The tarsal taste of honey bees: behavioral and electrophysiological analyses

Abstract

Taste plays a crucial role in the life of honey bees as their survival depends on the collection and intake of nectar and pollen, and other natural products. Here we studied the tarsal taste of honey bees through a series of behavioral and electrophysiological analyses. We characterized responsiveness to various sweet, salty and bitter tastants delivered to gustatory sensilla of the fore tarsi. Behavioral experiments showed that stimulation of opposite fore tarsi with sucrose and bitter substances or water yielded different outcomes depending on the stimulation sequence. When sucrose was applied first, thereby eliciting proboscis extension, no bitter substance could induce proboscis retraction, thus suggesting that the primacy of sucrose stimulation induced a central excitatory state. When bitter substances or water were applied first, sucrose stimulation could still elicit proboscis extension but to a lower level, thus suggesting central inhibition based on contradictory gustatory input on opposite tarsi. Electrophysiological experiments showed that receptor cells in the gustatory sensilla of the tarsomeres are highly sensitive to saline solutions at low concentrations. No evidence for receptors responding specifically to sucrose or to bitter substances was found in these sensilla. Receptor cells in the gustatory sensilla of the claws are highly sensitive to sucrose. Although bees do not possess dedicated bitter-taste receptors in the tarsi, indirect bitter detection is possible because bitter tastes inhibit sucrose receptor cells of the claws when mixed with sucrose solution. By combining behavioral and electrophysiological approaches, these results provide the first integrative study on tarsal taste detection in the honey bee.

Keywords: electrophysiology; gustation; gustatory receptors; honey bee; insect; proboscis extension reflex; tarsi; taste.

Figure 1

(A) Left: Honey bee with amputated antennae harnessed in a metal tube with its forelegs fixed wide open in order to allow tarsal gustatory stimulation. Right: Proboscis extension reflex (PER) upon tarsal stimulation with a drop of sucrose solution (red arrow) delivered to the left tarsus. (B) Scheme of the distal segments of a honey bee foreleg showing the tarsus and the pretarsus. The tarsus has five tarsomeres: a basitarsus (btr: 1), which is the largest tarsomere, and 4 smaller tarsomeres (2–5). The basitarsus presents a notch of antenna cleaner (at) and the tibia (Tb) a closing spine (cs). The distally situated pretarsus (pta) bears a pair of lateral bifid claws (cl) and an arolium (ar), a small pad used to increase adhesion. (C) Detail of the pretarsus (pta) of a foreleg: cl: claws; ar: arolium.

Figure 2

Experiment 1. (A) Bees with amputated antennae were stimulated along three trials with sucrose solution 1M (red arrow; red trace) on one fore-tarsus (either left or right) in order to elicit PER, and 5 s later with quinine, salicin, or caffeine (“bitter”, green arrow, green trace) of different concentrations on the contralateral tarsus to determine whether these substances induce proboscis retraction. (B) Percentage of proboscis extension responses (% PER) upon stimulation with sucrose 1M (red bars) and quinine (1, 10, and 100 mM; green bars). (C) % PER upon stimulation with sucrose 1M (red bars) and salicin (1, 10, and 100 mM; green bars). Different letters above bars indicate significant differences (p < 0.05). (D) % PER upon stimulation with sucrose 1M (red bars) and caffeine (1, 10, and 100 mM; green bars). (E) Percentage of bees into the invariance (black bars), increase (white bars), or decrease (gray bars) categories upon stimulation with sucrose 1M and quinine 1, 10, and 100 mM. “Decrease” means that sucrose induced PER and quinine induced retraction, “increase” means that sucrose did not induce PER but quinine did. “Invariance” means that no response change was induced by quinine with respect to sucrose. (F) Percentage of bees into the invariance, increase, or decrease categories upon stimulation with sucrose 1M and salicin 1, 10, and 100 mM. (G) Percentage of bees into the invariance, increase, or decrease categories upon stimulation with sucrose 1M and caffeine 1, 10, and 100 mM.

Figure 3

Experiment 2. (A) Bees with amputated antennae were stimulated along three trials with quinine or salicin (“bitter”, green arrow, green trace) of different concentrations on one fore-tarsus, and with sucrose solution 1M (red arrow; red trace) on the contralateral fore-tarsus to determine whether sucrose overcomes the potential inhibitory effect of the bitter substance and elicits PER. (B) % PER upon stimulation with quinine (1, 10, and 100 mM; green bars) and sucrose 1M (red bars). (C) % PER upon stimulation with salicin (1, 10, and 100 mM; green bars) and sucrose 1M (red bars). Different letters above bars indicate significant differences (p < 0.05). (D) % of bees into the invariance (black bars), increase (white bars), or decrease (gray bars) categories upon stimulation with sucrose 1M and quinine 1, 10, and 100 mM. (E) % of bees into the invariance (black bars), increase (white bars), or decrease (gray bars) categories upon stimulation with sucrose 1M and salicin 1, 10, and 100 mM. (F) % PER upon stimulation with water and sucrose 1M (diagonally hatched bar), quinine 100 mM and sucrose 1M (horizontally hatched bar) and salicin 100 mM and sucrose 1M (vertically hatched bar). Different letters above bars indicate significant differences (p < 0.05).

Figure 4

Experiment 2. (A) Bees with intact antennae were stimulated along three trials with quinine or salicin (“bitter,” green arrow, green trace) of different concentrations on one fore-tarsus, and with sucrose solution 1M (red arrow; red trace) on the contralateral fore-tarsus to determine whether sucrose overcomes the potential inhibitory effect of the bitter substance and elicits PER. (B) % PER upon stimulation with quinine (1, 10, and 100 mM; green bars) and sucrose 1M (red bars). (C) % PER upon stimulation with salicin (1, 10, and 100 mM; green bars) and sucrose 1M (red bars). Different letters above bars indicate significant differences (p < 0.05). (D) % of bees into the invariance (black bars), increase (white bars), or decrease (gray bars) categories upon stimulation with sucrose 1M and quinine 1, 10, and 100 mM. (E) % of bees into the invariance (black bars), increase (white bars), or decrease (gray bars) categories upon stimulation with sucrose 1M and salicin 1, 10, and 100 mM.

Figure 5

(A) Experiment 3: Examples of extracellular recordings performed at the level of chaetic sensilla located on the third and fourth tarsomeres of the foreleg, upon 2-s gustatory stimulation. The contact electrolyte used for all solutions was KCl 0.1 mM. The black arrow indicates the start of stimulation. Vertical scale: 1 mV; horizontal scale: 50 ms. (B) Experiment 6: Examples of extracellular recordings performed at the level of chaetic sensilla located on the claws, upon 5-s gustatory stimulation.

Figure 6

(A) Mean normalized spike frequencies (±SE) obtained upon stimulation with the different tastants assayed. Responses (action potentials per second) were normalized to those recorded for KCl 0.1 mM (hatched horizontal line). Data were obtained from 6 bees in which responses for the six tastants assayed were recorded 4 times (n = 144) in 7 different sensilla. Different letters above bars indicate significant differences (p < 0.05). (B) Temporal analysis of mean normalized spike frequencies along four consecutive bins of 500 ms each, starting at the onset and finishing at the offset of stimulation. Asterisks indicate significant differences between tastants within a bin (p < 0.05).

Figure 7

(A) Experiment 4. Mean normalized spike frequencies (±SE) obtained upon stimulation with 5 different concentrations of KCl solution: 0.01, 0.1, 1, 10, and 100 mM. Responses (action potentials per second) were normalized to those recorded for KCl 100 mM (hatched horizontal line), which yielded maximal responsiveness. Data were obtained from 6 bees in which responses to the 5 KCl concentrations were recorded 4 times in 6 different sensilla (n = 120). Different letters above bars indicate significant differences (p < 0.05). (B) Experiment 5. Mean normalized spike frequencies (±SE) obtained upon stimulation with KCl 0.1 mM and 4 different concentrations of quinine solution including KCl 0.1 mM as contact electrolyte: 0.1, 1, 10, and 30 mM. Responses (action potentials per second) were normalized to those recorded for KCl 0.1 mM (hatched horizontal line). Data were obtained from 8 bees in which responses to the 5 substances assayed were recorded 4 times in 9 different sensilla (n = 180). Different letters above bars indicate significant differences (p < 0.05). (C) Experiment 5: Mean normalized spike frequencies (±SE) obtained upon stimulation with KCl 0.01 mM and 4 different concentrations of sucrose solution—1 mM, 10 mM, 100 mM and 1 M—including KCl 0.01 mM as contact electrolyte. Responses (action potentials per second) were normalized to those recorded for KCl 0.01 mM (hatched horizontal line). Stimulations lasted 1 s. The graph shows the responses of 14 different sensilla of 5 bees to the 5 substances assayed, each one being tested once (n = 70). No differences between stimuli were found; NS, non-significant.

Figure 8

Experiment 6. (A) Chaetic sensilla of the 3rd and 4th tarsomeres. Mean normalized spike frequencies (±SE) obtained upon stimulation with the different tastants assayed. Responses (action potentials per second) were normalized to those recorded for KCl 0.01 mM (hatched horizontal line). Data were obtained from 12 tarsomere sensilla of 5 bees in response to the 6 stimuli assayed, each one being tested once (n = 72). NS, non significant. (B) Chaetic sensilla of the claws. Mean normalized spike frequencies (±SE) obtained upon stimulation with the different tastants assayed. Responses (action potentials per second) were normalized to those recorded for KCl 0.01 mM (hatched horizontal line). Data were obtained from 13 claw sensilla of 4 bees in response to the 6 stimuli assayed, each one being tested once (n = 78). Different letters above bars indicate significant differences (p < 0.05).

Figure 9

Experiment 6. (A) Chaetic sensilla of the 3rd and 4th tarsomeres. Temporal analysis of mean normalized spike frequencies along five consecutive seconds of stimulation, starting at the onset and finishing at the offset of stimulation. The hatched line shows the reference response to the contact electrolyte KCl 0.01 mM. Besides KCl, the curves show the temporal responses upon stimulation with sucrose 1 M, quinine 10 mM, and amygdalin 10 mM. There were significant differences between seconds of stimulation (p < 0.0005) but within each second, the responses obtained for the different tastants were statistically similar. (B) Chaetic sensilla of the claws. Temporal analysis of mean normalized spike frequencies along five consecutive seconds of stimulation, starting at the onset and finishing at the offset of stimulation. The hatched line shows the reference response to the contact electrolyte KCl 0.01 mM. Besides KCl, the curves show the temporal responses upon stimulation with sucrose 1 M, quinine 10 mM, and amygdalin 10 mM. Responses varied significantly with the substance assayed as responses to sucrose 1 M were higher than those to the other tastants all along the stimulation period (p < 0.0001). Differences were neither found for the factor “second” of stimulation nor for the interaction “substance × second”.