,2 4.00 0 0,2,2 0 4.00 1,0,0 Geraniol alone (4.00) 0,0,0 * Roundabout with eight arms; n = 14 trials; other details as in Table 1.

Experiment if When geraniol was also present (Table 6), the mixtures containing equal proportions of the components and in which geraniol was the predominant component were preferred.

TABLE 6 Attractiveness of various ratios of (E) and (Z)-citrals: nerolic acid: geraniol Amounts present {lig/ll/J on each roundabout arm* (E)- and (Z) citrals Nerolic acid Geraniol Clusters 2.0 2.0 2.0 5,4,3 1.0 1.0 4.0 6,5,0 0.5 0.5 5.0 3,3,3 1.0 4.0 1.0 1,2,2 0.5 5.0 0.5 2,4,1 4.0 1.0 1.0 2,2,1 5.0 0.5 0.5 2,1,1 6.0 0 0 1,0,0 0 6.0 0 0,0,0 0 0 6.0 0,0,0 * Roundabout with ten arms; n = 22 trials; other details as in Table 1.

Discussion of Experiments 7a to 7f In combination with the (E)-9-oxo-2-decenoic acid each of the components tested induced clustering, but nerolic acid, the citrals and geraniol were the most effective, (E,E)-farnesol the least (Table 1). Absence of individual components, other than (E)-citral, geraniol and nerolic acid, from the total mixture did not significantly decrease its attractiveness (Table 2). This is surprising, especially because (E,E)-farnesol is a major component of the natural Nasonov secretion. That a mixture of components in equal proportions was as effective as one with natural proportions (Table 3) is also surprising.

Example 2 Clustering experiments were performed using the method of Example 1 except that the Nasonov components were replaced by (i) a set of Nasonov components in which some of the components were presented as unpurified preparations or mixtures equivalent to unpurified preparations, all components except geraniol being in admixture with other geometric isomers, (ii) a synthetic pheromone containing Nasonov components in approximately the same proportions as in the Nasonov pheromone secretion except that nerolic acid was present at about 1/75 of its natural proportion and (iii) a synthetic pheromone containing the set of components (i) in equal proportions by weight.In (i) the components were a commercial mixture of (Z)- and (E)-citrals in a 1 1 ratio; a commercial mixture of nerol and geraniol in a 1 1 ratio; geranic and nerolic acids prepared by oxidation of the citrals mixture by the method of Pickett petal., J.

Chemical Ecology 6, 425-433 (1980) and in a ratio of approximately 1: 2 nerolic: geranic acids; 99% pure geraniol obtained commercially, and commercial farnesol containing 4 isomers containing 42% (E,E). Each was presented in an amount of 2001lug. In (ii), the synthetic modified Nasonov pheromone, the components were (Z)-citral (1 part), (E)-citral (1 part), nerol (1 part), nerolic acid (1 part), geranic acid (8 parts), (E,E)-farnesol (44 parts) and geraniol (100 parts = 200,ug = 100 worker bee equivalents). In (iii) each "unpurified preparation" was present in an amount of 2001lug.

The worker bees were not accompanied by a queen, but, as in Example 1, each polyethylene block was dosed with 100yg of the queen pheromone component (E)-9-oxo-2-decenoic acid ("902" for short).

Experiment 2a In this experiment seven treatments were used (Table 7), namely (A) synthetic pheromone (iii) of unpurified components in equal proportions, (B) synthetic pheromone (ii) of Nasonov components in natural proportions (except as regards nerolic acid) and (C-G), the equal proportion synthetic pheromone (iii), one of each of the unpurified preparations being omitted in turn. Treatment C, without farnesol and treatment A, with unpurified components in equal porportions, were chosen first most frequently. Treatment D (without nerol) received most second choices. Thesethree treatments were also favoured in general.

It is surprising that the synthetic pheromone (iii), having unpurified components in equal proportions, was favoured to the synthetic pheromone (ii) having 6 of the components in approximately natural proportions.

TABLE 7 Number of choices made by clustering bees for groups of four "unpurified components" of the Nasonov pheromone "Unpurified components" present in treatment (+ = present) nerolic plus nerol plus Treatments citral geranic acid geraniol geraniol farnesol A + + + + + (in equal proportions) B + + + + + (in modified natural proportions C + + + + D + + + + E + + (+) + + F + + + + G + + + + (+): some geraniol present via the nerol and geraniol mixture TABLE 7 (cont'd.) Number of choices made by clustering bees for groups of four "unpurified components" of the Nasonov pheromone No. lst2nd3rdchoices Total number Treatments in clustering of clusters 1 sot 2nd 3rd A 4 1 5 10 B 1 0 0 1 C 6 4 3 13 D 1 7 2 10 E 2 3 1 6 F 2 0 2 4 G 0 1 2 3 Experiment 2b In this experiment, five replicates of each of two treatments were arranged alternately (Table 8). In one treatment all the "commercial preparations" were present in equal proportions, and in the other there were equal proportions of citral, geranic plus nerolic acid and geraniol but no farnesol or nerol. The latter was preferred.

TABLE 8 Choices made in 15 tests by clustering bees for partial and complete synthetic pheromone containing Nasonov components Order of clustering Total number of clusters 1 sot 2nd 3rd Synthetic pheromone (iii), i.e. all "unpurified 3 8 3 14 preparations" present in equal proportions Synthetic pheromone (iii) omitting farnesol and nerol. 12 7 8 27 Citral, geranic plus nerolic acid, and geraniol present Example 3 A solution was made up containing a mixture of (E)- and (Z)-citrals (1.25 g), geraniol (1.25 g), and a mixture of nerolic and geranic acids at a weight ratio of about 1: 3(1.25 g), in 12.5 ml of hexane. Polyethylene vials (WP/5, Fisons Ltd.) were charged with 100 microlitres of this solution.Each vial therefore contained 10 mg., i.e. 5,000 worker bee equivalents, of geraniol, 10 mg. of citrals, and 10 mg. of nerolic and geranic acids, providing a synthetic Nasonov pheromone composition. Each vial weighed 2.75 g. and had an average wall thickness of 1.6 mm. The vials were closed immediately and stored at -20 C in closed glass bottles. Other polyethylene vials were charged with 100 microlitres of the same solution plus 100 microlitres of a solution of 10 mg. of re-sublimed (E)-9-oxo-2-decenoic acid (queen pheromone component, known as "902" for short) made up to 10 ml. with ethanol.

Experiments 3a and3b An unoccupied hive was put in each corner of a nylon screen cage (3 x 2 x 2 m high) or at the centre of each side. In Experiment 3a, a synthetic Nasonov pheromone lure (without "902") was hung just inside the entrance of each of two opposite hives (A,B). The two other hives (C,D) in each cage received no treatment. A natural swarm (containing a queen) was deposited in the centre of the floor of the cage; the hives at which bees fanned and released Nasonov pheromone and the hive chosen by the swarm were recorded.

Experiment 3b was similar to 3a, except that one pair of opposite hives (A,B) contained the synthetic Nasonov component pheromone lure and queen pheromone component lure, "902" and the other pair (C,D) contained only the synthetic Nasonov component lure, without "902". During a single test all the hives were either: type I Dark brown plastic bins (approximately 30 litre capacity) each with a hole, cm. diameter, drilled 30 cm.

above the floor and 12 cm. belowthe lid; type II New British Standard nucleus hives (approximately 18 litres capacity) that had not previously housed bees); type III As II but had previously been occupied by colonies; type IV As Ill but contained 2 empty combs.

Five tests were made with each type of hive. One hive of each pair had a tube of the synthetic pheromone lure just inside its entrance. After five weeks' exposure in the hive lures contained 42% (E)-citral, 63% geraniol and 68% nerolic acid, percentages being with reference to unexposed lures.

Within a few minutes of a natural swarm being introduced to a cage in Experiment 3a, bees were releasing Nasonov pheromone and fanning at the entrance to one or both of the hives with synthetic Nasonov component pheromone lure. Usually within an hour the swarm had entered the hive. The hive chosen was always one with the lure (Table 9); in each test the other hive containing the lure also often attracted bees.

The hives not chosen by the swarm were more likely to- be visited by bees if they had previously housed a honeybee colony (63% visited; hive types Ill and IV) than if the hive was new (23% visited; hive types I and II).

In experiment 3b the swarms preferred hives with the synthetic Nasonov component plus queen pheromone component lure. Even after the swarm had entered a hive, the other hive with the synthetic Nasonov component plus queen pheromone component lure usually retained many bees, irrespective of whether it had housed a colony previously.

Experiment 3c The attraction of the synthetic pheromone lure to swarms was also investigated in natural conditions.

Pairs of Langstroth hives each consisting of roof, floorboard and shallow box (approximately 25 litres capacity) with 10 empty combs, were placed 2 to 10 m. apart in 50 locations in Southern England (Surrey, Sussex and Hampshire).

One hive in each of the 50 pairs of empty hives was provided with a synthetic pheromone lure on 14 or 15 May. These lures were renewed on 9 and 10 June, when five hives had been occupied by swarms. An additional seven hives were occupied by 11 August. All 12 swarms occupied hives with synthetic pheromone lures.

TABLE 9 Choices made by natural swarms for hives with and without synthetic pheromone lure (Experiment 3a) Approximate no. bees present after swarm had entered hive Hive type Test In hives with lure In hives without lure Hive'A' Hive'B' Hive'C' Hive'D' Swarm 1 Swarm 10+ 0 0 2 Swarm 0 0 0 3 Swarm 60+ 0 0 4 Swarm 0 0 0 5 Swarm 0 0 0 II 6 Swarm 0 0 0 7 Swarm 20+ 0+ 0 8 Swarm 0 0 0 9 Swarm 0+ 0 0 10 Swarm 10+ 0 0 Ill 11 Swarm 5+ 0 0 12 Swarm 0 0 0 13 Swarm 0+ 0 0 14 Swarm 6 0 0 15 Swarm 0 0 0 + More than 10 bees present together at hive entrance before swarm occupied a hive.

TABLE 9 (cont'd.) Approximate no. bees present after swarm had entered hive Hive type Test In hives with lure In hives without lure Hive'A' Hive'B' Hive'C' Hive'D' IV 16 Swarm 100+ 0 0 17 Swarm 0 0 0 18 Swarm 0 0 0 19 Swarm 0 0+ 0 20 Swarm 50+ 250+ 0 + More than 10 bees present together at hive entrance before swarm occupied a hive.

TABLE 10 Choices made by natural swarms for hives containing synthetic pheromone lure with and without queen pheromone component "902" (Experiment 3b) Approximate no. bees present after swarm had entered hive In hives with lure In hives without lure not Hive type Test containing "902" containing "902" Hive'A' Hive'B' Hive'C' Hive'D' Swarm 1 Swarm 0 2 0+ 2 Swarm 0 15+ 0 3 Swarm 1+ 1 0 4 Swarm 0 0 0 5 Swarm 0 0 0 lil 6 Swarm 0 0 0 7 Swarm 4+ 0+ 0 8 Swarm 10+ 2 0 9 Swarm 5 4 0 10 Swarm 1+ 0 0 + More than 10 bees present together at hive entrance before swarm occupied a hive.

Example 4 In this Example honey bees were allowed to choose between two hives in the manner of Experiments 3a and 3b, the hives or lures of each pair being stationed mid-way along opposite sides of a cage. A number of trials were made, in each of which bees were released into the cage. The preferences of the bees for a particular lure or hive were noted.

Experiment 4a These experiments used hives with small entrances (they were circular, diameter 2.7 cm) followed by hives with larger entrances (slots, 2.5 x 10 cm). Lures were prepared consisting of a mixture of 10 mg each of (1) (Z)- and (E)-citrals, (2) geraniol and (3) a mixture of a nerolic and geranic acids in an approximately 1: 2 weight ratio sealed into a polyethylene vial. One or more lures was hung just inside each hive and comparisons made between hives with differing numbers of lures. A number of trials were made in each of which bees were released into the cage and given a choice of hives. The results are shown in Table 11 below.

TABLE 11 Number of trials in Number of lures which the bees chose placed in the hives a particular hive Hive A Hive B Hive A Hive B (i) Hives with small entrances 1 lure 2lures 8 5 1 lure 4lures 2 2 (ii) Hives with large entrances 1 lure 2lures 2 8 1 lure 4lures 2 18 4lures 10 lures 4 6 These experiments suggest that when the hive entrance was large enough to allow the bees to sense differences in concentration of pheromone in the atmosphere, they were lured to the hives with the higher concentration (those containing the greater number of lures).

Experiment 4b In this experiment the amount of active ingredients in each vial of lure was varied. The standard amount for comparison (lures A) was as in the lures of Experiment 4a and this amount was decreased or increased (lures B). The bees could choose between a hive containing lure A and a hive containing a lure B. The results are shown in Table 12 below.

TABLE 12 Number of trials in Factor by which which the bees chose the amount of active a hive containing a ingredients in lure particular lure B differs from lure A LureA LureB B=2xA 5 5 B=lOxA 14 9 B=lOxA 5 5 both A and B having been stored at room temperature for 2 weeks B=0.5xA 14 6 B=0.25xA 8 12 B=0.1xA 9 1 These results suggest that a smaller amount of active ingredient per lure than the standard is less preferred, but that there is no marked preference for a greater amount.

Claims (21)

1. A method of luring a colony of honey bees to a hive or trap which comprises positioning at or near the entrance to the hive or trap a synthetic pheromone lure comprising (1) (E)-citral or a mixture of (E)- and (Z)-citrals, (2) geraniol and (3) nerolic acid and in which nerol and (E,E)-farnesol are substantially absent.

2. A method according to Claim 1 in which the weight ratio of components (1), (2) and (3) is from 1: 0.1: 0.1: to 1:10:10, component (1) being reckoned as total citrals if (Z) and (E) isomers are both present.

3. A method according to Claim 2 in which the weight ratio of components (1), (2) and (3) is from 1: 0.25: 0.3 to 1: 10 : 2.

4. A method according to Claim 3 in which the weight ratio of components (1), (2) and (3) is from 1: 0.5: 0.5to 1:2:2.

5. A method according to Claim 4 in which the weight ratio of components (1), (2) and (3) is about 1:1:1.

6. A method according to any preceding claim in which the lure also contains geranic acid in a weight ratio to nerolic acid not greater than 4:1.

7. A method according to any preceding claim in which the lure also contains 9-oxo-2-decenoic acid.

8. A method according to any preceding claim in which the lure is formulated to release pheromone as vapour in amounts attractive to the bees for at least one month.

9. A method according to Claim 8 in which the lure is formulated as a composition contained in a closed plastics vial having walls of low permeability to the vapour of the composition.

10. A composition useful as a synthetic pheromone lure for a colony of honey bees, which comprises (1) E-citral or a mixture of (E)- and (Z)-citrals, (2) geraniol and (3) nerolic acid and in which nerol and (E,E)-farnesol are substantially absent, but excluding a composition consisting of (E)- and (Z)-citrals, geraniol and nerolic acid in a weight ratio of 1: 0.5 : 2.

11 .A composition according to Claim 10 in which the weight ratio of components (1), (2) and (3) is from 1 :0.1:0.1 to 1:10:10.

12. A composition according to Claim 11 in which the weight ratio of components (1), (2) and (3) is from 1 :0.25: 0.3to 1:10:2.

13. A composition according to Claim 12 in which the weight ratio of components (1), (2) and (3) is above 1 : 0.5 : 0.5 up to :2:2.

14. A composition according to any one of claims 10 to 13 also containing geranic acid.

15. A composition according to any one of claims 10 to 14 also containing 9-oxo-2-decenoic acid.

16. A container containing a composition defined in any one of Claims 1 to 7 and constructed to provide a slow release of vapour of the active ingredients in amounts attractive to the bees for at least one month.

17. A container according to Claim 16 in the form of a closed plastics vial having walls of low permeability to the vapour of the composition.

18. A container according to Claim 17 in which the closed plastics vial is of polyethylene.

19. A beekeeping kit comprising a hive and a composition as defined in any one of Claims 1 to 7.

20. A kit according to Claim 19 in which the composition is provided within at least one container defined in Claim 16,17 or 18.

21. A kit according to Claim 20 which further comprises means for fixing the container to the hive.