1. Introduction

Maintenance of integrity of a honey bee colony is highly dependent on a sophisticated communication system that is largely dependent on chemical cues. Due to the crowded environment within the hive, bees appear to communicate to large extent through chemicals, of low volatility, vibrations, and other near-contact or contact modalities (Blum and Fales, 1988; Breed et al., 1988; Naumann et al., 1991; Breed, 1998; Slessor et al., 2005). However, volatiles form a small but important part of the signalling chemicals (semiochemicals), that mediate interactions between colony members (for reviews, see Free, 1987; Pankiw, 2004; Slessor et al., 2005). Some volatiles are used as releaser pheromones to rapidly communicate information to the rest of the colony (Pankiw, 2004), including alarm pheromones for defence (Boch et al., 1962; Hunt et al., 2003) and Nasanov pheromones for colony cohesion (Boch and Shearer, 1964; Pickett et al., 1980). Other volatile pheromones, such as (Z)-β-ocimene, have long-term primer effects on physiology, development and fertility (Maisonnasse et al., 2009; Maisonnasse et al., 2010). A few volatile pheromones, such as the alarm pheromone component isoamyl acetate (IAA), have both releaser effects on behaviour and primer effects on physiology (Alaux and Robinson, 2007). On a more localized scale, both volatiles and non-volatile contact cues (e.g. cuticular hydrocarbons) serve as signalling cues for intimate interactions of workers with their immediate colony environment such as hygienic behaviour (Masterman et al., 2001; Gramacho and Spivak, 2003; Swanson et al., 2009; Schöning et al., 2012). Volatiles also mediate interactions of bees with non-nestmates.

Like many social hymenopterans, honey bees use non-volatile to volatile acquired colony chemicals to distinguish between nestmates and non-nestmates (Breed, 1998). Natural enemies of the honey bee such as the small hive beetle Aethina tumida and the parasitic mite Varroa destructor also use colony odours as kairomone cues (Nazzi et al., 2004; Torto et al., 2005; Torto et al., 2007a; Nazzi et al., 2009; see the BEEBOOK papers on small hive beetles (Neumann et al., 2013) and varroa mites (Dietemann et al., 2013) for more details on these organisms).

This paper describes basic methods essential in elucidating chemically-mediated behavioural interactions among honey bees, and between honey bees and other arthropods. These range from bioassay methods used to demonstrate the role of specific behaviours, techniques and equipment used to collect and analyse semiochemicals (both volatiles and non-volatiles) from individual honey bees, groups of bees or an entire colony in its native environments. This paper is subdivided into 5 main sections; collection and analysis of honey bee volatiles in the natural environment (Section 2), collection and analysis of bee volatiles out of their natural environment and their antennal detection (Section 3), collection and analysis of non-volatile semiochemicals (Section 4), bioassays with queen pheromone (Section 5) and the in vitro bioassays as a tool for elucidation of mechanisms regulating pheromone gland activity (Section 6). While this paper intends to provide simple easy to follow and replicate guidelines when working on the semiochemically-mediated interactions of honey bees, readers must bear in mind that chemical ecology requires basic understanding of behavioural biology and analytical chemistry, which are two very broad fields of study, that cannot be exhaustively dealt with in this BEEBOOK paper. Therefore, before embarking on any chemical ecology experiments, it is advised to consult a chemist (if a biologist) or a biologist (if a chemist) and the other BEEBOOK papers (e.g. for behaviour, Scheiner et al., 2013)).