Increased survival of honeybees in the laboratory after simultaneous exposure to low doses of pesticides and bacteria

Abstract

Recent studies of honeybees and bumblebees have examined combinatory effects of different stressors, as insect pollinators are naturally exposed to multiple stressors. At the same time the potential influences of simultaneously occurring agricultural agents on insect pollinator health remain largely unknown. Due to different farming methods, and the drift of applied agents and manure, pollinators are most probably exposed to insecticides but also bacteria from organic fertilizers at the same time. We orally exposed honeybee workers to sub-lethal doses of the insecticide thiacloprid and two strains of the bacterium Enterococcus faecalis, which can occur in manure from farming animals. Our results show that under laboratory conditions the bees simultaneously exposed to the a bacterium and the pesticide thiacloprid thiacloprid had significant higher survival rates 11 days post exposure than the controls, which surprisingly showed the lowest survival. Bees that were exposed to diet containing thiacloprid showed decreased food intake. General antibacterial activity is increased by the insecticide and the bacteria, resulting in a higher immune response observed in treated individuals compared to control individuals. We thus propose that caloric restriction through behavioural and physiological adaptations may have mediated an improved survival and stress resistance in our tests. However, the decreased food consumption could in long-term also result in possible negative effects at colony level. Our study does not show an additive negative impact of sub-lethal insecticide and bacteria doses, when tested under laboratory conditions. In contrast, we report seemingly beneficial effects of simultaneous exposure of bees to agricultural agents, which might demonstrate a surprising biological capacity for coping with stressors, possibly through hormetic regulation.

Fig 1. Effect of exposure to single and combined agricultural agents on honeybee survival.

Survival is expressed as the percentage of cumulated number of surviving bees during the monitoring period of 11 days for each treatment group with n = 180 (MMH = E. faecalis strain MMH594, FLY = E. faecalis strain FLY1). Asterisks indicate differences in the survival rate after 11 days of individuals exposed to different stressors, * ≤ 0.005, ** ≤ 0.001, *** ≤ 0.0001 (Kaplan-Meier survival analysis, log Rank test for overall comparison).

Fig 2. Quantity of food uptake by honeybees depending on the diet.

Food consumed representing the average mean amount of diet consumed in ml per bee per day during the 11-day monitoring period. Diets were inoculated with potential agricultural stressors (C = control, MMH = E. faecalis strain MMH594, FLY = E. faecalis strain FLY1, THIA = thiacloprid) with n = 1341 bees in total after 11 days (THIA n = 167, MMH n = 164, FLY n = 169, CONTROL n = 157, MMH+THIA n = 161, FLY+THIA n = 173, MMH+FLY n = 175, MMH+FLY+THIA n = 175; food consumption was controlled for bees per diet). Results represent mean values ± 2 s.e.m. Asterisks indicate differences in average food consumed per bee, * ≤ 0.005, ** ≤ 0.001, *** ≤ 0.0001; post hoc comparisons adjusted with Bonferroni (alpha = 0.005). Bars are indicated in: black = control, dark-grey = MMH, light-grey = FLY, red = THIA, shaded black = MMH+FLY, shaded dark-grey = MMH+THIA, shaded light-grey = FLY+THIA, shaded red = MMH+FLY+THIA.

Fig 3. Lysozyme-like activity in the haemolymph of honeybees after being exposed to thiacloprid and E. faecalis.

General antibacterial activity measured as the diameter of the lytic zone on agar plates, transformed to lysozyme equivalents (μg/ml). Different enzyme activity in the haemolymph as a result of all possible different diet combinations as well as single stress exposure (C = control, MMH = E. faecalis strain MMH594, FLY = E. faecalis strain FLY1, THIA = thiacloprid). Asterisks indicate differences in the lysozyme-like activity, * ≤ 0.005, ** ≤ 0.001, *** ≤ 0.0001. Results represent mean values ± 2 s.e.m, Kurskal-Wallis test, n = 16 for each treatment group. Bars are indicated in: black = control, dark-grey = MMH, light-grey = FLY, red = THIA, shaded black = MMH+FLY, shaded dark-grey = MMH+THIA, shaded light-grey = FLY+THIA, shaded red = MMH+FLY+THIA.

Fig 4. ROS concentration in the gut of honeybees after being exposed to different agricultural agents.

Differences in the mean concentration of reactive oxygen species in honeybee midguts, adjusted to the units of protein, when feeding on different diets. Diets were inoculated with potential agricultural stressors (C = control, MMH = E. faecalis strain MMH594, FLY = E. faecalis strain FLY1, THIA = thiacloprid). Results represent mean values ± 2 s.e.m, ANOVA, n = 16 for each treatment group. Bars are indicated in: black = control, dark-grey = MMH, light-grey = FLY, red = THIA, shaded black = MMH+FLY, shaded dark-grey = MMH+THIA, shaded light-grey = FLY+THIA, shaded red = MMH+FLY+THIA.