Interaction of field realistic doses of clothianidin and Varroa destructor parasitism on adult honey bee (Apis mellifera L.) health and neural gene expression, and antagonistic effects on differentially expressed genes

Abstract

While many studies have examined the effects of neonicotinoid insecticides and the parasitic mite Varroa destructor on honey bees (Apis mellifera), more information on the combined effects of such stressors on gene expression, including neural related genes, and their impact on biological pathways is needed. This study analyzed the effects of field realistic concentrations of the neonicotinoid clothianidin on adult bees infested and not infested with V. destructor over 21 consecutive days and then determined bee survivorship, weight, deformed wing virus (DWV) levels and gene expression. V. destructor parasitism with or without clothianidin exposure was significantly associated with decreased survivorship, weight loss and higher DWV levels, while clothianidin exposure was only associated with higher levels of DWV. Expression analysis of the neural genes AmNlg-1, BlCh and AmAChE-2 showed that V. destructor caused a significant down-regulation of all of them, whereas clothianidin caused a significant down-regulation of only AmNrx-1 and BlCh. An interaction was only detected for AmNrx-1 expression. RNAseq analysis showed that clothianidin exposure resulted in 6.5 times more up-regulated differentially expressed genes (DEGs) than V. destructor alone and 123 times more than clothianidin combined with V. destructor. Similar results were obtained with down-regulated DEGs, except for a higher number of DEGs shared between V. destructor and the combined stressors. KEGG (Kyoto Encyclopedia of Genes and Genomes) biological pathway analysis of the DEGs showed that the stressor linked to the highest number of KEGG pathways was clothianidin, followed by V. destructor, and then considerably fewer number of KEGG pathways with the combined stressors. The reduced numbers of DEGs and KEGG pathways associated with the DEGs for the combined stressors compared to the stressors alone indicates that the interaction of the stressors is not additive or synergistic, but antagonistic. The possible implications of the antagonistic effect on the number of DEGs are discussed.

Fig 1. Kaplan-Meier survival curves of adult bees exposed to field realistic doses of clothianidin and/or V. destructor (Vd) for 21 consecutive days.

Log rank (Mantel-Cox) tests were used to determine significant differences between survival curves (adjusted p value of 0.001).

Fig 2. Weight of 21-day old bees after exposure to clothianidin and/or V. destructor.

Mean weight (mg ±S.E.) of adult bees exposed to field realistic doses of clothianidin and/or V. destructor (V) for 21 consecutive days. Bars with different letters above them represent significant differences using the Tukey’s HSD test after a two-way ANOVA showed significant differences. Non-transformed data are presented.

Fig 3. Box plots of DWV levels of 21-day old bees exposed to clothianidin and/or V. destructor.

DWV gc per μg of RNA. Different letters above the boxes represent significant differences using the Conover-Iman procedure after a Kruskal Wallis test showed a significant effect.

Fig 4. Box plots of the of relative gene expression versus ng of clothianidin.

(A) AmNlg-1 relative gene expression, calculated using the Livak 2^ΔΔ method with AmRPS5 as reference gene and 0 ng as calibrator. (B) AmNlg-1 relative gene expression, calculated using the Livak 2^ΔΔ method with AmRPS5 as reference gene and 0 ng as calibrator. (C) BlCh relative gene expression, calculated using the Livak 2^ΔΔ method with AmRPS5 as reference gene and 0 ng as calibrator. (D) AChE-2 relative gene expression, calculated using the Livak 2^ΔΔ method with AmRPS5 as reference gene and 0 ng as calibrator.

Fig 5. Venn diagram.

Number of DEGs in the Differential Expression Analysis (DEA), and the genes in common between the pairwise comparisons of 0 ng/μl of clothianidin vs 1x10^-2 ng/μl of clothianidin (0vs1x10^-2), 0 ng/μl vs 0 ng plus V. destructor (0vsVd) and 0 ng/μl vs 1x10^-2 ng/μl of clothianidin plus V. destructor (0vs1x10^-2+Vd). (A) Venn diagram showing the number of up-regulated (↑) DEGs. (B) Venn diagram showing the number of down-regulated (↓) DEGs.

Fig 6. Summary of the effects of clothianidin and/or V. destructor.

Effect of clothianidin (Cloth) and/or V. destructor (Vd) on honey bee survivorship, weight, DWV levels, the expression of AmNrx-1, AmNlg-1, BlCh and AmAChE-2, number of DEGs and KEGG pathways (↑ indicates an increase or up-regulation, ↓ indicates a decrease or a down-regulation).