,Units:,Materials and methods. Two experiments were conducted, the first from August 2023 – February 2024, and the second a year later with new colonies. In August of each year ten honey bee colonies of each of three bee stocks: Pol-line, Russian and unselected Italian, for a total of thirty colonies, were selected. The colonies had been established from single box colonies received in mid-April. Bee colonies were housed in painted, two-box, 10-frame, wooden Langstroth hives (43.7 Liter (L) capacity per box). The hives were located at the University of Arizona Red Rock Agricultural Station where they were placed on stainless steel electronic scales (Tekfa model B-2418 and Avery Weigh-Tronix model BSAO1824-200, max. capacity: 100 kg, precision: ±20 grams (g) ; operating temperature: -30ºC to 70ºC) and linked to dataloggers (Hobo UX120-006M External Channel datalogger, Onset Computer Corporation, Bourne, MA) with weight recorded every 5 min. Temperature sensors (HOBO MX2201, resolution ±0.04°C, accuracy ±0.5°C) were attached to the center of the top bar on the middle frame in the bottom box and set to record every 5 min. Carbon dioxide probes (model GMP251, Vaisala Inc., Helsinki, Finland), calibrated for 0-20% concentrations, were placed on top of the center frames in the top box of each hive and linked to dataloggers (HOBO UX120-006M) set to record every 5 min.,The total weight of all hive components and frames was subtracted from the total hive weight recorded during the night pre-assessment, to calculate the adult bee mass. Hives were periodically assessed to determine adult bee mass and total capped brood area. Briefly, each hive frame was lifted out sequentially, gently shaken to dislodge adult bees, photographed using a 16.3 megapixel digital camera (Canon Rebel SL1, Canon USA, Inc., Melville, NY), weighed on a portable scale (model EC15, OHaus Corp., Parsippany, NJ), and replaced in the hive. During the first assessment all hive components (i.e. lid, inner cover, box, bottom board, frames, entrance reducer) were also shaken free of bees and weighed to yield an initial mass of all hive components. The area of sealed brood per frame was measured from photographs using image analysis software, and frame values were summed to estimate colony values. Assessments took place in mid-August (pre-cold storage treatment period), late September (after the cold storage and mite treatments), mid-November (pre-winter period), and a final assessment in mid-February (post-winter period). In November and at the end of January all colonies were fed 200 g pollen patty.,Half of the hives were moved into a cold storage unit (30 meters cubed (m3) internal volume, with CO2 and temperature monitors, PolarKing, Fort Wayne, IN) set to 5°C with a dehumidifier and a roof-mounted exhaust fan operating 5 min per hour for ventilation, in late-August for 18 days to induce a break in brood production. Immediately after hives were moved from the cold storage unit (CSU) back to the Red Rock apiary, all colonies were treated with thymol miticide (Apiguard, Vita Bee Health, Basingstoke, UK) at 25g/hive/week for 3 weeks. Thereafter colonies were managed in an identical manner throughout the fall and winter, concluding the following February.,Samples of 100 adult bees were collected from the brood nest area of the hive in August (pre-treatment), in November and in late January for gene expression analysis, and an additional approximately 150 adult bees were collected from the brood nest area in August pre-treatment and in late January to determine phoretic mite density using the alcohol wash method. Just prior to placing hives in cold storage in August, and again in October, slick paperboard coated with petroleum jelly and covered with mesh were placed onto the hive floor to monitor Varroa mite fall within the hive. The paperboards were removed after 3 days and the number of mites counted on each board.,Gene expression analysis. For each colony and time point,

,Pollination services provided by the honey bee, Apis mellifera (Hymenoptera: Apidae, Linnaeus, 1758) have broad economic impacts and are necessary for production of a diversity of important crops. Hives may be transported multiple times per year to provide pollination. To test how temperature may contribute to transportation stress, temperature sensors were placed in hives in different locations and orientations on the trailer during shipping. Colony size prior to shipping significantly contributed to loss of population immediately after shipping which contributed to colony failure with smaller colonies more likely to fail and fail faster. Colony size also affects thermoregulation and temperature stress. Internal hive temperature varies significantly based on location and orientation. While colonies near the front and rear of the trailer and those oriented toward the center aisle had significantly different average internal temperatures, colony size best predicts loss of thermoregulation. Additionally, we profiled gene expression at departure, on arrival, and after a recovery period to identify transcriptional responses to transportation. Functional and enrichment analysis identified increased methylation and decreased ribosomal and protein-folding activity. Pheromone and odorant-binding transcripts were up-regulated after transportation. After recovery, transcripts associated with defense response, immune activity, and heat shock decreased, while production of antibiotic peptides increased. We conclude that hives experience considerable temperature stress possibly caused by turbulent airflow in exposed locations. Transportation stress should be considered an important component of annual colony losses which can be mitigated with improved management strategies.,Supplementary Tables S1–S8 are presented. Sequence reads associated with the mRNA sequencing analysis are archived at NCBI under BioProject PRJNA495845.,,

,Nutritional deprivation is known to contribute to increased honey bee mortality, physiological stress, aberrant behaviors, and disease incidence. To investigate the effect of a realistic nutritional protein deficiency, we simulated a pollen dearth in half of our experimental colonies by robbing incoming foragers of their pollen loads, the primary source of dietary protein, at the colony entrance. We then conducted temperament assays on each colony weekly for pollen deprived and control counterparts. We also identified the plant species bees foraged from and took various physiological measures of honey bee nutritional status including gland size, lipid quantification, and gene expression to further investigate and explain our behavioral results. We found that colonies deprived of pollen reacted by becoming more defensive and that immature bees likely receive cues during rearing which prime their gene expression and behavior as adults, ultimately suggesting that environmental stress caused significant behavioral changes. Temperament is primarily associated with genotype, but there are environmental cues which are less acknowledged and still important. As droughts become increasingly frequent and resource availability therefore changes over time, the impacts on behaviors of agricultural keystone species need additional consideration in order to form scientifically driven best management practices.,

,In all files, temperature data are in degrees centigrade and CO2 data are in the original data in mA as generated by the CO2 sensors themselves. Colony weights are in kg.,,For spreadsheet “Ag Commons Fall 2020 and Fall 2021” file, in October 2020 fifty bee colonies in painted, 10-frame, wooden Langstroth boxes with marked European queens and at least 2 frames of sealed brood, were identified. This experiment was originally part of a study on the use of cold storage in Varroa management (see Meikle et al. (2023) Cold storage as part of a Varroa management strategy: Effects on honey bee colony performance, mite levels and stress biomarkers. Scientific Reports 13:11842. doi: 10.1038/s41598-023-39095-5). Temperature sensors (iButton Thermochron) were placed in plastic cassettes and stapled to the center of the top bar on the middle frame in the bottom box and set to record every 30 min. in 2020 and every 15 min. in 2021. CO2 probes (model GMP251, Vaisala Inc., Helsinki, Finland) were placed on top of the center frames in the top box of each hive and linked to HOBO UX120-006M dataloggers set to record every 5 min. On 1 October migratory entrance screens were placed on half of the colonies. Colonies were evaluated to estimate colony size (for a description of protocol see Meikle W.G., Weiss M. (2017) Monitoring colony-level effects of sublethal pesticide exposure on honey bees. J. Vis. Exp. 129, e56355. https://doi.org/10.3791/56355) and then placed in a cold storage unit (30 m3 internal volume, with CO2 and temperature monitors) set to 5°C with a dehumidifier and a roof-mounted exhaust fan operating 4 minutes per hour. Those hives remained in the completely darkened unit with the entrance covers until 22 October. The remaining hives were kept outside in the original apiary during that period. The experiment was repeated in October 2021.,,For “Ag Commons Summer 2023” file, experimental protocol is as follows: In May 2023 16 bee colonies were obtained with marked queens of approximately the same size and in the same hives as described above. CO2 probes and temperature sensors (HOBO Pendant Temp MX2201) were installed under the top bar of the middle frame in the bottom box and set to record every 5 min. Colonies were evaluated as described above on 15 June. On 22 June eight of the colonies were moved into the cold storage unit (30 m3 internal volume, with a dehumidifier and a roof-mounted exhaust fan operating 3 min per h, hereafter “CSU”) set to 5°C. Large mesh screens were placed over the entrance of each hive to provide space for the colony to dispose of dead bees but prevent bees from flying. After 6 d darkness, light cycles were turned on for 12 h from 6PM to 6AM each d for the remaining 12 d, after which colonies were moved back to their original apiary. On 11 July the brood frame photographs were taken but the adult bee mass was estimated visually as frames of bees. On 14 July the remaining eight colonies were moved into the CSU and subjected to the same treatment: 6 d darkness followed by 12 d of 12:12 light:dark cycle, with light from 6PM to 6AM. Those colonies were removed from the CSU to the outdoor apiary on 2 August and assessed on 3 August as described above for the first group of colonies. To estimate the adult bee mass for the post-CSU assessments, adult bee mass in kg was regressed on frames of bees using data from the pre-CSU evaluation, and the resulting equation used to convert frames of bees to adult bee mass.,,For “Ag Commons Winter 2023” file, on 7 December 2023 eight bee colonies with marked queens were assessed by taking photographs of brood frames with a visual count of frames of bees (a full assessment was contra-indicated because of cold weather). CO2 probes and temperature sensors (HOBO Pendant Temp MX2201) were installed under the top bar of the middle frame in the bottom box and set to record every 5 min. On 8 December the large mesh screens were placed over hive entrances and the hives placed in the

,Note: Updated on 02/28/25 to include Ag Commons Winter 2024 data file.,,In all files, temperature data are in degrees centigrade and CO2 data are in the original data in mA as generated by the CO2 sensors themselves. Colony weights are in kg.,,For spreadsheet “Ag Commons Fall 2020 and Fall 2021” file, in October 2020 fifty bee colonies in painted, 10-frame, wooden Langstroth boxes with marked European queens and at least 2 frames of sealed brood, were identified. This experiment was originally part of a study on the use of cold storage in Varroa management (see Meikle et al. (2023) Cold storage as part of a Varroa management strategy: Effects on honey bee colony performance, mite levels and stress biomarkers. Scientific Reports 13:11842. doi: 10.1038/s41598-023-39095-5). Temperature sensors (iButton Thermochron) were placed in plastic cassettes and stapled to the center of the top bar on the middle frame in the bottom box and set to record every 30 min. in 2020 and every 15 min. in 2021. CO2 probes (model GMP251, Vaisala Inc., Helsinki, Finland) were placed on top of the center frames in the top box of each hive and linked to HOBO UX120-006M dataloggers set to record every 5 min. On 1 October migratory entrance screens were placed on half of the colonies. Colonies were evaluated to estimate colony size (for a description of protocol see Meikle W.G., Weiss M. (2017) Monitoring colony-level effects of sublethal pesticide exposure on honey bees. J. Vis. Exp. 129, e56355. https://doi.org/10.3791/56355) and then placed in a cold storage unit (30 m3 internal volume, with CO2 and temperature monitors) set to 5°C with a dehumidifier and a roof-mounted exhaust fan operating 4 minutes per hour. Those hives remained in the completely darkened unit with the entrance covers until 22 October. The remaining hives were kept outside in the original apiary during that period. The experiment was repeated in October 2021.,,For “Ag Commons Summer 2023” file, experimental protocol is as follows: In May 2023 16 bee colonies were obtained with marked queens of approximately the same size and in the same hives as described above. CO2 probes and temperature sensors (HOBO Pendant Temp MX2201) were installed under the top bar of the middle frame in the bottom box and set to record every 5 min. Colonies were evaluated as described above on 15 June. On 22 June eight of the colonies were moved into the cold storage unit (30 m3 internal volume, with a dehumidifier and a roof-mounted exhaust fan operating 3 min per h, hereafter “CSU”) set to 5°C. Large mesh screens were placed over the entrance of each hive to provide space for the colony to dispose of dead bees but prevent bees from flying. After 6 d darkness, light cycles were turned on for 12 h from 6PM to 6AM each d for the remaining 12 d, after which colonies were moved back to their original apiary. On 11 July the brood frame photographs were taken but the adult bee mass was estimated visually as frames of bees. On 14 July the remaining eight colonies were moved into the CSU and subjected to the same treatment: 6 d darkness followed by 12 d of 12:12 light:dark cycle, with light from 6PM to 6AM. Those colonies were removed from the CSU to the outdoor apiary on 2 August and assessed on 3 August as described above for the first group of colonies. To estimate the adult bee mass for the post-CSU assessments, adult bee mass in kg was regressed on frames of bees using data from the pre-CSU evaluation, and the resulting equation used to convert frames of bees to adult bee mass.,,For “Ag Commons Winter 2023” file, on 7 December 2023 eight bee colonies with marked queens were assessed by taking photographs of brood frames with a visual count of frames of bees (a full assessment was contra-indicated because of cold weather). CO2 probes and temperature sensors (HOBO Pendant Temp MX2201) were installed under the top bar of the middle frame in the bottom box and set to record every 5 min. On 8 December the large

We measured nesting rates and nesting success for a community of cavity-nesting bees across gradients of floral abundance, floral richness, and land-cover in the Prairie Pothole Region. Variables in this dataset include a unique transect identifier, a count of the occupied nests per transect, total floral species richness per transect, total floral stem abundance per transect, bee species richness, number of emerged adult bees per transect, and the proportion of those emerged bees which were female. In addition, there are land-cover measurements of the total area, in hectares, of grasslands, wetlands, and woodlands within 500m and 1500m of each transect. We binned land-covers from the 2019 National Agricultural Statistics Service Cropland Data Layer into our definitions of grasslands (Other Hay/Non Alfalfa, Sod/Grass Seed, Fallow/Idle Cropland, and Grassland/Pasture), wetlands (Woody Wetlands and Herbaceous Wetlands), and woodlands (Deciduous Forest, Evergreen Forest, and Shrubland).

We measured nesting rates and nesting success for a community of cavity-nesting bees across gradients of floral abundance, floral richness, and land-cover in the Prairie Pothole Region. Variables in this dataset include a unique transect identifier, a count of the occupied nests per transect, total floral species richness per transect, total floral stem abundance per transect, bee species richness, number of emerged adult bees per transect, and the proportion of those emerged bees which were female. In addition, there are land-cover measurements of the total area, in hectares, of grasslands, wetlands, and woodlands within 500m and 1500m of each transect. We binned land-covers from the 2019 National Agricultural Statistics Service Cropland Data Layer into our definitions of grasslands (Other Hay/Non Alfalfa, Sod/Grass Seed, Fallow/Idle Cropland, and Grassland/Pasture), wetlands (Woody Wetlands and Herbaceous Wetlands), and woodlands (Deciduous Forest, Evergreen Forest, and Shrubland).

,A native solitary bee to North America, the blue orchard bee (Osmia lignaria Say, Hymenoptera: Megachilidae) is a crucial pollinator for orchard crops such as apples, almonds, and cherries. Osmia lignaria is often managed commercially and sold to complement honey bee pollination services.,We collected data following an accidental drop of developing immature bees inside their cocoons. These bees were part of a larger experiment performed in 2020. On June 17, 2020, bees were dropped approximately one meter onto a linoleum floor at the USDA-ARS-PWA Pollinating Insect Research Unit in Logan, Utah, USA. Developing bees were in gelatin capsules and attached to a sticky board for X-ray imaging. Using a board from the same study that had not fallen, we compared survival, life stages, and bodily injuries to document the effects of dropping immature O. lignaria a short distance.,Our research highlights the risks of handling immature O. lignaria during metamorphosis. Our data provides valuable information for bee managers and researchers about the risks of physical disturbances during critical developmental stages, which could affect bee survival and pollination services in orchards.,Key findings include: (1) Near-complete mortality of developing bees before the adult molt stage, (2) Insights into the vulnerability of O. lignaria during immature developmental stages, even when inside cocoons, and (3) Documentation of how mechanical injury during immature development impacts survival.,The dataset provides counts of bees in different life stages and conditions, including: (1) Life status (alive or dead) at cocoon completion, pupation, and adult molt stages, (2) Sex determination for bees that reached adulthood (male or female), (3) Final life stage reached (prepupa, pupa, or adult), and (4) Body condition after the fall (malformed, melanized, no observable change, or partially melanized).,Additional variables in the dataset include: (1) Sample identifiers, treatment groups, and X-ray board identifiers from the original experiment and (2) Whether the board was dropped or not.,Abbreviations and acronyms in the dataset,,

,A mathematical model is constructed to quantify the loss of resilience in collapsing honey bee colonies due to the presence of a strong Allee effect. In the model, recruitment and mortality of adult bees have substantial social components, with recruitment enhanced and mortality reduced by additional adult bee numbers. The result is an Allee effect, a net per-individual rate of hive increase that increases as a function of adult bee numbers. The Allee effect creates a critical minimum size in adult bee numbers, below which mortality is greater than recruitment, with ensuing loss of viability of the hive. Under ordinary and favorable environmental circumstances, the critical size is low, and hives remain large, sending off viably-sized swarms (naturally or through beekeeping management) when hive numbers approach an upper stable equilibrium size (carrying capacity). However, both the lower critical size and the upper stable size depend on many parameters related to demographic rates and their enhancement by bee sociality. Any environmental factors that increase mortality, decrease recruitment, or interfere with the social moderation of these rates has the effect of exacerbating the Allee effect by increasing the lower critical size and substantially decreasing the upper stable size. As well, the basin of attraction to the upper stable size, defined by the model potential function, becomes narrower and shallower, indicating the loss of resilience as the hive becomes subjected to increased risk of falling below the critical size. Environmental effects of greater severity can cause the two equilibria to merge and the basin of attraction to the upper stable size to disappear, resulting in collapse of the hive from any initial size. The model suggests that multiple proximate causes, among them pesticides, mites, pathogens, and climate change, working singly or in combinations, could trigger hive collapse. This data supplement provides a text file containing 7 scripts written in the R programming language for reproducing Figures 1–7.,,