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Mucignat-Caretta C, editor. Neurobiology of Chemical Communication. Boca Raton (FL): CRC Press/Taylor & Francis; 2014.

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Chapter 5Chemical Communication in the Honey Bee Society

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5.1. PHEROMONES OF THE HONEY BEE COLONY

Together with the honey bee dance, honey bee pheromones represent one of the most advanced ways of communication among social insects.

Pheromones are chemical substances secreted by an animal’s exocrine glands that elicit a behavioral or physiological response by another animal of the same species. In honey bees the targets of pheromonal messages are usually members of the same colony, but there are some exceptions in which the target can be a member of another colony (Free 1987).

The composite organization of the honey bee society, which consists of three adult castes (queen, worker, and male) and non-self-sufficient brood, provides for many coordinated activities and developmental processes and thus needs a similar elaborate way of communication among the colony members. Pheromones are the key factor in generating and maintaining this complexity, assuring a broad plasticity of functions that allow the colony to deal with unforeseen events or changing environmental conditions.

Pheromones are involved in almost every aspect of the honey bee colony life: development and reproduction (including queen mating and swarming), foraging, defense, orientation, and in general the whole integration of colony activities, from foundation to decline. Pheromones allow communication among all the honey bee castes: queen–workers, workers–workers, queen–drones, and between adult bees and brood (Trhlin and Rajchard 2011; Winston 1987).

In honey bees, as in other animals, there are two types of pheromones: primer pheromones and releaser pheromones. Primer pheromones act at a physiological level, triggering complex and long-term responses in the receiver and generating both developmental and behavioral changes. Releaser pheromones have a weaker effect, generating a simple and transitory response that influences the receiver only at the behavioral level.

Most of the pheromones known in insects are of the releaser type; they are classified into several categories based on their function (e.g., sexual, aggregation, dispersal, alarm, recruitment, trail, territorial, recognition) (Ali and Morgan 1990). Primer pheromones are especially developed in social insects, where they represent the major driving force in the evolution of social harmony and in maintaining colony homeostasis (Le Conte and Hefetz 2008). Among honey bee pheromones, the queen signal and the brood pheromones (described in detail below) are principally primer pheromones (having also some releaser functions), while most worker pheromones are to be considered releaser pheromones.

In the following paragraphs the main honey bee pheromones are described, based on the honey bee caste to which they belong and the glands responsible for their production. In the first part of the chapter the effect (or the effects) exerted by each pheromone on the receivers and on the bee colony will be illustrated, while the neurophysiologic and molecular mechanisms of the response to the chemicals will be discussed in the second part of the chapter.

5.1.1. Queen Pheromones

The honey bee queen represents the main regulating factor of the colony functions. This regulation is largely achieved by means of pheromones, which are produced by different glands and emitted as a complex chemical blend, known as the “queen signal.”

The queen signal acts principally as a primer pheromone, inducing several physiological and behavioral modifications in the worker bees of the colony that result in maintenance of colony homeostasis through establishment of social hierarchy and preservation of the queen’s reproductive supremacy. More specifically, the effects of the queen signal are maintenance of worker cohesion, suppression of queen rearing, inhibition of worker reproduction, and stimulation of worker activities: cleaning, building, guarding, foraging, and brood feeding (Figure 5.1). It is known that when the queen is old or sick (low pheromonal signal) or it dies (no pheromonal signal), workers are driven to rear new queens from young brood within 12–24 hours; the removal of the queen in absence of young brood soon leads to the decline of the colony: the workers stop performing their activities and start to lay unfertilized eggs that develop into male adults (drones); the colony becomes disorganized, unfit, dirty, susceptible to diseases and prey of predators; it rapidly depopulates and goes toward a certain death.

FIGURE 5.1. Releaser and primer effects of the queen signal, which regulates colony functions and development.

FIGURE 5.1

Releaser and primer effects of the queen signal, which regulates colony functions and development. Stimulating effects are indicated as “+” and inhibiting effects as “–.” (Adapted from Winston, M.L. (1987) The Biology (more...)

In addition to its primer effect, the queen signal exerts an attractive releaser effect: it calls workers around the queen in a retinue group, which is stimulated to feed and groom her; in young premating queens it acts as attractant for drones during the mating flights; during swarming it keeps the swarm together.

5.1.1.1. Main Component of the Queen Signal: The Queen Mandibular Pheromone

The queen mandibular pheromone (QMP) is by far the most studied and well-known chemical signal in the honey bee society. Its first identification dates back to 1960, when (E)-9-oxodec-2-enoic-acid, more simply known as 9-ODA, was detected as the substance secreted by the queen mandibular glands (Barbier and Lederer 1960; Callow and Johnston 1960). The secreting organs are a pair of saclike glands located inside the head above the base of the mandible (Figure 5.2). The glands open through a short duct at the base of the mandible and their secretion runs along a deeper channel surrounded by hairs (Billen 1994).

FIGURE 5.2. Exocrine glands of the honey bee queen.

FIGURE 5.2

Exocrine glands of the honey bee queen. The pheromone-producing glands that concur to the formation of the queen signal are highlighted in bold. (Adapted from Goodman, L. (2003) Form and Function in the Honey Bee. Cardiff, UK: IBRA.)

In 1988, Slessor et al. discovered four other compounds secreted by the mandibular glands that act synergistically with 9-ODA: the two enantiomers of 9-hydroxydec-2-enoic acid (9-HDA), methyl p-hydroxybenzoate (HOB) and 4-hydroxy-3-methoxy-phenylethanol (homovanillyl alcohol [HVA]). The five components together were more active than any of the single substances, alone or in combination, in forming the retinue of worker bees (see Section 5.1.1.1.1). It was concluded that these five chemicals together form the base of QMP secretion, which represents the main constituent of the queen signal.

Several authors analyzed the evolution of QMP components during queen aging, from emergence until the full dominant status. Generally, the amount of volatiles was found to increase with age, but the findings concerning the different compounds and their relative amounts were inconsistent among authors, as illustrated by the studies described below.

Engels et al. (1997) identified three different ontogenetic patterns of QMP in queens: (1) virgin premating queens presented a weak signal, with oleic acid (OLA) as the main component, (2) mating queens intensified the signal, mainly consisting of 9-ODA along with OLA and small amounts of 9-HDA, and (3) postmating dominant queens exhibited a strong signal with high concentrations of 9-ODA combined with medium proportions of 9-HDA, less OLA, and small amounts of oxygenated aromates. The authors suggested that these oxygenated aromates, especially HOB and the late appearing HVA, could be the typical signal of old egg-laying and dominant queens.

Plettner et al. (1997) compared the quantities of the QMP components between 6-day-old virgins and 1-year-old mated egg-laying queens of several species; they found that mated Apis mellifera queens had significantly higher levels of 9-ODA, 9-HDA, HOB, and HVA, while an opposite trend was found for 10-HDA and 10-HDAA, which are typical components of worker mandibular glands (see Section 5.1.2.1) and are produced in higher amounts by virgin queens.

Slessor et al. (1990), who compared virgin with mated queens of different ages, found slightly different results, with levels of 9-ODA almost constant in the different groups, 9-HDA levels higher in mated than in virgin queens, and HOB and HVA levels higher in the oldest mated queens compared to the virgin and the young mated ones. In all cases pheromone levels were highest in mature, mated, laying queens.

On the contrary, Rhodes et al. (2007), comparing 7-day-old virgin and mated queens, found that the former had higher levels of 9-HDA, 9-ODA, and 10-HDA than the latter. In a similar comparison, Richards et al. (2007) found that the quantities of 9-ODA, 9-HDA, and HVA were all significantly lower in mated queens compared to virgins, although the extract of mated queens’ mandibular glands was more attractive towards workers than the one of virgin queens.

Finally, Strauss et al. (2008), analyzing the mandibular gland compounds of virgin, drone-laying, and mated queens, found similar amounts of 9-ODA in the three groups and increasing amounts of all the other components (9-HDA, 10-HDA, 10-HDAA, and HVA) except HOB, from virgin to mated queens. The constant level of 9-ODA, which corresponds to a higher relative proportion in the virgin queen, suggests that 9-ODA plays a greater role in drone attraction of virgin queens than of retinue attraction in mated queens; on the contrary, 9-HDA, 10-HDA, 10-HDAA, and HVA are positively correlated with the reproductive potential and the ovarian activation of the queen (Strauss et al. 2008).

These contradictory results suggest that the role of single QMP components in queen signal is not fully understood and that there could be additional unknown compounds in mated queens’ mandibular glands that synergize with 9-ODA and 9-HDA in the attractive function.

5.1.1.1.1. Attracting the Workers: The Retinue

The first functions of QMP to be discovered were due to its attractant properties toward the workers: the formation of the queen retinue and the constitution and maintenance of the swarm cluster (Kaminsky et al. 1990; Winston et al. 1989).

When the queen is stationary on the comb she is surrounded by a circle of workers known as “court” or “retinue” that face toward her and feed, palpate, and lick her. Usually the retinue is composed of eight or 10 workers. Several studies demonstrated that QMP and its components are accountable for the formation of the retinue (Free 1987), and this is supported by the fact that worker attraction towards the queen could be related to the modifications in the QMP pattern. The attention paid by retinue workers increases when the virgin queen becomes mated and lays eggs, and then decreases as she grows old; the degree of attractiveness of the queen is null at 0–1 day old, medium from 2 to 4 days old, and high from 5 days to 18 months old (De Hazan et al. 1989). Richards et al. (2007) tested the mandibular gland extracts of virgin and inseminated queens on worker retinue responses and found that gland extracts of the inseminated queens were more attractive than those of the virgin queens, and those of queens inseminated with more than one drone were more attractive than those of queens inseminated with a single drone. This suggests that mating is a crucial factor for the development of the chemical signal of the queen and its attractive effect on workers.

In 2003, Keeling et al. identified four additional compounds produced by the queen that act synergistically with QMP in attracting workers to form the retinue group: coniferyl alcohol (CA), methyl oleate (MO), hexadecane-1-ol (PA), and linoleic acid (LA); the first is secreted by the mandibular glands, while the others are produced in different parts of the queen’s body. These substances were inactive alone, but in combination with QMP they were found to greatly increase activity of the queen’s retinue. Furthermore, in a recent study, Maisonnasse et al. (2010a) showed that queens artificially deprived of mandibular glands can still attract workers in the retinue, suggesting that QMP was not the only pheromone able to attract workers and that in its absence other substances can take its role.

5.1.1.1.2. Attracting the Workers: The Swarm

Swarming is the way in which the colony reproduces itself; workers rear new queens and the first emerging one will kill the others, and after mating will become the new colony regnant, while the old queen drives the swarm toward a new nest. The presence of the queen is essential to keep the swarming bee cluster together: if the queen dies or is unable to fly, the swarm soon returns to the parental hive. The queen’s attractiveness towards the swarm cluster is triggered by means of pheromonal signals, mainly the QMP. In 1989, Winston et al. compared the effects of the queen, the mandibular gland extracts, and the five-component-blend on the swarming and thus demonstrated that the component blend and the gland extract showed comparable effects, while the queen alone always had the strongest attractiveness. This suggested, as with induction of retinue behavior, that other extra mandibular components could be involved in formation of the swarm cluster.

5.1.1.1.3. Attracting the Drones: QMP as a Sexual Pheromone

Soon after its discovery it became clear that the QMP is used by virgin queens to attract drones during mating flights (Gary 1962); more specifically, by using queen dummies 9-ODA was clearly shown to attract drones (Gary and Marston 1971).

In further experiments, different combinations of 9-HDA, 10-HDA, and HOB were also found to increase the number of drones making contact with the queen dummy; 9-HDA and 10-HDA in particular seemed to be responsible for the increased mating contacts, although they were active only at short range, contrary to 9-ODA, which also acted at a higher distance (Brockmann et al. 2006; Loper et al. 1996). Comparing QMP components in virgin and mated queens it emerges that 10-HDA is more represented in the former while it is highly reduced in quantity in the latter (Plettner et al. 1997). The fact that 10-HDA is produced in large amounts by virgin queens suggests its role as a sex pheromone in mating behavior.

An increase in the frequency of mating behavior was observed also when tergal gland extracts were added to 9-ODA (Renner and Vierling 1977). This suggests that several glandular sources could cooperate in increasing the effectiveness of the pheromonal stimulus, leading to a stronger response and a more complete performance of the mating behavior sequence.

Thus, the relative contribution of different components of QMP and other glands on the sex pheromone blend is not yet completely clear.

5.1.1.1.4. Unique Queen: Suppression of Queen Rearing and Swarming

Many insect societies are monogynous, which means that a single queen is present in each colony. In small and primitive social species the maintenance of queen dominance is achieved by fight and physical competition among females; in contrast, in large monogynous colonies a physical dominance is not possible and a more efficient system has evolved for the maintenance of queen dominance that is based on pheromonal signals.

As previously stated, the removal of the queen from a colony of A. mellifera results in worker bees building special cells (queen cups) for rearing new queens (Winston 1992), but the precise way in which this happens is still in part unclear.

The rearing of new queens in a colony has two main scopes: reproduction of the colony through swarming and replacement of the queen when it is old or weak (this phenomenon is known as supersedure) or if it dies for some apicultural or pathological reason. QMP suppresses both queen supersedure and swarming by its dispersal throughout the colony (Winston et al. 1989). Several studies were addressed to elucidate the mechanisms of dispersal of QMP inside the colony and its transfer among workers. In 1991 Naumann et al. identified the group of retinue workers as the first actors in transferring queen pheromones toward the other workers and self-grooming as the means by which the pheromone is translocated from the mouthparts and the head to the abdomen of the workers (Naumann 1991). The distribution of QMP seems to be influenced by the size of the colony, since in populous colonies workers at the periphery gain a lower amount of pheromone than in unpopulous nests (Naumann et al. 1993). This explains why populous colonies swarm: the pheromone signal “the queen is present” tends to decline when the colony grows because pheromone dispersal is reduced and workers thus perceive a lower amount of pheromone, and the result is colony reproduction through queen rearing and colony swarming. When the queen dies or is removed, the pheromone signal disappears completely and workers are stimulated to rear new queens.

The role of QMP in the suppression of initiating queen rearing was confirmed by several studies that showed that administration of synthetic QMP to queenless colonies (i.e. colonies without a queen) suppresses the production of queen cups (Pettis et al. 1995) if the administration occurs within 24 hours from queen loss; in fact if synthetic QMP is applied 4 days after queen loss no effect is observed, indicating that QMP inhibits the initiation of queen-rearing but not the maintenance of established cells (Melathopolous et al. 1996).

5.1.1.1.5. Unique Mother: Suppression of Worker Reproduction

One of the main features of the honey bee society is the presence of two female castes (queen and workers) among which the queen is the only reproductive one. Workers are anatomically equipped with ovaries (which contain a lower number of ovarioles compared to queens) but development of the oocytes is inhibited by presence of the queen. If the queen is absent (and the colony and its workers are termed “queenless” as opposite to “queenright”), the workers’ ovaries can become active and workers can thus lay eggs (Butler and Fairey 1963; de Groot and Voogd 1954; Jay 1968; Velthuis et al. 1990). However, they can only produce haploid unfertilized eggs that give rise to male offspring, since they have not mated and do not have a spermatheca. A further confirmation of the inhibiting role of queen presence on ovarian development is provided by the findings of Malka et al. (2007), who showed that queenless egg-laying workers reintroduced into queenright colonies showed a clear regression in ovary development, whereas this did not happen if they were reintroduced into queenless colonies. This indicates that ovarian regression was not due to the change of colony but to the presence of a queen and her signals.

The specific role of QMP in the suppression of worker reproduction has been hotly debated for a long time. The first hypothesized mechanism for inhibition of ovarian development is that QMP acts by lowering the titer of juvenile hormone (JH) in workers and that this low titer corresponds to a low level of ovarian development and vice versa, as demonstrated in other social insects (Hartfelder 2000; Robinson and Vargo 1997). However, different studies exploring this hypothesis in honey bees gave contrasting results. First, the correlation between JH level and ovarian development in adult hemolymph is not confirmed (Robinson et al. 1991, 1992a). Furthermore, the administration of synthetic QMP alone was able to lower the titer of JH but not able to suppress the development of worker ovaries and egg-laying (Kaatz et al. 1992; Willis et al. 1990).

Disregarding the involvement of JH, the work of Hoover et al. (2003) clearly demonstrated that synthetic QMP alone is active in suppressing ovary development in workers and that its effect is comparable to that of the whole queen extract, thus excluding the involvement of other queen-produced substances. The components of queen pheromone identified by Keeling et al. in 2003 failed in suppressing ovary development and did not improve the efficacy of QMP when applied in combination, suggesting that they play a role in retinue behavior but not in workers’ ovary development.

More recently Katzav-Gozansky et al. (2006) observed that QMP alone or in combination with the secretion of Dufour’s gland inhibited ovarian development, with the combination of the two pheromones being the most effective treatment, but always less effective than the presence of a live queen, redrawing attention to the hypothesis of a queen multisignal in the regulation of worker reproduction.

It thus appears that the regulation of worker ovary development is probably an even more complex process, involving both queen and brood signals, since two components of the brood pheromone, ethyl palmitate and methyl linoleate, also play a role in suppressing worker ovary development (Mohammedi et al. 1998). Possibly the queen regulation alone becomes essential when no brood is present in the colony, such as during interruption of egg-laying due to environmental conditions (in winter or in summer in southern climates).

5.1.1.1.6. Regulation of Worker Activity and Behavioral Development

In highly advanced insect societies there is a typical organization of the infertile caste that determines an age-dependent division of labor, called temporal polyethism, in which workers progress from tasks performed inside the nest (cleaning, building, feeding) during the first 2–3 weeks of life, to those performed outside it (ventilating, guarding, foraging) in the last 1–3 weeks (Robinson 1992). This behavioral progress seems to be driven by endogenous factors, as it is linked to the amount of JH in worker hemolymph, which increases with the increasing age (Huang et al. 1991). Nevertheless, the task allocation of each worker cohort (group of workers of the same age) can be modulated by environmental factors that modify the requirements of the colony: a loss of older workers (e.g., forager bees that die in the field) can result in faster development of young bees into foragers, while a lack of young bees (e.g., for a natural or artificial interruption of queen egg-laying) can result in a slower behavioral development or a reverse from foragers to nest bees (Huang and Robinson 1992; Robinson et al. 1992b).

This plasticity in worker task allocation is at least partly regulated by QMP, via suppression of JH titers in workers; in colonies supplemented with synthetic QMP workers showed a reduced level of JH associated with a delay in behavioral development and a reduction of foraging activity (Pankiw et al. 1998). This mechanism could have an adaptive significance, such that the presence of the queen prevents workers from developing too rapidly into foragers, thus preserving a stock of young workers in case of loss of foragers due to adverse environmental causes (Winston and Slessor 1998).

Nevertheless, it is likely that the regulation of worker behavioral development is primarily modulated by the workers themselves, since the artificial alteration of worker demography is effective in changing age polyethism development even with constant presence of the queen (Huang and Robinson 1996; Robinson and Huang 1998). It is more likely that QMP acts as an auxiliary inhibitor on the workers’ division of labor rather than as a primary motor; this modulating role can be exerted by the queen’s strict contact with nurse bees in the nest, which determines an augmented inhibiting effect on the behavioral development of this group of bees.

The influence of QMP has been demonstrated on the activity of single workers, such as comb building. In the presence either of a mated queen or of artificial QMP, workers are stimulated to produce a higher amount of wax for the comb than in the presence of a virgin queen or in queen absence. In the latter case, furthermore, workers tend to produce male cells, demonstrating that the presence of the mated queen or QMP inhibits the production of male brood (Ledoux et al. 2001). A small difference was observed in the effects produced by presence of a mated queen and application of synthetic QMP, suggesting that QMP is not the only queen pheromone that influences comb building. Among the QMP components, HVA and HOB seem to play a major role in comb building, confirmed by the fact that virgin queens produce very low amounts of this component in their mandibular glands and do not stimulate comb building (Ledoux et al. 2001).

QMP also has an effect on stimulating foraging behavior and brood rearing. Higo et al. (1992) observed that in newly established spring colonies treated with synthetic QMP the number of foragers and the weight of pollen loads increased. Brood rearing also increased, but not significantly. In contrast, no effects were observed on large established colonies at their summer population peak, suggesting that QMP affects foraging, but its effect is influenced by colony conditions and environmental factors.

The defensive behavior is one of the best known features of a honey bee colony and consists in recognition of predators, alerting nestmates, and enacting antipredator behavior (from threat postures to buzzing and finally stinging). The defensive behavior will be illustrated more in detail in the description of the alarm pheromones but we mention it here because the presence of the queen seems to be important for its regulation, since it was observed that queenless colonies exhibit an increased aggressive behavior compared to queenright ones, and that synthetic QMP decreases stinging response in caged honey bees (Kolmes and Njehu 1990). The effect of QMP on the colony’s defensive behavior was recently confirmed by Gervan et al. (2005), who showed that the administration of synthetic QMP significantly reduces defensive behavior in both queenright and queenless colonies, with a decrease in the number of bees that respond to a simulated danger and a slight reduction of sting reaction, and by Vergoz et al. (2007) who found that QMP blocks aversive learning in young workers.

5.1.1.2. Beyond the QMP: Other Queen Pheromones

Mandibular glands are not the only source of chemicals with a role in social cohesion and colony homeostasis. For many years researchers presumed that QMP alone could account for the regulation of all colony functions. Later on, other pheromone sources were discovered that were in agreement with a multicomponent nature of the queen signal. Already in 1970, Velthuis already found that queens from which mandibular glands were removed were still able to exert some regulatory functions on workers (retinue behavior, inhibition of queen cup construction, suppression of worker ovary development). But it was not until 2010 that Maisonnasse et al. confirmed these results, showing that demandibulated queens retain their full regulatory role on the above mentioned functions (Maisonnasse et al. 2010a). The authors discovered that the levels of QMP components were similar in demandibulated and control queens, with the exception of 9-ODA, which was not detected in the former. This suggests that only 9-ODA is uniquely produced and stored in the mandibular glands, while the other substances (HOB and 9-HDA) appear to have another source of production in the queen’s body. 9-ODA has always been considered the main substance acting on retinue behavior but this is conserved in demandibulated queens, suggesting that other queen substances have the potential to substitute 9-ODA in evoking this behavior.

Alternative sources of the queen signal have been identified in the tergal, tarsal, Dufour’s, and Koschevnikov glands (Figure 5.2). Their secretions can either cooperate with QMP in the composition of the queen signal or be responsible for a single or few specific regulatory functions.

5.1.1.2.1. QMP Assistants: Tergal Gland Pheromones

Tergal glands, also known as Renner and Bumann glands, are located underneath the abdominal tergites and their ducts open through the cuticle in the tergites’ posterior edge region (Renner and Baumann 1964). In queens, numerous big gland cells occur, mainly in tergites III to V, whereas workers only have very few and considerably reduced cells (Billen et al. 1986).

The role of tergal glands as the source of pheromones with a supporting function to QMP has been postulated by several authors, in particular in African honey bee races (Velthuis 1970, 1985). De Hazan et al. (1989) found that the exocrine secretions of both mandibular and tergal glands in A. mellifera contribute to the attraction of worker bees, although the first to a greater extent than the second. On the contrary Moritz and Crewe (1991) in A. m. capensis (a honey bee race that occurs in the Cape province of South Africa) found that tergal and mandibular secretions contribute equally to the total pheromone blend. According to Wossler and Crewe (1999a) the major compound of the tergal gland secretion in workers and virgin and mated queens (studied in A. m. capensis and A. m. scutellata) is (Z)-9-octadecenoic acid, while Espelie et al. (1990) had found that the major components in virgin honey bee queens of 3–10 days old were decyl decanoate and longer chain-length esters of decanoic acid.

Just like the QMP, the tergal gland secretion shows both primer and releaser properties. In A. m. scutellata Wossler and Crewe (1999b) showed that queen mandibular gland secretions were more effective than tergal gland secretions in formation of the retinue, but the two secretions together were even more efficient, indicating a releaser function of queen tergal gland pheromone in evoking the worker retinue behavior. On the other hand, the tergal gland extracts of virgin queens of both A. m. capensis and A. m. scutellata showed a significant effect in the inhibition of ovarian development in their own workers, indicating that the secretions from the tergal glands can also operate as primer pheromone (Wossler and Crewe 1999c). Furthermore, in A. m. capensis the secretions of the queen tergal glands are used by workers as kin recognition signals (Moritz and Crewe 1988).

In A. m. ligustica the glandular production was suggested to be a specific signal of the mated queen, since the production of tergal gland alkenes is stimulated by natural mating and not by instrumental insemination (Smith et al. 1993).

5.1.1.2.2. Footprint Pheromone: Tarsal Gland Pheromones

The tarsal glands (Arnhart 1923) are present in queens, workers, and drones and consist of a unicellular layer of glandular epithelium located in the sixth tarsomere of each of the six legs. The secretory products accumulate in a saclike reservoir inside the tarsus, which communicates with the exterior at the level of an articular slit located between the fifth tarsomere and the arolium (Lensky et al. 1985); these secretions are oily, colorless substances that are extruded through openings when the bee is walking, from which comes the name footprint pheromones. It is assumed that the secrete of tarsal glands can serve different purposes in the three bee castes, since some differences in the chemical composition in queens, workers, and males were observed (Lensky et al. 1984).

Lensky and Slabezki (1981) observed that tarsal gland secretions deposited by the mated queen on the comb inhibit queen cup construction by workers; this hypothesis is supported by the observation that in overcrowded colonies the queens’ movements are restricted to the central parts of the comb, thus they are almost absent from the bottom edges of the combs where queen cups are usually built. A blend of the mandibular and tarsal pheromones is able to inhibit this behavior, giving an example of coregulation by these two pheromones.

5.1.1.2.3. Fertility Signal: Dufour’s Gland Pheromones

Dufour’s gland, first described in honey bees by Dufour in 1841, is a tubular gland associated with the sting apparatus together with the venom, sting sheath, and Koschevnikov glands (Figure 5.3). It opens into the dorsal vaginal wall (Billen 1987) close to the setosa membrane, a hairy region of cuticle that surrounds the entire sting bulb and acts as a platform for pheromone release (Lensky et al. 1995; Martin et al. 2005). The peculiar position of Dufour’s gland suggested different possible functions for its secretions, linked mainly to reproduction and egg-laying in queens (production of an egg coating or egg marking) and to defense in workers (production of a sting lubricant, neutralization of the remains of the acid secretion in the sting).

FIGURE 5.3. Pheromone-producing glands and organs and their main products in the honey bee worker, and their effect in the different worker activities.

FIGURE 5.3

Pheromone-producing glands and organs and their main products in the honey bee worker, and their effect in the different worker activities. Stimulating effects are indicated as “+” and inhibiting effects as “–.” (more...)

One of the main explored theories is the role of Dufour’s gland secretion as a caste-specific egg-marking pheromone applied by the queen during deposition, which could allow the workers to distinguish between queen-laid or worker-laid eggs (Ratnieks 1988; Ratnieks and Visscher 1989). Egg recognition is fundamental to the mechanism of worker policing, by which workers kill eggs laid by fellow workers but leave queen-laid eggs (a small percentage of workers lays eggs even in presence of the queen) (Ratnieks 1993). The existence of an egg-discriminatory pheromone was postulated by Ratnieks in 1988, and in subsequent experiments he found evidence that the Dufour’s gland secretion could be a source for this pheromone (Ratnieks 1995). However, in later studies it was found that neither the glandular secretion nor its ester or hydrocarbon constituents were able to protect worker-born eggs from policing; in fact, treated worker eggs were removed significantly faster than queen eggs, and at the same rate as nontreated worker eggs (Katzav-Gozansky et al. 2001; Martin et al. 2002). These results lead to the rejection of the hypothesis that the Dufour’s gland secretion serves as an egg-marking pheromone.

An alternative hypothesis for the role of the Dufour’s gland secretions is a fertility signal. This is supported by the caste-specific composition of its secretions: in workers they consist of a series of long-chain hydrocarbons, whereas in queens there is in addition a series of waxlike esters; these queen-specific esters are tightly correlated with ovarian development (Katzav-Gozansky et al. 1997, 2000). Queenless workers, which are likely to become egg layers, produce a queenlike secretion with an augmented level of the ester fraction that is correlated with worker ovarian development (Dor et al. 2005; Katzav-Gozansky et al. 2003). In workers of A. m. capensis, which are known to be social parasites, this mimicry of queen secretion profile by egg-laying workers is even more developed (Sole et al. 2002).

The Dufour’s gland secretion seems to act similarly to the QMP in attracting workers, which form a retinue around the scented source. Bioassays reveal that the active constituents are the queen-specific esters rather than the hydrocarbons. The queenlike glandular secretions of egg-laying workers are also attractive to nestmates, although to a lesser degree than those of the queen, while the secretions of non-egg-laying workers are totally inactive (Katzav-Gozansky et al. 2002, 2003). Moreover, workers were more attracted to Dufour’s gland extract from inseminated queens compared with virgins and to multidrone inseminated queens compared with single-drone inseminated ones, confirming the relation between Dufour’s gland secretions and reproductive potential (Richard et al. 2011).

Finally, as previously stated, the combination of QMP and Dufour’s gland secretions was effective in inhibiting ovarian development in workers, although neither was as effective as the presence of the queen (Katzav-Gozansky et al. 2006).

All this evidence suggests that Dufour’s gland secretion is a component of the queen signal, both in its releaser effects, as revealed by the stimulation of retinue behavior, and in its primer effects, correlated with reproductive dominance and fertility, through the inhibition of ovarian development. The specific roles of the Dufour’s gland secretion and QMP in regulating the physiological development and the behavior of workers still have to be elucidated.

5.1.1.2.4. Aging of the Queen Signal: Koschevnikov Gland Pheromones

The Koschevnikov gland is located near the sting shaft (Figure 5.3) and is composed of glandular units, each consisting of a secretory cell and a duct cell connected to the epidermis. Secretions are emitted onto the entire surface of the setosa membrane (Grandperrin and Cassier 1983), where they are released together with the alarm pheromones originating from the glandular part of the sting sheaths.

In honey bee workers the gland produces an alarm pheromone that is released when a bee stings (see Section 5.1.2.5.1, worker alarm pheromones). In queens the gland seems to play a different role, concurring to the already described queen signal. This is supported also by a caste-specific chemical composition of its extracts: 28 compounds including acids, alcohols, alkanes, and alkenes were found in queen Koschevnikov glands, which are not present in worker alarm pheromone (Lensky et al. 1991). Other studies highlighted that topical treatment of worker bees with extracts of queen Koschevnikov glands induced aggressive reaction by other workers, the so-called “balling behavior,” which is usually generated by a high-concentration QMP treatment (Pettis et al. 1998).

The gland starts degenerating after the queen is 1 year of age and this contributes to the loss of signal in old queens (Grandperrin and Cassier 1983).

5.1.2. Worker Pheromones

Caste-specific secretion is an important feature of the honey bee pheromonal system. Some honey bee glands are typically developed in only one of the two female castes (e.g., the tergal glands in queens or the Nasonov gland in workers); nevertheless, most glands are developed in both queens and workers, but their secretion is caste-specific, as is the case of mandibular, Dufour’s, tarsal, and Koschevnikov glands.

The glandular plasticity in honey bees is linked to two processes of caste determination: the one that results in the differences between queens and workers, and the one that results in differentiation among workers, the behavioral development referred to as temporal polyethism. The development of the glands in workers follows a temporal pattern linked to the activities connected with the gland secretions: for example, wax glands and hypopharyngeal glands (with secretions correlated to building and feeding activities) develop sooner and are more active in young bees, while the sting alarm pheromone production is low in young bees and rises as the workers become guard bees. However, gland secretion does not necessarily show a caste-specific rigidity, but it can be rather plastic and adaptive, thus supporting the changing needs of the colony (Katzav-Gozansky et al. 2002).

5.1.2.1. Regulation of Worker Reproduction: The Mandibular Gland Pheromones

Honey bee mandibular glands represent a clear model of caste-specific secretion. Queens and workers produce a caste-related blend of functionalized 8- and 10-carbon fatty acids, which match the queen’s reproductive and the worker’s non-reproductive roles in the colony (Plettner et al. 1996). While the queen mandibular glands produce mainly 9-ODA, 9-HDA, HOB, and HVA, in worker mandibular glands the prevailing components are 10-hydroxy-2(E)decenoic acid (10-HDA), 10-hydroxydecanoic acid (10-HDAA), and their respective diacids. Both castes produce the other caste’s aliphatic compounds in small quantities: queens have some 10-HDA and 10-HDAA and workers have a trace of 9-HDA (Plettner et al. 1995, 1997). Occasionally, traces of 9-ODA can be found in the glands of queenless workers, more frequently in African than European subspecies (Crewe and Velthius 1980; Plettner et al. 1993).

In almost all honey bee subspecies, removal of the queen leads to the development of a certain numbers of egg-laying workers that are then able to suppress ovary development in the other workers by means of pheromones, just as the queen does; for this reason these egg-laying workers are also called false queens (Sakagami 1958; Velthuis et al. 1990) or pseudoqueens (Moritz et al. 2000). Thus, if necessary, worker mandibular glands are able to produce a set of chemicals very similar to those of queen glands and with a comparable action. In queenright colonies this secretion is suppressed by the presence of the queen by means of the complex pheromonal secretion formerly illustrated, among which the QMP plays the major role. Conversely, in queenless colonies, where the queen inhibition fails, the secretion of worker mandibular pheromone plays an important role in the regulation of reproductive dominance among workers. This phenomenon is particularly evident in A. m. capensis workers, in which the transformation from infertile worker to false queen is rapid and recognizable by the formation of a retinue of worker bees surrounding the false queen (Crewe and Velthuis 1980). Furthermore, workers of this subspecies show intermediate characteristics between queens and workers—they present a functional spermatheca and are able to produce female offspring by thelytokous parthenogenesis (Onions 1912). For these reasons the role of worker mandibular pheromones in the regulation of worker reproduction has been extensively studied in A. m. capensis, where for example, Simon et al. (2001) found that while the mandibular gland secretions of newly emerged workers are mainly composed of 10-HDA and 10-HDAA, the secretion of 4-day-old queenless workers is dominated by the queen substance 9-ODA. Tan et al. (2012), in a comparative study of A. mellifera and A. cerana, observed that 9-ODA, HOB, HVA, 9-HDA, 10-HDA, and 10-HDAA levels are higher in mandibular glands of egg-laying workers than in non-egg-laying ones.

Other studies demonstrated a direct correlation between worker ovary development and the amount of pheromone produced by worker mandibular (and occasionally tergal) glands (Velthuis et al. 1990). In A. m. capensis Moritz et al. (2000) observed that the amount of pheromone produced by workers represents the main selection criterion for pseudoqueens: the ones producing higher amounts of pheromones have better chances of becoming pseudoqueens since they inhibit the production of pheromones in the other workers. Studies involving the subspecies A. m. ligustica also showed that the development of egg-laying workers appears to be mediated by mandibular pheromone production; old bees whose mandibular glands were removed were significantly less inhibitory towards young bees compared to unoperated bees (Huang et al. 1998). These results are consistent with the hypothesis that worker mandibular glands contain an inhibitor of behavioral development acting similarly to QMP (Robinson and Huang 1998).

Other authors found that not only the secretion of the mandibular gland is involved in worker reproductive development, but also that of Dufour’s gland, since both glands show higher activity in egg-laying workers (Katzav-Gozansky et al. 2000). As for queens, it is likely that the Dufour’s gland secretion acts as a fertility signal, since it has a very strict correlation with ovarian development (Katzav-Gozansky et al. 2004, 2006), while the mandibular gland secretion is involved in the establishment of a reproductive dominance (Plettner et al. 2003). The two signals seem to act independently: observations carried out in queenless groups established that workers that show a precocious aggressive behavior but undeveloped ovaries have a more queenlike pheromone in mandibular glands (dominance signal), while workers with late aggressive behavior and larger oocytes have a more queenlike pheromone in the Dufour’s gland (fertility signal), but not in the mandibular ones (Malka et al. 2008).

Mandibular gland secretions also have additional specific functions in honey bee workers. When a worker becomes a forager its mandibular glands produce a very odorous compound, 2-heptanone, which acts as a releaser alarm pheromone. Its role will be discussed later, together with the other alarm pheromone signals.

5.1.2.2. Regulation of Worker Activity: Ethyl Oleate

When talking about the regulation of worker polyethism, it has been said that the queen is only an auxiliary factor in driving the onset of behavioral development, which is primarily modulated by the workers themselves (Huang and Robinson 1996). Indeed, Pankiw in 2004 observed that administration to young workers of substances extracted from the surface of foraging bees increased their foraging age, whereas extracts of young preforaging workers decreased it (Pankiw 2004a). This confirmed the role of substances produced by adult forager bees as primer pheromones.

The chemical substance that acts as an inhibiting factor delaying onset of foraging age was identified by Leoncini et al. (2004) as ethyl oleate. This substance was found in high concentrations on the body of adult forager bees. Further studies demonstrated that it is produced in the epithelium of the honey crop through the transformation of ethanol derived from fermented nectar, then it exudes to the esoskeleton where it is transmitted among workers as a low-volatile at close range or by physical contact, and diffused in the hive by evaporation (Castillo et al. 2012; Muenz et al. 2012).

The discovery of this new pheromone elucidates how workers are able to regulate their own task allocation: when a high number of foragers are present in the colony their secretion inhibits the development of young bees, which can devote themselves to nest occupations; when foragers grow old or are lost, the inhibition fails and young bees develop into new foragers.

Interestingly, ethyl oleate was also found as a component of the pheromone blend of queens and brood, and therefore, classified as a colony pheromone (Keeling and Slessor 2005; Slessor et al. 2005).

5.1.2.3. Orientation and Recruitment: The Nasonov Gland Pheromones

The Nasonov gland secretion is the most well known worker-exclusive pheromone in honey bees. The gland consists of a mass of cells located beneath the intersegmental membrane, between the sixth and seventh tergites (Figure 5.3). Each cell has a small duct that transports the secretion and opens through the cuticle (Cassier and Lensky 1994). The glandular secrete is released under the posterior part of the sixth tergite and the workers free it by flexing the tip of the abdomen downward; during the secretion the bee usually stands with the abdomen elevated and fans its wings to facilitate volatile dispersion (Free 1987). Thus it is very easy to recognize workers bees while they are secreting the Nasonov pheromone (Figure 5.3c).

The Nasonov secretion is composed of seven volatile compounds: geraniol, nerolic acid, geranic acid, (E)-citral, (Z)-citral, (E-E)-farnesol, and nerol (Pickett et al. 1980). It has a general attractive effect and is used by workers in several different situations, the principal being marking of hive entrance, swarm clustering, and marking of foraging sources.

Workers secrete and disperse the Nasonov pheromone by fanning at the hive entrance to orientate other members of the colony toward the nest. It is also released by young workers during their first orientation flights. The pheromone release is especially evident after colony disturbance and can be elicited by nest odors such as comb, honey, pollen, propolis, and QMP (Ferguson and Free 1981).

Recent studies revealed a new possible role of the Nasonov pheromone within the hive: workers selecting young larvae to rear as new queens expose their Nasonov gland to attract other workers toward the selected larval cell. In an experimental test with queenless workers, the cells where a higher amount of pheromone was released had a higher chance of developing into queen cups, confirming the involvement of the Nasonov pheromone in recruiting workers to queen rearing (Al-Kahtani and Bienefeld 2012).

5.1.2.3.1. Swarm Clustering

Together with the QMP, the secretion of workers’ Nasonov gland functions as a cohesion factor for swarm clustering. When the swarm leaves the hive it settles in a temporary location; the first bees reaching the site immediately expose their Nasonov gland to call the rest of the swarm. Here a certain number of workers, called scout bees, are in charge of finding a new suitable place to establish the nest. When a scout bee finds a potential nest site, it communicates the location to the other workers by the waggle dance, then returns to the site and starts to release the Nasonov pheromone to drive the swarm exactly to the new nest entrance (Free 1987; Seeley 2010).

The potential of Nasonov gland components in attracting clustering bees was demonstrated in behavioral assays (Free et al. 1981a,b). Among the various components, geraniol, (E)-citral, and nerolic acid were the most attractive ones. In further studies a synthetic pheromone blend was able to attract swarms to the artificial nest cavities where it was applied (Schmidt 1994, 1999).

5.1.2.3.2. Foraging Recruitment

The function of the Nasonov gland in recruiting workers toward foraging sites has been known for some time, but its precise mechanism is still debated. When a forager finds a profitable food source, it exposes its Nasonov glands to orientate nestmates and stimulates them to land on it (Free 1987). Foragers, however, were seldom observed to expose the Nasonov gland while visiting flowers, whereas this behavior was more frequently observed during water collection. In an experimental trial with artificial nectars, the release of Nasonov pheromone was stimulated only by sugar concentrations much greater than those of natural nectars and the pheromone release was limited when the colony had already abundant nectar supplies (Pflumm and Wilhelm 1982). This suggests that the Nasonov pheromone is mainly used to recruit workers toward water sources and is involved in nectar source location only when the reward is very high or the nest storages are particularly scarce. In agreement with this, Fernandez et al. (2003) in experimental tests observed that the duration of Nasonov gland exposure at the feeding place was higher when the bees exploited the highest sugar reward.

5.1.2.4. Marking and Recruiting in Foraging Behavior: Tarsal Glands and Other Pheromones

The secretion of worker tarsal glands, also known as the worker footprint pheromone, is thought to have an auxiliary role in marking the hive entrance and food sources. It is deposited on the hive entrance by landing workers and probably also on visited flowers, enhancing the attractiveness of the Nasonov pheromone (Williams et al. 1981). In this shared function, the footprint pheromone probably acts as a proximity signal, being active at short distances, while the Nasonov pheromone is a broad attractive, with its volatile compounds being effective also at higher distances (Ferguson and Free 1981).

Thom et al. (2007) found that waggle-dancing bees produce and release four cuticular hydrocarbons (two alkanes, tricosane and pentacosane, and two alkenes, Z-(9)-tricosene and Z-(9)-pentacosene) from their abdomens into the air. These compounds are produced subcutaneously and secreted on the surface of the cuticle. When they are injected into a hive, the number of foragers leaving the hive increases significantly, suggesting a pheromonal role in worker recruitment.

Another pheromone involved in foraging behavior is 2-heptanone secreted by workers’ mandibular glands. It exerts a repellent effect and therefore seems to be correlated to a repellent forage marking scent; its role is discussed in Section 5.1.2.5.2.

5.1.2.5. Defensive Behavior: Alarm Pheromones

The defensive response is one of the most well known honey bee behaviors, especially after the introduction of the African honey bee A. m. scutellata, also named “killer bee,” to Brazil and its subsequent spread through tropical and subtropical New World habitats (Breed et al. 2004).

Defensive behavior is the first out-nest task performed by workers and is thus initiated at a younger age than foraging. There are two different kinds of workers involved in the defensive behavior: the guards and the defenders. Guard bees are workers that patrol the entrance of the hive in search of bees, insects, animals, or any other object or creature that approaches the colony (Arechavaleta-Velasco and Hunt 2003). They also inspect all bees that land at the hive entrance through antennation to recognize nestmates and reject non-nestmates (Breed et al. 1992). Defenders, also called stingers, are bees that respond to a danger or a disturbance by flying out, stinging, and sometimes pursuing intruders (Breed et al. 1990).

Alarm pheromones integrate the defensive response, the signaling potential, or actual dangers to the members of the colony. They can be released by bees while extruding the sting without stinging, during stinging, and also from stings left in the victim. The receiver bees are activated to a defensive behavior in the form of dispersal, or more often, attack against the source of danger. Defensive response is an example of collective action based on recruitment and amplification processes (Millor et al. 1999).

The possibility of modulating this response largely depends on the peculiar attributes of alarm pheromones, which are represented by a multicomponent and multisource pheromonal blend. Two main groups of substances with alarm effect have been identified in honey bees: the sting apparatus alarm pheromones, which have as a main component isopentyl acetate (Blum et al. 1978; Boch et al. 1962), and the mandibular gland alarm pheromone, with its single-component 2-heptanone (Shearer and Boch 1965). Both substances elicit defensive behavior against intruders at the hive entrance. Nevertheless many studies suggest that the two substances have different functional values even if both are capable of evoking deterrent responses in a defensive context (Balderrama et al. 2002).

5.1.2.5.1. Sting Apparatus

The alarm pheromones are mainly produced in worker honey bees by the Koschevnikov gland and by the glandular areas of the sting sheaths (Figure 5.3); the secretions are quickly volatilized on the hairs of the setosa membrane at the base of the sting bulb (Cassier et al. 1994; Lensky et al. 1994).

During defensive behavior guard bees appear at the hive entrance, raising their abdomen and exposing the sting chamber to release alarm pheromones; at the same time they fan their wings, dispersing the volatile components.

Over 40 compounds (including precursor, intermediate, and final biosynthetic products) have been identified from extracts of the worker sting apparatus, among which about 15 components stimulate one or more alarm behaviors (flying from the nest to locate the source of disturbance, pursuing, biting, and stinging) (Pankiw 2004b; Wager and Breed 2000).

Isopentyl acetate (IPA, or isoamyl acetate) is the principal active component of the alarm pheromone blend and is responsible for the majority of sting-releasing activity. Its main functional value seems related to alerting and eliciting defensive responses at the hive entrance.

It is absent in queens and young workers and increases as a worker bee ages, reaching its highest level when the worker is about 2–3 weeks old, just at the time she begins to perform guarding tasks. The amount then decreases as she becomes a forager (Allan et al. 1987; Boch and Shearer 1966).

Tests with IPA show that it is effective in alerting bees and inducing them to sting, but not as effective as the complete alarm pheromone blend (Boch et al. 1962; Free and Simpson 1968). Hence, the sting apparatus appears to be a multicomponent gland in which at least some component is specialized for a different function or can be more usefully used in one context than another. The most effective components in alerting bees are IPA and 2-nonanol (Collins and Blum 1982), while many other components stimulate attack and stinging (Free et al. 1983). In laboratory tests, honey bee workers stimulated with an electric shock increased their threshold of responsiveness after the application of IPA (Nuñez et al. 1998).

Pickett et al. (1982) identified a less volatile component, (Z)-11-eicosenol, as another effective alarm pheromone component for inducing stinging behavior. (Z)-11-eicosenol prolongs the activity of the more volatile IPA, presumably by slowing down its evaporation; as a consequence the blend of IPA and (Z)-11-eicosenol is active for a longer time than IPA alone.

There is evidence that honey bee races which differ for the intensity of defensive behavior can show differences in the amount and composition of alarm pheromones. Collins et al. (1989) found that Africanized honey bees have higher levels for nine of 12 alarm pheromone components compared to European honey bees and twice as much IPA; they also have a much more intense defense reaction when artificial pheromones are released at the hive entrance, indicating a lower threshold of pheromone response (Collins et al. 1982). Hunt et al. (2003) analyzed the alarm pheromone components from colonies of Africanized honey bees and they found a specific unsaturated derivative of IPA (3-methyl-2-buten-1-yl acetate, 3M2BA) that was able to recruit worker bees as efficiently as IPA; the two substances act synergistically and a mixture of these two compounds recruited bees more efficiently than either of the compounds alone. These two characteristics of Africanized bees can partly account for their higher defensive behavior rates.

5.1.2.5.2. Mandibular Glands: 2-Heptanone

The role of 2-heptanone (2HPT) produced by worker mandibular glands in colony defense is less clear than IPA. Close to the nest there is a strong response to it by guard bees (Shearer and Boch 1965) but in general it shows a much lower ability to recruit bees and to induce stinging than IPA: small amounts of IPA are 20–70 times more efficient than equivalent amounts of 2HPT in eliciting alarm behavior at the hive entrance (Boch et al. 1970; Lensky and Cassier 1995). Furthermore it has been observed that the level of 2HPT in workers did not differ significantly between docile and aggressive colonies or between Africanized and European honey bees (Sakamoto et al. 1990a,b; Vallet et al. 1991). Finally, in laboratory tests on the aversive stinging extension reflex (SER) small amounts of IPA led to an increase of responsiveness to the electric shock, while the same response is achieved by large amounts of 2HPT, confirming that the two pheromones are capable of evoking deterrent responses in a defensive context, but at different concentrations (Balderrama et al. 2002). A recent study by Papachristoforou et al. (2012) showed a further role of 2HPT in defensive behavior: it acts as an anesthetic in small arthropods such as wax moth larva (WML) and Varroa mites, which are paralyzed after a honey bee bite (honey bees use their mandibles to bite invaders that are too small to sting).

From these results it can be deduced that while IPA is a true defensive substance, 2HPT can have another principal role in the colony. This is supported also by the observation that the amount of 2HPT in workers progressively increases with age, its level being higher in foragers than in guard bees (Boch and Shearer 1967; Vallet et al. 1991), which suggests that the main function of 2HPT could be associated with foraging.

The hypothesis of a correlation between 2HPT and foraging behavior has been examined in behavioral assays, which showed a repulsive effect of 2HPT when added to sucrose solution visited by workers and a temporary, repulsive effect on the visitation of flowers by foraging bees. Hence it seems to act as a repellent forage-marking pheromone that may aid honey bee foragers in quickly discarding recently visited flowers (Giurfa 1993; Vallet et al. 1991).

5.1.2.6. Nestmate Recognition: The Cuticular Hydrocarbons

Cuticular lipids are a complex mixture of compounds in which aliphatic long-chain hydrocarbons are generally the major component. They evolved primarily as a water-impermeable layer for protecting the insect from desiccation thanks to their chemical composition, which also gives them a resistance to high temperatures. In social insects the cuticular lipids show an unusual richness of branched hydrocarbons, which increase insect vulnerability to desiccation, because they have a considerably lower cuticular break point compared to straight-chain hydrocarbons. This apparently meaningless disadvantage can be justified by the supposition that in the course of evolution cuticular hydrocarbons (CHCs) shift their role from insect protection to communication, in particular in those insects showing a social behavior. The highly diversified composition of branched hydrocarbons, in fact, enables the creation of highly specific blends, which can serve as communication cues (Le Conte and Hefetz 2008).

Nestmate recognition is the capability of an individual belonging to a social group (namely a colony) to discriminate between nestmates (members of the same colony) and conspecific non-nestmates (members of other colonies). This function requires that nestmates present a uniform chemical pattern and tolerate individuals that present the same pattern, while non-nestmates, which have a different pattern, are recognized as invaders, eliciting an aggression response. In social insects, nestmate recognition is the base of defense behavior against parasites or conspecific invaders. It is mainly based on olfactory signals, and many studies have demonstrated that such chemical cues are contained within the lipid layer covering the insect cuticle.

The specific chemical profile that characterizes individuals of a same colony is achieved partly by genetic inheritance and partly by the environment (e.g., nest odors, diet, colony environment). In honey bees, heritable self-produced cues appear to be of minor importance in nestmate recognition; in fact guard bees are unable to discriminate between related and unrelated conspecifics if they have been living within the same hive during adulthood (Downs and Ratnieks 1999). Among the environmental factors, nest material—in particular, wax components—rather than food source and flower scent seem to be the most important source of recognition cues (Breed et al. 1998; Downs et al. 2000, 2001). Page et al. (1991) demonstrated that variability in hydrocarbons of individual workers is determined at least in part genetically, as they found the highest correlations of cuticular hydrocarbon extracts among closely related individuals.

Evidence of the importance of cuticular hydrocarbons as nestmate recognition pheromones derives from the common observation that their composition is less variable among nestmates than among individuals belonging to different colonies (Breed 1998). Moreover, young bees, which have fewer hydrocarbons in their cuticle, are accepted more readily into an unrelated colony, while the removal of the hydrocarbons from older bees improves acceptance (Breed et al. 2004b).

Among the aliphatic compounds that make up the honey bee cuticular hydrocarbons, alkenes and alkanes seem to be the most effective ones in nestmate recognition. In a conditioning proboscis extension reflex (PER) test, Chaline et al. (2005) observed that honey bees learn and discriminate alkenes better than alkanes, suggesting that the former may constitute the main compounds used as cues in the social recognition processes. Dani et al. (2005) found that honey bees treated with alkenes were attacked more intensively than bees treated with alkanes, and they conclude that the two different classes of compounds have a different effect on acceptance and this may correspond to a differential importance in the recognition signature.

Finally, exposure to the queen mandibular pheromone (QMP) significantly alters cuticular hydrocarbon patterns of worker bees; Fan et al. (2010) showed that QMP-treated nestmates are no longer recognized as nestmates by untreated bees, and vice versa.

It appears that nestmate recognition is a complex phenomenon triggered by several different cues and strictly connected with the already described process of colony cohesion and organization.

5.1.3. Drone Pheromones

Very few pheromonal signals are known in the honey bee drones and most are linked to sexual features. This reflects the minor role of males in honey bee society, almost entirely limited to the mating function.

Lensky et al. (1985) verified the role of drone mandibular gland secretions in attracting other flying drones to congregation areas. Drone mandibular glands are much smaller than those of queens and workers and their size varies according to age. The secretory activity increases from 0–3 days old to a maximum at 7 days of age, while after 9 days the glands were no longer active.

The drone tarsal gland secretion also differs chemically from the female’s, and its biological effects are still obscure (Lensky et al. 1984).

5.1.3.1. Drone Acceptance in the Colony

Similarly to workers, honey bee drones show features that determine their acceptance or refusal in the colony. This depends on whether they belong to the colony or not and on their age. According to Free (1957), in late summer young drones of 7 days of age are fed and cleaned by workers, while older, sexually mature drones (average age 23 days) are rejected and attacked. The mechanism that might be used by the workers to distinguish between drones of different ages could be the perception of chemical signals on the drone surface. Wakonigg et al. (2000) in Apis cerana observed that drone cuticular hydrocarbons have an age-related profile; although only small differences exist in cuticular composition among colonies, there is an evident trend in their profiles during drone aging.

Kirchner and Gadagkar (1994) observed that honey bee drones undergo a similar nestmate recognition process at the hive entrance as honey bee workers, suggesting the presence of an analogous recognition mechanism based on a cuticular pheromonal signal. The cuticular profile is sex-specific, since there are several compounds that are barely present or totally absent in the female but exist on the male cuticle. Compared with workers, drones of A. dorsata and A. cerana had higher proportions of branched alkanes and short-chain alkenes and lower proportions of normal alkanes and long-chain alkenes (Francis et al. 1989). This supports the hypothesis that worker bees are able to distinguish between the two sexes by chemical cues.

5.1.4. Brood Pheromones

Larvae of A. mellifera produce a complex mixture of compounds that act both as primer and releaser pheromones, regulating worker development and colony growth. This brood pheromone (BP) is a blend of 10 fatty-acid esters: methyl palmitate, methyl oleate, methyl stearate, methyl linoleate, methyl linolenate, ethyl palmitate, ethyl oleate, ethyl stearate, ethyl linoleate, and ethyl linolenate (Le Conte et al. 1990). All together these compounds form the brood primer pheromone, but each component, alone or in combination, shows one or more releaser effects on adult bees (Le Conte et al. 2001).

5.1.4.1. Regulation of Brood Development and Care

BP is secreted by larval salivary glands, which are better known in honey bee larvae for their function in the silk secretion necessary for the construction of the pupal cocoon. The epithelial cells of the larval salivary glands secrete the fatty acids into the lumen of the glands, which act as a reservoir of the ester components of BP (Le Conte et al. 2006). The production of the different pheromone constituents varies in function of caste and larval age (Le Conte et al. 1994/1995) and this modulation of the signal is functional to guarantee the proper response to the needs of any larval age and caste by receiver nurse bees. For instance, methyl linolenate, methyl linoleate, methyl oleate, and methyl palmitate, which are produced in large quantities by the larvae during the cell capping, were found to induce the worker bees to cap the cells (Le Conte et al. 1994). Methyl palmitate and ethyl oleate increase the activity of the workers’ hypopharyngeal glands, which produce proteinaceous material (royal jelly) that is fed by nurse bees to young larvae (Mohammedi et al. 1996).

During queen rearing, methyl stearate increases the acceptance of the queen cups, methyl linoleate enhances the production and administration of royal jelly, and methyl palmitate increases the weight of the queen larvae (Le Conte et al. 1995).

Several studies demonstrated that colonies treated with BP rear more brood and more adults than controls, thus regulating colony growth. The BP treatment lead to an increased brood area and an augmented number of bees; the amount of protein consumption was augmented, as well as the amount of extractable protein from hypopharyngeal glands, indicating an enriched nutritional environment (Pankiw et al. 2008). Moreover, the queen in BP-treated colonies was fed longer, was more active, and laid more eggs; the workers spent more time cleaning cells and rearing the brood, resulting in a larger brood area in BP-treated colonies compared nontreated colonies (Pankiw et al. 2004; Sagili and Pankiw 2009).

5.1.4.2. Regulation of Worker Reproduction

Beside its releaser effects on workers linked to larval development, BP components act as primer pheromones regulating, in synergy with QMP, worker ovarian development. In particular, ethyl palmitate and methyl linolenate were found to act as worker ovary development inhibitors (Mohammedi et al. 1998), probably lowering worker JH titers, since the administration of high doses of BP resulted in lower JH levels in both laboratory and field experiments (Le Conte et al. 2001). This is consistent with JH inhibitory effect on behavioral development: JH titers and rates of JH biosynthesis are low in nurse bees and high in foragers, and JH treatments cause precocious foraging (Robinson 1992a; Robinson and Vargo 1997). Moreover, BP shunts vitellogenin transport to the hypopharyngeal gland rather than to the ovaries, thus redirecting worker metabolism from reproduction to brood care (Le Conte and Hefetz 2008).

All the above described BP components are nonvolatile substances and their distribution is probably mediated by worker to worker contact. Recently a new highly volatile pheromone, E-β-ocimene, has been identified in honey bee larvae (Maisonnasse et al. 2009); it belongs to the terpene family and has an aerial transmission, being easily dispersed within the colony. Compared to BP, E-β-ocimene has a prevalent effect in the regulation of adult worker physiology and development. In particular it exerts two main effects: inhibition of worker ovaries and modulation of worker behavioral maturation (Maisonnasse et al. 2010b). It seems that BP and E-β-ocimene act in a synergistic manner to repress the activation of the workers’ ovaries; this influence also has a consequence on brood care, since reproductive workers do not work as hard as sterile workers, showing a reduction in both larval care and foraging tasks.

5.1.4.3. Regulation of Worker Behavioral Development

In addition to the effects on larval development, BP induces an increase in colony growth also through a modulation of worker behavioral development. The treatment of colonies with BP caused an increased number of pollen foragers and augmented the weight of pollen load they transport (Pankiw et al. 2004). Pollen intake is also modulated by BP by altering the ratio of pollen to nonpollen foragers. Treatment with BP significantly decreased pollen forager turnaround time in the hive, increasing the ratio of pollen to non-pollen foragers entering the colony (Pankiw 2007).

Later studies demonstrated that BP acts in a dose-dependent manner in altering the demographics of colony foraging behavior: a low amount decreases the foraging age, resulting in a higher proportion of pollen foragers compared to nurse bees; high doses slow down the development of young bees from nest to foraging duties, so the foraging age increases, resulting in a lengthened nursing phase (Le Conte et al. 2001; Sagili et al. 2011). Moreover, pollen foragers exposed to a low amount of BP return to the nest with larger pollen loads compared to those treated with a higher amount (Sagili et al. 2011). This dose-dependent action is functional to the regulation of worker tasks on the basis of brood presence and age, as will be described.

As for worker ovarian development, E-β-ocimene cooperates with BP in the regulation of worker activity; in particular E-β-ocimene induces an earlier worker development toward foraging tasks, thereby optimizing food collection. Maisonnasse et al. (2010b) tried to explain how these two pheromones act synergically in maintaining colony homeostasis through the retention of a proper nurse/forager ratio and the inhibition of worker reproduction. It is known that in presence of brood, workers initiate foraging earlier compared to broodless colonies (Amdam et al. 2009; Tsuruda and Page 2009), thus assuring adequate food collection; however, an overabundance of foragers could lead to a lack of and a decline in brood care. Conversely, too many nurses cause a decrease in food collection and storage in the colony and a subsequent decline in brood nourishment. BP and E-β-ocimene are able to control this equilibrium since young and old larvae emit different types and quantities of these two pheromones: young larvae emit principally E-β-ocimene, while BP is produced in a growing amount during larval growth, reaching the highest concentrations during the capping stage.

In this way the young larvae, which have lower nursing needs, promote foraging and pollen collection by emitting a low quantity of BP and a large amount of E-β-ocimene, whereas old larvae, which have higher nursing needs, delay foraging and promote an increased brood care by producing a high quantity of BP, which also stimulates the development of worker hypopharyngeal glands and brood care tasks like cleaning, nourishment, and cell capping (Figure 5.4). Thus, young and old larvae play opposite roles in the behavioral maturation of worker bees according to their specific needs: young larvae promote foraging and old larvae promote brood care (Maisonnasse et al. 2010b).

FIGURE 5.4. Effect of brood pheromones on the regulation of worker behavioral and sexual development.

FIGURE 5.4

Effect of brood pheromones on the regulation of worker behavioral and sexual development. Large arrows indicate effect of the pheromones on development; small black arrows indicate effect of the pheromones on activities. In the lower part of the figure: (more...)

It is clear that worker behavioral development, which leads to the typical honey bee age polyethism, is a complex and flexible process, involving more than one stimulus. The combined effect of queen signals, worker pheromones (ethyl oleate), and brood pheromones results in a plastic modulation of worker activity that is able to adapt worker response to the needs of the colony, which vary depending on colony developmental stage and environmental factors (Castillo et al. 2012).

5.1.5. Pheromone Complexity and Evolution of Sociality in Bees

The deep diversification of chemical signaling in the honey bee society is strictly linked to the progression toward an increasing social complexity that evolved during the development of eusociality. In fact the success of social insect colonies lies in the capacity of all members of the society to act concertedly and in a well-organized and context-dependent manner. This ability is mainly based on the sophisticated means of communication represented by pheromones.

The study of pheromones in social bees, and in general in the superfamily Apoidea, is a viable means to understand the evolution of sociality in these insects, thanks to the gradual development of pheromonal regulation from the more simple bee societies to the highly organized A. mellifera (Bloch and Grozinger 2011). Similarly, the development of exocrine glands is much greater in social Apoidea compared to solitary ones (Billen and Morgan 1998). Some of these glands have a role in producing building substances (like wax glands) or digestive enzymes (like salivary glands) but most of them have a function related to the social organization of the colony, as we saw in the preceding paragraphs.

For classification purposes the different pheromones were described separately here according to the producing caste and the gland source, but they must be considered as the single components of a unique and multifaceted language of chemical communication. The most clarifying example is the queen signal with its main component QMP, whose main aim is to establish the reproductive dominance over male parentage with respect to the other females. Why should such a complex signal, with such a complex blend and multiple glandular sources, have evolved?

In support of the evolutionary theory is the finding that other Apis species show fewer components in their mandibular gland extract compared to Apis mellifera. For example, the QMP of the more primitive open-nesting species A. dorsata, A. florea, and A. andreniformis contain only the three acid components, 9-ODA and +/– 9-HDA, and lack the aromatic HOB and HVA compounds. A. cerana, the other cavity-nesting species besides A. mellifera, has four of the five QMP components but lacks HVA. In addition, mandibular gland secretions from open-nesting species show less differentiation between workers and queens (Plettner et al. 1997; Winston and Slessor 1998).

According to some authors, the complex blend of the queen signal may have evolved as a result of a social struggle for reproduction, an evolutionary “arms race” in which the queen signals and the workers’ attempts to overcome this signal have coevolved, giving rise to the increasing chemical complexity (Katzav-Gozansky 2006). The capacity of egg-laying workers to mimic, at least partly, the queen signal, and the existence of workers able to overcome the queen signal and to reproduce even in queenright colonies (Hoover et al. 2005) give support to this theory. The end result is that the queen developed multiple pheromones, none of which is individually sufficient to obtain the full effect, but whose combination may act either additively or synergistically in establishing the complete reproductive dominance.

Another interesting feature of honey bee pheromones is the evident redundancy of signals, so that several chemical substances act in synergy or in cooperation in regulating one or more processes. This complementarity is responsible for a special chemical syntax that is probably functional to fine-tuning social regulation: more than one substance can regulate the same function, exerting similar effects but with a slightly different and specific target, location, or time lapse, thus assuring both a reinforcement and a modulation of the signal. An example is the regulation of worker behavioral development, mediated by the queen, workers, and two different brood signals. Again, why are four different pheromones produced by three separate sources needed to regulate this function? The queen, the forager workers, and the old brood produce three different pheromone blends—respectively, QMP, ethyl oleate, and BP—which slow down the behavioral development of workers toward foraging tasks, whereas young larvae produce E-β-ocimene, which has the opposite effect. A possible explanation for this multiple source signal is that the regulation of honey bee behavioral maturation, whose outcome strongly affects colony growth and development, and in ultimate analysis, colony survival, needs a multilevel feedback control network.

Some specific worker tasks appear to be influenced by multiple chemical signals, as is the case of foraging. This activity is regulated by worker-worker signals, which act both in an attractive (Nasonov and tarsal gland pheromones) and in a repulsive (2-heptanone) way, to guide and recruit nestmates to the most profitable food source.

Even from these few examples it is evident that the pheromonal system has evolved to fulfill the needs of efficiency and modulation typical of a complex insect society, acting according to cooperation, synergy, or complementarity schemes. The whole comprehensive mechanism of pheromone communication and its role in the regulation of sociality will be further elucidated in the next chapter, where we will describe the decoding process of the multiple pheromonal signals, the neurophysiological changes resulting from their processing, and the final effects on worker behavioral modules and colony functions.

5.2. NEUROPHYSIOLOGY OF CHEMICAL COMMUNICATION: PHEROMONE PROCESSING IN THE BEE BRAIN

The study of honey bee pheromones started in the 1960s and since then many advancements have been made in the knowledge of composition of pheromone blends, their glandular origin, and their colony target. But while we knew the effects of many of these pheromones, for a long time we could only speculate as to the neuronal mechanisms that mediate between pheromone and function. Only recently, with the development of molecular and genetic tools, some progress has been achieved in this direction. Thus, we are just starting to gain some awareness of the neurophysiological pathways of pheromone reception and processing in the bee brain, and only a few mechanisms have been entirely elucidated.

As previously reported, releaser and primer pheromones exert different effects on the receiver, the former being immediate and transitory and the latter delayed and long-termed. This difference suggests that two different mechanisms may exist by which pheromones influence the receiver: a direct effect on neural transmission for releaser pheromones against an effect on physiological processes (e.g., hormonal, metabolic, or genetic changes) for primer pheromones.

A common question regarding queen primer pheromones is their mode of action in regulating worker reproduction and behavioral development: is it by means of a controlling mechanism (queen pheromone as a suppressive agent) or a signaling one (queen pheromone as an “honest” signal) (Strauss et al. 2008)? In the “control” hypothesis the queen pheromones manipulate workers coercively by inhibiting their ovarian and behavioral development. In the signal hypothesis, also called the cooperation hypothesis, queen pheromones simply act to signal to workers the queen presence and its egg-laying potential, rather than to manipulate worker behavior and/or physiology. In the presence of a strong and healthy queen, workers refrain themselves from reproducing and prevent nestmate workers from reproducing (worker policing) in order to maximize colony fitness. When workers perceive a decline in the fecundity of the queen, they can activate their ovaries to produce their own male offspring (Keller and Nonacs 1993; Kocher and Grozinger 2011; Le Conte and Hefetz 2008; Strauss et al. 2008).

Both hypotheses make sense from an evolutionary point of view, and several authors tried to collect evidence to support one or the other theory, but without giving a definite and unquestionable response. Both theories could explain the richness and variety of queen pheromones, whose components increase with increasing level of sociality, such as both theories could support the variability of response given by different workers to the colony pheromones (Kocher and Grozinger 2011; Strauss et al. 2008). In either case, the way the pheromone is detected and processed in the brain of different receiver workers seems to play a crucial role in the regulation mechanism.

Different pheromones use different ways of transmission from the producer to the receiver. Volatile substances, such as the alarm and Nasonov pheromones produced by the workers, and the components of the QMP that attract drones for mating and workers for swarm clusters, use a dispersal mechanism; footprint pheromones, BP, and most components of QMP are transmitted primarily by contact, and the same is true for the esters produced in other queen glands (e.g., tergal and Dufour’s), which are delivered as an integral part of the queen signal together with QMP; for this reason some authors have named them “passenger pheromones” (Keeling and Slessor 2005; Slessor et al. 2005).

Whatever the way of transmission of the pheromone, the reception process starts in the receiver olfactory system.

5.2.1. Reception of the Pheromonal Signal

5.2.1.1. Olfactory Receptor Neurons

In insects, the peripheral odour detection starts in the peripheral chemosensory system with the detection of the chemical signal by olfactory receptor neurons (ORNs), which express specific olfactory receptors (ORs). ORs are seven-transmembrane domain proteins coupled to G proteins; following the binding with odorant molecules cellular transduction cascades are activated, implicating the production of cAMP, leading to depolarization and action potentials.

These receptors are located mainly in the antennae, where they are organized in olfactory sensilla of various shapes; the poreplate sensilla are the most frequent sensilla in the honey bee antennae (Figure 5.5). A poreplate sensillum is formed by an oval-shaped thin cuticular plate with numerous minute pores and is innervated by 5 to 35 ORNs with their corresponding ORs. Each poreplate contains the whole range of ORs and thus represents a whole miniature system (Brockmann and Brueckner 1995; Sandoz 2011).

FIGURE 5.5. Schematic representation of the reception pathway for general odors and social pheromones in the worker honey bee (left) and for sexual pheromones in the honey bee drone (right) from the antenna poreplate sensilla to the antennal lobe, and the following transmission of the signal in the central nervous system.

FIGURE 5.5

Schematic representation of the reception pathway for general odors and social pheromones in the worker honey bee (left) and for sexual pheromones in the honey bee drone (right) from the antenna poreplate sensilla to the antennal lobe, and the following (more...)

Odorant molecules reach the dendrites of ORNs by diffusing through an extracellular fluid, called sensillum lymph, filling the sensillum cavity. In this fluid, odorant binding proteins (OBPs) transport the odorants to the ORNs (Sandoz 2011). While OBPs bind general odorants, a specific class of OBPs, the PBPs (pheromone binding proteins) are specialized in binding sexual pheromones and are present mainly in male insect sensilla (Laughlin et al. 2008; Leal 2005). OBPs and PBPs play an essential role in the detection of general odors and pheromonal molecules and in their transduction, passing the molecules to the sensory neuron membrane protein, which then delivers it to the olfactory receptor (Pesenti et al. 2008, 2009). Another class of soluble chemosensory proteins (CSPs), which shares no sequence homology with either PBPs or general OBPs, has been described in honey bees (Danty et al. 1998). However, in honey bees only 21 genes coding for OPBs and six coding for CSPs have been found in the genome, so that the relative importance of these molecules in the process of odor perception is still unclear (Forêt and Maleska 2006). The results of a recent study using a proteomic approach show that 12 of the 21 OBPs and 2 of the 6 CSPs predicted in the honey bee genome are present in the foragers’ antennae (Dani et al. 2010) and some OBPs are found to be more highly expressed in the mandibular glands of different honey bee castes, suggesting their involvement also in solubilization and release of semiochemicals (Iovinella et al. 2011). Three main subclasses of OBPs are defined in honey bees on the basis of antennal specific proteins (ASPs), namely ASP1, ASP2, and ASP3 (Danty et al. 1997, 1998). ASP1 is thought to be associated with QMP because of its higher abundance in drone sensilla and the ability to bind 9-ODA and 9-HDA, the most active components of the queen pheromone blend, while ASP2 and ASP3 bind general odorants (Danty et al. 1999). One of the CSPs, called ASP3c, specifically binds brood pheromone components and not general odorants or other pheromones (Briand et al. 2002).

The wide range of pheromones described in honey bees, together with the great number of environmental odors they encounter, suggests a highly developed olfactory system that must be able to discriminate a large number of volatile substances. Indeed, the sequencing of the honey bee genome allowed the identification of an exceptionally high number of OR types (160–170), compared to the already known ORs of Drosophila melanogaster (62 ORs) and Anopheles gambiae (79 ORs) (Robertson and Wanner 2006). This high number is evidently linked to the extraordinary olfactory abilities of honey bees, whose social life requires the perception of several pheromone blends as well as kin recognition signals and numerous floral odors.

It is presumable that different ORs are differentially expressed according to caste and function; indeed, among the identified antennal ORs, the AmOR11, which is upregulated in drones, was recently demonstrated in male antennae to specifically detect 9-ODA and to respond to all the main QMP components (Wanner et al. 2007). On the contrary, a number of other receptors (OR63, OR81, OR109, OR150, OR151, OR152) are more highly expressed in worker bees than in drones and are probably linked to floral odorant reception, being differentially expressed in bees which live in different environments and thus experience diverse floral scents (Reinhard and Claudianos 2013).

5.2.1.2. Antennal Lobes and the Glomeruli

The ORNs project their axons to a specific area of the deutocerebrum called antennal lobe, which is organized in densely packed nervous structures termed glomeruli (Figure 5.5). The axons of several ORNs converge to the glomeruli through four sensory tracts (T1–T4), which define four subpopulations of glomeruli, two containing about 70 glomeruli each (T1 and T3) and two with seven glomeruli each (T2 and T4). The ORNs of an individual poreplate project to all four glomerular subpopulations and are therefore distributed across the whole antennal lobes (Brockmann and Brueckner 1995; Flanagan and Mercer 1989; Kelber et al. 2006).

The arrangement and number of glomeruli are largely species-specific and vary from about 32 in the mosquito Aedes aegypti to more than 1000 in locusts and social wasps. In honey bees the workers possess 166 glomeruli and the drones 103. The latter also have four large glomerular complexes exclusively committed to processing sexual pheromones, probably with a functional specialization for a specific pheromone substance in each of the four complexes (Arnold et al. 1985).

It has to be noted that the number of glomeruli in the honey bee antennal lobe is almost equal to the number of ORN types expressing a given OR in the antennae, supporting the hypothesis of a linear relationship one-receptor/one-neuron/one-glomerulus.

Within the glomeruli the ORN axons synapse with two other kinds of neurons: the local neurones and the projection neurones (Figure 5.5). The former are mainly GABAergic neurons with an inhibitory output, while the latter are cholinergic neurons that show either excitatory or inhibitory responses to odors. The local neurons can be classified into two main types: the homogeneous local neurons, which innervate most if not all glomeruli in a uniform manner, and the heterogeneous local neurons, which innervate only a small subset of glomeruli with one dominant glomerulus that is densely innervated and a few others with very sparse processes (Flanagan and Mercer 1989; Sandoz 2011). The function of local interneurons is to interconnect the glomeruli and modulate the signal coming from ORs.

Projection neurons leave the antennal lobe via a variable number of pathways called antennocerebral tracts, connecting it with different areas of the protocerebrum, mostly the calyces of the mushroom bodies and the lateral protocerebrum (Hansson and Anton 2000; Kay and Stopfer 2006). Projection neurons can also be classified into two types: uniglomerular projection neurons branch in a single glomerulus within the antennal lobe and project to the mushroom body or the lateral horn through two major antennocerebral tracts, while multiglomerular projection neurons branch in most glomeruli and their axons follow three lesser antennocerebral tracts leading to other areas of the protocerebrum surrounding the α-lobe of the mushroom body or extending toward the lateral horn (Sandoz 2011, 2013).

5.2.1.3. Pheromone Processing in the Glomeruli

Within the glomeruli the olfactory signal undergoes an important integration and encoding before being transmitted to the higher centers. Glomeruli are the anatomical and functional units of the antennal lobes and constitute sites of synaptic interaction between different neuron types. The activity patterns of antennal lobes in response to odors was studied in the honey bee by optical imaging techniques (Galizia et al. 1997, 1998). Axons of ORNs expressing the same odorant receptor or with similar odor specificities converge onto the same glomerulus. Considering that a single type of molecule interacts with a number of different ORNs, which activate a similar number of glomeruli, an odor blend is represented by the activation of a variable number of glomeruli, resulting in a spatial representation of the odor within the antennal lobe (Galizia et al. 1999; Joerges et al. 1997; Sachse and Galizia 2003; Sachse et al. 1999). This representation is variable in time and depends on the olfactory experience; therefore, odorants are represented in the antennal lobe as changing spatiotemporal patterns of glomerular activity (Sandoz et al. 2003). The early olfactory learning during young adulthood enhances glomerular activity and modifies the spatiotemporal response patterns; these changes affect neural activity until the time when bees initiate foraging activities (Arenas et al. 2009; Galizia and Vetter 2005).

Social (nonsexual) pheromones, like citral and geraniol (components of the Nasonov gland), IPA (the major component of the alarm pheromone associated with the sting apparatus), and the worker mandibular gland pheromone 2-heptanone, are coded in the antennal lobe as “general” odors since they elicit activity in the same brain region as environmental odors (Galizia et al. 1999; Joerges et al. 1997; Sachse et al. 1999). IPA elicits strong responses in several glomeruli that also exhibit strong responses to orange, clove oil, limonene, and several plant extracts (Galizia and Menzel 2001). Nevertheless, Sandoz et al. (2001) found that IPA and 2-heptanone, which share an alarm role but have a different chemical structure and source, induce a reciprocal generalization in olfactory conditioning tests, suggesting that a similarity in the neural representation of odor could rely not only on the chemical structure but also on their functional value (Sandoz et al. 2001).

Wang et al. (2008) investigated the neural activity elicited by eight components of the sting pheromone, compared with the whole bee sting apparatus, at the level of the antennal lobes of honey bee workers. They found that the sting preparation evokes a clearly distinct glomerular pattern compared to those of individual pheromone components (e.g., IPA-activated glomeruli in the medial part of the antennal lobes), whereas the stings activated the lateral dorsal part. It seems that the sting apparatus pheromone is processed in a similar way to general odors, since the main determinant of glomerular activation is its chemical structure rather than its pheromonal value. However, in contrast to the elemental strategy used for processing nonpheromonal mixtures, where the neural representation of mixtures of two to four nonpheromonal odors could be linearly predicted based on the neural representation of each component (Deisig et al. 2006), pheromonal blends do not follow such a linear representation, revealing more complex strategies for the processing of pheromonal mixtures in the honey bee antennal lobe (Wang et al. 2008).

5.2.1.4. Sexual Communication: Drone Reception of QMP in Macroglomerular Complexes

Male insects, including honey bee drones, have a specialized olfactory subsystem to detect female sexual pheromones even at long distances. This subsystem is characterized by a large number of ORs and ORNs sensitive to the components of the female pheromones. Their axons converge to hypertrophied glomerular subunits called macroglomerular complexes that are located in the antennal lobes. In honey bees the sexual dimorphism of the reception system is evident (Figure 5.5); compared with worker bees, drones have larger antennae, with a flagellum surface twice as large as that of the workers, and about seven times as many poreplate sensilla (around 18,000 versus 2700) and ORNs (around 340,000 versus 65,000) (Esslen and Kaissling 1976). In addition, the female antennal lobe is composed of isomorphic glomeruli (about 160 in workers and 150 in queens), whereas in drones there is a reduced number of these “ordinary” isomorphic glomeruli (about 100) but there are four voluminous macroglomeruli (Arnold et al. 1985).

The sexual dimorphism of the reception system corresponds to different neuronal strategies to detect and respond to the pheromone signals. Electroantennographic studies showed that while worker antennae have a very similar response to the various QMP components, suggesting that there is no antennal specialization with regard to the number of sensory neurons, in contrast, drone antennae showed marked responses to 9-ODA and to synthetic QMP compared to the other QMP components. This high antennal response is characteristic of sexual pheromones that elicit a long-distance reaction and is attributed to a much higher number of sensory neurons in the male antennae (Brockmann et al. 1998).

These results confirm that worker antennae have a kind of generalized antennal tuning with no significant differences in the number of sensory neurons for individual mandibular pheromone components, while drone antennae are specialized in the perception of one component of the mandibular pheromone, 9-ODA. This scenario is in accordance with the above described differential morphology in the olfactory system between workers and drones, and confirmed by the finding that drones of the more primitive honey bee species, Apis florea, have only 1200 poreplate sensilla per antenna and only two macroglomeruli in their antennal lobes, corresponding to a minor complexity of the sex pheromone mixture in this species compared to A. mellifera (Brockmann and Bruchner 2001).

Wanner et al. (2007) identified four candidate sex pheromone ORs from the honey bee genome based on their higher expression in drone antennae compared to worker antennae. This number coincides with the number of macroglomeruli in the drone antennal lobe, but only one of them, the already cited AmOr11, specifically responds to 9-ODA, while the other three could not be linked to any queen pheromone component.

Further analysis of drone antennal lobes led to the discovery that the ventral part carries only ordinary glomeruli while the dorsal part shows two of the four macroglomeruli, one located dorsomedially (MG1) and the other on the dorso-lateral side (MG2). Optical imaging of the antennal lobe showed that floral odors and blend mixtures induced focal responses on the ventromedial side of the antennal lobe, a region rich in ordinary glomeruli. In contrast MG2 is clearly and specifically devoted to the reception of the QMP main component 9-ODA, which does not induce signals in regions other than MG2. Among the other QMP components, HOB and HVA induced activity mostly in two ordinary glomeruli in the center of the frontal region, which showed responses also to floral odorants 1-hexanol, limonene, clove oil, and orange oil blends, while 9-HDA and 10-HDA induced only very low and diffuse signals in ordinary glomeruli that could not be measured (Sandoz 2006, 2007). The fact that HVA and HOB are detected in drones by the general olfactory system and not by the pheromonal subsystem can be explained by their different pheromonal role: in fact they are produced mainly by mated queens and not (or very little) by virgin queens, suggesting that they are used for the induction of workers’ retinue behavior and not for drone attraction by virgin queens (Plettner et al. 1997). The role of 9-HDA and 10-HDA as sex attractants remains unclear.

The different organization of the olfactory system between workers and drones reflects their diverse role in the honey bee society: drones exhibit a clear olfactory specialization for the sexual pheromone 9-ODA consistent with their exclusive reproductive role in the hive, while workers show a broader range and less specific response for both pheromonal and nonpheromonal odors consistent with the use of these different signals in different behavioral contexts (Sandoz et al. 2007).

5.2.1.5. Pheromone Processing in Higher Centers

Processed olfactory information leaves the antennal lobe by the projection neurons, towards higher-order brain centers, especially the mushroom bodies and the lateral horn (Figure 5.5).

Olfactory inputs project to a specific area of the mushroom bodies, the Kenyon cells, which form two cup-shaped regions called calyces in each brain hemisphere. The calyces are anatomically and functionally subdivided into the lip, the collar, and the basal ring. The lip region and the inner half of the basal ring receive olfactory input, whereas the collar and outer half of the basal ring receive visual input. The axons of Kenyon cells project in bundles into the midbrain, forming the peduncle and the vertical and horizontal lobes, also called α and β lobes (Strausfeld 2002).

The mushroom bodies receive not only olfactory and visual signals, but also mechanosensory and gustatory inputs. They play an important role in the process of associative learning of olfactory stimuli but also act as a multisensory integration center with a feedback and modulatory function (Mercer and Erber 1983). They are also involved in higher nervous functions, such as learning, memory, and cognitive processes.

In contrast, the processing of olfactory stimuli in the lateral horn are still mostly unknown, including the topography of neurons leaving the lateral horn and the descending pathways involved in behavioral output. In Drosophila this region is divided in two main subregions that separately process pheromones and fruit odors (Jefferis et al. 2007); since the honey bee’s lateral horn shows a specific compartmentalization with at least four subcompartments, an organization similar to that of Drosophila could exist in the honey bee, with a specific pheromone processing region in the lateral horn.

Given that no dedicated glomeruli have been found in workers for the processing of pheromones, Sandoz et al. (2007) hypothesized that specific recognition of pheromones, especially the social ones, may take place at higher processing levels downstream from the antennal lobes. It is conceivable that particular Kenyon cells could recognize specific combinations of activated projection neurons, which would indicate that the detected stimulus is a pheromone.

5.2.2. Processing and Modulation of the Pheromonal Signal

The reception and processing of pheromones lead to a response in the receiver that corresponds to a behavioral and physiological change. But how does this process work? The response to pheromones involves both environmental and physiological factors, since pheromones induce a behavioral plasticity in the receiver through a shift in neural response thresholds to environmental conditions.

Releaser pheromones act through a direct and unambiguous pathway in which one pheromone evokes one response in the receivers. In contrast, primer pheromones induce more deep and prolonged effects that can be modulated by the receiver to give a different behavioral response according to its physiological state. These different patterns suggest a different way of action for these two types of pheromones, but until now evidence suggests that the two pathways are partly overlapping and involve similar neuronal and physiological mechanisms.

Study of the mode of action of pheromones should first take into account that many factors affect their reception and processing. The same chemicals can be perceived and processed in a different manner according to the physiological state of the receiver, which in turn is influenced by both genetic and environmental factors correlated to the social environment and the individual developmental stage.

A well-known example is the response to QMP by workers of different ages: Pham-Delègue et al. (1993) demonstrated that there is an age-dependency and experience-dependency in the attraction effect of QMP toward workers. Furthermore, they showed that the olfactory environment experienced in the first day of adult life can strongly modify the functioning of the olfactory nervous system and thus worker behavioral responses (De Jong and Pham-Delègue 1991; Pham-Delègue et al. 1991). This was observed both for general olfactory sensitivity and for pheromonal stimuli, suggesting that age and experience induce different behavioral responses linked to the plasticity of the olfactory system at a peripheral or central level. The relationships between peripheral sensitivity, signal processing, and behavioral responses have only recently started to be elucidated.

The behavioral development from nurses to foragers is accompanied by a brain plasticity that involves in particular the antennal lobes and the mushroom bodies. This transition from life inside the hive to activities outside the hive is associated with a distinct increase in antennal lobe and mushroom body size: the volume of glomeruli changes with the shift to foraging duties, and forager bees have a larger mushroom body calyx than nurse bees of the same age (Brown et al. 2004; Farris et al. 2001; Maleszka et al. 2009). This increase is due to a growing number of neural connections, driven by the richer sensory experience of the outside life.

Another useful approach to uncover the physiological mechanism of pheromone effects exploits genetic differences in worker responses. For instance, some workers are highly responsive to QMP, while others respond poorly or not at all in laboratory bioassays (Kaminski et al. 1990; Pankiw et al. 1994, 1995). There may be genetic and physiological differences between high and low responding workers in receiving or responding to the queen pheromonal message and these differences could provide a powerful tool to dissect the neurochemical pathways of QMP effects (Winston and Slessor 1998).

Pheromones could act by modulating sensory response thresholds which affect the probability of workers performing certain behaviors, such as nursing, foraging, or defence. Besides QMP, alarm pheromones also show this modulating effect, for example on appetitive and aversive learning, which are important behaviors in forager and guard bee workers (Hunt 2007; Urlacher et al. 2010).

The different substances that are possibly involved in the neuromodulation of pheromone signals in the bee brain will be described in the following section, together with some interesting discovered cases of the pheromonal effect on specific functions.

5.2.2.1. Modulation of the Signal: The Role of Biogenic Amines and Juvenile Hormones

5.2.2.1.1. Brain Amines as Neuromodulators

In the honey bee brain several biogenic amines with potential modulatory function have been detected both in the central and peripheral nervous system. These molecules function as neurotransmitters, neuromodulators, and neurohormones, mediating a diversity of physiological and behavioral functions. In particular, dopamine (DA), serotonin (5-hydroxy-tryptamine, 5-HT) and octopamine (OA), which are all neurotransmitters and long-term brain modulators, seem to be involved in the modulation of behavior, which is functionally linked to pheromone activity (Mercer 1987; Mercer and Menzel 1982).

Biogenic amines in the honey bee brain are synthesized by a relatively small number of modulatory neurons, which often possess widespread projections. The mushroom body calyces in particular receive input from OA and DA neurons, which play an important role in associative learning (Bicker 1999).

DA neurons are present in most parts of the bee brain and in the subesophageal ganglion, representing about 0.1% of the entire neuronal population. Most are located in the mushroom bodies below the lateral calyx and in the anterior-ventral protocerebrum. DA neurons occupy large volumes of neuropil and DA fibers synapse onto the antennal lobes and the Kenyon cell bodies, suggesting a role in mediating distant rather than local neural interactions (Schaefer and Rehder 1989; Schuermann et al. 1989).

5-HT neurons are found in all areas of the brain, in particular the optic lobes, but 5-HT-immunoreactive fibers innervate the mushroom bodies outside the calyces, the antennal lobes, and almost all parts of the central body (Gauthier and Grünewald 2013). Antennal glomeruli contain 5-HT fibers restricted around the margin (Schuermann and Klemm 1984) and a large 5-HT interneuron interconnects the deutocerebral antennal and dorsal lobes with the subesophageal ganglion and descends into the ventral nerve chord (Rehder et al. 1987).

OA neurons are represented in most of the cerebral ganglion, but mainly in five brain regions: in the pars intercerebralis, mediodorsal to the antennal lobes, on both sides of the protocerebrum midline, between the lateral protocerebral lobes and the dorsal lobes, and on either side of the central body. Fine networks invade the antennal lobes, the calyces, and a small part of the α-lobes of the mushroom bodies, the protocerebrum, and all three optic ganglia (Kreissl et al. 1994). Another unpaired median cluster of OA neurons is located within the subesophageal ganglion, where the VUM neurons were identified (see Section 5.2.2.1.2).

The level of these three biogenic amines (5-HT, DA, OA) in the honey bee brain has been shown to vary during worker development, namely active foragers had significantly higher levels of amines than younger bees working in the hive. These variations are age- and task-dependent and can be correlated to the behavioral development of workers (Schulz and Robinson 1999; Taylor et al. 1992; Wagener-Hulme et al. 1999). This variability thus reflects a differential responsiveness to stimuli associated respectively with brood care or with foraging, such as optical cues (nurse bees live in the dark while foragers need light to orientate), odorant signals (flower and environmental scent), and also learning and memory, since foraging tasks demand cognitive functions for orientation, flower handling, and communication. Furthermore, high levels of DA in the honey bee brain were found to be correlated with ovarian development (Sasaki and Nagao 2001) and the dietary administration of dopamine is able to activate ovaries in queenless workers, suggesting a role of dopamine in the regulation of the reproductive status of honey bee workers (Dombroski et al. 2003).

The levels of amines can vary also independent of age: a different level of DA and 5-HT was found in the optic lobes of nectar foragers and pollen foragers, behaviors that are typically performed at similar ages (Taylor et al. 1992), and between food storers and comb builders, the former having significantly lower levels of DA (Wagener-Hulme et al. 1999). This non-age-dependent difference can be correlated to a differential development of specific brain functions correlated to the performed tasks. There is a different modulation of amine levels in the two brain regions involved in the division of labor, the optical lobes, and the mushroom bodies. In the optical lobes the amounts of DA, 5-HT and OA vary significantly with worker age, but not with task, whereas in mushroom bodies they vary significantly with worker behavior, but not with age (Schulz and Robinson 1999).

Among the three amines, OA is the one that exhibits the most robust association with behavior: foragers had significantly higher brain levels of OA compared to bees performing in-hive tasks, such as nursing or food storing, independent of age (Schulz and Robinson 1999; Wagener-Hulme et al. 1999). The strong correlation between OA concentration in the antennal lobes and worker task suggests that it plays a causal role in the regulation of honey bee behavioral development. In particular, its increase in the antennal lobes seems to be involved in the release and maintenance of the foraging state since the administration of OA to workers at the foraging age results in an earlier onset of foraging, but when administered to younger workers it produces no effects (Schulz and Robinson 2001; Schulz et al. 2002a).

The influence of OA on foraging behavior probably acts through the regulation of response to foraging-related stimuli that involve learning and memory. This is supported both by anatomical and experimental findings: OA fibers were found in all neuropils that contain pathways for proboscis extension learning (Kreissl et al. 1994); OA administration enhances worker responsiveness to unconditioned olfactory stimuli, probably producing a central excitatory state in which the effectiveness of sensory stimuli is improved (Mercer and Menzel 1982); furthermore, while both DA and 5-HT injected into the bee brain reduce the response to a conditioned olfactory stimulus, OA-treated bees do not have a reduced response. The application of DA in the mushroom body causes a reduction of potentials after antennal stimulation that can account for the reduced response (Mercer and Erber 1983). Further studies confirmed the role of OA in appetitive olfactory learning in bees: injections of this amine in the honey bee brain provide a substitute for sucrose reward and induce olfactory learning (Hammer and Menzel 1998); last, blocking OA receptors disrupts olfactory conditioning (Farooqui et al. 2003). Recent research has examined in depth the role of DA neurons in aversive learning and of OA neurons in appetitive learning (see Sections 5.2.2.3.1 and 5.2.2.3.2).

5.2.2.1.2. Brain Amines and Pheromones

It is known that queen pheromones act as typical tranquillizer signals, suppressing perception and stabilizing emotional agitation especially of young worker bees (Lipinski 2006). For instance, workers in queenless colonies tend to be agitated, nervous, and aggressive; it seems that queen pheromones act on workers as a sort of social peacemaker. This effect is achieved through different physiological and hormonal mechanisms. In queenright colonies young workers have significantly lower levels of all three main biogenic amines and JH titers compared to queenless colonies: the calming effect is probably exerted by lowering the level of neurotransmitters and by decreasing the excitation of corpora allata, which results in a reduced arousal to external stimuli. A similar calming effect is exerted by brood pheromones and by mandibular pheromones of older workers (Lipinski 2006).

To understand the role of brain amines in the modulation of pheromonal signals, the relationship between their level in the worker brain and the worker response to pheromones was investigated in several studies. For example, Harris and Woodring (1999) found that in honey bees the ingestion of 5-hydroxytryptophan, a precursor of 5-HT, causes a reduction of the worker response to IPA, measured as buzzing response. On the contrary, the ingestion of L-DOPA, precursor of DA, has no effect on the buzzing response stimulated by IPA, suggesting that response to alarm pheromone in honey bees is regulated only by 5-HT metabolism, while it is known that DA and 5-HT are both involved in the neuromodulation of aggressive behavior in many vertebrates and invertebrates (Hunt 2007).

OA has been shown to be quite strictly involved in the response to pheromones linked to behavioral development, which we know to be regulated by the demographic composition of the colony and by the presence of brood, through worker and brood pheromones. Barron et al. (2002) showed that OA is able to enhance worker responsiveness to brood pheromones and to decrease responsiveness to social inhibition exerted by adult bees. OA thus acts as a modulator of pheromonal communication by regulating the response thresholds to worker and brood pheromones. However, the modulation of brood pheromone response is selective for the foraging stimuli, since other functions regulated by this pheromone are not enhanced by OA, like capping behavior (Barron and Robinson 2005). Furthermore, OA does not enhance the response to other pheromonal signals, like retinue response to QMP. The specific mechanism by which OA achieves these results is not yet clear; it may act by modulating ORNs in the antennal lobes or by modulating the neuronal circuits involved in the processing of the olfactory stimulus within the mushroom bodies (Schulz et al. 2002a). Neurons of the octopaminergic VUM family may be involved in this modulating function: the VUM mx1 neuron projects from the subesophageal ganglion, where it gets gustatory input from sucrose receptors, to the brain, meeting the olfactory pathway in three areas: the antennal lobes, the mushroom bodies calyces, and the lateral horn; thus it may act by combining olfactory and gustatory stimuli with higher functions (Hammer 1993; Schröter et al. 2006).

5.2.2.1.3. Juvenile Hormone and Pheromones

Similar to brain amines, the level of JH is functionally correlated to worker behavioral development: JH levels are higher in foragers compared to nurses, and treatment with JH or JH analogues results in precocious foraging (Huang et al. 1991; Robinson 1987a). It has been demonstrated that QMP is able to reduce the titer of JH in workers (Kaatz et al. 1992; Pankiw et al. 1998), which results in the lower level of JH in nurse bees, which are in strict contact with the queen and thus with QMP, compared to foragers.

There is a strict relation between the level of OA in the honey bee brain and the level of JH in the hemolymph: OA stimulates production of JH in vitro (Kaatz et al. 1994) and treatment with the JH analog methoprene results in increased forager-like levels of OA in the antennal lobes of preforager workers (Schulz et al. 2002b). The regulation of foraging behavior probably passes through an increase in OA levels in the brain, since allatectomized bees (no JH production) can still initiate foraging after an OA treatment. The timing of OA and JH presence is consistent with the hypothesis that JH acts earlier in the process of forager development as a trigger factor, while OA acts later but more rapidly as a releaser factor of foraging behavior (Schulz et al. 2002b). These findings suggest that the variability in JH and OA levels between workers of different age and task are a key factor in modulating the worker behavioral response to pheromones, but it is not fully established whether JH and OA act through the same or different neural pathways.

The hypothesis that JH influences age-dependent olfaction was tested by examining the effect of the JH analog methoprene on alarm pheromone perception (Robinson 1987b). Worker sensitivity to alarm pheromone increases with age (Collins 1980) and with increasing group size (Southwick and Moritz 1985), indicating a strong influence of the social context on pheromone processing. Methoprene strengthens the behavioral response to alarm pheromone at every age, but is strongest between 5 and 8 days of age. Contrary to behavioral assays, the electroantennographic response to alarm pheromone did not increase in workers after day 5 and was not affected by methoprene: this shows that the honey bee peripheral olfactory system reaches full maturity 4 days after adult emergence and suggests that hormonal modulating effects on pheromone perception occur in the central nervous system (Masson and Arnold 1984; Robinson 1987b).

5.2.2.2. Direct Modulation of Worker Behavior: HVA Mimic of Dopamine

Another interesting cue in the study of pheromone processing in the bee brain came from the observation that one of the components of QMP, HVA, has a similar structure to DA, one of the biogenic amines that plays a role in honey bee behavioral regulation (Beggs et al. 2007). The presence of this compound within the pheromonal blend suggested that exposure to HVA might affect DA function, modulating dopaminergic neural pathways.

Three DA receptor genes Amdop1, Amdop2, and Amdop3 were identified in the honey bee mushroom bodies; the receptor density, their gene transcript, and levels of gene expression have been found to change during the lifetime of the adult worker bee (Humphries et al. 2003; Kokay et al. 1999; Kurshan et al. 2003; Mustard et al. 2003). Beggs and Mercer (2009) demonstrated that HVA selectively activates the D2-like DA receptor Amdop3.

The application of QMP to worker honey bees alters DA receptor gene expression, mainly lowering Amdop1 transcript levels; consistently, the DA-evoked response, measured as intracellular cAMP level, is lower in mushroom bodies of workers exposed to QMP or HVA (Beggs et al. 2007). This finding is in agreement with the hypothesis that HVA plays a direct role on the modulation of DA levels in the brain. Further confirmation came from an experiment in which workers exposed to a mated queen (which produces higher levels of HVA) showed significantly lower brain DA levels than workers of the same age exposed to a virgin queen (low or absent HVA production); HOB, the other QMP component produced by mated and virgin queens, showed no effect on DA levels of worker brain. Finally, activity levels in bees treated with QMP are reduced, but this effect can be reversed by a treatment with L-dopa, a precursor of DA (Beggs et al. 2007). Taken together, all these results confirm that HVA alone is able to mimic the effects of QMP on DA levels in the honey bee brain and that DA pathways are not affected by other components of the QMP blend.

Another possible role of HVA in the QMP blend focuses on the inhibition of ovarian reproduction in workers: since the treatment of queenless workers with dopamine enhances ovarian development in workers (Dombroski et al. 2003), HVA may inhibit ovarian activation by acting agonistically on the dopamine pathway. However, a direct effect of HVA on ovarian development has not yet been confirmed.

5.2.2.3. Worker Attraction and Aversion: The Role of Pheromones on Appetitive and Defense Behavior

The appetitive learning conditioning in honey bees is a well-known experimental technique in which bees rewarded with sucrose on particular stimuli become able to respond to the same stimulus or to a similar one even without sucrose reward; the response is typical and measurable, consisting in the proboscis extension reflex (PER) (Giurfa 2007). On the contrary, the aversive learning conditioning consists in training bees to a defensive response, namely the SER, in response to potentially noxious stimuli. This is achieved through a modified protocol for the PER, in which the stimulus is not associated with a sucrose reward, but to a mild electric shock (Carcaud et al. 2009). The PER and SER tests were used to reveal the modulating role of some pheromones on worker appetitive and aversive learning.

5.2.2.3.1. QMP and Queen Attraction

Vergoz et al. (2007a) demonstrated that while OA mediates appetitive learning, as already shown by other authors, aversive learning in honey bees is mediated by DA; in fact it is suppressed by blocking of DA, but not OA, receptors. Since it has been demonstrated that HVA can mimic DA function, Vergoz et al. (2007a) postulated that QMP, through its component HVA, is responsible for blocking aversive learning in young workers. This hypothesis was proved in a further study (Vergoz et al. 2007b), which showed that QMP does block aversive learning in young bees while leaving appetitive learning intact. The authors postulate that QMP production by the mated queen gives her an advantage by preventing young workers, which are in close contact with her and on which she depends for feeding, form an aversion to her pheromonal bouquet.

During their studies on appetitive and aversive behavior, Vergoz et al. (2009) observed that worker responsiveness to QMP is strongly age-dependent, since 2-day-old workers are more strongly attracted to QMP than 6-day-old ones, while foragers are even repelled by QMP. They also showed that this behavior is likely to be modulated by receptors in honey bee antennae: those of 2-day-old workers strongly attracted by QMP have a higher expression level of OA receptor Amoa1 and of DA receptor Amdop3 compared to 6-day-old workers; the level of Amdop3 transcript decreases during the first week of adult life, together with the attraction towards QMP. However, this pattern is true only for bees that have been exposed to QMP since adult emergence, while young bees that have not been exposed previously to QMP are not attracted to it and show a higher expression level of the DA receptor Amdop1. Thus it seems that the queen possesses several ways to modulate worker behavior through QMP at the level of the antennal sensory neurons: by suppressing avoidance behavior (by blocking the DA signal) and by enhancing the attractiveness of her pheromone (by increasing the OA signal). This is supported by the fact that high expression levels of OA receptor gene Amoa1 and DA receptor Amdop3 in the antennae augment the attractive qualities of QMP, while suppression of DA receptor Amdop1 also enhances attraction to QMP by reducing worker sensitivity to unattractive components of the pheromone.

Similar results were found by McQuillan et al. (2012), who analyzed OA and DA antennal receptors in workers of different age and task commitment. The expression levels of the receptors Amoa1 and Amdop2 show an increase with age, being higher in older workers, while the opposite trend is shown for Amdop3 expression levels, which clearly decrease with age. Furthermore, expression levels of Amoa1 are higher in same-age pollen foragers than in nurses, consistently with the higher OA brain level in foragers (Schulz and Robinson 1999; Wagener-Hulme et al. 1999). Although the physiological significance of this variability in receptor gene expression has not been fully determined, the dynamics of gene expression in the antennae are indicative of a functional role of the periphery in the behavioral changes of honey bee workers.

5.2.2.3.2. Alarm Pheromones and Defense Behavior

Several studies have shown that alarm pheromones, besides their important role in triggering bee defense behavior, can act as modulators of the sensitivity to environmental stimuli.

Stress-induced analgesia is a mechanism that increases the threshold of responsiveness to external stimuli that elicit innate defensive responses by activating endogenous opioid pathways. In honey bees the threshold of stinging response (the main defense behavior) was artificially increased with injection of morphine, and this effect was antagonized by naloxone, demonstrating the presence of an endogenous opioid system in the honey bee and its involvement in the modulation of the stinging response (Nuñez et al. 1983). The exposure of workers to IPA causes a reduction in the responsiveness to a nociceptive stimulus (electrical stimulation) by increasing the threshold of responsiveness. This effect is antagonized by naloxone, indicating the involvement of an opioid system, as a typical opioid analgesia is induced. The social meaning of this analgesic effect is to increase the worker defensive efficiency by reducing the probability of withdrawal when facing an enemy (Nuñez et al. 1998).

In the experiments of Balderrama et al. (2002) IPA exposure led to a decrease in responsiveness to sucrose and an increase of responsiveness to a noxious stimulus (i.e., an electric shock). In a followup study the exposure to alarm pheromones or IPA showed a strong effect on appetitive learning by decreasing the learning success of exposed bees (Urlacher et al. 2010). These effects are not in contrast with the main hypothesized role of alarm pheromones, as the depression of foraging activity, through the decrease in sucrose responsiveness and the appetitive learning, allows workers to freely contrast a potential danger or enemies signaled by the release of alarm pheromones. This can strengthen worker’s commitment to their role in colony guarding and defense.

The physiological mechanism subtending this modulating effect could involve biogenic amines, which are known to regulate aversive and appetitive learning, respectively, through DA and OA pathways (Giurfa 2007; Vergoz et al. 2007a). Alternatively, the activation of an opioid-like system, which was shown to be affected by this pheromone, could lead to a general learning impairment for its analgesic effects (Nuñez et al. 1998).

5.2.2.4. Modulation of Worker Metabolism: The Effect of Pheromones on Nutrient Stores

We saw that two pheromones have a prevalent role in the regulation of worker development by slowing the worker transition from nurse to foragers: QMP and brood pheromone. In the previous sections we showed that QMP acts through a central or peripheral modulation of brain amines, which influences the subsequent behavioral and physiological pathways, including the reception level of the pheromone itself. Moreover, OA modulates worker responsiveness to brood pheromone by regulating worker response thresholds.

Another way of action of these two pheromones seems to be the regulation of worker metabolism. Nurse bees have higher lipid stores than foragers and isolated worker bees have lower lipid levels than bees kept in a colony, regardless of food availability (Toth and Robinson 2005; Toth et al. 2005); thus pheromones may partly exert their effects by regulating workers’ nutrient storage. Moreover, among worker proteins, vitellogenin (Vg), an egg yolk protein, is produced in higher levels by the fat bodies of nurse bees than forager bees (Fluri et al. 1982) and thus can serve as a molecular marker for the nurselike physiological state.

In an experiment by Fischer and Grozinger (2008), the administration of QMP on caged workers increased protein and Vg level in the fat bodies. According to the authors, this effect could be achieved by behavioral, physiological, or molecular mechanisms: QMP modulates feeding behavior, inducing treated bees to consume more food or to reduce activity; it decreases level of JH, which is known to increase metabolism (Sullivan et al. 2003), and this reduction in turn increases Vg levels and potential lipid storage; finally, it can modulate metabolic pathways through regulation of the genes involved in the insulin signaling pathway, which is associated with nutrient storage (Fischer and Grozinger 2008).

A confirmation of the role of pheromones in regulating worker metabolism comes from the researches of Smedal et al. (2009), which demonstrated that BP also regulates Vg accumulation in the fat body. Beside its role in oogenesis Vg is utilized by workers for food production and is involved in the regulation of foraging behavior and the enhancing of worker lifespan, possibly by scavenging free radicals and enhancing honey bee immunity (Amdam et al. 2003, 2004, 2005; Nelson et al. 2007; Seehuus et al. 2006). Exposure to synthetic BP blend causes an increase in the amount of Vg in the fat bodies of young bees (3–4 days old) and a decrease in older workers (23–24 days old). This is consistent with the results of Pankiw et al. (2008), who showed that brood pheromone stimulates pollen consumption, leading to an increase of protein content in hypopharyngeal glands, but also showed that workers of different ages are affected in an opposite manner by the pheromone, confirming the differential perception of pheromones according to worker age and task. In this case brood pheromone acts on young workers by enhancing their capacity to produce brood food and to store a surplus from Vg synthesis, and on older workers by inhibiting an extensive Vg storage, ensuring that more protein remains free in the hemolymph to be converted into brood food (Smedal et al. 2009).

5.2.3. From Signal to Behavior: Pheromones and Gene Expression

Pheromone processing in the bee brain leads to neurophysiological changes that result in the production of a specific behavior or to changes in sensory thresholds that result in altered behavior under different contexts. In either case, the molecular mechanisms by which pheromones are transduced in the brain to influence behavior are only beginning to be understood. A great breakthrough was made with the completion of the honey bee genome (Honey Bee Genome Sequencing Consortium 2006) and the development of a genome-wide honey bee microarray, which enabled to search for differences associated with variation in responsiveness to pheromones.

A number of authors found that worker division of labor is based, in addition to the already mentioned age and environmental factors, on genetic differences among workers performing different tasks. Thus the probability of performing a particular task within a specific age caste would be determined not only by the endogenous and exogenous environment, but also by the genotype of the worker (Calderone and Page 1988; Frumhoff and Baker 1988). These genetic differences could influence, for example, the probability of a worker to become a guard, a nectar forager, a pollen forager, or a nest-site scout (Robinson and Page 1988, 1989).

The natural variation in honey bee pheromone response, observed by several authors (Pankiw et al. 1994) may be potentially adaptive, because it creates variability in task performance that supports colony plasticity and thus productivity. Kocher et al. (2010) found variability in worker attraction to QMP and consequently in the retinue response of adult workers, which appears to be associated with brain gene expression patterns and linked to the reproductive potential in honey bees. The authors found 960 gene transcripts that are differentially expressed between high and low responder workers, and a negative correlation between individual retinue response and ovariole number, a trait strongly linked to reproductive potential (Makert et al. 2006). This indicates that workers with the highest reproductive potential (e.g., the greatest number of ovarioles) avoid the queen, while those with lower reproductive potential are attracted to her. Under queenless condition workers with high reproductive potential would activate their ovaries, whereas the ones with low reproductive potential would be in charge of rearing a new queen (Kocher et al. 2010). This would confirm the observations by Moritz et al. (2002) that in A.m. capensis, workers that are likely to become reproductively active are indeed more likely to avoid the queen.

One way that a pheromone can influence behavior is by orchestrating large-scale changes in brain gene expression. In recent years several authors demonstrated that a differential gene expression exists between workers performing different tasks (Whitfield et al. 2003, 2006) and that exposure of honey bee workers to pheromones causes changes in brain gene expression that are associated with downstream changes in behavior. Therefore, it should be possible to investigate the mode of action of pheromones by correlating the changes in gene expression and the resulting behavioral expression. The first attempt in this direction was made by Grozinger et al. (2003) with QMP.

5.2.3.1. Insights into the Pheromone-Mediated Genetic Mechanism Underlying Worker Behavioral Development

We know that QMP has a delaying effect on the transition from hive tasks to foraging in workers. Several genes have been identified as correlated to nursing or foraging conditions (Whitfield et al. 2003) and the exposure of young honey bee workers to QMP was found to activate genes associated with nursing and to repress genes associated with foragers. In the study by Grozinger et al. (2003) the gene that was more robustly and chronically regulated was found to be an ortholog of the Drosophila transcription factor krüppel homolog 1 (Kr-h1). This gene encodes for a zinc finger transcription factor that plays an important role in orchestrating development and cell differentiation. Although the different components of QMP taken individually were thought to elicit limited responses, two of them, 9-HDA and 9-ODA, were both able to produce a strong QMP-like gene activation. In particular, they were able to downregulate expression of Kr-h1, suggesting that 9-ODA and 9-HDA are the QMP components that influence the timing of the transition from hive work to foraging (Grozinger et al. 2007).

From all the reported observations about the role of pheromones in modulating worker behavior, it is interesting to investigate the functional relation between QMP, which regulates the transition from nurse to forager, and OA and JH, which have levels with strong correlation to these behavioral stages. Grozinger and Robinson (2007) studied the effects of these three factors on the modulation of the gene Kr-h1. JH analog, methoprene, or OA are unable, alone, to modulate Kr-h1 expression, demonstrating that these molecules do not have a direct influence on the gene expression. Conversely, methoprene, but not OA, significant reduces the effect of QMP on Kr-h1 brain expression in young bees, suggesting that high JH titers, typical of foragers, prevent downregulation of Kr-h1 expression by QMP in older bees (Grozinger and Robinson 2007). The authors’ interpretation is that QMP affects workers’ transition to foragers partly via JH regulation, since the pheromone is able to lower JH levels, and JH levels in turn modulate pheromone response, but other mechanisms must be involved, since a JH analog is not able to affect gene (namely Kr-h1) expression.

Together with QMP, BP is responsible for the regulation of worker behavioral development, delaying the transition of workers from nurses to foragers. Its way of action seems to be even more complex than QMP, since it has a dose- and age-dependent effect, and in addition to a primer effect on behavioral maturation, it acts as a releaser, stimulating the foraging activity of older bees that are competent to forage (Pankiw 2004c; Pankiw and Page 2001).

Alaux et al. (2009) showed that BP effect on foraging ontogeny is linked to a variation in gene expression, since BP treatment upregulates brain genes that are highly expressed in workers specialized in brood care, and downregulates genes that are highly expressed in foragers. According to its age-related effect, the exposure to BP for 5 days caused a brain gene expression profile similar to the profile of nurse bees, while this similarity to nurse bees was absent in bees exposed to BP for 15 days. In fact, although there was a significant overlap between the gene sets controlled by BP in young and old bees, many were regulated in opposite directions. For example, the gene malvolio (mvl), which is activated in precocious foragers (Ben-Shahar et al. 2004), was upregulated by BP in 15-day-old bees, but not in 5-day-old bees, suggesting that mvl represents a key component of the regulation of foraging behavior by BP. This differential effect on brain gene expression of 15-day-old bees is consistent with the role of BP as releaser pheromone, triggering foraging behavior in older bees (Alaux et al. 2009).

Comparing these results with those obtained by Grozinger et al. (2003) with QMP, which exerts a similar effect on behavioral development, it emerges that some genes are regulated by both BP and QMP, probably because of the different chemical composition of the two pheromones, which are also found to use different peripheral receptors (Robertson and Wanner 2006; Wanner et al. 2007).

5.2.3.2. Alarm Pheromone and the Expression of Immediate Early Genes

It has been demonstrated that primer pheromones exert their effects partly by causing changes in brain gene expression (Alaux et al. 2009; Grozinger et al. 2003). Releaser pheromones, which cause immediate and short-term responses, are thought to act through more direct neurophysiologic modulation systems. Today, the study of the mode of action of these two kinds of pheromones has changed this rigid distinction. For example, the exposure of honey bee colonies to IPA, originally classified as a releaser pheromone, caused a significant increase in expression of the gene c-Jun (Alaux and Robinson 2007). C-Jun belongs to the group of immediate early genes (IEGs), which are activated transiently and rapidly in response to a wide variety of stimuli. They are activated at the transcription level before any new proteins are synthesized and are known as early regulators of cell growth and differentiation signals, but are also involved in synaptic plasticity. The correlation between IPA exposure and c-Jun expression in honey bees blurs the long-standing distinction between primer and releaser pheromones and highlights the importance of brain gene expression in social regulation (Robinson et al. 2005).

5.3. CONCLUDING REMARKS

If one compares the very high number of pheromonal substances identified in the honey bee colony with the relative scarcity of uncovered physiological mechanisms subtending pheromonal effects, it clearly emerges that there is still much study required to fully understand the pathways from pheromone production to pheromonal output.

Pheromone processing starts in the peripheral sensory system where the chemical signal is transduced, and partially elaborated in the glomeruli of the antennal lobes (Figure 5.6).

FIGURE 5.6. Schematic representation of the pheromone processing pathway from its reception to its behavioral and physiological effects.

FIGURE 5.6

Schematic representation of the pheromone processing pathway from its reception to its behavioral and physiological effects. Pheromones are transduced by the antennal odorant reception neurons (ORNs) and processed in the antennal lobes, respectively, (more...)

The pheromonal signal is probably elaborated in the mushroom bodies and in the lateral horn during its transmission from the neuronal fibers leaving the antennal lobes to the central nervous system. At this level, the outcome of the signal is modulated by biogenic amines, which act as neurotransmitters and neuromodulators in several neuronal functions, and whose fibers are well represented in these two neuropils. The precise role of biogenic amines in the transmission of pheromonal signals has not been clearly elucidated, but it is quite certain that they are involved in the process of worker behavioral development, which is triggered by the queen pheromones. This is confirmed by the fact that one component of QMP, HVA, which shares a similar chemical structure with DA, is able to skip the reception step in antennal lobes and directly affect worker behavior by modulating DA neuronal pathway and DA levels in the brain (Figure 5.6).

The recent advances in genomics have strongly contributed to understanding the mechanisms that regulate pheromone communication by showing a direct correlation between pheromonal signal and expression of genes involved in worker behavior. This was shown in particular for two primer pheromones, QMP and BP, but also for a releaser one, the sting alarm pheromone, thus questioning the old distinction between primer and releaser pheromones, at least for their operating mechanism (Figure 5.6).

In 1998, Winston and Slessor, in their article “Honey bee primer pheromones and colony organization: gaps in our knowledge,” stated that “we know a considerable amount about the functions in which these pheromones are active, but the modes of action, physiological effects and precise chemical nature of specific pheromonal activities remain as subjects for future research”. Today, after 15 years of research, we can say that several of these gaps have been filled, but still our understanding of neuronal and molecular mechanisms in pheromone processing is represented by separate pieces of an extremely more complex puzzle. A complete comprehension of the mechanism of pheromone communication in honey bees needs to put all the pieces together in an organic and all-inclusive picture that is able to display the complex routes and the multiple connections of this sophisticated chemical communication system.

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