3. Analysis of juvenile hormone and ecdysteroid levels in honey bees

Juvenile hormone (JH) and ecdysteroids are lipid signalling molecules playing fundamental roles in postembryonic development and the reproductive physiology of insects (Nijhout, 1994). In social insects, where these hormones are also involved in regulating caste development, reproductive dominance and division of labour, the role of these hormones has been extensively reviewed (de Wilde and Beetsma, 1976; Nijhout and Wheeler, 1982; Robinson, 1992; Robinson and Vargo, 1997; Hartfelder and Engels, 1998; Hartfelder and Emlen, 2012) ever since their biochemical characterization.  

While many of these insights into the roles played by these hormones in social insects have been gained through application of synthetic hormones or hormone analogs, such experiments require confirmation through analyses of circulating hormone titres or rates of hormone synthesis by the respective endocrine glands or peripheral tissues. Conclusions based solely on hormone treatment experiments are frequently and justifiably subject to critique (Zera, 2007) as the applied doses usually exceed endogenous hormone levels by three to six orders of magnitude, thus introducing the possibility of pharmacological effects masking their truly physiological ones. Employing sensitive detection methods is thus paramount to fully understand the role of these lipid signalling molecules.

As a key regulator of insect development, reproduction, and behaviour (Goodman and Cusson, 2012), juvenile hormone (JH) also plays a major role in the social organization  of bees, wasps, ants and termites (Hartfelder and Emlen, 2012). In honey bees, JH has been shown to drive caste development in the larval stages (Hartfelder and Engels, 1998), and in adult workers it plays an important role in division of labour (Robinson and Vargo, 1997; Amdam et al., 2007) and sensory modulation (Pankiw and Page, 2003). There are several isoforms of JH in different insects (e.g. JH I, JH II, JH III, bis-epoxy JH III). These differ slightly in their side chains and unsaturated bonds, but in honey bees, as in most insects, JH III is the only isoform produced by the corpora allata (Hagenguth and Rembold, 1978).

Whereas insects can synthesize JH de novo from relatively simple compounds (acetyl CoA or proprionyl CoA) they cannot do this for ecdysteroids. Rather, they require dietary steroids for conversion to physiologically active hormone. This conversion occurs in the prothoracic glands of larvae and pupae and in the gonads of the adults. In honey bees, the predominant ecdysteroid moiety in haemolymph of larvae and pupae and ovaries of adult females is makisterone A, quantitatively followed by 20-hydroxyecdysone and ecdysone (Feldlaufer et al., 1985, 1986; Rachinsky et al., 1990). These are the physiologically active ecdysteroids, whereas others, especially a series of different conjugates, are either metabolites or storage forms (Lafont et al., 2012). In honey bee caste development, haemolymph ecdysteroid titres differ between queen and worker larvae and pupae (Rachinsky et al. 1990; Pinto et al., 2002), but they do not seem to play a major role in reproduction or division of labour (Hartfelder et al., 2002).

With the importance of these hormones in honey bee biology in mind, we will focus in this section primarily on currently used and firmly established analytical methods. We detail radioimmunoassay (RIA) and physicochemical detection methods, such as gas chromatography coupled with mass spectroscopy (GC-MS), for hormone titration, as well as a radiochemical in vitro assay for determining the JH-synthetic activity of the corpora allata (CA). It is important to note that while radioimmunoassays have frequently been substituted by enzyme-linked immunosorbant assays (ELISAs), due to restrictive regulations for the use of radioisotopes, there are no ELISAs of sufficient sensitivity available for the quantification of insect ecdysteroids and JH.

Older methods, such as the Galleria bioassay, will not be described herein. While important tools in the early days of JH quantification, including in honey bees (Fluri et al., 1982), these older methods are extremely laborious, and provide only relative measures (e.g. Galleria units ) rather than absolute quantities (ng JH per ml haemolymph). We also do not present recently developed analysis methods employing liquid chromatography mass spectrometry (LC-MS) that have been developed (Westerlund and Hoffmann, 2004; Li et al., 2006). Although these have been validated for use in honey bees (Zhou et al., 2011) and are comparable in terms of sensitivity to radioimmunoassays and GC-MS (Chen et al., 2007), they are not yet in common use. A recently developed, very elegant and highly sensitive method for quantifying JH based on tagging the epoxy group of JH with a fluorescent tags, with subsequent analysis by reverse phase high performance liquid chromatography coupled to a fluorescent detector (HPLC-FD) (Rivera-Perez et al., 2012) may, however, eventually become an option. Ultimately, the method of choice for a laboratory will, of course, essentially depend on available equipment

3.5. Measuring the rate of juvenile hormone biosynthesis by the paired corpora allata