Sudden deaths and colony population decline in Greek honey bee colonies

During June and July of 2009, sudden deaths, tremulous movements and population declines of adult honey bees were reported by the beekeepers in the region of Peloponnesus (Mt. Mainalo), Greece. A preliminary study was carried out to investigate these unexplained phenomena in this region. In total, 37 bee samples, two brood frames containing honey bee brood of various ages, eight sugar samples and four sugar patties were collected from the affected colonies. The samples were tested for a range of pests, pathogens and pesticides. Symptomatic adult honey bees tested positive for Varroa destructor,Nosema ceranae, Chronic bee paralysis virus (CBPV), Acute paralysis virus (ABPV), Deformed wing virus (DWV), Sacbrood virus (SBV) and Black queen cell virus (BQCV), but negative for Acarapis woodi. American Foulbrood was absent from the brood samples. Chemical analysis revealed that amitraz, thiametoxan, clothianidin and acetamiprid were all absent from symptomatic adult bees, sugar and sugar patty samples. However, some bee samples, were contaminated with imidacloprid in concentrations between 14 ng/g and 39 ng/g tissue. We present: the infection of Greek honey bees by multiple viruses; the presence of N. ceranae in Greek honey bees and the first record of imidacloprid (neonicotonoid) residues in Greek honey bee tissues. The presence of multiple pathogens and pesticides made it difficult to associate a single specific cause to the depopulation phenomena observed in Greece, although we believe that viruses and N. ceranae synergistically played the most important role. A follow up in-depth survey across all Greek regions is required to provide context to these preliminary findings.     

Predictive markers of honey bee colony collapse

Across the Northern hemisphere, managed honey bee colonies, Apis mellifera, are currently affected by abrupt depopulation during winter and many factors are suspected to be involved, either alone or in combination. Parasites and pathogens are considered as principal actors, in particular the ectoparasitic mite Varroa destructor, associated viruses and the microsporidian Nosema ceranae. Here we used long term monitoring of colonies and screening for eleven disease agents and genes involved in bee immunity and physiology to identify predictive markers of honeybee colony losses during winter. The data show that DWV, Nosema ceranae, Varroa destructor and Vitellogenin can be predictive markers for winter colony losses, but their predictive power strongly depends on the season. In particular, the data support that V. destructor is a key player for losses, arguably in line with its specific impact on the health of individual bees and colonies.     

A quantitative model of honey bee colony population dynamics

Since 2006 the rate of honey bee colony failure has increased significantly. As an aid to testing hypotheses for the causes of colony failure we have developed a compartment model of honey bee colony population dynamics to explore the impact of different death rates of forager bees on colony growth and development. The model predicts a critical threshold forager death rate beneath which colonies regulate a stable population size. If death rates are sustained higher than this threshold rapid population decline is predicted and colony failure is inevitable. The model also predicts that high forager death rates draw hive bees into the foraging population at much younger ages than normal, which acts to accelerate colony failure. The model suggests that colony failure can be understood in terms of observed principles of honey bee population dynamics, and provides a theoretical framework for experimental investigation of the problem.     

Emergency queen cell production in the honey bee colony

Summary Emergency queen cell production was examined in honey bee colonies of mixed European races. Thirteen colonies were dequeened and followed on a daily basis until after queen emergence. Observations were made on the number of cells, the temporal sequence of queen cell construction, cell location within the nest, the age of larvæ selected for queen rearing, mortality of immature queens and the scenting behavior of workers in queenless colonies.Queen loss was detected within 6–12 hours and was first indicated by an increase in scenting behavior (on colony disturbance) and queen cup construction. The number of scenting workers reached a peak in 12–24 hours and then declined, as queen cell numbers increased. The time of queen cell initiation varied from 12–48 hours in different colonies. Emergency queen cells were usually started over worker larvæ less than 2 days of age (64.7%), but cells were built over 3 (25.3%) and 4 (10.0%) day old larvæ. Only 2 of 268 cells (0.8 %) were started over eggs; one survived and developed into a drone larva. In 6 colonies emergency queen cells were started over drone larvæ but these were destroyed immediately before or shortly after capping. The overall rate for queen cell construction over drone larvæ was 9.3%.The rate at which new queen cells were started after queen loss was high for two to four days, but then declined although new queen cells were started as late as eight or nine days after queen removal. The number of cells produced by a colony usually peaked by the third or fourth day and then leveled. Slight declines in total cell number often occurred because of cell mortality. The number of queen cells started by colonies varied from 11–49 with a mean of 20.4; cell mortality averaged 39.1%. Queen cells were well distributed throughout the brood nest but placement was biased toward the bottom of the frames and away from the entrance.

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Production de cellules royales après orphelinage accidentel dans des colonies d'abeilles à miel

Resume La production de cellules royales après orphelinage accidentel fut examinée dans des colonies d'abeilles de différentes races européennes. Treize colonies ont été quotidiennement placées dans un orphelinat expérimental après l'apparition d'une nouvelle reine. Des observations ont été faites sur le nombre de cellules, le timing de la reconstruction des cellules royales, l'emplacement des cellules à l'intérieur du nid, l'âge des larves sélectionnées en vue de l'élevage des reines, le taux de mortalité des cellules et le phénomène d'exhibition de la glande de Nassanoff des ouvrières dans les colonies orphelines.On a pu détecter la perte d'une reine après 6 à 12 heures; celle-ci fut tout d'abord indiquée par le fait qu'un certain nombre d'abeilles exhibent leur organe odorant lors de l'ouverture de la ruche, et l'élaboration de la cupule royale. Le nombre des ouvrières exposées a atteint son record entre 12 et 24 heures puis s'est mis à décroître, alors que les cellules royales augmentaient. Le temps requis pour l'initiation des cellules royales a varié entre 12 et 48 heures, selon les colonies. Les cellules royales de remplacement ont commencé ordinairement à se former sur des larves d'ouvrières de moins de 2 jours (64,7%), mais des cellules se sont développées sur des larves âgées de 3 (25,3%) à 4 jours (10,0%). Sur 268 cellules, 2 étaient uniquement formées à partir d'ufs, dont un seul survivait et devenait une larve mâle. Dans six des colonies, des cellules royales se sont développées à partir de larves mâles, mais celles-ci furent immédiatement détruites soit avant, soit juste après l'operculation. Le taux de développement de cellules royales était de 9,3% par rapport aux cellules mâles.Le taux de développement de nouvelles cellules royales après la perte d'une reine a été assez élevé pendant une période de 2 à 4 jours, mais s'est mis à décroître bien que de nouvelles cellules royales se formaient entre 8 et 9 jours après le début de l'orphelinage. Nous avons noté un taux record de cellules produites par une colonie vers le 3^e ou 4^e jour, qui s'est ensuite réparti de façon plus égale. Le taux de mortalité des cellules a alors provoqué la baisse du nombre total des cellules. Le nombre des cellules royales des colonies a varié entre 11 et 49, c'est-à-dire une moyenne de 20,4; le taux de mortalité des cellules s'est avéré de 39,1%. Les cellules royales étaient bien distribuées dans tout le nid à couvain, mais surtout vers le fond du cadre, et loin de l'entrée de la ruche.

     

Non-destructive genotyping and genetic variation of fanning in a honey bee colony

The relationship between workers from different patrilines in a naturally mated queen honey bee colony is very complex due to queen polyandry, and still poorly characterized. Here, we report a means of determining the genotype of living workers in a natural honey bee colony by a new non-destructive method, which makes it possible to observe the relationship between behaviours and genotypes. DNA was extracted from the exuvia, found at the bottom of each brood cell, and confirmed to be identical to the DNA extracted from the thorax muscle of the bee emerging from that particular cell. The genotypes were thus determined using DNA from the exuviae without having to hurt or kill the organisms. The emerging workers were marked with coloured, numbered tags to enable behavioural observations over their entire life. Using this new method, we determined 20 patrilines in a naturally mated queen colony, and discovered that the patriline composition of bees exhibiting fanning behaviour was significantly different from the patriline composition of the whole colony. Our results confirm that the genetic structure of a natural insect society plays a fundamental role in the division of labour. The new non-destructive method reveals a novel avenue for the determination of relationships between the behaviours and genes of social insects.     

不同人工代花粉对蜂群群势和生产性能的影响

早春将群势、蜂王年龄和质量基本一致的25群意大利蜜蜂Apis melliferaL.,随机分为5个处理,分别饲喂纯花粉和4种不同的人工代用花粉,观测它们对蜂群群势和生产性能的影响。结果表明,含有添加剂的各代用花粉组与纯花粉组的蜂群的蜂子数量差异均不显著,但均显著高于纯豆粕组(P<0.01);各组产蜜量随着纯花粉所占比例的减少而呈现递减的趋势,但饲喂含有60%花粉+40%豆粕+添加剂1组与纯花粉组没有明显的差异;含有添加剂的各代用花粉组的王台接受率均显著高于纯花粉组(P<0.01);饲喂含有添加剂的各代用花粉组与饲喂纯花粉组相比,王浆产量及蜂王浆中的10-HDA、水分、蛋白质的含量及酸度均没有差异。     

Functional flexibility of the honey bee hypopharyngeal gland in a dequeened colony

The role of the worker honey bee Apis mellifera L. changes depending on age after eclosion (age polyethism): young workers (nurse bees) take care of their brood by synthesizing and secreting brood food (royal jelly), while older workers (foragers) forage for nectar and process it into honey. Previously, we showed that the major proteins synthesized in the hypopharyngeal gland of the worker change from brood food proteins to alpha-glucosidase at the single secretory cell level in parallel with this age polyethism [Kubo et al., J. Biochem. 119, 291-295 (1996); Ohashi et al., Eur. J. Biochem. 249, 797-802 (1997)]. Here, we examined whether the function of the hypopharyngeal gland has flexibility depending on the colony conditions, by creating a dequeened colony in which the older workers were compelled to feed the drone larvae. It was found that most of the older workers in the dequeened colony synthesized brood food proteins as did nurse bees. Furthermore, the percentage of workers that synthesized brood food proteins was maintained at 80-90% of the total workers for at least two months, as in a normal colony. These results indicate that the function of the hypopharyngeal gland cells of the worker has flexibility and can, if necessary, be maintained as that of the nurse bee, depending on the condition of the colony.     

A spatial model of honey bee colony collapse due to pesticide contamination of foraging bees

Journal of Mathematical Biology - We develop a model of honey bee colony collapse based on contamination of forager bees in pesticide contaminated spatial environments. The model consists of...     

Pesticides linked to bee deaths

For the first time, a chemicals company has paid compensation to beekeepers for loss of colonies. Michael Gross reports.     

Multiple virus infections in the honey bee and genome divergence of honey bee viruses

Using uniplex RT-PCR we screened honey bee colonies for the presence of several bee viruses, including black queen cell virus (BQCV), deformed wing virus (DWV), Kashmir bee virus (KBV), and sacbrood virus (SBV), and described the detection of mixed virus infections in bees from these colonies. We report for the first time that individual bees can harbor four viruses simultaneously. We also developed a multiplex RT-PCR assay for the simultaneous detection of multiple bee viruses. The feasibility and specificity of the multiplex RT-PCR assay suggests that this assay is an effective tool for simultaneous examination of mixed virus infections in bee colonies and would be useful for the diagnosis and surveillance of honey bee viral diseases in the field and laboratory. Phylogenetic analysis of putative helicase and RNA-dependent RNA polymerase (RdRp) encoded by viruses reveal that DWV and SBV fall into a same clade, whereas KBV and BQCV belong to a distinct lineage with other picorna-like viruses that infect plants, insects and vertebrates. Results from field surveys of these viruses indicate that mixed infections of BQCV, DWV, KBV, and SBV in the honey bee probably arise due to broad geographic distribution of viruses.     

Spatial foraging patterns and colony energy status in the African honey bee,Apis mellifera scutellata

The relationship between changes in foraging patterns (inferred from waggle dance activity) and colony energy status (inferred from brood rearing activity, food storage, and colony weight) was examined for the African honey bee during a period of relative resource abundance and resource dearth. When resources were more abundant mean foraging distances (about 400 m) and foraging areas (4–5 km²) were small, and colonies recruited to 12–19 different sites per day. Colony foraging ranges and sites visited increased slightly during the dearth period, yet foraging continued to be concentrated within less than 10 km². The degree to which fluctuations in foraging patterns were correlated with colony energy status varied with the availability of floral resources. During periods of relative forage abundance, increases in foraging range and number of sites visited were significantly correlated with increases in brood rearing and colony weight. In contrast, colonies examined during periods of resource dearth exhibited no correlations between foraging areas, foraging distances, and fluctuations in brood rearing, food storage, or colony weight. Thus, during dearth periods colonies may not be able to coordinate foraging patterns with changes in colony energy status.     

Toxicity of cryoprotectants to honey bee semen and queens

Given the threats to the intraspecific biodiversity of Apis mellifera and the pressure on bee breeding to come up with disease-tolerant lines, techniques to cryopreserve drone semen are of great interest. Freeze-thawed drone semen of high viability and/or motility has repeatedly been obtained, but fertility of such semen, when it was measured, was always low. The cryoprotective agent (CPA) most frequently used with drone semen is dimethyl sulfoxide (DMSO), although this substance has been suspected of causing genetic damage in sperm. No form of sperm washing is currently performed. Using a membrane permeability assay, we measured the short-term toxicity of four possible replacements for DMSO, 1,3-propane diol, 2,3-butane diol, ethylene glycol, and dimethyl formamide. We also tested whether the practice of inseminating queens with CPA-containing semen affects sperm numbers in the storage organs of queens, or sperm fertility. Finally, we tested whether CPA-toxicity in vivo can be reduced by using mixtures of two CPAs, DMSO, and ethylene glycol. Our results show that, although short-term toxicity of all CPAs tested was low, the presence of single CPAs in insemination mixtures at concentrations required for slow freezing greatly reduced the number of sperm reaching the spermatheca. Contrary to earlier reports, this was also true for DMSO. Ethylene glycol was additionally shown to reduce the viability of spermatozoa reaching the storage organ. Mixtures of DMSO and EthGly performed better than either substance used singly at the same concentration. We conclude that the toxicity of CPAs, including DMSO, on honey bee semen and/or queens has been underestimated in the past. This could partly explain the discrepancy between in vitro and in vivo quality of cryopreserved drone semen, described by others. Combinations of several CPAs and techniques to partly remove CPAs after thawing could help to solve this problem.     

Genetic variation in worker temporal polyethism and colony defensiveness in the honey bee, Apis mellifera

To test the hypothesis that colonies of honey bees composedof workers with faster rates of adult behavioral developmentare more defensive than colonies composed of workers with slowerbehavioral development, we determined whether there is a correlationbetween genetic variation in worker temporal polyethism andcolony defensiveness. There was a positive correlation for thesetwo traits, both for European and Africanized honey bees. Thecorrelation was larger for Africanized bees, due to differencesbetween Africanized and European bees, differences in experimentaldesign, or both. Consistent with these results was the findingthat colonies with a higher proportion of older bees were moredefensive than colonies of the same size that had a lower proportionof older bees. There also was a positive correlation betweenrate of individual behavioral development and the intensityof colony flight activity, and a negative correlation betweencolony defensiveness and flight activity. This suggests thatthe relationship between temporal polyethism and colony defensivenessmay vary with the manner in which foraging and defense dutiesare allocated among a colony's older workers. These resultsindicate that genotypic differences in rates of worker behavioraldevelopment can influence the phenotype of a honey bee colonyin a variety of ways.     

Widespread dispersal of the microsporidian Nosema ceranae, an emergent pathogen of the western honey bee, Apis mellifera

The economically most important honey bee species, Apis mellifera, was formerly considered to be parasitized by one microsporidian, Nosema apis. Recently, [Higes, M., Martín, R., Meana, A., 2006. Nosema ceranae, a new microsporidian parasite in honeybees in Europe, J. Invertebr. Pathol. 92, 93-95] and [Huang, W.-F., Jiang, J.-H., Chen, Y.-W., Wang, C.-H., 2007. A Nosema ceranae isolate from the honeybee Apis mellifera. Apidologie 38, 30-37] used 16S (SSU) rRNA gene sequences to demonstrate the presence of Nosema ceranae in A. mellifera from Spain and Taiwan, respectively. We developed a rapid method to differentiate between N. apis and N. ceranae based on PCR-RFLPs of partial SSU rRNA. The reliability of the method was confirmed by sequencing 29 isolates from across the world (N =9 isolates gave N. apis RFLPs and sequences, N =20 isolates gave N. ceranae RFLPs and sequences; 100% correct classification). We then employed the method to analyze N =115 isolates from across the world. Our data, combined with N =36 additional published sequences demonstrate that (i) N. ceranae most likely jumped host to A. mellifera, probably within the last decade, (ii) that host colonies and individuals may be co-infected by both microsporidia species, and that (iii) N. ceranae is now a parasite of A. mellifera across most of the world. The rapid, long-distance dispersal of N. ceranae is likely due to transport of infected honey bees by commercial or hobbyist beekeepers. We discuss the implications of this emergent pathogen for worldwide beekeeping.     

Worker's genetic diversity has no relation to pollen diversity in a honey bee colony (Apis mellifera L.)

No differences were observed between the species diversity of pollen gathered by colonies with artificially reduced worker genetic diversity and those derived from naturally mated queens. Such a result does not support the hypothesis that polyandry increases colony fitness in which a genetic basis is created for using a wider range of food sources.     

The effects of honey bee (Apis mellifera L.) queen reproductive potential on colony growth

Reproduction in species of eusocial insects is monopolized by one or a few individuals, while the remaining colony tasks are performed by the worker caste. This reproductive division of labor is exemplified by honey bees (Apis mellifera L.), in which a single, polyandrous queen is the sole colony member that lays fertilized eggs. Previous work has revealed that the developmental fate of honey bee queens is highly plastic, with queens raised from younger worker larvae exhibiting higher measures in several aspects of reproductive potential compared to queens raised from older worker larvae. Here, we investigated the effects of queen reproductive potential (“quality”) on the growth and winter survival of newly established honey bee colonies. We did so by comparing the growth of colonies headed by “high-quality” queens (i.e., those raised from young worker larvae, which are more queen-like morphologically) to those headed by “low-quality” queens (i.e., those raised from older worker larvae, which are more worker-like morphologically). We confirmed that queens reared from young worker larvae were significantly larger in size than queens reared from old worker larvae. We also found a significant positive effect of queen grafting age on a colony’s production of worker comb, drone comb, and stored food (honey and pollen), although we did not find a statistically significant difference in the production of worker and drone brood, worker population, and colony weight. Our results provide evidence that in honey bees, queen developmental plasticity influences several important measures of colony fitness. Thus, the present study supports the idea that a honey bee colony can be viewed (at least in part) as the expanded phenotype of its queen, and thus selection acting predominantly at the colony level can be congruent with that at the individual level.     

Purification and characterization of honey bee vitellogenin

A protocol has been developed for the purification of vitellogenin from the honey bee, Apis mellifera. Purification allows for the first characterization of a vitellogenin from the large order Hymenoptera. Hymenopteran vitellogenins are unusual among insect vitellogenins in that they contain only one type of apoprotein. The honey bee vitellogenin was isolated from hemolymph of honey bee queens by a combination of density gradient ultracentrifugation, ion-exchange chromatography, and affinity chromatography. The native vitellogenin particle is a very high density glycolipoprotein containing approximately 91% protein, 7% lipid, and 2% carbohydrate. Phospholipid and diacylglycerol are the major lipid components. The equilibrium density (1.28 g/ml) is the same as that for Manduca sexta vitellogenin, which contains a much higher proportion of lipid. The covalently bound carbohydrate moiety of the particle is high in mannose. The amino acid composition of vitellogenin is similar to those of vitellogenins from other insect species. The N-terminal amino acid sequence of the apoprotein was determined, the first such sequence for any insect vitellogenin. When analyzed by sodium dodecyl sulfate (SDS)-gel electrophoresis, A. mellifera vitellogenin resolved into a single band with an apparent Mr of 180,000. Gel filtration under reducing and native conditions yielded estimated Mr values of about 300,000.