Exposure to sublethal doses of fipronil and thiacloprid highly increases mortality of honeybees previously infected by Nosema ceranae

Abstract

Background: The honeybee, Apis mellifera, is undergoing a worldwide decline whose origin is still in debate. Studies performed for twenty years suggest that this decline may involve both infectious diseases and exposure to pesticides. Joint action of pathogens and chemicals are known to threaten several organisms but the combined effects of these stressors were poorly investigated in honeybees. Our study was designed to explore the effect of Nosema ceranae infection on honeybee sensitivity to sublethal doses of the insecticides fipronil and thiacloprid.

Methodology/finding: Five days after their emergence, honeybees were divided in 6 experimental groups: (i) uninfected controls, (ii) infected with N. ceranae, (iii) uninfected and exposed to fipronil, (iv) uninfected and exposed to thiacloprid, (v) infected with N. ceranae and exposed 10 days post-infection (p.i.) to fipronil, and (vi) infected with N. ceranae and exposed 10 days p.i. to thiacloprid. Honeybee mortality and insecticide consumption were analyzed daily and the intestinal spore content was evaluated 20 days after infection. A significant increase in honeybee mortality was observed when N. ceranae-infected honeybees were exposed to sublethal doses of insecticides. Surprisingly, exposures to fipronil and thiacloprid had opposite effects on microsporidian spore production. Analysis of the honeybee detoxification system 10 days p.i. showed that N. ceranae infection induced an increase in glutathione-S-transferase activity in midgut and fat body but not in 7-ethoxycoumarin-O-deethylase activity.

Conclusions/significance: After exposure to sublethal doses of fipronil or thiacloprid a higher mortality was observed in N. ceranae-infected honeybees than in uninfected ones. The synergistic effect of N. ceranae and insecticide on honeybee mortality, however, did not appear strongly linked to a decrease of the insect detoxification system. These data support the hypothesis that the combination of the increasing prevalence of N. ceranae with high pesticide content in beehives may contribute to colony depopulation.

Figure 1. Infection monitoring.

(A) Semi-thin sections of midgut epithelium of control (left) and infected (right) honeybees stained with toluidin blue. Arrows indicate N. ceranae foci. Bar = 25 µm. (B) Effect of N. ceranae infection on sucrose consumption. Data represent the mean of cumulative sucrose consumption (mg/bee +/− standard deviation, sd) from 9 replicates, each containing 50 honeybees.

Figure 2. Effect of N. ceranae infection on ECOD (A) and GST (B) activities at 10 days p.i. in midgut and fat body of honeybees.

Data represent the mean of specific activity (nmoles/min/mg of proteins +/− standard deviation, sd) from 6 replicates (45 honeybees/replicate) of control and infected honeybees. Asterisks indicate the level of significance at p<0.01 (**). ECOD: 7-ethoxycoumarin-O-deethylase; GST: Glutathione-S-Transferase.

Figure 3. Effect of N. ceranae infection on honeybee sensitivity to insecticides.

(A) Percentage of honeybee mortality of (i) uninfected control (light green square), (ii) N. ceranae-infected (dark green square), (iii) uninfected and chronically exposed to fipronil (light red circle), (iv) uninfected and chronically exposed to thiacloprid (light blue triangle), (v) N. ceranae-infected then chronically exposed to fipronil (dark red circle) and (vi) N. ceranae-infected then chronically exposed to thiacloprid (dark blue triangle). The arrow indicates the time of exposure to insecticides (10 days p.i.). Data represent the percentage of cumulative mortality calculated from 3 cages, each containing 50 honeybees. The means of fipronil (B) and thiacloprid (C) consumptions (pg or ng/bee/day +/− standard deviation, sd) were daily-monitored until 20 days p.i. for both infected (dark red or blue) and uninfected (light red or blue) honeybees.

Figure 4. Effect of exposure to insecticides on N. ceranae spore production.

The spore production in the digestive tract (midgut and rectum) was assessed at 20 days p.i. Data represent the mean numbers of spores/honeybee (×10⁶ +/− standard deviation, sd) from 15 honeybees, 20 days p.i. Asterisks indicate the level of significance at p<0.05 (*) and p<0.01 (**).