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.     

The influence of a honeybee (Apis mellifera) colony on egg-laying by its queen

Exchanging the queens of honeybees (Apis mellifera L.) between colonies with few and many eggs influenced the number of eggs the queens laid, but the queens did not completely adapt to the previous egg-laying rates of their recipient colonies.

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Zusammenfassung Der Austausch von Königinnen zwischen Völkern mit wenigen und Völkern mit vielen Eiern beeinflußte die Anzahl der von den Königinnen abgelegten Eier, doch paßten sich die Königinnen den bisherigen Eiablageraten ihrer Empfängervölker nicht vollständig an.

     

A modeling approach to swarming in honey bees (Apis mellifera)

Identifying the mechanisms of colony reproduction is essential to understanding the sociobiology of honey bees. Although several proximate causes leading to the initiation of queen rearing – an essential prerequisite to swarming – have been proposed, none have received unequivocal empirical support. Here we model the main proximate hypotheses (colony size, brood comb congestion, and worker age distribution) and show that all proposed swarming triggers occur as a function of the ultimate cause of a colony reaching replacement stability, the point at which the queen has been laying eggs at her maximal rate. We thus present a reproductive optimization model of colony swarming based on evolutionary principles. All models produce results remarkably similar both to each other and to empirically-determined swarming patterns. An examination of the fit between the individual models and swarm-preventing techniques used by beekeepers indicates that the reproductive optimization model has a relatively broad explanatory range. These results suggest that an examination into the behavioral correlates of a queen’s maximum egg laying rate may provide a unified proximate mechanistic trigger leading predictably to colony fission. Generating a predictive model for this very well studied animal is the first step in producing a model of colony fission applicable to other swarm-founding eusocial animals. Received 16 November 2004; revised 31 May 2005; accepted 27 June 2005.     

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.     

Carbohydrate utilization in the thoraces of honey bees (Apis mellifera) during early times of flight

Trehalose levels in the thoraces of honey bees did not change significantly during the first 2–4 min of flight. This effect was seen both for bees flown shortly after removal from the hive and for bees which were flown after a 2 hr starvation period. There was also no detectable activation of the trehalose in isolated mitochondria from bees flown for periods of up to 2 min. However, the glucose content of the thoraces of bees flown shortly after removal from the hive dropped dramatically during the first 15 sec of flight. There was no evidence of a transient increase in the glucose content in the thorax at any of the times studied as would be expected if trehalose were hydrolized. The drop in glucose content at early times of flight was not detected if the bees were starved for 2 hr before flight was started. The changes in fructose content of the thoraces with time were similar to those observed for glucose, but were not statistically significant due to variation among individual bees. The sucrose content of bee thoraces varied greatly and no meaningful conclusions could be reached about how it changed as a function of time of flight.     

The dynamics of sleep-like behaviour in honey bees

At night, honey bees pass through a physiological state that is similar to mammalian sleep. Like sleep in mammals, sleep-like behaviour in honey bees is an active process. This is expressed most clearly in these insects by spontaneous antennal movements which appear at irregular intervals throughout the night and interrupt episodes of antennal immobility. Here we present a newly developed video technique for the continuous recording of the position and movements of the bee's antennae. The same technique was used to record head inclination and ventilatory movements. Despite the constancy of the ambient temperature, the magnitudes of antennae-related parameters, as well as head inclination and ventilatory cycle duration, displayed dynamic unimodal time-courses which exhibited a high degree of temporal covariance. The similarity between these time-courses and the nightly time-course of the reaction threshold for a sensory stimulus, investigated previously, indicates that, in honey bees, deepest "sleep" and least ventilatory activity occur at the same time (in the 7th hour of the rest phase).Abbreviations DD continuous darkness - EMG electromyogram - LD periodic alternation between light (L) and darkness (D) - MEST Middle European Summer Time (UTC+2 h); - UV ultraviolet This paper is dedicated to Professor Martin Lindauer, who—to our knowledge—was the first to meticulously record the nightly behaviour of honey bees (Lindauer 1952) and who also inspired one of us (W.K.) to investigate antennal motility during nightly rest in these animals.     

A multiplex PCR assay to diagnose and quantify Nosema infections in honey bees (Apis mellifera)

Correct identification of the microsporidia, Nosema apis and Nosema ceranae, is key to the study and control of Nosema disease of honey bees (Apis mellifera). A rapid DNA extraction method combined with multiplex PCR to amplify the 16S rRNA gene with species-specific primers was compared with a previously published assay requiring spore-germination buffer and a DNA extraction kit. When the spore germination-extraction kit method was used, 10 or more bees were required to detect the pathogens, whereas the new extraction method made it possible to detect the pathogens in single bees. Approx. 4-8 times better detection of N. ceranae was found with the new method compared to the spore germination-extraction kit method. In addition, the time and cost required to process samples was lower with the proposed method compared to using a kit. Using the new DNA extraction method, a spore quantification procedure was developed using a triplex PCR involving co-amplifying the N. apis and N. ceranae 16S rRNA gene with the ribosomal protein gene, RpS5, from the honey bee. The accuracy of this semi-quantitative PCR was determined by comparing the relative band intensities to the number of spores per bee determined by microscopy for 23 samples, and a high correlation (R² = 0.95) was observed. This method of Nosema spore quantification revealed that spore numbers as low as 100 spores/bee could be detected by PCR. The new semi-quantitative triplex PCR assay is more sensitive, economical, rapid, simple, and reliable than previously published standard PCR-based methods for detection of Nosema and will be useful in laboratories where real-time PCR is not available.     

The effect of diet on protein concentration, hypopharyngeal gland development and virus load in worker honey bees (Apis mellifera L.)

Elucidating the mechanisms by which honey bees process pollen vs. protein supplements are important in the generation of artificial diets needed to sustain managed honeybees. We measured the effects of diet on protein concentration, hypopharyngeal gland development and virus titers in worker honey bees fed either pollen, a protein supplement (MegaBee), or a protein-free diet of sugar syrup. Workers consumed more pollen than protein supplement, but protein amounts and size of hypopharyngeal gland acini did not differ between the two feeding treatments. Bees fed sugar syrup alone had lower protein concentrations and smaller hypopharyngeal glands compared with the other feeding treatments especially as the bees aged. Deformed wing virus was detected in workers at the start of a trial. The virus concentrations increased as bees aged and were highest in those fed sugar syrup and lowest in bees fed pollen. Overall results suggest a connection between diet, protein levels and immune response and indicate that colony losses might be reduced by alleviating protein stress through supplemental feeding.     

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.     

Colony founding and initial nest design of honey bees, Apis mellifera L

Thirty-two colonies of bees with varying populations were established in 1·2-m³ boxes for 24 days. At the end of this time, the bees were killed, and the comb structure, design, and placement were examined to obtain information about the initiation of bee nests. The analysis of variance of three dimensions of the comb (height, width, and length) showed no statistical differences when all thirty-two colonies were considered as a group.Comb built by queenless bees differed from any described in the literature. We think it is comb characteristically built by queenless bees that is neither worker nor drone in size but intermediate.The occurrence of multiple clusters building comb with and without queen in the same box is recorded but unexplained at present.An explanation is offered for the curl appearing in the lower edges of straight combs.     

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.     

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.     

Differential gene expression of the honey bees Apis mellifera and A. cerana induced by Varroa destructor infection

Varroa destructor mite is currently the most serious threat to the world bee industry. Differences in mite tolerance are reported between two honey bee species Apis mellifera and Apis cerana. Differential gene expression of two honey bee species induced by V. destructor infection was investigated by constructing two suppression subtractive hybridization (SSH) libraries, as first steps toward elucidating molecular mechanisms of Varroa tolerance. From the SSH libraries, we obtained 289 high quality sequences which clustered into 132 unique sequences grouped in 26 contigs and 106 singlets where 49 consisted in A. cerana subtracted library and 83 in A. mellifera. Using BLAST, we found that 85% sequences had counterpart known genes whereas 15% were undescribed. A Gene Ontology analysis classified 51 unique sequences into different functional categories. Eight of these differentially expressed genes, representative of different regulation patterns, were confirmed by qRT-PCR. Upon the mite induction, the differentially expressed genes from both bee species were different, except hex 110 gene, which was up-regulated in A. cerana but down-regulated in A. mellifera, and Npy-r gene, which was down-regulated in both species. In general, most of the differential expression genes were involved in metabolic processes and nerve signaling. The results provide information on the molecular response of these two bee species to Varroa infection.     

Host-seeking behaviour of tracheal mites (Acari: Tarsonemidae) on honey bees (Hymenoptera: Apidae)

The behaviour of the endoparasitic tracheal mite, Acarapis woodi (Rennie) on honey bees (Apis mellifera L.) is a challenge to observe because of its small size. Through a microscope, we videotaped this mite's movement on young bees, dead bees and bees exposed to vegetable oil. Previous studies have shown that solid vegetable oil decreases mite infestations in a bee colony. We hypothesized that the oil alters mite behaviour to the detriment of the parasite, thus helping to safeguard the host. Habitat-seeking behaviour, identified as necessary for mites to locate a new host environment, was disrupted on both dead and oil-treated bees. Questing behaviour, which is associated with transfer between hosts, increased significantly on the dead and oily bees. The behaviours of mites were significantly different between all three treatments (x ²=494.96, p<0.001 on dead bees and x ²=851.11, p<0.001 on oily bees). Both questing and seeking behaviours were significantly different on each of the thoracic treatments (F _2,66=7.88, p<0.001 and F _2,66=21.28, p<0.001) and mite questing behaviour was not altered between males and females on live or oily bees (F _1,22=0.25, p<0.62), but habitat seeking was (F _1,22=7.42, p<0.012). The male questing and habitat-seeking behaviours were observed. We conclude that oil-treated bees gained protection from habitat-seeking mites because the normal behaviour of the mites seeking an oviposition site is interrupted.