Phenotypic variation among individuals of the same species triggered by environmental action is an intriguing biological phenomenon that can be found in quite striking manifestations in members of different insect orders [1
]. In highly eusocial bees (Hymenoptera) one or a few females (queens) specialize in reproductive tasks, whereas a large number of quasi-sterile individuals (workers) engage in colony maintaining activities [2
]. This polyphenism is generally determined by discrete switches during postembryonic development, and commences with the differential feeding of female larvae [4
]. The nutritional stimuli trigger an endocrine response that is manifested by an elevated juvenile hormone (JH) titer in queen larvae when compared to workers [for review see [5
]]. The queen-inducing properties of JH were first demonstrated by Wirtz and Beetsma [6
], who topically applied JH on fourth and early fifth instar worker larvae [for similar results in stingless bees see [7
]]. However, the molecular mechanisms underlying this phenomenon are not yet understood. In particular, we are largely ignorant of how nutritional factors affect the endocrine system and alter JH synthesis rates of queens and workers, and how these changes drive caste-specific developmental pathways during metamorphosis.
In Apis mellifera
, a model system for caste development and division of labor in social Hymenoptera, young larvae of both castes are fed with royal jelly, a secretion produced by glands in the head of adult workers. Whereas these nurse bees feed copious amounts of royal jelly to queen larvae until they enter metamorphosis, they switch the diet for late instar worker larvae from pure royal jelly to a mixture of glandular secretions with honey and pollen (worker jelly). In addition, prospective queen larvae receive 10 times more food than worker larvae [9
]. As a consequence of this differential feeding regime the two types of larvae follow two very different developmental trajectories, in spite of having exactly the same genetic background. Conceptually, this process of caste differentiation involves two kinds of alterations in the original developmental pattern (or ground plan present in ancestral solitary bees): one type, which we can call incremental alterations, affects the general growth of the body or specific organs, especially the ovaries. The other type can be considered as character state alterations that result in the presence or absence of entire specific structures, such as the pollen-collecting apparatus on the hind legs, wax glands, etc. Both types of alterations can be envisaged as JH threshold responses controlling the expression of genes involved in the development of specific organs and in specifying the general body plan (Figure ).
Figure 1 Reaching of the juvenile hormone (JH) threshold in developing females is proposed not only to allow for the general body growth and ovary development, but also to act by negatively regulating the development of some organismal systems that are characteristics (more ...)
The first large-scale study on the molecular biology of caste differentiation was done in A. mellifera
by Severson et al. [10
]. These authors demonstrated by in vitro
translation analyses that queens and workers differ in their mRNA profiles during larval and prepupal stages. Later studies by Corona et al. [11
] and Evans and Wheeler [12
] found that most of the differentially expressed genes between prospective queens and workers were related to metabolic processes, and specifically, that queens up-regulate metabolic enzymes. Conversely, workers were shown to up-regulate a member of the cytochrome P450 family, hexamerin 2, dihydrodiol dehydrogenase and a fatty-acid binding protein. In addition, these studies revealed that several regulatory genes such as the mitochondrial translation initiation factor (AmIF-2mt), a member of the Ets family of transcription factors with a DNA binding domain, were also up-regulated in worker larvae [11
In a recent study, Cristino et al. [14
] examined the up-stream regulatory elements associated with all transcripts previously found to be differentially expressed in worker and queen larvae. They confirmed that the majority of the annotated differentially expressed genes (DEGs) are related to metabolic processes, with an interesting dichotomy for enzymes with hydrolase and oxidoreductase activities, which were found to be up-regulated in workers and queens, respectively. Genes up-regulated in workers were also shown to share more common (or conserved) overrepresented cis
-elements when compared to genes up-regulated in queens.
While the aforementioned studies support the notion that incremental alterations are associated with the differential expression of physiometabolic genes, the nature of genetic mechanisms underlying the development of worker distinctiveness or character state alterations remains to be understood.
Here we used cDNA microarrays to monitor differential gene expression in honeybee queen and worker larvae and to identify cis-acting elements associated with these two developmental trajectories. Graph theory and complex networks concepts were adopted to attain an objective visual representation of the connectivity between motifs and genes in both castes. We identify groups of genes responsible for the development of queen and worker singularities and describe how their expression is co-regulated during critical stages of larval development. We also discuss the role of morphogenetic hormones in the developmental process of queen-like character and finally, propose a model of caste differentiation in A. mellifera.