Nutritionally Driven Differential Gene Expression Leads to Heterochronic Brain Development in Honeybee Castes

Abstract

The differential feeding regimes experienced by the queen and worker larvae of the honeybee Apis mellifera shape a complex endocrine response cascade that ultimately gives rise to differences in brain morphologies. Brain development analyzed at the morphological level from the third (L3) through fifth (L5) larval instars revealed an asynchrony between queens and workers. In the feeding phase of the last larval instar (L5F), two well-formed structures, pedunculi and calyces, are identifiable in the mushroom bodies of queens, both of which are not present in workers until a later phase (spinning phase, L5S). Genome-wide expression analyses and normalized transcript expression experiments monitoring specific genes revealed that this differential brain development starts earlier, during L3. Analyzing brains from L3 through L5S1 larvae, we identified 21 genes with caste-specific transcription patterns (e.g., APC-4, GlcAT-P, fax, kr-h1 and shot), which encode proteins that are potentially involved in the development of brain tissues through controlling the cell proliferation rate (APC4, kr-h1) and fasciculation (GlcAT-P, fax, and shot). Shot, whose expression is known to be required for axon extension and cell proliferation, was found to be transcribed at significantly higher levels in L4 queens compared with worker larvae. Moreover, the protein encoded by this gene was immunolocalized to the cytoplasm of cells near the antennal lobe neuropiles and proximal to the Kenyon cells in the brains of L4 queens. In conclusion, during the larval period, the brains of queens are larger and develop more rapidly than workers’ brains, which represents a developmental heterochrony reflecting the effect of the differential feeding regime of the two castes on nervous system development. Furthermore, this differential development is characterized by caste-specific transcriptional profiles of a set of genes, thus pointing to a link between differential nutrition and differential neurogenesis via genes that control cell proliferation and fasciculation.

The ontogenetic saga of a social brain

“…The brain of a recently hatched honeybee larva is similar to that of other insects and does not seem to exhibit morphological differences between the castes much before the fourth larval instar, L4 (Nelson 1924;Moda et al 2013). This lack of observed differences, however, might be due to methodological limitations and the challenge involved in dissecting such small organs.…”

“…L3: third larval instar; L4: fourth larval instar; L5: fifth larval instar; Pw: white-eyed pupa, unpigmented cuticle; Pbm: brown-eyed pupa, intermediary pigmented cuticle; Pbd: brown-eyed pupa, dark pigmented cuticle; NE: newly emerged adult. Values on the y-axis are arbitrary units only to demonstrate the relative developmental trend (modified from Moda et al 2013; see also Section 6. Honeybee brain development during the pupal and pharate-adult stages).…”

“…Knowledge of the development of the MBs and other honeybee brain regions such as the optic lobes (Marco Antônio and Hartfelder 2016) has broad scientific interest given that, for example, pesticides used in agriculture to control insects target the brains of larval bees (Tomé et al 2012;Tavares et al 2015). The ontogenetic saga of a social brain Using phalloidin and DAPI staining of wholemount brains, Moda et al (2013) showed clear peduncular organisation in worker larvae in the L5F phase. In contrast, this organisation was already observed to the end of L3 in queen larvae (Figure 2a).…”

“…This neuropil differential morphogenesis is also accompanied by a marked difference in MB cell proliferation. Cell proliferation rates, as assessed by EdU staining, increasingly differ between castes from L4 until L5S, difference that favours queen larvae (Moda et al 2013). Higher levels of brain neuron proliferation in queen larvae were previously demonstrated by Roat and Cruz Landim (2008).…”

“…Studies on the ontogenetic development of the brain diphenism found in adult honeybees have revealed an interesting phenomenon. The differential nutritional inputs received by developing larvae boost the development of the Bbipotentialb rain found in embryos and the initial phases of larval development (primarily L1-2) into that of presumptive queens thus resulting in a bigger brain compared to that of presumptive workers (Moda et al 2013). This picture, nevertheless, shifts during subsequent developmental stages before adult emergence.…”