Neonicotinoids disrupt memory, circadian behaviour and sleep

Abstract

Globally, neonicotinoids are the most used insecticides, despite their well-documented sub-lethal effects on beneficial insects. Neonicotinoids are nicotinic acetylcholine receptor agonists. Memory, circadian rhythmicity and sleep are essential for efficient foraging and pollination and require nicotinic acetylcholine receptor signalling. The effect of field-relevant concentrations of the European Union-banned neonicotinoids: imidacloprid, clothianidin, thiamethoxam and thiacloprid were tested on Drosophila memory, circadian rhythms and sleep. Field-relevant concentrations of imidacloprid, clothianidin and thiamethoxam disrupted learning, behavioural rhythmicity and sleep whilst thiacloprid exposure only affected sleep. Exposure to imidacloprid and clothianidin prevented the day/night remodelling and accumulation of pigment dispersing factor (PDF) neuropeptide in the dorsal terminals of clock neurons. Knockdown of the neonicotinoid susceptible Dα1 and Dβ2 nicotinic acetylcholine receptor subunits in the mushroom bodies or clock neurons recapitulated the neonicotinoid like deficits in memory or sleep/circadian behaviour respectively. Disruption of learning, circadian rhythmicity and sleep are likely to have far-reaching detrimental effects on beneficial insects in the field.

Figure 1

Field relevant concentrations of neonicotinoids or knockdown of Dα1 or Dβ2 in the mushroom bodies reduced 1 h memory. 1 h memory was reduced in flies exposed to field relevant concentrations of 1 or 10 μg/L (a) imidacloprid (IM) (χ²₂) = 7.3, p = 0.026), (b) clothianidin (CLO) (χ²₂ = 12.4, p = 0.002), (c) thiamethoxam (TMX) (χ²₂ = 9.6, p = 0.008) and not in (d) thiacloprid (TCL) (χ²₂ = 5.0, p = 0.084). (e) Likewise, 1 h memory was reduced in flies with RNAi mediated knockdown of Dα1 (OK107-Gal4 > uas-nAChR-Dα1) or Dβ2 (OK107-Gal4 > uas-nAChR-Dβ2) throughout the mushroom body (F_2,20 = 4.6, p = 0.023) and compared to genotype controls. Each data point represents ~ 100 flies, n ≥ 4 per treatment. Graphs show mean ± standard error of the mean (SEM) (post hoc pairwise comparisons: p ≤ 0.05*, p ≤ 0.01**, p ≤ 0.001***, p ≤ 0.0001****). The same tests, error bars and p values were used throughout.

Figure 2

Field relevant concentrations of neonicotinoids reduced behavioural rhythmicity. (a) Representative actograms of the activity of single flies for control or 50 μg/L imidacloprid, clothianidin, thiamethoxam or thiacloprid. Blue bars indicate activity, grey background indicates lights off, white background lights on. Mean rhythmicity for flies exposed to 1, 10 or 50 μg/L (b) IM (F_3,112 = 2.5, p = 0.060), (c) CLO (F_3,116 = 14.2, p < 0.001), (d) TMX (F_3,118 = 23.7, p < 0.001) and (e) TCL (F_3,118 = 0.05, p = 0.987). Each data point represents a single fly, n = 28–32 flies per treatment. Pie charts show the increase in the proportion of the population who were arrhythmic (rhythmicity statistic (RS) ≤ 1.5) for 50 μg/L: (f) IM, (g) CLO, (h) TMX and (i) TCL, compared to controls.

Figure 3

Field relevant concentrations of neonicotinoids disrupted sleep behaviour. Sleep plots showing the total sleep achieved per 30 min bin over the 24 h period (zeitgeber time (ZT)) for flies exposed to 1, 10 or 50 µg/L of (a) imidacloprid, (b) clothianidin, (c) thiamethoxam or (d) thiacloprid. The number of (no.) of sleep episodes initiated in (e) IM, day (F_3,114 = 1.2, p = 0.320) and night (F_3,114 = 5.5, p = 0.001), (f) CLO, day (F_3,120 = 11.5, p < 0.001) and night (F_3,120 = 25.0, p < 0.001), (g) TMX, day (F_3,124 = 1.1, p = 0.344) and night (F_3,124 = 17.0, p < 0.001) or (h) TCL, day (F_3,120 = 0.2, p = 0.872) and night (F_3,120 = 3.0, p = 0.034). Mean length (in minutes) of sleep episodes initiated in (i) IM, day (F_3,114 = 0.2, p = 0.889) and night (F_3,114 = 4.5, p = 0.005), (j) CLO, day (F_3,120 = 9.9, p < 0.001) and night (F_3,120 = 21.8, p < 0.001), (k) TMX, day (F_3,124 = 2.5, p = 0.061) and night (F_3,124 = 15.7, p < 0.001) or (l) TCL, day (F_3,120 = 5.2, p = 0.002) and night (F_3,120 = 2.0, p = 0.121). Each data point represents a single fly, n = 28–32 flies per treatment. CLO and TMX were run together with one set of controls, whilst IM and TCL were run together with another control group.

Figure 4

Knockdown of Dα1 or Dβ2 in the clock bearing cells disrupts circadian rhythmicity and sleep with no further effect by addition of neonicotinoids. Representative actograms for (a) Dα1 knock down (tim-Gal4 > uas-nAChR-Dα1) and (b) Dβ2 knockdown (tim-Gal4 > uas-nAChR-Dβ2. Effects of knocking down Dα1 in clock bearing cells on (c) rhythmicity (RS) (F_2,79 = 11.8, p < 0.001), (d) number (no.) of sleep episodes in day (F_2,79 = 2.9, p = 0.063) and night (F_2,79 = 12.3, p < 0.001) and (e) mean episode length in day (F_2,79 = 5.1, p = 0.008) and night (F_2,79 = 8.3, p = 0.001). Effects of knocking down Dβ2 in clock bearing cells on (f) rhythmicity (F_2,79 = 31.5, p < 0.001), (g) no. of sleep episodes in day (F_2,79 = 1.6, p = 0.211) and night (F_2,79 = 28.2, p < 0.001) and (h) mean episode length in day (F_2,79 = 11.2, p < 0.001) and night (F_2,79 = 9.4, p < 0.001). Each data point represents a single fly, n = 19–32 flies per treatment. There was no additive effect of 50 µg/L of IM and CLO on (i) tim > α1 (F_2,85 = 0.4, p = 0.677) and (j) tim > β2 (F_2,89 = 1.1, p = 0.336). Each data point represents a single fly, n = 24–32 flies per treatment.

Figure 5

Field relevant concentrations of neonicotinoids disrupt the day/night remodelling and PDF cycling in the s-LNv clock neuron dorsal terminals. (a) Representative confocal images of the s-LNv dorsal terminals for control and treated (50 µg/L IM or CLO) flies in the day (ZT2 i.e. 11am) and night (ZT14 i.e. 11 pm). (b) s-LNv dorsal terminal branching complexity is greater in the day than at night for control flies (t₁₇ = 2.3, p = 0.036). The day/night differences in complexity is removed in flies exposed to 50 µg/L of IM (t₁₄ = 2.1, p = 0.055) or CLO (t₁₅ = 2.1, p = 0.052). (c) Accumulation of PDF in dorsal terminals is greater in the day than at night in control flies (t₁₇ = 2.9, p = 0.010), treatment with 50 µg/L IM (t₁₃ = 1.0, p = 0.332) or CLO (t₁₄ = 2.1, p = 0.054) removed this day/night difference in PDF levels. Each data point represents a single brain, n = 6–15 brains.

Figure 6

Knockdown of Dα1 or Dβ2 disrupted the day/night remodelling and PDF cycling of the s-LNv dorsal terminals. (a) Representative confocal images of the s-LNv dorsal terminals of control flies (PDF-Gal4/ +) and flies with Dα1 (PDF-Gal4 > uas-nAChR-Dα1-RNAi) and Dβ2 (PDF-Gal4 > uas-nAChR-Dβ2-RNAi) knocked down in LNv clock neurons taken in the day (ZT2) and night (ZT14). (b) The s-LNv dorsal terminals of control flies showed greater branching complexity in the day than at night (t₁₇ = 2.3, p = 0.036), this day/night difference in terminal complexity was removed in PDF > Dα1-RNAi (t₁₉ = 1.4, p = 0.183) and PDF > Dβ2-RNAi (t₁₃ = -0.7, p = 0.515) flies. (c) PDF accumulation in the s-LNv dorsal terminals was greater in the day than at night for control flies (t₁₇ = 2.9, p = 0.010), but not in PDF > Dα1-RNAi flies (t₁₉ = 1.8, p = 0.089) and PDF > Dβ2-RNAi flies (t₁₄ = 1.3, p = 0.218). Each data point represents a single brain, n = 6–15 brains.