Apoptosis In The Nerve Cells Of Honey Bee(Apis Mellifera L.)Brain Induced By The Sublethal Doses Of Imidacloprid

Honey bees are important pollinators, but they are currently threatened by noxious substances in the environment. Neonicotinoid is one of the most widely used pesticides. It was reported that sublethatl dose of neonicotinoid pesticides could induce apoptosis in the cells of midgut, ovaries and salivary glands of bee larvae. While the target site of these pesticides was at the brain of insets, the study on the neuron of brain was well-focused, on which the information was limited. Moreover, neonicotinoid pesticides can also affect the learning and memory capacity of the honey bees. However, very few reports have been known in this aspect. We discussed the effect of neonicotinoid pesticides on the apoptosis of brain neurons of the honey bees. By testing the climbing ability of the honey bees, the neural toxicity of neonicotinoid pesticides on honey bees as a non-target insect was evaluated.Imidacloprid, the representative of neonicotinoid pesticides, was used as the pesticide against the adult Apis mellifera L.. The brain tissues were harvested, and the effect of the sublethal dose of imidacloprid on the apoptosis of brain neurons of the honey bees was analyzed on the molecular and submolecular levels. The neural toxicity of imidacloprid was evaluated by combining with testing apoptosis and the climbing ability of the honey bees. The results were obtained as follows:1. Under laboratory conditions, the honey bees were daily fed with imidacloprid at the dose of 0.5, 1.5 and 4.5ng/individual, respectively. Samplings were done on 3, 6, 9 and 12 d of treatment. TUNEL assay was performed to detect the apoptosis of brain neurons of adult honey bees. Results showed that the sublethal dose of imidacloprid could induce the apoptosis of brain neurons. The cell apoptosis rate increased with the dose and the prolonging of treatment time, indicating an obvious dose and time effect.2. Under the same treatment methods and feeding conditions, immunofluorescence method was adopted to detect the apoptosis of brain neural cells in adult honey bees by measuring the level of Caspase-3. It was found that the sublethal dose of imidacloprid could induce the activation of Caspase-3 in the brain neurons. The positive cell rate increased with the dose and the prolonging of the treatment time, showing a dose and time effect. Moreover, a positive correlation was observed with the apoptosis of brain neurons.3. Fluorescence quantitative method was used to detect the transcriptional level of Caspase-1 gene. Results showed that the sublethal dose of imidacloprid caused the m RNA expression of Caspase-1 in brain neural cells to increase in a dose- and time-dependent manner. Combining with the detection of the Caspase-3 expression, it is inferred that the apoptosis of brain neural cells induced by sublethal dose of imidacloprid is associated with the Caspase-dependent apoptotic pathway.4. Each bee was fed with 1.5ng imidacloprid every day. At 9d of feeding, transmission electron microscopy was employed to observe the ultrastructural changes of brain neurons. The treatment group and the blank control group showed obvious differences in submolecular morphology. In the treatment group, the chromatin was condensed and concentrated on the nuclear membrane with the formation of apoptotic bodies and reduced size of nuclei. These were typical apoptotic signs, except for the delayed degradation of nuclei. The signs of autophagy included the formation of autolysosomes and autophagosomes and the swelling of mitochondria, endoplasmic reticula and Golgi apparatuses.5. The effect of sublethal dose of imidacloprid on the climbing behavior and survival of adult honey bees was evaluated, and the relationship between apoptosis of brain neurons, climbing ability and survival rate of the bees was tested. According to the results, although there was no significant difference in survival rate under the given treatment dose and time, the climbing ability of bees in the treatment group was obviously impaired. Other conditions being constant, a statistically significant difference between the two groups was observed at an earlier time in the apoptosis rate of brain neuons under low dose than in the indicators of climbing ability and survival rate.