In Situ Metabolomics of the Honeybee Brain: The Metabolism of l-Arginine through the Polyamine Pathway in the Proboscis Extension Response (PER).

The proboscis extension response (PER) reflex may be used to condition the pairing of an odor with sucrose, which is applied to the antennae, in experiments to induce learning, where the odor represents a conditioned stimulus, while sucrose represents an unconditioned stimulus. A series of studies have been conducted on honeybees, relating learning and memory acquisition/retrieval using the PER as a strategy for accessing their ability to exhibit an unconditioned stimulus; however, the major metabolic processes involved in the PER are not well known. Thus, the aim of this investigation is profiling the metabolome of the honeybee brain involved in the PER. In this study, a semiquantitative approach of matrix-assisted laser desorption ionization (MALDI) mass spectral imaging (MSI) was used to profile the most abundant metabolites of the honeybee brain that support the PER. It was reported that execution of the PER requires the metabolic transformations of arginine, ornithine, and lysine as substrates for the production of putrescine, cadaverine, spermine, spermidine, 1,3-diaminopropane, and γ-aminobutyric acid (GABA). Considering the global metabolome of the brain of honeybee workers, the PER requires the consumption of large amounts of cadaverine and 1,3-diaminopropane, in parallel with the biosynthesis of high amounts of spermine, spermidine, and ornithine. To exhibit the PER, the brain of honeybee workers processes the conversion of l-arginine and l-lysine through the polyamine pathway, with different regional metabolomic profiles at the individual neuropil level. The outcomes of this study using this metabolic route as a reference are indicating that the antennal lobes and the calices (medial and lateral) were the most active brain regions for supporting the PER.

MALDI-imaging analyses of honeybee brains exposed to a neonicotinoid insecticide.

BACKGROUND: Toxicological studies evaluating the possible harmful effects of pesticides on bees are important and allow the emergence of protection and pollinator conservation strategies. This study aimed to evaluate the effects of exposure to a sublethal concentration of imidacloprid (LC50/100 : 0.014651 ng imidacloprid µL-1 diet) on the distribution of certain proteins identified in the brain of Apis mellifera worker bees using a MALDI-imaging approach. This technique enables proteomic analysis of tissues in situ by monitoring the spatiotemporal dynamics of the biochemical processes occurring at a specific time in specific brain neuropils. For this purpose, foraging bees were exposed to an 8-day diet containing a sublethal concentration of imidacloprid corresponding to the LC50/100 . Bees were collected on day 8 of exposure, and their brains analyzed using protein density maps. RESULTS: The results showed that exposure to imidacloprid led to a series of biochemical changes, including alterations in synapse regulation, apoptosis regulation and oxidative stress, which may adversely impair the physiology of these colony bees. CONCLUSION: Worker bee contact with even tiny amounts of imidacloprid had potent effects leading to the overexpression of a series of proteins related to important cellular processes that were possibly damaged by the insecticide. © 2018 Society of Chemical Industry.

MALDI Imaging Analysis of Neuropeptides in Africanized Honeybee ( Apis mellifera) Brain: Effect of Aggressiveness.

Aggressiveness in honeybees seems to be regulated by multiple genes, under the influence of different factors, such as polyethism of workers, environmental factors, and response to alarm pheromones, creating a series of behavioral responses. It is suspected that neuropeptides seem to be involved with the regulation of the aggressive behavior. The role of allatostatin and tachykinin-related neuropeptides in honeybee brain during the aggressive behavior is unknown, and thus worker honeybees were stimulated to attack and to sting leather targets hung in front of the colonies. The aggressive individuals were collected and immediately frozen in liquid nitrogen; the heads were removed and sliced at sagittal plan. The brain slices were submitted to MALDI spectral imaging analysis, and the results of the present study reported the processing of the precursors proteins into mature forms of the neuropeptides AmAST A (59-76) (AYTYVSEYKRLPVYNFGL-NH2), AmAST A (69-76) (LPVYNFGL-NH2), AmTRP (88-96) (APMGFQGMR-NH2), and AmTRP (254-262) (ARMGFHGMR-NH2), which apparently acted in different neuropils of the honeybee brain during the aggressive behavior, possibly taking part in the neuromodulation of different aspects of this complex behavior. These results were biologically validated by performing aggressiveness-related behavioral assays using young honeybee workers that received 1 ng of AmAST A (69-76) or AmTRP (88-96) via hemocele. The young workers that were not expected to be aggressive individuals presented a complete series of aggressive behaviors in the presence of the neuropeptides, corroborating the hypothesis that correlates the presence of mature AmASTs A and AmTRPs in the honeybee brain with the aggressiveness of this insect.

Exposure to a sublethal concentration of imidacloprid and the side effects on target and nontarget organs of Apis mellifera (Hymenoptera, Apidae).

The use of insecticides has become increasingly frequent, and studies indicate that these compounds are involved in the intoxication of bees. Imidacloprid is a widely used neonicotinoid; thus, we have highlighted the importance of assessing its oral toxicity to Africanized bees and used transmission electron microscopy to investigate the sublethal effects in the brain, the target organ, and the midgut, responsible for the digestion/absorption of food. In addition, the distribution of proteins involved in important biological processes in the brain were evaluated on the 1st day of exposure by MALDI-imaging analysis. Bioassays were performed to determine the Median Lethal Concentration (LC50) of imidacloprid to bees, and the value obtained was 1.4651 ng imidacloprid/µL diet. Based on this result, the sublethal concentration to be administered at 1, 4 and 8 days was established as a hundredth (1/100) of the LC50. The results obtained from the ultrastructural analysis showed alterations in the midgut cells of bees as nuclear and mitochondrial damage and an increase of vacuoles. The insecticide caused spacing among the Kenyon cells in the mushroom bodies, chromatin condensation and loss of mitochondrial cristae. The MALDI-imaging analysis showed an increase in the expression of such proteins as vascular endothelial growth factor receptor, amyloid protein precursor and protein kinase C, which are related to oxygen supply, neuronal degeneration and memory/learning, and a decrease in the expression of the nicotinic acetylcholine receptor alpha 1, which is fundamental to the synapses. These alterations demonstrated that imidacloprid could compromise the viability of the midgut epithelium, as well as inhibiting important cognitive processes in individuals, and may be reflected in losses of the colony.

Profiling the proteomics in honeybee worker brains submitted to the proboscis extension reflex.

The proboscis extension reflex (PER) is an unconditioned stimulus (US) widely used to access the ability of honeybees to correlate it with a conditioned stimulus (CS) during learning and memory acquisition. However, little is known about the biochemical/genetic changes in worker honeybee brains induced by the PER alone. The present investigation profiled the proteomic complement associated with the PER to further the understanding of the major molecular transformations in the honeybee brain during the execution of a US. In the present study, a quantitative shotgun proteomic approach was employed to assign the proteomic complement of the honeybee brain. The results were analyzed under the view of protein networking for different processes involved in PER behavior. In the brains of PER-stimulated individuals, the metabolism of cyclic/heterocyclic/aromatic compounds was activated in parallel with the metabolism of nitrogenated compounds, followed by the up-regulation of carbohydrate metabolism, the proteins involved with the anatomic and cytoskeleton; the down-regulation of the anatomic development and cell differentiation in other neurons also occurred. SIGNIFICANCE: The assay of proboscis extension reflex is frequently used to access honeybees' ability to correlate an unconditioned stimulus with a conditioned stimulus (such as an odor) to establish learning and memory acquisition. The reflex behavior of proboscis extension was associated with various conditioned stimuli, and the biochemical/genetic evaluation of the changes occurring in honeybee brains under these conditions reflect the synergistic effects of both insect manipulations (training to answer to an unconditioned stimulus and training to respond to a conditioned stimulus). Little or no information is available regarding the biochemical changes stimulated by an unconditioned stimulus alone, such as the proboscis extension reflex. The present investigation characterizes the proteomic changes occurring in the brains of honeybee workers submitted to proboscis extension reflex. A series of metabolic and cellular processes were identified to be related to the reflex of an unconditioned stimulus. This strategy may be reproduced to further understand the processes of learning and memory acquisition in honeybees.

MALDI imaging analysis of neuropeptides in the Africanized honeybee (Apis mellifera) brain: effect of ontogeny.

The occurrence and spatial distribution of the neuropeptides AmTRP-5 and AST-1 in the honeybee brain were monitored via MALDI spectral imaging according to the ontogeny of Africanized Apis mellifera. The levels of these peptides increased in the brains of 0-15 day old honeybees, and this increase was accompanied by an increase in the number of in-hive activities performed by the nurse bees, followed by a decrease in the period from 15 to 25 days of age, in which the workers began to perform activities outside the nest (guarding and foraging). The results obtained in the present investigation suggest that AmTRP-5 acts in the upper region of both pedunculi of young workers, possibly regulating the cell cleaning and brood capping activities. Meanwhile, the localized occurrence of AmTRP-5 and AST-1 in the antennal lobes, subesophageal ganglion, upper region of the medulla, both lobula, and α- and ß-lobes of both brain hemispheres in 20 to 25 day old workers suggest that the action of both neuropeptides in these regions may be related to their localized actions in these regions, regulating foraging and guarding activities. Thus, these neuropeptides appear to have some functions in the honeybee brain that are specifically related to the age-related division of labor.