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

We used the proboscis extension reflex of honeybees to test their ability to 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 their chemical composition, and an ability to discriminate between them may aid the orientation of the bees in the nest. To train the bees, we used whole extracts of waxes and four different fractions of the whole extract based on different elutions of solid-phase extractions (extract I, fraction A eluted with hexane and fraction B with diethylether; 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 whole extracts or with the different fractions, we paired one type of wax with a reward and another with no reward. The bees learned to discriminate between all tested pairs of whole extracts. The two subfractions (fractions A and B) gave different results: the bees could discriminate between waxes of 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 only fraction D contained the elements that enabled bees to discriminate between old and new wax. Fraction D makes up only 5?8 % of the total wax mass and contains hydroxy alkyl esters (5?6 % of the total wax mass), primary alcohols (0.3?0.5 % of the 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 with a mixture of chloroform, methanol and water (13:5:1) as fraction E.