Impact of dietary supplementation of ascorbic acid on reproductive genotoxicity in Apis cerana indica F. exposed to imidacloprid

Abstract

The Indian bee, Apis cerana indica F., is one of the most important bee species domesticated in India for honey production and crop pollination. It is an effective pollinator with high survivability and is responsible for maintaining global biodiversity and food production. In addition to providing crucial pollination services to agricultural crops, it has significant economic potential for both small and large-scale beekeeping. Population declines of A. cerana indica are affecting ecosystem services and food security. Worker bees are exposed to various stressors while foraging and are vulnerable to pesticides due to a decline in the number of genes encoding key enzyme families responsible for detoxification. Neonicotinoids are one of the major stresses contributing to the decline of honey bee populations. They are a group of systemic insecticides with different application methods that are increasingly used for crop protection. Imidacloprid was the first neonicotinoid introduced to the market and is one of the fastest growing neonicotinoids. Because of its systemic properties, they are highly toxic to the non-target organisms including beneficial insects.In the present study, it was hypothesized that dietary supplementation with ascorbic acid (VIT C) could reduce the toxicity induced by a sublethal concentration of imidacloprid (IMD) in worker bees of Apis cerana indica. To conduct the study, honey bee colonies were provided with sugar syrup for six months for the control group and sugar syrup containing 0.2% VIT C for the experimental group. Worker bees from both groups were randomly collected and exposed to 1μg/mL IMD. The activities of antioxidant enzymes were examined to evaluate the effect of ascorbic acid supplementation on peroxidative damage induced by imidacloprid. The expression of nicotinic acetylcholine receptors (nAChRα1 and nAChRα2 subunit), which are the targets of imidacloprid, and the activities of acetylcholinesterase (AChE) were analyzed to determine the toxicity of imidacloprid on neuronal transmission. To investigate the possible effects of imidacloprid on reproduction, ovarian expression of the multifunctional gene vitellogenin and expression of CYP336A1 were analyzed. Histological analysis was performed to determine the effect of ascorbic acid supplementation on the modification of cyto architecture of midgut and ovarian tissues on exposure to imidacloprid.The activities of antioxidant enzymes including catalase, superoxide dismutase, glutathione S-transferase and glutathione peroxidase in the hemolymph and midgut tissues of worker bees were significantly decreased by exposure to IMD as a single agent. However, their activities showed a significant increase when supplemented with ascorbic acid. When worker bees were exposed to imidacloprid (IMD) alone, there was a significant decrease in AChE activity and a significant upregulation of nAChRα1 and nAChRα2 subunit expression in brain tissue. Our results indicated that the dietary supplementation with ascorbic acid has the potential to maintain AChE activity and expression of nAChRs at normal levels to some extent against the toxic effects of imidacloprid. The expression of vitellogenin and CYP336A1 was found to be down-regulated in the ovaries of queen bees upon exposure to IMD, while the supplementation of ascorbic acid maintained the ovarian expression of vitellogenin and CYP336A1 at normal levels. Histological examination of midgut tissue revealed cytoarchitectural alterations and rupture of the peritrophic membrane in workers and queen bees exposed to IMD. The damage was alleviated and the peritrophic membrane was intact in worker bees treated with ascorbic acid. Histological examination of the ovaries of queen bees showed degeneration of oocytes and trophocytes upon exposure to IMD, whereas no complete degeneration was observed in queen bees supplemented with ascorbic acid.Our results suggest that dietary supplementation with ascorbic acid is able to maintain the redox status of tissues and to defend against the peroxidative damage induced by the sublethal concentration of imidacloprid. Ascorbic acid has the potential to maintain the redox status in the ovary, as shown by the upregulation of vitellogenin and CYP336A1, which protect the organism from the oxidative damage induced by imidacloprid. Ascorbic acid supplementation may provide protection against peroxidative damage induced by sublethal concentrations of imidacloprid, as evidenced by the maintenance of normal tissue morphology. Therefore, we propose that ascorbic acid can be used as an effective supplement to alleviate the toxic aspects of the pesticide-induced oxidative stress.