DOI: 10.5061/dryad.51c59zwgz
title: Data from: Age dominates flight distance and duration, while body size shapes flight speed in Bombus terrestris L. (Hymenoptera: Apidae)

description: Flight plays crucial role in the fitness of insect pollinators, such as
 bumble bees. Despite their relatively large body size compared to their
 wings, bumble bees can fly under difficult ambient conditions, such as
 cooler temperatures. While their body size is often positively linked to
 their foraging range and flight ability, the influence of age remains less
 explored. Here, we studied the flight performance (distance, duration, and
 speed) of aging bumble bee workers using tethered flight mills.
 Additionally, we measured their intertegular distance (ITD) and dry mass
 as proxies for their body size. We found that flight distance and duration
 was predominantly influenced by age, challenging assumptions that age does
 not play a key role in foraging and task allocation. From the age of 7 to
 14 days, flight distance and duration increased six-fold and five-fold,
 respectively. Conversely, body size primarily impacted the maximum and
 average flight speed of workers. Our findings indicate that age
 substantially influences flight distance and duration in bumble bee
 workers, affecting foraging performance and potentially altering task
 allocation strategies. This underscores the importance of considering
 individual age and physiological changes alongside body size/mass in
 experiments involving bumble bee workers.
description: <strong>Experimental overview</strong>
 The experiment was designed based a pilot study, in which the
 flight performance of differently aged workers from two colonies had been
 examined in the previous year. In this study, we repeatedly measured the
 individual flight performance of 61 workers from four colonies (n = 14,
 18, 8 and 21 workers per colony) at the age of 7, 14 and 21 days. Due to
 occasional difficulties in locating every marked worker on each day of
 measurement or potential mortality (n = 13/77), or instances where a few
 workers had lost their tags during flight measurement (n = 5/77), these
 individuals were excluded from data analysis. We selected workers with a
 wet body mass ranging from 100 mg to 320 mg, primarily during the colony’s
 exponential growth phase.  <strong>Bumble bee
 husbandry</strong> We used four commercial,
 queenright <em>Bombus terrestris</em> colonies (Natupol
 Research Hives, Koppert B.V., Netherlands) with approximately 20-30
 workers. They were housed in their standard plastic nest boxes (27 [l] x
 24 [w] x 14 [h] cm). Each colony was connected to a small foraging arena
 (60 x 40 x 28 cm), where bees were required to perform foraging flights to
 access <em>ad libitum</em> 40% w/v sucrose solution provided
 through 50 mL gravity feeders. Additionally, colonies were provided with
 3-6 g of pollen candy (2:1 honey bee collected organic pollen: 75% sucrose
 syrup) daily, depending on their consumption. Colonies were kept under
 laboratory conditions with a relative humidity of about 40 % RH and an
 average room temperature of 23 ± 1 °C. Foraging areas were illuminated by
 two flicker free daylight-like LEDs (each 2400 lm, CRI98, 5500 K,
 True-Light International GmbH, Germany) under 14:10 h light:dark regime,
 but nest boxes were kept dark by covering them with cardboard. The
 foraging arenas served to allow aging bees to ‘practice’ flying and become
 accustomed to the same lighting conditions used during tests (described
 below). <strong>Tagging and
 marking</strong> Newly emerged workers (&lt;
 1 d old), identified by their silvery appearance and unfurled wings [43],
 were collected from their colonies. Their sex was determined by counting
 antennal segments (females have 12, males 13), under a stereo microscope.
 Then bees were immobilized with metal pins without harming them to
 carefully shave off the thorax hairs between the tegula, where a circular
 stainless-steel tag (Æ 2 mm, thickness = 0.1 mm, weight (mean ± sd) = 2.43
 ± 0.02 mg, n = 20, Sartorius Micro SC2, Sartorius AG, Germany) was
 attached using superglue (Supergel, UHU GmbH & Co. KG, Germany).
 The weight of the tag accounted for only 1% of the mean weight of all
 tested bees (231.84 ± 52.62 mg, analytic balance A210P-OD1, Sartorius AG,
 Germany), significantly lighter than typical pollen and nectar loads
 carried by foragers (up to 90% of body weight [28]). Therefore, we assume
 that this tagging method is unlikely to significantly affect flight
 performance as measured here. These metal tags were colour coded to
 differentiate cohorts. For reliable identification of each individual bee,
 the middle legs were colour coded using water-based permanent paint
 markers (5M Uni-Posca, Mitsubishi pencil, Japan). Afterwards, tagged bees
 were placed in a separate plastic cup for 30-60 min before returning them
 to their natal colony. Although precautions were taken
 to guarantee that attached tags would not interfere with wing movements,
 tag positions were measured using a digital microscope (figure
 S1<em>a</em>, CHX-500F, Keyence GmbH, Germany) on frozen
 specimens at the end of the experiment. The tag deviation from the centre
 between the tegulae was calculated, but tag position did not significantly
 affect their flight performance (figure
 S1<em>b,c</em>). <strong>Flight mill
 setup</strong> We used four tethered air flight
 mills (figure <em>S2a</em>), similar to previously described
 setups and methods [25, 41, 42]. The core of each flight mill is a
 lightweight and counter-weighted arm (length 32 cm) that floats by
 magnetic levitation and a needle that is inserted into a low-friction
 Teflon bearings at the centre of the arm. Individual bees were attached to
 a magnet (Æ 2 mm, 4 mm long) on one end of the arm and counter-balanced on
 the other arm, enabling tethered flights with their own power. An optical
 sensor transmitted a voltage pulse every half rotation (flight distance of
 50 cm), recorded to a PC using the software <em>guiBee</em>
 [44]. Data extraction and calculations of flight distance, duration, and
 speeds were executed using the RScript
 <em>FlightMillDataExtraction</em> [45] in R (version 4.3.0).
 Each flight mill was positioned at the centre of a plastic cylinder (Æ 46
 cm), keeping about 7 cm distance between the bee and the cylinder wall.
 The inside was decorated with 2.5 cm wide black and white vertical stripes
 continuously printed on paper to provide consistent visual feedback [46].
 The walls also prevented interference from neighbouring flight mills and
 reduced any potential impacts of air currents [47]. The flight mills were
 illuminated by four flicker free daylight-like LEDs (each 2400 lm, CRI98,
 5500 K, True-Light International GmbH, Germany) from a height of 70
 cm. <strong>Flight
 trials</strong> Marked workers were gently
 collected from each colony in the morning using tweezers at the age 7, 14
 and 21 days (i.e. each bee was repeatedly measured). They were kept
 separately per colony in a metal cage (9.5 x 8.5 x 5 cm) with <em>ad
 libitum</em> access to 40% w/v sucrose solution. After collection,
 workers were individually separated into flat-bottom glass vials (10 mL,
 50 x 22 mm) with mesh lids, containing a 45 x 15 mm piece of cardboard to
 absorb any faeces. For 20 min each bee was individually fed with 40% w/v
 sucrose solution to satiation through the mesh of the lid. Subsequently,
 individual bees were weighed (d = 0.1 mg, analytic balance A210P-OD1,
 Sartorius AG, Germany) and attached to the magnet on the one side of the
 flight mill arm and kept in place on a launch platform. A counterweight
 (to ± 10 mg) was attached on the other side of the flight mill arm. The
 bees were then allowed to calm down and rest in the dark for 20 min by
 covering each flight mill with thick cardboard. Each bee was then
 positioned in the direction of flight and the launch platform was quickly
 removed to initiate flight (figure <em>S2b,c</em>; video
 <em>S1</em>). When bees stop flying, they would raise their
 wings and extend their legs, which resulted in a slowing of flight mill
 rotation. In addition to direct observations, stops can be detected in the
 raw data using our R script described above [45]. When a bee stopped
 flying (or did not initiate flight), it was allowed to rest on a handheld
 plastic Petri dish (Æ 46 cm) for approximately 20 s. Each bee was allowed
 4 stops, i.e. 5 flight starts. We decided to allow multiple stops and pool
 individual flight data based on field observations showing that foragers
 periodically pause [48], and previous flight mill studies indicating that
 flight durations tend to be short after three stops [25, 49]. Our pilot
 study was consistent with those findings. To avoid biases in our dataset,
 we refrained from setting minimum or maximum flight durations or
 distances, even if a bee would not fly (e.g. one bee aged 21 days flew 0
 m; see data in electronic supplementary material). The temperature during
 all flight was recorded at 5 min intervals (RC-5 temperature data logger,
 Elitech Ldt., UK) to calculate the average flight temperature and account
 for slight room temperature differences. At the end of each flight trial,
 bees returned to their natal colonies. After the last flight trial at age
 of 21 d, bees were frozen and stored at -20°C until further
 analysis. <strong>Measuring intertegular distance
 and dry mass</strong> In addition to the
 evaluation of the tag position, the intertegular distance (ITD) for each
 bee was measured using a digital microscope (figure
 S1<em>a</em>, CHX-500F, Keyence GmbH, Germany). The ITD serves
 as a proxy for workers body size, along with their dry weight (figure
 <em>S1b</em>) [50]. Prior to measuring their dry weight, the
 sternites (ventral abdominal segments) of each individual bee were cut
 open from the stinger to the fourth sternite without damaging their guts.
 The bees were then individually dried at 60°C for 3 d in drying cabinet
 (U40, Memmert GmbH & Co. KG, Germany) and subsequently weighed (d
 = 0.1 mg, analytic balance M-Pact AX224, Sartorius GmbH,
 Germany). <strong>Statistical
 analyses</strong> All statistical analyses and
 data visualizations were performed using R version 4.3.2 [51]. The
 complete code and output are provided in the electronic supplementary
 material. Briefly, to analyse the effects of the fixed factors age and
 body size (using dry mass as a proxy, figure <em>S1b</em>) as
 fixed factors and their potential interactions on the response variables
 flight distance, flight duration, average and maximum flight speed,
 generalized linear mixed effect models (GLMMs) were ran using the
 <em>glmmTMB</em> package. Flight distances were
 log-transformed using log<sub>10 </sub>(x + 1) to improve
 model fit based on gaussian data distribution. To account for repeated
 measures of individual bee flight performance, Bee ID was included as a
 random factor. The covariates colony (figure S4) and temperature during
 flight (figure S3) were additionally included as random factors. Model
 selection was performed based on the Akaike information criterion (AIC)
 and likelihood ratio tests. The final models were compared with their
 respective null-models. Model assumptions and dispersion of the data were
 checked using the <em>DHARMa</em> package [52]. Significance
 (p &lt; 0.05) of model terms was determined using the
 <em>Anova</em> function of the <em>car
 </em>package [53]. Pairwise comparisons between age groups were
 conducted using the function <em>emmeans </em>[54] with
 Bonferroni correction. The individual flight improvement was analysed
 using <em>chisq-test</em> function to perform a Pearson’s
 <em>χ</em><sup>2</sup> test for count data with
 Yate’s continuity correction. The <em>cor.test</em>function
 was used to further describe the relationship between the flight duration
 and distance, dry mass and flight distance, dry mass and flight duration,
 and dry mass with mean and maximum speeds within each age class.
  
description: # Age dominates flight distance and duration, while body size shapes
 flight speed in *Bombus terrestris* L. (Hymenoptera: Apidae) ##
 Description of the data and file structure The data table
 contains the entire dataset on which our statistical analysis is based.
 **Data2023.csv** Variables are as follows: Date = date on which the bee
 was tested in flight mill Bee = bee ID Age = bee age (days) as numerical
 AGE = bee age (days) as factor Colony = colony name BeeMass = bee weight
 before flight (mg) Start = flight start time (h:min:s) Distance = flight
 distance (m) Duration = flight duration (s) Speed = mean flight speed
 (m/s) throughout the entire flight MaxSpeed = maximum flight speed (m/s)
 throughout the entire flight Temp = mean room temperature throughout the
 entire flight  ITD = intertegular distance (mm) TagPos = deviation of tag
 position from the center (%) (see methods for a detailed description)
 WetMass = sample bee weight (mg) after storage at -20°C DryMass = sample
 bee weight (mg) after drying sample at 60°C for 3 days Count = number of
 flights performed by each bee (used for filtering the data set) ##
 Code/Software The RMD file includes the complete R code and output,
 ensuring full transparency of our statistical analysis.