DOI: 10.5061/dryad.2jm63xspz
title: Specialized metabolites in floral resources: effects and detection in buff-tailed bumblebees

description: The selection of appropriate food resources by bees is a critical aspect
 for the maintenance of their populations, especially in the current
 context of global change and pollinator decline. Wild bees have a
 sophisticated ability to forage selectively on specific resources, and can
 assess the quality of pollen using contact chemosensory perception
 (taste). While numerous studies have investigated the detection
 of pollen macronutrients in bees and their impact on bee health and
 reproductive success, only a few studies have described the gustatory
 responses of bees towards specialized metabolites. In addition, these
 studies mostly focused on the response to nectar and neglected pollen,
 which is the main food resource for both bee imagines and
 larvae. Whether bees have the ability to detect specialized toxic
 metabolites in pollen and then rapidly adapt their foraging behavior to
 avoid them is very little studied. In this study, we tested
 whether pollen specialized metabolites affect bumblebees at both the
 micro-colony and individual levels (i.e. bioassays using
 supplemented pollen), and whether foragers detect these specialized
 metabolites and potentially display an avoidance behavior
 (i.e. preference tests using supplemented syrup). Bumblebees were
 fed with either amygdalin-, scopolamine- or sinigrin-supplemented
 pollen diets in ratios that mimic 50%, 100% and 200%
 of naturally occurring concentrations. We found no effect of
 these specialized metabolites on resource collection, reproductive success
 and stress response at the micro-colony level. At the individual level,
 bumblebees fed on 50%-amygdalin or 50%-scopolamine diets displayed the
 highest scores for damage to their digestive systems. Interestingly,
 during the preference tests, the solution with 50%-scopolamine displayed a
 phagostimulatory activity, whereas solution with 50%-amygdalin had a
 deterrent effect and could trigger an active avoidance behavior in
 bumblebees, with a faster proboscis retraction. Our results suggest that
 regulation of toxin intake is not as well-established and effective as the
 regulation of nutrient intake in bees. Bees are therefore not equally
 adapted to all specialized pollen metabolites that they can come into
 contact with.
description: Developmental_parameter.txt and
 Histo_scoring.txt How specialized metabolites
 can impact pollinator behavior, performance and health was investigated by
 the use of a control diet as well as amygdalin-, scopolamine- and
 sinigrin-supplemented diets (<i>i.e.</i> test diets). The test
 diets contained chemicals mixed with the control diet in ratios that mimic
 50%, 100% and 200% of the naturally occurring concentration. The control
 diet consisted of ground pollen loads with a dominance of <i>Salix
 </i>sp. mixed with inverted sugar syrup (BIOGLUC®,
 Biobest).  The experiments were conducted at the
 University of Mons from February 2015 to May 2016. A first run of
 bioassays was performed in 2015 for amygdalin (<i>i.e.</i>
 four treatments; control, 50%-amygdalin, 100%-amygdalin and
 200%-amygdalin), and a second run in 2016 for scopolamine and sinigrin
 (<i>i.e. </i>seven treatments; control, 50%-scopolamine,
 100%-scopolamine, 200%-scopolamine, 50%-sinigrin, 100%-sinigrin and
 200%-sinigrin). Ten queenless <i>B. terrestris
 </i>micro-colonies were established for each treatment using workers
 from five different colonies (Biobest <i>bvba, </i>Westerlo,
 Belgium) that were equally distributed among the treatments to ensure
 homogeneity of origin. A total of 110 micro-colonies were then monitored
 for all experiments. Each micro-colony was composed of five two-day-old
 workers placed in different plastic boxes (10 cm × 16 cm × 16 cm) in a
 dark room at 27°C and 76% relative humidity. The micro-colonies were fed
 <i>ad libitum</i> with sugar syrup (BIOGLUC®, Biobest) and
 pollen candies that were freshly prepared and renewed every two days (0.5
 g, 1.0 g or 1.5 g depending on the age of the micro-colony) to avoid
 nutrient alteration and drying out during the experiment. Pollen and syrup
 collections were measured by weighing pollen candies and syrup container
 before their introduction into the micro-colony and after their removal.
 Ejected larvae were removed from the micro-colony; workers that died during the
 experiment were removed and replaced. Syrup and pollen
 supplies as well as micro-colonies monitoring were done in the darkroom
 under red light during the 35-day period following the first episode of
 egg laying of a worker. At the end of the experiment,
 workers were weighed. The total mass of workers was expressed as the sum
 of the weights of the five workers in each micro-colony, taking into
 account the time they spent in the micro-colony in case of death and
 replacement. The nest was then carefully dissected, and the number and
 mass of individuals were recorded for each brood
 stage. 
 Developmental_parameter.txt<strong>
 - </strong>Feeding response and micro-colony development were
 evaluated based on: (i) composition (<i>i.e.</i> number of
 eggs, non-isolated larvae, isolated larvae, pupae, non-emerged and emerged
 drones) and fresh weight of offspring, (ii) larval ejection
 (<i>i.e.</i> number of larvae, alive and dead, removed from
 the nest by workers), (iii) pollen collection (<i>i.e.
 </i>amount of pollen consumed and stored) (fresh matter), (iv)
 pollen efficiency (i.e. the weight of hatched offspring divided by the
 total pollen collected per micro-colony), (v) syrup collection (i.e.,
 amount of syrup consumed and stored) and (vi) pollen dilution (i.e. the
 total syrup collected divided by the total pollen collected per
 micro-colony). All weight parameters (<i>i.e.</i> brood
 weight, pollen collection, and syrup collection) were standardized by the
 total mass of workers in the micro-colonies to avoid potential bias from
 worker activities (<i>i.e.</i> consumption and brood
 care).
 Histo_scoring.txt<strong>
 - </strong>Tissues for histological evaluation were prepared
 following the method described by Vanderplanck <i>et al.</i>
 (2020). For each treatment, four bumblebee individuals were randomly
 collected from the different micro-colonies and cold-anesthetized. Their
 abdomens were cut and incised to facilitate the fixation (Duboscq–Brazil
 fluid), dehydration and paraffin-embedding processes. Transverse serial
 sections of 5 µm thicknesses were performed with a microtome
 (Reichert-Jung® 2040 microtome) with the use of a softening agent
 (Mollifex<sup>TM</sup>), and placed on silane-coated glass
 slides. After rehydration, the sections were stained with Masson's
 Trichrome staining method. A single-blind
 microscopic evaluation was carried out using a research optical microscope
 (Leitz® Orthoplan). This allowed for eliminating biases due to knowledge
 of treatment. The parameters evaluated for damage score were the common
 histopathological alterations in the digestive tract, namely: (i)
 disorganization or loos of the brush-like border, (ii) vacuolization of
 the epithelial cells (hydropic degeneration), (iii) interstitial edema,
 (iv) apoptosis, and (v) necrosis. All parameters were scored from 0 (no
 damage) to 5 (extensive changes), except necrosis parameter that was
 scored from 0 to 6 (see original publication for criteria and score
 details). When necrosis parameter was set to at least 4 (i.e. sublethal
 damage), all other parameters were automatically set to the maximal value
 (5). Analysis was made of the damage score for each of the parameters on
 one hand, and of the total sum of damage scores (TDS) of the five
 parameters on the other hand. Thus, the TDS had a minimum possible total
 damage score of 0 and a maximum possible total damage score of
 26. Detection.txt We tested the hypothesis that bumblebees can detect the specialized metabolites using preference tests following the protocol from Ma <i>et al.</i> (2016).<b> </b>For each treatment, 15 bumblebee individuals were randomly collected from five different colonies (<i>i.e.</i> three bumblebees per colony) and starved for 2-4 h in plastic vials (70 mm long, 25 mm inner diameter) in the rearing dark room at 27°C and 76% relative humidity.<b> </b>After this starvation period, bumblebees were transferred into a holding tube where they were able to move freely. The holding tube consisted in a modified 15 mL centrifuge tube fixed on a polystyrene holder as described in Ma <i>et al.</i> (2016). After a habituation phase of 3 min, the trial started and was recorded with a digital Dino-lite USB microscope camera fixed 5 cm above the tip of the holding tube. The trial was recorded using the software Dinocapture 2.0, with a 26.7 frames.sec<sup>-1</sup> and a 25X magnification rate. A drop of sugar syrup (BIOGLUC®, Biobest) was presented to the bumblebee using a 1-mL syringe. Individuals that did not consume the syrup within 5 min were discarded. For responsive individuals, test solutions were presented using a 100 µL micro-capillary tube connected to a pumping system to ensure the presence of a permanent droplet of test solution at the top of the micro-capillary tube (Ma <i>et al.</i>, 2016). Test solutions were prepared by diluting the commercial powders directly in sugar syrup (50%, 100% and 200% of the naturally occurring concentrations). The control solutions consisted of pure sugar syrup (negative control) and a 1mM quinine solution (positive control) that was proven to have a deterrent effect (Ma <i>et al.</i>, 2016). A total of 165 workers (<i>i.e. </i>15 workers per treatment and 11 treatments namely, negative control, positive control, 50%-amygdalin, 100%-amygdalin, 200%-amygdalin, 50%-scopolamine, 100%-scopolamine, 200%-scopolamine, 50%-sinigrin, 100%-sinigrin and 200%-sinigrin) have been tested. The 2-min test phase started as soon as the bumblebee's proboscis contacted the test or control solution inside the micro-capillary tube. The lengths of liquid inside the micro-capillary tube were measured before and after the test phase to calculate the volume of solution consumed. The volume of solution consumed as well as the number of feeding bouts, the cumulative duration of the feeding bouts, the total duration of effective feeding (<i>i.e.</i> contact with test or control solution) and the duration of the first contact (<i>i.e.</i> before the first proboscis retraction) were used to evaluate the phagostimulatory or the deterrent activity of the compounds tested. A feeding bout was defined as a contact between the extended proboscis and the test solution for at least 5 sec (French <i>et al.</i>, 2015).