Ostwald_etal_WaterHomeostasis2016_ReadMe.text File Data Files: Ostwald_etal_WaterHomeostasis2016_Data.xlsx* Ostwald_etal_WaterHomeostasis2016_Data.zip, which contains - Ostwald_etal_WaterHomeostasis2016_Fig1Data.csv - Ostwald_etal_WaterHomeostasis2016_Fig2Data.csv - Ostwald_etal_WaterHomeostasis2016_Fig3-1Data.csv - Ostwald_etal_WaterHomeostasis2016_Fig3-2Data.csv - Ostwald_etal_WaterHomeostasis2016_Fig3-3Data.csv - Ostwald_etal_WaterHomeostasis2016_Fig4Data.csv - Ostwald_etal_WaterHomeostasis2016_Fig5Data.csv - Ostwald_etal_WaterHomeostasis2016_SuppFigData-1.csv - Ostwald_etal_WaterHomeostasis2016_SuppFigData-2.csv - Ostwald_etal_WaterHomeostasis2016_SuppFigData-3.csv * The Microsoft Excel file was written in Office2016, and contains worksheets with the same data as in all the csv files. Publication Title: The behavioral regulation of thirst, water collection, and water storage in honey bee colonies Short Title: Water homeostasis in honey bee colonies Authors: Madeleine M. Ostwald, Michael L. Smith, and Thomas D. Seeley* *Corresponding Author Email: tds5@cornell.edu Alternate Contact Email: mls453@cornell.edu Summary Statement: When a honey bee colony experiences broodnest hyperthermia, its water collectors quickly spring into action after being begged for fluid. Keywords: Apis mellifera, social thirst, water collection, water storage Abstract: This study investigates how a honey bee colony develops and quenches its collective thirst when it experiences hyperthermia of its broodnest, the nest region whose temperature and humidity are precisely regulated. We found that in this emergency situation a colony must strongly boost its water intake because evaporative cooling is critical to avoiding broodnest hyperthermia, and it must rapidly boost its water intake because a colony maintains only a small water reserve. We also shed light on how the worker bees functioning as water collectors know when to spring into action—by sensing either more frequent requests for fluid or greater personal thirst, or both—and we demonstrate that this sensing mechanism is sufficiently fine-tuned to prevent overheating and desiccation of the colony's brood. Finally, we found that the impressive behavioral flexibility of a colony's water collectors enables them not only to satisfy their colony's current water needs, but also to buffer their colony somewhat against future extreme water stresses by storing water in their crops and in the combs. Funding: This study was supported by a Rawlings Cornell Presidential Research Scholarship (to M.M.O), by a National Science Foundation Graduate Research Fellowship (DGE- 1144153 to M.L.S.), and by Hatch Funds administered by the Cornell University Agricultural Experiment Station (to T.D.S.). Research Site: All experiments were performed with colonies living in a two-frame, glass-walled observation hive (46.0 x 50.5 x 4.5 cm) that was installed in a 6 m x 8 m greenhouse room at the Liddell Field Station at Cornell University in Ithaca, NY (42 degrees 27.6’ N, 76 degrees 26.7’ W). Statement of Data Use: These data are free and available for public use. The authors would appreciate if you would cite the original publication and the published dataset. Suggested citation (manuscript) Ostwald, Madeleine M., Michael L. Smith and Thomas D. Seeley (2016). The behavioral regulation of thirst, water collection and water storage in honey bee colonies. J Exp. Bio. 219: 2156-2165. doi: 10.1242/jeb.139824 Suggested citation (dataset) Ostwald, Madeline M., Smith, Michael L. and Seeley, Thomas D. (2016). Data from: The behavioral regulation of thirst, water collection, and water storage in honey bee colonies (Dataset). eCommons Digital Repository at Cornell University. Retrieved from: http://hdl.handle.net/1813/43783 Figure Legends: The data sheets are organized according to the figures in the manuscript. Fig. 1. Two tests of the importance of water to honey bee colonies in resisting broodnest hyperthermia. Shown are measurements from two colonies (left: Colony A, observed 3 July 2015; right, Colony B, 22 July 2015) in which the broodnest was heated with 100-W lamp throughout the experiment. Water was provided ad libitum at a source outside the hive except during a 2.5-h period in the middle of the experiment. For the statistical analyses, we define: Colony A "Before" = 11:30-12:30 Colony A "During" = 14:00-15:00 Colony A "After" = 17:00-18:00 Colony B "Before" = 10.5-11.5 Colony B "During" = 13:00-14:00 Colony B "After" = 16:00-17:00 Fig. 2. The multifaceted response of a honey bee colony to broodnest hyperthermia. When a colony's broodnest overheats, the workers increase their fanning to ventilate the nest, they partially evacuate the nest (forming a "beard" outside its entrance), and they raise their water intake for evaporative cooling. Shown are measurements from two colonies (left: Colony A, observed 1 July 2015; right, Colony B, 15 July 2015) in which the broodnest was heated with 100-W lamp for 2 h in the middle of the experiment. Water was provided ad libitum at a source outside the hive throughout the experiment. For the statistical analyses, we define: Colony A "Before" = 9:15-10:15 Colony A "During" = 11:50-12:00 Colony A "After" = 13:15-14:15 Colony B "Before" = 13:25-14:25 Colony B "During" = 15:25-16:25 Colony B "After" = 16:5-17:25 Fig. 3. Changes in the behavior of the water collectors in a honey bee colony when it experiences broodnest hyperthermia but is deprived of water so it becomes extremely thirsty. Shown are measurements from Colony A on two days: Left, control treatment on 30 June 2015, when the broodnest was not heated and water was provided ad libitum; Right, experimental treatment on 2 July 2015, when broodnest was heated and water was withheld for 2 h, and then the heating ended and water was provided ad libitum outside the hive. For the statistical analyses, we define: "Before" = 9:00-10:00 "During" = 11:00-12:00 "After" = 13:00-14:00 Fig. 4. Analyses of the crop contents of "hive bees" (bees in the broodnest) and water collectors in various contexts. The sugar concentrations of the bees' crop contents vary depending on how much each bee had fed on the honey stores inside the hive and how much she had obtained water, directly or indirectly, from the water source outside the hive. A. Comparison of hive bees and water collectors at end of a day with broodnest hyperthermia and water available ad libitum. B. Comparison of hive bees at ends of days with and without broodnest hyperthermia; water available ad libitum. C. Comparison of hive bees in the evening and the next morning, following a day with broodnest hyperthermia plus a 2-h period of water deprivation. D. Same comparison as in C, but for water collectors. Fig. 5. Sugar concentrations of liquids found in cells within the broodnest on evenings following various treatments during the day. The sugar concentrations of the cells vary depending on how much the bees who deposited the liquid in each cell had fed on the honey stores inside the hive and how much they had obtained water, directly or indirectly, from the water source outside the hive. A. No broodnest hyperthermia and water available ad libitum. B. Broodnest hyperthermia for 3 h and water available ad libitum. C. Broodnest hyperthermia for 3 h and water withheld during hyperthermia (combined data for three separate treatment days). Supplementary Fig. 1: Changes in the behavior of the water collectors in a honey bee colony when it experiences broodnest hyperthermia but is deprived of water so it becomes extremely thirsty. Shown are measurements from Colony B on two days: Left, control treatment on 17 July 2015, when the broodnest was not heated and water was provided ad libitum; Right, experimental treatment on 16 July 2015, when the broodnest was heated and water was withheld for 2 h, and then the heating ended and water was provided ad libitum outside the hive. For the statistical analyses, we define: "Before" = 10:00-11:00 "During" = 12:00-13:00 "After" = 14:00-15:00 Descriptions of column headers: * Note that not all measurements were taken at all time periods or for all dates and colonies. Colony ID Colony identification used for the two colonies in this study, A and B. Date Date that the data was collected. All data were collected in 2015. YYYY-MM-DD “Before” “During” “After” In performing the statistical tests, we used only the data collected during the last hour of each phase, hence when enough time had passed for each treatment to have an effect on the colony. These values are highlighted in yellow in the excel file. We have also noted the time of each of these three periods for each experiment. Time Time of the data collection, HH:MM, EDT. Unloading Area Temperature (degrees C) Temperature of the unloading area in the observation hive. Broodnest Temperature (degrees C) Temperature of the broodnest area in the observation hive. To do so, we mounted thermocouples (copper-constantan, type T, Omega Engineering, Stamford, Connecticut) in the center of the front side of the lower comb (broodnest). Ambient Temperature (degrees C) Ambient temperature in the greenhouse where the observation hive was kept. To do so, we mounted thermocouples (copper-constantan, type T, Omega Engineering, Stamford, Connecticut) just outside the hive (ambient). Fanning_Avg (average count of the number of bees, from Fanning Count 1, 2, 3) To quantify a colony's responses to hyperthermia, we measured every 15 min its number of bees fanning their wings for nest ventilation and its number of active water collectors. This was done at three locations (Fanning Count 1, 2, and 3). We made three counts every 15 min of the number of bees fanning their wings (part of the nest-cooling process) within an area 20 cm wide x 4 cm high just inside the hive's entrance. Beard (count of the number of bees) We also measured every 30 min the size of the colony's "beard" (the cluster of bees that have evacuated the hive and assembled outside the hive entrance). We counted the number of bees in the beard-like cluster of bees hanging just outside the hive entrance. Counts greater than 100 bees are estimates instead of exact counts. Feeder Weight (g) The weight of the water feeder. Water Collected (g) We measured the mass of water collected by placing the water source (feeder) on a scale (Ohaus triple-beam balance, 0.1 g precision) and recording its weight loss every 30 min. Number of Collectors (count of the number of bees) We made a roll call of the water collectors (all labeled with paint marks for individual identification) that visited the water source during each 15-min segment of an experiment. Thirst (seconds) The time for bees in the colony to drink 0.2 mL of water (a measure of the colony's thirst). We squirted 200 microL of water dyed blue (for better visibility) onto the hive floor 5 cm inside the hive entrance and measured how long it took the bees to drink up this tiny puddle of water. The dye came from a McCormick "Food Color and Egg Dye" set. Behavioral Observations: Walking, Standing, Getting Begged (number of observations) For each trial, we made repeated observations on 5-6 focal water collectors over a period of 4-5 hours; each focal bee had been seen collecting water on the previous day and had been labeled for individual identification. Throughout the 5 h of the experiment, we made a series of 30-s observations of focal water collectors, tracking them one at a time. We cycled through the focal bees, completing one or two 30-s observations of each bee every 10 min except when we were unable to find some individuals, because they were outside the hive or on the back of the comb. During each 30-s observation period, we recorded the in-hive activities of the water-collector bee using the following nine categories: standing, walking, grooming self, grooming other, waggle dancing, begging for food/water, being begged for food/water, giving or receiving food/water, and inserting head into a cell. We defined standing as the act of a bee remaining motionless for at least 5 s, and begging as the extension of a bee’s proboscis toward another bee’s mouthparts for 2 s or less. We counted only contacts lasting 2 s or less because those lasting 3 s or longer are likely to involve transfer of food or water (Farina and Wainselboim, 2001) and we wanted to record instances in which a bee received a request for fluid but she did not respond to it by regurgitating fluid, presumably because she did not have any. We distinguished begging and getting begged by noting which bee (the focal water collector or the bee interacting with her) extended her proboscis; the beggar extends her tongue. We then calculated for each of the nine categories of behavior the proportion of the 30-s observation periods made during a half-hour period in which a particular behavior was seen at least once, i.e., in what fraction of the 30-s observation periods made over a 30- min period that behavior occurred. Thus for each category of behavior the proportion could vary between 0.00 and 1.00. Number of observations in which BEHAVIOR was seen Summary table of the counts that each behavior was seen during the before, during, and after period of the experiment. Number of observations in which BEHAVIOR was NOT seen Summary table of the counts that each behavior was not seen during the before, during, and after period of the experiment. (Number of times behavior was seen + number of times behavior was not seen = total number of observations). Figure Which figure from the manuscript the data refer to. If the figure has multiple parts, the data are organized by part (if necessary). Treatment (each class) There are four classifications for treatment: heat (Yes or No) and water (Yes or No). To heat the broodnest of the hive (Yes Heat), we positioned a 100-W incandescent bulb in a Luxo lamp 10-20 cm from the glass covering the front side of the lower comb. We adjusted the distance between the lamp and the glass throughout the heating period to maintain a broodnest temperature of 40 +/- 2 degrees C, except for in Experiment 1, in which the lamp was kept at a fixed distance, ca. 10 cm from the glass. In some experiments we allowed the bees to collect water (Yes Water), and in other experiments we prevented them from collecting water (No Water). We supplied the bees with a water source (see Seeley, 1995, Fig. 4.5 and 4.6) that was positioned 1 m from the hive and that could be visited by 20+ bees simultaneously. There were no other sources of water in the greenhouse. We trained water collectors to the water source according to standard techniques (von Frisch, 1967). Each bee that collected water was labeled for individual identification with a paint mark on her thorax or abdomen, or both. Bees Sampled We sampled two types of bees, hive bees and water collectors. Hive bees are those that are living in the colony, but have not been observed to collect water. Water collectors are bees that have been observed collecting water (and marked for individual identification). Percent Sugar Concentration of Crop Contents We analyzed the contents of workers’ crops over a range of conditions to determine whether bees store water in their crops in response to broodnest hyperthermia. Bees were sampled in the evening, starting at approximately 20:00 h, on days when we had performed an experiment. We opened the observation hive by removing the glass on the front side, located bees with distended abdomens—generally indicative of a full crop— and removed them one at time from the hive. Using a pair of flat-tipped forceps, we gently squeezed each bee's swollen abdomen until she regurgitated a droplet of liquid onto the glass sample surface of a handheld refractometer (Atago Model No. 050106) and then we measured the % sugar of the liquid. Percent Sugar Concentration of Cells In Colony B, on the evenings of the days when the colony experienced the three treatments described above for the analysis of crop contents, we also analyzed the contents of cells holding liquids in the lower comb. This comb contained most of the colony's broodnest and originally it had contained some of the colony's honey stores. By the time we collected data on comb contents, however, the honey stores were gone in the lower comb because we had not given this colony access to sugar solution for more than 10 days. (There remained, however, large honey stores in the upper comb.) Therefore, when we sampled the cells containing liquid on this frame, we were confident that we were not simply sampling the colony’s honey stores. We sampled up to 20 cells (fewer if we could not find 20 cells containing liquid). We extracted a droplet of the liquid in each cell with a disposable glass pipette and measured the liquid's sugar content using the handheld refractometer.