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Effects of lethal and subletal doses of pesticides on Apis mellifera bees transcriptomes


In recent years, honey bees population decline has been observed, impacting wild and managed colonies for production and pollination services. One of the probable causes of this decline is related to the indiscriminate use of agrochemicals and the consequent exposure of bees to these molecules, which may affect the maintenance and survival of the colony. Although the LD50 values are important to demonstrate the acute toxicity for bees, these tests do not consider the effects of bees exposure to sublethal doses (lower than LD50) which the bees are exposed in the field, which can compromise several physiological pathways, affecting the colony. Studies have shown that among chemicals used, insecticides are the most harmful to bees; however, fungicides and herbicides can also affect colonies, and may promote behavioral and physiological changes. Thus, the aim of this research project will be evaluate different agrochemicals (Fipronil, Imidacloprid, Piraclostrobin and Glyphosate) lethal and sublethal doses for africanized Apis mellifera bees, in forager phase, analyzing cerebral transcriptomes. The diagnosis of the presence of diseases in the colonies selected for the present project will be carried out, as these may affect the transcriptome results, for the acute paralysis virus (VPA), wing deforming virus (VDA), Melissococcus pluton, Nosema apis , Nosema ceranae, Paenibacillus larvae. After confirming the absence of these diseases newly emerged bees from ten distinct colonies will be marked in the pronotum region and returned to their respective colonies; after 21 days, 10 of the previously marked bees will be captured from each colony, randomly, totalizing five repetitions per product and concentration. For the ingestion tests, these bees will be kept without food for a period of 3 hours, to empty the contents of their nectariferous vesicle. Then, 1 ml of honey syrup containing the lethal (DL50) and sublethal (DL50 / 100) dose of agrochemicals will be provided. For the contact tests, 10 field bees from each experimental colony will receive 2 µL containing the lethal (DL50) and sublethal dose (DL50 / 100) of the agrochemicals. The bees will be placed and kept in perforated Petri dishes to ensure ventilation and kept in an incubator (33 ± 1.0ºC and 75% U), totaling five repetitions.Forager bees will be kept as controls, without contamination with the products. After 1 and 4 hours of exposure, 20 bees will be collected randomly for the analysis of cerebral transcriptome. The bees collected in each treatment will be immediately frozen in an ultra-freezer at -80 ° C, where they will be kept until processing. The total RNA of the brain tissue of the bees from each replica will be extracted. The calculations of gene expression measures from mRNA sequences and the generation of gene expression profiles will be performed on the CLC Genomics Workbench 7.01 platform (CLC Bio, Denmark). The Rnaseqgene package of the R program will be used for the statistical analysis of data generated from sequencing on the Illumina platform. Differences between treatments will be compared by ANOVA, followed by the Bonferroni test (p <0.01) for multiple comparisons. Fisher's exact test will be considered the statistical method (p <0.05), with correction of the false discovery rate (<0.05) for Gene Ontology analyzes (GO). This project is expected to obtain important information on how the exposure of Apis mellifera bees to the studied agrochemicals can alter the profile of gene expression in the brain tissue of bees and which metabolic and physiological pathways that can change in these conditions. (AU)

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