Exp 1: chemo-tactile conditioning of the proboscis extension response (PER)
Bee foragers may assess the quality of floral nectars through chemo-sensilla located on their antennae47. In this first experiment, we asked whether nectar-relevant concentrations of GABA, β-alanine, taurine, citrulline and ornithine can be detected by bees through their antennae. To this aim, we used a chemo-tactile differential conditioning of PER protocol48 in which different groups of bees were trained to discriminate one of the five NPAAs from water. Briefly, tethered bees experienced five pairings of a neutral stimulus (either NPAA-laced water or water) (CS+) with a 30% sucrose solution reinforcement (US) and five pairings (either water or NPAA-laced water) (CS−) with a saturated NaCl solution (US) used as punishment. The results showed that bees increased their response to both the rewarded (CS+) and the punished (CS−) stimuli over the ten conditioning trials (GLMM, trial: GABA: n = 76, χ2 = 65.75, df = 1, p < 0.0001; β-ALA: n = 81, χ2 = 98.15, df = 1, p < 0.0001; ORN: n = 72, χ2 = 39.23, df = 1, p < 0.0001; TAU: n = 69, χ2 = 59.24, df = 1, p < 0.0001; CIT: n = 79, χ2 = 60.70, df = 1, p < 0.0001, Fig. 1). In all cases, the responses to the CSs did not differ over the course of the training, suggesting that bees were not able to discriminate any dissolved NPAAs from water with their antennae (GLMM, CS: GABA: χ2 = 3.01, df = 1, p = 0.08; β-ALA: χ2 = 2.68, df = 1, p = 0.10; ORN: χ2 = 3.31, df = 1, p = 0.07; TAU: χ2 = 0.20, df = 1, p = 0.65; CIT: χ2 = 1.89, df = 1, p = 0.17, Fig. 1).
Honey bees are not able to detect NPAAs when using only their antennae. Proportion of bees showing a conditioned PER during the chemo-tactile conditioning assay. Blue lines represent PER to the reinforced stimulus (CS+). Pink lines represent PER to the punished stimulus (CS−). NPAA-laced water solutions and pure water were used as CS+ and CS−. No significant differences were found in the proportion of bees showing PER to the CS+ and the CS− for any of the NPAAs (GLMM, GABA: p = 0.08; β-ALA: p = 0.10; TAU: p = 0.65; CIT: p = 0.17; ORN: p = 0.07).
Exp 2: taste aversion/preference assay
Besides using their antennae, bees may detect nectar constituents through the chemo-sensilla located on their mouthparts44,45. We thus further investigated the gustatory responses of bees to nectar NPAAs using a binary choice protocol adapted from49. Freely moving bees confined into custom-modified plastic tubes were presented with two microcapillaries, one containing 100 µl of 30% sucrose solution and the other containing 100 µl of NPAA-laced 30% sucrose solution (GABA, n = 56; β-ALA, n = 46; TAU, n = 55; CIT, n = 57; ORN, n = 58). A feeding preference index for each NPAA was calculated by subtracting the residual volume of the liquid inside the two capillaries at the end of test. Bees did not exhibit a feeding preference nor avoidance for any of the nectar NPAAs (One-Sample Wilcoxon test, GABA: V = 796, p = 0.99; β-ALA: V = 512, p = 0.76; TAU: V = 855, p = 0.48; CIT: V = 925, p = 0.44; ORN: V = 911, p = 0.67, Fig. 2).
NPAAs are not preferred or avoided by honey bees in the feeding assay. Median, quartiles, minimum and maximum consumption indexes observed for each of the NPAAs in the feeding preference test. A higher Δ value indicates a preference for the NPAA-laced sucrose solution. A lower Δ value indicates a preference for plain sucrose solution. Dots represent individual bees. Bees did not exhibit any clear preference or avoidance for any of the NPAAs (One-Sample Wilcoxon test, GABA: p = 0.99; β-ALA: p = 0.76; TAU: p = 0.48; CIT: p = 0.44; ORN: p = 0.67).
Exp 3: influence of NPAAs on feeding and mortality
Bees can easily learn to associate the food with the post-ingestive consequences of consuming it. Food aversion or preference may therefore change over time and arise from malaise or phagostimulation caused by SM ingestion28,44,46. To test this hypothesis, we housed a total of 900 bees in groups of 10 individuals each in plastic cages equipped with two food dispensers (syringes) and monitored them until all bees were dead50. For each NPAA, six replicates for three experimental conditions were set up: (1) two syringes providing plain sucrose solution (S–S); (2) two syringes providing NPAA-laced sucrose solution (NPAA-NPAA); (3) one syringe providing plain sucrose solution and another one providing NPAA-laced sucrose solution (S-NPAA). The results showed that bees did not consume more NPAA-laced solutions compared to control ones (GLMM, Syringe content: GABA: χ2 = 1.02, df = 1, p = 0.31; β-ALA: χ2 = 0.13, df = 1, p = 0.71; TAU: χ2 = 0.91, df = 1, p = 0.34; CIT: χ2 = 0.14, df = 1, p = 0.71; ORN: χ2 = 0.38, df = 1, p = 0.53, Fig. 3). Per capita food consumption increased over time in all groups of bees (GLMM, Days: all groups, P < 0.001). NPAAs did not induce phagostimulation or loss of appetite in bees kept under the three different regimes (GLMM, Experimental condition: GABA: χ2 = 1.11, df = 2, p = 0.58; β-ALA: χ2 = 0.34, df = 2, p = 0.84; TAU: χ2 = 0.89, df = 2, p = 0.64; CIT: χ2 = 0.79, df = 2, p = 0.67; ORN: χ2 = 1.16, df = 2, p = 0.56, Fig. 3).
Nectar NPAAs did not affect food consumption by bees kept in caged conditions under three different feeding regimes for up to 10 days. Plots represent fitted means for the factors “syringe content” (NPAA vs Sucrose) and “experimental condition” (S–S, NPAA-NPAA, S-NPA) and the covariate time “day”. Overall, bees did not consume more NPAA-laced solutions compared to control ones (GLMM, Syringe: GABA: p = 0.31; β-ALA: p = 0.71; TAU: p = 0.34; CIT: p = 0.71; ORN: p = 0.53). Per capita food consumption increased over time in all groups (GLMM all group p < 0.001). NPAAs did not induce phagostimulation or loss of appetite (GLMM, Experimental condition: GABA: p = 0.58; β-ALA: p = 0.34; TAU: p = 0.64; CIT: p = 0.67; ORN: p = 0.56).
A statistical evaluation of bees’ survival during the experiment revealed that NPAAs were not a significant predictor of mortality in any feeding regime (Log-rank Mantel Cox test, GABA: χ2 = 0.77, df = 2, p = 0.68; β-ALA: χ2 = 1.32, df = 2, p = 0.52; TAU: χ2 = 0.78, df = 2, p = 0.68; CIT: χ2 = 3.19, df = 2, p = 0.20; ORN: χ2 = 2.80, df = 2, p = 0.25, Fig. S1).
Exp 4: contextual absolute olfactory learning
Nectar metabolites may enhance or inhibit bees’ ability to learn floral cues by directly acting on the nervous system, regardless of their ability to trigger gustatory and olfactory receptor neurons of bees’ antennae and mouthparts14,31. Thus, we investigated whether the presence of NPAAs in sucrose rewards altered honey bees’ learning and memory performance during an olfactory absolute conditioning task42,43. Briefly, for each of the five NPAAs, tethered bees were presented with four pairings of a neutral odorant (CS, either 1-Hexanol or Nonanal) with a reinforcement (US, either 30% sucrose solution (control paired group) or NPAA-laced 30% sucrose solution (experimental paired group)). In addition, for each of the five NPAAs, we trained two explicitly unpaired groups of bees to control for true associative learning42. All bees in the paired groups increased their responses to the conditioned odorant over the four training trials (GLMM, trial: GABA: χ2 = 57.9, df = 1, p < 0.0001; β-ALA: χ2 = 52.3, df = 1, p < 0.0001; TAU: χ2 = 42.1, df = 1, p < 0.0001; CIT: χ2 = 53.3, df = 1, p < 0.0001; ORN: χ2 = 52.1, df = 1, p < 0.0001, Fig. 4). GABA and β-alanine significantly enhanced acquisition performances in bees (GLMM, treat: GABA: χ2 = 4.87, df = 1, p = 0.027; β-ALA: χ2 = 6.86, df = 1, p = 0.009, Fig. 4). Conversely, no effect on learning was observed for taurine, citrulline and ornithine (GLMM, trial: TAU: χ2 = 0.003, df = 1, p = 0.95; CIT:, χ2 = 0.84, df = 1, p = 0.36; ORN: χ2 = 0.58, df = 1, p = 0.45). Bees that received GABA or β-alanine as reward also had significantly higher acquisition scores (ACQS, calculated as the number of PER exhibited over the 4 trials) than controls (Mann–Whitney U test, ACQS: GABA, W = 922, p = 0.041; β-ALA, W = 1495, p = 0.006). No difference was observed for any of the other NPAAs (Mann–Whitney U test, ACQS: TAU, W = 707, p = 0.87; CIT: W = 1007, p = 0.34; ORN: W = 799, p = 0.39). Our analysis showed that the conditioned stimulus (CS, either Nonanal or 1-Hexanol) significantly affected responses of both experimental and control bees in both the paired and unpaired GABA groups. In particular, bees in the paired groups showed a significantly higher number of responses toward 1-Hexanol than to Nonanal (GLMM, CS: χ2 = 27.22, df = 1, p < 0.0001), whereas the contrary was true in the unpaired groups (GLMM, CS: χ2 = 4.4, df = 1, p = 0.036). Bees in the unpaired groups did not differ in acquisition scores for any of the NPAAs (see Suppl. Mat. and Fig. 4) confirming the occurrence of a true associative learning phenomenon in the paired groups.
Effects of NPAAs dissolved in US on appetitive olfactory learning and memory retention in harnessed bees. Acquisition trials: Percentages of PER showed by experimental (blue lines) and control (red lines) bees in the paired (solid lines) and unpaired (dotted lines) groups during the contextual conditioning experiment for each of the NPAAs. In the paired groups, GABA and β-alanine significantly enhanced the acquisition performances of bees (GLMM, GABA: p = 0.027; β-ALA: p = 0.009). None of the other NPAAs affected learning performances (all cases: p > 0.4). Unpaired groups did not learn the US-CS association (all cases: p > 0.7). Memory test: Proportions of PER showed by experimental (blue) and control (red) bees in the paired and unpaired groups during the memory test performed two hours after conditioning. GABA significantly enhanced the specific memory of bees for the trained odorant (χ2 test, p = 0.03). No difference has been found in the responses to the CS+, to the NOd or in CS−specific memory in all other cases (all cases: p > 0.1). Unpaired groups did not differ in the memory performances (all cases: p > 0.2).
Two hours after conditioning, bees were tested for memory retention and generalization to a novel odour (NOd)43. Experimental and control bees in all the paired groups did not differ in their responses to the conditioned odorant for any of the NPAAs (χ2 test, CS: GABA, χ2 = 2.3, p = 0.13; β-ALA, χ2 = 1.9, p = 0.16; TAU, χ2 = 0.68, p = 0.41; CIT, χ2 = 0.01, p = 0.91; ORN, χ2 = 0.19, p = 0.66, Fig. 4). Neither did responses to the novel odorant (NOd) differ between experimental and control bees (χ2 test, NOd: GABA, χ2 = 1.21, p = 0.27; β-ALA, χ2 = 0.47, p = 0.49; TAU, χ2 = 0.52, p = 0.47; CIT, χ2 = 0.71, p = 0.40; ORN, χ2 = 0.27, p = 0.87, Fig. 4). GABA significantly enhanced specific memory for the conditioned odorant (χ2 test, specific memory: χ2 = 4.63, p = 0.03, Fig. 4). No such difference was found for any of the other nectar NPAAs (χ2 test, specific memory: β-ALA, χ2 = 0.40, p = 0.53; TAU, χ2 = 0.053, p = 0.82; CIT, χ2 = 0.48, p = 0.49; ORN, χ2 = 0.001, p = 0.98, Fig. 4). In the unpaired groups, the response to the CS, to the NOd or the CS-specific memory did not differ for any NPAAs (see Suppl. Mat. and Fig. 4).
Exp 5: post-feeding absolute olfactory learning
To explore the post-ingestive mechanisms of NPAAs on bees’ learning and memory performance, bees were fed 5 µL of either NPAA-laced 30% sucrose solution (experimental groups) or plain 30% sucrose solution (control groups) two hours before conditioning. Bees were then confronted with a conditioning procedure identical to that described above (Exp. 4), except that we always used plain sucrose as US. Bees in all the paired groups significantly increased their responses to the conditioned odorant over the four training trials (GLMM, trial: GABA, χ2 = 39.5, df = 1, p < 0.0001; β-ALA, χ2 = 35.35, df = 1, p < 0.0001; TAU, χ2 = 46.8, df = 1, p < 0.0001; CIT, χ2 = 51.7, df = 1, p < 0.0001; ORN, χ2 = 56.3, df = 1, p < 0.0001, Fig. 5). Bees pre-fed GABA and taurine had significantly worse acquisition performances than controls (GLMM, treat: GABA, χ2 = 5.79, df = 1, p = 0.016; TAU, χ2 = 3.91, df = 1, p = 0.048, Fig. 5). β-alanine had a similar almost significant effect (GLMM, treat: β-ALA, χ2 = 2.73, df = 1, p = 0.098). Neither ornithine nor citrulline had an effect on learning performances (GLMM, trial: CIT, χ2 = 1.83, df = 1, p = 0.18; ORN, trial: χ2 = 0.01, df = 1, p = 0.99, Fig. 5). Accordingly, ACQS were significantly lower in bees pre-fed GABA than controls (Mann–Whitney U test, W = 656, p = 0.035). A non-significant tendency in the same direction was found for both β-alanine (Mann–Whitney U test, W = 817, p = 0.077) and taurine (Mann–Whitney U test, W = 833, p = 0.057). No difference in ACQS values was found between experimental and control bees pre-fed citrulline (Mann–Whitney U test, W = 967, p = 0.13) or ornithine (Mann–Whitney u test, W = 807, p = 0.94). In the unpaired groups, pre-feeding did not alter the responses to the CS and bees did not differ in acquisition scores for any of the NPAAs (see Suppl. Mat. and Fig. 5) confirming the occurrence of a true associative learning phenomenon in the paired groups.
Effects of pre-feeding NPAAs on appetitive olfactory learning and memory retention in harnessed bees. Acquisition trials: Percentages of PER exhibited by experimental (blue lines) and control (red lines) bees in the paired (solid lines) and unpaired (dotted lines) groups during the post-feeding absolute olfactory learning experiment. Pre-ingestion of GABA and taurine impaired acquisition performance in honey bees (GLMM, GABA: p = 0.016; TAU: p = 0.048). β-alanine had a similar almost significant effect (p = 0.098). Ingestion of citrulline and ornithine did not affect associative learning (CIT: p = 0.18; ORN: p = 0.99). Unpaired groups did not learn the US-CS association (all cases: p > 0.17). Memory test: proportions of PER showed by experimental (blue) and control (red) bees in the paired and unpaired groups during the memory retention test performed two hours after the post-feeding conditioning assay. In the paired groups, bees pre-fed GABA exhibited a lower number of PER to the CS (χ2 test, p = 0.01) and to the novel odorant (p = 0.047). However, GABA did not affect bees’ specific memory (p = 0.83). β-alanine and taurine enhanced bees’ specific memory (β-ALA: p = 0.003; TAU: p = 0.02). In all other cases there were no significant differences in the response to the CS+, to the NOd and in the specific memory (all cases, p > 0.1). Unpaired groups did not differ in the memory performances (all cases: p > 0.2).
In the memory retention test bees pre-fed GABA showed significantly less appetitive responses to the CS (χ2 test, χ2 = 5.96, p = 0.01) and to the novel odorant (χ2 test, χ2 = 3.95, p = 0.047) than controls (Fig. 5). However, their CS-specific memory was not affected compared to control bees (χ2 test, χ2 = 0.49, p = 0.83, Fig. 5). Bees that were pre-fed β-alanine exhibited a significantly higher number of appetitive responses to the CS than controls (χ2 test, χ2 = 4.52, p = 0.03) but not to the novel odorant (χ2 test, χ2 = 2.28, p = 0.13, Fig. 5). Accordingly, a higher proportion of bees pre-fed β-alanine exhibited CS-specific memory (χ2 test, χ2 = 8.63, p = 0.003, Fig. 5). Taurine, citrulline and ornithine did not alter bees’ response to the CS (χ2 test, TAU: χ2 = 0.40, p = 0.53; CIT: χ2 = 2.72, p = 0.1; ORN: χ2 = 2.78, p = 0.09) or to the novel odorant (χ2 test, TAU: χ2 = 1.74, p = 0.19; CIT: χ2 = 0.36, p = 0.55; ORN: χ2 = 1.01, p = 0.31, Fig. 5). However, a significantly higher proportion of bees pre-fed taurine exhibited CS-specific memory during the test (χ2 test, χ2 = 5.41, p = 0.02). Citrulline and ornithine had no effect on memory retention (χ2 test, CIT: χ2 = 0.41, p = 0.52; ORN: χ2 = 3.71, p = 0.054). In the unpaired groups, no NPAA altered the responses to the CS or to the NOd and experimental and control bees did not differ in CS-specific memory for any of the NPAAs (see Suppl. Mat. and Fig. 5).
Source: Ecology - nature.com
