Long-lived Winter Research Articles

Characterisation of proteomic alterations in worker bees in response to amitraz treatment during summer to winter transition in Apis mellifera colonies

The application of treatments to reduce/control the number of Varroa destructor mites within Apis mellifera colonies is a common apicultural practice. The effectiveness of the treatments in reducing Varroa mites from colonies has been extensively studied, however, the effects of these treatments on the bees within the colonies are poorly characterised. This work utilised label-free quantitative proteomic analysis to investigate how the presence of the anti-Varroa treatment amitraz, affected worker bees. Samples of A. mellifera were isolated from colonies one week before amitraz treatment (T0) and 1, 3, 6 and 8 weeks post-treatment application. The trial was conducted during the transition period from short-lived summer workers to long-lived winter workers. The results highlight two large proteomic shifts; a decrease in the abundance of cuticular proteins and an increase in ribosomal protein abundance in Weeks 3, 6, and 8. The changes in the protein abundance in Week 8 may not be a response to amitraz exposure alone but may also be attributed to a potential change in the population of short-lived to long-lived winter bees. The work provides insight into the effect of amitraz on the honey bee proteome during the transition period from summer to winter colony and how it may contribute to inducing a stress response in individual bees.

1H NMR Profiling of Honey Bee Brains across Varying Ages and Seasons.

Honey bees (Apis mellifera) provide a useful model for studying aging because of the differences in longevity between the relatively short-lived summer and long-lived winter bees, as well as bees lacking signs of cognitive senescence as they age. Bee brains were dissected from newly emerged, 14-day-, and 28-day- old bees in mid- and late summer, as well as brood nest bees in fall, winter, and spring, before, during, and after overwintering, respectively. Brains were examined with nuclear magnetic resonance (NMR) spectroscopy to analyze their metabolome. Nine variable importance in projection (VIP) variables were identified, primarily amino acids and choline derivatives. Differences in metabolite concentrations were found with different ages of summer bees, mostly between newly emerged and 14-day- old bees, such as a decrease in phenylalanine and an increase in β-alanine, but there were also changes in older adults, such as o-phosphocholine that declined in 28-day- old bees. Differences in brood nest bees were observed, including a decline in tryptophan and an increase in β-alanine. These may provide distinct metabolomic signatures with age and season. Such research holds promise for a better understanding of the complex interplays between bee physiology, development, and aging, which has implications for improving bee health and management.

Evaluating the role of social context and environmental factors in mediating overwintering physiology in honey bees (Apis mellifera).

In temperate climates, honey bees show strong phenotypic plasticity associated with seasonal changes. In summer, worker bees typically only survive for about a month and can be further classified as young nurse bees (which feed the developing brood) and older forager bees. In winter, brood production and foraging halt and the worker bees live for several months. These differences in task and longevity are reflected in their physiology, with summer nurses and long-lived winter bees typically having large fat bodies, high expression levels of vitellogenin (a longevity-, nutrition- and immune-related gene), and large provisioning glands in their head. The environmental factors (both within the colony and within the surrounding environment) that trigger this transition to long-lived winter bees are poorly understood. One theory is that winter bees are an extended nurse bee state, brought on by a reduction in nursing duties in autumn (i.e. lower brood area). We examined that theory here by assessing nurse bee physiology in both the summer and autumn, in colonies with varying levels of brood. We found that season is a better predictor of nurse bee physiology than brood area. This suggests that seasonal factors beyond brood area, such as pollen availability and colony demography, may be necessary for inducing the winter bee phenotype. This finding furthers our understanding of winter bee biology, which could have important implications for colony management for winter, a critical period for colony survival.

Seasonal changes in ultrastructure and gene expression in the fat body of worker honey bees

The anatomical, physiological, and behavioral characteristics of honey bees are affected by the season as well as division of labor. In this study, we examined the structure, ultrastructure, and gene expression of fat body cells in both long-lived winter and short-lived summer worker bees (the youngest stage of hive bees and forager bees). In contrast to hive bees, foragers and winter bees have a higher metabolism due to intensive muscle activity during their flight (foragers) or endothermic heat production (winter bees). These workers differ from hive bees in the biology of their mitochondria, peroxisomes, and lysosomes as well as in the expression of the genes involved in lipid, carbohydrate, amino acid metabolism, insulin, and TGF- β signaling. Additionally, the expression of genes related to phospholipid metabolism was higher in the hive bees. However, we found no differences between workers in the expression of genes controlling cell organelles, such as the Golgi apparatus, endoplasmic reticulum, ribosomes, nucleus, and vacuoles, as well as genes for DNA replication, cell cycle control, and autophagy. Furthermore, lysosomes, autophagic processes and lipofuscin particles were more frequently observed in winter bees using electron microscopy.

Bee year: Basic physiological strategies to cope with seasonality

Worker honey bees are subject to biochemical and physiological changes throughout the year. This study aimed to provide the reasons behind these fluctuations. The markers analysed included lipid, carbohydrate, and protein levels in the haemolymph; the activity of digestive enzymes in the midgut; the levels of adipokinetic hormone (AKH) in the bee central nervous system; the levels of vitellogenins in the bee venom and haemolymph; and the levels of melittin in the venom. The levels of all the main nutrients in the haemolymph peaked mostly within the period of maximal bee activity, whereas the activity of digestive enzymes mostly showed a two-peak course. Furthermore, the levels of AKHs fluctuated throughout the year, with modest but significant variations. These data suggest that the role of AKHs in bee energy metabolism is somewhat limited, and that bees rely more on available food and less on body deposits. Interestingly, the non-metabolic characteristics also fluctuated over the year. The vitellogenin peak reached its maximum in the haemolymph in winter, which is probably associated with the immunoprotection of long-lived winter bees. The analysis of bee venom showed the maximal levels of vitellogenin in autumn; however, it is not entirely clear why this is the case. Finally, melittin levels showed strong fluctuations, suggesting that seasonal control was unlikely.

A multivariate perspective on lithic technological organization at Housepit 54, Bridge River Site (EeRl4), British Columbia

The management of lithic raw materials is a significant concern to anthropological archaeologists interested in hunter-gatherer socio-economies. A wide range of studies have implicated quarry distance, work needs, occupational longevity, and consumer population to variation in tactics for reduction, use, and maintenance of chipped stone tools. Fewer studies have examined socio-political factors in the organization of stone tool technology. This study examines the interactive effects of population, occupational longevity, favored prey, and socio-political complexity on the tactics by which lithic raw materials were managed in a long-lived winter pithouse in North America’s interior Pacific Northwest. Results of bivariate and multivariate analyses implicate variability in toolstone economies as related to different classes of lithic items. Lithic cores and flake tools were more extensively reduced in contexts of productive subsistence economies, high populations, long winter occupations, and material wealth-based inequality. Bifaces were produced and maintained in relation to needs associated with intensity of deer hunting. The creation and maintenance of slate tools was affected at least in part by the intensity of salmon fishing. Combined, these data suggest that the organization of lithic tool technology responded to multiple factors across the lifespan of the house.

Gut microbiota structure differs between honeybees in winter and summer

Adult honeybees harbor a specialized gut microbiota of relatively low complexity. While seasonal differences in community composition have been reported, previous studies have focused on compositional changes rather than differences in absolute bacterial loads. Moreover, little is known about the gut microbiota of winter bees, which live much longer than bees during the foraging season, and which are critical for colony survival. We quantified seven core members of the bee gut microbiota in a single colony over 2 years and characterized the community composition in 14 colonies during summer and winter. Our data show that total bacterial loads substantially differ between foragers, nurses, and winter bees. Long-lived winter bees had the highest bacterial loads and the lowest community α-diversity, with a characteristic shift toward high levels of Bartonella and Commensalibacter, and a reduction of opportunistic colonizers. Using gnotobiotic bee experiments, we show that diet is a major contributor to the observed differences in bacterial loads. Overall, our study reveals that the gut microbiota of winter bees is remarkably different from foragers and nurses. Considering the importance of winter bees for colony survival, future work should focus on the role of the gut microbiota in winter bee health and disease.

Neonicotinoids and ectoparasitic mites synergistically impact honeybees

The Western honeybee, Apis mellifera, is the most important managed pollinator globally and has recently experienced unsustainably high colony losses. Synergistic interactions among stressors are believed to be primarily responsible. However, despite clear evidence of strong effect on honeybee longevity of widely-employed neonicotinoid insecticides and of the ubiquitous ectoparasitic mite Varroa destructor, no data exist to show synergistic effects between these two stressors. Even though neonicotinoids had no significant impact by themselves, we here show for the first time a synergistic time-lag interaction between mites and neonicotinoids that resulted in significantly reduced survival of long-lived winter honeybees. Even though these mites are potent vectors of viruses, the virus-insecticide interaction had no significant impact. The data suggest a previously overlooked mechanism possibly explaining recent unsustainably high losses of managed A. mellifera honeybee colonies in many regions of the world. Future mitigation efforts should concentrate on developing sustainable agro-ecosystem management schemes that incorporate reduced use of neonicotinoids and sustainable solutions for V. destructor mites.

The novel pesticide flupyradifurone (Sivanto) affects honeybee motor abilities.

Honeybees and other pollinators are threatened by changing landscapes and pesticides resulting from intensified agriculture. In 2018 the European Union prohibited the outdoor use of three neonicotinoid insecticides due to concerns about pollinators. A new pesticide by the name of "Sivanto" was recently released by Bayer AG. Its active ingredient flupyradifurone binds to the nicotinic acetylcholine receptor (AchR) in the honeybee brain, similar to neonicotinoids. Nevertheless, flupyradifurone is assumed to be harmless for honeybees and can even be applied on flowering crops. So far, only little has been known about sublethal effects of flupyradifurone on honeybees. Intact motor functions are decisive for numerous behaviors including foraging and dancing. We therefore selected a motor assay to investigate in how far sublethal doses of this pesticide affect behavior in young summer and long-lived winter honeybees. Our results demonstrate that flupyradifurone (830 µmol/l) can evoke motor disabilities and disturb normal motor behavior after a single oral administration (1.2 µg/bee). These effects are stronger in long-lived winter bees than in young summer bees. After offering an equal amount of pesticide (1.0-1.75 µg) continuously over 24 h with food the observed effects are slighter. For comparisons we repeated our experiments with the neonicotinoid imidacloprid. Intriguingly, the alterations in behavior induced by this pesticide (4 ng/bee) were different and longer-lasting compared to flupyradifurone, even though both substances bind to nicotinic acetylcholine receptors.

Conventional and Organic Options for the Control of Woolly Distaff Thistle (Carthamus lanatus)

Woolly distaff thistle is a long-lived winter annual that threatens the ranching and dairy industries within the North Coast counties of California, particularly the organic producers. No peer-reviewed publications have documented effective control options or integrated management approaches for this species. We conducted two experiments, each replicated, in Marin County, California. The first compared several conventional herbicides at two timings and rates, while the second compared a conventional herbicide treatment with organic and integrated organic control methods, including an organic herbicide (mixture of capric and caprylic acids). Results of the conventional herbicide treatments showed most spring applications (March or April) of aminopyralid, aminocyclopyrachlor, clopyralid, and combinations of aminopyralid + triclopyr, or aminocyclopyrachlor + chlorsulfuron had greater than 99% control of woolly distaff thistle with fewer than 1.5 seedlings per 27-m2 plot by the end of the growing season. Higher rates were generally necessary to achieve the same level of control with winter (January) applications. In the organic herbicide treatments, the most consistent treatment was a combination of mowing followed by 9% (v/v) or the organic herbicide. This treatment was slightly less effective compared with aminopyralid but did have better than 95% control of woolly distaff thistle. The results of this study provide control options for both conventional and organic ranching practices where woolly distaff thistle is a problem.

Ancient Duplications Have Led to Functional Divergence of Vitellogenin-Like Genes Potentially Involved in Inflammation and Oxidative Stress in Honey Bees.

Protection against inflammation and oxidative stress is key in slowing down aging processes. The honey bee (Apis mellifera) shows flexible aging patterns linked to the social role of individual bees. One molecular factor associated with honey bee aging regulation is vitellogenin, a lipoglycophosphoprotein with anti-inflammatory and antioxidant properties. Recently, we identified three genes in Hymenopteran genomes arisen from ancient insect vitellogenin duplications, named vg-like-A, -B, and -C. The function of these vitellogenin homologs is unclear. We hypothesize that some of them might share gene- and protein-level similarities and a longevity-supporting role with vitellogenin. Here, we show how the structure and modifications of the vg-like genes and proteins have diverged from vitellogenin. Furthermore, all three vg-like genes show signs of positive selection, but the spatial location of the selected protein sites differ from those found in vitellogenin. We show that all these genes are expressed in both long-lived winter worker bees and in summer nurse bees with intermediate life expectancy, yet only vg-like-A shows elevated expression in winter bees as found in vitellogenin. Finally, we show that vg-like-A responds more strongly than vitellogenin to inflammatory and oxidative conditions in summer nurse bees, and that also vg-like-B responds to oxidative stress. We associate vg-like-A and, to lesser extent, vg-like-B to the antiaging roles of vitellogenin, but that vg-like-C probably is involved in some other function. Our analysis indicates that an ancient duplication event facilitated the adaptive and functional divergence of vitellogenin and its paralogs in the honey bee.

What Is the Main Driver of Ageing in Long-Lived Winter Honeybees: Antioxidant Enzymes, Innate Immunity, or Vitellogenin?

To date five different theories compete in explaining the biological mechanisms of senescence or ageing in invertebrates. Physiological, genetical, and environmental mechanisms form the image of ageing in individuals and groups. Social insects, especially the honeybee Apis mellifera, present exceptional model systems to study developmentally related ageing. The extremely high phenotypic plasticity for life expectancy resulting from the female caste system provides a most useful system to study open questions with respect to ageing. Here, we used long-lived winter worker honeybees and measured transcriptional changes of 14 antioxidative enzyme, immunity, and ageing-related (insulin/insulin-like growth factor signaling pathway) genes at two time points during hibernation. Additionally, worker bees were challenged with a bacterial infection to test ageing- and infection-associated immunity changes. Gene expression levels for each group of target genes revealed that ageing had a much higher impact than the bacterial challenge, notably for immunity-related genes. Antimicrobial peptide and antioxidative enzyme genes were significantly upregulated in aged worker honeybees independent of bacterial infections. The known ageing markers vitellogenin and IlP-1 were opposed regulated with decreasing vitellogenin levels during ageing. The increased antioxidative enzyme and antimicrobial peptide gene expression may contribute to a retardation of senescence in long-lived hibernating worker honeybees.

Somatic Maintenance Resources in the Honeybee Worker Fat Body Are Distributed to Withstand the Most Life-Threatening Challenges at Each Life Stage

In a global transcriptome analysis of three natural and three manipulated honeybee worker phenotypes at different ages, we have investigated the distribution of investment in somatic maintenance of the fat body. Gene expression is modulated so that the bees are able to resist the most life-threatening challenges at the actual life stage. Different modes of maintenance and repair are regulated, apparently to meet the environmental challenges most detrimental to survival and reproductive potential for the hive. We observed a broad down-regulation of genomic and cellular maintenance in the short-lived foragers and nurse bees compared to the long-lived winter bees. Our results show that survival and reproduction of the entire hive is given priority over the individual bees, hence supporting the idea of the honeybee society as a superorganism. Our results also fit the disposable soma theory of aging.

Aging and its modulation in a long-lived worker caste of the honey bee

Highly social animals provide alternative aging models in which vastly different lifespan patterns are flexible, and linked to social caste. Research in these species aims to reveal how environment, including social cues, can shape the transition between short-lived and extremely long-lived phenotypes with negligible senescence. Among honey bee workers, short to intermediate lifespans are typical for summer castes, while the winter caste can live up to 10 times longer. For summer castes, experimental interventions could predictably accelerate, slow or revert functional senescence. In contrast, little is known about the partic ular conditions under which periods of negligible senescence in winter castes can be disrupted or sustained. We asked how manipulation of social environment in colonies with long-lived winter bees might alter the pace of functional senescence, measured as learning performance, as well as of cellular senescence, measured as lipofuscin accumulation. We show that behavioral senescence becomes rapidly detectable when the winter state is disrupted, and changes in social task behaviors and social environment (brood) are induced. Likewise, we found that cellular senescence was induced by such social intervention. However, cellular senescence showed marked regional differences, suggesting that particular brain regions age slower than others. Finally, by preventing post-winter colonies from brood rearing, behavioral senescence became undetectable, even after transition to the usually short-lived phenotypes had occurred. We envision that social regulation of negligible functional senescence and highly dynamic accumulation of a universal symptom of cellular aging (lipofuscin) offers rewarding perspectives to target proximate mechanisms of slowed aging.

Winter Survival of Individual Honey Bees and Honey Bee Colonies Depends on Level of Varroa destructor Infestation

BackgroundRecent elevated winter loss of honey bee colonies is a major concern. The presence of the mite Varroa destructor in colonies places an important pressure on bee health. V. destructor shortens the lifespan of individual bees, while long lifespan during winter is a primary requirement to survive until the next spring. We investigated in two subsequent years the effects of different levels of V. destructor infestation during the transition from short-lived summer bees to long-lived winter bees on the lifespan of individual bees and the survival of bee colonies during winter. Colonies treated earlier in the season to reduce V. destructor infestation during the development of winter bees were expected to have longer bee lifespan and higher colony survival after winter.Methodology/Principal FindingsMite infestation was reduced using acaricide treatments during different months (July, August, September, or not treated). We found that the number of capped brood cells decreased drastically between August and November, while at the same time, the lifespan of the bees (marked cohorts) increased indicating the transition to winter bees. Low V. destructor infestation levels before and during the transition to winter bees resulted in an increase in lifespan of bees and higher colony survival compared to colonies that were not treated and that had higher infestation levels. A variety of stress-related factors could have contributed to the variation in longevity and winter survival that we found between years.Conclusions/SignificanceThis study contributes to theory about the multiple causes for the recent elevated colony losses in honey bees. Our study shows the correlation between long lifespan of winter bees and colony loss in spring. Moreover, we show that colonies treated earlier in the season had reduced V. destructor infestation during the development of winter bees resulting in longer bee lifespan and higher colony survival after winter.

Vanishing honey bees: Is the dying of adult worker bees a consequence of short telomeres and premature aging?

Einstein is often quoted to have said that without the bee, mankind would have but 4years to live. It is highly unlikely that he made this comment, which was even mentioned in a Lancet article on honey bees. However, the current vanishing of the bees can have serious consequences for human health, because 35% of the human diet is thought to benefit from pollination. Colony collapse disorder (CCD) in honey bees is characterized by the rapid decline of the adult bee population, leaving the brood and the queen poorly or completely unattended, with no dead bodies in or around the hive. A large study found no evidence that the presence or amount of any individual pesticide or infectious agent occurred more frequently or abundantly in CCD-affected colonies. The growing consensus is that honey bees are suffering from comprised immune systems, which allow various infectious pathogens to invade. The question remains, what causes immunosuppression in many colonies of Apis mellifera in North America and Europe? Telomeres are protective DNA structures located at eukaryotic chromosome tips that shorten in the somatic tissues of animals with age. Lifelong tissue regeneration takes place in Apis mellifera, and worker bees have been shown to senesce. In humans, a vast amount of literature has accumulated on exhausted telomere reserves causing impaired tissue regeneration and age-associated diseases, specifically cancer and immunosuppression. Therefore, we propose a new causative mechanism for the vanishing of the bees: critically short telomeres in long-lived winter bees. We term this the telomere premature aging syndrome.

Manipulating pollen supply in honey bee colonies during the fall does not affect the performance of winter bees

AbstractEach fall, honey bee (Apis mellifera L. (Hymenoptera: Apidae)) colonies in northern temperate regions rear a population of long-lived winter bees that maintains a broodless nest throughout the winter and resumes brood-rearing activities in the spring. Pollen supply in colonies is closely tied to this phenomenon; winter bees sequester large reservoirs of pollen-derived nutrients in their bodies and the brood-rearing capacity of colonies is dictated by the availability of pollen. We determined the effects of manipulating pollen supply during the fall on the number of winter bees present in colonies by spring, their mass and protein content before and after winter, and their capacity to rear brood during the spring. Colonies were either supplemented with or partially deprived of pollen during the fall, while a third group of colonies was not manipulated (control). We found that the performance of winter bees was not enhanced by supplementing colonies with pollen in the fall, nor did worker function suffer if pollen supply was restricted. Similar numbers of winter bees survived to spring in colonies and workers had similar physiology and brood-rearing efficiencies. These results suggest that beekeepers would not benefit by investing in fall pollen supplements to maximize colony growth in early spring.

The Regulatory Anatomy of Honeybee Lifespan

Honeybee workers (Apis mellifera) may be classified as either short-lived summer bees or long-lived winter bees in temperate zones. The protein status appears to be a major determinant of honeybee lifespan, and the lipoprotein vitellogenin seems to play a crucial role. Here, we give a review of the role of the vitellogenin in honeybee workers, and present a data-driven mathematical model describing the dynamics of this representative protein in the individual bee as a function of its task profile under various regimes. The results support the hypothesis that vitellogenin is a true storage protein that is utilized for various metabolic purposes including the synthesis of brood food. Except for workers having been foragers for many days, they also suggest that the previous life histories of workers do not constrain them from becoming winter bees as long as they get ample food and time to build up their protein reserves before wintering. The results also indicate that it may not be necessary to introduce the ovary as a storage organ for vitellogenin in order to generate normal winter bees. The insights gained from these results are then discussed in a broader gerontological and life history context. Remarkably similar features concerning regulation of ageing in Caenorhabditis elegans, Drosophila melanogaster and honeybees are pointed out and discussed. Furthermore, we show that in contrast to the “mutation accumulation” and the “antagonistic pleiotropy” evolutionary theories of ageing, the “disposable soma” theory is capable of explaining the bimodal longevity distribution of honeybees when interpreted in a group selection context. Finally, by showing that depletion of nutrient stores can be actively controlled by pathways connected to regulation of ageing, we strengthen the claim that age-based division of labour, with performance of risky tasks delayed until late in life by workers with depleted nutrient stores, may have evolved as an energy-saving mechanism in insect colonies.