Sep 5, 2023 / Developmental Biology

Mapping collective aggression in honeybee colonies with single cell tools

Jeanene Swanson

There are a variety of animals that show group behavior. It’s fascinating to watch fish move as a school or birds fly as a flock. Honeybee colonies model collective behavior in many ways, including when certain bees defend the hive. While it’s known that honeybees have different roles depending on their age—it’s the older bees that either forage for food or defend the hive—it isn’t known what drives this particular division of labor at the molecular level. Until now. Recently, researchers used single cell technologies to explore how group behavior affects individual bees at the genetic level. Read more in this blog post.

Adult Apis mellifera scutellata (African honey bee) in Florida. CREDIT: Jeffrey W. Lotz, Florida Department of Agriculture and Consumer Services,
Adult Apis mellifera scutellata (African honey bee) in Florida. CREDIT: Jeffrey W. Lotz, Florida Department of Agriculture and Consumer Services,

In honeybees, it’s not size but age that matters when it comes to being the meanest. Younger bees care for the hive while older bees move on to either foraging for food (also called foragers) or defending the colony (also called soldiers). The soldiers are the first bees in the hive to react to an attack by becoming aggressive and stinging the invader. Some hives are more aggressive than others, causing scientists to wonder, what drives this collective behavior at the individual level?

A previous genome-wide association study (GWAS) of a honeybee species from Puerto Rico that had recently evolved to become less aggressive showed hundreds of genes associated with different levels of colony aggression, also called “colony aggression genes” (1). In a recent study led by Ian Traniello, PhD, at the University of Illinois Urbana-Champaign, they used this GWAS data alongside single cell gene expression and gene regulatory network analysis to explore the differences in forager and soldier bee brains from colonies of varying levels of aggression (2). They found that it was gene regulation that mattered, not gene expression, and, depending on an individual bee’s susceptibility to become more (or less) aggressive, colonies can become more (or less) aggressive when faced with different environmental triggers.

Gene expression not the difference maker for bee aggression

To test their original hypothesis that differences in gene expression should cause differences in colony aggression, Dr. Traniello first compared whole brain transcriptomic profiles of soldiers and foragers from nine colonies of “gentle” African honeybees from Puerto Rico. Though they found about 4,000 differentially expressed genes (DEGs) between the two types of bees, these DEGs did not significantly overlap with the colony aggression genes.

They went on to perform the same experiment using 10x Genomics Single Cell Gene Expression, but this time, due to logistical issues, they used brain cells from soldiers and foragers from four different colonies (of different levels of aggression) of European honeybees (they noted that the gentle African bees are genetically similar to the European bees [1,3]). Again, they found some DEGs but not a large number that overlapped with the colony aggression genes, suggesting that gene expression differences at the individual level couldn’t account for genetic variation predictive of increased aggression at the group level.

The missing link between genetic differences in colony aggression and individual gene expression regulation

Maybe, the authors thought, if gene expression differences weren’t measurably different, then differences in regulation of gene expression might offer a clue. Several recent studies point to gene regulatory networks as influencing behavior (4,5). However, studying the inferred relationships between interacting transcription factors (TFs) and their target genes (TGs) at the single cell level in the brain, as it pertains to collective behavior traits, had not been done yet.

Taking the single cell transcriptomic data, they were able to model (using the SimiC algorithm) a single cell gene regulatory network that was not only different between soldiers and foragers, but more different between soldiers and foragers from more aggressive colonies. This dissimilarity stood out most in two cell types associated with honeybee aggression and behavioral plasticity, respectively mushroom body non-compact Kenyon cells and astrocyte-like glia.

To find out more about individual changes in the regulatory network, they turned back to the GWAS analysis for colony aggression genes, identifying what they called eGenes—genes that contain single nucleotide polymorphisms in soldiers and foragers that were also found in the colony aggression genes. They were able to describe a subnetwork of 15 TF–TG relationships with strong soldier–forager differences. One gene they identified as a prominent target gene was tryptophanyl-tRNA synthetase mitochondrial (TrpRS-m), which has been linked to genes associated with tryptophan availability and reuptake (6). Tryptophan turns into serotonin, which helps regulate aggression in honeybees (7). Because the biggest differences in TF–TG relationships for TrpRS-m between foragers and soldiers were in cell types influencing vision, these results suggest that honeybee aggression, as an inherited trait, is partly based on variable environmental cues affecting vision (and/or smell) that in turn trigger aggression.

Single cell and the future of mapping collective behavioral changes

Serotonin metabolism is just one example of how the genetics of a colony can impact individual behavior. Learning more about how collective behavior can be influenced by environmental cues is especially crucial for an important species like the honeybee—and will go a long way toward understanding how collective traits adapt a colony or group to a changing environment.


  1. Avalos A, et al. Genomic regions influencing aggressive behavior in honey bees are defined by colony allele frequencies. Proc Natl Acad Sci USA 117: 17135–17141 (2020).
  2. Traniello IM, et al. Single-cell dissection of aggression in honeybee colonies. Nat Ecol Evol 7: 1232–1244 (2023).
  3. Avalos A, et al. A soft selective sweep during rapid evolution of gentle behaviour in an Africanized honeybee. Nat Commun 8: 1550 (2017).
  4. Fagny M & Austerlitz F. Polygenic adaptation: integrating population genetics and gene regulatory networks. Trends Genet 37: 631–638 (2021).
  5. Boyle EA, Li YI, Pritchard JK. An expanded view of complex traits: from polygenic to omnigenic. Cell 169: 1177–1186 (2017).
  6. Boccuto L, et al. Decreased tryptophan metabolism in patients with autism spectrum disorders. Mol Autism 4: 16 (2013).
  7. Hunt GJ. Flight and fight: a comparative view of the neurophysiology and genetics of honey bee defensive behavior. J Insect Physiol 53: 399–410 (2007).