Box 1.

Genes and molecular pathways commonly described in the literature as being involved in honeybee worker task division compared to present findings in B. terrestris and T. angustula. For—foragers; Nur—Nurses. Symbols indicate whether evidence suggests that the expression is higher (↑) or lower (↓) in one group compared to the other. Blue indicates higher expression levels in foragers than in nurses and orange indicates the opposite; (≈) in black, no changes identified or controversial evidence; and (↑↓) in red, indicates a mixed pattern, with some genes in the pathway being upregulated or downregulated in one of the two subcastes. A. mellifera by A. Wide, T. angustula by L. Costa—images reproduced with permission from the original authors.

Juvenile hormone (JH)	These hormones are important regulators in honeybee maturation affecting the task division system in workers46. In honeybees, foragers have higher levels of JH than nurses4,5,46, but in primitively eusocial bees, changes in JH appear not to affect worker behaviour37. This observation led to the hypothesis that JH might only be involved with age-related task division47,48. In the present dataset, we did not find any direct evidence of the involvement of JH in the age-related task division of T. angustula workers. This result is in agreement with previous studies about JH in stingless bees, which demonstrated that JH expression differences are important in differentiating queens and workers but not nurses and foragers. Notably, significantly reduced JH titer levels in foragers have been reported49. One transcript in our dataset, highly expressed in B. terrestris foragers, was indirectly related to JH pathways and predicted to be a “takeout-like” gene. This gene family has been associated with multiple processes in insects, including eusocial insects, in which it has been shown to be strongly sensitive to queen pheromone50
Vitellogenin (vg)	This yolk precursor protein is related to egg production in many insects51. In honeybees, it interacts with JH in a double repressor network, and its expression is reduced in foragers4,5,51. For bumblebees, this double repressor network apparently does not exist; instead, this protein gene has been associated with worker aggression37 and reproductive status when expressed in the fat body52. Our B. terrestris data identified two highly expressed genes in foragers with vg transcription factor domains. As a primitively eusocial species, bumblebee workers may dispute reproductive status with queens in later stages of the colony cycle53. In this sense, it would be interesting to determine if the augmented expression of these vg associated genes in foragers could be related to this behavior. Similar to honeybees, we found a higher expression of one vg receptor gene in T. angustula nurses, indicating the relevance of this protein in this subcaste. It has been proposed that since stingless bee workers usually produce trophic eggs54, vg might be involved in this process or even have alternative and/or unknown roles55
Foraging (for)	This gene has been reported as highly expressed in honeybee56 and bumblebee38 foragers. In honeybees, although the gene expression of this gene was not among the best predictors of the subcaste division of workers 5,7, its association with foraging is well established in the literature56,57. In bumblebees, the results about its effects are more controversial58, as its expression was higher in nurses than foragers in one study36. In our datasets, this gene was not differentially expressed
Period (per)/circadian rhythm	The gene period is related to circadian rhythm and has been reported as overexpressed in honeybee foragers59,60. This specific gene does not appear among the ones differentially expressed in our study. However, B. terrestris foragers have other highly expressed rhythm genes such as protein quiver or sleepless that are related to sleep, rhythmic process, and regulation of circadian sleep/wake cycles. Conversely, none of the differentially expressed superTranscripts of T. angustula were associated with rhythm genes. This result suggests that in B. terrestris, and Apis, rhythm genes are more relevant to nurse/forager behavioral differences than in T. angustula
Insulin/Insulin-like signaling (IIS)	In bees and other insects, genes involved in this pathway are important regulators of metabolism and feeding-related behavior58,61,62. In Apis mellifera, this energetic pathway is related to the subcaste division of workers and with lipid storage (lower levels of lipid storage increase IIS gene expression)61. We identified differentially expressed genes between nurses and foragers in both species studied herein, and some were related to insulin metabolism (genes containing insulin domains, transcription factor and regulators). These observations, taken together, indicate that the regulation of the insulin signaling pathway is essential to worker subcaste specialization in all these eusocial bees
Energetic metabolism	In general, since feeding circuits are basal pathways to different bee activities, genes related to energetic metabolism are expected to be involved in worker bee behavior58,63. Indeed, many genes related to energetic metabolism are differentially expressed in nurses and foragers of both species, with some of the common GO enriched terms related to this pathway. Specific examples of genes involved in energetic pathways (besides JH and IIS) studied in honeybees include malvolio and major royal jelly proteins64,65. The first was not differentially expressed in our data, and the second was related to differentially expressed superTranscripts in B. terrestris. In B. terrestris nurses, two highly expressed genes were predicted as protein yellow genes (which have a major royal jelly protein family domain), and in foragers, two other overexpressed genes had major royal jelly protein family domains
Transcription factors (TF)	Different TFs are believed to be involved in the dynamic changes related to behavior in eusocial bees62. Indeed, we identified differentially expressed TF superTranscripts in both species. However, it should be pointed out that the ultraspiracle (usp) TF, which is known to participate in the honeybee worker task division transition via its interaction with JH66, was not among them
DNA methylation/epigenetic modifications	DNA methylation is known to participate in the nursing to foraging transition in honeybees17,18. In the two species investigated in the present study, genes possibly related to epigenetic changes were also differentially expressed. Histone genes (H3 and H2B) and a methyltransferase in T. angustula were differentially expressed, and histone H3-K4 demethylation was differentially expressed, and lncRNAs were detected in B. terrestris. Except for one lncRNA overexpressed in B. terrestris foragers, all of these genes were highly expressed in nurses