A neuroprpteomic approach on worker africanized honeybee Apis mellifera submitted to proboscis extension reflex assay

Author(s)

Anally Riberio da Silva Menegasso

• Document type: Doctoral Thesis
• Press: Rio Claro. 2018-10-24.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. Rio Claro
• Defense date: 2017-06-30
• Advisor: Mario Sergio Palma

Abstract

Nowadays, bees have been used as models in studies of learning and memory, highlighting their utility for neuroscience, in particular for a better understanding of the bases of cognition. For this, proboscis extension reflex (PER) is an unconditioned stimulus (US) widely used to access bees' ability to correlate it with a conditioned stimulus (CS) during learning and memory acquisition. In the present study, PER was used for proteomic studies of bees brain through several innovative strategies. Proteomic shotgun was applied to the study of soluble proteins in μLC-ESI-micrOToF-QIII system and label-free quantification in LTQ-Orbitrap XL ETD system, being the first proteomic analysis study that draws attention to the fact that reflex behavior (US) alone activates various metabolic cascades, including biological processes related to memory. The comparative analysis of the proteomic profiles for the workers' brains from control group and REP group demonstrated metabolism of cyclic / heterocyclic / aromatic compounds in parallel with the metabolism of nitrogen compounds. This process was followed by the negative regulation of proteins involved in metabolism of phosphorylated metabolites and the positive regulation of proteins related to the metabolism of carbohydrates. This has probably occurred to provide metabolic energy for the cellular processes needed to adapt the brain to PER. In addition, the transcriptome analysis of foraging bees brain was performed, which was used in the creation of a database that gathered the deposited sequences in the Uniprot, NCBI and sequences obtained by the transcriptome analysis. The study of membrane proteome in the brain of PER and PER-conditioned bees with label quantification in the LTQOrbitrap XL ETD system allowed the identification of 6,882 proteins, of which 1,582 were membrane proteins and among them 658 presented differential expression between control, REP and REP-conditioned groups. Several important processes of the nervous system have been detected, such as proteins involved in olfactory transduction, synapses GABAergic, cholinergic, dopaminergic, serotonergic e glutamatergic, among other important neuronal receptors. It is clear that this is a multicomplex process that requires the expression / synthesis of proteins and different transduction signals. It is suggested that the routes most correlated with the associative memory process (PER-conditioned vs PER) compared to PER vs control are olfactory transduction, dopaminergic synapse, glutamatergic synapse and GABAergic synapse. In the case of previously acquired memory recovery, the cholinergic synapse (acetylcholine) seems to be of fundamental importance for the PER group. Validation studies are being used to better understand these processes. Finally, it was applied the in situ proteomic analysis of neuropeptides by MALDI Imaging. For the neuropeptides apidaecin, NPLP-1, SIFamide the results obtained suggest the low detection of peptides in REP individuals with the rapid release and use of these biomolecules in the modulation of biological processes studied, corroborating with the hypothesis that, probably, memory and olfactory learning are more closely related to the chalices. For the neuropeptides Alatostatin and Tachykinins, the intense labeling of these neuropeptides in PER group suggests the synthesis of neuropeptides through the enzymatic degradation of new protein precursors present in these regions of the brain. From different methodological strategies applied it is expected to have contributed to the understanding of neuroscience at the level of proteins and peptides, and their role in molecular interactions and memory formation in bees. (AU)

• FAPESP's process: 13/13542-6 - A neuroproteomic approach of the brain from Africanized Apis mellifera submitted to the assay proboscis extension reflex
• Grantee: Anally Ribeiro da Silva Menegasso
• Support Opportunities: Scholarships in Brazil - Doctorate