Chapter I ATTRACTANTS FOR HONEYBEES ATTRACTANTS FOR HONEYBEES 1.1 INTRODUCTION In the anim^' kingdom, the largest class is of insects- Among the insects which we c0me across. most are harmful but honeybees are c^ of a few insects, which are useful t0 mankind. Honeybees Pot on|y provide us honey but also give tne important products like beesv/ax> propolis, royal jelly and pollen. Bee stinQ is certainly painful for human being, nevertheless, bee venom is found to be useful as a medicine for arthritic Honeybees also serve us by assisting pollination of crops. They transfer P°l|en grains from one flower to another during their visits to flowers for collecting nectar. They are looked upon 0s one °ftne important pollinators and are employed for pollination of economically The genus AP'S t0 which honeybees belong includes four well-defined species viz. Apis florea, A. dorsata, A. cerana and A. me0era Other species belonging t0 tnis genus are being evolved and identified in the course of time. In order to Use honeybees more efficiently and fruitfully efforts were made to understand tneir behaviour in more details. Like in all otner insects communication is one of tne most important factors determining tne pattern of 2 behaviour of these species Honeybees communicate with each other by means of vision, taste, touch and smell, pfieromones, the chemicals used by insects for tne purpose of cotttcwjKucaUocv <?,aY a vita| role in it. Pheromones are norm^'ly classified on the basis of function they serve. Accordingly, pheromones <$ honeybees, such as attractant, repellent, trail, alarm, sting, queen, nuptial pheromones, are well known3 There are various glandular sources of pheromones in the body of honeybee 3 (^'9- 1 •1 )• 1 Figure 1.1 Glands in honeybees Tergite Glands Poison Sac Head Labial Gland Nasonov Gland Hypopharyngeal Gland KoschevnikoVs Gland Mandibular Gland Gland of Sting Sheath Thoracic Labial Wax Gland Glands Dufour's Gland Poison Gland 1.2 NASONOV GLAND PHEROMONE: ATTRACTANT PHEROMONE IN HONEYBEES The pheromone secreted by Nasonov gland is of much significance in communication 4 It was found to attract the other members of the species. The message of attraction is important in order to ensure the visits of other bees 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. The function of Nasonov gland was first described by Russian scientist Zoubareff s Sladen was the first to suggest that the odour dispersed by fanning and scenting by bees, attracted other members of their colony 6r 7. He suggested 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 companions 8. Workers were found to release Nasonov gland pheromone at the hive entrance when virgin queen was about to leave the hive to mate 9 10 This, probably, served the purpose of guiding her to the hive entrance on her return. Nasonov gland pheromone also facilitated the reorientation of the members of colony11 The conditions under which foraging bees expose their Nasonov gland were eventually studied 12 The scout bees were seen using this pheromone in searching activity 13 Usually Nasonov gland pheromone release was mainly found to be associated with the collection of water 14 The factors determining the release of Nasonov gland pheromone by honeybees at the hive entrance were also studied 15 Pflumm while studying the factors releasing scent-marking behavior in foraging honeybees found that scent marking with the Nasonov gland was dependent on "the flux of sugar per unit time"16 Influence of the composition of the diet on the scent marking behavior and honey-sack load of a foraging honeybee was also studied 17 It was found that the release of Nasonov gland pheromone was more conspicuous if the source of food was odourless. The foraging bees were found to mark the food with a scent 18 To demonstrate this, 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 steeped in absolute alcohol and the extract were found to be very attractive to bees. Thus, as per the preliminary observations it could be hypothesised that the honeybees mainly released this pheromone during colony movement, reorientation, foraging, collection of water and at the time of nuptial flight of virgin queen. Nasonov gland 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 reorientation. All these reports showed the main function of Nasonov gland pheromone was to attract and to gather the members of species. 1.3 CHEMICAL COMPOSITION OF NASONOV GLAND PHEROMONE Efforts to know the chemical composition of Nasonov gland pheromone began in 1960s. Nasonov scent organ and the physiological role 3 of its secretion were initially studied by Renner19. He obtained the scents by wiping the exposed scent glands of bees by filter papers and demonstrated the strongly attractive effect of the scent gland substance. However, he found it was not specific to a colony or a race. Boch and Shearer used the wiping method for isolation of pheromone of A. mellifera 20. Chromatography of carbon disulfide extracts of the wipes on a vapour fractometer equipped with a hydrogen flame ionisation detector showed characteristic major fractions 20. To identify these components extracts were gas chromatographed and the effluents were trapped in carbon disulfide. Infra Red (IR) spectrum of this solution was identical with that of geraniol 21 (1, Fig. 1.2). As the chemical constituent of Nasonov gland pheromone, geraniol was expected to possess attractive properties. Accordingly, the attractiveness of geraniol to foraging European honeybees was demonstrated 22. The geraniol content of bees of various ages was also studied 23. 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 citral as a minor component which added attractiveness to major components geraniol and nerolic acid (6, Fig. 1.2) was reported by Shearer and Boch 24. They also found citral was not present initially in freshly prepared wipes, and formed when wipes were kept at room temperature. Further examination of the extracts of Nasonov gland led to the identification of geranic acid, nerolic acid (3 and 6, Fig. 1.2) and both the isomers of citral (2 and 5, Fig. 1.2) as its constituents25. 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 column 26,27. The presence of seven terpenoids (Fig. 1.2) was shown in Nasonov gland extracts including the two new compounds, nerol (4, Fig. 1.2) and (E, E)-farnesol (7, Fig. 1.2). The approximate proportions of these terpenoids were also determined 26 (Table 1.1). 4 Figure 1.2 Terpenoids constituting Nasonov gland pheromone of Apis mellifera XH2OH CH2OH (1) Geranio! (4) Nerol CHO CHO (2) Geranial, (E)-Citral (5) Neral, (Z)-Citral COOH ^V COOH (3) Geranic acid (6) Nerolic acid (7) (E, E)-Famesol 5 Table 1.1 Approximate proportions of terpenoids constituting Nasonov gland pheromone of Apis mellifera Terpenoid Proportion ; . __j Geraniol 100 parts | Nerolic acid 75 parts (E, E)-Farnesol j 50 parts Geranic acid 12 parts i Geranial 1 part Neral 1 part Nerol 1 part 1.4 BIOEVALUATION OF CONSTITUENTS OF NASONOV GLAND PHEROMONE Identification of the chemical constituents of Nasonov gland pheromone initiated the experiments to examine their attractiveness on honeybees. These experiments subsequently led to the development of attractant formulations, which involved combinations of these compounds. Attractiveness of citral to foraging honeybees was demonstrated 28 by Weaver et al. Even though citral was a minor component of Nasonov gland pheromone, it was found to be much more attractive than geraniol, the major component of Nasonov gland secretion 28. Sedin studied the influence of some aromatic substances on bees 29. He found synthetic scent, geraniol, was a less effective marker for "novice bees" than the natural secretion. Geraniol and nerol, singly or combined, did not influence bee behavior unless bees were foraging. 6 Attractiveness of geraniol, citral and anise oil to honeybees on plots of Lucerne (Medicago sativa) was studied by Waller 30. It was found that geraniol was generally more attractive than citral when it was applied in a solution containing 5 or 10% sucrose, but less attractive than citral when applied in water. Study of response of honeybees to the chemical constituents of Nasonov gland pheromone continued in 1970s and 1980s also. Ferguson et al. described the techniques for studying honeybee pheromone involved in clustering and experiments on the effect of Nasonov and queen pheromones 31. Williams ef al. carried out bioassay of seven individual components of Nasonov pheromone (Fig. 1.2). Each component attracted honeybees foraging on syrup. The mixture of components in the proportions present in the secretion of honeybees was found to be as attractive as natural secretion. They also found that the presence of 'footprint' pheromone enhanced the attractiveness of the synthetic Nasonov mixture 32. Eventually a 'lure' for A. mellifera was evolved. Pickett ef al. patented the results obtained on the basis of the application of synthetic bee pheromone in attracting colony of bees to a hive or a trap. The synthetic 'lure' contained (E)-citral, or a mixture of (E)- and (Z)-citrals, geraniol and nerolic acid in ratio 1: 0.1: 0.1 and 1: 10: 10.