We have demonstrated that differences in the mating process are associated with differences in ovary activation and pheromone composition and production. Furthermore, workers could discriminate between reproductive states and were more attracted to the extracts of queens with higher levels of ovary activation. These results support a model in which queen pheromone composition is linked to her reproductive status and fecundity, such that queen pheromone serves as an honest signal to the workers. However, it is important to note that other studies (Moritz et al. 2002
; Hoover et al. 2003
; Kocher et al. submitted) indicate that workers modulate their responses to queen pheromone, supporting a model in which workers may be attempting to escape queen control. Thus, both models may be operating in this system, and queen-worker chemical communication in honeybees appears to be a complex process.
Previous studies have suggested that pheromone production in honeybees is linked to reproductive status, but the direct link between changes in reproductive status and behavioral consequences has not been demonstrated. For example, reproductive workers produce queen-like blends of Dufour's glands extracts (Malka et al. 2007
), dominant workers (which are more likely to ultimately become reproductive workers) produce queen-like blends of the mandibular glands (Moritz et al. 2002
; Malka et al. 2007
), and mated, laying queens produce different mandibular gland blends compared with virgin queens (Plettner et al. 1993
; Kocher et al. 2008
; Strauss et al. 2008
). Furthermore, naturally mated, laying queens produce different mandibular gland blends compared with instrumentally inseminated, laying queens (Al-Qarni et al. 2005
). We have also previously demonstrated that insemination quantity modulates mandibular and Dufour's gland composition (Richard et al. 2007
, in preparation), and queens collected during the transition from virgin to laying also have different pheromone blends (Kocher et al. 2008
). This current study provides a crucial link between ovarian status, pheromone production, and worker responses. However, it is important to note that these particular queens were analyzed only 2 days after insemination or mating (and differed at most by 2 days in age) in order to accentuate differences in ovary activation, and thus, although these differences represent a link between ovary activation and worker preference, a definitive link between variation in queen fecundity and worker preference still needs to be demonstrated. However, coupled with previous work demonstrating that there are significant differences in the pheromonal blends of naturally mated queens compared with instrumentally inseminated queens at 1 and 2 weeks after the initiation of egg laying, and that workers prefer the extracts from the naturally mated queens (Al-Qarni et al. 2005
), it appears that the observed differences in pheromone composition are stable over time and may function as an accurate indicator of queen fecundity in a natural colony. Furthermore, long-term studies comparing laying queens inseminated with low versus high volumes of semen in colonies demonstrate that these queens have significantly different longevity and that worker behavior and physiology is affected by queen insemination volume (Niño EL, Richard FJ, Tarpy DR, Grozinger CM, submitted). Studies to examine the chemical profiles of laying queens inseminated with low versus high volumes of semen are ongoing (Niño EL, Richard FJ, Tarpy DR, Grozinger CM, unpublished results).
It is apparent that the chemical composition of the mandibular gland extract is quite complex and that subtle changes in the proportion or quantity of the chemical components can be detected by worker bees. Most research on queen pheromone has focused on characterizing quantities of compounds found in QMP. QMP consists of 5 components: 9-ODA, 9-HDA ((RE
)-(−)- and (S
)-(+)-), HOB, and HVA (Slessor et al. 1988
). However, an additional 4 components have been identified that improve the retinue response (Keeling et al. 2003
), and there are several more compounds that are present but have not yet been characterized in terms of behavioral responses by workers. The levels of QMP and associated compounds are affected by mating status and age (Plettner et al. 1993
). Previous studies have sought to determine if specific compounds are associated with reproductive state and fecundity by comparing virgin queens, queens treated with CO2
(which stimulates ovary development and laying of unfertilized drone eggs), and naturally mated, egg-laying queens (Strauss et al. 2008
). In that study, there were no overall differences in the proportions of the 5 QMP compounds or 2 additional compounds (10-HDA: 10-hydroxy-2-decenoic acid and 10-HDAA: 10-hydroxydecanoic acid) between naturally mated and drone-laying queens, although there were significant differences in the levels of some individual compounds (i.e., 9-ODA levels were lower and 10-HDA levels were higher in naturally mated queens). Al-Qarni (2005) found that naturally mated queens had significantly higher levels of 9-ODA than instrumentally inseminated queens. In our studies, we found no significant differences in the relative proportions or quantities of these specific compounds, but when all of the components of the mandibular glands were considered, the samples separated into a distinct hierarchy with virgin and naturally mated queen extracts most distant and the inseminated queen extracts most similar to each other and clustering near the virgin extract chemical profiles. Similarly, in previous studies comparing differentially inseminated queens (Richard et al. 2007
) or virgin, partially mated, and laying queens (Kocher et al. 2008
), there was also little variation in the QMP components but there was significant overall variation in the proportions of the blend when the majority of compounds were considered. Our results suggest that the mandibular gland composition is quite complex, subtle changes in proportion or quantities may have significant behavioral consequences, and other components in addition to the 5 main QMP chemicals play a significant role in regulating worker behavior. Furthermore, several other pheromone-producing glands exist in queens, such as Dufour's gland (reviewed in Katzav-Gozansky et al. 2002
) and tergal glands (Wossler and Crewe 1999a
), and there are also significant differences in volatile chemical production between virgin and mated queens (Gilley et al. 2006). Thus, fully understanding how queen reproductive state alters chemical communication between queens and workers will require a complete analysis of all chemicals produced by the queen.
Although workers clearly could distinguish between these chemical extracts in our assay, it remains to be determined if this has consequences for worker behavior and physiology in a functioning colony. In this study, we monitored the responses of workers to different queen pheromone extracts using a retinue-response choice assay. The underlying assumption is that the stronger attraction of the workers for a particular pheromone extract indicates that this is a higher “quality” extract. However, the retinue response is only one of many behaviors regulated by queen pheromone; more important to colony and individual fitness are the effects of queen pheromone on inhibiting worker reproduction and inhibiting the rearing of new queens (Winston et al. 1989
; Hoover et al. 2003
). Previous studies have demonstrated that a 9-component queen pheromone blend that produces a stronger retinue response than QMP was not more effective at inhibiting ovary development in caged workers (Hoover et al. 2003
). Thus, improved retinue response may not necessarily be correlated with improved inhibition of worker reproduction. Obviously, to fully demonstrate that the pheromone extracts produced by differentially inseminated queens have different behavioral and physiological consequences, it will be necessary to demonstrate that both worker reproduction and queen rearing are differentially inhibited by live queens in full colonies. However, the retinue response serves as a useful assay to probe for differences in the biological activity of these pheromone blends.
It is also apparent from our results that instrumental insemination and natural mating have different effects on queens. The naturally mated queens had the most activated ovaries and the most divergent pheromone blend, whereas the instrumentally inseminated queens were more similar to virgins. The saline- and semen-inseminated queens were not significantly different in terms of their pheromone profiles, ovary activation, or worker responses. These results suggest that instrumental insemination is not a perfect mimic of natural mating, and that instrumentally inseminated queens may transition to the fully mated state more slowly. Indeed, previous studies have found that instrumentally inseminated queens take longer to initiate egg-laying than naturally mated queens (Kaftanoglu and Peng 1982
; Al-Qarni et al. 2003
), and have significantly different pheromonal blends after the initiation of egg-laying (Al-Qarni et al. 2005
). It is somewhat surprising that there are no strong differences between saline- and semen-inseminated queens, but previous studies have demonstrated that both insemination volume and substance (i.e., seminal proteins or sperm) can affect postmating responses in female insects (Nijhout 1984
; Ringo 1996
; Klowden 2006
; Wolfner 2007
). Our studies suggest that at least for the early postmating changes, insemination volume may be the dominant regulatory factor, or other aspects of the insemination procedure (such as anesthetization with carbon dioxide or physical manipulation of the oviducts) may cause changes which obscure any differences caused by insemination substance.
These studies and others suggest that pheromonal communication in honeybees involves modulation of both the production of the signal and response of the receiver. In this particular case, queen pheromone appears to be acting as a signal for workers to monitor queen fecundity, but it remains to be determined if variation in these pheromone blends also alters other aspects of worker behavior and physiology. However, other studies suggest that workers can modulate how they respond to the pheromone (Moritz et al. 2002
; Hoover et al. 2005
; Kocher et al., submitted), which would be more consistent with a queen-control model. Thus, pheromonal communication in honeybees and other social insects may have evolved as an intricate dialog between the queen and workers, rather following a simple signal-receiver model.