Neonicotinoid Clothianidin reduces honey bee immune response and contributes to Varroa mite proliferation

Abstract

The neonicotinoid Clothianidin has a negative impact on NF-κB signaling and on immune responses controlled by this transcription factor, which can boost the proliferation of honey bee parasites and pathogens. This effect has been well documented for the replication of deformed wing virus (DWV) induced by Clothianidin in honey bees bearing an asymptomatic infection. Here, we conduct infestation experiments of treated bees to show that the immune-suppression exerted by Clothianidin is associated with an enhanced fertility of the parasitic mite Varroa destructor, as a possible consequence of a higher feeding efficiency. A conceptual model is proposed to describe the synergistic interactions among different stress agents acting on honey bees.

Fig. 1. Effect of Clothianidin treatment on encapsulation and melanization of a nylon thread implanted in the body cavity of larvae and adults of honey bee.

For each experimental condition, 3 replicates of 5 honey bees each were considered. Range (from minimum to maximum value), 1st and 3rd quartile and median are reported in the box-plots; the horizontal bars in the scatter-jittered plots represent the sample average; different letters denote significant differences. a Melanization in mature bee larvae (0 vs 0.01 ppm and 0 vs 0.05 ppm: Mann–Whitney U test: n₁ = 15, n₂ = 15, U = 0, adjusted, one tailed P < 0.001). b Encapsulation in mature bee larvae (0 vs 0.01 ppm and 0 vs 0.05 ppm: Mann–Whitney U test: n₁ = 15, n₂ = 15, U = 0, adjusted, one tailed P < 0.001). c DWV RNA copies recorded in experimental honey bee larvae (0 vs 0.01 ppm and 0 vs 0.05 ppm: Mann Whitney U test: n₁ = 15, n₂ = 15, U = 0, adjusted, one tailed P < 0.001; 0.01 vs 0.05 ppm: Mann–Whitney U test: n₁ = 15, n₂ = 15, U = 60, adjusted, one tailed P = 0.044). d Melanization in adult bees (Kruskal–Wallis: adj H = 70.17, df = 4, P < 0.001). e Encapsulation in adult bees (Kruskal–Wallis: adj H = 68.34, df = 4, P < 0.001). f DWV titer recorded in adult bees used for the implantation experiment (Mann–Whitney U test: n₁ = 15, n₂ = 15, U = 4, one tailed P < 0.001).

Fig. 2. Relative expression over time of two genes involved in humoral and cellular immune response of honey bee larvae.

At each time point, 15 biologically independent larvae per treatment were sampled. The asterisk denotes a significant difference (Mann–Whitney U test: n₁ = 15, n₂ = 15, U = 58, one tailed P = 0.012) for the mean values recorded at a specific time point; error bars represent the standard deviation of the mean, calculated according to the ∆∆Ct method. a Relative expression over time of Amel\102. b Relative expression over time of Dorsal 1A.

Fig. 3. Clothianidin effect on DWV infection level and Varroa reproduction.

Asterisks denote significantly different mean values (one asterisk: P < 0.05; three asterisks: P < 0.001). a DWV infection level in honey bees eclosed from larvae reared in vitro on a diet with or without Clothianidin (the experiment was run in triplicate, for a total of 28 and 27 individuals for Clothianidin treated and untreated controls, respectively; the horizontal bars represent the sample average; Mann–Whitney U test: n₁ = 28, n₂ = 27, U = 140, one tailed P < 0.001). b Fertility of Varroa mites on honey bee larvae treated with Clothianidin or on untreated controls (the experiment was run in triplicate, for a total of 111 and 120 individuals for Clothianidin treated and untreated controls, respectively; the proportion of reproducing mites in each replicate along with the average fertility and relative standard deviation are reported; Mantel–Haenszel test: df = 2, M–H Chi-2 = 3.970, P = 0.046).

Fig. 4. Comparison between predicted and observed mite infestation of hives at different sampling times during the season.

a Mite infestation as predicted using the discrete time model we developed and the reproduction data of Varroa mites as affected by Clothianidin exposure. b Mite infestation observed in hives close to fields treated with neonicotinoids.

Fig. 5. The network of the observed interactions among different stress agents.

Clothianidin (CLT) contamination decreases NF-κB activation, impairing the immune response and enhancing mite’s fitness, as a possible consequence of a higher feeding efficiency. The decrease in NF-κB activation relaxes immune control on DWV, which, above a certain threshold, impacts NF-κB, reinforcing the negative effect on immune response. Arrows indicate positive (i.e., stimulation or upregulation) interactions; bar-headed lines mark negative interactions (i.e., inhibition or downregulation); dashed lines indicate that the effect can be impaired by Clothianidin treatment.