Bacteria and fungi play key roles in social insect colonies

Brazilian researchers show how certain leafcutter ant and stingless bee species depend on microorganisms for food, communication, defense against pathogens, and the completion of their development cycle.

By Karina Toledo  |  Agência FAPESP – A line of worker ants returning to their nest carrying pieces of plants up to 100 times their own body weight is a scene that may astonish at first sight but is commonplace in colonies of leafcutter ants. They are guided without deviation to the nest by aromatic chemical compounds called trail pheromones. 

In the case of Atta sexdens rubropilosa, the main substances used in this geolocation process belong to the class of heterocyclic compounds known as pyrazines. 

Researchers working on the Ribeirão Preto campus of the University of São Paulo (USP) in Brazil have discovered that a bacterium found in the microbiota associated with this leafcutter ant species produces the same pyrazines used by the ants to lay a trail to their nest. An article on their findings has been published in the journal Scientific Reports.

“We’ve observed this phenomenon in more than one ant colony. It raises an interesting question. Does the bacterium Serratia marcescens produce trail pheromones for the ants or contribute to this process in some way? We intend to investigate in search of answers,” said Mônica Tallarico Pupo, a professor in the University of São Paulo’s Ribeirão Preto School of Pharmaceutical Sciences (FCFRP-USP) and the principal investigator for the project.

The study was part of Eduardo Afonso da Silva Junior’s PhD research and was conducted in partnership with scientists at Harvard University in the United States under the aegis of a Thematic Project supported by FAPESP and the US National Institutes of Health (NIH).

According to Pupo, the main aim of the project is to explore the microbiota of Brazilian ants in search of natural molecules that can give rise to new drugs (read more at: agencia.fapesp.br/19498). However, another goal related to chemical ecology is investigating the dependency between social insects and the symbiotic microorganisms with which they interact in a mutually beneficial relationship.

The pyrazine-producing bacterium was discovered by chance when the scientists were looking for microorganisms capable of protecting ant colonies from parasitic fungi.

“The leaves carried to their nests by these leafcutters actually serve as a substrate for the cultivation of Leucoagaricus gongylophorous, the fungal species on which they feed. However, this system is susceptible to infections,” Pupo explained.

“In some cases, another pathogenic species that may impair the ant colony’s viability grows on the fungus they eat. The symbiotic bacteria produce compounds that can kill the parasitic fungi without damaging the food source. We set out to identify these compounds.”

The experiments described in the article involved colonies collected on USP’s Ribeirão Preto campus. When the scientists succeeded in collecting the queen, part of the colony was transported to the laboratory, and all the bacteria found on the insects’ surface and inside their bodies were isolated, characterized, and placed in culture medium.

During this process, Silva Junior realized that when S. marcescens was grown in the laboratory, it released a strong aroma that closely resembled the smell of the ants kept in the same lab.

“We decided to investigate the volatile compounds produced by this bacterium and discovered the pyrazines, among which there was a molecule not described in the scientific literature,” Pupo said.

The researchers used a type of fiber specifically indicated for absorbing the aromatic compounds from the culture plates. Later, the material was analyzed by gas chromatography-mass spectrometry (GC-MS).

“We found both pyrazines and bacteria in the ants’ poison glands,” Pupo said. “We don’t know for sure if their synthesis is shared: maybe the microorganism produces the aromatic compounds and the ants store them in their glands. In future studies, we plan to test techniques to remove the bacteria from the ants and observe whether the compounds continue to be produced.”

Another line of research the group plans to pursue consists of determining whether similar phenomena can be observed in other ant species. Nothing of the kind has been described in the literature.

Bee metamorphosis

The cultivation of fungi in the nest for food or defense seems to be a widespread practice among social insects. According to an article by Brazilian researchers published in 2015 in the journal Current Biology, the newborn larvae of Scaptotrigona depilis, a species of stingless bee native to Brazil, feed on filaments of a fungus cultivated in the brood cells (read more at: agencia.fapesp.br/22225). Without this food, few larvae survive to become adults.

This symbiosis was recently studied in greater depth by Pupo’s group during Camila Paludo’s PhD research as part of the same Thematic Project. The results were published in Scientific Reports in January 2018.

“We know insects can’t synthesize hormones, so they must obtain precursor substances from their food,” Pupo said. “Our hypothesis was that the fungus supplied a precursor for the molting and pupating hormone required for larvae to complete the metamorphosis into adult bees.” 

The first step of the investigation consisted of isolating the fungus from brood cells and characterizing it in the laboratory. The group found it to be a fungus belonging to the genus Zygosaccharomyces.

“We aren’t sure how this fungus gets into brood cells. The bees lay eggs and then fill the cells with a liquid called larval food. Some three days later, the fungus begins to grow inside the cells,” Pupo said.

Using fluorescence microscopy, the researchers found an accumulation of lipids in the fungal cytoplasm from samples grown in vitro as well as samples extracted directly from bee colonies.

“Steroids, the precursors of molting hormones, are lipids. Using GC-MS, we found that the predominant compound among the lipids in this fungus was ergosterol,” Pupo said.

Via in vitro experiments, the researchers proved that most larvae completed pupal morphogenesis when the larval food was inoculated with the fungus and when only ergosterol was added. 

“The results were statistically equivalent for these two situations,” Pupo explained. “When the larvae received only larval food without the fungus, they failed to reach the adult stage. We therefore concluded that ergosterol was in fact being used by the larvae to produce molting hormone, which reinforces the dependency between these bees and the fungus.”

The group now plan to investigate whether similar phenomena occur in other species of stingless and stinging bees.

The article “Pyrazines from bacteria and ants: convergent chemistry within an ecological niche” can be read at nature.com/articles/s41598-018-20953-6. 

The article “Stingless bee larvae require fungal steroid to pupate” can be read at nature.com/articles/s41598-018-19583-9.