Bees prefer foods containing neonicotinoid pesticides

Abstract

The impact of neonicotinoid insecticides on insect pollinators is highly controversial. Sublethal concentrations alter the behaviour of social bees and reduce survival of entire colonies. However, critics argue that the reported negative effects only arise from neonicotinoid concentrations that are greater than those found in the nectar and pollen of pesticide-treated plants. Furthermore, it has been suggested that bees could choose to forage on other available flowers and hence avoid or dilute exposure. Here, using a two-choice feeding assay, we show that the honeybee, Apis mellifera, and the buff-tailed bumblebee, Bombus terrestris, do not avoid nectar-relevant concentrations of three of the most commonly used neonicotinoids, imidacloprid (IMD), thiamethoxam (TMX), and clothianidin (CLO), in food. Moreover, bees of both species prefer to eat more of sucrose solutions laced with IMD or TMX than sucrose alone. Stimulation with IMD, TMX and CLO neither elicited spiking responses from gustatory neurons in the bees' mouthparts, nor inhibited the responses of sucrose-sensitive neurons. Our data indicate that bees cannot taste neonicotinoids and are not repelled by them. Instead, bees preferred solutions containing IMD or TMX, even though the consumption of these pesticides caused them to eat less food overall. This work shows that bees cannot control their exposure to neonicotinoids in food and implies that treating flowering crops with IMD and TMX presents a sizeable hazard to foraging bees.

Extended Data Figure 1:. The proportion of bees surviving after 24 h in the two-choice assay presented in Figure 1

a, Bumblebees given a choice between sucrose and sucrose laced with 1k nM TMX or CLO were less likely to survive after 24 h (lreg: IMD: χ₄² = 4.36, P = 0.359; TMX: χ₄² = 62.3, P < 0.001; CLO: χ₄² = 79.7, P < 0.001). b, Honeybees given a choice between sucrose and sucrose laced with 1k nM TMX or CLO were less likely to survive after 24 h (lreg: IMD: χ₄² = 5.18, P = 0.269; TMX: χ₄² = 577, P < 0.001; CLO: χ₄² = 243, P < 0.001). Cohort (cov) accounted for a significant portion of the variance in survival for all three treatment groups (lreg: IMD: χ₁² = 22.0, P < 0.001; TMX: χ₁² = 32.4, P < 0.001; CLO: χ₁² = 70.2, P < 0.001). * indicates P < 0.05 in least squares post hoc comparisons against sucrose in each treatment

Extended Data Figure 2:. Antennal proboscis extension response (PER) and mouthparts assay of honeybees to solutions containing neonicotinoids

a, Stimulation of the antennae with 1 M sucrose solutions containing neonicotinoids did not affect the elicitation of PER. b, Honeybees did not refuse to consume solutions containing neonicotinoids; only one bee in the CLO treatments failed to drink the solutions. N = 40/neonicotinoid treatment for antennal stimuli and N = 10/each concentration of each neonicotinoid for the mouthparts taste assay.

Extended Data Figure 3:. Young bees avoid solutions containing neonicotinoids

a, Newly-emerged worker bumblebees (N = 30 bees/treatment) and honeybees (N = 20 boxes/treatment) were tested in the behavioural choice assay with 1 nM and 10 nM IMD in sucrose solution as in Figure 2. Bumblebees avoided consuming both solutions containing IMD (one-sample t-test against 0, 1nM: P < 0.001, 10nM: P = 0.001), whereas honeybees avoided only the 1 nM concentration (one-sample t-test against 0, 1nM: P = 0.003, 10nM: P = 0.773). Error bars represent ± SE. b, The presence of IMD did not alter the spike frequency of gustatory neurons in the galeal sensilla of newly-emerged honeybees (repeated-measures ANOVA, stimulus: F_{1, 47} = 0.207, P = 0.653). Recordings were made from the basiconic sensilla on the galea as in Figure 1. Boxplots represent the frequencies of responses to 50 mM sucrose or to 50 mM sucrose solutions containing 1 nM or 10 nM IMD. N = 5 bees, 10 sensilla/bee. Boxplots represent the median (black bars), the 1.5 interquartile range (whiskers) and outliers (circles). Stimuli on x-axis are in order of presentation during the experiment.

Extended Data Figure 4:. Spike-sorted recordings for four of the honeybees in Figure 2

a, To verify that the spike rates we observed in Figure 2 were not a result in changes in the rates of firing of individual neurons, we spike sorted recordings from 4 honeybees. b, Spike sorting revealed 2 potential spiking neurons (units) with different amplitudes that responded by eliciting spikes during sucrose stimulation. (This was also observed previously by Wright et al. 2010). One neuron is labelled in green, the other in red. Spike doublets (indicated in pink as ‘d’) where both neurons spiked nearly simultaneously were also observed. c, d. These same two spiking neurons continued to respond when stimulated with sucrose containing 1uM IMD. e, Boxplots reveal that the rate of spiking was lower on average for one of the neurons (repeated-measures ANOVA, unit: F_1,36 = 596, P < 0.001). The rate of firing of both neurons was not affected by IMD concentration (repeated-measures ANOVA, unit: F_1,36 = 0.369, P = 0.547). Spikes from additional neurons (units) were not detected, and so we concluded that no other neurons were recruited during stimulation with IMD. ‘S’ indicates stimulation with sucrose. Boxplots represent the median (black bars), the 1.5 interquartile range (whiskers) and outliers (circles). Stimuli on x-axis are in order of presentation during the experiment.

Figure 1. Foraging-age bees prefer to eat food containing neonicotinoids

a, Bumblebees and (b) honeybees given a choice of sucrose or sucrose containing a neonicotinoid pesticide chose to eat solutions containing IMD and TMX (Extended Data Table 2, bumblebees: GLM: χ₂² = 12.1, P = 0.002; honeybees: χ₂² = 11.1, P = 0.004). Data represent the difference in the amount consumed over 24 h; positive values indicate a preference for solutions containing neonicotinoids. White bars indicate the sucrose control. Asterisks indicate P ≤ 0.002 (Bonferroni-adjusted critical value) for one-sample t-tests against the ‘0’ value (indicating no preference, see Extended Data Table 3). Bumblebees: IMD: 1nM = 57, 10nM = 66, 100nM = 65, 1μM = 66; TMX: 1nM = 38, 10nM = 39, 100nM = 36, 1μM = 40; CLO: 1nM = 57, 10nM = 59, 100nM = 48, 1μM = 62; honeybees: N = 40 cohorts of 25 bees/treatment. (c) The total amount of food eaten from both tubes by bumblebees was affected by the concentration and the presence of a neonicotinoid pesticide (GLM: χ₆² = 47.7, P < 0.001, Extended Data Table 2) in one of the food tubes. (d) Honeybees ate less total food only when it contained 1k nM TMX or CLO (GLM: χ₆² = 10.5, P = 0.005, Extended Data Table 2). White diamonds indicate amount eaten by sucrose control group. * indicates P < 0.05 in post hoc comparisons against sucrose. Error bars represent ± SE.

Figure 2. Electrophysiological recordings of the gustatory receptor neurons from the mouthparts of bumblebees and honeybees during stimulation with neonicotinoids

a, Scanning electron micrographs (SEM) of the galea (white arrows) of bumblebees and (b) honeybees. Recordings were made from the basiconic sensilla of the galea (white arrows); inserts are higher resolution SEM of individual sensilla. c, d. Spike trains recorded from both species reveal responses to NHT and to sucrose, but not to IMD. Boxplots of the spiking responses of gustatory neurons of the mouthparts of bumblebees (e) and honeybees (f) to KCl, NHT and two concentrations of each of the neonicotinoids. Dashed lines represent the median response to 50 mM sucrose. Solutions of the three neonicotinoids did not elicit activity from gustatory neurons greater than the response to water (indicated as ‘0’ on x-axis) (Extended Data Table 5, ANOVA: bumblebees: F_2,77 = 0.935, P = 0.397; honeybees: F_2,144 = 2.38, P = 0.096). (Note: NHT elicited spike frequencies in gustatory neurons greater than those elicited by water in only 7/17 of the bumblebees we tested, whereas NHT elicited spike frequencies greater than water in all of the honeybees tested). Bumblebees: N_IMD = 5; N_TMX = 7; N_CLO = 5. Honeybees: N_IMD = 5; N_TMX = 5; N_CLO = 6. g, h. The spiking response to sucrose was not reduced by the presence of the neonicotinoids at concentrations in the nectar-relevant range (Extended Data Table 5, ANOVA: bumblebees: F_1,86 = 0.579, P = 0.449; honeybees: F_1,127 = 2.00, P = 0.053). Bumblebees: N_IMD = 8; N_TMX = 5; N_CLO = 6. Honeybees: N_IMD = 6; N_TMX = 5; N_CLO = 6. Boxplots represent the median (black bars), the 1.5 interquartile range (whiskers) and outliers (circles). Stimuli on x-axes of e-h are in order of presentation during the experiment.