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.