Introduction
Diapause is a genetically programmed state of dormancy, which is
triggered by environmental cues in advance of adverse conditions, such
as winter in temperate areas or hot and dry conditions in tropical areas
(Denlinger, D. L. & Armbruster, P. A., 2014, Saulich, A. & Musolin,
D., 2017; Sim, C. & Denlinger, D. L., 2013). Many insect species
exhibit a diapause phenotype, which typically involves a reduction or
arrest in physical activity, development and/or reproduction. Different
insect species diapause in different developmental stages, from eggs to
adults. Several studies have evaluated the transcriptional and
physiological processes underpinning diapause initiation, maintenance,
and termination in different species (Amsalem, E. et al., 2015; Ragland,
G. J. & Keep, E., 2017; Ragland, G. et al., 2019; Santos, P. K. F. et
al., 2018; Treanore, E. D. et al., 2020; Yocum, G. & Rinehart, J.,
2015). Common physiological processes have been found to be associated
with the diapause phenotype, across developmental stages and insect
species. These include changes in regulation of energetic resources
(e.g., increased feeding, reduced metabolism, upregulation of fatty acid
synthesis), altered stress and immune responses, changes in circadian
rhythm, and altered hormonal profiles. Transcriptomic studies of
diapause phenotypes across species have demonstrated that there is no
common set of genes underpinning these processes: rather, species use
different genes, albeit in similar functional categories, to generate a
diapause phenotype (Amsalem, E. et al., 2015; Koštál, V. et al., 2017;
Ragland, G. J. & Keep, E., 2017; Treanore, E. D. et al., 2020).
However, the full range of variation in molecular processes underpinning
the diapause phenotype has not been evaluated, since studies largely
focus on whole-body or single tissue expression patterns: within the
same individual, different tissues may span the range from quiescent to
active.
Honey bees (Apis mellifera ) are a unique species in which to
study the molecular and physiological pathways underpinning diapause.
Honey bees live in perennial colonies, with a single reproductive female
queen and tens of thousands of facultatively sterile female workers
(Winston, M. L., 1987). Honey bees evolved in tropical regions, and
later expanded into temperate Europe (reviewed in Dogantzis, K. A. &
Zayed, A., 2019). In both temperate and tropical regions, bees
experience periods of season dearth during which they reduce or cease
brood rearing, requiring that the adult workers exhibit an increased
lifespan. This long-lived, diapause-like phenotype has been best studied
in winter conditions (reviewed in Döke, M. A. et al. 2015 and Grozinger,
C.M. et al., 2014). During the temperate winter, honey bees feed on
stored honey and pollen and form a thermoregulating cluster. Colonies
cease brood rearing in the winter, and winter worker bees exhibit a
unique physiological phenotype and significantly extended lifespan
compared to summer bees. In tropical regions, honey bees will cease
brood production during hot and dry conditions, and can leave their
hives to migrate long distances before establishing a new colony
(Grozinger, C. M., et al., 2014). Change in worker longevity has not
been studied in these migrating colonies. However, in the absence of
young bees to replace the older bees, the existing adult workers
presumably must live longer for the colony to survive. Indeed, our
recent studies of honey bees on the tropical island of Puerto Rico
demonstrated that worker bees’ lifespan changes throughout the year and
long-lived worker bee phenotype can be induced by removing the brood
from colonies at any time in the year (Feliciano-Cardona, S. et al.,
2020). Other studies have demonstrated that caging the queen and
preventing her from laying eggs can result in long-lived worker bees,
even in summer months in temperate climates (reviewed in Döke, M. A. et
al., 2015).
The physiology of winter worker bees (and long-lived summer bees)
suggest that they are in a diapause state. Winter worker bees will live
up to eight months, whereas summer worker bees typically live for only a
few (~6) weeks (Fluri et al. 1982). In the summer
months, worker bees will transition between different physiological and
behavioral states (reviewed in Robinson, G. E., 1992). When they are
young, honey bees act as nurses, feeding developing brood from
secretions produced by specialized glands in their heads. When they are
middle-aged (~2 weeks old), worker bees will transition
to other tasks in the colony, such as comb-building or guarding. In
their final behavioral phase, worker bees will transition to become
foragers, where they collect resources from the surrounding landscape.
Nurse bees are characterized by having large nutritional (fat) stores in
their abdominal fat bodies, and the loss of these stores is associated
with (and can accelerate) the transition to foraging (Toth, A. L. &
Robinson, G. E., 2005; Toth, A. L. et al., 2005). Compared to foragers,
nurses also have significantly higher hemolymph levels of vitellogenin
(Vg), a nutritional storage protein that is converted into brood food
(Amdam, G. V. et al., 2003). Vg also negatively regulates levels of a
key hormone, juvenile hormone (JH), and JH levels are significantly
lower in nurse bees than in forager bees (reviewed in Amdam, G. V. et
al., 2009). Decreasing levels of Vg or increasing levels of JH can
accelerate the transition to foraging behavior in worker bees. Like
summer nurse bees, long-lived winter honey bees exhibit high nutritional
stores, high levels of Vg, and low levels of JH (reviewed in Döke, M. A.
et al., 2015).
However, winter bees also exhibit similarities to summer foragers, in
terms of their use of their thoracic flight muscles. Winter bees
actively use their wing muscles to generate heat when exterior
temperatures drop below 10°C; this process is so effective that worker
bees can increase the internal temperatures of the hive to 34°C in late
winter/early spring to support brood rearing (Döke, M. A. et al., 2015;
Seeley, T. D. & Visscher, P. K., 1985; Currie, R. W. et al., 2015).
Forager bees are active fliers and can travel several kilometers from
the colony during foraging trips (Couvillon, M. J. et al., 2015). Thus,
it could be hypothesized that winter bees and foragers may have flight
muscles that have distinct transcriptional, physiological or biochemical
profiles than nurse bees. Indeed, studies have found differences in the
transcriptional profiles of nurse and forager bees is primarily affected
by age, and not by behavioral state or even foraging experience
(Margotta, J. W. et al., 2012; Schippers, M-P. et al., 2010). The
improved flight ability of foragers may be because they have lost
considerable mass during the transition to foraging, by reducing their
nutritional stores (Vance, J. T. et al., 2009). However, there are
differences in oxidative damage which are generated by flight muscle use
(Margotta, J. W. et al., 2018) which may be associated with differences
in expression patterns in flight muscles based on activity (Oskay, D.,
2007).
The genome-wide transcriptional profile associated with the winter bee
physiological state has not been characterized. One challenge with such
studies is that it is unclear which “physiological state” can serve as
a non-diapausing comparison, since summer worker bees have two distinct
physiological states which vary significantly in physiological processes
classically associated with diapause (nurses versus foragers). It could
be expected that winter bees and nurse bees would resemble each other,
as states with considerable nutritional resources and the ability to
enter a long-lived phenotype. However, winter bees and forager bees are
both capable of extended use of their flight muscles, while nurses
typically are not active fliers. Interestingly, k -means
clustering analysis of candidate gene expression profiles (n = 9 in fat
body tissues and n = 5 in flight muscle tissues) demonstrated that
winter bee fat body fat body tissue profiles clustered with nurses,
while winter bee flight muscle tissue profiles clustered with foragers
(Döke, M. A. et al., in preparation). Similarly, brain expression
patterns of long-lived summer bees collected from brood-less colonies
resembled nurse bees more than foragers (Münch, D. et al., 2013;
Whitfield, C. et al., 2003). Overall, these studies would suggest that
there are expression profiles in winter bees that are consistent with
physiological processes that differ between summer nurses and foragers,
some of which are classically associated with the diapause phenotype.
However, these results also suggest there is considerable variation
across tissues.
Here, we evaluated and compared the transcriptional profiles of winter
bees (collected in December), summer nurse and forager bees (collected
in July), in abdominal samples containing fat body tissue and thoracic
samples containing flight muscles. First, we determined the extent to
which these two tissues varied in expression across these different
phenotypes. Based on previous studies, we expected large differences in
fat body expression patterns and much fewer differences in flight muscle
tissue. Second, we determined if and how the expression patterns of
winter bees corresponded to those of summer bees, predicting that winter
bee fat body profiles will be more similar to summer nurse fat body
tissue profiles versus foragers, while winter bee flight muscle tissue
will be more similar to foragers. Third, we determined if the fat body
and flight muscle transcriptional profiles showed regulation of
functional categories of genes previously associated with diapause in
different insects species – e.g., metabolic processes, stress response,
circadian rhythm (Amsalem, E. et al., 2015; Denlinger, D. L., &
Armbruster, P. A., 2014; Koštál, Š, T. et al., 2017; Kunk, M. et al.,
2019; Ragland, G. J. & Keep, E., 2017; Santos, P, K. F. et al., 2018;
Treanore, E. D. et al., 2020; Yamada, H. & Yamamoto, M. T., 2011;
Yocum, G. D. et al., 2018). We predicted the fat body tissues will show
classic signatures of diapause, but the flight muscle tissue will not.