Genetic differences in learning behavior in honeybees (Apis mellifera capensis)

Workers from colonies of Cape honeybees show marked phenotypic differences in performance in proboscis extension reflex (PER) conditioning. Analysis of these differences using parthenogenetic offspring groups permitted the estimation of genotypic values and revealed a high degree of genetic variability that is evident among related as well as unrelated bees. The results obtained from related groups are of particular importance, since they demonstrated the existence of strong genetic variability among individuals of the same colony. Quantitative analysis yielded high estimates of additive genetic effects and low estimates of dominance effects. Selection of individual workers resulted in an explicit increase in genetic variance of the next generation (G1). However, selection of bees from the parthenogenetic G1 generation, which was done to obtain parthenogenetic G2 offspring, did not lead to further improvement in selection. This observation suggests that recombination of linked genes underlying proboscis extension reflex was neglible during selection in parthenogenetic groups. Taken together with further behavioral analysis (Brandes and Menzel, 1990; Brandes et al., 1988), results from these quantitative genetic experiments suggest that additive genetic factors contribute significantly to variability among individuals for associative learning.This work was supported by a fellowship from the Deutsche Forschnungsgemmeinschaft (Br 827/1, Br 827/2).     

Estimation of heritability of learning behavior in honeybees (Apis mellifera capensis)

The learning behavior of Cape honeybees (Apis mellifera capensis) is examined using conditioning of the proboscis extension reflex. The bees are tested cumulatively with several learning tests. The genetic influence on differences in the learning scores is calculated by means of heritability (h ²) estimations obtained by different methods. Theh ² values of the sum of the trials derived by each method range between 0.39 and 0.54. This shows that the proboscis extension reflex paradigm is suitable for the characterization and selection of genetic differences in learning behavior. Heritabilities in the narrow sense and in the broad sense are in the same range. The small differences obtained are discussed with respect to the type of selection existing in natural populations.This study was supported by the Deutsche Forschungsgemeinschaft (Br 827/1-2).     

Transfer of paternal mitochondrial DNA during fertilization of honeybee (Apis mellifera L.) eggs

Strict maternal inheritance of mitochondrial (mt) DNA is believed to be the rule in most eukaryotic organisms because of exclusion of paternal mitochondria from the egg cytoplasm during fertilization. In honeybees, polyspermic fertilization occurs, and many spermatozoa, including their mitochondria-rich flagellum, can completely penetrate the egg, thus allowing for a possibly high paternal leakage. In order to identify paternal mtDNA in honeybee eggs, restriction fragment length polymorphisms (RFLP) of different subspecies were used. Total DNA extracts of different developmental stages of an Apis mellifera carnica x Apis mellifera capensis hybrid brood were tested with a radioactively-labelled diagnostic mtDNA probe. Densitograms of autoradiographs indicated that the male contribution represents up to 27% of the total mitochondrial DNA in the fertilized eggs 12 h after oviposition. In subsequent developmental stages the portion of paternal mtDNA slowly decreased until hatching of the larvae when only traces were found. Although rapid disintegration of paternal mtDNA does not occur, the initially high paternal mitochondrial contribution is not maintained in the adult animal.     

Analysis of the complete genome sequence of two Korean sacbrood viruses in the Honey bee, Apis mellifera

The complete genomic RNAs of two Korean sacbrood virus (SBV) strains, which infect the honey bee, Apis mellifera, were sequenced. The two sequences (AmSBV-Kor19, AmSBV-Kor21) were distinguished by the presence or absence of a PstI restriction site. These strains comprised of 8784 bp and 8835 bp; contained a single large ORF (179-8707 and 179-8758) encoding 2843 and 2860 amino acids, respectively. Deduced amino acid sequences comparison with some insect viruses showed that regions of helicase, protease and RdRp domains; structural genes were located at the 5′ end and non-structural genes at the 3′ end. Multiple sequence alignment showed that AmSBV-Kor19 was missing a section between nucleotides 2311 and 2361 (present in SBV-UK and CSBV) but was similar to that of the Korean SBV strain that infects A. cerana (AcSBV-Kor). The differences in the AmSBV-Kor19 strain may be the result of the virus adapting to a different host.     

Antibacterial Activity of a Cardanol from Thai Apis mellifera Propolis

Background: Propolis is a sticky, dark brown resinous residue made by bees that is derived from plant resins. It is used to construct and repair the nest, and in addition possesses several diverse bioactivities. Here, propolis from Apis mellifera from Nan province, Thailand, was tested for antibacterial activity against Gram^+ve (Staphylococcus aureus and Paenibacillus larvae) and Gram^-ve (Escherichia coli) bacteria.Materials and methods: The three bacterial isolates were confirmed for species designation by Gram staining and analysis of the partial sequence of 16S rDNA. Propolis was sequentially extracted by methanol, dichloromethane and hexane. The antibacterial activity was determined by agar well diffusion and microbroth dilution assays using streptomycin as a positive control. The most active crude extract was further purified by quick column and adsorption chromatography. The apparent purity of each bioactive fraction was tested by thin layer chromatography. The chemical structure of the isolated bioactive compound was analyzed by nuclear magnetic resonance (NMR).Results: Crude methanol extract of propolis showed the best antibacterial activity with a minimum inhibition concentration (MIC) value of 5 mg/mL for S. aureus and E. coli and 6.25 mg/mL for P. larvae. After quick column chromatography, only three active fractions were inhibitory to the growth of S. aureus and E. coli with MIC values of 6.25 and 31.3 µg/mL, respectively. Further adsorption chromatography yielded one pure bioactive fraction (A1A) with an IC₅₀ value of 0.175 µg/mL for E. coli and 0.683 µg/mL for P. larvae, and was determined to be cardanol by NMR analysis. Scanning and transmission electron microscopy analysis revealed unusual shaped (especially in dividing cells), damaged and dead cells in cardanol-treated E. coli.Conclusion: Thai propolis contains a promising antibacterial agent.     

The nicotinic acetylcholine receptor gene family of the honey bee, Apis mellifera

Nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission and play roles in many cognitive processes. They are under intense research as potential targets of drugs used to treat neurodegenerative diseases and neurological disorders such as Alzheimer's disease and schizophrenia. Invertebrate nAChRs are targets of anthelmintics as well as a major group of insecticides, the neonicotinoids. The honey bee, Apis mellifera, is one of the most beneficial insects worldwide, playing an important role in crop pollination, and is also a valuable model system for studies on social interaction, sensory processing, learning, and memory. We have used the A. mellifera genome information to characterize the complete honey bee nAChR gene family. Comparison with the fruit fly Drosophila melanogaster and the malaria mosquito Anopheles gambiae shows that the honey bee possesses the largest family of insect nAChR subunits to date (11 members). As with Drosophila and Anopheles, alternative splicing of conserved exons increases receptor diversity. Also, we show that in one honey bee nAChR subunit, six adenosine residues are targeted for RNA A-to-I editing, two of which are evolutionarily conserved in Drosophila melanogaster and Heliothis virescens orthologs, and that the extent of editing increases as the honey bee lifecycle progresses, serving to maximize receptor diversity at the adult stage. These findings on Apis mellifera enhance our understanding of nAChR functional genomics and provide a useful basis for the development of improved insecticides that spare a major beneficial insect species.     

Quantitative Trait Loci Associated with Reversal Learning and Latent Inhibition in Honeybees (Apis mellifera)

A study was conducted to identify quantitative trait loci (QTLs) that affect learning in honeybees. Two F₁ supersister queens were produced from a cross between two established lines that had been selected for differences in the speed at which they reverse a learned discrimination between odors. Different families of haploid drones from two of these F₁ queens were evaluated for two kinds of learning performance—reversal learning and latent inhibition—which previously showed correlated selection responses. Random amplified polymorphic DNA markers were scored from recombinant, haploid drone progeny that showed extreme manifestations of learning performance. Composite interval mapping procedures identified two QTLs for reversal learning (lrn2 and lrn3: LOD, 2.45 and 2.75, respectively) and one major QTL for latent inhibition (lrn1: LOD, 6.15). The QTL for latent inhibition did not map to either of the linkage groups that were associated with reversal learning. Identification of specific genes responsible for these kinds of QTL associations will open up new windows for better understanding of genes involved in learning and memory.     

Pilocarpine improves recognition of nestmates in young honey bees

Honey bees can distinguish nestmates from non-nestmates, directing aggressive responses toward non-nestmates and rarely attacking nestmates. Here we provide evidence that treatment with pilocarpine, a muscarinic agonist, significantly reduced the number of aggressive responses directed toward nestmates. By contrast, treatment with scopolamine, a muscarinic antagonist, significantly increased attacks on nestmates. Locomotor activity was not altered by these pharmacological treatments. When interpreted in light of known cholinergic pathways in the insect brain, our results provide the first evidence that cholinergic signaling via muscarinic receptors plays a role in olfaction-based social behavior in honey bees.     

Phylogenetic analysis of the RNA-dependent RNA polymerase (RdRp) and a predicted structural protein (pSP) of the Chronic bee paralysis virus (CBPV) isolated from various geographic regions

Chronic bee paralysis virus (CBPV) is responsible for chronic paralysis, an infectious and contagious disease of adult honey bees (Apis mellifera L.). The full-length nucleotide sequences of the two major RNAs of CBPV have previously been characterized. The Orf3 of RNA1 has shown significant similarities to the RNA-dependent RNA polymerase (RdRp) of positive single-stranded RNA viruses, whereas the Orf3 of RNA2 encodes a putative structural protein (pSP). In the present study, honey bees originating from 9 different countries (Austria, Poland, Hungary, Spain, Belgium, Denmark, Switzerland, Uruguay and France) were analysed for the presence of CBPV genome. The complete genomic nucleotide sequence of the RdRp (1947 bp) and of the pSP (543 bp) from 24 honey bee positive samples was determined and the phylogenetic relationship among isolates was investigated. Four distinct genotypes of CBPV were observed.     

A review of neurohormone GPCRs present in the fruitfly Drosophila melanogaster and the honey bee Apis mellifera

G protein-coupled receptor (GPCR) genes are large gene families in every animal, sometimes making up to 1-2% of the animal's genome. Of all insect GPCRs, the neurohormone (neuropeptide, protein hormone, biogenic amine) GPCRs are especially important, because they, together with their ligands, occupy a high hierarchic position in the physiology of insects and steer crucial processes such as development, reproduction, and behavior. In this paper, we give a review of our current knowledge on Drosophila melanogaster GPCRs and use this information to annotate the neurohormone GPCR genes present in the recently sequenced genome from the honey bee Apis mellifera. We found 35 neuropeptide receptor genes in the honey bee (44 in Drosophila) and two genes, coding for leucine-rich repeats-containing protein hormone GPCRs (4 in Drosophila). In addition, the honey bee has 19 biogenic amine receptor genes (21 in Drosophila). The larger numbers of neurohormone receptors in Drosophila are probably due to gene duplications that occurred during recent evolution of the fly. Our analyses also yielded the likely ligands for 40 of the 56 honey bee neurohormone GPCRs identified in this study. In addition, we made some interesting observations on neurohormone GPCR evolution and the evolution and co-evolution of their ligands. For neuropeptide and protein hormone GPCRs, there appears to be a general co-evolution between receptors and their ligands. This is in contrast to biogenic amine GPCRs, where evolutionarily unrelated GPCRs often bind to the same biogenic amine, suggesting frequent ligand exchanges ("ligand hops") during GPCR evolution.     

The chemoreceptor superfamily in the honey bee, Apis mellifera: expansion of the odorant, but not gustatory, receptor family

The honey bee genome sequence reveals a remarkable expansion of the insect odorant receptor (Or) family relative to the repertoires of the flies Drosophila melanogaster and Anopheles gambiae, which have 62 and 79 Ors respectively. A total of 170 Or genes were annotated in the bee, of which seven are pseudogenes. These constitute five bee-specific subfamilies in an insect Or family tree, one of which has expanded to a total of 157 genes encoding proteins with 15%-99% amino acid identity. Most of the Or genes are in tandem arrays, including one with 60 genes. This bee-specific expansion of the Or repertoire presumably underlies their remarkable olfactory abilities, including perception of several pheromone blends, kin recognition signals, and diverse floral odors. The number of Apis mellifera Ors is approximately equal to the number of glomeruli in the bee antennal lobe (160-170), consistent with a general one-receptor/one-neuron/one-glomerulus relationship. The bee genome encodes just 10 gustatory receptors (Grs) compared with the D. melanogaster and A. gambiae repertoires of 68 and 76 Grs, respectively. A lack of Gr gene family expansion primarily accounts for this difference. A nurturing hive environment and a mutualistic relationship with plants may explain the lack of Gr family expansion. The Or family is the most dramatic example of gene family expansion in the bee genome, and characterizing their caste- and sex-specific gene expression may provide clues to their specific roles in detection of pheromone, kin, and floral odors.     

Quantitative trait loci that influence the expression of guarding and stinging behaviors of individual honey bees

This study was conducted to test for the effect of three stinging behaviors QTLs (sting-1, sting-2 and sting-3) on the expression of guarding and stinging behavior of individual honey bees, and to determine if results of defensive behavior QTLs found in studies with Africanized honey bees could be extended to other populations of bees. Samples of guards, stingers, foragers and nurse bees were taken from two backcross colonies derived from a defensive colony and a gentle colony. The genotype of each bee for both types of colonies was determined for two sequence tagged site (STS) markers linked to sting-1 and for another two STSs, one linked to sting-2 and one linked to sting-3. Results showed that sting-1 had an effect on the expression of both stinging and guarding behaviors, sting-2 and sting-3 influenced the expression of guarding behavior. These results indicate that division of labor is influenced by specific QTLs. Results also show that QTLs mapped in a population of Africanized honey bees using colony level phenotypes also influenced the expression of guarding and stinging behavior of individual bees of other populations.     

Production, structure and in vitro degradation of electrospun honeybee silk nanofibers

Honeybees produce silken cocoons containing four related fibrous proteins. High levels of each of the honeybee silk proteins can be produced recombinantly by fermentation in Escherichia coli. In this study we have used electrospinning to fabricate a single recombinant honeybee silk protein, AmelF3, into nanofibers of around 200 nm diameter. Infrared spectroscopy found that the molecular structure of the nanofibers was predominantly coiled coil, essentially the same as native honeybee silk. Mats of the honeybee nanofibers were treated with methanol or by water annealing, which increased their β-sheet content and rendered them water insensitive. The insoluble mats were degraded by protease on a time scale of hours to days. The protease gradually released proteins from the solid state and these were subsequently rapidly degraded into small peptides without the accumulation of partial degradation products. Cell culture assays demonstrated that the mats allowed survival, attachment and proliferation of fibroblasts. These results indicate that honeybee silk proteins meet many prerequisites for use as a biomaterial.     

Predictive value of venom-specific IgE, IgG and IgG subclass antibodies in patients on immunotherapy with honey bee venom

Sixty-seven patients with a history of severe systemic reactions following honey bee stings were treated by immunotherapy (IT) with honey bee venom. During maintenance therapy all were submitted to a sting challenge under clinical conditions. 15 developed mostly minor symptoms of a systemic reaction while 52 showed only a local swelling at the sting site. Phospholipase A2-specific IgE, IgG and IgG subclass serum antibodies were estimated in samples obtained before IT and immediately before the challenge. Specific IgE decreased in reactors and in non-reactors. There was no difference between the two groups at any time. Specific total IgG, IgG1 and IgG4 increased in both reactors and non-reactors during IT. An early increase of specific IgG1, was observed while specific IgG4 remained elevated throughout the treatment. Specific total IgG was higher in reactors than non-reactors before the challenge, specific IgG1 higher in reactors before treatment and specific IgG4 higher in reactors than non-reactors both before treatment and before challenge. In the individual patient, no single antibody estimation or combination of various antibodies was predictive of the outcome of a sting challenge.     

Triclosan treatment to pregnant albino rats affects offspring numbers and the liver of both the pregnant rats and their offspring,and these effects are ameliorated by co-treatment with bee honey

We have assessed the effects of the broad-spectrum bactericide triclosan on the liver of pregnant albino rats and their offspring, and evaluated the protective potential of bee honey, which has radical-scavenging properties. The study involved treatment of 72 pregnant rats followed by examination of the pregnant rats and their offspring. The pregnant rats were divided equally into six groups (I–VI), each of which was subdivided equally into two Subgroups (A and B). Rats in the A subgroups were gavaged with a daily dose of 1.26 ml distilled water (IA), 1 ml corn oil (IIA), 1.68 ml aqueous solution of Clover Blossom honey (IIIA), 0.3 mg triclosan (IVA), 13 mg triclosan (VA), or 1.68 ml aqueous solution of honey with 13 mg triclosan (VIA), throughout pregnancy. Rats in the B subgroups received the same treatments throughout pregnancy and for 14 days after delivery. At the end of the experiments, the offspring's numbers were recorded and blood samples were taken from the pregnant rats for analysis. The livers of the studied groups were subjected for; histological study, morphometric analysis, and biochemical estimation of markers of oxidative stress. The results showed that the acceptable daily intake of triclosan did not induce significant pathological changes in the liver while high dose of triclosan induced pathological changes in the livers and reduced the numbers of offspring. Co-administration of honey with triclosan ameliorated most pathological change. Therefore, decrease the exposure of the pregnant women to triclosan is encouraged or co-supplementation with bee honey if exposure could not be avoided.     

Function and evolution of a gene family encoding odorant binding-like proteins in a social insect, the honey bee (Apis mellifera)

The remarkable olfactory power of insect species is thought to be generated by a combinatorial action of two large protein families, G protein-coupled olfactory receptors (ORs) and odorant binding proteins (OBPs). In olfactory sensilla, OBPs deliver hydrophobic airborne molecules to ORs, but their expression in nonolfactory tissues suggests that they also may function as general carriers in other developmental and physiological processes. Here we used bioinformatic and experimental approaches to characterize the OBP-like gene family in a highly social insect, the Western honey bee. Comparison with other insects shows that the honey bee has the smallest set of these genes, consisting of only 21 OBPs. This number stands in stark contrast to the more than 70 OBPs in Anopheles gambiae and 51 in Drosophila melanogaster. In the honey bee as in the two dipterans, these genes are organized in clusters. We show that the evolution of their structure involved frequent intron losses. We describe a monophyletic subfamily of OBPs where the diversification of some amino acids appears to have been accelerated by positive selection. Expression profiling under a wide range of conditions shows that in the honey bee only nine OBPs are antenna-specific. The remaining genes are expressed either ubiquitously or are tightly regulated in specialized tissues or during development. These findings support the view that OBPs are not restricted to olfaction and are likely to be involved in broader physiological functions.     

The behavioral genetics of colony defense in honeybees: Genetic variability for guarding behavior

Guard honeybees stand at the entrance of colonies and facilitate the exclusion of nonnestmates from the colony. In this study, we examined the hypothesis that genetic variability among individuals in colonies might explain variability in guarding activity. To do this, we cross-fostered honey bees between colonies with high-defensive responses and colonies with low-defensive responses in alarm pheromone tests. Individuals from high-defensive colonies were more likely to guard in their own colonies (controls) than cross-fostered bees from low-defensive colonies. Cross-fostered high-defensive bees also were more likely to guard in low-defense colonies. These results support the hypothesis that interindividual differences in guarding behavior are at least partially under genetic control. A positive correlation between number of guards and response to alarm pheromone demonstrates a link between behaviorally separated components of the overall defensive response.This work was supported by NSF Grant BNS 8605604.     

Genotype,Task Specialization,and Nest Environment Influence the Stinging Response Thresholds of Individual Africanized and European Honeybees to Electrical Stimulation

This study was conducted to analyze the stinging response thresholds of individual European and Africanized worker honeybees (Apis mellifera L.) to electrical stimulation. Newly emerged workers were identified, and either were placed into an incubator, into their natal colonies, or cross-fostered in common colonies of European or Africanized ancestry. Nest and guard bees of each type were collected and exposed to an electric stimulus of 0.5 mA, and the time they took to sting a leather substrate was recorded. Africanized bees consistently had significant lower thresholds of defensive response than European bees across all of the environments tested. Guards were faster to sting than nest bees only for the Africanized genotype, suggesting that alleles of African origin have pleiotropic effects on guarding and stinging. This is the first study that shows that single individuals specialized in guarding also may have a lower response threshold for stinging. Environmental effects were also evident. In all cases, bees responded faster to the electrical stimulation after being kept in environments other than their natal nest. Moreover, significant genotype by environment and genotype by task specialization interactions were found. Our results fit a model of division of labor based on differences in response thresholds to stimuli among workers of different genotypes and task groups that result in non-additive effects on colony behavior. Edited by Yong-Kyu Kim.     

Development of the chemosensory apparatus of human tongue in postnatal ontogeny

The development of the chemosensory apparatus of the tongue and taste perception in infants is studied by morphological (scanning electron microscopy) and physiological (vegetative and motor responses) methods. The chemosensory elements on the radix and body of the tongue develop, more rapidly than those on the tip of the tongue. Heterochrony in the development of the sensitivity of the tongue tip to various gustatory stimuli (sweet, bitter, salty, and sour is demonstrated. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 12, pp. 673–677, December, 1996