Pesticides effects on stingless bees (Hymenoptera, Meliponini) imune system

Abstract

Bees play a fundamental role as pollinators of native and cultivated plants. Among these insects, the Africanized bee Apis mellifera (Hymenoptera: Apidae) is considered an essential pollinator for several crops, mainly due to its generalist habit. However, besides this species, stingless bees deserve attention, as they can pollinate equally or even more efficiently than A. mellifera. Bee populations have been threatened by different factors, including the increased use of pesticides in agriculture that can cause mortality and adverse effects on the morphology of organs. Furthermore, the bee's immune system can be affected after exposure to pesticides, which can directly impact the survival of these pollinators. Apis mellifera is used as a pollinator model in pesticide risk assessment tests for bees in Brazil and other countries. However, considering the ecological and economic importance of stingless bees, toxicity tests for these bees should be carried out to understand whether pesticides affect both bee species in the same manner. In this sense, the project aims to evaluate the interactions and effects of different classes of insecticides on the immune system of two species of native bees and compare them with the Africanized hybrid. Worker bees of the species Melipona scutellaris, Scaptotrigona postica and Africanized A. mellifera from at least three different non-parental colonies will be studied. These bees will be exposed to realistic concentrations (ng/microliter) of a neonicotinoid insecticide (imidacloprid), a fungicide (pyraclostrobin), and a herbicide (glyphosate). For each experiment, groups of 30 bees from three different colonies will be exposed in the laboratory to the sublethal concentrations offered in the food, both in an isolated and combined way. Bees will be collected, anesthetized under refrigeration, and dissected every 24 hours, and the material will be prepared according to specific protocols for each technique used to investigate the effects of sublethal doses of different pesticides in isolated and combined ways on: the morphology of hemocytes and trophocytes through light microscopy and atomic force microscopy; the expression of Heat Shock Proteins (HSPs), autophagy, and cell death by immunohistochemistry, using primary antibodies anti-HSP 70 and 90, anti-LC3 A/B and anti-cleaved caspase 3; the number of immune system cells present in the hemolymph by flow cytometry; the encapsulation rate by the mean gray value per filament; the activity of the enzymes Superoxide Dismutase (SOD), Catalase (CAT), Glutathione S-Transferase (GST) and Phenoloxidase (PO); the expression pattern of defense-related proteins of the organism (abaecin, apidaecin, defensins, glucose dehydrogenase, and hymenoptaecin) by PCR-Real Time; the walking behavior and the mean velocity of the workers through the Ethoflow software. (AU)