Transcriptome analyses of the honeybee response to Nosema ceranae and insecticides

Abstract

Honeybees (Apis mellifera) are constantly exposed to a wide variety of environmental stressors such as parasites and pesticides. Among them, Nosema ceranae and neurotoxic insecticides might act in combination and lead to a higher honeybee mortality. We investigated the molecular response of honeybees exposed to N. ceranae, to insecticides (fipronil or imidacloprid), and to a combination of both stressors. Midgut transcriptional changes induced by these stressors were measured in two independent experiments combining a global RNA-Seq transcriptomic approach with the screening of the expression of selected genes by quantitative RT-PCR. Although N. ceranae-insecticide combinations induced a significant increase in honeybee mortality, we observed that they did not lead to a synergistic effect. According to gene expression profiles, chronic exposure to insecticides had no significant impact on detoxifying genes but repressed the expression of immunity-related genes. Honeybees treated with N. ceranae, alone or in combination with an insecticide, showed a strong alteration of midgut immunity together with modifications affecting cuticle coatings and trehalose metabolism. An increasing impact of treatments on gene expression profiles with time was identified suggesting an absence of stress recovery which could be linked to the higher mortality rates observed.

Figure 1. Effect of N. ceranae and fipronil, alone or in combination, on honeybee survival.

Data give the cumulative proportion of surviving honeybees exposed to no treatment (blue), N. ceranae (red), fipronil (green), or a N. ceranae-fipronil combination (pink). N. ceranae-treated honeybees were individually infected at their emergence (day 0) and fipronil-treated ones were chronically and orally exposed to fipronil (1.3 μg/L) from day 0 to day 7. Data from 165 honeybees per experimental condition were analysed using the Kaplan-Meier method.

Figure 2. Principal component analysis of RNA-Seq data.

Gene expression changes were investigated at day 1 (circles) or 7 (squares) in honeybees exposed to no treatment (blue), N. ceranae (red), fipronil (green), or a N. ceranae-fipronil combination (pink). The PCA was performed using normalized RNA-Seq data of a set of 3001 genes showing an adjusted p-value <0.1 in at least one pairwise comparison.

Figure 3. Impact of N. ceranae and fipronil on gene expression or enzyme activity in the honeybee.

The figure presents a selection of genes or enzymes whose expression or activity has been shown to significantly increase (↑) or decrease (↓) under exposure to N. ceranae (red), to fipronil (green), or to a N. ceranae-fipronil combination (purple) in the present work or in previous studies: ¹Antunez et al., 2009; ²Chaimanee et al., 2012; ³Dussaubat et al., 2012; ⁴Vidau et al., 2011.