By Clarence Collison
Primer pheromones affect the physiological state of the individual resulting in longterm changes in behavior which may take days to manifest, that are likely produced by restructuring neural networks in the brain, potentially by modifying gene expression.
Pheromones are defined as chemicals that are released by one individual of a species that alter behavior or physiology of another individual of the same species. Honey bee pheromones are classified as releasers or primers. Releaser pheromones stimulate a rapid response within seconds or minutes that are short lived, a behavioral response mediated by the nervous system (Pankiw 2004a). Primer pheromones affect the physiological state of the individual resulting in long-term changes in behavior which may take days to manifest, that are likely produced by restructuring neural networks in the brain, potentially by modifying gene expression (Grozinger and Robinson 2007). Depending on adult worker response state and social context, primer pheromones may change reproductive, endocrine, and neuro-sensory systems and associated behaviors. Primer pheromones are relatively nonvolatile, acting within a short-volatile space and primarily moving through the colony by inter-individual contact (Pankiw 2004a).
The best-known and characterized releaser responses to honey bee pheromones are stinging (alarm pheromone) and orientation behaviors (Nasonov pheromone). Pheromone regulated defensive behavior is a classical mass-action response to a releaser pheromone demonstrating individual and colony level decision-making (Pankiw 2004a).
Queen mandibular pheromone (QMP) elicits multiple distinct behavioral and physiological responses in worker bees, as both a releaser and primer pheromone, and thus produces responses on vastly different time scales. Grozinger et al. (2007) demonstrated that releaser and primer responses to QMP can be uncoupled. First, treatment with the juvenile hormone analog methoprene leaves a releaser response (attraction to QMP) intact, but modulates QMP’s primer effects on sucrose responsiveness. Secondly, two components of QMP (9-ODA and 9-HDA) do not elicit a releaser response (attraction) but are as effective as QMP at modulating a primer response, down regulation of foraging-related brain gene expression. These results suggest that different responses to a single pheromone may be produced via distinct pathways.
Queen pheromones elicit a combination of releaser and primer effects. Retinue response of workers to the queen is a releaser effect of queen pheromones from the mandibular and tergal glands (Pankiw 2004a). Queen mandibular pheromone (QMP), as a primer pheromone inhibits worker behavioral maturation (Pankiw et al. 1998), increases worker fat stores (Fischer and Grozinger 2008), and alters worker brain gene expression (Grozinger et al. 2003). It also increases foraging activity (Higo et al. 1992), attracts workers to a swarm (Winston et al. 1989), and inhibits rearing of new queens (Pettis et al. 1995). Lastly, QMP inhibits worker ovary development (Hoover et al. 2003), as well as the associated production of queen-like esters in the Dufour’s gland of workers (Katzay-Gozansky et al. 2006).
Grozinger et al. (2003) demonstrated that queen mandibular pheromone (QMP) causes changes in expression levels of many genes in the brain of adult honey bees, and that these changes correlate with some of the downstream behavioral effects of the pheromone. QMP effects on gene expression were detected both in a controlled laboratory environment and in bee colonies in the field, which represent a more natural environment. QMP causes transient changes in expression of several hundred genes, but causes chronic changes in only a small subset of genes in the brain. The effects of QMP on brain gene expression changed over time.
To evaluate the hypothesis (that pheromone regulated changes in gene expression are correlated with pheromone-induced changes in behavior), Grozinger et al. (2003) focused on one function of QMP: delaying the transition from working in the hive (e.g., brood care or “nursing”) to foraging. They compared the list of QMP regulated genes with the lists of genes differentially regulated in nurse and forager brains generated in a separate study. QMP consistently activated “nursing genes” and repressed “foraging genes,” suggesting that QMP may delay behavioral maturation by regulating genes in the brain that produce these behavioral states. The fact that many QMP-regulated genes were not found to be associated with nursing or foraging behavior may reflect the fact that QMP also is involved in the regulation of several other behavioral and physiological processes besides age-related division of labor, such as inhibiting ovary development in workers and the rearing of new queens.
The influence of the queen and her pheromonal signal on comb construction has also been examined. Ledoux et al. (2001) tested four treatments with newly hived packages of bees containing: 1) a mated queen, 2) a virgin queen, 3) no queen but with a dispenser containing synthetic queen mandibular pheromone (QMP), and 4) no queen and no pheromone. After 10 days, the comb produced by each colony was removed, comb measurements made, bees from the comb-building area collected, the size of the scales on the wax mirrors of the collected bees ranked on a scale of 0-4 and the queens removed and analyzed for QMP components. Queenless workers built substantially less comb and the comb they did build had significantly larger, drone-sized cells than for the other three treatments, indicating that both cell size and the quantity of comb built are mediated through the queen, particularly QMP. The observations of wax scale size suggested that QMP influenced comb building behavior rather than wax scale production. These results support the idea that queenless honey bees can adopt a strategy of constructing drone-sized cells in order to increase reproductive fitness through male production following queen loss.
Honey bees are social insects and one of the characteristics of eusociality is cooperative brood care. Brood rearing labor is divided between individuals working in the nest tending the queen and larvae, and foragers collecting food outside the nest. To understand brood rearing division of labor, Sagili and Pankiw (2009) investigated the relationships between individuals in the nest engaged in brood care and colony growth. They examined responses of the queen, queen-worker interactions, and nursing behaviors to an increase in the brood rearing stimulus environment using brood pheromone. Colony pairs were derived from a single source and were headed by open-mated sister queens, for a total of four colony pairs. One colony of a pair was treated with 336 µg of brood pheromone, and the other a blank control. Queens in the brood pheromone treated colonies laid significantly more eggs, were fed longer, and were less idle compared to controls. Workers spent significantly more time cleaning cells in pheromone treatments. Increasing the brood rearing stimulus environment with the addition of brood pheromone significantly increased the tempo of brood rearing behaviors by bees working in the nest resulting in a significantly greater amount of brood reared.
Alaux et al. (2009) studied the effects of brood pheromone on brain gene expression. Brood pheromone (BP) caused changes in the expression of hundreds of genes in the bee brain in a manner consistent with its known effects on behavioral maturation. Brood pheromone exposure in young bees causes a delay in the transition from working in the hive to foraging, and they found that BP treatment tended to upregulate genes in the brain that are upregulated in bees specialized on brood care but downregulate genes that are upregulated in foragers. However, the effects of BP were age dependent; this pattern was reversed when older bees were tested, consistent with the stimulation of foraging by BP in older bees already competent to forage. These results support the idea that one way that pheromones influence behavior is by orchestrating large-scale changes in brain gene expression.
In contrast to primer pheromones, it is not known whether the quicker-acting releaser pheromones can also affect brain gene expression. Alaux and Robinson (2007) found that isopentyl acetate (IPA), a releaser pheromone associated with the stinger, that communicates alarm, not only provokes a quick defensive response but also influences behavior for a longer period of time and affects brain gene expression. Exposure to IPA affected behavioral responsiveness to subsequent exposures to IPA and induced the expression of the immediate early gene and transcription factor c-Jun in the antennal lobes. Their findings blur the long-standing distinction between primer and releaser pheromone and highlight the pervasiveness of environmental regulation of brain gene expression.
Queen mandibular pheromone and e-beta-ocimene (eß), emitted by young worker larvae, have both releaser and primer effects. Both QMP and eß have been shown to affect worker physiology and behavior, but it has not yet been determined if these two key pheromones have interactive effects on hypopharyngeal gland (HPG) development and ovary activation, components of worker reproductive physiology. Experimental results demonstrate that both QMP and eß significantly suppress ovary activation compared to controls but that the larval pheromone is more effective than QMP. The underlying reproductive anatomy (total ovarioles) of workers influenced HPG development and ovary activation, so that worker bees with more ovarioles were less responsive to suppression of ovary activation by QMP. These bees were more likely to develop their HPG and have activated ovaries in the presence of eß (Traynor et al. 2014).
Pankiw (2004b) demonstrated how substances extracted from the surface of foraging and young pre-foraging worker bees regulated age at onset of foraging, a developmental process. Hexane-extractable compounds washed from foraging workers increased foraging age compared with controls, whereas extracts of young pre-foraging workers decreased foraging age. This represents the first known direct demonstration of primer pheromone activity derived from adult worker bees.
Leoncini et al. (2004) reported on the identification of a substance produced by adult forager honey bees, ethyl oleate, that acts as a chemical inhibitory factor to delay age at onset of foraging. Ethyl oleate is present in highest concentrations in the bee’s crop. These results suggest that worker behavioral maturation is modulated via trophallaxis, a form of food exchange that also serves as a prominent communication channel in insect societies. Their findings provide critical validation for a model of self-organization explaining how bees are able to respond to fragmentary information with actions that are appropriate to the state of the whole colony.
Le Conte et al. (2001) reported that the blend of 10 fatty-acid esters found on the cuticles of honey bee larvae (brood pheromone), are already known to be a kairomone (a pheromone emitted by an organism which mediates interactions that benefit an individual of another species that receives it, i.e. Varroa mite), a releaser pheromone and a primer pheromone, also act as a primer pheromone in the regulation of division of labor among adult workers. Bees in colonies receiving brood pheromone initiated foraging at significantly older ages than did bees in control colonies in five out of five trials. Laboratory and additional field tests also showed that exposure to brood pheromone significantly depressed blood titers of juvenile hormone. Brood pheromone exerted more consistent effects on age at first foraging than on juvenile hormone. These results bring the number of social factors known to influence honey bee division of labor to three: worker-worker interactions, queen mandibular pheromone and brood pheromone.
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Clarence Collison is an Emeritus Professor of Entomology and Department Head Emeritus of Entomology and Plant Pathology at Mississippi State University, Mississippi State, MS.