\title{Variation in complex olfactory stimuli and its influence on odour recognition}

\author{Geraldine A. Wright and Brian H. Smith \\
Department of Entomology, Ohio State University, 318 West 12th Avenue, Columbus, OH 43235, USA}

\begin{abstract}
Natural olfactory stimuli are often complex and highly variable. The olfactory systems of animals are likely to have evolved to use specific features of olfactory stimuli for identification and discrimination. Here, we train honeybees to learn chemically defined odorant mixtures that systematically vary from trial to trial and then examine how they generalize to each odorant present in the mixture. An odorant that was present at a constant concentration in a mixture becomes more representative of the mixture than other variable odorants. We also show that both variation and intensity of a complex olfactory stimulus have affect the rate of generalization by honeybees to subsequent olfactory stimuli. These results have implications for the way that all animals perceive and attend to features of olfactory stimuli.
\end{abstract}

Keywords: olfactory; honeybee; generalization; natural stimulus; odour recognition

\section{Introduction}

Olfactory stimuli are used by animals for many important tasks crucial to their fitness. Naturally occurring olfactory stimuli, such as floral perfumes or animal pheromones, are typically complex and often highly variable combinations of many chemical compounds \cite{Dobson_1994,Laurent_2002,Pichersky_Gershenzon_2002}. Each compound in an odour stimulus may differ from the others by several orders of magnitude in concentration \cite{Dobson_1994,Levin_2001}. Differences in the ratios of concentration of these compounds can produce distinct perceptual changes that affect odour recognition \cite{Laska_Hudson_1992,Olsson_Cain_2000}. Odour recognition is made even more complex by the ephemeral nature of odour mixtures \cite{Crimaldi_2002}. Odour stimuli emitted by the same type of `object' often differ slightly from one encounter to the next, and this variability makes odour objects difficult to classify such that they can be recognized and distinguished.

The olfactory system of an animal must, therefore, form a memory representation for a class of objects, such as the odour of a flower species that presents nectar to a honeybee, which accounts for variability across objects and allows for identification of all objects that mean the same thing. Several behavioural mechanisms have been identified that may be involved in the extraction of features that can be used to classify stimuli. The features that olfactory stimuli have in common, such as the number and types of odorants present and their concentrations, are likely to be important in classifying stimuli \cite{Chandra_Smith_1998,Hosler_Smith_2000,Wise_2000,Laurent_2002,Wilson_2003}. Without et al. Specific features that dominate a mixture, however, such as an odorant that is present at a higher concentration than other odorants, could perceptually overshadow other features. In addition, studies of olfactory blocking indicate that olfactory systems of a variety of animals are capable of perceptually isolating individual odorants or submixtures from a blend \cite{Hosler_Smith_2000,Giannaris_2002} when the individual odorants were more reliably associated with reinforcement than the other odorants present.

Our study addresses how variability across experiences with an odour stimulus affects the subsequent identification of odour objects. Trial-to-trial variability could lead to consolidation of an olfactory memory that reflects an average of all experiences of odour stimuli. However, we predicted that the subsequent identification of odour objects, in our case odour mixtures, would reflect features that are most reliably associated with reinforcement. We conditioned honeybees to mixtures of odorants that varied qualitatively from trial to trial as they were reinforced with an appetitive reward, and then tested them with each odorant present in the mixture. Generalization from conditioning with a mixture to each of the test odorants was interpreted as a measure of how similar the test odorant was to the mixture.

\section{Material and Methods}

Worker honeybees (\textit{Apis mellifera}) were collected from colonies at the Rothenbuhler Honeybee Laboratory, Columbus, OH, USA. Each subject was placed individually in a restraining harness as described by Smith (1998). All experiments used the proboscis extension response assay to evaluate the olfactory learning behaviour of individual subjects (see Smith (1998) for details). Odour stimuli were delivered by placing 5~$\mu$l of odour solution on a small strip of filter paper that was then placed in a modified, 1 ml tuberculin glass syringe attached to an air supply and controlled by a solenoid valve (see Smith (1998) for details).

Our odour stimuli were mixtures of pure monomolecular odorants at specific molarities created using hexane as a solvent. To examine the effect of qualitative variables on generalization from mixtures to the odorants present, we developed two types of odour mixtures: mixtures composed of perceptually `similar' odorants, and mixtures composed of perceptually `dissimilar' odorants \cite{Stoffer_1997,Laska_1999,Daly_Smith_2000}. The similar odours, 1-hexanol, 1-heptanol and 1-octanol (Sigma, 99.0\% + purity), all aliphatic alcohols, differed only with respect to their carbon chain length. The dissimilar odours,