A systematic review of research conducted by pioneer groups in ecotoxicological studies with bees in Brazil: advances and perspectives.

Brazil presents the most threatened endemic or rare species among neotropical regions, with the Hymenoptera order, to which bees belong, classified as a high-risk category. In Brazil, the main cause of bee death is the indiscriminate use of pesticides. In this context, groups such as Bee Ecotoxicology and Conservation Laboratory (LECA in Portuguese) and Bees and Environmental Services (ASAs in Portuguese) have become a reference in studies evaluating the impacts of pesticides on bees since 1976. Thus, the objective of this review was to conduct a quantitative and qualitative review of the studies conducted by these groups to evaluate and compile the advances made over the years, identify potential knowledge gaps for future studies, and support the sensitivities of stingless bees when compared to the species Apis mellifera. The quantitative analyses showed that most studies were carried out in the genus Apis, under laboratory conditions. However, more recently (since 2003), studies have also focused on stingless bees and the neonicotinoid class of insecticides. The most relevant gaps identified were the lack of studies under field conditions and on bee biology. The qualitative analyses indicated that Brazilian stingless bees are more susceptible to pesticides than A. mellifera and require a much lower average dose, concentration, or lethal time to display morphological and behavioral damage or decreased lifespan. Thus, future studies should work towards establishing more representative protocols for stingless bees. Furthermore, public policies must be created for the protection and conservation of bees native to Brazil.

Optimization of in vitro culture of honeybee nervous tissue for pesticide risk assessment.

The most used pesticides have neurotoxic action on the neurotransmitter system of target and non-targeted insects, such as honeybees. However, honeybees have foremost importance worldwide, which has encouraged the development of tools to evaluate the action of specific pesticide molecules on their nervous system, providing accurate data on damage to their brain. In this sense, our study aimed to optimize in vitro honeybee nervous tissue culture to assess pesticide risks. To this end, six forager honeybee brains were dissected and transferred to different combinations of Leibovitz-15 (L-15) culture medium supplemented with Fetal Bovine Serum (FBS), Hank's Balanced Salt Solution (HBSS), and Insect Medium Supplement (IMS). Nervous tissues were collected after different incubation times (1, 6, 12, and 24 h) for morphology and Kenyon cell analyses. Our results showed that L-15 medium supplemented with HBSS and with HBSS plus FBS were the best media for culturing honey nervous tissue for 24 h, as they resulted in less tissue spacing and cell disarrangement. Therefore, they may be assessed in future ecotoxicological tests.

Standardization of in vitro nervous tissue culture for honeybee: A high specificity toxicological approach.

Bees are important pollinators that help to maintain the biodiversity of wild and cultivated plants. However, the increased and inappropriate use of agrochemicals has caused an imbalance in the populations of these insects visiting flowers for pollen and nectar collection. Therefore, new research methods for understanding the mechanisms of action of pesticides and their impacts on the brains of bees, such as neurotoxicity and cellular changes, in response to different active characteristics and dosages of insecticides are necessary. Thus, with the aim of developing tests with greater specificity at the level of cells or tissues, this study sought to standardize a method for the in vitro culture of the nervous tissue of Apis mellifera. For this purpose, the brains of six foragers bees were transferred to three different insect cell culture media and it supplementation with 10% foetal bovine serum (FBS): Grace, Schneider, Leibovitz, Grace + FBS, Schneider + FBS and Leibovitz + FBS media for each collection time. Nervous tissue was collected after 1, 6, 12 and 24 h of incubation in a humidified CO2 incubator at 32 °C, and histological sections of the organs were analysed. The results showed that Leibovitz medium and Leibovitz medium + serum are potential culture media for the cultivation of nervous tissue, since they resulted in less tissue spacing and tissue disarrangement. Therefore, additional supplements are necessary to obtain an ideal medium for the cultivation of A.mellifera nervous tissue.