DOI: 10.5061/dryad.w3r22814f
title: Age and atitude: How longevity influences cognitive biases in honeybee workers

description: This study examines how life expectancy influences cognitive biases in
 honeybee workers (Apis mellifera) and their emotional states affecting
 decision-making. Recent research indicates that emotions impact behavior
 and cognition in various species, including invertebrates. We conducted
 three experiments to manipulate honeybee lifespan and assess its effects
 on judgment biases using a classic judgment bias paradigm. In the first
 experiment, we shortened the lifespan of bees through CO2 anesthesia or
 thoracic puncture. The second experiment involved feeding workers Nosema
 sp. spores to induce infection and evaluate its impact on survival and
 judgment. The final experiment focused on "rebel workers," who
 have naturally longer life expectancies. We conditioned bees to associate
 specific odors with rewards and punishments, then measured their responses
 to ambiguous stimuli. Results showed that bees subjected to
 life-shortening treatments exhibited more optimistic cognitive biases, as
 indicated by an increased likelihood of extending their proboscis to
 ambiguous odors. Conversely, rebel workers with longer lifespans displayed
 more pessimistic biases, indicating a cautious foraging approach. Overall,
 our findings suggest that honeybee decision-making is closely tied to
 their longevity, with shorter-lived individuals adopting riskier
 strategies compared to their longer-lived counterparts.
description: # Age and atitude: How longevity influences cognitive biases in honeybee
 workers Dataset DOI: [10.5061/dryad.w3r22814f](10.5061/dryad.w3r22814f) ##
 Description of the data and file structure Lifespan-dependent cognitive
 biases in honeybee workers **Summary** This study investigates how
 honeybee lifespan influences their cognitive biases and emotional states,
 focusing on decision-making processes. The research was conducted at two
 locations in Krakow, Poland—the Institute of Environmental Sciences and
 the University of Agriculture—between May 2018 and June 2023. A total of
 14 honeybee colonies, each containing 20,000 to 40,000 workers, were used
 across three experiments. The first experiment aimed to shorten the
 bees' lifespan through two methods: CO2 anesthesia and thoracic
 injury. Worker bees were collected from subunit B of their colonies during
 the afternoon, after being separated from non-foraging bees in the
 morning. The bees were then randomly assigned to three groups: untreated
 controls, CO2-anesthetized for 30 minutes, or injured with a needle
 puncture in the thorax. The bees underwent lifespan assessment in
 laboratory cages, followed by judgment bias tests, and control tests to
 observe spontaneous responses. These experiments took place at the apiary,
 with sample collection and testing occurring in designated laboratory
 settings. The second experiment involved infecting worker bees with Nosema
 spores to induce a reduction in lifespan. Similar to the first, bees were
 collected from the colonies, and their survival was monitored after
 infection. The purpose was to evaluate how infection-related lifespan
 shortening affects cognitive biases. The third experiment focused on
 "rebel workers," a naturally longer-lived group within the
 colony, to compare their behavior and decision-making patterns with those
 of shorter-lived bees. These rebel workers were also subjected to judgment
 bias assessments. The judgment bias paradigm was employed to measure
 emotional states indirectly. Bees were trained to associate specific odors
 with rewards (positive stimuli) or punishments (negative stimuli). During
 testing, responses to ambiguous stimuli—intermediate odors—were recorded.
 Bees that responded optimistically (proboscis extension to ambiguous
 stimuli) were interpreted as exhibiting positive or risk-taking biases,
 while more cautious responses indicated negative biases. Results showed
 that bees with shortened lifespans, whether through injury or infection,
 exhibited more optimistic biases, tending to respond more readily to
 ambiguous stimuli. Conversely, rebel workers with longer lifespans
 displayed more pessimistic biases, suggesting a cautious foraging
 approach. Overall, the findings demonstrate that honeybee decision-making
 and emotional states are closely tied to their lifespan. Shorter-lived
 bees tend to adopt riskier strategies, possibly favoring immediate gains,
 whereas longer-lived bees are more cautious, reflecting different
 affective states influenced by their life expectancy. These insights
 highlight the complex relationship between longevity and cognition in
 social insects. **How to read the files from the Supplementary Data:** 1)
 Each file in the Supplementary Data corresponds to a separate experiment.
 Specifically, the data for: Experiment 1 is in *data_file_Experiment
 1_longevity.csv*, *data_file_Experiment 1_jugment_bias_test.csv,
 data_file_Experiment 1_spontanous_responses.csv*, Experiment 2 in
 *data_file_Experiment 2_longevity.csv*, *data_file_Experiment
 2_jugment_bias_test.csv, data_file_Experiment 2_spontanous_responses.csv,
 data_file_Experiment 2_nosema_infection.csv*, and Experiment 3 in
 *data_file_Experiment 3_longevity.csv, data_file_Experiment
 2_jugment_bias_test.csv, data_file_Experiment 3_spontanous_responses.csv,
 data_file_Experiment 2_ovariole_number.csv* 2) In each file, the
 "Longevity" - *data_file_Experiment 1_longevity,
 data_file_Experiment 2_longevity, data_file_Experiment 3_longevity* 
 presents lifespan data for individual bees from different colonies and
 treatments. The columns include: the colony number from which each bee
 originated, the treatment applied (for Experiment 1: control, injury, and
 anesthesia with CO₂; for Experiment 2: control and infected workers; for
 Experiment 3: control [normal workers] and rebel workers), the individual
 number (with 50 bees per colony and treatment in each case), the lifespan
 in days and censored data were 0 means that bess alive at the end of the
 study or lost to follow-up, while 1 usually indicates that the event of
 interest (e.g., death) has occurred. 3) In each file the
 "Judgment_Bias_Test" - *data_file_Experiment
 1_jugment_bias_test* , *data_file_Experiment 2_jugment_bias_test*,
 *data_file_Experiment 3_jugment_bias_test* presents data from the last
 conditioning session as well as the results of the judgment bias test. The
 columns include (from left to right): the colony from which the bees
 originated, the individual bee number, and the treatment to which the bee
 was subjected (see point above). The next column, "Odour CS+,"
 describes the scent that was paired with the reward (ratio 9:1). The
 following two columns display the results from the last conditioning
 sessions, where "A+" (with a scent combination of 9:1) indicates
 a response to the signal associated with the reward, and "B-"
 (with a scent combination of 1:9) indicates a response to the signal
 associated with an unpleasant event. The columns labeled with ratios (9:1,
 7:3, 1:1, 3:7, 1:9) show the bees' responses (proboscis extension
 reflex) to the exposed scents during the judgment bias test. Additionally,
 there is a side section showing the number of individuals responding to
 each scent by extending their proboscis in each colony and treatment, as
 well as the total responses across all colonies. 4) Each file contains a
 "Spontaneous responses” *data_file_Experiment 1_spontanous_responses,
 data_file_Experiment 2_spontanous_responses, data_file_Experiment
 3_spontanous_respons* which serves as a control for the bias tests
 themselves. In this section, bees are exposed to scent mixtures without
 prior conditioning to CS+ and CS-. The purpose of this control is to
 determine whether the bees respond spontaneously to any of the scents used
 during conditioning, possibly due to previous exposure. The first column
 provides information about each individual bee (bee number), followed by
 the treatment it received (see point 3), and the colony from which it
 originated. The "Odour CS+" column describes the scent that was
 used in a higher proportion during the test (ratio 9:1). The subsequent
 columns, ranging from 9:1 to 1:9, show the responses of individual bees to
 the exposed scents in the form of binary data (0 or 1), indicating whether
 the proboscis extension reflex was observed. 5) The file
 "*data_file_Experiment 2_nosema_infection*" presents data on how
 individuals in both groups were infected with Nosema sp. The first column
 contains information about the colony of origin of the tested workers. The
 second column indicates the treatment (whether the individual was a
 control or infected with Nosema through feeding). The third column
 specifies the individual’s number. The last column reports the number of
 Nosema spores, which is an estimate of the spores per individual after
 normalization. 6) The file "*data_file_Experiment
 3_ovariole_number*" includes data on the number of ovarioles in the
 tested control and rebel individuals. These data were collected to ensure
 that we are dealing with two distinct groups of bees. The table provides,
 from left to right, the individual bee's number, the colony it
 originated from, the treatment (whether it was assigned to control workers
 or rebel workers), and the number of ovarioles in the ovaries of each
 individual. ### Files and variables #### File:
 data_file_Experiment_1_longevity.csv **Description:**  ##### Variables *
 colony: number of colony * treatment: the treatment applied: control,
 injury, and anesthesia with CO₂; * no. of bee: number of tested bee *
 longevity (days): the number of days the bee survived * censored data:
 were 0 means that bess alive at the end of the study or lost to follow-up,
 while 1 usually indicates that the event of interest (e.g., death) has
 occurred. #### File: data_file_Experiment_3_ovariole_number.csv
 **Description:**  ##### Variables * nr of bees : number of tested bee *
 colony: number of colony * treatment: normal or rebel workers  * no. Of
 ovarioles: the sum of ovarioles from two ovaries #### File:
 data_file_Experiment_2_longevity.csv **Description:**  ##### Variables *
 colony: number of colony * treatment: the treatment applied: healthy bees
 and infected with Nosema apis spores * no. of bee: number of tested bee *
 longevity (days): the number of days the bee survived * censored
 data: number of colony: were 0 means that bess alive at the end of the
 study or lost to follow-up, while 1 usually indicates that the event of
 interest (e.g., death) has occurred. #### File:
 data_file_Experiment_2_nosema_infection.csv **Description:**  #####
 Variables * colony: number of colony * treatment: the treatment applied:
 healthy bees and infected with Nosema apis spores * no. of
 individuals: number of tested bee * no. Of nosema spores per digestive
 track: number of spores in the digestive track #### File:
 data_file_Experiment_2_spontanous_responses.csv **Description:**  #####
 Variables * no.of bees: number of tested bee * treatment: the treatment
 applied: healthy bees and infected with Nosema apis spores *
 colony: number of colony * odour: type of odour tested as CS+  * 9:1: show
 the responses of individual bees to the exposed scents in the form of
 binary data (0 or 1) * 7:3: show the responses of individual bees to the
 exposed scents in the form of binary data (0 or 1) * 1:1: show the
 responses of individual bees to the exposed scents in the form of binary
 data (0 or 1) * 3:7: show the responses of individual bees to the exposed
 scents in the form of binary data (0 or 1) * 1:9: show the responses of
 individual bees to the exposed scents in the form of binary data (0 or 1)
 #### File: data_file_Experiment_3_jugment_bias_test.csv **Description:** 
 ##### Variables * colony: number of colony * no. Of bees:  number of
 tested bee * treatment:  the treatment applied: normal and rebel workers 
 * odour CS+ (9:1): type of odour tested as CS+  * CS+: show the responses
 of individual bees in the last session * CS-:  show the responses of
 individual bees in the last session * 9:1: show the responses of
 individual bees to the exposed scents in the form of binary data (0 or 1)
 * 7:3: show the responses of individual bees to the exposed scents in the
 form of binary data (0 or 1) * 1:1: show the responses of individual bees
 to the exposed scents in the form of binary data (0 or 1) * 3:7: show the
 responses of individual bees to the exposed scents in the form of binary
 data (0 or 1) * 1:9: show the responses of individual bees to the exposed
 scents in the form of binary data (0 or 1) #### File:
 data_file_Experiment_3_longevity.csv **Description:**  ##### Variables *
 colony: number of colony * treatment:  the treatment applied: normal and
 rebel workers  * no. of bee: number of tested bee * longevity (days): the
 number of days the bee survived * censored data: were 0 means that bess
 alive at the end of the study or lost to follow-up, while 1 usually
 indicates that the event of interest (e.g., death) has occurred. ####
 File: data_file_Experiment_3_spontanous_responses.csv **Description:** 
 ##### Variables * no.of bees: number of tested bee * treatment: the
 treatment applied: normal and rebel workers * odour: type of odour tested
 as CS+  * 9:1: show the responses of individual bees to the exposed scents
 in the form of binary data (0 or 1) * 7:3: show the responses of
 individual bees to the exposed scents in the form of binary data (0 or 1)
 * 1:1: show the responses of individual bees to the exposed scents in the
 form of binary data (0 or 1) * 3:7: show the responses of individual bees
 to the exposed scents in the form of binary data (0 or 1) * 1:9: show the
 responses of individual bees to the exposed scents in the form of binary
 data (0 or 1) #### File: data_file_Experiment_2_jugment_bias_test.csv
 **Description:**  ##### Variables * colony: number of colony * no. Of
 bees:  number of tested bee * treatment:  the treatment applied:  healthy
 bees and infected with Nosema apis spores * odour CS+ (9:1): type of odour
 tested as CS+  * CS+: show the responses of individual bees in the last
 session * CS-:  show the responses of individual bees in the last session
 * 9:1: show the responses of individual bees to the exposed scents in the
 form of binary data (0 or 1) * 7:3: show the responses of individual bees
 to the exposed scents in the form of binary data (0 or 1) * 1:1: show the
 responses of individual bees to the exposed scents in the form of binary
 data (0 or 1) * 3:7: show the responses of individual bees to the exposed
 scents in the form of binary data (0 or 1) * 1:9: show the responses of
 individual bees to the exposed scents in the form of binary data (0 or 1)
 #### File: data_file_Experiment_1_jugment_bias_test.csv **Description:** 
 ##### Variables * colony: number of colony * no. Of bees:  number of
 tested bee * treatment:  the treatment applied: control, injury, and
 anesthesia with CO₂; * odour CS+ (9:1): type of odour tested as CS+  *
 CS+: show the responses of individual bees in the last session *
 CS-:  show the responses of individual bees in the last session *
 9:1: show the responses of individual bees to the exposed scents in the
 form of binary data (0 or 1) * 7:3: show the responses of individual bees
 to the exposed scents in the form of binary data (0 or 1) * 1:1: show the
 responses of individual bees to the exposed scents in the form of binary
 data (0 or 1) * 3:7: show the responses of individual bees to the exposed
 scents in the form of binary data (0 or 1) * 1:9: show the responses of
 individual bees to the exposed scents in the form of binary data (0 or 1)
 #### File: data_file_Experiment_1_spontanous_responses.csv
 **Description:**  ##### Variables * no.of bees: number of tested bee *
 treatment: the treatment applied: control, injury, and anesthesia with
 CO₂; * odour: type of odour tested as CS+  * 9:1: show the responses of
 individual bees to the exposed scents in the form of binary data (0 or 1)
 * 7:3: show the responses of individual bees to the exposed scents in the
 form of binary data (0 or 1) * 1:1: show the responses of individual bees
 to the exposed scents in the form of binary data (0 or 1) * 3:7: show the
 responses of individual bees to the exposed scents in the form of binary
 data (0 or 1) * 1:9: show the responses of individual bees to the exposed
 scents in the form of binary data (0 or 1) ## Code/software **How the
 analyses were conducted in Statistica 13.** **Longevity** The data were
 sourced from the following files: data_file_Experiment 1_longevity for
 Experiment 1, data_file_Experiment 2_longevity for Experiment 2, and
 data_file_Experiment 3_longevity for Experiment 3. From the Statistica 13
 interface, the following steps were taken: select the tab – Statistics –
 Survival Analysis – Multiple Sample Comparison – Variables (survival
 times, censor indicator, grouping variable), where we input data from the
 "longevity" column as the lifespan. The censored data is the
 censor indicator, and the grouping variable is the treatment or family for
 all three experiments – then click OK. We assign codes for complete
 observations – our censoring data as 1, and for censored observations
 (i.e., organisms that survived the experiment but are not included in our
 study) as 0. Then, click on "Codes for layers," select
 "All," and click OK. Next, click OK again to obtain the results.
 If more than two curves were compared, a post hoc test should be
 performed, i.e., compare each curve with every other. In this case, go to
 – Statistics – Survival Analysis – Compare Two Samples. We provide the
 data similarly as described above, only adding group codes such as
 Control, Injury, etc., then click OK. From the available options, select
 the log-rank test for comparing the curves, and the results will be
 generated. **Jugment bias test** The data were sourced from the following
 files: data_file_Experiment 1_judgment_bias_test for Experiment 1,
 data_file_Experiment 2_judgment_bias_test for Experiment 2, and
 data_file_Experiment 3_judgment_bias_test for Experiment 3. From the
 Statistica 13 interface, the following steps were taken: select the tab –
 Statistics – Advanced Models – Generalized Linear and Nonlinear Models –
 Logit Model – Variables – .Dependent Variable (PER responses at the
 appropriate concentration in the test, e.g., 9:1 or 3:7, depending on
 which we wanted to test at that moment); for qualitative predictor
 variables, select the two "colony" and "treatment" (as
 named in each experiment) – click OK. Next, select the codes for the
 dependent variable, click "All" and then OK, and do the same for
 the qualitative predictor codes – click "All" and then OK.
 Finally, click on "Summary of All Effects" to obtain the
 statistics. **Nosema spores infection** The data were sourced from the
 following files: Experiment 2, and data_file_Experiment 2\_
 nosema_infection From the Statistica 13 interface, the following steps
 were taken: select the tab – Statistics – Nonparametric – Multiple
 Independent Sample Comparison. The dependent variable chosen was “Number
 of Nosema spores per digestive tract,” and the independent variables were
 colony and treatment. Then, click OK. Click on "Codes" – select
 all, then OK. Next, click on the ANOVA Kruskal-Wallis summary and the
 median test. When the results turn out to be statistically significant, go
 back to the previous interface and perform multiple comparisons of rank
 means for all samples, and obtain the results. **Ovariole Number** The
 data were sourced from the following files: data_file_Experiment 3\_
 ovariole number. From the Statistica 13 interface, the following steps
 were taken: select the tab – Statistics – Advanced Models – Generalized
 Linear and Nonlinear Models – Log Poisson Model – then click OK. The
 variables included the dependent variable – number of ovarioles, and the
 qualitative predictors were colony and treatment. Next, select the factor
 codes – all, then click OK. Then, click OK again, provide a summary of all
 effects, and obtain the results.