Attractants for Honeybees Attractants for Honeybees

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

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

(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. Geranic acid and queen mandibular gland pheromone, (E)-9-oxo-2-decenoic acid (8), were also present in this lure. A kit consisting of a hive and the 'lure' is described 33.

(8) (E)-9-Oxo-2-decenoic acid

Free et al. found that a synthetic pheromone 'lure' induced and stimulated worker honeybees A. mellifera to consume water and artificial forage 34. Winston et al. tested the attractiveness of the constituents of Nasonov gland pheromone and their mixtures. Citral, geraniol plus nerolic acid was

7 found to be the most attractive than citral, geraniol plus queen mandibular gland pheromone followed by citral, geraniol plus hexanal (9) and citral, geraniol alone35. o

(9) Hexanal

Fine structure of Nasonov gland of the worker honeybee A. mellifera was examined using electron microscopy and cytochemical techniques. It was proved that the gland secreted volatile terpenoids and protein compounds. The bioassay indicated that the protein fraction enhanced the attractiveness of the volatile fraction to workersx. In the review on honeybee Nasonov pheromone 'lure', Free et al. have described the development of 'lure' containing components of the honeybee Nasonov pheromone. They have taken an account of experimental results obtained and the possible uses of 'lures' 37.

1.5 APPLICATIONS OF ATTRACTANTS BASED ON NASONOV GLAND PHEROMONE

Experiments on attracting A. mellifera bees using components of Nasonov pheromone on the plots of strawberry are reported in the recent past. The most attractive components were found to be nerol, neral and citral x. It was found that geraniol, geranic acid and nerolic acid were the least attractive. A mixture of neral, citral, nerol, geraniol as well as geranic and nerolic acid in the proportions 1: 2: 4: 2: 2 proved to be more attractive than any one of the individual components alone. The increased number of visits of honeybees to the plots sprayed with Nasonov pheromone suggested that it could be used to attract honeybees and, thus, to improve pollination 38.

8 Use of chemicals like citral, geraniol, limonene (10) as the secondary attractants to improve the pollination and yields of the onion crop were also reported»

\**)

(10) Limonene

Application of citral, geraniol and anise was made to attract honeybees to increase onion seed production in Poland 40. Ohe et al. found that citral and geraniol, when added to the fungicide, pallinal-endosulfan, masked the repellency of fungicide and improved pollination 41.

1.6 APPLICATION OF 'LURES' AS SWARM ATTRACTANTS

1.6.1 Phenomenon of Swarming In the flowering season, when food is abundantly available, queen lays more eggs resulting into the increase in bee population. The number of workers increases to such an extent that the bees cannot be accommodated in the original colony. A phenomenon called 'swarming' takes place under such circumstances. In swarming the original queen flies out of the colony along with some worker bees and settles elsewhere. The worker bees left behind develop other queen. Thus, the natural division of colony occurs due to swarming. Nasonov gland of worker is reported to have an important role in the process of swarming 42~44. Swarming bees were seen to be guided by queen's substance and pheromone released by some worker bees respectively 45.

9 1.6.2 Swarm Attractants Use of attractants to capture the honeybee swarms is the area of active interest since long. Burgett in his article on, the use of lemon balm (Melissa officinalis) for attracting honeybee swarms 46, has mentioned the report on the use of "balme" as swarm attractant by Parkinson in 1629 and also drew the attention of researchers to the records which showed that Melissa oil was apparently used as a swarm attractant in the 1930s. Witherell reviewed the scientific literature relating to honeybee bait hives and swarm attractants 47. Effectiveness of bait hives and 'lures' to attract honeybee swarms were reported as at survey tools for use in Africanised honeybee eradication programs. Mixtures of citral and geraniol were found to be the most effective among the 'lures' tested48. It was found that synthetic (E)-citral, geraniol, nerolic acid, geranic acid components of Nasonov gland pheromone induced worker honeybees (A. mellifera) to release Nasonov pheromone at their hive entrance. Lacking of any one of these components stimulated pheromone release less effectively but the absence of nerol or (E, E)-farnesol increased effectiveness. This result has implication in the development of 'lure' for 48 49 swarms ' . Use of Nasonov 'lure' to attract Africanised colonies to hives was shown by Ferguson 50. Kigatiira et al. carried out the experiments using synthetic pheromone 'lures' to attract honeybee colonies in Kenya. They concluded that 'lures' should be placed in hives only at those times of years when migrating colonies usually settle in the area concerned 51. Mixture of citral, geraniol and nerolic acid in equal proportions in the traps was found to be useful in swarm capture52"54. The effect of 'lure', described by Pickett et al., on attracting the swarms was also studied55. It was found that, of the eight hives occupied by swarms, seven contained 'lures'. Honey mixed with citral and geraniol was effective to attract honeybees. The trials were also carried out with baiting system. It was found

10 to be effective for attracting bees during nectar dearths and in favourable flight conditions. However, it was observed that, this method was not effective during strong nectar flows56. When the efficacy of queen and Nasonov pheromones in the capture of honeybee A. mellifera swarms was compared, it was found that the Nasonov pheromone treated traps were more attractive than queen pheromone and queen and Nasonov pheromone combinations 57. Effectiveness of Nasonov pheromone in long distance swarm attraction was shown by Villa and Schmidt58. In the experiments to determine the criteria for nest site selection by honeybees between pheromone attractants and cavity shape, it was observed that nest cavity shape was apparently not an important factor for the selection of site by honeybee swarms. The detailed investigation indicated that both Africanised and European honeybees preferred synthetic pheromone blends containing nerolic acid in combination with citral and geraniol to those containing only citral and geraniol. Nerolic acid, the second most abundant component in Nasonov gland pheromone of European honeybees, was found to exert an important synergistic effect on the attractiveness of other Nasonov components 59. When further tests were performed using bait hives treated with different materials, it was observed that the addition of queen pheromone did not increase the attractiveness of Nasonov pheromone treated cavities. However, in the study of short range attractiveness swarms preferred cavities with Nasonov pheromone along with queen pheromone to those with Nasonov pheromone alone60. Experiments to examine the attraction of reproductive honeybee swarms to artificial nests by Nasonov pheromone indicated that it played a key role in the attraction of honeybee swarms to nest cavities61. Visscher and Khan proposed inexpensive traps which contain pheromone 'lures' and insecticidal soap solution for trapping and killing the bees remaining after swarm and colony removal. It was found that these traps were useful especially where Africanised honeybees were established62.

11 Recently, Schmidt designed the experiments to determine if Nasonov pheromone serves as a true pheromone or it is simply a general attractant for A. mellifera swarms. He compared synthetic Nasonov secretion with linalool (11), oil of clove, skatole and Galleria mellonella sex pheromone.

(11) Linalool

The results of these experiments confirmed that Nasonov pheromone was a true pheromone in the context of attracting swarms to nest cavities and that odours naturally present in the environment or in the cavity play little or no role in nest seeking behaviour63.

1.7 CONTRADICTION TO ATTRACTIVE NATURE OF NASONOV GLAND PHEROMONE

Since the beginning of 20th century Nasonov gland pheromone of honeybees has been studied extensively. The attractive nature of this pheromone has been observed and demonstrated by most of the researchers. However, note must be taken of the reports which do not support the theory that Nasonov gland secretion contains an attractant pheromone. Wells and Wenner observed that when unscented food was used, recruitment of honeybee foragers failed in spite of much dancing and Nasonov gland exposure. They further observed the recruitment of new foragers was increased by the addition of clove scent to the food. They found no evidence supporting the attractive nature of Nasonov gland secretion 64.

12 A series of experiments using marked A. mellifera foragers was performed in which the behaviour of these foragers towards scented and unscented sugar syrup was observed. The scent compounds such as components of Nasonov gland secretion and other odours like cinnamon, anise, and bay were used for the experiments. It was found that honeybees did not prefer Nasonov gland components to other odours. The results of the experiments did not support the hypothesis that Nasonov gland secretion could act as a 'forager attractant' pheromone. A question was, therefore, raised, 'Does honeybee Nasonov pheromone attract foragers?'65.

1.8 STUDIES ON NASONOV GLAND PHEROMONE OF INDIAN HONEYBEES

The pheromone chemistry of Indian honeybees had not been studied till late 1980s. The first report indicated the presence of only one major compound in the extract of Nasonov gland pheromone of the Indian honeybee A. cerana indica 66. A 'lure' to attract these honeybees was eventually developed67. By using similar strategies a 'lure' to attract the other Indian honeybee A. florea was also developed subsequently68. It is interesting to note that although Nasonov gland pheromone of European honeybee A. mellifera is a mixture of seven terpenoids in different proportions, there is only one major constituent in the Nasonov gland pheromone of Indian honeybees A. cerana. Honeybees A. mellifera have been introduced in India in 1960s. They are now established and being used for improved beekeeping. Nasonov gland pheromone of honeybee A. mellifera maintained in India was initially examined in 1999. Surprisingly it was found that nerol, the minor constituent of Nasonov gland pheromone of European honeybee, was the only major component of these honeybees maintained in India 69. Examination of the chemical composition of Nasonov gland pheromone extracts of the

13 honeybees collected from cooler parts of India showed that, neral was the minor component, along with nerol as the major one70

1.9 VARIATION IN PHEROMONE COMPOSITION

The chemical compositions of the pheromones of the same species of honeybees are known to vary with the change in place, meteorological conditions and other factors. Nasonov gland pheromone is not an exception to this observation. Naik et al. reported the only major constituent of Nasonov gland pheromone of A. cerana from India as neral66. Abdullah et al. identified the Nasonov pheromone of Asiatic honeybee A. cerana as (E)-citral, geraniol and (Z)-citral71, while Matsuyama et al. reported occurrence of linalool oxide (12) as a constituent of Nasonov gland pheromone of A. cerana from Japan 72, which was not the replication of the either of the earlier results reported.

(12) Linalool oxide

It is noteworthy that even the trace occurrence of neral was not detected in the bees from Japan and linalool oxide was not found even in minor quantities in the bees from India. Similarly, the significant variation is seen between Nasonov gland pheromone composition of European honeybee A. mellifera and that of the same bees maintained in India69,70. Nasonov gland pheromone of A. mellifera in India is reported to have nerol as the major constituent69. Furthermore, the presence of other major compounds reported from Nasonov gland pheromone of these bees in Europe is not observed in those maintained in India. This type of variations in the pheromone composition demands further study.

14 1.10 PLANT BASED ATTRACTANTS

Since ancient times the relations between plants, flowers, nectar and insects are known. Especially honeybees are well known to collect nectar from flowers to produce honey. Amongst the factors those invite honeybees to flowers are the attractive colors of the flowers, but it was proved later that the smell of aromatic compounds of the plant parts played an important role in attracting honeybees to the plants. Butler studied the importance of perfume in the discovery of food by the worker honeybees A. mellifera 73. He carried out the experiments using colored discs of paper and the perfumes of white clover. (Trifolium repens), hawthorn {Crataegus oxyacantha), red clover (7. pratense), oil of thyme and Spiraea arguta dissolved in odourless paraffin oil. In conclusion, he reported that the untrained bees were dependent upon the sight to find the flowers from a distance but on close approach they entered flowers more readily if they were attractively scented. Wykes studied the preferences of honeybees for solutions of various sugars, which occur in nectar. From these experiments, it was found that the sugars occurring in nectar were not equally attractive to bees. The attention was drawn to the possible biological significance of the selective responses by bees and it is reported that the sugar composition of nectar might be a factor influencing the visits of bees to flowers74. Value of medicinal plants to beekeeping was recognised by Youngken. The observations indicated that certain plants cultivated for medicinal or flavouring purposes were also valuable to bees. In this report he listed the plants those were most visited by bees out of over 50 species he selected for the study (Table 1.2)75

15 Table 1.2 Plants most visited by the bees

Common name ! Botanical name

1 Rosemary Rosmarinus officinalis Sage j Salvia officinalis

Fennel Foeniculum vulgare, F. dulee

i— -• • "• i Hyssop Hyssopus officinalis \

Lavender Lavendula spica, L. vera I

Hoarhound Marrubium vulgare j

Peppermint ! Mentha piperita

Spearmint Mentha spicata \

The feeding responses of honeybees in the hive were examined, using sugar paste, and pollens of Melilotus alba, Echium lycopsis (plantagineum) and Trifolium pratense. The comparative study of attractiveness of these pollens, their extracts in chloroform, methanol and water was reported. The failure of honeybees to consume non-pollen supplements was observed. This could be due to a lack of specific feeding stimulants, which are needed to release feeding behaviour in honeybees76 Attractiveness of pollen to bees was evaluated by Doull. It was seen that the response of bees to pollen was due to the chemicals whose molecules are diffused in the air77. The benzene extract of almond pollen was found to be attractive to bees78. While studying taste and aroma attractants in honeybee (A. mellifera) feeds, Maliszewska and Szymas made the use of geraniol in the tests. They found honey as most attractive to bees, next was the pollen followed by geraniol79. While studying the A. mellifera swarms in Ithaca, NY, Ambrose found that some substrates such as liliac bushes seemed to be particularly

16 attractive to the swarms He observed that the interim site was apparently not predetermined by the bees before emergence and beeswax pellets containing a 2:1 mixture of citral : geraniol were effective in attracting stable clusters. This supports the theory that scenting with the Nasonov gland is important in initiating a swarm cluster. Use of lemon balm (Melissa officinalis) for attracting honeybee swarms is reported by Burgett. At least 23 compounds have been identified which contribute to the fragrance of the oil in the green leaves of M. officinalis, which include citral, geraniol and nerol46. He made the mention of the report on the use of "balme" as swarm attractant by Parkinson in 1629 and also drew the attention of researchers to the records which show that Melissa oil was apparently used as a swarm attractant in the 1930s. Wilhelm and Pflumm investigated the effect of oil from lily of the valley, rose oil or geraniol as a scented substance on the exposure of Nasonov glands by honeybees 81. However, they found the period of Nasonov gland exposure was not significantly different from that of the bee feeding in an unscented atmosphere. Honeybees were found to be attracted to the flowers and fruits of Evodia hupehensis. The crude extract of dried fruits was attractive to honeybees when sprayed on Lucerne plots. Two compounds, 2-keto- tridecan-1-yl acetate (13) and 2-keto-tridecanol (14) were identified in the fruit extracts82.

I 4 6 8 10 12 o

(13) 2-Keto-tridecan-1-yl-acetate

1 3 5 7 9 11 13

(14)2-Ketotridecanol

17 Pinzauti evaluated different substances that attract pronubial insects including honeybees. Among the various solutions sprayed on the plots of safflower (Carthamus tinctorious), oil of lemon and orange were also used along with a commercial 'lure', Beeline83. In a review on spatial management of honeybees on crops by Jay, evaluation of systems for maintaining honeybees on insect-pollinated crops is discussed. An account of the management techniques for attracting honeybees to target crops is given. It is concluded that the modification of bee behaviour by the use of pheromones and pollen attractants offer the best method for maintaining honeybees on a specific crop84. The flower aroma of oriental orchid, Cymbidium pumilum was found to attract drones, individual workers as well as swarms of Japanese honeybee A. cerana japonica that did not, however, attract A. mellifera85. Neira and Barriga studied the pollinating behaviour of honeybees, A. mellifera, on flowering raspberry plots. They found that, the extract of Lavandula lalifolia (lavender) when applied to plots gave more number of bee visits. However, it did not have any significant effect on yields of fruits 86,87. Zvedenok reported the use of the secondary attractants such as citral, geraniol, limonene and carrot seed extract to improve the pollination of onion crop in presence of the competitors like sunflower or buckwheat. He also suggested sowing the strips of nectar bearing plants like, Phacelia, along the sprayer access strips left every 20-25 meters in the onion fields 39. Alluring substances for honeybee, A. mellifera, were reported from the nectar of the flowers of Angelica gigas Nakai and Fagopyrum esculentum Monech and their pollinating effects were also noted. More than 20 aromatic compounds were identified in the nectar of these plants M. Volatile chemical emissions evolved from the pollen were stated to influence bee foraging 89. Similarly, the use of pollen cues in research location by a pollinator and a pest was demonstrated 90. It was found that the restrained A. mellifera were able to learn the odours of Brassica napus and Vicia faba pollens associatively and could discriminate between them in a conditioned proboscis extension bioassay.

18 1.11 SCREENING OF NON-PHEROMONAL CHEMICALS AS ATTRACTANTS

Although screening of the chemical constituents of Nasonov gland pheromone is a logical step to evolve bee attractants, attempts were also made to screen chemicals and materials other than Nasonov gland pheromone constituents randomly for studying their attractiveness. Bolwig made the use of the perfume, Eau-de-Cologne, while studying the role of scent as nectar guide for honeybees on flowers and concluded that strongly scented parts of flowers can guide the visiting bees 91. This suggested a possibility of getting non-pheromonal attractants for honeybees. While carrying out the tests for Japanese beetles Popillia japonica, Ladd et al., found that honeybees were also attracted to the traps baited with 'lures' for Japanese beetles 92. Anethole (15), geraniol (1) and phenethyl butyrate (16), with and without eugenol (17); eugenol alone; methyl cyclohexanepropionate (18) with eugenol were used as test 'lures'. Anethole + eugenol (9:1) attracted the most honeybees, followed by anethole alone. However, the other 'lure' for these beetles consisting of phenethyl propionate + eugenol + geraniol (3:7:3) + Japonilure (female sex attractant for the beetles) was not able to attract honeybees 93. Amongst the individual components of this 'lure', anethole attracted significantly more number of honeybees than other 'lures'.

OCH3

(15) Anethole (16) Phenethyl butyrate

19 OH

,OCH3

(17) Eugenol (18) Methylcyclohexane propionate

Free et al. discovered that (Z)-11-eicosen-1-ol (19), a volatile component of the sting apparatus, was attractive to foragers trained to collect sugar syrup and to scout bees 94 . It did not enhance attractiveness of synthetic Nasonov pheromone. This suggested that the mode of action of pheromone based 'lures' and 'lures' based on the random screening of the non-pheromonal chemicals might be different.

(19) (Z)-11 -Eicosen-1 -ol

Gupta studied 16 ketones / essential oils for their effect on honeybees A. florea under semi-field conditions. Ethyl benzyl ketone (20) and o-amino acetophenone (21) were found to attract least number of bees while acetyl acetone (22) was found to be most attractive 95. NH2 O

X^ o

(20) Ethyl benzyl ketone (21) o-Amino acetophenone

O O

(22) Acetyl acetone

20 Goodwin took an account of the use of feeding sugar syrup to improve pollination by honeybees. This review deals with the aspects regarding, attracting bees to a crop by spraying it with sugar syrup, feeding bees with scented sugar syrup for directing them to a crop and feeding syrup inside the hive to increase the number of pollen foragers96. Wax is used by the honeybees in constructing the cells of hive. It was found that apart from this use it might be involved in the attraction between bees of a colony, possibly in the same way that pheromones are 97.

1.12 PROTOCOLS FOR BIOASSAYS OF ATTRACTANTS

As the idea of using different plant extracts, oils, components of Nasonovgland, random chemical compounds for controlling the behaviour of honey bees originated, a question that, how could the use of these compounds be made properly and effectively as the attractants for honeybees, needed to be answered. This need led to the development of various methods or protocols for testing the compounds to evaluate their effect on honeybees. Bolwig, while studying the role of scent as a nectar guide for honeybees on flowers and effect of colour on recruits, designed an ingenious experiment91. He arranged six petals made up of paper in the form of a flower and placed a scent on the petals at center or at circumference. It was found that the bees were attracted to the scented part of the flower. They preferred to approach the paper bearing the scent to the unscented coloured piece of paper. In another experiment, three pairs of small glass tubes were set out on a rotating stand. During training, the scent was placed in one tube covered with netting and the other tube of this pair was filled with syrup. One tube of each of the other pairs contained the solvent only (for threshold experiments) or another scent (for discrimination experiments). The other tube of these pairs contained water. During experiments the three uncovered tubes were empty. Using this technique it was shown that honeybees could perceive pure

21 chemical scents in great dilution. They were also found to distinguish between mixtures, which contain only slightly different proportions of the same two scents98. Ferguson et al. designed the experiment to study honeybee pheromones involved in clustering and to study the effect of Nasonov and queen pheromones 31. They used wire mesh cages in which the test pheromone mixtures were kept. These cages were suspended from the arms having length 50 cm of a roundabout fixed on the top of the honeybee flight cage and rotated at one revolution/ minute. The clustering of workers released from the cage was observed. The formation of stable clusters in response to synthetic Nasonov pheromone mixed with 9-oxo-2-decenoic acid was seen. In another method, the attractiveness of the test chemical was estimated quantitatively by counting the number of bees visiting the dish containing the formulation and those visiting the dish containing only the solvent of the test formulation. The difference between the two numbers was a measure of attractiveness of the test chemical67. Maliszewska and Szymas studied the taste and aroma attractants in feeds for honey bees Apis mellifera L79. For this study, they tested different attractants by adding them to sugar candy. They found that bees preferred candy moistened with water to powdered sugar. While evaluating the new compounds as bee attractants, the choice of method / protocol depends upon the conditions of experiment and the nature of the compound(s) to be tested.

1.13 COMMERCIAL'LURES'

The fact that Nasonov gland of honey bees secretes a pheromone which gives message of attraction to the members of species led to an idea of using attractants for controlling the behaviour of honey bees for better and improved beekeeping. The search for an ideal attractant began and

22 continued with the testing of synthetic components of Nasonov gland in various compositions, plant extracts and random chemicals. The need of beekeepers induced researchers and manufacturers to develop commercial 'lures' / attractant formulations for application in fields which was expected to improve pollination and crop yields. Commercial 'lures' have been developed on the principle that foraging honeybees search for food, nectar, pollen and they are tempted to the attractive smells, like components of Nasonov gland. These 'lures' are either food-based attractants containing sugars and other constituents such as flavour and colour or they are pheromone mimics. Sometimes they contain proteins and pollen supplements or they are based on queen's substance. Various protocols / methods for application of the 'lures' have bee's standardised. They basically mention the use of inert support on which the attractant formulation is loaded. Sometimes the sugar syrup, sugar candy or pieces of sugar cane have also been used. Some of the commercial 'lures' and their components have been summarised in Table 1.3.

1.14 REPORTS ON THE APPLICATIONS OF COMMERCIAL 'LURES*

Contradictory reports on the applications of the commercial 'lures' in the fields to improve the pollination of crops are available in the recent past. The increase in the number of bees visiting the sites marked with 'lures' is observed in most of the cases. However, it is important to note that although the 'lures' are commercialised for improving the efficiency of pollination, some of the 'lures' fail to do so. The unexpected results of the application of the 'lures' are not yet explained satisfactorily.

23 Table 1.3 Commercial bee 'Lures' and their chemical constituents"

— _ 'Lure' Pheromones Others Company Nutritional j i Substances . . i i BEELINE Saccharose, - Fatty acids, Custom Lactose, Pollen Chemical Protein (USAL BEELURE 39% Strawberry Helena i Glucose, aroma, Red Chemical I : 28% Maltose, colourant, (USA) f: I 10% Preservatives I Maltotriose, j 22% other sugars f

BEESCENT 42.5% 9.5% 48.5% Inert Yellowston j ! Sugars Nasonov ingredients e Intl. (USA) ; POLLENAID Gluconic acid j Crop King Chemical (USA) POLLINUS | 45% Nasonov - Calliope (France)

API-FIX 6% Reducing - - Coda glucose, 25% (France) Proteins

1.14.1 Beeline When Beeline containing sugar and protein was applied in carrot fields, it was found that it did not attract more insects or enhance pollen transfer between the plants in order to improve pollination 100

24 Similarly, Margalith et al. concluded that Beeline was totally ineffective in attracting bees under the experimental conditions101. Neira ef al. investigated the effect of application of Beeline on number and size of raspberry fruits. Number of drupelets/ fruits or fruit weight, length, width or volume was found to be unaffected by using the 'lures' in the fields87. When Beeline was tried as an attractant on plots of flowering raspberry, the number of honeybee visits to flowers and the average time spent by the bees on the flowers were found to be more than on untreated plots with the highest dose (5 kg / ha)86. Pinzauti assessed the attractiveness of different attractants to foraging insects by spraying them on plots of safflower. Beeline with lemon and orange oil was found to be most effective83.

1.14.2 Bee-Scent Mayor et al. applied Bee-Scent on flowering apple, cherry, pear and plum. The treated trees were found to attract significantly more number of honeybees. Moreover, the fruit set was found to be higher in these trees102. As against this, Loper and Roselle showed that there was no significant increase in the yield of watermelon when Bee-Scent was applied. The 'lure' was found to be ineffective as an attractant to honeybees under the experimental conditions103. Neira et al. treated raspberry field with Bee-Scent. The number of bee visits and the average length of visit were found to be increased, but there was no significant increase in fruit weight, size or drupelet number compared with freely pollinated controls104.

1.14.3 Bee Lure When Bee Lure was sprayed on apple trees it was found that there was no significant increase in the number of foraging bees on treated and untreated plants105.

25 In another experiment, application of Bee Lure was shown to increase the mean numbers of foraging honeybees on apple trees. However, the 106 increase was not significant. There was no increase in fruit set also

1.14.4 Bee-here Efficiency of Bee-here as a honeybee attractant was tested on marrow crop cultivated under green house conditions. Bees were not found to exhibit preferences for any experimental treatment indicating ineffectiveness of Bee- here as a honeybee attractant under the given conditions 107. Tsirakoglou ef a/, described the techniques to increase the attractiveness of Kiwi flowers to honeybees. However, they reported that use of Bee-here is one of the ineffective methods out of the methods reported108. Although the field evaluation of Bee-here was not found to be encouraging, the results of in vitro studies were exactly opposite! Bee-here was found to be the best honeybee attractant when tested with other commercial 'lures' on the chopped sugar cane109. These results indicated that the optimum potential of the 'lure' might not be getting utilised in the field experiments.

1.14.5 Bee-Q No increase in the number of honeybee visits or in percentage seed set of hybrid sunflower was found when the plots were sprayed with a commercial attractant, Bee-Q110.

1.14.6 Beeline and Bee-Scent Beeline and Bee-Scent were not found to be effective in improving the bee visitations as well as yields of cucumber and watermelon crops 111,112. These attractants did not cause a significant increase in bee activity and the harvests. It was, therefore, recommended that instead of using these attractants additional honeybee colonies should be rented for the crops. Although the effect of Beeline and Bee-Scent was discouraging on watermelon and cucumber, their effect on pear trees was found to be

26 encouraging. The number of bees was found to be significantly more for up to three days between 14-16 hours. Bee-Scent was found to attract more bees than Beeline113. It was, however, reported that the treatment had no effect on fruits. Nakamura found that Beeline, a food attractant, and Bee-Scent, a pheromone mimic, attracted honeybees to feeding sites and to wild plants offering nectar114. He suggested that these products could be used to attract bees to area small enough to be sprayed with one of them. Recently, Sotomayor while evaluating different pollination modes in almonds sprayed the branches with bee attractants Beeline or Bee-Scent. He reported that the bee pollination is essential for almond production. He also added that the best fruit set was achieved with these attractants indicating the improved efficiency of pollination due to 'lures'115.

1.14.7 Bee-here and Bee-Q Patil ef al. studied the effect of Bee-Q and Bee-here on pollinators and yield of sesamum 116. They found both these attractants were effective in attracting higher number of pollinators {Apis spp.) and resulted in more yields. They reported that the best results were obtained when the rates applied were 12.5 g Bee-Q/ L and 4ml Bee-here/ L.

1.14.8 Beeline and Poilenaid-D Screening of Beeline and Pollenaid-D for their attractiveness to honeybees showed that none of them caused an increase in honeybee activity on the flowering apple trees117.

1.14.9 Evaluation of Six Commercial Attractants Vaissiere and Serpeille have discussed six commercial products, Api- Fix, Beeline, Bee Lure, Bee-Scent, Pollenaid and Pollinus, tryase claim to attract pollinators to crops. It is concluded that the manufacturers' claims have, in general, not been proved118.

27 1.15 COMMENTS

The application of 'lures' as swarm attractants is well established and can be a strong reason for their commercialisation. Another purpose of using 'lures' is to attract honeybees to the desired crops to increase efficiency of pollination. It is observed that even though in many cases these 'lures' may help in increasing the number of honeybee visits in most of the cases they fail in increasing crop yields indicating that the improved efficiency of pollination is not achieved. To arrive at the effective commercial 'lure' more research on the methods of application of the 'lure' in fields needs to be carried out. The modifications in the protocol to lengthen the active time of 'lure', to affect the slow release of it, use of proper medium / solvent are needed. The efficacy of the already established 'lures' also needs to be optimised. The type of crop, climatic conditions or the type of formulation might turn out to be the crucial factors. The positive results obtained with some of the commercial 'lures' give a glimpse of expected success in such cases. It has been a century since the research on attractants and the methods of their implementation is being carried out but still there is a vast scope to arrive at the effective, ready to use, commercial 'lures' for honeybees.

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