DOI: 10.5061/dryad.34tmpg4v1
title: Social control of egg-laying in independently nest-founding bumble bee queens

description: Background: Evolution has shaped diverse reproductive investment
 strategies, with some organisms integrating environmental cues into their
 reproductive decisions. In animal societies, social cues can further
 influence reproductive decisions in ways that might support the survival
 and success of the social group. Bumble bees are a lineage of eusocial
 insects wherein queens initiate nests independently. Bumble bee queens
 enter their eusocial phase only after successfully rearing their first
 offspring and thereafter exhibit an increased rate of egg-laying. We
 tested the idea that during bumble bee nest initiation, queen reproduction
 is socially context-dependent and under the control of social conditions
 in the nest. Results: Our findings reveal that in the bumble bee
 Bombus impatiens, queen egg-laying follows a dynamic, stereotypical
 pattern and is also heavily influenced by social group members. During the
 initial stages of nest initiation, accelerated egg-laying in queens is
 associated with the presence of workers or older larvae and pupae.
 Moreover, workers are required for queens to maintain increased levels of
 egg laying across the nest initiation stage. We also confirmed a
 previously-described pattern where queens temporarily decelerate
 egg-laying early in nest-founding, only to increase it again when the
 first adult workers are soon to emerge. This “pause” in egg-laying was
 observed in all B. impatiens queens and an additional species examined.
 Conclusions: Our results support the idea that eusocial systems
 can employ socially context-dependent control of queen egg-laying as a
 reproductive strategy. In some solitary-founding lineages, including
 bumble bees, queens may reach their full reproductive potential only after
 the emergence of the first adult workers, who then take over brood care.
 This stands in contrast to the hyper-reproductivity observed in some
 eusocial species. The presence of workers and older brood (who will soon
 eclose) not only alleviates queen brood care responsibilities but may also
 provide signals that cause queens to increase their reproductive output.
 These phenomena may allow queens to adapt their reproductive output to the
 conditions of the colony. Broadly, these findings highlight the dynamic
 interplay between social conditions and reproduction in bumble bees.
description: We reared <em>Bombus impatiens </em>queens in the lab
 and collected data on queen egg-laying patterns across the early nesting
 developmental stage. We also explored questions regarding the social
 control of developing brood on queen reproductive behavior. Data was
 transcribed from paper to Microsoft Excel sheets and analyzed in
 R. 
description: # Social control of egg-laying in independently nest-founding bumble bee
 queens
 [https://doi.org/10.5061/dryad.34tmpg4v1](https://doi.org/10.5061/dryad.34tmpg4v1) ## Description of the data and file structure: Contains all analyses performed on datasets regarding the social control of egg-laying for *Bombus impatiens* queens.  Synopsis: We examined the egg-laying patterns of *Bombus impatiens* queens and demonstrated that social group members influence it. We believe queens rely on social cues from adult workers and brood to optimize reproductive output. Sample Information: See Social_Control_Data_3_25.xlx for all data related to egg-laying patterns, nest development, worker body size, brood manipulation, ovarian status Analysis: See Social_Control.R for codes ### Files and variables: #### File: Social\_Control\_Raw\_Data.zip **Description:** Compressed folders of raw daily photos and videos (necessary if taking photos was not possible, .MOV and .mp4 files) of nest development. Each folder is an individual queen's colony development labeled with both the Queen ID and Random ID (see below for more information). #### File: Social\_Control\_Data\_3\_25.xlsx **Description:** Excel document with multiple sheets depending on analyses performed. Descriptions below: **File:** Social_Control_Data_3_25, Full.Data.Days **Variables:** * **Random.ID**: Random 4- or 5-digit unique identifier assigned to individual queens (4 digits for Experiment 1; 5 digits for Experiment 2). * **QID**: Unique identifier for individual queens. * **Natal.Colony**: Colony source for the individual queen. * **Experiment**: Indicates the experiment number (1 for Experiment 1, 2 for Experiment 2). * **Date.Pulled**: Date the callow queen was sourced from the natal colony. * **Last.Date.Gassed**: Final day of CO2 narcosis treatment before entering an experiment. * **Date.First.Egg**: Date the first egg cup was present in the nest (i.e., nest initiation). * **Days.Until.First.Egg**: Number of days until the first egg cup was present in the nest (calculated as Date.First.Egg - Last.Date.Gassed). * **Days.til.Pause**: Number of days the queen laid eggs before ceasing egg-laying for 2 or more consecutive days (pause). * **Age.of.Queen.First.Egg:** Number of days after the queen was sourced from the natal colony at the time of nest initiation. * **Age.Queen.at.Pause**: Number of days after the queen was sourced from the natal colony at the time the pause began. * **Last.Days.Laid**: Number of days queens laid eggs before entering the “pause” * **Length.of.Pause**: Number of consecutive days of no new egg cups present in the nest until re-initiation (new eggs laid by the queen). * **Brood.Type.At.Pause**: Oldest brood type present (visually identified) in the nest at the start of the pause (E=eggs, SLandE = young larvae and eggs). * **Number.Egg.by.Pause**: Number of egg cups present in the nest at the start of the pause. * **First.Days.New.Eggs**: Number of days from initial nest initiation until new egg cups were present (end of the pause or re-initiation). * **Brood.Type.With.Eggs.Category**: Oldest brood type present (visually identified) in the nest at the time of re-initiation (Donut.BL = older larva, Oval.PL.or.P = late-stage brood, Small.Larvae = younger larvae). * **Days.First.Brood**: Number of days from initial nest initiation until the first offspring were present in the nest. * **Date.First.Brood**: Date of the first offspring present in the nest. * **Queen.Marginal.Cell.Avg**: Average wing length in millimeters (n/a if not recorded). * **Keep.Or.Remove**: Indicates the treatment performed on the nest ("keep" = workers remain in the nest; "remove" = workers removed once present). * **Total.Offspring**: Total number of offspring present in the nest at the end of the experiment (44 days after nest initiation). * **Worker.in.Week**: Number of workers within 7 days after the first offspring emergence. **File:** Social_Control_Data_3_25, FullEggRates **Variables:** * **Queen.ID**: Unique identifier for individual queens. * **Natal.Colony**: Colony source for the individual queen. * **Day**: Day of the experiment (1–45). * **Number.Egg.Clumps**: Count of new egg cups observed in the nest (unadjusted for missing days). * **Alt.Number.Egg.Clumps**: Approximate daily average of new egg cups, accounting for missing days. * **Type_Status**: Nest treatment description; "with" (with workers) or "none" (workers removed). * **Type**: Treatment of the nest; "keep" (with workers) or "remove" (workers removed for related experiments). **File:** Social_Control_Data_3_25, CombinedEggRates **Variables:** * **Type:** Treatment of the nest; "keep" (workers present, Experiment 2), "remove" (workers removed, Experiment 2), or “worker-only” (Experiment 1). * **Day:** Day of the experiment (1–45). * **Mean:** Mean daily number of new egg cups (not accumulated) * **SD:** Standard deviation of the mean number of new egg cups per day. * **SE:** Standard error of the mean number of new egg cups per day. **File:** Social_Control_Data_3_25, Exp1 **Variables:** * **Random.ID**: Random 4- or 5-digit unique identifier assigned to individual queens (4 digits for Experiment 1; 5 digits for Experiment 2). * **QID**: Unique identifier for individual queens. * **Natal.Colony**: Colony source for the individual queen. * **Experiment**: Indicates the experiment number. * **Date.Pulled**: Date the callow queen was sourced from the natal colony. * **Last.Date.Gassed**: Final day of CO2 narcosis treatment before entering an experiment. * **Date.First.Egg**: Date the first egg cup was present in the nest (i.e., nest initiation). * **Days.Until.First.Egg**: Number of days until the first egg cup was present in the nest (calculated as date.first.egg - last.date.gassed). * **Days.til.Pause**: Number of days the queen laid eggs before ceasing egg-laying for 2 or more consecutive days (pause). * **Age.of.Queen.First.Egg:** Number of days after the queen was sourced from the natal colony at the time of nest initiation. * **Age.Queen.at.Pause**: Number of days after the queen was sourced from the natal colony at the time the pause began. * **Last.Days.Laid**: Number of days queens laid eggs before entering the “pause” * **Length.of.Pause**: Number of consecutive days of no new egg cups present in the nest until re-initiation (new eggs laid by the queen). * **Brood.Type.At.Pause**: Oldest brood type present (visually identified) in the nest at the start of the pause (E=eggs, SLandE = young larvae and eggs). * **Number.Egg.by.Pause**: Number of egg cups present in the nest at the start of the pause. * **First.Days.New.Eggs**: Number of days from initial nest initiation until new egg cups were present (end of the pause or re-initiation). * **Brood.Type.With.Eggs.Category**: Oldest brood type present (visually identified) in the nest at the time of re-initiation (Donut.BL = older larva, Oval.PL.or.P = late-stage brood, Small.Larvae = younger larvae). * **Days.First.Brood**: Number of days from initial nest initiation until the first offspring were present in the nest. * **Date.First.Brood**: Date of the first offspring present in the nest. * **Queen.Marginal.Cell.Avg**: Average wing length in millimeters (n/a if not recorded). * **Keep.Or.Remove**: Indicates the treatment performed on the nest ("keep" = workers remain in the nest; "remove" = workers removed once present). * **Total.Offspring**: Total number of offspring present in the nest at the end of the experiment (44 days after nest initiation). * **Worker.in.Week**: Number of workers within 7 days after the first offspring emergence. **File:** Social_Control_Data_3_25, Exp2 **Variables:** * **Random.ID**: Random 4- or 5-digit unique identifier assigned to individual queens (4 digits for Experiment 1; 5 digits for Experiment 2). * **QID**: Unique identifier for individual queens. * **Natal.Colony**: Colony source for the individual queen. * **Experiment**: Indicates the experiment number. * **Date.Pulled**: Date the callow queen was sourced from the natal colony. * **Last.Date.Gassed**: Final day of CO2 narcosis treatment before entering an experiment. * **Date.First.Egg**: Date the first egg cup was present in the nest (i.e., nest initiation). * **Days.Until.First.Egg**: Number of days until the first egg cup was present in the nest (calculated as date.first.egg - last.date.gassed). * **Days.til.Pause**: Number of days the queen laid eggs before ceasing egg-laying for 2 or more consecutive days (pause). * **Age.of.Queen.First.Egg:** Number of days after the queen was sourced from the natal colony at the time of nest initiation. * **Age.Queen.at.Pause**: Number of days after the queen was sourced from the natal colony at the time the pause began. * **Last.Days.Laid**: Number of days queens laid eggs before entering the “pause” * **Length.of.Pause**: Number of consecutive days of no new egg cups present in the nest until re-initiation (new eggs laid by the queen). * **Brood.Type.At.Pause**: Oldest brood type present (visually identified) in the nest at the start of the pause (E=eggs, SLandE = young larvae and eggs). * **Number.Egg.by.Pause**: Number of egg cups present in the nest at the start of the pause. * **First.Days.New.Eggs**: Number of days from initial nest initiation until new egg cups were present (end of the pause or re-initiation). * **Brood.Type.With.Eggs.Category**: Oldest brood type present (visually identified) in the nest at the time of re-initiation (Donut.BL = older larva, Oval.PL.or.P = late-stage brood, Small.Larvae = younger larvae). * **Days.First.Brood**: Number of days from initial nest initiation until the first offspring were present in the nest. * **Date.First.Brood**: Date of the first offspring present in the nest. * **Queen.Marginal.Cell.Avg**: Average wing length in millimeters (n/a if not recorded). * **Keep.Or.Remove**: Indicates the treatment performed on the nest ("keep" = workers remain in the nest; "remove" = workers removed once present). * **Total.Offspring**: Total number of offspring present in the nest at the end of the experiment (44 days after nest initiation). * **Worker.in.Week**: Number of workers within 7 days after the first offspring emergence **File:** Social_Control_Data_3_25, Exp1e5KRE **Variables:** * **Random.ID:** Random 4- or 5-digit unique identifier assigned to individual queens (4 digits for Experiment 1; 5 digits for Experiment 2). - **QID**: Unique identifier for individual queens. - **Treatment**: Treatment of the nest; "remove" (workers removed for related experiments). - **Natal.Colony**: Colony source for the individual queen. - **Timing**: Five-day sequential segments over the first 25 days of the experiment. - **TotEggs**: Total number of egg cups present in the nest across the five-day period **File:** Social_Control_Data_3_25, 45DaysEggs **Variables:** * **QID**: Unique identifier for each individual queen. * **Type**: Treatment of the nest; "keep" (with workers) or "remove" (workers removed for related experiments). * **ECS**: Identifier for each individual egg cup within a nest, numbered sequentially (1 through total number of egg cups in the nest). * **EGG**: Count of eggs present in the corresponding egg cup. **File:** Social_Control_Data_3_25, 45DaysEggCups **Variables:** * **QID**: Unique identifier for each individual queen. * **Type**: Treatment of the nest; "keep" (with workers) or "remove" (workers removed for related experiments). * **NEC**: Total number of egg cups present in nest **File:** Social_Control_Data_3_25, PropOVsub **Variables:** * **QueenID**: Unique identifier for each individual queen. * **Colony.ID**: Source colony for the individual queen. * **Treatment:** Treatment performed on queens during the experiment: (a) **before pause** – newly initiated (collected on the second day of egg-laying after initiation); (b) **UUP** – pre-pause (collected on the last day of egg-laying before ceasing egg-laying during the pause); (c) **add brood** – pause manipulation (brood artificially added to nests); (d) **during pause** – collected three days into the pause; (e) **after pause** – collected five days after queens re-initiated egg-laying; (f) **AWE** – after worker emergence (collected 10 days after the first worker emerged). - **Avg**.**Wing**: Average wing length of the offspring in millimeters (n/a if not recorded). * **PropOvaries**: Proportion of ovaries resorbed, ranging from 0 (none resorbed) to 1 (fully resorbed). **File:** Social_Control_Data_3_25, VAROVSUB **Variables:** * **QueenID**: Unique identifier for each individual queen. * **ReproAge**: Number of days after the queen was sourced from the natal colony at the time of treatment * **Colony**: Source colony for the individual queen. * **Treatment**: Treatment performed on queens during the experiment. * **OVaryNR**: Binomial description of ovary resorption status (e.g., resorbed or not resorbed). * **OVarySummary**: Simplified ovary resorption status, categorized as "yes" (resorbed) or "no" (not resorbed). * **Ovary.Status**: Detailed categorical description of ovary resorption status: * **R**: Fully resorbed, **N**: Not resorbed, **PR**: Partially resorbed, **N/A**: Not measured. * **Ovary.Size**: Length of the terminal oocyte (measured in millimeters, n/a if not recorded). **File:** Social_Control_Data_3_25, Altered.Egg.Cups.Pre.Pause **Variables:** * **Queen**: Unique identifier for each individual queen. * **EggClump**: Identifier for each individual egg cup within a nest, numbered sequentially (1 through total number of egg cups in the nest). * **Number**: Count of eggs present in the corresponding egg cup. **File**: Social_Control_Data_3_25, wosmall **Variables:** * **Queen.ID**: Unique identifier for individual queens. * **Colony.ID**: Source colony for the individual queen. * **Treatment.Type**: Treatment performed on queens during the experiment (a_control = control queens, no manipulations, c_biglarvae = older brood added, d_pupae = late-stage brood added). * **Date.Added**: Date of manipulation/artificial brood addition. * **Date.New.EC**: Date a new egg cup was present in the nest. * **Days.Till.EC**: Number of days until a new egg cup was present in the nest (calculated as date.new.ec - date.added). * **No.Brood.Added**: Number of brood added to the nest as part of the manipulation. * **Y.Brood.Dis**: Youngest brood present at the time of manipulation. * **O.Brood.Dis**: Oldest brood present in the nest when queens re-initiated egg-laying (either the added brood from manipulation or as part of normal nest development in the control). **File**: Social_Control_Data_3_25, Worker.Body.Size.Week **Variables:** * **Bee.ID**: Unique identifier for individual bee offspring. * **Natal.Colony**: Source colony for the individual queen. * **Colony.ID**: Unique identifier for colonies established by queens. * **Queen.BS**: Average wing length of the queen in millimeters (n/a if not recorded). * **Emerged.Date**: Date the offspring emerged in the nest. * **Date.Week**: Day of the week the worker emerged (1 = first day of worker emergence, 2 = second day after first worker emergence, etc.). * **Week.Use**: Indicates if the offspring emerged within the first week and was included in analyses ("yes") or not ("no"). * **Left.Wing**: Left-wing length of the offspring in millimeters (n/a if not recorded). * **Right.Wing**: Right-wing length of the offspring in millimeters (n/a if not recorded). * **Avg.Wing**: Average wing length of the offspring in millimeters (n/a if not recorded). * **Caste**: Caste of the offspring (male or worker). * **Use.In.Exp**: Indicates if the individual was used in analyses ("yes" or "no"). * **Length.of.Pause**: Length of the queen’s egg-laying “pause” for the nest (total days of a queen's pause). **File:** Social_Control_Data_3_25, PostWorker **Variables:** * **Random.ID:** Random 4- or 5-digit unique identifier assigned to individual queens (4 digits for Experiment 1; 5 digits for Experiment 2). - **QID**: Unique identifier for individual queens. - **Treatment**: Treatment of the nest; "remove" (workers removed for related experiments). - **Natal.Colony**: Colony source for the individual queen. - **Treatment:** Treatment of the nest; "keep" (with workers) or "remove" (workers removed for related experiments). - **Totegg**: Total number of egg cups present in the nest across period - **Day**: Five-day sequential segments after worker emergence **File:** Social_Control_Data_3_25, KeepRemoveEggs **Variables:** * **Random.ID**: Random 4- or 5-digit unique identifier assigned to individual queens (4 digits for Experiment 1; 5 digits for Experiment 2). * **QID**: Unique identifier for individual queens. * **Treatment**: Treatment performed on queens during the experiment. * **Natal.Colony**: Source colony for the individual queen. * **Total.Brood**: Total count of brood in the nest, regardless of developmental stage. **Note on Missing Data:** Missing values are represented as `"n/a"` in the dataset, indicating that data is not available for those entries. The presence of `"n/a"` may vary across different sheets depending on the dataset. ## Code/software **Code/software** All statistical analyses were performed in R version 4.3.1 (R Developmental Core Team, 2023). This R script is part of a research analysis pipeline designed to evaluate the behavioral and biological characteristics of bumble bee queens and their reproduction in early-stage nests. It primarily focuses on data from laboratory-reared bumble bee queen egg-laying patterns and colony development, data collected between March 2022 and December 2023. **Structure of the Script:** **Packages Imported**: The script uses several R packages for data manipulation, visualization, statistical analysis, and modeling, including: * **plyr**: Tools for splitting, applying, and combining data. * **stats**: Basic statistical functions and models. * **tidyverse**: A collection of packages for data science, including dplyr, tidyr, ggplot2, and more. * **hrbrthemes**: Themes for creating publication-quality ggplot2 visuals. * **viridis**: Color scales for data visualization. * **readr**: Tools for reading rectangular data. * **ggplot2**: Data visualization. * **gridExtra**: Arranging multiple ggplot2 plots. * **ggpubr**: Publication-ready ggplot2 visualizations and statistical annotations. * **lme4**: Linear and generalized linear mixed-effects models. * **multcomp**: Multiple comparison procedures. * **car**: Companion functions for regression analysis and hypothesis testing. * **AICcmodavg**: Model selection and comparison based on AIC. * **lmtest**: Diagnostic tests for linear regression models. * **dplyr**: Data manipulation. * **tidyr**: Data tidying. * **cowplot**: Enhanced plots and arrangement of ggplot2 visuals. * **outliers**: Outlier detection tests. * **MuMIn**: Multi-model inference. * **lattice**: Multivariate data visualization. * **lubridate**: Date-time manipulation. * **readxl**: Reading Excel files. * **lmerTest**: Mixed-effects models with p-values. * **emmeans**: Estimated marginal means and pairwise comparisons. * **rstatix**: Statistical summaries and tests. * **DHARMa**: Residual diagnostics for hierarchical models. * **RColorBrewer**: Color palettes for visualization. * **did**: Difference-in-differences analysis. * **glmmTMB**: Generalized linear mixed models. * **goft**: Goodness-of-fit tests. * **brms**: Bayesian regression models using Stan. * **tmbstan**: Tools for Stan-based modeling with TMB. * **fitdistrplus**: Fitting distributions to data. * **multcompView**: Compact letter displays for multiple comparisons. * **patchwork**: Combining multiple ggplot2 plots. **Purpose and Applications:** The script is designed for behavioral and biological studies, particularly focusing on: • Queen egg-laying rates during early nest development. • The impact of nest development and offspring on queen reproduction and biology. • Effects of artificial brood addition on queen egg-laying behavior. This analysis can be used to infer the effects of nest dynamics, such as brood addition or offspring presence, on queen reproduction and behavior. Let us know if you'd like detailed explanations for any specific part of the script or if you want any additional modifications or analyses.