Neonicotinoids disrupt circadian rhythms and sleep in honey bees

Abstract

Honey bees are critical pollinators in ecosystems and agriculture, but their numbers have significantly declined. Declines in pollinator populations are thought to be due to multiple factors including habitat loss, climate change, increased vulnerability to disease and parasites, and pesticide use. Neonicotinoid pesticides are agonists of insect nicotinic cholinergic receptors, and sub-lethal exposures are linked to reduced honey bee hive survival. Honey bees are highly dependent on circadian clocks to regulate critical behaviors, such as foraging orientation and navigation, time-memory for food sources, sleep, and learning/memory processes. Because circadian clock neurons in insects receive light input through cholinergic signaling we tested for effects of neonicotinoids on honey bee circadian rhythms and sleep. Neonicotinoid ingestion by feeding over several days results in neonicotinoid accumulation in the bee brain, disrupts circadian rhythmicity in many individual bees, shifts the timing of behavioral circadian rhythms in bees that remain rhythmic, and impairs sleep. Neonicotinoids and light input act synergistically to disrupt bee circadian behavior, and neonicotinoids directly stimulate wake-promoting clock neurons in the fruit fly brain. Neonicotinoids disrupt honey bee circadian rhythms and sleep, likely by aberrant stimulation of clock neurons, to potentially impair honey bee navigation, time-memory, and social communication.

Figure 1

Neonicotinoid ingestion alters circadian locomotor rhythms of honey bee foragers in LD and DD. Representative actograms of forager bees showing (A) control rhythmic activity, (B) loss of rhythms following ingestion of clothianidin (140 ppb) and altered locomotor rhythm patterns after ingestion of (C) thiamethoxam (140 ppb) and (D) clothianidin (140 ppb). Contingency plots reporting the percent of arrhythmic individuals for bees exposed to (E) thiamethoxam (Pearson X² = 13.14; p = 0.0043, **) or (F) clothianidin (Pearson X² = 10.59; p = 0.0142, *). 30 min binned activity profiles of control bees vs. bees exposed to either 140 ppb of (G) thiamethoxam (Two-way RM ANOVA, Time p < 0.0001, ****; Dose p = 0.7150; Interaction p < 0.0001, ****) or (H) clothianidin (Two-way RM ANOVA, Time p < 0.0001, ****; Dose p = 0.1014; Interaction p < 0.0001, ****). Average time of activity offset following lights off for (I) thiamethoxam-exposed bees (One-way ANOVA F = 11.23; p < 0.0001, ****; Dunnett’s multiple comparison 70 ppb p = 0.0054, **; 140 ppb p < 0.0001, ****) and (J) clothianidin-exposed bees (One-way ANOVA F = 9.664; p < 0.0001, ****; Dunnett’s multiple comparison 70 ppb p = 0.0012, **; 140 ppb p < 0.0001, ****). (K) Offset of activity during the first day of constant darkness relative to time of lights off in previous LD for clothianidin-exposed bees (One-way ANOVA F = 5.805; p < 0.0009, ***; Dunnett’s multiple comparison 70 ppb p = 0.0077, **; 140 ppb p = 0.0063, **). (L) Free-running period of bees under constant darkness after 4 days of LD and ingestion of different doses of thiamethoxam (One-way ANOVA F = 7.323; p < 0.0002, ***; Dunnett’s multiple comparison 70 ppb p = 0.0374, *; 140 ppb p = 0.0028, **). Dunnett’s multiple comparisons were performed, only significant values are shown for panels (I–L).

Figure 2

Constant light and thiamethoxam ingestion act synergistically to disrupt circadian rhythmicity of honey bees. (A) Representative actogram of foragers showing rhythmic activity of a bee exposed to 70 ppb thiamethoxam in constant darkness (DD). (B) Representative actogram of arrhythmic behavior after exposure to 70 ppb thiamethoxam in constant light (LL). Contingency plots showing the percentage of honey bees that were arrhythmic after exposure to thiamethoxam at various concentrations (C) under constant darkness (DD) (X² = 2.101 p = 0.3498, ns), or (D) constant light (LL) (250 lx; X² = 12.16 p = 0.0023, **).

Figure 3

Neonicotinoids disrupt sleep in honey bee foragers. Four day average sleep profiles for control bees and bees exposed to either (A) thiamethoxam (Two-way RM ANOVA, Time p < 0.0001, ****; Dose p = 0.0004, ***; Interaction p = 0.0015, **) or (B) clothianidin (Two-way RM ANOVA, Time p < 0.0001, ****; Dose p = 0.0054, **; Interaction p < 0.0001, ****). Significant decreases in total sleep duration for exposure to (C) thiamethoxam (One-way ANOVA F = 6.436; p = 0.0004, ***; Dunnett’s multiple comparison 70 ppb p = 0.0038, **; 140 ppb p = 0.0026, **) or exposure to (D) clothianidin (One-way ANOVA F = 4.205; p = 0.0067, **; Dunnett’s multiple comparison 140 ppb p = 0.0115, *). In addition, there were significant decreases in the number of sleep episodes for exposure to (E) thiamethoxam (One-way ANOVA F = 3.020; p = 0.0314*; Dunnett’s multiple comparison 140 ppb p < 0.0233, *) and for (F) clothianidin (One-way ANOVA F = 2.586; p = 0.0549, ns; Dunnett’s multiple comparison 140 ppb p < 0.0396, *) dosed bees. Significant values only are shown for Dunnett’s post hoc multiple comparisons (C–F).

Figure 4

Clothianidin induces excitatory increases in [Ca²⁺]_i in Drosophila l-LN_v clock neurons. (A) Representative images showing the baseline and peak fluorescence in Drosophila PDF⁺ l-LN_v neurons expressing GCaMP5G fluorescent calcium indicator, in response to 4.1 ppb clothianidin (cloth, top) or vehicle (bottom). (B) Fluorescence intensity traces showing individual responses of Drosophila PDF⁺ l-LN_v neurons to clothianidin (pale orange) or vehicle (pale blue). Averaged traces are shown for explants exposed to clothianidin (orange) and vehicle (blue). (C) Average peak fluorescence responses of neurons in explants exposed to vehicle (blue) or clothianidin (orange). Error bars represent SEM. ****Indicates p < 0.0001.