DOI: 10.5061/dryad.9ghx3ffdv
title: Data from: Bumble bees damage plant leaves and accelerate flower production when pollen is scarce

description: Maintaining phenological synchrony with flowers is a key ecological
 challenge for pollinators that may be exacerbated by ongoing environmental
 change. Here, we show that bumble bee workers facing pollen scarcity
 damage leaves of flowerless plants and thereby accelerate flower
 production. Laboratory studies revealed that leaf-damaging behavior is
 strongly influenced by pollen availability and that bee-damaged plants
 flower significantly earlier than undamaged or mechanically damaged
 controls. Subsequent outdoor experiments showed that the intensity of
 damage inflicted varies with local flower availability; furthermore,
 workers from wild colonies of two additional bumble bee species were also
 observed to damage plant leaves. These findings elucidate a feature of
 bumble bee worker behavior that can influence the local availability of
 floral resources.
description: Plants and insects:<em> Bombus terrestris</em>
 colonies were obtained commercially from the Biobest group. These included
 queenless microcolonies and founding queenright hives. Each queenless
 “mini-hive” box contained approximately 30 workers. <br>
 <em>Brassica nigra, B. oleracea, Solanum elaeagnifolium, S.
 melongena and S. lycopersicum </em>plants were grown from seed in a
 climate chamber (Kälte 3000, RH 60–80%, LD 16:8). <br>
 Flowering time studies: Plants (<em>B.
 nigra </em>and <em>S. lycopersicum</em>) in each
 flowering-time experiment were assigned to one of three treatment groups:
 control (undamaged), bee-damaged, and mechanically damaged (S.
 lycopersicum, n=20 per treatment; B. nigra, n=10 per treatment). At the
 start of the experiment plants were of uniform age (S. lycopersicum, 6
 weeks old; B. nigra, 9 weeks old). Plants in the bee damaged treatments
 were placed together with a pollen-deprived B. terrestris colony inside
 a<br> mesh enclosure  inside a climate chamber (Kälte 3000, RH
 60–80%, LD 16:8). To standardize damage, plants were regularly monitored
 and removed when bees had made a target number of leaf holes (5 for S.
 lycopersicum; 5–10 for B. nigra). Each plant in the mechanical damage
 treatments was then paired with a plant in thecorresponding bee-damage
 treatment, and we used a metal forceps and razor to replicate the damage
 pattern observed in the bee-damaged plant as closely as possible. Plants
 from both damage treatments, as well as undamaged controls, were then
 placed at randomly selected positions within a climate chamber (Kälte
 3000, 60–80% RH, LD 16: 8) and moved to new<br> random positions
 every two days. Plants were monitored daily, and flowering time was
 reported as days elapsed since treatment (i.e., bee-damage, mechanical
 damage, no-damage). <br> Laboratory pollen deprivation
 study: We assessed damage behavior by Bombus terrestris
 microcolonies that were either given abundant pollen resources within the
 hive (pollensatiated) or deprived of pollen (pollen-deprived). These
 microcolonies were queenless, but contained psuedoqueens that produced
 haploid larvae. Queenless microcolonies (with psuedoqueens) are commonly
 used for behavioral experiments with B. terrestris and have been shown to
 serve as good models for investigating a variety of behavioral,
 developmental, and ecological questions. Colonies were then given three
 days (days 2–4) to adjust to the treatments prior to being exposed to
 plants. During<br> this adjustment period (and throughout the
 experiment). On day 5, two non-flowering B.nigra plants (5-7 weeks old)
 were placed into each cage. Plants were replaced every 24h over three days
 (days 5–7) and we recorded the proportion of leaves damaged for each
 plant. On day8, all plants were removed and the treatment (pollen satiated
 vs pollen deprived) for each colony was reversed. Colonies were then given
 three days (days 9–11) to adjust to the new treatments, before again being
 exposed to plants (on days 12–14, during which we replaced plants and
 recorded damage as described above). Hives were weighed on days when the
 treatments were implemented or reversed (days 1 and 8) and monitored
 during the subsequent adjustment periods. At the end of the experiment
 (Day 15), final measurements were taken and hives were frozen and kept for
 dissection.<br> Roof study 2018:<br> Phase
 one (March 26th – May 25th): Bombus terrestris microcolonies were placed
 on a rooftop (on the Zentrum campus of ETH Zurich; Zurich CH) near a focal
 patch of 36 non-flowering plants of 6 different plant species (initially,
 Alliaria petiolata, Alyssum montanum, Aurinia saxatilis, Brassica nigra,
 Brassica oleracea, and Isatis tinctoria) (Fig. S5a). A. montanum was
 replaced by Fragaria vesca early in the experiment (13th April 2018)
 because bee-inflicted damage was not visible on the very small leaves of
 A. montanum. Bumblebee colonies were deprived of pollen for 3 days prior
 to the experiments, and received no supplemental pollen during the
 experiment, but had access to the Biogluc sugar solution within the hive
 throughout the study. Colonies were replaced approximately every 3 weeks,
 in accordance with the timeframe for effective pollination estimated by
 the commercial distributor. Plants were replaced at the same time. A total
 of three colonies were used during phase one. Damage was scored as the
 number of new leaf-holes produced each day (to facilitate tracking,
 damaged leaves, having known numbers of pre-existing holes, were
 individually marked with small green metal rings).<br> On sampling
 days (all weekdays except when it rained) we also recorded the number of
 bumblebees entering and exiting the hive and the number of returning
 foragers with and without pollen during three 60-minute periods per day
 (beginning at ~09:00, ~13:00, and ~16:00).<br> Phase two (June 4th-
 July 20th): Starting on June 4, we repeated the experimental design of
 phase one, except that we additionally placed a patch of 100 flowering
 plants, comprising four species (Brassica nigra, Fragaria vesca, Isatis
 tinctoria, Alliaria petiolata), separated by ~1 m<br> from the focal
 patch of 36 non-flowering plants. Additionally, on sampling days we
 recorded the number and species identity of any bees visiting our
 flowering patch, as well as those visiting the non-flowering patch, during
 three 60-minute periods per day (beginning at ~09:00, ~13:00, and ~16:00),
 along with the other observations described for phase one. Plants in the
 flowering patch that started fruiting were replaced. As in phase one, bee
 colonies (and plants in the non-flowering patch) were replaced every three
 weeks; two colonies were employed during this phase.<br>
 2019 Rooftop experiment (May 29th – July
 13th):<br> Sixteen founding queenright Bombus terrestris
 colonies were equally divided between two treatments (non-flowering and
 flowering) and placed on separate rooftops, including the rooftop used
 during the 2018 study (Roof 1) and another nearby (Roof 2; ~200m from Roof
 1). Colonies on both Rooftops (n = 8 per roof) were placed near focal
 patches of 300 non-flowering plants comprising 7 different species
 (Armoracia rusticana, Aurinia saxatilis, Brassica nigra, Fragaria vesca,
 Isatis tinctorial, Solanum lycopersicum and Solanum melongena). No
 flowering plants were present on Roof 1, but Roof 2 had a rooftop garden
 planted with wildflowers (4.5 x 7m; ~20m from the focal patch); in
 addition, we placed 30 additional flowering border plants (Antirrhinum ca.
 3 months old) near the focal patch on Roof 2 (Fig. S5b). Colonies were not
 given access to any supplemental pollen or nectar resources, except on
 June 17th and 27th, when all colonies on both rooftops were provided with
 Biogluc sugar solution within the hive for 24h<br> to mitigate the
 effects of heavy rain and hot weather (respectively). Additionally,
 colonies were provided with external water feeders to provide relief to
 the hive during hot weather. Colonies were weighed and measured twice a
 week (after sunset) to monitor queen presence and hive development. The
 rooftop garden (on Roof 2) was mowed on June 29, and we also removed the
 other flowering plants on Roof 2 on this date, after which point no
 flowering plants were present on either roof. Colonies and focal
 (non-flowering) plant patches were kept in place until the onset of the
 reproductive switch point (measured as the appearance of the first drone).
 All hives were removed for continued monitoring in climate chambers on
 experimental day 45. Damage was monitored, as in the 2018 experiment, for
 150 randomly selected non-flowering plants in<br> each focal patch.
 Plants in the focal patches were replaced after three weeks to ensure they
 remained in a non-flowering state. Concurrently, we conducted a transect
 study between 11th March- 26th July to roughly estimate surrounding
 flowering resources. Flowering plants were identified along two 1 km long
 transects (with North-South and East-West axes) originating at Roof 1.
 Surveys were conducted every two weeks, during which we identified all
 flowering<br> plants visible within 5m of the transect (using the
 iNaturalist app).  <br> Statistical
 analyses: All statistical analyses were carried out using R: A
 language and Environment for Statistical Computing version 2.15.3 (R Core
 Team 2013). To test for effects of the three damage treatments on
 flowering time, we used generalized additive models (GAMs) using the “gam”
 function in the R package mgvc. To account for the<br> fact that six
 plants in the B. nigra experiment (4 in the undamaged control treatment
 and 2 in the mechanically damaged treatment) did not flower by day 40
 (termination of experiment), we converted flowering data into a binary
 response variable, with each plant classified as “flowering” or
 “not-flowering” on each experimental day. After initial data exploration,
 we decided to model flowering time as a function of treatment, time
 itself, and plant ID, with an interaction between time and plant.
 Treatment and time were entered as parametrically estimated explanatory
 variables, with the smoothed term including time by plant (Table. S3). We
 set the data family to binomial and the basic dimension of the smoothing
 function to ‘re’ in order to account for random factors (34).
 Additionally, we used the package itsadug to handle the autocorrelation
 within the time series. Models were validated by plotting Pearson versus
 Fitted residuals for response variables and covariates, to ensure that
 homoscedasticity and normality assumptions were met. Model selection was
 made using the ANOVA function and AIC to get the best fit. This approach
 allowed us to explore the simultaneous effects of treatment and time and
 thereby explain a high proportion of deviance within the model. Following
 this analysis, we used a generalized linear model (GLM) with a binomial
 error distribution and logistic-link function to calculate the least
 square means comparisons between the treatments (35). Additionally, we
 used the mean least squares coefficients to estimate the estimated time to
 reach 50% of flowering plants for each treatment. To test for effects of
 pollen availability on damaging behavior, we used a generalized
 linear<br> model with discrete error distribution (Poisson), with
 diet as a fixed effect and colony as a 7 random effect. We used backwards
 stepwise model simplification based on likelihood ratio tests to reduce
 model complexity as far as possible. To test for differences among damage
 treatments over the course of our rooftop studies, we<br> used a
 series of generalized linear mixed effect models (GLMMs). For the 2018
 rooftop study, we used a GLMM with Poisson error fitted by maximum
 likelihood, in order to look at differences between the phases. Date was
 included to account for temporal non-independence of data. We used
 backwards stepwise model simplification based on likelihood ratio tests to
 reduce model complexity as far as possible . We also calculated a rolling
 7-day average for plant damage, using weekday data to estimate the
 weekends, using the “zoo” package. In 2019, we also used a GLMM to look at
 the effect of roof treatment, in addition to using a general linear model
 to test for differences before and after the roof treatment was cut. These
 models were also validated using backwards stepwise model
 simplification.