Ovarian development and amount of esters in Dufour's gland of α- and β-workers in 3 different group-sizes.

<p>The experiments were performed in 5-day-old workers that were kept in queenless groups of 3, 5 and 10 (12 groups for each group-size). Data are presented as mean ± SE for the α-workers and β-workers in each group. Different letters denote statistical differences using two-way ANOVA test.</p

The percentage of workers that lost their Dufour's gland esters in queenless groups and colonies.

<p>Workers with less than 1% esters per total secretion were defined as workers that had lost their ester-sterility signal. The workers were 5-day-old and were kept in queenless groups of 3, 5 and 10. Workers' age in colonies was distributed normally (for more details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018238#pone.0018238-Amsalem1" target="_blank">[24]</a>). The numbers in brackets denote the numbers of workers per colony and the size of the sample (number of groups or colonies). All the workers in each group were dissected, therefore the n describes the number of Dufour glands as well. Letters above the columns denote statistical differences at p<0.05. Data are presented as mean ± SE.</p

Percentage of aggression exhibited by the α-worker towards the other females in the group.

<p>Aggression level includes cumulative aggression (attack and darting) during 5 days. Humming was excluded because of inability to determine directionality with certainty. Each group was observed for 120 minutes. Workers were kept in queenless groups of 3, 5 and 10 (12 groups for each group-size). Workers are presented in accordance with the amount of aggression each has received from the α-worker (e.g. 2′ female received the highest amount of aggression).</p

Aggression level per worker in α-workers and β-workers in 5-day-old workers.

<p>Workers were kept in queenless groups of 3, 5 and 10 (12 groups for each group-size). Each group was observed for a total of 120 minutes. Data are presented as mean ± SE for the α-workers and β-workers in each group. Different letters denote statistical differences using two-way ANOVA test.</p

Effect of Bombus impatiens queen-produced cues on worker behavior, physiology, and gene expression

This file contains all the data associated with the manuscript. We include data on Bombus impatiens worker oocyte activation, egg-laying, aggressive behavior, and expression of the candidate genes vitellogenin and kr-h1. The data set allows comparison of these parameters in queenless workers, queenright workers, and workers exposed to volatile and contact cues generated by the queen

Supplementary Figure 1 from Chemical communication is not sufficient to explain reproductive inhibition in the bumblebee <i>Bombus impatiens</i>

Reproductive division of labour is a hallmark of eusociality, but disentangling the underlying proximate mechanisms can be challenging. In bumblebees, workers isolated from the queen can activate their ovaries and lay haploid, male eggs. We investigated if volatile, contact, visual or behavioural cues produced by the queen or brood mediate reproductive dominance in <i>Bombus impatiens.</i> Exposure to queen-produced volatiles, brood-produced volatiles and direct contact with pupae did not reduce worker ovary activation: only direct contact with the queen could reduce ovary activation. We evaluated behaviour, physiology and gene expression patterns in workers that were reared in chambers with all stages of brood and a free queen, caged queen (where workers could contact the queen, but the queen was unable to initiate interactions) or no queen. Workers housed with a caged queen or no queen fully activated their ovaries, whereas ovary activation in workers housed with a free queen was completely inhibited. The caged queen marginally reduced worker aggression and expression of an aggression-associated gene relative to queenless workers. Thus, queen-initiated behavioural interactions appear necessary to establish reproductive dominance. Queen-produced chemical cues may function secondarily in a context-specific manner to augment behavioural cues, as reliable or honest signal

Supplementary Table 1. Primer sequences from Chemical communication is not sufficient to explain reproductive inhibition in the bumblebee <i>Bombus impatiens</i>

Reproductive division of labour is a hallmark of eusociality, but disentangling the underlying proximate mechanisms can be challenging. In bumblebees, workers isolated from the queen can activate their ovaries and lay haploid, male eggs. We investigated if volatile, contact, visual or behavioural cues produced by the queen or brood mediate reproductive dominance in <i>Bombus impatiens.</i> Exposure to queen-produced volatiles, brood-produced volatiles and direct contact with pupae did not reduce worker ovary activation: only direct contact with the queen could reduce ovary activation. We evaluated behaviour, physiology and gene expression patterns in workers that were reared in chambers with all stages of brood and a free queen, caged queen (where workers could contact the queen, but the queen was unable to initiate interactions) or no queen. Workers housed with a caged queen or no queen fully activated their ovaries, whereas ovary activation in workers housed with a free queen was completely inhibited. The caged queen marginally reduced worker aggression and expression of an aggression-associated gene relative to queenless workers. Thus, queen-initiated behavioural interactions appear necessary to establish reproductive dominance. Queen-produced chemical cues may function secondarily in a context-specific manner to augment behavioural cues, as reliable or honest signal

Relative proportions of compounds found in mandibular glands of queens inseminated with different volumes and substances.

<p>The mean ± SE is shown for the raw data, but to meet the assumptions of the ANOVA the data for HOB, Unk 4, 10HDAA, HVA, tetradecanoic acid, decanedioic acid, Unk 13 and octadecanoic acid were arcsine square root transformed prior to statistical analysis. Statistical differences in the relative proportions of each individual compound across the three groups of queens were determined using an ANOVA with treatment as the main factor. Post hoc analysis was performed with a Tukey's HSD all pairs comparison and different letters annotate significant differences between groups.</p><p>#  =  compound number, Unk  =  unknown. Retention times and the information on m/z and intensities of the ten most abundant ions for all unknown compounds are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078637#pone.0078637.s006" target="_blank">Table S4</a> and fragmentation patterns for all of the compounds are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078637#pone.0078637.s002" target="_blank">Figure S2</a>.</p

Chemical Profiles of Two Pheromone Glands Are Differentially Regulated by Distinct Mating Factors in Honey Bee Queens (<i>Apis mellifera</i> L.)

<div><p>Pheromones mediate social interactions among individuals in a wide variety of species, from yeast to mammals. In social insects such as honey bees, pheromone communication systems can be extraordinarily complex and serve to coordinate behaviors among many individuals. One of the primary mediators of social behavior and organization in honey bee colonies is queen pheromone, which is produced by multiple glands. The types and quantities of chemicals produced differ significantly between virgin and mated queens, and recent studies have suggested that, in newly mated queens, insemination volume or quantity can affect pheromone production. Here, we examine the long-term impact of different factors involved during queen insemination on the chemical composition of the mandibular and Dufour's glands, two of the major sources of queen pheromone. Our results demonstrate that carbon dioxide (an anesthetic used in instrumental insemination), physical manipulation of genital tract (presumably mimicking the act of copulation), insemination substance (saline vs. semen), and insemination volume (1 vs. 8 µl) all have long-term effects on mandibular gland chemical profiles. In contrast, Dufour's gland chemical profiles were changed only upon insemination and were not influenced by exposure to carbon dioxide, manipulation, insemination substance or volume. These results suggest that the chemical contents of these two glands are regulated by different neuro-physiological mechanisms. Furthermore, workers responded differently to the different mandibular gland extracts in a choice assay. Although these studies must be validated in naturally mated queens of varying mating quality, our results suggest that while the chemical composition of Dufour's gland is associated with mating status, that of the mandibular glands is associated with both mating status and insemination success. Thus, the queen appears to be signaling both status and reproductive quality to the workers, which may impact worker behavior and physiology as well as social organization and productivity of the colony.</p></div

Assay of Behavioral Responses to Mandibular Gland Extracts.

<p>Caged groups of 30 five-day-old workers were presented queen mandibular gland extracts from two different queen groups. The number of workers in each cage antennating and/or licking the two extracts were counted every five minutes over a 40-minute period and for two consecutive days. Statistical differences in the preferences for one extract versus the other in the pairwise comparisons were determined via repeated measures ANOVA with treatment as the main effect and day as a repeated variable. The mean ± SE is shown for the raw data, an asterisk above the bars indicates statistically significant differences; n is the number of cages used in comparison. <b>A</b>) In the first experiment we tested workers' preference for extracts of Virg, CO₂, and CPM queens. <b>B</b>) In the second experiment we tested workers' preference for extracts of Virg, SA1, SA8, SE1, and SE8 queens.</p