Neonicotinoid clothianidin adversely affects insect immunity and promotes replication of a viral pathogen in honey bees

Abstract

Large-scale losses of honey bee colonies represent a poorly understood problem of global importance. Both biotic and abiotic factors are involved in this phenomenon that is often associated with high loads of parasites and pathogens. A stronger impact of pathogens in honey bees exposed to neonicotinoid insecticides has been reported, but the causal link between insecticide exposure and the possible immune alteration of honey bees remains elusive. Here, we demonstrate that the neonicotinoid insecticide clothianidin negatively modulates NF-κB immune signaling in insects and adversely affects honey bee antiviral defenses controlled by this transcription factor. We have identified in insects a negative modulator of NF-κB activation, which is a leucine-rich repeat protein. Exposure to clothianidin, by enhancing the transcription of the gene encoding this inhibitor, reduces immune defenses and promotes the replication of the deformed wing virus in honey bees bearing covert infections. This honey bee immunosuppression is similarly induced by a different neonicotinoid, imidacloprid, but not by the organophosphate chlorpyriphos, which does not affect NF-κB signaling. The occurrence at sublethal doses of this insecticide-induced viral proliferation suggests that the studied neonicotinoids might have a negative effect at the field level. Our experiments uncover a further level of regulation of the immune response in insects and set the stage for studies on neural modulation of immunity in animals. Furthermore, this study has implications for the conservation of bees, as it will contribute to the definition of more appropriate guidelines for testing chronic or sublethal effects of pesticides used in agriculture.

Keywords: Apis mellifera; DWV; NLR (CLR); neuroimmunity; toxicology.

Fig. 1.

Dmel\LRR inhibits NF-κB activation and negatively modulates the immune response. The transcriptional down-regulation of Dmel\LRR upon immune challenge (A) was associated with a concurrent increase of the transcription rate of the genes encoding the antimicrobial peptides Defensin and Drosomycin, under the control of the Toll pathway (B). The RNAi-mediated knockdown of Dmel\LRR (C) determined a significant increase in the basal level of transcription of the defensin and drosomycin genes (D). The mean ± SD of the fold change in gene expression is represented. The observed trends for Dmel\LRR (A) and defensin and drosomycin (B) are significant at P < 0.05, P < 0.001, and P < 0.001, respectively; significant (P < 0.05) differences between treatments (C and D) are shown with an asterisk.

Fig. 2.

Effect of insecticides on NF-κB signaling in D. melanogaster. The Toll pathway activation in the larvae of the transgenic strain expressing drosomycin-GFP was assessed by scoring the fluorescence as low (Inset 1) or high (Inset 2) in experimental individuals treated with the neonicotinoid clothianidin or the organophosphate chlorpyriphos, at a standard LD₅₀ dose, and immune-challenged with S. cerevisiae. The large majority of the larvae exposed to clothianidin showed low or barely visible fluorescence, whereas the opposite was observed both for controls and for larvae treated with chlorpyriphos. The mean percentage of D. melanogaster larvae with high fluorescence ± normalized SD is represented; significant differences are shown with asterisks (***P < 0.001) (A). The Dmel\LRR transcript was up-regulated in the experimental larvae exposed to clothianidin. The mean ± SD of the fold change in Dmel\LRR expression is represented; significant differences are shown with asterisks (*P < 0.05) (B).

Fig. 3.

Effect of insecticides on NF-κB signaling in A. mellifera. The Toll pathway activation, assessed by measuring the transcript level of the antimicrobial peptide Apidaecin gene, was evaluated in honey bees treated with the neonicotinoid clothianidin or the organophosphate chlorpyriphos, at a standard LD₅₀ dose, and immune-challenged with S. cerevisiae. Clothianidin treatment significantly reduced the transcription level of apidaecin gene, which, in contrast, was not affected by chlorpyriphos. The mean ± SE of the fold change in apidaecin expression is represented; significant differences are shown with asterisks (*P < 0.05) (A). The immune-challenged larvae exposed to clothianidin showed an increased transcription of the gene Amel\LRR, which has 53% sequence identity with Dmel\LRR. The mean ± SE of the fold change in Amel\LRR expression is represented; significant differences are shown with asterisks (***P < 0.001) (B). A sustained transcriptional up-regulation of Amel\LRR was evident in honey bees starting from 4 h after the treatment with clothianidin and in absence of immune challenge. The mean ± SD of the fold change in Amel\LRR expression is represented; the observed trend is significant at P < 0.05 (C).

Fig. 4.

Effect of insecticides on DWV replication in honey bees bearing covert infections. The number of DWV genome copies was assessed in honey bees treated with increasing amounts of different insecticides; topical application (A and B) and oral uptake by feeding (C–E) were adopted to deliver insecticides to the experimental honey bees. In bees treated topically, viral replication was assessed after 24 h; in bees treated orally, viral replication was assessed over time, at 24-h time intervals. Viral replication was promoted, in both cases, in a dose-dependent manner by treatments with the neonicotinoid insecticides clothianidin and imidacloprid (A, C, and D), whereas this was not the case for chlorpyriphos (B and E). The ratio: (DWV genome copies at dose x)/(DWV genome copies at dose 0) ± SD is represented. The graphical schemes summarize how neonicotinoids, unlike chlorpyriphos, up-regulate the transcription of Amel\LRR, which results in the reduced activation of NF-κB and of the downstream antiviral barriers. Arrows indicate positive (e.g., stimulation or up-regulation) interactions; bar-headed lines mark negative interactions (e.g., inhibition or down-regulation); and dashed lines mark reduced effects.