Winter Bees Research Articles

Impacts of chronic sublethal exposure to clothianidin on winter honeybees.

A wide application of systemic pesticides and detection of their residues in bee-collected pollen and nectar at sublethal concentrations led to the emergence of concerns about bees' chronic exposure and possible sublethal effects on insect pollinators. Therefore, special attention was given to reducing unintentional intoxications under field conditions. The sensitivity of winter bees throughout their long lifespan to residual exposure of pesticides is not well known, since most previous studies only looked at the effects on summer bees. Here, we performed various laboratory bioassays to assess the effects of clothianidin on the survival and behavior of winter bees. Oral lethal and sublethal doses were administered throughout 12-day. The obtained LD50 values at 48, 72, 96h and 10days were 26.9, 18.0, 15.1 and 9.5ng/bee, respectively. Concentrations <20µg/kg were found to be sublethal. Oral exposure to sublethal doses was carried out for 12-day and, the behavioral functions were tested on the respective 13th day. Although slight reductions in the responses at the concentrations 10 and 15µg/kg were observed, all tested sublethal concentrations had showed non-significant effects on the sucrose responsiveness, habitation of the proboscis extension reflex and olfactory learning performance. Nevertheless, chronic exposure to 15µg/kg affected the specificity of the early long-term memory (24h). Since the tested concentrations were in the range of field-relevant concentrations, our results strongly suggest that related-effects on winter and summer bees' sensitivity should also be studied under realistic conditions.

Prolonged effects of in-hive monoterpenoids on the honey bee Apis mellifera.

Honey bees are exposed in their environment to contaminants but also to biological stressors such as Varroa destructor that can weaken the colony. Preparations containing monoterpenoids that are essential oil components, can be introduced into hives to control Varroa. The long-term sublethal effects of monoterpenoids used as miticides have been poorly investigated. Analysis of behavior of free-moving bees in the laboratory is useful to evaluate the impact of chemical stressors on their cognitive functions such as vision function. Here, the walking behavior was quantified under a 200-lux light intensity. Weeks and months after introduction of the miticide (74% thymol) into the hives, decreases of phototaxis was observed with both summer and winter bees. Curiously, in spring, bees collected in treated hives were less attracted by light in the morning than control bees. The survival of bees collected in spring was increased by treatment. After a 1-year period of observation, the colony losses were identical in treated and non-treated groups. Colony loss started earlier in the non-treated group. In public opinion, natural substances as essential oils are safer and more environmentally friendly. We demonstrated that a monoterpenoid-based treatment affects bee responses to light. The latter results have notable implications regarding the evaluation of miticides in beekeeping.

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.

Supporting local diversity of habitats and species on farmland: a comparison of three wildlife-friendly schemes.

Summary Restoration and maintenance of habitat diversity have been suggested as conservation priorities in farmed landscapes, but how this should be achieved and at what scale are unclear. This study makes a novel comparison of the effectiveness of three wildlife‐friendly farming schemes for supporting local habitat diversity and species richness on 12 farms in England.The schemes were: (i) Conservation Grade (Conservation Grade: a prescriptive, non‐organic, biodiversity‐focused scheme), (ii) organic agriculture and (iii) a baseline of Entry Level Stewardship (Entry Level Stewardship: a flexible widespread government scheme). Conservation Grade farms supported a quarter higher habitat diversity at the 100‐m radius scale compared to Entry Level Stewardship farms. Conservation Grade and organic farms both supported a fifth higher habitat diversity at the 250‐m radius scale compared to Entry Level Stewardship farms. Habitat diversity at the 100‐m and 250‐m scales significantly predicted species richness of butterflies and plants. Habitat diversity at the 100‐m scale also significantly predicted species richness of birds in winter and solitary bees. There were no significant relationships between habitat diversity and species richness for bumblebees or birds in summer.Butterfly species richness was significantly higher on organic farms (50% higher) and marginally higher on Conservation Grade farms (20% higher), compared with farms in Entry Level Stewardship. Organic farms supported significantly more plant species than Entry Level Stewardship farms (70% higher) but Conservation Grade farms did not (10% higher). There were no significant differences between the three schemes for species richness of bumblebees, solitary bees or birds. Policy implications. The wildlife‐friendly farming schemes which included compulsory changes in management, Conservation Grade and organic, were more effective at increasing local habitat diversity and species richness compared with the less prescriptive Entry Level Stewardship scheme. We recommend that wildlife‐friendly farming schemes should aim to enhance and maintain high local habitat diversity, through mechanisms such as option packages, where farmers are required to deliver a combination of several habitats.

Honeybee Venom Proteome Profile of Queens and Winter Bees as Determined by a Mass Spectrometric Approach

Venoms of invertebrates contain an enormous diversity of proteins, peptides, and other classes of substances. Insect venoms are characterized by a large interspecific variation resulting in extended lists of venom compounds. The venom composition of several hymenopterans also shows different intraspecific variation. For instance, venom from different honeybee castes, more specifically queens and workers, shows quantitative and qualitative variation, while the environment, like seasonal changes, also proves to be an important factor. The present study aimed at an in-depth analysis of the intraspecific variation in the honeybee venom proteome. In summer workers, the recent list of venom proteins resulted from merging combinatorial peptide ligand library sample pretreatment and targeted tandem mass spectrometry realized with a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS/MS). Now, the same technique was used to determine the venom proteome of queens and winter bees, enabling us to compare it with that of summer bees. In total, 34 putative venom toxins were found, of which two were never described in honeybee venoms before. Venom from winter workers did not contain toxins that were not present in queens or summer workers, while winter worker venom lacked the allergen Api m 12, also known as vitellogenin. Venom from queen bees, on the other hand, was lacking six of the 34 venom toxins compared to worker bees, while it contained two new venom toxins, in particularly serine proteinase stubble and antithrombin-III. Although people are hardly stung by honeybees during winter or by queen bees, these newly identified toxins should be taken into account in the characterization of a putative allergic response against Apis mellifera stings.

Overwintering Is Associated with Reduced Expression of Immune Genes and Higher Susceptibility to Virus Infection in Honey Bees.

The eusocial honey bee, Apis mellifera, has evolved remarkable abilities to survive extreme seasonal differences in temperature and availability of resources by dividing the worker caste into two groups that differ in physiology and lifespan: summer and winter bees. Most of the recent major losses of managed honey bee colonies occur during the winter, suggesting that winter bees may have compromised immune function and higher susceptibility to diseases. We tested this hypothesis by comparing the expression of eight immune genes and naturally occurring infection levels of deformed wing virus (DWV), one of the most widespread viruses in A. mellifera populations, between summer and winter bees. Possible interactions between immune response and physiological activity were tested by measuring the expression of vitellogenin and methyl farnesoate epoxidase, a gene coding for the last enzyme involved in juvenile hormone biosynthesis. Our data show that high DWV loads in winter bees correlate with reduced expression of genes involved in the cellular immune response and physiological activity and high expression of humoral immune genes involved in antibacterial defense compared with summer bees. This expression pattern could reflect evolutionary adaptations to resist bacterial pathogens and economize energy during the winter under a pathogen landscape with reduced risk of pathogenic viral infections. The outbreak of Varroa destructor infestation could have overcome these adaptations by promoting the transmission of viruses. Our results suggest that reduced cellular immune function during the winter may have increased honey bee’s susceptibility to DWV. These results contribute to our understanding of honey bee colony losses in temperate regions.

Dynamics of Apis mellifera Filamentous Virus (AmFV) Infections in Honey Bees and Relationships with Other Parasites.

Apis mellifera filamentous virus (AmFV) is a large double stranded DNA virus of honey bees, but its relationship with other parasites and prevalence are poorly known. We analyzed individual honey bees from three colonies at different times post emergence in order to monitor the dynamics of the AmFV gut colonization under natural conditions. Prevalence and loads of microsporidia and trypanosomes were also recorded, as well as five common honey bee RNA viruses. The results show that a high proportion of bees get infected with AmFV during the first week post-emergence (75%) and that AmFV DNA levels remained constant. A similar pattern was observed for microsporidia while trypanosomes seem to require more time to colonize the gut. No significant associations between these three infections were found, but significant positive correlations were observed between AmFV and RNA viruses. In parallel, the prevalence of AmFV in France and Sweden was assessed from pooled honey bee workers. The data indicate that AmFV is almost ubiquitous, and does not seem to follow seasonal patterns, although higher viral loads were significantly detected in spring. A high prevalence of AmFV was also found in winter bees, without obvious impact on overwintering of the colonies.

The fraction haemolymph vitellogenin of a honey bee colony, derived from a pooled haemolymph sample, a colony vitality parameter

The number of bees, amount of brood and haemolymph vitellogenin titre are parameters to establish the vitality of a honey bee colony. Increasing numbers of bees during summer until autumn; increasing amounts of brood in spring towards summer followed by a decrease; and low haemolymph vitellogenin titres in foraging bees and high vitellogenin titres in nurse bees and winter bees are typical courses over the bee season. In contrast to the number of bees and amount of brood, determined at the colony level, vitellogenin is determined in individual bees. To turn the factor vitellogenin from the mean of individual data into a colony parameter, a protocol was developed to determine the colony fraction vitellogenin in a pooled haemolymph sample of 30 bees, sampled from a defined location in the colony. This colony fraction vitellogenin is a parameter for honey bee colony vitality.

Performance of Bee Colonies Headed by Queens Instrumentally Inseminated with Semen of Drones Who Come from a Single Colony or Many Colonies

Abstract The aim of the study was to determine the effect of honey bee worker diversity within the colony on: development, honey productivity, and wintering. Two different levels of diversity within the colony were tested. The appropriate levels of diversity within the colony were obtained by selecting drones for inseminating the queens. Lower genetic diversity was obtained in the colonies headed by a queen inseminated with semen collected from drones originating from a single colony. Higher genetic diversity was obtained in the colonies with queens inseminated with semen from drones of 30 different colonies. Colonies with a higher genetic variation of workers in the colonies had greater levels of functional characteristics. However, apart from the number of dead bees in winter, the genetic diversity level of the workers on the colony development and honey production, did not have a significant influence. There was an averaging effect observed concerning that male component in the colonies with a higher genetic variation of workers - on honey yield, when compared to the non-additive effect of the best drones.

Winter Bees &amp; Other Poems of the Cold by Joyce Sidman (review)

Winter Bees &amp; Other Poems of the Cold by Joyce Sidman (review)

Delayed and time-cumulative toxicity of imidacloprid in bees, ants and termites.

Imidacloprid, one of the most commonly used insecticides, is highly toxic to bees and other beneficial insects. The regulatory challenge to determine safe levels of residual pesticides can benefit from information about the time-dependent toxicity of this chemical. Using published toxicity data for imidacloprid for several insect species, we construct time-to-lethal-effect toxicity plots and fit temporal power-law scaling curves to the data. The level of toxic exposure that results in 50% mortality after time t is found to scale as t1.7 for ants, from t1.6 to t5 for honeybees, and from t1.46 to t2.9 for termites. We present a simple toxicological model that can explain t2 scaling. Extrapolating the toxicity scaling for honeybees to the lifespan of winter bees suggests that imidacloprid in honey at 0.25 μg/kg would be lethal to a large proportion of bees nearing the end of their life.

Chronic Bee Paralysis Virus and Nosema ceranae Experimental Co-Infection of Winter Honey Bee Workers (Apis mellifera L.)

Chronic bee paralysis virus (CBPV) is an important viral disease of adult bees which induces significant losses in honey bee colonies. Despite comprehensive research, only limited data is available from experimental infection for this virus. In the present study winter worker bees were experimentally infected in three different experiments. Bees were first inoculated per os (p/o) or per cuticle (p/c) with CBPV field strain M92/2010 in order to evaluate the virus replication in individual bees. In addition, potential synergistic effects of co-infection with CBPV and Nosema ceranae (N. ceranae) on bees were investigated. In total 558 individual bees were inoculated in small cages and data were analyzed using quantitative real time RT-PCR (RT-qPCR). Our results revealed successful replication of CBPV after p/o inoculation, while it was less effective when bees were inoculated p/c. Dead bees harbored about 1,000 times higher copy numbers of the virus than live bees. Co-infection of workers with CBPV and N. ceranae using either method of virus inoculation (p/c or p/o) showed increased replication ability for CBPV. In the third experiment the effect of inoculation on bee mortality was evaluated. The highest level of bee mortality was observed in a group of bees inoculated with CBPV p/o, followed by a group of workers simultaneously inoculated with CBPV and N. ceranae p/o, followed by the group inoculated with CBPV p/c and the group with only N. ceranae p/o. The experimental infection with CBPV showed important differences after p/o or p/c inoculation in winter bees, while simultaneous infection with CBPV and N. ceranae suggesting a synergistic effect after inoculation.

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.

Antibacterial Immune Competence of Honey Bees (Apis mellifera) Is Adapted to Different Life Stages and Environmental Risks

The development of all honey bee castes proceeds through three different life stages all of which encounter microbial infections to a various extent. We have examined the immune strength of honey bees across all developmental stages with emphasis on the temporal expression of cellular and humoral immune responses upon artificial challenge with viable Escherichia coli bacteria. We employed a broad array of methods to investigate defence strategies of infected individuals: (a) fate of bacteria in the haemocoel; (b) nodule formation and (c) induction of antimicrobial peptides (AMPs). Newly emerged adult worker bees and drones were able to activate efficiently all examined immune reactions. The number of viable bacteria circulating in the haemocoel of infected bees declined rapidly by more than two orders of magnitude within the first 4–6 h post-injection (p.i.), coinciding with the occurrence of melanised nodules. Antimicrobial activity, on the other hand, became detectable only after the initial bacterial clearance. These two temporal patterns of defence reactions very likely represent the constitutive cellular and the induced humoral immune response. A unique feature of honey bees is that a fraction of worker bees survives the winter season in a cluster mostly engaged in thermoregulation. We show here that the overall immune strength of winter bees matches that of young summer bees although nodulation reactions are not initiated at all. As expected, high doses of injected viable E.coli bacteria caused no mortality in larvae or adults of each age. However, drone and worker pupae succumbed to challenge with E.coli even at low doses, accompanied by a premature darkening of the pupal body. In contrast to larvae and adults, we observed no fast clearance of viable bacteria and no induction of AMPs but a rapid proliferation of E.coli bacteria in the haemocoel of bee pupae ultimately leading to their death.

Effects of Extended Prepupal Storage Duration on Adult Flight Physiology of the Alfalfa Leafcutting Bee (Hymenoptera: Megachilidae)

The alfalfa leafcutting bee, Megachile rotundata (F.), is a solitary, cavity-nesting bee and is the primary pollinator for alfalfa seed production. Bee management practices include cold storage during the prepupal stage. Fluctuating thermal regimes during cold storage increases survival of cold storage and allows a doubling of the cold storage period with no decrease in survival. However, survival, characterized as successful adult emergence, is not qualitative. In this study, we determined whether extended storage affects adult bee respiration or flight physiology. We overwintered bees for a single winter (current management protocol) or for 12 mo longer (extended storage). We used resting and tethered flight metabolic rates and resting critical PO2 (the oxygen partial pressure below which metabolism can no longer be sustained) as indices of adult bee quality. We found no significant differences in body mass, resting or flight metabolic rates, or critical PO2 between the two groups. Together these data indicate that extended storage of M. rotundata produces bees of similar respiratory capacity and flight ability. These findings could increase the use of M. rotundata as an alternative pollinator, allowing for extended storage to time adult emergence with early blooming crops.

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.

Extending the honey bee venome with the antimicrobial peptide apidaecin and a protein resembling wasp antigen 5

Honey bee venom is a complex mixture of toxic proteins and peptides. In the present study we tried to extend our knowledge of the venom composition using two different approaches. First, worker venom was analysed by liquid chromatography-mass spectrometry and this revealed the antimicrobial peptide apidaecin for the first time in such samples. Its expression in the venom gland was confirmed by reverse transcription PCR and by a peptidomic analysis of the venom apparatus tissue. Second, genome mining revealed a list of proteins with resemblance to known insect allergens or venom toxins, one of which showed homology to proteins of the antigen 5 (Ag5)/Sol i 3 cluster. It was demonstrated that the honey bee Ag5-like gene is expressed by venom gland tissue of winter bees but not of summer bees. Besides this seasonal variation, it shows an interesting spatial expression pattern with additional production in the hypopharyngeal glands, the brains and the midgut. Finally, our immunoblot study revealed that both synthetic apidaecin and the Ag5-like recombinant from bacteria evoke no humoral activity in beekeepers. Also, no IgG4-based cross-reactivity was detected between the honey bee Ag5-like protein and its yellow jacket paralogue Ves v 5.

Two-dimensional proteomic analysis of honeybee, Apis mellifera, winter worker hemolymph

Honeybee (Apis mellifera Linnaeus) colonies in temperate zones produce either summer bees, which have a lifespan of 15 to 48 days, or winter bees, which emerge in late summer and live up to 8 months. Winter bees develop unique physiological conditions characterized by changes in protein composition that appear to be major determinants of honeybee lifespan. We analyzed winter honeybee worker hemolymph using a proteomic approach for the first time. Hemolymph collected from the dorsal vessel of winter honeybees using a glass capillary tube was analyzed using two-dimensional gel electrophoresis followed by MALDI TOF/TOF protein identification. Overall, 93 spots were assigned significance (P < 0.05). Many identified proteins corresponded well with extended lifespan. Vitellogenin subunits (mainly ∼180 and ∼100 kDa) comprised the major portion of the proteins; however, vitellogenin dominance repressed the signals of the lower-abundance proteins. Future physiological studies related to overwintering bees, including health, immunity, longevity, nutrition, and/or colony losses, can benefit from these results.

Insulin Modifies Honeybee Worker Behavior.

The insulin signaling pathway has been hypothesized to play a key role in regulation of worker social insect behavior. We tested whether insulin treatment has direct effects on worker honeybee behavior in two contexts, sucrose response thresholds in winter bees and the progression to foraging by summer nurse bees. Treatment of winter worker bees with bovine insulin, used as a proxy for honeybee insulin, increased the bees’ sucrose response threshold. Treatment of summer nurse bees with bovine insulin significantly decreased the age at which foraging was initiated. This work provides further insight into the role of endocrine controls in behavior of in honeybees and insects in general.

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.