Comb-wax discrimination by honeybees tested with the proboscis extension reflex

We used the proboscis extension reflex of honeybees to test their ability t o discriminate between comb waxes of different ages (wax scales, 1-week-old wax, 2- to 3-year-old wax, 8- to 10-year-old wax). Such waxes differ in th eir chemical composition, and an ability to discriminate between them may a id the orientation of the bees in the nest, To train the bees, we used whole extracts of waxes and four different fract ions of the whole extract based on different elutions of solid-phase extrac tions (extract I, fraction A eluted with hexane and fraction B with diethyl ether; extract II, fraction B further subdivided into fraction C by elution with isopropylchloride and fraction D by elution with diethylether), In a differential training regime (six learning and six test trials) with w hole extracts or with the different fractions, we paired one type of wax wi th a reward and another with no reward. The bees learned to discriminate be tween all tested pairs of whole extracts, The two subfractions (fractions A and B) gave different results: the bees could discriminate between waxes o f different ages when fraction B was used but not when fraction A was used, A further subdivision of fraction B into fractions C and D showed that onl y fraction D contained the elements that enabled bees to discriminate betwe en old and new wax. Fraction D makes up only 5-8 % of the total wax mass and contains hydroxy a lkyl esters (5-6 % of the total wax mass), primary alcohols (0.3-0.5 % of t he total wax mass) and acids (0.06-1.0 % of the total wax mass). Fractions A and C (together forming 62-64 % of the total wax mass), which consist of unbranched and branched aliphatic hydrocarbons and alkyl esters, could not be discriminated by the bees. The remaining wax mass (25-29 %) was eluted w ith a mixture of chloroform, methanol and water (13:5:1) as fraction E.