Thymol is a natural substance increasingly used as an alternative to pesticides in the fight against the Varroa destructor mite. Despite the effectiveness of this phenolic monoterpene against Varroa, few articles have covered the negative or side effects of thymol on bees. In a previous study, we have found an impairment of phototaxis in honeybees following application of sublethal doses of thymol-lower or equal to 100 ng/bee-under laboratory conditions. The present work shows the same behavioral effects on bees from hives treated with Apilife Var®, a veterinary drug containing 74 % thymol, with a decrease in phototactic behavior observed 1 day after treatment. Thus, thymol causes disruption of bee phototactic behavior both under laboratory conditions as well as in beehives. The bee exposure dose in treated hives was quantified using gas chromatography coupled to mass spectrometry (GC-MS), giving a median value of 4.3 μg per body 24 h after treatment, with 11 ng in the brain. The thymol level in 20 organic waxes from hives treated with Apilife Var® was also measured and showed that it persists in waxes (around 10 mg/kg) 1 year after treatment. Thus, in the light of (1) behavioral data obtained under laboratory conditions and in beehives, (2) the persistence of thymol in waxes, and (3) the high load on bees, it would appear important to study the long-term effects of thymol in beehives.
Rearing pests or parasites of very small size in the absence of their living host is a challenge for behavioural, physiological and pathological studies. For feeding Varroa destructor, an ectoparasitic mite of Apis mellifera, a confinement space with a membrane separating the nutritive solution and the space was designed. The mite measures less than 2 mm and bears a perforating apparatus with a length of 15 μm. The membrane, an essential element of the chamber, has a thickness of 0.1 μm, and is made of chitosan. It closes one face of the individual confinement chamber and allows piercing and the ingestion of the nutritive solution. Factors inducing feeding can be applied on the inner walls or on the membrane. In the particular case of Varroa, the highest percentages of feeding mites are obtained by addition of host haemolymph to the nutritive solution, suggesting the kairomonal role of haemolymph in addition to its nutritional one. The membrane concept can be easily applied to several mites or other micro-pests.
Essential oils are used by beekeepers to control the Varroa mites that infest honeybee colonies. So, bees can be exposed to thymol formulations in the hive. The effects of the monoterpenoid thymol were explored on olfactory memory and gene expression in the brain of the honeybee. In bees previously exposed to thymol (10 or 100 ng/bee), the specificity of the response to the conditioned stimulus (CS) was lost 24 h after learning. Besides, the octopamine receptor OA1 gene Amoa1 showed a significant decrease of expression 3 h after exposure with 10 or 100 ng/bee of thymol. With the same doses, expression of Rdl gene, coding for a GABA receptor subunit, was not significantly modified but the trpl gene was upregulated 1 and 24 h after exposure to thymol. These data indicated that the genes coding for the cellular targets of thymol could be rapidly regulated after exposure to this molecule. Memory and sensory processes should be investigated in bees after chronic exposure in the hive to thymol-based preparations.
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