The chemical language of insects

Within colonies, bees and ants recognize each other and organize themselves through compounds that cover their bodies

LÉO RAMOS CHAVES In colonies of the stingless bee Tetragonula carbonaria, the queens express their fertile status through cuticular hydrocarbons, which also suppress the reproductive drive of the worker beesLÉO RAMOS CHAVES

How do social insects like bees, wasps, ants, and termites recognize each other, organize themselves, and divide tasks in the complete darkness of their colonies? In 2003, when planning his post-doctorate research at the University of São Paulo’s Riberão Preto School of Philosophy, Sciences, and Languages and Literature (FFCLRP-USP), the biologist Fábio Santos do Nascimento found that genetic analyses and behavioral studies did not provide a satisfactory answer to this question. In search of alternatives, he began to study a group of chemical compounds produced by insects known as cuticular hydrocarbons (CHCs), which had already attracted attention from research groups in the United States and Europe. Nascimento and the chemist Norberto Peporine Lopes, a professor at the University of São Paulo’s Ribeirão Preto School of Pharmaceutical Sciences (FCFRP-USP), soon found that CHCs indicate whether a bee, wasp, ant, or termite is male or female, worker or queen. Each individual, species, and colony exhibits subtle variations in the composition of the CHCs that differentiate them. These compounds also proved to be crucial for the division of tasks between the insect castes and the cohesion of colonies.

When they release CHCs, the queens indicate that they are fertile and inhibit the workers’ drive to mate, according to a study by the USP group published in the June 2017 issue of Nature Ecology & Evolution. “It is the chemical signaling induced by queens that keeps the workers dedicated to cleaning and guarding the nest or searching for food,” says Nascimento, who was hired as a professor at FFCLRP-USP in 2009. The teams in Ribeirão Preto also found that queen Melipona scutellaris bees spread CHCs on the cells where they lay their eggs, signaling that the workers should not touch them.

The CHCs, which are produced by subcutaneous glands, form the yellowish wax that covers the external skeleton of the insects. These substances are comprised exclusively of carbon and hydrogen atoms arranged into long, linear structures with single or double bonds between the carbon atoms. “The position of the double bonds between the carbon atoms varies according to the insect species or genus,” says Lopes. “And the variation in the structures of these molecules allows them to recognize individuals from the same hive, and permits communication between them.” In 2003, when he began to work with Nascimento, their chemical analysis equipment was suitable for hydrocarbons with up to 40 carbon atoms, but today they use a new mass spectrometry technique in their lab that allows them to identify even longer carbon chains (60 atoms), which were also seen to differ between males and females and between queens and workers.

Revealing contact
This form of communication depends on physical contact between the insects. For example, one ant can recognize that another ant is of the same species or from the same colony by touching its body (particularly the head) with its antennae, which have pores or receivers specifically for identifying CHCs. This is why the more than one thousand CHCs which have already been identified are known as superficial or contact pheromones. This classification differentiates them from sex pheromones, which are released into the air by females who are able to reproduce.

“In their hives, social insects mainly communicate through chemical signals,” says Lopes. “Outside the colony, the primary form of communication between species is visual. If an insect of the same species or another species attempts to invade the anthill, the ants will recognize it as an enemy and attack it immediately.” When light is present, Polistes satan wasps also recognize each other through unique signs on their faces, according to a study conducted by the biologist Ivelize Tannure Nascimento of USP in Ribeirão Preto, and published in 2008 in the Proceedings of the Royal Society B.

léo ramos chaves The ant Dinoponera australis uses chemical compounds captured by its antennae to recognize whether another ant of the same species is male or femaleléo ramos chaves

Two days after hatching, the wasps already produce the colony’s specific CHC because of contact with the other members of the group. The composition of these substances may change, for example, in response to variations in diet. Under the guidance of Nascimento, the biologist Lohan Valadares divided a colony of leafcutter ants into two groups: one group was fed rose leaves and petals, and the other group was given leaves from crepe myrtles (Lagerstroemia sp.), trees with pink flowers which are planted in urban settings. Valadares then put ants from one group into the other group; the newcomers were attacked. The analyses indicated that the ants’ smell had changed after their diets were altered. “As the chemical profile of the cuticular hydrocarbons changed, the ants that had been part of the same colony ceased to recognize each other,” says Nascimento.

The ability to produce these compounds must have arisen even before the insects started to live in colonies approximately 100 million years ago. The biologists Ricarda Kather and Stephen Martin of the University of Salford, Manchester in England examined the chemical profile of the CHCs of 241 species of insects, including 164 with social habits from the order Hymenoptera, which is the largest in this group with 130,000 species. As they explained in a 2015 study in the Journal of Chemical Ecology, the CHC profiles of solitary species were seen to be as complex as those of social species.

Another group from England showed that the antennae (at least in Iridomyrmex purpureus ants) not only receive but also transmit chemical signals, extending the conclusion made by Swiss psychiatrist and entomologist Auguste-Henri Forel (1848–1931). At the end of the nineteenth century, Forel showed that antennae function like organs which are capable of capturing chemical signals by removing antennae from four species of ants and observing that the insects became disoriented and massed together, regardless of species.

Disoriented ants
Similarly, without CHCs the insects become disoriented and social organization breaks down. In the behavior and evolution laboratory at Rockefeller University in the United States, biologist Daniel Kronauer’s team disabled a gene known as orco that produces CHC receptors in Ooceraea biroi ants, which are native to Japan. As soon as they emerged from the larval phase and became adults, the genetically altered ants immediately exhibited strange behavior for this species: they no longer walked in a line, but moved aimlessly, as described in a December 2016 article in the Proceedings of the National Academy of Sciences (PNAS). The researchers also observed changes in the ants’ brain structures, indicating that these insects require odor receptors for the brain to develop properly.

CHCs explain intriguing social behaviors in insects, far beyond the fact that they are constantly touching each other with their antennae. “After they get dirty or emerge from water, the ants clean themselves or rinse themselves with their legs as a way of recovering the layer of hydrocarbons that covers their bodies. If they didn’t, the colony’s guards would not recognize them and would not let them enter,” explains Nascimento. Another solved mystery relates to the fact that worker bees of the species Melipona scutellaris decapitate seven-day-old virgin queens, when they could attract males interested in copulation. By touching the body—mainly the head—of the virgin queens, the workers perceive that their CHCs differ from those of fertilized queens. This perceived difference leads the workers to kill, concludes biologist Edmilson Souza, a professor at the Federal University of Viçosa in Minas Gerais. No major damage is caused to the hive, since queens in stingless bee colonies frequently produce eggs which generate queens.

By combining biology and chemistry, these studies complement work on bee genetics begun by the geneticist from São Paulo, Warwick Kerr, in the 1950s, and other biological studies on the behavior of social insects conducted by the biologist Vera Imperatriz Fonseca starting in the 1970s, and require a multidisciplinary vision from researchers. “Here in the laboratory,” says Nascimento, “every student and researcher, even biologists, must be a bit of a chemist, learn to use the chromatograph and interpret the results it produces.”


The bright colors of dragonfly wings

The dragonfly Zenithoptera lanei, which is found in the Cerrado dryland region of northeastern Brazil and is known as the morpho because of its similarity to the genus of predominantly blue butterflies with the same name, may have become the first case of an adult insect with wings comprised of tissue which is alive, not dead, as was previously thought.

The biologist Rhainer Guillermo Ferreira, a professor at the Federal University of São Carlos (UFSCAR), used electron microscopy to identify a network of tubes known as tracheae within the membranes of the intense blue wings of this species.

ANA COTTA / WIKIMEDIA …is responsible for the blue wing coloration of Zenithoptera laneiANA COTTA / WIKIMEDIA

As described in an article published in the September 2017 issue of Biology Letters, the tracheae vary from 3 to 200 nanometers in diameter and supply oxygen to cells which produce a thick wax that covers the wings. According to Ferreira, the wax reflects ultraviolet radiation, simultaneously accentuating the blue color of the wings and protecting the insect from excessive sunlight. “One of the indications that the wing cells are alive is that the blue quickly loses its brightness after the dragonfly dies,” he says.

The network of tracheae also helps support the wings and control the temperature of these insects. “So far, this is the only species with this type of structure,” he adds. “We looked at 40 other species of dragonflies and did not find anything like this.”

1. Evaluation of exogenous and endogenous mechanisms that influence the variability of cuticular hydrocarbons in neotropical social insects (No. 15/25301-9); Grant Mechanism Regular Research Grant; Principal Investigator Fábio Santos do Nascimento; Investment R$191,870,92.
2. Metabolism and distribution of natural and synthetic xenobiotics: From understanding reactional processes to generating tissue images (No. 14/50265-3); Grant Mechanism Thematic Project; Principal Investigator Norberto Peporine Lopes; Investment R$1,137,805.87.

Scientific articles
KATHER, R. e MARTIN, S. J. Evolution of Cuticular Hydrocarbons in the Hymenoptera: a Meta-AnalysisJournal of Chemical Ecology. v. 41, n. 10, p. 871-883, 2015.
McKENZIE, S. K. et al. Transcriptomics and neuroanatomy of the clonal raider ant implicate an expanded clade of odorant receptors in chemical communicationPNAS. v. 113, n. 49, p. 14091-6, 2016.
NUNES, T. M. et alEvolution of queen cuticular hydrocarbons and worker reproduction in stingless beesNature Ecology & Evolution. v. 1, 0185, 2017
TANNURE-NASCIMENTO, I.C. et alThe look of royalty: visual and odour signals of reproductive status in a paper waspProceedings of the Royal Society B. v. 275, p. 2555-61, 2008.
GUILLERMO-FERREIRA, R. et alThe unusual tracheal system within the wing membrane of a dragonflyBiology Letters. v. 13 (5), 20160960, 2017.