A Locomotor Deficit Induced by Sublethal Doses of Pyrethroid and Neonicotinoid Insecticides in the Honeybee Apis mellifera

Abstract

The toxicity of pesticides used in agriculture towards non-targeted organisms and especially pollinators has recently drawn the attention from a broad scientific community. Increased honeybee mortality observed worldwide certainly contributes to this interest. The potential role of several neurotoxic insecticides in triggering or potentiating honeybee mortality was considered, in particular phenylpyrazoles and neonicotinoids, given that they are widely used and highly toxic for insects. Along with their ability to kill insects at lethal doses, they can compromise survival at sublethal doses by producing subtle deleterious effects. In this study, we compared the bee's locomotor ability, which is crucial for many tasks within the hive (e.g. cleaning brood cells, feeding larvae…), before and after an acute sublethal exposure to one insecticide belonging to the two insecticide classes, fipronil and thiamethoxam. Additionally, we examined the locomotor ability after exposure to pyrethroids, an older chemical insecticide class still widely used and known to be highly toxic to bees as well. Our study focused on young bees (day 1 after emergence) since (i) few studies are available on locomotion at this stage and (ii) in recent years, pesticides have been reported to accumulate in different hive matrices, where young bees undergo their early development. At sublethal doses (SLD48h, i.e. causing no mortality at 48 h), three pyrethroids, namely cypermethrin (2.5 ng/bee), tetramethrin (70 ng/bee), tau-fluvalinate (33 ng/bee) and the neonicotinoid thiamethoxam (3.8 ng/bee) caused a locomotor deficit in honeybees. While the SLD48h of fipronil (a phenylpyrazole, 0.5 ng/bee) had no measurable effect on locomotion, we observed high mortality several days after exposure, an effect that was not observed with the other insecticides. Although locomotor deficits observed in the sublethal range of pyrethroids and thiamethoxam would suggest deleterious effects in the field, the case of fipronil demonstrates that toxicity evaluation requires information on multiple endpoints (e.g. long term survival) to fully address pesticides risks for honeybees. Pyrethroid-induced locomotor deficits are discussed in light of recent advances regarding their mode of action on honeybee ion channels and current structure-function studies.

Fig 1. Synthetic insecticides from three classes.

Chemical structure of 3 pyrethroids (cypermethrin, tau-fluvalinate, tetramethrin), a phenylpyrazole (fipronil) and a neonicotinoid (thiamethoxam).

Fig 2. Video tracking of bees using a vertical arena.

A, Examples of paths followed during 3 minutes by four individual young bees (day 1 after emergence). B, Superimposed paths followed by eighty individual bees. Overall, arena sides were more frequently visited. C, Locomotor ability measured at day 1, 2 and 6 after emergence (bees kept in an incubator). Mean distance (± S.E.M) covered by bees slightly increased from 3.2 to 3.8 meters between day 1 and 2 (p<0.01, n = 138 and 63 respectively) and did not significantly further increase as shown at 6 days after emergence (n = 38).

Fig 3. Evidence for locomotor deficits after exposure to a sublethal dose (SLD_48h) of a pyrethroid or a neonicotinoid but not a phenylpyrazole.

A, The average (± S.E.M) relative distance covered by young bees is significantly decreased 6±2h after exposure to either a SLD_48h of cypermethrin (2.5 ng/bee), tau-fluvalinate (33 ng/bee) or tetramethrin (70 ng/bee). B, A significant decrease in distance is observed after exposure to a SLD_48h of thiamethoxam (3.8 ng/bee) as well. C, The relative distance covered by bees after exposure to a SLD_48h of fipronil (0.5 ng/bee) is similar to the distance covered by control bees. In the case of fipronil, whereas early deleterious effects cannot be evidenced by the locomotion assay, an increased mortality is observed five days after exposure. For cypermethrin, n = 19 control and n = 20 exposed bees respectively. For tau-fluvalinate, n = 12 control and n = 19 exposed bees respectively. For tetramethrin, n = 20 control and n = 20 exposed bees respectively. For thiamethoxam, n = 19 control and n = 19 exposed bees respectively. For fipronil, n = 19 control and n = 20 exposed bees respectively.