Energetic stress in the honeybee Apis mellifera from Nosema ceranae infection

Parasites are dependent on their hosts for energy to reproduce and can exert a significant nutritional stress on them. Energetic demand placed on the host is especially high in cases where the parasite-host complex is less co-evolved. The higher virulence of the newly discovered honeybee pathogen, Nosema ceranae, which causes a higher mortality in its new host Apis mellifera, might be based on a similar mechanism. Using Proboscis Extension Response and feeding experiments, we show that bees infected with N. ceranae have a higher hunger level that leads to a lower survival. Significantly, we also demonstrate that the survival of infected bees fed ad libitum is not different from that of uninfected bees. These results demonstrate that energetic stress is the probable cause of the shortened life span observed in infected bees. We argue that energetic stress can lead to the precocious and risky foraging observed in Nosema infected bees and discuss its relevance to colony collapse syndrome. The significance of energetic stress as a general mechanism by which infectious diseases influence host behavior and physiology is discussed.     

Cuticular sclerotization in the honeybee (Apis mellifera adansonii)

Summary A micromethod has been developed for quantitative determinations of ketocatechols released by acid hydrolysis from samples of sclerotized insect cuticle, weighing from 0.1 to 5 mg. The method has been used to follow the changes in yield of ketocatechols from five different types of cuticle during honeybee development. Samples from workers, drones, and queens have been analyzed. The increase in yield of ketocatechols is most pronounced during the period of pharate development when abdominal cuticle is stiffened and the cuticular proteins become insoluble.     

Linkage analysis of sex determination in the honey bee (Apis mellifera)

A colony-level phenotype was used to map the major sex determination locus (designatedX) in the honey bee (Apis mellifera). Individual queen bees (reproductive females) were mated to single drones (fertile males) by instrumental insemination. Haploid drone progeny of an F1 queen were each backcrossed to daughter queens from one of the parental lines. Ninety-eight of the resulting colonies containing backcross progeny were evaluated for the trait low brood-viability resulting from the production of diploid drones that were homozygous atX. DNA samples from the haploid drone fathers of these colonies were used individually in polymerase chain reactions (PCR) with 10-base primers. These reactions generated random amplified polymorphic DNA (RAPD) markers that were analyzed for cosegregation with the colony-level phenotype. One RAPD marker allele was shared by 22 of 25 drones that fathered low brood-viability colonies. The RAPD marker fragment was cloned and partially sequenced. Two primers were designed that define a sequence-tagged site (STS) for this locus. The primers amplified DNA marker fragments that cosegregated with the original RAPD marker. In order to more precisely estimate the linkage betweenX and the STS locus, another group of bees consisting of progeny from one of the low-brood viability colonies was used in segregation analysis. Four diploid drones and 181 of their diploid sisters (workers, nonfertile females) were tested for segregation of the RAPD and STS markers. The cosegregating RAPD and STS markers were codominant due to the occurrence of fragment-length alleles. The four diploid drones were homozygous for these markers but only three of the 181 workers were homozygotes (recombinants). Therefore the distance betweenX and the STS locus was estimated at 1.6 cM. An additional linked marker was found that was 6.6 cM from the STS locus.     

Hyperalgesic and edematogenic effects of peptides isolated from the venoms of honeybee (Apis mellifera) and neotropical social wasps (Polybia paulista and Protonectarina sylveirae)

Stings by bees and wasps, including Brazilian species, are a severe public health problem. The local reactions observed after the envenoming includes typical inflammatory response and pain. Several studies have been performed to identify the substances, including peptides that are responsible for such phenomena. The aim of the present study is to characterize the possible nociceptive (hyperalgesic) and edematogenic effects of some peptides isolated from the venoms of the honeybee (Apis mellifera) and the social wasps Polybia paulista and Protonectarina sylveirae, in addition to characterize some of the mechanisms involved in these phenomena. For this purpose, different doses of the peptides mellitin (Apis mellifera), Polybia-MP-I, N-2-Polybia-MP-I (Polybia paulista), Protonectarina-MP-NH2 and Protonectarina-MP-OH (Protonectarina sylveirae) were injected into the hind paw of mice. Hyperalgesia and edema were determined after peptide application, by using an electronic von Frey apparatus and a paquimeter. Carrageenin and saline were used as controls. Results showed that melittin, Polybia-MP-I, N-2-Polybia-MP-I, Protonectarina-MP-NH₂ and Protonectarina-MP-OH peptides produced a dose- and time-related hyperalgesic and edematogenic responses. Both phenomena are detected 2 h after melittin, Polybia-MP-I, N-2-Polybia-MP-I injection; their effects lasted until 8 h. In order to evaluate the role of prostanoids and the involvement of lipidic mediators in hyperalgesia induced by the peptides, indomethacin and zileuton were used. Results showed that zileuton blocked peptide-induced hyperalgesia and induced a decrease of the edematogenic response. On the other hand, indomethacin did not interfere with these phenomena. These results indicate that melittin, Polybia-MP-I, N-2-Polybia-MP-I, Protonectarina-MP-NH₂, and Protonectarina-MP-OH peptides could contribute to inflammation and pain induced by insect venoms.     

The effect of induced changes in sexual asymmetry of honey bees (Apis mellifera) on swarming behaviour

This study was built on the assumption that mother (queen) and workers (nurses) distribute their genes either through swarms (female biomass) or through the drones (male biomass). The swarming mood of the bee colonies was suppressed by an exactly defined increase in drone rearing. We studied the efficiency of reproductive investments (on genetic and energetic levels) of the mother and workers to the next generations. The equalization of fitness of the mother and nurses was achieved by a deliberately induced change in numerically stable sex asymmetry of a bee colony. A swarm was compensated with its energy demand and a volume ratio of distributed genetic information. The newly introduced term “reproductive investment complex” (RIC) includes the reproductive potential of the mother and reproductive energy of workers into care for the mother and for the brood. The number of individuals of one sex was closely connected with the weight of individuals of the oppposite sex. The described method of suppression of swarming mood was successfully tested on 60 honey bee colonies over seven years (2003–2009). A number of beekeepers that were acquainted with this method confirmed the success.     

The honeybee queen influences the regulation of colony drone production

Social insect colonies invest in reproduction and growth, buthow colonies achieve an adaptive allocation to these life-historycharacters remains an open question in social insect biology.Attempts to understand how a colony's investment in reproductionis shaped by the queen and the workers have proved complicatedbecause of the potential for queen–worker conflict overthe colony's investment in males versus females. Honeybees,in which this conflict is expected to be minimal or absent,provide an opportunity to more clearly study how the actionsand interactions of individuals influence the colony's productionand regulation of males (drones). We examined whether honeybeequeens can influence drone regulation by either allowing orpreventing them from laying drone eggs for a period of timeand then examining their subsequent tendency to lay drone andworker eggs. Queens who initially laid drone eggs subsequentlylaid fewer drone eggs than the queens who were initially preventedfrom producing drone eggs. This indicates that a colony's regulationof drones may be achieved not only by the workers, who buildwax cells for drones and feed the larvae, but also by the queen,who can modify her production of drone eggs. In order to betterunderstand how the queen and workers contribute to social insectcolony decisions, future work should attempt to distinguishbetween actions that reflect conflict over sex allocation andthose that reflect cooperation and shared control over the colony'sinvestment in reproduction.     

The influence of queen age and quality during queen replacement in honeybee colonies

Honeybee, Apis mellifera, colonies replace their queens by constructing many queen cells and then eliminating supernumerary queens until only one remains. The ages of the queens and the variation in their reproductive potential are important factors in the outcome of such events. Selection would favour colonies that requeen as quickly as possible to minimize the brood hiatus, therefore selecting for queens reared from older larvae. Conversely, reproductive potential (queen 'quality') is maximized by rearing queens from younger larvae. This potential trade-off was tested during two phases of queen replacement, namely queen rearing and polygyny reduction. Our results suggest that queen age is a significant element during both queen rearing and polygyny reduction, whereas queen quality, at least to the magnitude tested in this experiment, has little impact on the outcome of either process. The rate of queen replacement therefore appears to be an important factor in the honeybee life cycle, and further mechanisms of potential importance during this life history transition are discussed. Copyright 2000 The Association for the Study of Animal Behaviour.     

Dependence of the life span of the honeybee (Apis mellifica) upon flight performance and energy consumption

Summary The life span of worker-honeybees is determined by the duration of the hive-period and of the foraging period (Figs. 1,2). The duration of the forgaing period is regulated in the following way: Total flight performance of the individual bee seems to be fixed. Daily flight performance strongly affects total flight duration. High daily flight performance decreases maximal flight duration and vice versa.Foragers accumulate the highest glycogen reserves in the flight muscles compared to other stages (Figs. 3, 4). They use these reserves to overcome starvation or when growing old. Young foragers are able to restore glycogen reserves after sugar intake, whereas old foragers were found to have a reduced glycogen synthesizing ability (Fig. 5).The results indicate that bees exhaust their energysupplying mechanisms after a definite total flight performance.     

The influence of social hunger on food distribution and its implications for disease transmission in a honeybee colony

Social insect colonies are characterized by extensive interactions among individuals, exchanges that can also potentially transmit pathogens. The large majority of these social interactions in a honeybee colony result from food transfer among individuals. Since colony hunger is likely to have a significant influence on these interactions, we investigated its effect on the distribution of food within the colony. By pulsing two colonies having different amounts of stored food with a radioactive label, we found that a starved colony sent out a larger number of foragers, brought in more food, and stored more of it than the satiated colony. We also found that the food brought into a starved colony was distributed more uniformly within each age class than that in the satiated colony. The queen and the young individuals received the lowest exposure to the label even though the label entered different regions of the colony at the same rate. The satiation level of the colony did not influence the relative exposures of different age groups to the label but a higher amount of it was stored in the hungry colony. We discuss the significance of these results in terms of the role played by the organizational structure of the honeybee colony on the transmission dynamics of an infectious disease.     

Muscle potentials and temperature acclimation and acclimatization in flight muscles of workers and drones of Apis mellifera

1. 1.Muscle potentials in fibrillar flight muscles of worker and drone honeybees were recorded extracellularly at thoracic temperatures from 30 to 10°C.

2. 2.Extinction temperatures for muscle potentials were higher in drones for all treatments.

3. 3.Cold acclimation (15°C) lowered extinction temperatures significantly in workers and drones. Acclimitization changed extinction temperatures significantly only in drones.

4. 4.Cold acclimitization had a bigger effect on the rate of muscle potential amplitude decline with decreasing temperature than acclimation.

5. 5.Acclimation and acclimitization had no effect on the increase of muscle potential duration with falling temperature.

6. 6.Muscle potential frequency during shivering was not much different between cold and warm treated bees.

The inhibiting effect of the queen bee (Apis mellifera L.) foot-print pheromone on the construction of swarming queen cups

Experiments were conducted to determine the rôle of population density of queenright honey bee colonies, and that of the queen bee pheromonal secretions, on the induction and inhibition of swarming queen cup construction during swarming and non-swarming seasons. Construction of queen cups was induced experimentally in overcrowded queenright colonies, during winter, which is a non-swarming season. This construction was induced by high population density of bee workers: above a threshold of 2.3 bee workers/ml there was a relationship between the number of cups constructed and the colony density. During the swarming season a relationship was established between the free volume of a hive (population density) and the number of queen cups constructed: 1.5 cups in a colony that occupied 80,960 ml, compared with 77 cups in a colony hived within a volume of 20, 240 ml. Observations of the queen's movements upon combs in colonies of high and normal population densities showed that in an overcrowded colony the queen bee was almost absent from the bottom edges of the comb, where queen swarming cups and cells are constructed. The tarsal glands of queens are located in the fifth tarsomere and the glandular oily secretion is deposited by the foot-pads upon the combs surface. The rate of secretion by the queen's tarsal glands was about 13 times higher than by those of the workers. A bioassay for testing the inhibitory effects of the queen's glandular extracts on the construction of queen cups was developed. It was based on increasing worker bee population densities, and can be used effectively throughout the year in a subtropical climate.The application of tarsal and mandibular glands' secretion to comb bottom edges in overcrowded colonies (bioassay) caused the inhibition of queen cup construction. None of these two secretions affected construction of these cups when applied separately. We presume that due to colony overcrowding the queen bee is unable to deposit the non-volatile secretions from tarsal glands along the comb edges and that the deficiency of the foot-print pheromone triggers the construction of swarming cups along the non-inhibited areas.     

The effect of the size of a honeybee colony on its foraging activity

Summary Deterioration in foraging conditions discourages foraging relatively more from large than from small colonies.

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Résumé Une détérioration des conditions de butinage décourage relativement plus le butinage chez les fortes colonies que chez les petites.

     

The chemical basis of honeybee, Apis mellifera, caste formation. Partial purification of queen bee determinator from royal jelly

On royal jelly, 1- to 2-day-old honeybee worker larvae have been reared in vitro to adults in a yield of 67±18 per cent. Up to 100 per cent and, on an average, 60 per cent of them were queens and intercaster. The preparation of a basic food from royal jelly by extensive alcohol extraction is described. With this control food, a survival rate of 47±18 per cent was achieved; 15 per cent of the adults were determined, 4·3 per cent of them were queens. Rearing of 1- to 2-day-old worker larvae on a basic food, to which unknown fractions may be added, was used as a biological test for the partial purification of queen bee determinator from royal jelly. By chromatography of the ethanol extract, previously treated with charcoal, on the cation exchanger Dowex 50 WX4 and rechromatography on silica gel, a 10⁵-fold purification of determinator was achieved. Chemical properties of the highly hydrophilic, low molecular active fraction are described.     

Molecular Phylogenetics of Honey Bee Subspecies (Apis melliferaL.) Inferred from Mitochondrial DNA Sequence

A mitochondrial DNA region encompassing part of the NADH dehydrogenase subunit 2 and isoleucine transfer RNA genes was PCR amplified, cloned, and sequenced for 14 morphometrically identifiedApis melliferasubspecies and the New World “Africanized” honeybee. Twenty different haplotypes were detected and phylogenetic analyses supported the existence of 3 or 4 major subspecies groups similar to those based on morphometric measurements. However, some discrepancies are reported concerning the subspecies composition of each group. Based on the sequence divergence ofDrosophila(2% per Myr) we found that the four lineages may have diverged around 0.67 Myr. The variability found in this region enables us to infer phylogenetic relationships and test hypotheses concerning subspecies origin, dispersion, and biogeography.     

Development of a user-friendly delivery method for the fungus Metarhiziumanisopliae to control the ectoparasitic mite Varroa destructor in honey bee, Apis mellifera, colonies

A user-friendly method to deliver Metarhizium spores to honey bee colonies for control of Varroa mites was developed and tested. Patty blend formulations protected the fungal spores at brood nest temperatures and served as an improved delivery system of the fungus to bee hives. Field trials conducted in 2006 in Texas using freshly harvested spores indicated that patty blend formulations of 10 g of conidia per hive (applied twice) significantly reduced the numbers of mites per adult bee, mites in sealed brood cells, and residual mites at the end of the 47-day experimental period. Colony development in terms of adult bee populations and brood production also improved. Field trials conducted in 2007 in Florida using less virulent spores produced mixed results. Patty blends of 10 g of conidia per hive (applied twice) were less successful in significantly reducing the number of mites per adult bee. However, hive survivorship and colony strength were improved, and the numbers of residual mites were significantly reduced at the end of the 42-day experimental period. The overall results from 2003 to 2008 field trials indicated that it was critical to have fungal spores with good germination, pathogenicity and virulence. We determined that fungal spores (1 × 10¹⁰ viable spores per gram) with 98% germination and high pathogenicity (95% mite mortality at day 7) provided successful control of mite populations in established honey bee colonies at 10 g of conidia per hive (applied twice). Overall, microbial control of Varroa mite with M. anisopliae is feasible and could be a useful component of an integrated pest management program.