Discussion
Our results demonstrate that winter bees and nurses have similar
transcriptional profiles in fat body tissues relative to foragers, while
winter bees and foragers have similar transcriptional profiles relative
to nurses. Thus, there are clearly tissue-specific expression patterns
associated with the winter bee phenotype. These patterns are consistent
with the differential functions of these tissues in bees. Winter bees
thus represent a “mix and match” phenotype between summer nurse bees
and summer forager bees, with winter fat bodies serving to store
nutrition (as is the case of nurse bees) and winter flight muscles
remaining active (as in forager bees) to generate heat. Our analysis of
biological processes demonstrated that genes differentially expressed
within both tissues corresponded to categories associated with the
diapause phenotype in other insect species.
We found that there were twice as many differentially expressed genes in
flight muscle tissue than in fat body tissues (Table 1). While many
studies have evaluated differences between nurses and foragers in fat
body tissue (Ament, S. A. et al., 2012; Khamis, A. M. et al., 2015;
Seehuus, S-C. et al., 2013), few have evaluated flight muscle tissue.
Interestingly, studies of flight muscle tissue report large changes in
muscle performance or physiology occurring in young, nurse-age, worker
bees, which are more related to maturation processes than the transition
from nursing to forager (Harrison, J., 1986; Herold, R., Borei, H.,
1963; Schippers, M-P. et al., 2010). This would suggest that few genes
would vary between older nurses and foragers, which were used in our
study. However, we found hundreds of differentially expressed genes in
the flight muscle of nurses, foragers and winter bees. In our samples,
bees were actively performing the behaviors (foragers were collected as
they returned to the hive, and winter bees were collected from
thermoregulating clusters) and thus expression patterns may be showing
differences between active versus quiescent muscle tissue. However,
overall transcriptional profiles of muscle tissue have not been broadly
investigated, and future studies should evaluate to what extent there
are “baseline” differences in expression patterns in flight muscle
tissue in these different phenotypes, which may prime the bees for
different levels of activity.
Based on total numbers of DEGs and clustering analyses, our results
suggest that winter bees’ transcriptional profiles in the fat bodies
that support the long-lived winter physiological state, while
transcriptional profiles in the flight tissues support (or are
reflective of) high levels of activity. Thus, winter bees appear to be
“mixing and matching” the gene expression profiles and underlying
physiological processes of nurse and forager bees, in a tissue-specific
way. These results highlight the importance of considering
tissue-specific expression patterns when evaluating processes involved
in diapause. It will be valuable to determine how this tissue-specific
phenotypic plasticity is controlled, both at the molecular and
behavioral level. Nurse-like transcription profiles in the fat body
tissue may be a result of reduced exposure to brood pheromone (since the
there are no developing larvae in the colony) and potentially the
presence of older forager-age bees which release ethyl oleate. Brood
pheromone can accelerate the transition from nursing to foraging, reduce
Vitellogenin levels and reduce longevity in worker bees (Amdam, G. V. et
al., 2009). Exposure to ethyl oleate can slow the behavioral maturation
from nursing to forager (Leoncini, I. et al., 2004). Forager-like
transcriptional profiles in the muscle tissue may be the result of
muscle activity, but it remains to be determined what neurophysiological
mechanism triggers the generation of muscle activity in these winter
bees.
When comparing the GO categories identified in our study with those from
previous studies, we found differential regulation of genes associated
with dendrite morphogenesis (GO:0048813) neurogenesis (GO:0022008) and
morphogenesis of an epithelium (GO:0002009). Several of the
differentially regulated dendrite morphogenesis-related genes
participate in mTOR and TGF-beta signaling. Interestingly, dendrite
morphological restructuring, driven in part by TGF-beta signaling,
underlies a related occurrence of phenotypic plasticity in C.
elegans : the Dauer phenotype. The Dauer larva is a stress-induced
developmental stage wherein several tissues (particularly nervous
tissue) undergo remodeling, producing a stress-resistant and long-lived
alternative developmental phenotype, akin to the diapause state
(Androwski, A. et al., 2017). Molecular analyses have established the
Dauer stage to be driven by genes encoding components of an
insulin-related pathway, a cyclic nucleotide pathway, and a
TGF-beta-related pathway. DAF-7 – Dauer larva development regulatory
growth factor 7 – is the TGF-beta-related ligand for the Dauer pathway.
The DAF-7 signal is transduced by the DAF-1 and DAF-4 TGF-beta-family
type 1 receptors as well as several SMAD-family transcription factors,
including DAF-8. The honey bee orthologs of genes encoding DAF-1, DAF-4,
and DAF-8 are TGF-beta receptor type 1 [LOC550930 ], activin
receptor type 2A [LOC412471 ], and mothers against
decapentaplegic homolog 3 [LOC412601 ], which were
differentially regulated between winter bees and foragers in fat bodies.
Similarly, the Dauer phenotype has been shown to promote neurogenesis of
mechanosensory neurons that were experimentally damaged in C.
elegans (Caneo, M. et al., 2019). Interestingly, studies in the
vertebrate African turquoise killifish (Nothobranchius furzeri )
(Chi-Kuo H. et al., 2020) and invertebrate cotton bollworm
(Helicoverpa armigera ) (Yu-Xuan L. et al., 2013), species which
both exhibit diapause phenotypes, reveal a role for
neurogenesis-associated Polycomb repressive complex members such as
histone-lysine N-methyltransferase E(z) [LOC552235 ] (which
was upregulated in winter bees and in foragers compared to nurses in the
flight muscles) in diapause maintenance. Thus, conserved signaling and
regulatory pathways might underly stress-induced long-lived
developmental phenotypes across taxa.
Overall, our results indicate that honey bees exhibit tissue-specific
transcriptional profiles associated with diapause as an adaptation to
different seasonal conditions. Differential regulation of genes
associated with dendrite morphogenesis and neurogenesis, including
members of the TGF-beta signaling pathway and the Polycomb repressive
complex suggest that conserved molecular pathways may underlie
stress-induced long-lived developmental phenotypes across taxa. These
studies lay the groundwork for future evaluations of the mechanisms,
evolution, and consequences of these interrelated phenomena.