DOI: 10.5061/dryad.bzkh189mq
title: Gustatory sensitivity to amino acids in bumblebee mouthparts

description: Bees rely on amino acids from nectar and pollen for essential
 physiological functions. While nectar typically contains low (&lt;1
 mM) amino acid concentrations, levels in pollen are higher but variable
 (10-200 mM). Behavioural studies suggest bumblebees have preferences for
 specific amino acids but whether such preferences are mediated via
 gustatory mechanisms remains unclear. This study explores bumblebees'
 (Bombus terrestris) gustatory sensitivity to two essential amino acids
 found in nectar and pollen, valine and lysine, using electrophysiological
 recordings from gustatory sensilla on their mouthparts. Valine elicited a
 concentration-dependent response from 0.1 mM, indicating that bumblebees
 could perceive valine at concentrations found naturally in nectar and
 pollen. In contrast, lysine failed to evoke a response across tested
 concentrations (0.1-500 mM). The absence of lysine detection raises
 questions about the specificity and diversity of amino acid-sensitive
 receptors in bumblebees. Bees responded to valine at lower concentrations
 than sucrose, suggesting comparatively higher sensitivity (EC50: 0.7 mM
 vs. 3.91 mM for sucrose). Our findings indicate that bumblebees can
 evaluate the amino acid content of pollen and nectar using pre-ingestive
 cues, rather than relying on post-ingestive cues or feedback from their
 nestmates. Such sensory capabilities likely impact foraging strategies,
 with implications for plant-bee interactions and pollination.
description: Bumblebee (<em>Bombus terrestris audax</em>) colonies
 were obtained from Biobest (Westerlo, Belgium) (<em>n</em>=2)
 and Koppert (Berkel en Rodenrijs, The Netherlands)
 (<em>n</em>=1). Colonies were maintained at the University of
 Sussex, U.K., housed either within a flight cage (73x73x65 cm) or
 connected to a feeding arena (40x40x35 cm) via a corridor (4x4x26 cm).
 Bees had <em>ad libitum</em> access to a nectar substitute
 (Biogluc, Biobest, Westerlo, Belgium) via both ground and suspended
 feeders, and finely ground honeybee collected pollen (Agralan, Swindon,
 U.K.), presented on chenille stems placed inside white plastic cups.
 Nectar was replenished as needed and pollen changed daily. Workers
 observed collecting pollen from the feeders were marked on the thorax with
 a small dot of white enamel paint (Humbrol, Hornby Hobbies Limited,
 Margate, U.K.). Only individuals observed collecting pollen at least once
 were used in testing (<em>n</em>=27).
 Bumblebees (<em>n</em>=27) were caught in the feeding
 arena and left at 4 °C overnight to immobilise them. On the day of
 recording, bees were inserted in small plastic tubes and harnessed using
 small strips of Parafilm M (American National Can, Greenwich, CT, U.S.A.).
 Bees were then transferred to a plate of sealing wax beneath a microscope
 (Nikon AZ100, Tokyo, Japan). Using wax, the head, antennae, and front legs
 of the bumblebee were immobilised, and the glossa and labial palps were
 manually extended and immobilised to the plastic tube. The galeae were
 rinsed in ultrapure water and dried with QL100 filter paper (Fisherbrand,
 Fisher Scientific, Loughborough, U.K.), and subsequently extended and
 fixed to the plastic tube using small strips of Parafilm to prevent heat
 damage. Extracellular tip recordings (figure
 1<em>a</em>) were performed on A-type sensilla chaetica on the
 left galea [27]. A 25 μm tungsten wire (Alfa Aesar, Ward Hill, MA, U.S.A.)
 was inserted into the galea at the proximal end and pushed gently down to
 ~1 mm from the sensilla from which the recording was to be made. This wire
 was used as the reference electrode. The recording electrode was a 250 μm
 silver/silver chloride wire, placed inside a borosilicate glass capillary
 (1x0.58x100 mm (ODxIDxL); Harvard apparatus, Holliston, MA, U.S.A.) pulled
 on a P-97 micropipette puller (Sutter Instrument Co., Novato, CA, U.S.A.)
 to a tip diameter of ~20-50 μm to fit comfortably over A-type gustatory
 sensilla on the galea (<em>n</em>=124) without deflecting the
 hair. Capillaries were filled with tastant solutions (see below) with no
 added electrolytes. The capillary was moved close to the galea using an
 LBM-7 manipulator (Scientifica, Uckfield, U.K.) and contact with the
 apical pore was made using an MO-203 micromanipulator (Narishige, Tokyo,
 Japan), mounted on the LBM-7. Electrical signals, which
 represent the ensemble activity of the four gustatory receptor neurons
 (GRNs) innervating each sensillum (figure 1<em>a</em>), were
 recorded through the tastant solutions. The electrodes were connected to a
 TasteProbe [28] headstage and TasteProbe DTP-02 10x amplifier (Syntech,
 Buchenbach, Germany). Recordings were made for 20 s with a 1 Hz high-pass
 filter. The signal was further amplified with 5x gain and band-pass
 filtered between 10 and 10k Hz (LHBF-48X filter-amplifier, npi electronic
 GmbH, Tamm, Germany). Recordings were digitised via a CED micro1401-3
 digital-to-analogue converter (Cambridge Electronic Design Ltd, Cambridge,
 U.K.) and acquired using CED Spike2 software (10.09) at 20k Hz.
 Gustatory sensilla on the galeae (<em>n</em>=3-6
 sensilla per bee) were stimulated with aqueous solutions of D-(+)-sucrose
 (Fisher Scientific, Loughborough, U.K.), L-valine (Thermo Fisher
 Scientific, Heysham, U.K.), or L-lysine (Thermo Fisher Scientific,
 Waltham, MA, U.S.A.), with no added electrolytes. For solutions with
 valine concentrations above 100 mM, hot (between 60 and 90°C) water was
 used to aid valine dissolution. Syringes filled with tastant solutions
 were prepared in advance and stored at -10 °C. Prior to the
 electrophysiological experiments, the syringes were thawed, and stored at
 4 °C. The capillary used for stimulation was filled immediately prior to
 use, to minimise water evaporation affecting solution concentration. Bees
 were allocated to one of two experimental groups, one stimulated with low
 amino acid concentrations and one with high concentrations (figure
 1<em>b</em>), to prevent slow adaptation over the course of
 the experiment. In both conditions, gustatory sensilla were tested
 initially with 5 mM sucrose and water, to ensure that sucrose sensitivity
 was comparable between GRN ensembles in all conditions. Bees in the
 low-concentration group were exposed to increasing concentrations of
 either lysine or valine from 0.1 to 50 mM. Then, bees in this group were
 tested with increasing concentrations of sucrose from 1 to 50 mM [27]. The
 high-concentration group was exposed to increasing concentrations of
 either lysine or valine from 50 to 500 mM. At least 3 minutes elapsed
 between exposure to different tastants and concentrations to reduce
 possible adaptation. Temperature was monitored every 30 s throughout the
 recording, using an automated temperature logger (EasyLog-USB-2-LCD,
 Lascar Electronics, Whiteparish, U.K.). Individual
 recordings were imported into Matlab (R2024a, MathWorks inc., Natick, MA,
 U.S.A.) for offline analysis based on [29]. Recordings were trimmed either
 using contact artefacts, or by the 20 s timestamp acquired from the
 amplifier, whichever was shortest; the entire trimmed length was used for
 data extraction. Filtered recordings were then created using a band-pass
 second-order Butterworth filter between 100 and 1000 Hz. Using these
 copies, a threshold was then selected manually for each recording to
 identify spikes. The peak of the spikes above the threshold was used to
 acquire 4 ms waveforms from the raw signal. The waveforms in each
 recording were inspected. Four measures were used to distinguish spikes
 and artefacts: maximum voltage reached, minimum voltage reached, waveform
 amplitude, and waveform half-width. Individual waveforms with unusual or
 irregular shapes were considered movement artefacts and removed. Spike
 frequency was calculated as the total number of spikes in the recording
 divided by the duration after trimming.
description: # Data from: Gustatory sensitivity to amino acids in bumblebees
 [https://doi.org/10.5061/dryad.bzkh189mq](https://doi.org/10.5061/dryad.bzkh189mq) ## Description of the data and file structure These spreadsheets contain the data used in the paper. Each spreadsheet contains a full dataset related to the substance in the file name (Valine/Lysine/Sucrose). In Valine.csv and Lysine.csv, the initial exposure to 5mM sucrose prior to testing is included at the beginning of the dataset; following this, sensilla were exposed to water and increasing concentrations of the amino acid indicated (0.1-500 mM). Sensilla used to test 0.1-50 mM and 50-500 mM are from different bees; a unique sensillum ID is included to differentiate between them. Sensilla used to test 0.1-50 mM valine were then exposed to water again and sucrose (1-50 mM); for this reason the sensillum ID numbers of the Sucrose.csv spreadsheet are the same as for Valine (between 0.1 and 50 mM). ### Files and variables #### File: Sucrose.csv **Description:** Spreadsheet containing the gustatory receptor neuron (GRN) firing frequency in response to water and sucrose (1-50 mM). ##### Variables * frequency_Hz: GRN firing frequency in spikes/second, or Hertz. * substance: name of the substance used (water/sucrose). * concentration_mM: concentration of the substance used, in millimolar (water is indicated as 0 mM). * temperature_C: temperature recorded during exposure, in degrees Celsius. * hair_ID: unique hair ID number, valid across spreadsheets. #### File: Lysine.csv **Description:** Spreadsheet containing the gustatory receptor neuron (GRN) firing frequency in response to sucrose (5 mM), water, and lysine (0.1-500 mM). ##### Variables * frequency_Hz: GRN firing frequency in spikes/second, or Hertz. * substance: name of the substance used (sucrose/water/lysine). * concentration_mM: concentration of the substance used, in millimolar (water is indicated as 0 mM). * temperature_C: temperature recorded during exposure, in degrees Celsius. * hair_ID: unique hair ID number, valid across spreadsheets. #### File: Valine.csv **Description:** Spreadsheet containing the gustatory receptor neuron (GRN) firing frequency in response to sucrose (5 mM), water, and valine (0.1-500 mM). ##### Variables * frequency_Hz: GRN firing frequency in spikes/second, or Hertz. * substance: name of the substance used (sucrose/water/valine). * concentration_mM: concentration of the substance used, in millimolar (water is indicated as 0 mM). * temperature_C: temperature recorded during exposure, in degrees Celsius. * hair_ID: unique hair ID number, valid across spreadsheets. ## Code/software Files are uploaded as comma separated value (csv) format. Files have been viewed and analysed by the authors using Microsoft Excel, MATLAB (version R2024a), and R studio (version 2024.04.2).