This study was conducted at University of California San Diego (UCSD), Division of Biological Sciences (La Jolla, CA, USA). We used six healthy honey bee colonies (A. mellifera ligustica Spinola, 1806, 10 frames per colony) housed at the UCSD Biology Field Station apiary. We tested a total of 78 bees.

The arena

We used a vertical arena illuminated from the top (Fig. 5, 30 × 30 × 5 cm) to assess forager locomotion and movement to light (Fig. 5). The arena was designed so that it would match a normal vertical comb environment. Previous studies tested bee walking through smaller spaces (petri dishes: 9 cm30; 15 × 1.5 cm31) or a similar apparatus (also vertical and artificially illuminated from above, 30 × 30 × 4 cm32,33,34,35). Our arena had white acrylic side walls and transparent acrylic back, front and top. The front could slide open to allow bee removal and cleaning after each test; a grid of white paper (36 squares, 5 cm in width) was placed on the outside of the back wall for measurements. The interior was completely composed of plastic to facilitate cleaning. Bees walked through a 1 cm diameter tubular entrance at the bottom right of the arena. We divided the arena in 36 cubes (Fig. 5, 5 × 5 × 5 cm) that we used to measure the location of the bee during the test. A LED light (luminous flux: 280 lm, 120° illumination angle, 6000 K color temperature) was centred at the top and pointed to the bottom (Fig. 5). During trials, no other lights were used. Light intensity was maintained at a constant 280 lm, because light intensity influences the phototactic behaviour of the bees25,26. We used a light intensity of 280 lm because this level of brightness provided a sufficient phototactic effect, attracting bees who travelled from the bottom towards the light within the trial duration of 3 min.

Figure 5 The arena used to test activity, motor functions, and movement to light of foragers. A bee is drawn approaching the light near the top of the arena. The amount of light (left: luminous flux (lux); right: Photosynthetic Photon Flux Density (µmol m−2 s−1); mean ± standard error) at the centre of the arena, directly below the light, is shown for each level. The oval at the base of the arena shows the position of the temperature (T) and relative humidity (RH) sensor. Full size image

Because the light levels inside the arena varied depending on the distance from the light source, we quantified the light levels (level 1–6, 10 replicates per each level) using a digital Lux meter (Dr. Meter, model LX1330B, measuring range of 0.1~200,000 Lux, resolution of 0.1 Lux, Fig. 5). To measure the Photosynthetic Photon Flux Density (PPFD, defined as the number of photons in the photosynthetic range received by a 1 m2 surface per s) we used a Photosynthetically Active Radiation (PAR) sensor (Vernier Software & Technology, PAR range 0–2000 µmol m−2 s−1, resolution 1 µmol m−2 s−1, spectral range 410–655 nm, Fig. 5). The wavelength range of this standard sensor is somewhat similar to the spectral range of honey bee vision (300–650 nm)57,58. In our apparatus, 280 lm corresponded to an illuminance range bottom-top of 89–710 lux and a PPFD range bottom-top of 89–1396 µmol m−2 s−1 (Fig. 5). These light levels span the intensities (14–560 lux25,26,27,34) used in previous phototactic experiments, and the range of light at our apiary (in shade (N = 10): 97 ± 1 µmol m−2 s−1; in full sun (N = 10): 1646 ± 26 µmol m−2 s−1).

The arena included a temperature and relatively humidity (RH) sensor (Fig. 5). Temperature and RH were at 25 ± 1 °C and between 50–80% RH during the experiments. We placed the video camera (Sony, model NXCAM Exmor R) directly in front of and perpendicular to the plane of the arena.

Honey bee preparation

We wished to study foragers, the bees most likely exposed to TMX, and therefore focused on bees returning to the nest with pollen in their corbiculae since these bees are, by definition, foragers. Returning pollen foragers were individually captured in vials at hive entrances. After collection, foragers were placed into plastic cages (11 × 11 × 9 cm, 10 bees/cage) and maintained in an incubator at 30 ± 1 °C and 50–80% RH with sucrose solution ad libitum for 1 h for the acute experiment or 48 h for the chronic experiment.

Forager behaviours in the Arena

To test the effect of TMX over time, each forager was tested inside the arena twice (30 min and 60 min after treatment). During each test, bee behaviours were recorded for 3 min, as in similar studies32,33,34. We chose this time interval because 3 min was more than sufficient for control bees to reach the light. To begin each trial, a bee was carefully captured from a plastic cage into a plastic vial that was then gently placed over a 1 cm long tube at the bottom, right side of the arena (Fig. 5). The room was completely dark, with the only light coming from the LED bulb at the top of the arena (Fig. 5). Bees then instinctively moved towards the light by entering the arena and then climbing up33. After reaching the light, bees (particularly those treated with TMX) would sometimes fall and could then climb again towards the light. Over the 3-min observation period, we measured 11 behavioural parameters (definitions in Table 2) related to bee activity, motor function and movement to light. Three parameters were related to the first path towards the light, which starts from the moment the bee enters the arena until the moment the bee reaches the light (time first path spent to reach the light for the first time, distance first path of the first path towards the light, and velocity first path during the first path towards the light). The other eight parameters referred to the whole 3-min period (overall velocity, time spent moving, time spent at the top, time spent at the bottom, overall number of falls, inability to reach the light, inability to ascend the arena walls, and exhibiting abnormal behaviours, Table 2). We calculated bee velocity by dividing the distance walked (number of 5 × 5 cm squares crossed) by time.

Table 2 List and definitions of parameters assessed during the phototaxis arena tests. Full size table

A preliminary analysis of a randomly selected sample of our data using video tracking (Tracker v4.96) yielded similar results (time to light: F 1,15 = 6.36, p = 0.024; distance to light: F 1,14 = 0.45, p = 0.515) as our simplified analysis of movements over squares. Measuring movement over squares facilitated the rapid analysis of the movements and the abnormal behaviours of more bees. It also created a simpler assay with greater potential for widespread use. We therefore use the analysis of movements over squares throughout this paper.

The inability to ascend the arena walls and the presence of general abnormal behaviours (trembling, loss of coordination, erratic movements) were scored if bees exhibited such behaviours for longer than 10 seconds throughout the 3-min test. We define trembling as the shaking or shivering of body, legs or antennae. A bee shows loss of coordination when she falls while walking or stumbles. We defined erratic movements as a bee walking in small circles or in other atypical patterns.

At the end of the trial, we opened the arena, carefully caught the bee in a vial and then thoroughly cleaned the arena with 100% ethanol to remove potential bee odours. We then allowed the arena to fully dry before reusing it.

Pesticide concentrations and doses

There is a wide range of field-relevant pesticide doses and concentrations, with variations across time and space59. Because we exposed foragers by feeding them TMX in sucrose solution, TMX levels in nectar provide the most realistic residue levels. However, we note that in relatively rare cases, foragers can contain higher TMX residues in their tissue (310 ppb60) and can consume higher concentrations of TMX (100 ppm61) by ingesting guttation droplets produced by TMX seed-treated plants such as corn and oilseed rape62.

The acute and chronic experiments and their respective analysis were based on the actual dose of TMX consumed by each bee (average per bee per cage). All TMX doses that we tested were lower than the worst case scenario thresholds, defined using calculations from the European Food Safety Authority (EFSA)63. The worst-case scenario calculations considered the field-relevant amount of nectar consumed by foragers, based upon the sucrose content of nectar and their energy requirements during foraging activity, and the highest TMX concentration found in nectar to which bees could be exposed. Because the worst-case scenarios were estimated depending on time of exposure, we compared our acute and chronic doses to calculated acute and chronic scenarios63.

In the acute exposure experiment, bees were fed a single dose of TMX (1.34 ng, 4.6 pmol). This same dose was used by prior studies that demonstrated an impact of TMX on forager homing41 and flight ability43. This dose is 3.7 times lower than the LD 50 of TMX64 and does not significantly increase mortality as compared to controls41. Although some authors consider 1.34 ng to not be field-relevant65, we calculated (based upon EFSA63) that foragers can acutely consume up to 1.80 ng TMX/bee in 1 h of foraging for nectar (10% sugar w/w, oilseed rape66,67 contaminated with 15 ppb of TMX (transplant-drip application68). Thus, 15 ppb68 is a fairly high TMX concentration, though even higher concentrations of TMX have been found in nectar by Sanchez-Bayo and Goka (17 ppb69), Dively and Kamel (19 ppb, including TMX metabolites68), and Stoner and Eitzer (20 ppb70). Reviews by Bonmatin et al.15 and Godfray et al.16 are informative. Because transplant-drip applications are typically a short-term contamination route for bees, we used this 15 ppb level to calculate the worst case acute exposure scenario (1 h short-term exposure63) only. Therefore, we tested an acute sublethal dose that was lower than the worst-case scenario (<1.8 ng/bee/1 h) in which bees foraged for 1 h on nectar that was contaminated by TMX after a transplant-drip application.

In the chronic exposure experiment, we exposed bees to a range of TMX daily doses (Range TMX daily doses = 1.42–3.48 ng/bee/day = 4.9–11.9 pmol/bee/day, Mean TMX daily doses = 2.56 ± 0.12 ng/bee/day, N TMX daily doses = 4). These daily doses reflected the actual TMX consumption per bee cage, and resulted from feeding bees a sucrose solution containing 45 ppb of TMX. EFSA estimated that foragers could consume up to 6.66 ng TMX/bee/day when foraging nectar from TMX seed-treated plants (i.e. oilseed rape) containing 5 ppb of TMX63. In our experiments, foragers consumed TMX daily doses that were always lower than 6.66 ng of TMX/bee/day.

Foragers consume less sucrose per day when maintained in cages as compared to colonies, perhaps because they have reduced activity in small cages. The foragers we reared in the lab consumed 61 ± 10 (control) and 57 ± 15 (TMX) mg/bee/day of sugar, while it’s been estimated that foragers could consume up to 128 mg/bee/day of sugar while foraging in the field63. To achieve field-relevant TMX daily doses approaching a realistic worst-case scenario, we provided foragers with a TMX solution that was more concentrated (45 ppb) than what is typically found in nectar after seed treatments. Therefore, we focused our analyses on the field-relevant TMX daily doses consumed by our bees.

We used analytical grade TMX (CAS#153719-23-43, Sigma Aldrich 37924-100MG-R). We prepared the stock solution with double-distilled H 2 0, and we maintained it at 4 °C inside a bottle completely wrapped in aluminium foil to avoid light degradation15. We prepared the solutions used to feed the bees daily, by diluting the stock solution with 1.8 M sucrose solution.

Acute exposure

After collection, foragers were incubated for 1 h with 0.5 M sucrose solution (pesticide-free, prepared with analytical grade sucrose and double-distilled water) ad libitum, to allow them to adjust to their new setting and to help equalize their hunger levels. After the 1 h incubation, bees were starved for 30 min to allow them to subsequently consume 10 µl of 1.8 M sucrose test solution. For feeding, each bee was individually inserted in a modified syringe (2.5 mL). During feeding, the plunger was gently pushed to coax the bee to the end of the syringe, in which a 2-mm diameter hole was cut and through which the bee was fed the test solution. The test solution was either pure sucrose or contained the same TMX dose used by Henry et al.41 and Tosi et al.43: 1.34 ng (corresponding to 118 ppb, 134 µg/L and 460 nmol/L). For these calculations, we took into account the density of 1.8 M sucrose solution at 20 °C and 1 ATM (1.230 kg/L71). After the individual feeding, we placed each bee into a separate cage to prevent food exchange with other bees. We maintained these cages in an incubator at 30 ± 1 °C, 50–80% RH, with no food, for 60 minutes after exposure, excluding the 3-min trial occurring in the arena 30 min after exposure. We tested 42 bees from three colonies.

Chronic exposure

Bees can be chronically exposed to neonicotinoids if they continue to forage over multiple days at a food source contaminated with the pesticide. We therefore tested the chronic effects of TMX. After collection, foragers were incubated for 2 days with 1.8 M sucrose solution ad libitum. The solutions were either pure sucrose or contained 45 ppb of TMX (corresponding to 55 µg/L and 189 nmol/L). The consumption of sucrose solution and TMX per bee was measured daily by weighing syringes. To measure potential evaporative loss, we separately used 10 cages maintained in identical conditions but without bees. We accounted for this evaporative loss (<1%) in our calculations. We tested 36 bees from four colonies.

Statistical analysis

In the acute exposure experiment, we tested bees at 30 and 60 minutes after exposure. We used Repeated-Measures Analysis of Variance (ANOVA) with a REML algorithm to test the effects of pesticide treatment (control vs. TMX) at 30 and 60 min after exposure upon the following continuous measures: number of falls (counts), velocity (squares/s), time at the top (s), time at the bottom (s), time moving (s), distance first path to reach the light (number of squares), time first path to reach the light (s), and mean velocity first path to reach the light (squares/s). Colony was included as a random factor. Significant effects were further analysed with post-hoc Least-Square Means contrast tests. We applied the Dunn-Sidak method72 to correct for multiple comparisons. We used a Repeated-Measures Multiway Frequency analysis73 to test the effect of pesticide treatment (control vs. TMX), time (30 vs. 60 min post-treatment) and their interaction on the following nominal measures: abnormal behaviours (Y/N), inability to ascend the arena (Y/N), and inability to reach the light (Y/N). All tested bees participated to both arena tests (30 and 60 min after treatment).

In the chronic exposure experiment, we tested bees at 60 min after exposure. We used Mixed-Model ANCOVA and tested TMX daily doses as a continuous effect (N TMX daily doses = 4) and colony as a nominal effect (random grouping variable, REML algorithm) on the same continuous parameters examined in the acute exposure experiment (see above). We used Nominal Logistic regression to test the effect of TMX daily doses (N TMX daily doses = 4) upon these nominal parameters: abnormal behaviours, inability to ascend the arena, and inability to reach the light. In these Nominal Logistic models, we included colony as a fixed effect. The sucrose consumption data were not normally distributed, and we therefore used the Kruskal-Wallis Rank Sum test to assess the effect of treatment on sucrose consumption.

We used R v3.3.274 and JMP v10.0 statistical software. We also used residuals analyses to confirm the appropriateness of our models. We report mean ± 1 standard error (s.e.m.). We used an alpha value of 0.05, corrected, as necessary, using the Dunn-Sidak method (see above). We only analysed bees that remained alive for the entire arena trial (180 seconds, 99% of bees). The analysis of the parameters related to the first path towards the light included only bees that managed to reach the light. Inability to reach the light was captured by the analysis of the inability to reach light variable.