Experimental setup. Workers were exposed to cuticular extracts of young, old and virgin queens in three settings that included no context (n = 38 cages), partial context using a live free-moving newly-emerged virgin queen (n = 37 cages), or full context using a live free-moving newly-emerged virgin queen and eggs (n = 47 cages). To control for the effect of eggs alone, workers were also kept with eggs without the queen or queen extracts (n = 13 cages). All cages contained a pair of newly emerged workers with the extract and the social context element/s. All pairs were kept for 7 days before they were terminated. Extracts containing 1 queen equivalent (1 Qeq) in 5 µl of hexane were applied daily to a glass coverslip (1x1 cm) in the context-free treatment or to the thorax of the live virgin queen in the partial context and full context treatments. The application was repeated for 7 days. In the full context setting and in cages containing only eggs, two batches of newly laid eggs were added to the cage on days 1,3,5 and 7, resembling the rate of egg laying by an active queen. At the end of the experiment all cages were frozen, workers were dissected to measure ovarian activation and presence of eggs and larvae in each cage was recorded. Cages in which the queen or one of the workers died prematurely were removed from the analysis. Cages in which no egg batches were placed (context-free and partial-context treatments) were inspected for egg laying at the end of the experiment. No eggs were by either the virgin queens or the workers within the duration of the experiment (workers typically lay eggs within 8–9 days 14 and virgin queens have inactivated ovaries). The sample size and the setup are summarized in Table 1.
Table 1
The experimental design and sample size used in the study. Worker reproduction was examined in three different contexts with 3 types of queen extracts. The numbers within the table denote numbers of cages. Each cage contained 2 newly-emerged workers that were frozen on day 7.
Queen extract / setting | Context-free (glass coverslip) | Partial context (live virgin queen) | Full context (live virgin queen and eggs) | Egg presence (eggs only) |
Virgin queen extract | 13 | 13 | 16 | 0 |
Young queen extract | 12 | 12 | 15 | 0 |
Old queen extract | 13 | 12 | 16 | 0 |
No extract | 0 | 0 | 0 | 13 |
Total | 38 | 37 | 47 | 13 |
Bees and housing. Colonies of Bombus impatiens were obtained from Koppert Biological Systems (Howell Michigan, USA) or reared in the lab using gynes from Koppert colonies as founders. Colonies maintained in the laboratory under constant darkness, a temperature of 28–30°C, 60% relative humidity, and supplied ad libitum with a 60% sugar solution and fresh pollen (Koppert Biological Systems). These colonies were used as a source of newly emerged workers (younger than 24 h) and queens.
Queen extracts. Cuticular extracts were collected from three types of queens (virgin, young and old) using a non-lethal method (see below). Newly-emerged virgin queens (n = 40) were collected from Koppert colonies and placed in separate cages upon emergence. Cuticular extracts from these queens were sampled every other day for the duration of 14 days since emergence. A portion of the newly-emerged virgin queens were aged for 6 days, mated in the laboratory with unrelated males and underwent CO2 treatment according to the protocol described in 21. They were housed in individual cages until they laid eggs and produced workers. Extracts from these young egg-laying queens (n = 20) were sampled every other day for the duration of 14 days since the emergence of their first daughter. Old egg-laying queens (n = 16) were obtained from lab-reared colonies several months following the emergence of the first worker. These colonies contained > 100 workers and were producing gynes and males. Extracts were sampled from these queens every other day for the duration of 30 days since the first gyne/male emergence.
Non-lethal sampling of queen cuticular extracts. Non-lethal sampling was performed as described in 20. Briefly, individual queens were placed in 20 ml glass vial for 10 minutes. Afterward, the vial was washed with 1 ml hexane to generate 1 queen equivalent (Qeq) extract. The extracts from 10 queens of the same type were pooled, evaporated and transferred to 2 ml glass vials fitted with a 200 ul glass insert. The pools were evaporated and reconstituted with 50 ul hexane, so that 5 ul solution contained 1 queen equivalent. Extracts were stored at -20° C to prevent evaporation.
Chemical analysis of extracts. Cuticular extracts of young, old and virgin queens were chemically analyzed in a previous publication 22. To confirm that the extracts are similar to previous data we analyzed 9 pools of queens (same pools as we used in the experiments, 2–4 pools per queen type). Pools were also used to calculate the queen equivalent amount in each of these queen types. To this end, 10 µl (2 Qeq) from each pool were mixed with 50 µl hexane containing 100 ng pentadecane (Sigma) as an internal standard. The mixture was evaporated to the volume of 10 µl out of which 1 µl containing 10 ng pentadecane and 0.2 Qeq of queen secretion were analyzed using GC/MS. In addition, we collected and analyzed 34 individual samples of young, old and virgin queens (8–17 extracts per queen type) and compared the individual queen equivalent to the one calculated from the pool. Extracts were collected as in 22 and the total amount of compounds was sum and averaged for each queen type.
Sample quantification was performed using gas chromatograph Trace 1310 (ThermoScientific) equipped with a TG-5ms column. The run was performed in splitless mode with temperature program from 60 ºC at 15 ºC/min for 4 min to 120 ºC, then at 4 ºC/min for 54 min to 300 ºC with a final hold of 5 min. All chromatograms were integrated using Chromeleon 7.0 software (ThermoScientific). Compounds were identified based on our previous work 22 and by matching retention times and spectra with authentic standards. Peak areas were normalized to the internal standards.
Measurement of ovarian activation. All workers were 7 days old upon freezing. Workers were dissected under a stereomicroscope. Ovaries were obtained and placed into drops of distilled water. The length of the terminal oocyte in the three largest ovarioles was measured with a micrometer eyepiece embedded into the lens. Workers possess four ovarioles per ovary and at least one oocyte per ovary was measured. Mean terminal oocyte length for each bee was used as an index of ovarian activation 23.
Statistical analysis. Statistical analyses were performed using SPSS v.21. Normal distribution of terminal oocyte size was confirmed using Kolmogorov-Smirnov test (p = 0.2). Generalized Linear Models analysis (hence GLM) was employed for comparisons of worker oocyte size. In all analyses we tested for the effect of setting (context-free, partial context and full context), treatment (extract type of young, old or virgin queens) and the interaction between them followed by post-hoc pairwise contrast estimation using Least Significant Difference (LSD) method.