Winter Bees 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.

Assessment of oxidative status, detoxification capacity and immune responsiveness in honey bees with ageing

The honey bee (Apis mellifera L.), as an eusocial insect species, is an important model organism in research focusing on ageing and longevity, due to prominent seasonal lifespan plasticity within the worker caste (summer and winter worker bees). In this study, we employed a screening approach to evaluate several molecular parameters, providing comprehensive insights into the antioxidative (superoxide dismutase and catalase activity, reduced glutathione and sulfhydryl group content, total antioxidative capacity), detoxifying (glutathione S-transferase and acetylcholinesterase activity), and immune (phenol oxidase and glucose oxidase activity) status, as well as vitellogenin content, in the summer and winter generation of honey bees, across ageing stages and in two body compartments: the whole abdomen and the head. Summer worker bees were collected weekly for six weeks, while winter bees were collected monthly for five months. The results of our study clearly indicate a reduced overall antioxidative capacity of older groups of worker bees from both generations, while the parameters of immune responsiveness mostly contributed to the separation between the two generations based on season rather than age categories. Detoxification ability appeared to be more susceptible to environmental factors. An age-dependent increase in vitellogenin content was recorded in the abdomen, but without seasonal differences. These findings provide an excellent starting point for further investigations into age-related changes, particularly within the context of honey bee sociality.

Acetylcholine and choline in honey bee (Apis mellifera) worker brood food are seasonal and age-dependent

Nursing honeybees produce brood food with millimolar concentrations of acetylcholine (ACh), which is synthesized through head gland secretions mixed with honey stomach contents. While we previously demonstrated the necessity of ACh for proper larval development, the dynamics of ACh levels throughout ontogenesis and their seasonal variations have remained unclear until now. Our HPLC analysis reveals dependencies of choline and ACh levels on larval development days (LDDs), influenced by seasonal (April–September) variations. Median ACh concentrations peak on LDD 2, declining significantly toward cell capping, while choline levels are lowest during the initial LDDs, rising markedly toward cell capping. Seasonal patterns show peak ACh levels from April to June and a low in August, paralleling choline's peak in July and low in August. This seasonality holds consistently across multiple years (2020–2022) and colonies, despite potential variations in colony performance and environmental conditions. Our analysis found no correlation between temperature, sunshine, precipitation, or favourable foraging days and ACh/choline levels, suggesting the involvement of additional factors. These findings underscore the seasonal fluctuation of ACh levels and its potential implications for the genetic programs governing winter bee development.

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.

Colony environment and absence of brood enhance tolerance to a neonicotinoid in winter honey bee workers, Apis mellifera

In eusocial insects, worker longevity is essential to ensure colony survival in brood-free periods. Trade-offs between longevity and other traits may render long-living workers in brood-free periods more susceptible to pesticides compared to short-lived ones. Further, colony environment (e.g., adequate nutrition) may enable workers to better cope with pesticides, yet data comparing long vs. short-living workers and the role of the colony environment for pesticide tolerance are scarce. Here, we show that long-living honey bee workers, Apis mellifera, are less susceptible to the neonicotinoid thiamethoxam than short-lived workers, and that susceptibility was further reduced when workers were acclimatized under colony compared to laboratory conditions. Following an OECD protocol, freshly-emerged workers were exposed to thiamethoxam in summer and winter and either acclimatized within their colony or in the laboratory. Mortality and sucrose consumption were measured daily and revealed that winter workers were significantly less susceptible than summer workers, despite being exposed to higher thiamethoxam dosages due to increased food consumption. Disparencies in fat body activity, which is key for detoxification, may explain why winter bees were less susceptible. Furthermore, colony acclimatization significantly reduced susceptibility towards thiamethoxam in winter workers likely due to enhanced protein nutrition. Brood absence and colony environment seem to govern workers’ ability to cope with pesticides, which should be considered in risk assessments. Since honey bee colony losses occur mostly over winter, long-term studies assessing the effects of pesticide exposure on winter bees are required to better understand the underlying mechanisms.

The Nutritional Year-Cycle of Italian Honey Bees (Apis mellifera ligustica) in a Southern Temperate Climate

Nutrition is a key aspect influencing honey bee health and overwintering. Since honey bee seasonality in southern temperate climates represents a significant research gap, this study conducted long-term monitoring of honey bees in the Mediterranean (Sassari, Italy). Specifically, individual weight, fat body, and size measurements (head, thorax, abdomen, and total body) were recorded monthly so to detect changes in the nutrient storage of worker bees during an annual cycle. Data were analysed according to sampling date, climate (temperature, precipitation, and daylength), and flower diversity and were conducted for nurse and forager bees separately. The nutritional honey bee year-cycle generally followed the nectar flow and showed two critical timepoints: summer and winter dearth. A short cessation of activities in late fall/early winter coupled with an increase in nutrient storage indicated the presence of winter bees. Precipitation was found to play an important role in honey bee nutrition in the study area through its impacts on colony demography and plants in particular illustrating how climate change could pose a threat to European honey bee populations in the future. These results provide valuable insights into the nutritional dynamics of Apis mellifera ligustica in the Mediterranean that could support management decisions to improve overwintering success and prevent unnecessary colony losses.

Indoor tent management for extending honey bee research season: benefits and caveats.

Honey bees are important organisms for research in many fields, including physiology, behavior, and ecology. Honey bee colonies are relatively easy and affordable to procure, manage, and replace. However, some difficulties still exist in honey bee research, specifically that honey bee colonies have a distinct seasonality, especially in temperate regions. Honey bee colonies transition from a large society in which workers have a strict temporal division of labor in the summer, to a group of behaviorally flexible workers who manage the colony over winter. Furthermore, opening colonies or collecting bees when they are outside has the potential to harm the colony because of the disruption in thermoregulation. Here, we present a simple and affordable indoor management method utilizing a mylar tent and controlled environmental conditions that allows bees to freely fly without access to outdoor space. This technique permits research labs to successfully keep several colonies persistently active during winter at higher latitudes. Having an extended research period is particularly important for training students, allowing preliminary experiments to be performed, and developing methods. However, we find distinct behavioral differences in honey bees managed in this situation. Specifically learning and thermoregulatory behaviors were diminished in the bees managed in the tent. Therefore, we recommend caution in utilizing these winter bees for full experiments until more is known. Overall, this method expands the research potential on honey bees, and calls attention to the additional research that is needed to understand how indoor management might affect honey bees.

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.

Exposure of honey bees to mixtures of microbial biopesticides and their effects on bee survival under laboratory conditions.

Biopesticides, having as active ingredients viruses, bacteria, or fungi, are developed to substitute or reduce the use of chemical plant protection products in different agrosystems. Though the application of mixtures containing several products is a common practice, interactions between microbial biopesticides and related effects on bees as non-target organisms have not been studied yet. In the current study, we exposed winter bees to five different microbial-based products and their combinations at the maximum recommended application rate to assess their responses. Laboratory oral exposure tests (acute/chronic) to single or binary products were conducted. Survival and food consumption of the tested bees were evaluated over the experimental duration. Our results show that some product combinations have potential additive or synergistic effects on bees, whereas others did not affect the bee's survival compared to the control. Exposure of tested bees to the most critical combination of products containing Bacillus thuringiensis aizawai ABTS-1857 and B. amyloliquefaciens QST 713 strongly resulted in a median lifespan of 4.5days compared to 8.0 and 8.5days after exposure to the solo products, respectively. The exposure to inactivated microorganisms by autoclaving them did not differ from their respective uncontaminated negative controls, indicating effects on bee mortality might originate in the treatment with the different microorganisms or their metabolites. Further investigations should be conducted under field conditions to prove the magnitude of observed effects on bee colonies and other bee species.

Modeling seasonal immune dynamics of honey bee (Apis mellifera L.) response to injection of heat-killed Serratia marcescens.

The honey bee, Apis mellifera L., is one of the main pollinators worldwide. In a temperate climate, seasonality affects the life span, behavior, physiology, and immunity of honey bees. In consequence, it impacts their interaction with pathogens and parasites. In this study, we used Bayesian statistics and modeling to examine the immune response dynamics of summer and winter honey bee workers after injection with the heat-killed bacteria Serratia marcescens, an opportunistic honey bee pathogen. We investigated the humoral and cellular immune response at the transcriptional and functional levels using qPCR of selected immune genes, antimicrobial activity assay, and flow cytometric analysis of hemocyte concentration. Our data demonstrate increased antimicrobial activity at transcriptional and functional levels in summer and winter workers after injection, with a stronger immune response in winter bees. On the other hand, an increase in hemocyte concentration was observed only in the summer bee population. Our results indicate that the summer population mounts a cellular response when challenged with heat-killed S. marcescens, while winter honey bees predominantly rely on humoral immune reactions. We created a model describing the honey bee immune response dynamics to bacteria-derived components by applying Bayesian statistics to our data. This model can be employed in further research and facilitate the investigating of the honey bee immune system and its response to pathogens.

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.

The Relation between MEGA-STN and Positron Wave Thermal Energy

Paul Dirac(1902-1984) theoretically predicted the existence of positrons for the first time in 1928, and Carl Anderson(1905-1991) found the positrons through cosmic ray observation experiments in 1932. Positrons are the antiparticle of electron, and their spin and mass are equal to electrons and they have the same electric charge but are different in sign. That is, their spin is ½ Fermion, their electric charge is base charge +e, and their mass is approximately 9.11×10−31 kg. Because positrons are the lightest particle with a positive charge, they have an infinite lifespan without collapsing into other particles. But if positron meets with another electron surrounding them, double extinction of positron and electron occurs, and positrons disappear with electrons, and then photons are generated. Because the earth has many electrons, double extinction occurs immediately in most cases even if positrons are created. There are gravity, electromagnetic force, weak interaction in the interaction of positrons, and the isotopes emitting positrons include carbon-11, nitrogen-13, oxygen-15, fluorine-18. For example, carbon11 turns into boron-11. These isotopes are used on positron emission tomography device. Electron capture is another way of decay that can occur competitively with positron emission, but the larger the difference of energy is, the higher the probability of decaying due to positron emission is. Researchers say that positrons are generated if there is collision between high energies by the interaction of positrons, but we think that this should be supplemented more. There is a hypothesis that positrons are generated when the unstable radioactive isotopes created during a supernova explosion collapse. It is inevitable that it will cost a lot of time and money when generating radioactive isotopes according to this hypothesis. This makes us try new methods about positrons emission breaking away from conventional fusion methods. Our new methods for generating positrons are to make artificially the fusion with micro/nanoparticles and isotopes emitting positrons and the interchangeability between them, and to create a great deal of heat energy by making micro/nanoparticles collide into each other with using thermal energy and waves between micro/ nanoparticles. We think that our new methods are the best way to generate high heat with a small energy source. To put our ideas into practice, we made a stainless steel rectangular plate using the combined materials of MEGA-STN, that is the new types fused with each of micro/nanoparticles and isotopes emitting positrons, and added MEGA-STN to materials of the existing stainless steel heater stick. We measured temperature changes depending on whether or not the materials are present and depending on the content of the materials, and checked even positrons emission in our study. Consequently, we found increasing to 200~300℃ compared to the general temperature when a constant temperature is created with minimal energy, making a marked change in temperature of our MEGA-STN and emitting positrons at a particular temperature. It is believed to have paved the way for the use of energy sources throughout the industry. Additionally, we realized that photon production caused by the double annihilation of positrons and electrons can affect plants (growth and yields) and insects (the activity of bees in winter) ecologically, cause changes in the environment such as the quality of air and water, and have a positive impact in all areas of our life.

Omics-based analysis of honey bee (Apis mellifera) response to Varroa sp. parasitisation and associated factors reveals changes impairing winter bee generation

The extensive annual loss of honey bees (Apis mellifera L.) represents a global problem affecting agriculture and biodiversity. The parasitic mite Varroa destructor, associated with viral co-infections, plays a key role in this loss. Despite years of intensive research, the complex mechanisms of Varroa – honey bee interaction are still not fully defined. Therefore, this study employed a unique combination of transcriptomic, proteomic, metabolomic, and functional analyses to reveal new details about the effect of Varroa mites and naturally associated factors, including viruses, on honey bees. We focused on the differences between Varroa parasitised and unparasitised ten-day-old worker bees collected before overwintering from the same set of colonies reared without anti-mite treatment. Supplementary comparison to honey bees collected from colonies with standard anti-Varroa treatment can provide further insights into the effect of a pyrethroid flumethrin. Analysis of the honey bees exposed to mite parasitisation revealed alterations in the transcriptome and proteome related to immunity, oxidative stress, olfactory recognition, metabolism of sphingolipids, and RNA regulatory mechanisms. The immune response and sphingolipid metabolism were strongly activated, whereas olfactory recognition and oxidative stress pathways were inhibited in Varroa parasitised honey bees compared to unparasitised ones. Moreover, metabolomic analysis confirmed the depletion of nutrients and energy stores, resulting in a generally disrupted metabolism in the parasitised workers. The combined omics-based analysis conducted on strictly parasitised bees revealed the key molecular components and mechanisms underlying the detrimental effects of Varroa sp. and its associated pathogens. This study provides the theoretical basis and interlinked datasets for further research on honey bee response to biological threats and the development of efficient control strategies against Varroa mites.

국내 꿀벌응애에서의 플루발리네이트 저항성 발달 현황과 해결 방안 제안

The beekeeping industry in Korea has been severely challenged by winter losses of honey bee colonies recently. Various factors such as climate changes, pesticides, pathogens, nutritional imbalance, parasitic mites and other environmental factors are argued as the causes of the large-scale losses of winter bees. However, it is more rational to suggest that a combination of these multiple stressors rather than a single factor is responsible for this damage. Among these factors, the ectoparasitic mites, particularly Varroa destructor, has been widely reported to significantly cause winter losses of honey bee colonies in various countries, and beekeepers in Korea complain that it is difficult to control mites with conventional methods. Considering that fluvalinate has been the most abundantly used synthetic acaricide in Korea, and intensive and repetitive use of fluvalinate has been suggested to be result in the development of resistant V. destructor populations in previous studies, we first focused on discussing the mode of action of fluvalinate and the mechanism of pyrethroid resistance. Current status of the fluvalinate resistance V. destructor in Korea and selection of alternative acaricides are further discussed in this review.

Overwintering in North American domesticated honeybees (Apis mellifera) causes mitochondrial reprogramming while enhancing cellular immunity.

Many factors negatively affect domesticated honeybee (Apis mellifera) health, causing a global decrease in their population year after year with major losses occurring during winter, and the cause remains unknown. Here, we monitored for 12 months North American colonies of honeybees enduring important temperature variations throughout the year, to assess the metabolism and immune system of summer and winter honeybee individuals. Our results show that in flight muscle, mitochondrial respiration via complex I during winter is drastically reduced compared with summer. However, the capacity for succinate and glycerol-3-phosphate (G3P) oxidation by mitochondria is increased during winter, resulting in higher mitochondrial oxygen consumption when complex I substrates, succinate and G3P were assessed altogether. Pyruvate kinase, lactate dehydrogenase, aspartate aminotransferase, citrate synthase and malate dehydrogenase tend to have reduced activity levels in winter, unlike hexokinase, NADH dehydrogenase and pyruvate dehydrogenase. Transcript abundance of highly important immunity proteins such as Vitellogenin and Defensin-1 were also increased in winter bees, and a stronger phagocytic response as well as a better hemocyte viability was observed during winter. Thus, a reorganization of substrate utilization favoring succinate and G3P while negatively affecting complex I of the ETS is occurring during winter. We suggest that this might be due to complex I transitioning to a dormant conformation through post-translational modification. Winter bees also have an increased response for antibacterial elimination. Overall, this study highlights previously unknown cellular mechanisms between summer and winter honeybees that further our knowledge about this important species.

Conflicting selection on workers underlies global honey bee colony losses

There is clear evidence for global and unsustainable high losses of the managed Western honey bee, Apis mellifera, colonies raising major concerns about the sustainability of pollination services and food security. Here, we address a conflicting selection scenario in workers to better understand life-history trade-offs underlying colony losses. Due to developmental plasticity, annual fluctuations in selection pressures result in long-living winter bees and short-living summer bees as adaptive responses to lack of food and ceased brood rearing in winter, or ample food and rapid brood turnover in spring and summer. Since trait evolution is governed by a balance of conflicting fitness advantages, trade-offs between longevity and other traits are inevitable (e.g. immune defense or detoxification). As worker longevity is essential to ensure colony functionality, these trade-offs render long-living workers more susceptible to the same stressors compared to short-lived ones. It seems as if trade-offs in workers play a previously overlooked key role, and the incorporation of long-living workers in research efforts is long overdue if our aim is to effectively mitigate declines and losses of social insect colonies globally. Indeed, managed honey bee colony losses in temperate regions predominantly occur throughout winter, but research efforts are almost exclusively conducted with short-lived summer bees, thereby sustaining the danger of false negative results. Thus, we should not shy away from meeting the respective logistic challenges. Studies of conflicting selection on workers will be informative about mechanisms underlying life history trade-offs, thereby fostering conservation efforts in the social insects.

Features of microsporidia Nosema apis and Nosema ceranae (Nosema species) development of winter bee (Apis mellifera L.) generation

Temperature is one of the main abiotic factors affecting the development of causative agents of nosemosis in the bee's body.
 The aim of the research. To determine the influence of the winter and summer bee nest temperature (20–22 °C and 35–36 °C, respectively) on the duration of Nosema species development of winter bee generation isolated in hoarding cages, and to trace the life cycle of Nosema species of bees with natural infection and overwintering in natural conditions, from November to the beginning of bee brood rearing (February).
 Materials and methods. For this, 200 bees Apis mellifera sossimai, selected from bee colony in November, were infected by syrup feeding containing Nosema species (5×104 spores per bee). Half of the bees were kept at 35–36 °C and half at 20–22 °C. The stages of Nosema species development were monitored daily for 13 days in midgut smears, stained according to Romanovsky-Giemsa (magnification 900x). Furthermore, with a 15 days frequency, from November to mid-February, 30 bees were selected from 20 bee colonies overwintered in natural conditions, and monitored the stages of Nosema species development at natural infection.
 Result. It was found that the Nosema species development of winter bee generation artificially infected by Nosema species, was suspended at meronts and sporonts stages until the 13th day from the moment of infection, regardless of the temperature at which the bees were kept in the experiment. In bees selected from bee colonies naturally infected with Nosema species prevailed meronts I, II and in an insignificant amount sporonts, until the end of December, active sporulation took place from the middle of January to the beginning of February.
 Conclusion. That is, the duration of the life cycle development of Nosema species depends little on temperature but is closely related to the life span of summer and winter bee generation and determined by the biochemistry of their relationships, which allow the parasite to save the host as its habitat

Proteomic analysis of summer and winter Apis mellifera workers shows reduced protein abundance in winter samples

Apis mellifera workers display two stages; short lived summer bees that engage in nursing, hive maintenance and foraging, and long lived winter bees (diutinus bees) which remain within the hive and are essential for thermoregulation and rearing the next generation of bees in spring before dying. Label free quantitative proteomic analysis was conducted on A. mellifera workers sampled in June and December to compare the proteomes of summer and winter bees. Proteomic analysis was performed on head, abdominal and venom sac samples and revealed an elevated level of protein abundance in summer bees. Head and abdominal samples displayed an increased abundance in cuticular proteins in summer samples whereas an increase in xenobiotic proteins was observed in winter samples. Several carbohydrate metabolism pathways which have been linked to energy production and longevity in insects were increased in abundance in winter samples in comparison to summer samples. Proteomic analysis of the venom sacs of summer samples showed an increased abundance of bee venom associated proteins in comparison to winter workers. These data provides an insight into the adaptions of A. mellifera workers in summer and winter and may aid in future treatment and disease studies on honeybee colonies. Data are available via ProteomeXchange with identifier PXD030483.

The dose makes the poison: feeding of antibiotic-treated winter honey bees, Apis mellifera, with probiotics and b-vitamins

Honey stores of Apis mellifera colonies are replaced with sugar water by beekeepers, which may result in malnutrition. Nutritional supplements have been developed, but the importance of bacterial probiotics and vitamins is poorly understood. Given that supplementary feeding with vitamins and probiotics may enhance worker weight and longevity, this might suggest a feasible approach to mitigate winter colony losses. Here, we conducted a laboratory hoarding cage study with freshly emerged winter bees, which were treated with the antibiotic tetracycline to reduce gut bacteria obtained post-emergence and subsequently assigned to feeding regimes: sucrose only, sucrose + pollen, probiotics (low and high dosage), probiotics + pollen (low and high dosage), or b-vitamins (low and high dosage) (N = 8 treatments, 29 workers/cage × 8 replicates). In parallel, another age cohort of bees remained on their frame (= Frame) to establish their gut microbiota and were subsequently fed with sucrose only or sucrose + pollen (N = 2 treatments, 29 workers/cage × 4 replicates). The most beneficial effects on body weights were found in workers given ad libitum access to pollen, notably in the Frame Sucrose + Pollen group, confirming the inherent importance of post-emergent gut flora inoculation and the role of gut bacteria in protein digestion. Furthermore, both Frame groups and the antibiotic-treated workers fed with probiotic low + pollen survived longer than all other groups, highlighting a fundamental host-microbial relationship. On the other hand, our current treatments alone, post-tetracycline, did not yield any positive results. In contrast, high dosages of both probiotic and b-vitamins significantly reduced lifespan compared to their low concentration counterparts, probably due to dysbiosis and toxicity, suggesting that the outcome was dose-dependent. These results highlight that bacterial and b-vitamin supplementation can alter longevity with advisable caution since harmful concentrations appear to exist.

Evaluation of the risks for animal health related to the presence of hydroxymethylfurfural (HMF) in feed for honey bees.

The European Commission has asked the EFSA to evaluate the risk for animal health related to the presence of hydroxymethylfurfural (HMF) in honey bee feed. HMF is a degradation product of particular sugars and can be present in bee feed. HMF is of low acute toxicity in bees but causes increased mortality upon chronic exposure. A benchmark dose lower limit 10% (BMDL10) of 1.16 μg HMF per bee per day has been calculated from mortalities observed in a 20‐day study and established as a Reference Point covering also mortality in larvae, drones and queens for which no or insufficient toxicity data were available. Winter bees have a much longer lifespan than summer bees and HMF shows clear time reinforced toxicity (TRT) characteristics. Therefore, additional Reference Point intervals of 0.21–3.1, 0.091–1.1 and 0.019–0.35 µg HMF/bee per day were calculated based on extrapolation to exposure durations of 50, 90 and 180 days, respectively. A total of 219 analytical data of HMF concentrations in bee feed from EU Member States and 88 from Industry were available. Exposure estimates of worker bees and larvae ranged between 0.1 and 0.48, and between 0.1 and 0.51 μg HMF/per day, respectively. They were well below the BMDL10 of 1.16 μg HMF/bee per day, and thus, no concern was identified. However, when accounting for TRT, the probability that exposures were below established reference point intervals was assessed to be extremely unlikely to almost certain depending on exposure duration. A concern for bee health was identified when bees are exposed to HMF contaminated bee feed for several months.