Metabolomics analysis of honeybee brain submitted to Proboscis Extension Reflex (PER) test

The honeybee Apis mellifera has long served as an invertebrate model organism for learning and memory research, contributing to a better understanding of cognition bases. In this context, different metabolites (especially neurotransmitters) were characterized by play distinct roles in learning process and in memory formation in insects. Whereas little is known about the metabolites in relation to the development of cognitive skills in A. mellifera, the present study aims to perform a metabolomic analysis of the honeybee brains submitted to the behavioral test of proboscis extension reflex (PER). For this, has been standardized a metabolomic analysis technique through the use of LC-ESI-MS and MSn system. Initially a low molecular weight compounds library was created, containing characteristic compounds of bee brain (neurotransmitters, free amino acids, polyamines, nucleotides, nucleosides, organic acids, etc.). In this first approach, from the library of 112 compounds, 48 compounds were identified and quantified; some of these compounds were only identified in the control group (cadaverine, spermine, glucose, uracil and N-acetyl-L-glutamate 5- semialdehyde), while others were only identified in PER group (phenylalanine, betaine, spermidine, serine and creatine). Among the compounds identified in both groups, only five compounds showed statistically significant differences in quantitative results (arginine, asparagine, guanosine monophosphate, putrescine and 4-guanidinebutanoate). In order to study the metabolic profiles by regions (within each brain region) it was also standardized an experimental protocol using a novel semi-quantitative method of MALDI Spectral Imaging strategy. This strategy allowed the study and the mapping of the spatial distribution of metabolites identified in honeybee brain sections, as well as a better understanding of the distribution of molecules in the different brain structures and their correlation with the behavior tested. Both strategies applied proved to be complementary and essential to the understanding of cognition in bees. Overall, in this study the PER test seems to stimulate intense brain activity, high energy expenditure, intense chemical signaling and activation of some specific metabolic pathways, such the arginine and proline metabolic pathway. In individuals of PER group arginine was probably catabolized in the synthesis of creatine, 4-guanidinobutanoato, putrescine and spermidine. This biochemical process were probably important to coordinate the recognition of sucrose molecule and associate this odor with a reflex behavior for the proboscis extension (unconditioned stimulus). (AU)