Substances associated with bee ferocity are discovered

Chemical compounds identified by Brazilian researchers may explain why less aggressive bees become ferocious. Study is published in Journal of Proteome Research.

By Peter Moon  |  Agência FAPESP – Brazilian researchers may have discovered why Africanized honeybees are so aggressive. The scientists detected higher levels of certain chemical substances in the brains of Africanized honeybees than in gentler strains of honeybees bred by beekeepers. 

According to a study published in Journal of Proteome Research, the chemical substances in question may trigger irascible behavior by normally placid bees. 

The same compounds had been detected in the brains of flies and mice, where they appeared to regulate food intake and digestion. This evidence provides an example of how behavior evolves differently in different species via similar molecular mechanisms.

The research was conducted by Professor Mario Sérgio Palma at the Center for Studies of Social Insects in São Paulo State University’s Bioscience Institute (IB-UNESP), Rio Claro, Brazil. FAPESP provides funding under a Thematic Project linked to its Research Program on Biodiversity Characterization, Conservation, Restoration & Sustainable Use (BIOTA-FAPESP). The research line relates to the identification, characterization and quantification of the proteins in animal systems. 

“We study the composition of proteins in the glandular systems of wasps, bees, ants, spiders and scorpions. We analyze the individual functions of each protein and its molecular interactions, as well as its molecular structures. That includes these animals’ venoms, their chemical communication systems via chemical compounds, and the neurochemistry of the regulation of arthropod behavior via the nervous system,” Palma told Agência FAPESP. 

The study set out to investigate the neurohormone production mechanism in the bee brain. Neurohormones are chemical compounds that regulate the nervous system and are involved in mediating the social behavior of these insects.

“We wanted to study the origin and metabolism of neuropeptide precursor proteins in the bee brain in order to understand how these hormones are produced,” Palma said. “We also wanted to identify the brain regions responsible for the action of these compounds. To this end, we used matrix-assisted laser/desorption ionization (MALDI) combined with mass spectrometry imaging (MSI).” 

The technique consists of breaking down the atoms or molecules in a sample so that they become charged with more or fewer electrons than the original (ionization) and then separating them by mass/charge ratio in order to identify and quantify them. It can be used to identify chemical compounds inside tissue without requiring cellular extracts and to locate the brain regions in which hormones are active.

“You can use it to investigate the chemical structure of neuropeptides and map the brain regions they affect,” Palma explained. 

The experiments were performed at IB-UNESP’s apiary. “Practically all our colonies consist of Africanized honeybees. We’ve been working with these insects for more than 20 years, and IB-UNESP’s Biology Department has studied them since its foundation 60 years ago,” Palma said. 

Combs containing larvae were incubated in the laboratory, and some 1,000 newly emerged workers were marked on the thorax using nontoxic paint. They were returned to the hive, and when the marked bees were 20 days old, the colony was subjected to what the researchers call an aggression or stinging assay. 

The bees, including marked individuals, were placed in an observation arena. A small black leather ball with a diameter of 5 cm was waved several times in front of them.

“Various kinds of warning and attack behavior were observed and recorded by the researchers,” Palma said.

In addition to defensive behavior of several types, some bees also stung the target. Their barbed lancets penetrated the leather material and stuck to the surface of the ball. Marked individuals were collected, immediately frozen in liquid nitrogen and dissected. Their brains were removed and cut into slices for proteomic analysis. Proteomics is a field of biotechnology that analyzes the entire protein complement of a cell, tissue or organism.

Worker bees that remained in the hive during the attack were also collected for comparison with the aggressive individuals.

The brain tissue samples were treated with laser radiation to ionize proteins and peptides and then analyzed using MALDI-MSI to characterize their chemical structure, focusing on short protein and peptide chains.

“When samples are prepared in the form of brain slices, the instrument creates a visual pattern for spatial orientation inside each tissue sample so that the presence of the spectrum typical of each molecule analyzed can be correlated with its spatial position in the tissue. Thus, the technique identifies the molecules and at same time estimates their concentrations and determines their location,” Palma said.

“The final images generated show the molecular topography of the histological sections and contour maps, highlighting the extent and boundary of each different brain region.” 

Hormones and aggression

Commenting on the comparison between the bees captured outside the hive and individuals who remained inside, Palma stressed that it was not a matter of location but of differences observed between worker bees that displayed the various kinds of alarm and aggressive behavior, especially stinging, and workers that did not become aggressive even when stimulated to do so. 

“The precursor proteins in the brains of the non-aggressive bees were found to be intact in their inactive form, meaning these substances didn’t stimulate aggressive behavior,” he said. 

On the other hand, only the mature forms of these proteins were found in the brains of the aggressive bees. They were small pieces of precursor proteins created by the action of enzymes known as proteases. These smaller pieces must undergo further chemical modification (proteolysis or cleavage) in order to become active neuropeptides that trigger aggressive behavior. Neuropeptides are hormones that play a key role in regulating the activity of the brain and other tissues so that the organism behaves in various different ways. 

“In practice, this means neuropeptides tell the organism to perform metabolic, physiological and pharmacological functions that prepare the individual to behave in this or that way. In the case of our study, the set of behaviors related to aggression,” Palma said.

“The neuropeptides we identified can be found with minor structural differences in several insects, but until now, they have rarely been chemically and functionally characterized. In the aggressive bees analyzed, we concluded that the functions of these neuropeptides were to regulate the energy metabolism, activate vigilance and spatial coordination of flight, and stimulate the production of alarm pheromones.” 

When the researchers observed that the neuropeptides stimulated aggressive behavior, they decided to synthesize these compounds in the laboratory and inject them into young worker bees, presumably too immature to perform aggressive roles in the colony. 

“The result was that these workers began displaying aggressive behavior only a short time after being injected with the neuropeptides. This included stinging the targets,” said Palma, who is principal investigator for the FAPESP Thematic Project. 

Origin of ferocity

Aggressiveness in bees is triggered as part of the colony’s defense mechanism. Aggressive behavior begins with a series of physical and chemical stimuli, such as sharp movements, loud noises, dark colors and strong odors, typically associated with the presence of intruders, invaders or predators. This sets off a cascade of reactions comprising a series of metabolic and physiological processes that enable aggressive bees to play their role as sentries or soldiers.

“Apparently the initial reaction to such stimuli leads to maturation of neurohormone precursors in the brains of these bees, leading in turn to the formation of mature neuropeptides, which are distributed in specific concentrated brain regions,” Palma said.

“These neuropeptides act on neurons in the brain regions concerned, triggering a series of metabolic and physiological processes that result in behaviors relating to alarm and aggression. The culmination is stinging.” 

The neuropeptide precursors are mature in the brains of adult workers, but in young workers, they have not yet been cleaved and cannot result in mature or active neuropeptides. 

By the time workers are between 15 and 20 days old, they have the molecular tools needed to catalyze precursor maturation. As a result, the neuropeptides in their brains are instantly activated in response to threatening physicochemical stimuli, and they begin to behave aggressively.

“When we injected synthetic neuropeptides in their mature form into these young workers, they were endowed with mature neuropeptides, and in a few moments, these compounds activated metabolic and physiological transformations that enabled the bees to behave very aggressively,” Palma said.

The article “MALDI imaging analysis of neuropeptides in Africanized honeybee (Apis mellifera) brain: effect of aggressiveness” by Marcel Pratavieira, Anally Ribeiro da Silva Menegasso, Franciele Grego Esteves, Kenny Umino Sato, Osmar Malaspina and Mario Sergio Palma can be retrieved from pubs.acs.org/doi/10.1021/acs.jproteome.8b00098.