The use of herbicides is increasingly controversial due to their different deleterious effects on the biodiversity out of their expected targets [1-6]. Among herbicides, glyphosate is the most widespread in the world, used to target a specific biosynthetic pathway existing only in plants and some microorganisms.

However, studies conducted in the field showed that this herbicide is able to persist in the soil for several weeks or months [7] ; [8] and therefore, it can lead to different neurotoxic effects in animals such as insects.

Indeed, different studies proved that low concentrations of glyphosate, similar to those found in the environment, can disturb the cognitive abilities such as learning and navigation in honey bees and could have a long-term negative impact on bee colonies [9] ; [10]. These changes in behaviour highlighted the role of these insects as bio-indicators to evaluate the impact of glyphosate spread in terrestrial environments.

Moreover, it was shown that glyphosate exposure can influence the vectorial capacity and survival of Aedes. aegypti and Aedes. albopictus mosquitoes [11] ; [12], the main vectors of several human diseases such as dengue, yellow fever, Zika or Chikungunya. Indeed, glyphosate exposure alters their life history traits such as larval development time and sex ratio [13], as well as the expression of genes conferring resistance to insecticides [14] which depends on mechanisms associated with behavioural, physiological and metabolic changes [15].

The preimaginal instars of mosquitoes develop in freshwater environments which may be exposed to pollutants such as glyphosate. In the absence of danger, larvae stay on the surface of the water to breathe atmospheric air. However, when mosquito larvae detect a potential danger over the water surface, they perform an escape response, causing an energy expenditure affecting their survival rate, and their chance to reach the adult stage [16].To reduce this type of escape behaviour and save their energy reserves, it is important to habituate larvae repeating expositions to a non-harmful visual stimulus which will be no longer perceived as a danger [17].

Using bioassay based on habituation, a team of the Insect Biology Research Institute from the University of Tours (France) studied the impact of glyphosate at field-realistic doses on learning in Ae. aegypti mosquito larvae. Their results are published in the Journal of Experimental biology [18].

First of all, researchers performed preliminary trials with a habituation protocol to familiarize larvae of Ae. aegypti to a non-harmful visual stimulus characterised by a black cardboard square. Larvae were placed individually in a Petri dish containing water, which was exposed under a spotlight. Therefore, it allowed it to project the shadow of the stimulus on the Petri dish (Figure 1).

Preliminary observations showed that larvae stopped performing spontaneous escape responses after 15 minutes.

Figure 1:

Stimulation apparatus. A single larvae was placed into a Petri dish. A servomotor delivered the visual stimulus moving it from position 1 to position 2. While in position 2 the visual stimulus covered the light source. Modified from Baglan et al. 2017 [17].

Source: Glyphosate impairs learning in mosquito larvae (Aedes aegypti) at field-realistic doses. ?(Baglan et al. 2018)

In a second part, the team tested the impact of glyphosate on the cognitive abilities of mosquito larvae. Researchers reared 5 groups of 20 mosquito larvae in water, whose 1 group corresponded to the control group which contained no herbicide (glyphosate) and the 4 others groups contained one of the 4 following field-realistic concentrations of glyphosate: 50 μg/l; 100 μg/l; 210 μg/l; 2 mg/l [19] ; [20]. Using the same habituation protocol than the one used for preliminary trials, researchers evaluated and compared learning performances of each group of larvae in the course of training.

Results showed that the escape response levels significantly dropped in the course of the habituation phase in all groups. However, the dynamic of the acquisition was different across groups. This dynamic corresponds to the time necessary to reduce a behavioural response after several repeated expositions to a stimulus, was different across groups. Generally, the lower is the intensity of learning, the slower is the acquisition. In other words, the time necessary for larvae to learn a stimulus in order to no longer perceive it as a danger was affected by the presence of glyphosate.

Indeed, the 3 groups with the higher concentrations of glyphosate showed a significantly lower intensity of learning and so, a slower acquisition than the control group and the one with the lower concentration. Moreover, the group reared with 100 μg/l of glyphosate, half the concentration that can mostly be found in the environment, showed a significantly faster

acquisition than the other two groups with higher glyphosate concentrations: 210 μg/l and 2 mg/l (Figure 2).

Figure 2:

Escape responses during habituation. Each concentration of glyphosate is represented with a different colour: blue (no glyphosate); red (50μg/l), yellow (100 μg/l); green (210 μg/l) and orange (2 mg/l).

Source: Glyphosate impairs learning in mosquito larvae (Aedes aegypti) at field-realistic doses. (Baglan et al. 2018)

Thus, this data proved that glyphosate affected habituation and learning performances of mosquito larvae at concentrations which can be found in the environment. The higher the glyphosate concentration, the slower the acquisition, therefore, the stronger the deleterious effects on learning abilities. However, in this experiment, it would have been interesting to use a positive control-group reared with a herbicide different from glyphosate.

Several studies have shown that exposure to glyphosate had an impact on the inhibition of acetylcholinesterase in the nervous system and on muscles in aquatic organisms [21-27]. Finally, the decrease in learning performances in mosquito larvae suggests that glyphosate impacts on their central nervous system by inhibiting acetylcholinesterase which would alter the number of neurotransmitters released at synapses and disrupt the habituation abilities of mosquitoes. However, further experiments are necessary to define the physiological paths which are linked to these behavioural modifications.

Recently, Vinauger et al. 2018 [28] showed that Ae. aegypti mosquitoes are able to develop a sort of avoidance memory against their potential « predator » host thanks to the learning of an association between a human odour with an aversive stimulus simulating a slap. This learning would be dependent on the dopamine receptors which would allow mosquitoes to better discriminate and avoid their potential « predator » host. In the future, it would be interesting to study the effects of glyphosate on the dopamine receptors of these mosquitoes as well as on their abilities to learn an aversive stimulus.

This work is one of the first evincing the effect of glyphosate at field-realistic concentrations on the cognitive system of a non-target species. It opens the possibility to use mosquito larvae as bio-indicators to evaluate the consequences of this herbicide on aquatic environments as well as on the biology of mosquitoes, which are among the major human vector-borne disease in order to better control the use of herbicides.

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