Several investigations have revealed the toxic effects that neonicotinoids can have on Apis mellifera, while few studies have evaluated the impact of these insecticides can have on the larval stage of the honeybee. From the lethal concentration (LC50) of thiamethoxam for the larvae of the Africanized honeybee, we evaluated the sublethal effects of this insecticide on morphology of the brain. After determine the LC50 (14.34 ng/μL of diet) of thiamethoxam, larvae were exposed to a sublethal concentration of thiamethoxam equivalent to 1.43 ng/μL by acute and subchronic exposure. Morphological and immunocytochemistry analysis of the brains of the exposed bees, showed condensed cells and early cell death in the optic lobes. Additional dose-related effects were observed on larval development. Our results show that the sublethal concentrations of thiamethoxam tested are toxic to Africanized honeybees larvae and can modulate the development and consequently could affect the maintenance and survival of the colony. These results represent the first assessment of the effects of thiamethoxam in Africanized honeybee larvae and should contribute to studies on honey bee colony decline.
Under laboratory conditions, the effects of thiamethoxam were investigated in larvae, pupae and emerging honey bees after exposure at larval stages with different concentrations in the food (0.00001 ng/μL, 0.001 ng/μL and 1.44 ng/μL). Thiamethoxam reduced the survival of larvae and pupae and consequently decreased the percentage of emerging honey bees. Thiamethoxam induced important physiological disturbances. It increased acetylcholinesterase (AChE) activity at all developmental stages and increased glutathione-S-transferase (GST) and carboxylesterase para (CaEp) activities at the pupal stages. For midgut alkaline phosphatase (ALP), no activity was detected in pupae stages, and no effect was observed in larvae and emerging bees. We assume that the effects of thiamethoxam on the survival, emergence and physiology of honey bees may affect the development of the colony. These results showed that attention should be paid to the exposure to pesticides during the developmental stages of the honey bee. This study represents the first investigation of the effects of thiamethoxam on the development of A. mellifera following larval exposure.
The use of insecticides on crops can affect non-target insects, such as bees. In addition to the adult bees, larvae can be exposed to the insecticide through contaminated floral resources. Therefore, this study aimed to investigate the possible effects of the exposure of A. mellifera larvae to a field concentration of thiamethoxam (0.001 ng/μL thiamethoxam) on larval and pupal survival and on the percentage of adult emergence. Additionally, its cytotoxic effects on the digestive cells of midgut, Malpighian tubules cells and Kenyon cells of the brain of newly emerged A. mellifera bees were analyzed. The results showed that larval exposure to this concentration of thiamethoxam did not influence larval and pupal survival or the percentage of adult bee emergence. However, this exposure caused ultra-structural alterations in the target and non-target organs of newly emerged bees. The digestive cell of bees that were exposed to the insecticide exhibited a basal labyrinth without long and thin channels and compromised mitochondria. In Malpighian tubules cells, disorganized basal labyrinth, dilated mitochondria with a deformed shape and a loss of cristae, and disorganized microvilli were observed. The results showed that the exposed bees presented Kenyon cells with alterations in the nucleus and mitochondria. These alterations indicate possible tissue degeneration, demonstrating the cytotoxicity of thiamethoxam in the target and non-target organs of newly emerged bees. Such results suggest cellular organelle impairment that can compromise cellular function of the midgut cells, Malpighian tubules cells and Kenyon cells, and, consequently, can compromise the longevity of the bees of the whole colony.
Several synthetic substances are used in agricultural areas to combat insect pests; however, the indiscriminate use of these products may affect nontarget insects, such as bees. In Brazil, one of the most widely used insecticides is imidacloprid, which targets the nervous system of insects. Therefore, the aim of this study was to evaluate the effects of chronic exposure to sublethal doses of imidacloprid on the brain of the Africanized Apis mellifera. The organs of both control bees and bees exposed to insecticide were subjected to morphological, histochemical and immunocytochemical analysis after exposure to imidacloprid, respectively, for 1, 3, 5, 7, and 10 days. In mushroom bodies of bees exposed to imidacloprid concentrations of LD50/10 and in optic lobes of bees exposed to imidacloprid concentrations of LD50/10, LD50/100, and LD50/50, we observed the presence of condensed cells. The Feulgen reaction revealed the presence of some cells with pyknotic nuclei, whereas Xylidine Ponceau stain revealed strongly stained cells. These characteristics can indicate the occurrence of cell death. Furthermore, cells in mushroom bodies of bees exposed to imidacloprid concentrations of LD50/10 appeared to be swollen. Cell death was confirmed by immunocytochemical technique. Therefore, it was concluded that sublethal doses of imidacloprid have cytotoxic effects on exposed bee brains and that optic lobes are more sensitive to the insecticide than other regions of the brain.
In Brazil, imidacloprid is a widely used insecticide on agriculture and can harm bees, which are important pollinators. The active ingredient imidacloprid has action on the nervous system of the insects. However, little has been studied about the actions of the insecticide on nontarget organs of insects, such as the Malpighian tubules that make up the excretory and osmoregulatory system. Hence, in this study, we evaluated the effects of chronic exposure to sublethal doses of imidacloprid in Malpighian tubules of Africanized Apis mellifera. In the tubules of treated bees, we found an increase in the number of cells with picnotic nuclei, the lost of part of the cell into the lumen, and a homogenization of coloring cytoplasm. Furthermore, we observed the presence of cytoplasmic vacuolization. We confirmed the increased occurrence of picnotic nuclei by using the Feulgan reaction, which showed the chromatin compaction was more intense in the tubules of bees exposed to the insecticide. We observed an intensification of the staining of the nucleus with Xylidine Ponceau, further verifying the cytoplasmic negative regions that may indicate autophagic activity. Additionally, immunocytochemistry experiments showed TUNEL positive nuclei in exposed bees, implicating increased cell apoptosis after chronic imidacloprid exposure. In conclusion, our results indicate that very low concentrations of imidacloprid lead to cytotoxic activity in the Malpighian tubules of exposed bees at all tested times for exposure and imply that this insecticide can alter honey bee physiology.
To explain losses of bees that could occur after the winter season, we studied the effects of the insecticide imidacloprid, the herbicide glyphosate and the fungicide difenoconazole, alone and in binary and ternary mixtures, on winter honey bees orally exposed to food containing these pesticides at concentrations of 0, 0.01, 0.1, 1 and 10 µg/L. Attention was focused on bee survival, food consumption and oxidative stress. The effects on oxidative stress were assessed by determining the activity of enzymes involved in antioxidant defenses (superoxide dismutase, catalase, glutathione-S-transferase, glutathione reductase, glutathione peroxidase and glucose-6-phosphate dehydrogenase) in the head, abdomen and midgut; oxidative damage reflected by both lipid peroxidation and protein carbonylation was also evaluated. In general, no significant effect on food consumption was observed. Pesticide mixtures were more toxic than individual substances, and the highest mortalities were induced at intermediate doses of 0.1 and 1 µg/L. The toxicity was not always linked to the exposure level and the number of substances in the mixtures. Mixtures did not systematically induce synergistic effects, as antagonism, subadditivity and additivity were also observed. The tested pesticides, alone and in mixtures, triggered important, systemic oxidative stress that could largely explain pesticide toxicity to honey bees.
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