When tested singly (E)-citral, (Z)-citral, geraniol, nerolic acid and geranic acid induced the bees to release Nasonov pheromone in the tunnel, and mixtures lacking only one of these components stimulated pheromone release less effecively. In contrast to the other components, (E,E)-farnesol and nerol did not encourage Nasonov exposure when tested singly, and omission of either from the Nasonov mixture increased the attractiveness of those that remained.
Pheromone Bioassay: foraging
The foraging bioassays for Nasonov pheromone are of three basic types that test the reactions of scout bees, recruits and foragers. In the ﬁrst type of bioassay petri dishes without food, but with either no odour or the odour of one or more pheromone components, are placed alternatively in a circle, or in a Latin Square arrangement, on a lawn or other area devoid of obvious orientation marks, and the scout bees that investigate each are counted (e. g. Free, 1962). In the second type of bioassay foragers are trained to a petri dish and, while bees are still foraging on it, other petri dishes with different treatments are placed nearer to the hive and the number of recruits that land on them are counted (e. g. Free, 1962 ; Boch and Shearer, 1964, 1966; Weaver et al., 1964).
In the third type of bioassay foragers are trained to collect sugar syrup from a petri dish which is then removed and replaced by empty dishes with two or more different treatments, and the foragers that land on each are counted (e.g. Free, 1962; Renner, 1960).
Because foraging bees approach an odour source from downwind, the location of the dishes, with different odour treatments, in relation to the wind direction is most important. If two dishes containing sucrose syrup are located 500 mm apart in line with the wind direction each will receive an approximately equal number of foragers, although the upwind dish may be slightly favoured. When the two dishes are empty, bees landing at the downwind dish tend to take ﬂight again and land at the upwind dish; those that take ﬂight from the upwind dish or overshoot it turn into the wind again and land at the upwind dish but rarely at the downwind dish, so the upwind dish accumulates most bees. Provided that bees have previously been trained to collect sucrose syrup from the dishes the same result is achieved even when the dishes are unscented (Free, 1981). Therefore it is most important that the treatment dishes are equally aligned to the wind direction, and because this continually ﬂuctuates the dishes are usually presented on the opposite sides of a circular sheet of glass supported on a turntable that is slowly rotated (Ribbands, 1955; Butler et al. , 1969; Ferguson and Free, 1979). Learn about pheromone attraction triggers.
Soon after geraniol had been identiﬁed as a component it was shown that its attractiveness to foragers was insufﬁcient to account for the response to Nasonov pheromone (Free, 1962; Boch and Shearer, 1964). A mixture of geraniol with geranic and nerolic acids was more effective but still failed to match the attractiveness of the Nasonov pheromone (Boch and Shearer, 1964). The citral component proved to be most attractive (Weaver et al., 1964). Butler and Calam (1969) used a mixture of citral and geraniol and found that, provided the amount of citral equalled or exceeded the amount of geraniol, it was almost as attractive as Nasonov pheromone wiped on to plaster blocks; they concluded that geranic and nerolic acid were of little consequence. Shearer and Boch (1966) reported that (E)- and (Z)-citrals were equally attractive, both on their own and in combination with geraniol. However, tests showed that nerolic acid on its own was more attractive than geranic acid on its own; the addition of nerolic acid to geraniol or the citrals increased their attractiveness, but the addition of geranic acid did not.
A mixture of geraniol (50 ug), citral (100 rig) and nerolic acid (200 pg) was as effective as small pieces of ﬁlter paper wiped over the Nasonov glands of 20-30 foraging bees. Preliminary tests by Free (1981) supported these results; either geraniol or (E)-citral alone were signiﬁcantly attractive to searching foragers, and removing either geraniol or nerolic acid from a synthetic mixture of Nasonov components (in approximately natural propor- tions) signiﬁcantly diminished its attraction. The presence of nerol and (E,E)-farnesol in a mixture of components sometimes diminished the collec- tion of pollen substitute in the hive and the collection of water and sucrose syrup both in the hive and the ﬁeld (Free et al., 1983a). Somewhat contrary results were obtained by Williams et al. (1981) who found that each of the seven components was attractive, although to varying extents, and each could contribute to the attractiveness of a mixture in natural proportions. The order of attractiveness of the single components was (E)-citral, geranic acid, nerolic acid, geraniol, nerol, (Z)-citral and (E,E)-farnesol. Removal of individual components from the mixture diminished attractiveness in the following descending order of effectiveness: (E)-citral, (E,E)-farnesol, gera- nic acid, nerol, nerolic acid, geraniol, (Z)-citral.
It is difﬁcult to summarize these results and clearly more experiments to resolve the differences are needed. All authors are agreed on the importance of (E)-citral and most on the importance of geraniol. Some tests suggest that nerolic acid is important and more so than geranic acid (this agrees with ﬁndings in another type of bioassay, page 123), while others suggest a reverse preference or that these acids have little or no attractiveness. These diverse results tend to emphasize the complexity of the ‘language’ involved and the difﬁculty in solving it by the means we have available.