HONEYBEE ATTRACTANTS AND REPELLENTS: REVIEW HONEYBEE ATTRACTANTS AND REPELLENTS: REVIEW Honeybees are amongst the few useful insects for mankind. The genus Apis to which honeybees belong, includes four major well defined species A. mellifera, commonly referred as the European honeybees, A. cerana, A. florea and A. dorsata, commonly referred as the rock bee1. In late sixties A. mellifera was also introduced in India and by now all the three species, A. cerana, A. florea and A. mellifera are found to be domesticated by Indian beekeepers. In India the main objective of beekeeping has always remained to be the honey production due to which the important function of pollination carried out by honeybees attained secondary importance. Although honeybees are the important pollinators of several crops, very little attention has been paid in getting the maximum benefit from their pollination. 1.1 HONEYBEE ATTRACTANTS Foraging is an important activity carried out by honeybees which is responsible for collection of nectar and transfer of pollen grains. When a honeybee visits a flower, pollens from that plant are collected. If, the subsequent visit of the honeybee happens to be to a different plant species, the pollens, even if transferred, get wasted. However if the honeybee visits the same plant species, pollination becomes successful. This can result into higher efficiency of pollination directly leading to the higher crop yields! Attempts were, therefore, made to regulate the visits of honeybees so that they can be made to visit only the plants of interest. Availability of honeybee attractants is very important from this point of view. Honeybees mainly communicate with each other by means of vision, taste, touch and smell. Pheromones are the chemicals used by insects for the purpose of communication2. Pheromones are normally classified on the basis of their function. Accordingly, pheromones of honeybees, such as attractant, repellent, alarm, trail, sting and queen nuptial pheromones are well known along with their glandular sources3. Amongst these, Nasonov gland pheromone is important for attraction. 1.1.1 NASONOV GLAND PHEROMONE: ATTRACTANT PHEROMONE IN HONEYBEES Pheromone secreted by Nasonov gland is very important for the purpose of communication4. Function of Nasonov gland was first described by a Russian Scientist Zoubareff 5 in 1883. Sladen6,7 subsequently suggested that the odour from glands of worker bees is dispersed by fanning which attracted other members of their colony. He hypothesized that scenting by the first worker of a colony to discover their hive rapidly initiates similar behaviour in their companions and pointed out the calling is infectious. It was, then, confirmed that this gland was the origin of odours by which honeybees distinguish their companions8. It was found that if the source of food was odourless, the release of Nasonov gland pheromone was more conspicuous. To understand this, an interesting experiment was done. The bees were allowed to forage on syrup in dishes containing glass balls. The glass balls from the dish at which the bees had foraged for one day were removed, dipped in absolute alcohol and the alcoholic extracts were screened on honeybees. The extracts were found to be very attractive to bees9. Foraging bees were found to mark the food with a scent. Worker honeybees were found to release Nasonov gland pheromone at the hive entrance when virgin queen was about to leave the hive for the nuptial flight10,11. This was thought to serve the purpose of guiding her to the hive entrance on her return. The conditions under which the foraging bees expose their Nasonov gland were being studied around the same time12. Nasonov gland pheromone release was also found to be associated with the collection of water by worker honeybees13. The scout bees were seen using this pheromone in the searching activity14. Worker bees which were denied access to their colony, especially after colony disturbance, were found to expose their Nasonov glands for releasing the pheromone at the entrance to their hive15. Pflumm16 while studying the factors releasing scent-making behaviour in foraging honeybees found that scent marking with the Nasonov gland was dependent on the „flux of sugar per unit time‟. Subsequently effect of composition of the diet on the scent marking behaviour and honey-sack load of a foraging honeybee was also demonstrated17. Thus, as per the preliminary observations, it could be hypothesized that honeybees mainly release the Nasonov gland pheromone during colony movement, reorientation, foraging, collection of water and at the time of nuptial flight of virgin queen. This pheromone was also seen to be released by queenless workers for nest entrance, forage and water source marking and also under artificial circumstances concerned with the reorientation3. Message of attraction is important in order to ensure the visits of honeybees to the desired site. It could be useful in getting unifloral honey, to increase the yield of economically valuable crops and also to attract honeybee swarms. 1.1.2 CHEMICAL COMPOSITION OF NASONOV GLAND PHEROMONE Nasonov scent organ and the physiological role of its secretion were initially studied by Renner18. He obtained the scents by wiping exposed scent glands of bees with small bits of filter paper and demonstrated strong attractive effect of the substance secreted by scent glands. However, he concluded that it was not specific to a colony. Boch and Shearer19 used the wiping method for isolation of pheromone of A. mellifera when they found geraniol to be the major constituent of the pheromone. In their study, the chromatography of carbon disulphide extracts of the wipes on a vapour fractometer equipped with a hydrogen flame ionization detector showed characteristic major fractions. To identify these components, gas chromatography (GC) of the extracts was carried out and the effluents were trapped in carbon disulphide20. Infra- red (IR) spectrum of this solution was identical with that of geraniol. As the major chemical constituent of Nasonov gland pheromone, geraniol was expected to possess attractive properties. Accordingly, the attractiveness of geraniol to foraging European honeybees was demonstrated21. The geraniol content of bees of different ages was subsequently determined22. It was found that the quantity of geraniol in Nasonov gland secretion was more closely related to the foraging activity of bees rather than the chronological age. Formation of two other minor components geranial and neral along with the other major compound nerolic acid was shown by Shearer and Boch23. Geranial and neral are double bond isomers of citral. These were shown to enhance the attractiveness of the major component, geraniol. They also found that isomers of citral were not present initially in freshly prepared wipes, and formed when wipes were kept at room temperature. Detailed examination of the extracts of Nasonov gland led to the identification of two isomers of citral, nerolic acid, and geranic acid as its constituents24. Chemical composition of Nasonov gland pheromone isolated from a single A. mellifera worker bee was determined by gas chromatography-mass spectrometry (GC/MS) using capillary column25,26. The presence of seven terpenoids was shown in Nasonov gland extracts including the two new compounds, nerol and (E, E)-farnesol (Figure 1.1). CH2OH CHO CHO Geraniol Geranial or (E)-Citral Neral or (Z)-Citral COOH COOH CH2OH Nerolic acid Geranic acid Nerol OH (E,E)-Farnesol Figure 1.1 Chemical constituents of Nasonov gland pheromone of A. mellifera It is noteworthy that the constituents are not present in equal quantities. It is also interesting that baring (E,E)-farnesol the constituents are oxidation products of monoterpene alcohols, geraniol and nerol. The relative proportions of these constituents were also determined25 (Table 1.1). Table 1.1 Relative proportions of the chemical constituents of Nasonov gland pheromone of A. mellifera in Europe Sr. No Compound Relative proportion 1 Geraniol 100 parts 2 Nerolic acid 75 parts 3 (E,E)-Farnesol 50 parts 4 Geranic acid 12 parts 5 Geranial 1 part 6 Neral 1 part 7 Nerol 1 part 1.1.3 BIOEVALUATION OF CONSTITUENTS OF NASONOV GLAND PHEROMONE Identification of the chemical constituents of Nasonov gland pheromone initiated the experiments to examine their attractiveness towards honeybees. These experiments subsequently led to the development of attractant formulations, which involved combination of these compounds. Although geraniol was the major compound present in the Nasnov gland pheromone of honeybee A. mellifera, attractiveness of citral to foraging honeybees was conspicuous27. It is very interesting to note that even though isomers of citral were minor components of Nasonov gland pheromone, they were found to be more attractive than geraniol, the major component of Nasonov gland secretion. Sedin28 studied the influence of some aromatic substances on bees. He found synthetic geraniol, was a less effective marker for „novice bees‟ than the natural secretion suggesting the role of other constituents in the process of attraction. Further, he observed that geraniol and nerol, singly or combined, did not influence the behaviour of honeybees unless they were foraging. This showed that the influence of Nasonov gland pheromone was predominant in the foraging activity. Attractiveness of geraniol, citral and anise oil to honeybees on plots of Lucerne (Medicago sativa) was studied by Waller29. It was interesting to note that geraniol was