Across all nests, we observed 410 eggs laid in 138 brood cells, spanning over 1,000 days of continuous video recording (Table 1). Consistent with previously published data for E. dilemma and other Euglossa species, we found that subordinate egg laying occurred primarily in the early afternoon, with dominant replacement occurring most frequently around 3.5-4 hours after subordinate egg laying, though egg replacement was also observed overnight and into the morning (Fig. 2). Of the 138 brood cells, a dominant bee laid the final egg in 136, or about 98.6%, of these brood cells. Of the two subordinate eggs that were not replaced, one was in a nest of three individuals (“small”), and one was in a nest of four individuals (“large”).
Table 1. Data summary for nests used in reproductive observations of E. dilemma.
Nest ID
|
Dates Observed
|
Days Observed
|
Brood Cells Completed
|
Egg Laid
|
Nest Size
|
2
|
05/19/2016 - 06/20/2016
|
32
|
9
|
20
|
Small
|
21_1
|
10/19/2021 - 06/13/2022
|
237
|
18
|
37
|
Small
|
21_50
|
12/01/2021 - 1/06/2022
|
36
|
9
|
18
|
Small
|
21_10
|
03/17/2022 - 05/26/2022
|
70
|
9
|
18
|
Small
|
21_12
|
06/12/2022 - 08/28/2022
|
77
|
9
|
22
|
Small
|
21_14
|
09/08/2022-11/12/2022
|
66
|
5
|
10
|
Small
|
8_47
|
07/03/2019 - 08/24/2019
|
52
|
12
|
43
|
Large
|
18_79
|
12/07/2021 - 08/07/2022
|
243
|
25
|
98
|
Large
|
P1
|
03/01/2022 - 05/29/2022
|
89
|
21
|
82
|
Large
|
21_44
|
04/01/2022 - 05/30/2022
|
59
|
11
|
31
|
Large
|
21_13
|
06/12/2022 – 08/28/2022
|
77
|
10
|
31
|
Large
|
Figure 2. Circadian rhythm of subordinate egg laying and dominant egg replacement. A density histogram is shown, with the X-axis showing hours from 0-23, with “0” representing midnight. The Y-Axis shows the frequency of observations falling into a particular hour, with each bar representing the percent of observations at that time. Subordinate eggs laid and dominant egg replacement frequencies were calculated from the total number of eggs laid.
Individual behavior in social groups
We sought to classify individual behavior in social groups to determine whether reproductive division of labor or other individual behaviors differ between groups of varying size (Table 2). We observed three broad categories of behavior across all nests. As expected from previous studies, we observed subordinate foragers, which collected pollen and resin and laid the first egg in brood cells. We also observed dominant females, which primarily remained in the nest without foraging and replaced eggs laid by other bees. Finally, in several large nests, we observed an additional category of behavior, that we term “stay-and-wait,” in which females were present in nests for multiple weeks without foraging or egg-laying, with these behaviors sometimes initiated after a turnover in nest membership. In “small” group nests, all individuals could be clearly categorized as “dominant” or “subordinate,” with all “small” groups having one dominant bee and one or two subordinate bees. No “stay-and-wait” females were observed in small nests.
The additional members of “large” social groups expressed several combinations of the three broad behavioral roles, thus elevating the number of nestmates compared to “small” groups. For example, in 2/5 “large” group nests, three subordinate foragers were simultaneously present along with at least one dominant. In 4/5 “large” nests, two dominant egg laying individuals were simultaneously present in the nest, with at least one of these corresponding to a bee that did not emerge from the focal nest and was not the foundress. In one case, the second dominant bee to join an existing social nest was formerly the dominant bee in a nearby nest, which had recently lost its subordinates and had no remaining live brood. In most cases, the two dominant bees persisted over time without obvious aggression. However, in one case, after a period of approximately three weeks in the same nest, one dominant bee became highly aggressive, stinging the other dominant bee until it died.
Table 2. Summary of behavioral roles observed in small and large nests.
Behavior
|
Observed at least once in “small” nests
|
Observed at least once in “large” nests
|
One dominant and one or more subordinates
|
6/6
|
5/5
|
Two dominants with one or more subordinates
|
0/6
|
4/5
|
Three provisioning subordinates
|
0/6
|
2/5
|
“Stay-and-wait” female(s)
|
0/6
|
3/5
|
Primary oophagy
|
6/6
|
5/5
|
Secondary oophagy
|
3/6 (7/136 events)
|
5/5 (129/136 events)
|
In 3/5 large nests, “stay-and-wait” females were observed. “Stay-and-wait” females differed substantially from one another in their duration in the nest and in the individual behaviors they exhibited. For example, in one nest, two “stay-and-wait” bees were simultaneously present for over four months before any foraging or reproductive activity was observed from these individuals. In this case, upon disappearance of the active dominant and subordinate pair, these two “stay-and-wait” females transitioned to behavior typical of a standard dominant and subordinate bee, with one “stay-and-wait” female initiating foraging while the other remained in the nest and replaced eggs. In another nest, one “stay-and-wait” female showed no foraging or reproductive activity for over one month, until she replaced two subordinate laid eggs and disappeared from the nest.
Egg replacement, oophagy, and brood cell completion
Next, we sought to evaluate possible deviations from expected egg replacement patterns in “small” versus “large” social nests. Considering the ratio of eggs laid per brood cell completed, we expect to see two eggs per brood cell—one first laid by the subordinate and one replacement egg laid by the dominant following oophagy (Saleh and Ramirez, 2019). In “large” nests, we see an elevated ratio of eggs laid per brood cell compared to “small” nests (mean: 3.26 vs 2.12 eggs per brood cell per nest, median: 3.58 vs 2.03, “large” nest n = 5, “small” nest n = 6, Mann Whitney U, p<0.01, Fig. 3A). No nest of either category had a ratio below two. We also saw differences in the timing of egg replacement by dominant bees between “small” and “large” nests, with dominant bees taking longer to replace eggs in “large” nests relative to “small” nests (mean: 9.42 vs 3.60 hours, median: 3.78 vs 2.41 hours, “large” nest n = 94, “small” nest n = 59, Mann Whitney U, p <0.001, Fig. 3B).
In addition, we examined patterns of oophagy between “small” and “large” nests. Typically, as observed in “small” nests, the first instance of oophagy in a brood cell is done by a dominant bee eating a subordinate’s egg, except in cases where the provisioning subordinate has disappeared before the brood cell was completed. In these cases, dominant bees laid eggs directly onto the available provision. Beyond the first occurrence of oophagy (primary oophagy), we observed 136 instances of secondary oophagy across all nests, where at least one additional round of oophagy occurred. These instances were divided into three categories of oophagy: “self-oophagy,” where a dominant bee ate her own egg, “dominant oophagy,” where a dominant bee ate the egg of another dominant egg-layer in the nest, and “subordinate oophagy,” where the dominant bee ate an egg laid by a subordinate bee an additional time after the first subordinate laid egg. In these “subordinate oophagy” cases, primary oophagy was not immediately followed by a dominant bee laying an egg, and this delay resulted in the subordinate restarting brood cell provisioning and then laying another egg in that brood cell. This egg was subsequently eaten by a dominant bee.
Of the 136 instances of secondary oophagy, seven occurred in “small” nests and 129 occurred in “large” nests. Of the seven instances in “small” nests, three of these consisted of “self-oophagy” by the dominant bee and four of these were categorized as “dominant oophagy.” In these four cases of “dominant oophagy,” outside bees, present in the nest for a period less than a day, opportunistically laid an egg in an open brood cell or replaced a recently laid egg, which was then replaced by the resident dominant bee. Of the 129 instances of secondary oophagy observed in large nests, 29/129 (22.5%) were “subordinate oophagy,” 37/129 (28.7%) were “dominant oophagy,” and 62/128 (48.8%) were “self-oophagy.”
Finally, we assessed the rate of brood cell completion between “large” and “small” nests, to evaluate possible effects of group size on productivity. To do this, we summarized productivity in each nest by dividing the number of days in a reproductive period (when foraging and egg laying were occurring) by the number of brood cells completed during that period. We found no significant differences in the rate of brood cell production between “large” and “small” nests (mean: 4.48 vs 4.42 days per brood cell, Mann Whitney U, p = 0.93), suggesting that the per-capita productivity is marginally lower in “large” nests compared to “small” nests. In other words, brood cell production was not faster in “large” nests, despite the presence of additional individuals.
Figure 3. Comparison of “small” nests and “large” nests based on (A) the ratio of eggs laid per number of brood cells provisioned in each nest and (B) the time between egg laying by a subordinate and egg laying of the corresponding replacement egg by a dominant. In Both A and B, the difference between the “small” nests and “large” nests is statistically significant in a Mann-Whitney U test.