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The Africanization of honeybees (Apis mellifera L.) of the Yucatan: a study of a massive hybridization event across time.

Until recently, African and European subspecies of the honeybee (Apis mellifera L.) had been geographically separated for around 10,000 years. However, human-assisted introductions have caused the mixing of large populations of African and European subspecies in South and Central America, permitting an unprecedented opportunity to study a large-scale hybridization event using molecular analyses. We obtained reference populations from Europe, Africa, and South America and used these to provide baseline information for a microsatellite and mitochondrial analysis of the process of Africanization of the bees of the Yucatan Peninsula, Mexico. The genetic structure of the Yucatecan population has changed dramatically over time. The pre-Africanized Yucatecan population (1985) comprised bees that were most similar to samples from southeastern Europe and northern and western Europe. Three years after the arrival of Africanized bees (1989), substantial paternal gene flow had occurred from feral Africanized drones into the resident European population, but maternal gene flow from the invading Africanized population into the local population was negligible. However by 1998, there was a radical shift with both African nuclear alleles (65%) and African-derived mitochondria (61%) dominating the genomes of domestic colonies. We suggest that although European mitochondria may eventually be driven to extinction in the feral population, stable introgression of European nuclear alleles has occurred.

Africa↗

Colony-level impacts of immune responsiveness in honey bees, Apis mellifera.

Social insects have evolved both communal and individual traits that reduce the impacts of their numerous parasites and pathogens. Among the individual traits, innate-immune responses have the potential to reduce both individual mortality and the spread of pathogens among colony members. An understanding of the costs and benefits of such responses can provide a more complete understanding of a primary risk of social life, horizontal disease transmission among colony members. Here we assess the impacts of individual immunity on colony-level disease in honey bee (Apis mellifera) colonies following exposure to an important bacterial pathogen (Paenibacillus larvae subsp. larvae, cause of the disease American foulbrood). Colony-level disease rates were negatively correlated with the immune responsiveness of colony members, as assessed by larval transcript levels for the gene encoding the antibacterial peptide abaecin. Concomitantly, colonies whose members mounted a stronger abaecin response showed significantly lower productivity, indicating a colony-level cost to this immune response. The results show considerable variation across colonies in an immune trait important for survival, and point toward a significant trade-off between this trait and colony productivity.

Animals↗

Sperm storage and antioxidative enzyme expression in the honey bee, Apis mellifera.

Honey bee (Apis mellifera) sperm remains viable in the spermatheca of mated female honey bees for several years. During this time, the sperm retains respiratory activity, placing it at risk of the damaging effects of reactive oxygen species common to many biological processes. Antioxidative enzymes might help reduce this damage. Here we use quantitative real-time RT-PCR to establish gene-expression profiles in male and female honey bee reproductive tissues for three antioxidative enzymes: catalase, glutathione-S-transferase (GST) and superoxide dismutase (SOD1, cytosolic). Catalase and GST showed ten- to twenty-fold transcript increases in the sperm storage organs of mated queens vs. unmated queens, whereas SOD1 levels are high in both mated and unmated queens. Male reproductive and somatic tissues showed relatively high levels of all three antioxidant-encoding transcripts. All three enzymes screened were higher in mature males vs. young males, although this effect did not appear to be confined to reproductive tissues and, hence, need not reflect a role in sperm longevity. Furthermore, antioxidative enzyme transcripts remained present, and apparently increased, in male tissues long after sperm had matured and seminal fluid was produced. We also found measurable levels of catalase transcripts in honey bee semen. The presence of catalase transcripts in both reproductive tissues and semen in bees suggests that this enzyme might play a key role in antioxidative protection.

Age Factors↗

Apis mellifera ultraspiracle: cDNA sequence and rapid up-regulation by juvenile hormone.

Two hormones, 20-hydroxyecdysone (20E) and juvenile hormone (JH) are key regulators of insect development including the differentiation of the alternative caste phenotypes of social insects. In addition, JH plays a different role in adult honey bees, acting as a 'behavioural pacemaker'. The functional receptor for 20E is a heterodimer consisting of the ecdysone receptor and ultraspiracle (USP) whereas the identity of the JH receptor remains unknown. We have cloned and sequenced a cDNA encoding Apis mellifera ultraspiracle (AMUSP) and examined its responses to JH. A rapid, but transient up-regulation of the AMUSP messenger is observed in the fat bodies of both queens and workers. AMusp appears to be a single copy gene that produces two transcripts ( approximately 4 and approximately 5 kb) that are differentially expressed in the animal's body. The predicted AMUSP protein shows greater sequence similarity to its orthologues from the vertebrate-crab-tick-locust group than to the dipteran-lepidopteran group. These characteristics and the rapid up-regulation by JH suggest that some of the USP functions in the honey bee may depend on ligand binding.

Amino Acid Sequence↗

Cloning, expression and immunological characterization of full-length timothy grass pollen allergen Phl p 4, a berberine bridge enzyme-like protein with homology to celery allergen Api g 5.

BACKGROUND: Timothy grass pollen is a common cause of respiratory allergy in the temperate regions. The major group 4 allergen, Phl p 4, has previously been purified and studied biochemically and immunologically, but has so far not been produced and characterized as a recombinant protein. OBJECTIVE: To clone and characterize timothy grass pollen allergen Phl p 4. METHODS: Full-length Phl p 4 cDNA was cloned using a PCR-based strategy including 3'-and 5'-RACE. Recombinant Phl p 4 was expressed in Escherichia coli and purified by immobilized metal ion affinity chromatography. Its immunological activity was investigated using experimental ImmunoCAP tests, sera from Phl p 4 sensitized individuals and Phl p 4 reactive polyclonal and monoclonal animal antibodies. RESULTS: Five full-length Phl p 4 cDNA clones were analysed. Sequence deviations between the clones were present at nine amino acid positions, and the consensus sequence comprised an open reading frame of 525 amino acids, including a predicted 25-residue signal peptide. The calculated molecular weight of the deduced mature protein was 55.6 kDa and the isoelectric point 9.9, both consistent with previously observed properties of purified nPhl p 4. Close sequence similarity was found to genomic clones from several other Pooideae grass species and to Bermuda grass pollen allergen BG60. Further, similarity was found to members of the berberine bridge enzyme (BBE) family, including celery allergen Api g 5. Recombinant Phl p 4 bound specific immunoglobulin (Ig)E from 31 of 32 nPhl p 4-reactive sera, and the IgE binding to rPhl p 4 could be inhibited by nPhl p 4 in a dose-dependent manner. CONCLUSIONS: Full-length Phl p 4 cDNA was cloned and showed sequence similarity to members of the BBE family. Recombinant Phl p 4 was produced and shared epitopes with natural Phl p 4.

Allergens↗

Immune pathways and defence mechanisms in honey bees Apis mellifera.

Social insects are able to mount both group-level and individual defences against pathogens. Here we focus on individual defences, by presenting a genome-wide analysis of immunity in a social insect, the honey bee Apis mellifera. We present honey bee models for each of four signalling pathways associated with immunity, identifying plausible orthologues for nearly all predicted pathway members. When compared to the sequenced Drosophila and Anopheles genomes, honey bees possess roughly one-third as many genes in 17 gene families implicated in insect immunity. We suggest that an implied reduction in immune flexibility in bees reflects either the strength of social barriers to disease, or a tendency for bees to be attacked by a limited set of highly coevolved pathogens.

Animals↗

Genome sequences of the honey bee pathogens Paenibacillus larvae and Ascosphaera apis.

Genome sequences offer a broad view of host-pathogen interactions at the systems biology level. With the completion of the sequence of the honey bee, interest in the relevant pathogens is heightened. Here we report the genome sequences of two of the major pathogens of honey bees, the bacterium Paenibacillus larvae (causative agent for American foulbrood disease) and the fungus Ascosphaera apis. (causative agent for chalkbrood disease). Ongoing efforts to characterize the genomes of these species can be used to understand and mitigate the effects of two important pathogens, and will provide a contrast with pathogenic, benign and freeliving relatives.

Animals↗

Evolutionary history of the honey bee Apis mellifera inferred from mitochondrial DNA analysis.

Variability of mitochondrial DNA (mtDNA) of the honey bee Apis mellifera L. has been investigated by restriction and sequence analyses on a sample of 68 colonies from ten different subspecies. The 19 mtDNA types detected are clustered in three major phylogenetic lineages. These clades correspond well to three groups of populations with distinct geographical distributions: branch A for African subspecies (intermissa, monticola, scutellata, andansonii and capensis), branch C for North Mediterranean subspecies (caucasica, carnica and ligustica) and branch M for the West European populations (mellifera subspecies). These results partially confirm previous hypotheses based on morphometrical and allozymic studies, the main difference concerning North African populations, now assigned to branch A instead of branch M. The pattern of spatial structuring suggests the Middle East as the centre of dispersion of the species, in accordance with the geographic areas of the other species of the same genus. Based on a conservative 2% divergence rate per Myr, the separation of the three branches has been dated at about 1 Myr BP.

Animals↗

The purification and characterisation of hyaluronidase from the venom of the honey bee, Apis mellifera.

Hyaluronidase has been purified from the venom of the honey bee, Apis mellifera. The purification proved remarkably difficult, requiring a large number of chromatographic steps culminating in the removal of traces of phospholipase A2 with an affinity purified rabbit anti-phospholipase A2 immunosorbent column. The purified enzyme showed a 1143-fold increase in specific activity and was homogeneous. Electrophoresis in polyacrylamide gels (12%) containing sodium dodecyl sulphate (pH 8.9) or urea (pH 2.8) and electrofocusing in polyacrylamide (5%) gave a single band. The final product contained less than 0.1% phospholipase A2 and less than 1.5% acid phosphatase and gave a single line of precipitation against rabbit anti-hyaluronidase but was not precipitated by rabbit anti-phospholipase A2. Previous reports of instability were not confirmed, and we found the enzyme to be highly stable over a wide range of temperature and pH, and to denaturing agents. Purified hyaluronidase was found to be 'sticky' when highly pure and at low concentration, and adhered strongly to Sephadex G-75. The relative molecular mass was estimated at 35 000-37 000 by gel filtration, and at 41 000 by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. A value of 50 000 was obtained by ultracentrifugation assuming a partial specific volume of 0.73 cm3/g. Hyaluronidase was found to be a minor allergen in bee venom allergic patients.

Amino Acids↗

Isolation and characterization of abaecin, a major antibacterial response peptide in the honeybee (Apis mellifera).

Honeybee (Apis mellifera) are frequently exposed to and likely to be infected by plant-associated bacteria. We mimicked this process by injecting bees with live bacteria and isolated five induced antibacterial substances by comparative liquid chromatographic mapping of the hemolymph. Three of these antibiotics belong to a unique family of small (18 amino acids) peptides: the apidaecins [Casteels et al. (1989) EMBO J. 8, 2387-2391]. We have now characterized a fourth bee immune response peptide. The complete sequence was established by Edman degradation of the peptide and fragments thereof. It is 34 amino acids long and contains 10 proline residues. The amino-terminal half is related to the apidaecins; similar proline motifs are also present in the amino-terminal quarter of the much longer fly diptericins. The newly identified peptide's broad spectrum, lower specific activities against Gram-negative plant pathogens and its inability to inhibit bacterial growth at medium ionic strength are different from the apidaecins. Moreover, the highest observed specific activity was against an apidaecin-resistant Xanthomonas strain. In contrast to the immediate action of apidaecins, bactericidal activity is delayed. We propose the name 'abaecin' for this new antibacterial response peptide.

Amino Acid Sequence↗

GDP-fucose: beta-N-acetylglucosamine (Fuc to (Fuc alpha 1----6GlcNAc)-Asn-peptide)alpha 1----3-fucosyltransferase activity in honeybee (Apis mellifica) venom glands. The difucosylation of asparagine-bound N-acetylglucosamine.

Incubation of honeybee (Apis mellifica) venom-gland extracts with GDP-[14C]fucose and GlcNAc beta 1----2Man alpha 1----6(GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----6)GlcNAc beta 1----N-Asn-peptide(NAc) gave a labeled product in 40% yield. Analysis by 500-MHz 1H-NMR spectroscopy indicated the transferred fucose-(Fuc) residue to be alpha 1----3-linked to the Asn-bound GlcNAc. Further proof was provided by one-dimensional and two-dimensional 1H-NMR analysis of the incubation mixture, after incubation with beta-N-acetylhexosaminidase. The established carbohydrate structure (formula; see text) proves the existence of a novel alpha 1----3-fucosyltransferase with the ability to effect difucosylation of the Asn-bound GlcNAc in N-glycans.

Acetylglucosamine↗

Characterisation of the ultraviolet-sensitive opsin gene in the honey bee, Apis mellifera.

The cDNA sequence of the ultraviolet-sensitive opsin in the honey-bee, Apis mellifera, with associated 5' and 3' untranslated regions, is presented. The analysis of genomic structure reveals seven introns in the coding region of the gene, with six at novel positions for an insect opsin gene. The equivalent site to the counterion in vertebrate opsins is occupied by a Tyr residue. This contrasts with the presence of Phe at this site in the ultraviolet-sensitive opsins of Drosophila sps. A comparison of the amino acid sequence within the seven alpha-helical transmembrane regions of insect ultraviolet/blue-sensitive opsins identifies substitution at five sites that involve either replacement of a polar with a non-polar residue, or a change in charge. Such changes are known to result in spectral shifts in vertebrate pigments. Phylogenetic analysis indicates that the ultraviolet-sensitive pigments represent an ancient class of insect opsins.

Amino Acid Sequence↗

The nitric oxide/cGMP system in the antennal lobe of Apis mellifera is implicated in integrative processing of chemosensory stimuli.

The high concentration and the localization of nitric oxide synthase in the olfactory system of both vertebrates and invertebrates suggest that the diffusible messenger nitric oxide plays a central role in the processing of chemosensory information. This paper describes the nitric oxide releasing system in the antenna and the antennal lobes of Apis mellifera using the NADPH diaphorase technique, and analyses the contribution of the nitric oxide system in the neuronal processing of chemosensory signals using a behavioral assay in vivo. In the antenna the strongest NADPH diaphorase staining is found in non-neuronal auxiliary and/or epithelial cells, while the sensory cells and the antennal nerve are stained at a low level. At the major site of chemosensory signal integration, the antennal lobes, the highest nitric oxide synthase activity is located in the glomeruli, which are ideally suited to act as diffusion compartments. We demonstrate that inhibition of nitric oxide synthase in the antennal lobes specifically interferes with neuronal processing of repetitive chemosensory stimuli but does not affect the response to single stimuli, and is independent of parameters such as satiation level, stimulus strength, interstimulus interval and duration of sensory stimuli. Since inhibition of the soluble guanylate cyclase, a major target of nitric oxide, also particularly affects the adaptive component, the physiological effects of nitric oxide appear to be mediated by the action of cGMP. These findings suggest that the nitric oxide/cGMP system in the antennal lobes is a component of the molecular machinery involved in adaptive and/or integrative mechanisms during chemosensory information processing in vivo.

Animals↗

Odour coding is bilaterally symmetrical in the antennal lobes of honeybees (Apis mellifera).

The primary olfactory neuropil, the antennal lobe (AL) in insects, is organized in glomeruli. Glomerular activity patterns are believed to represent the across-fibre pattern of the olfactory code. These patterns depend on an organized innervation from the afferent receptor cells, and interconnections of local interneurons. It is unclear how the complex organization of the AL is achieved ontogenetically. In this study, we measured the functional activity patterns elicited by stimulation with odours in the right and the left AL of the same honeybee (Apis mellifera) using optical imaging of the calcium-sensitive dye calcium green. We show here that these patterns are bilaterally symmetrical (n=25 bees). This symmetry holds true for all odours tested, irrespective of their role as pheromones or as environmental odours, or whether they were pure substances or complex blends (n=13 odours). Therefore, we exclude that activity dependent mechanisms local to one AL determine the functional glomerular activity. This identity is genetically predetermined. Alternatively, if activity dependent processes are involved, bilateral connections would have to shape symmetry, or, temporal constraints could lead to identical patterns on both sides due to their common history of odour exposure.

Acyclic Monoterpenes↗

Am5-HT7: molecular and pharmacological characterization of the first serotonin receptor of the honeybee (Apis mellifera).

The biogenic amine serotonin (5-HT) plays a key role in the regulation and modulation of many physiological and behavioural processes in both vertebrates and invertebrates. These functions are mediated through the binding of serotonin to its receptors, of which 13 subtypes have been characterized in vertebrates. We have isolated a cDNA from the honeybee Apis mellifera (Am5-ht7) sharing high similarity to members of the 5-HT(7) receptor family. Expression of the Am5-HT(7) receptor in HEK293 cells results in an increase in basal cAMP levels, suggesting that Am5-HT(7) is expressed as a constitutively active receptor. Serotonin application to Am5-ht7-transfected cells elevates cyclic adenosine 3',5'-monophosphate (cAMP) levels in a dose-dependent manner (EC(50) = 1.1-1.8 nm). The Am5-HT(7) receptor is also activated by 5-carboxamidotryptamine, whereas methiothepin acts as an inverse agonist. Receptor expression has been investigated by RT-PCR, in situ hybridization, and western blotting experiments. Receptor mRNA is expressed in the perikarya of various brain neuropils, including intrinsic mushroom body neurons, and in peripheral organs. This study marks the first comprehensive characterization of a serotonin receptor in the honeybee and should facilitate further analysis of the role(s) of the receptor in mediating the various central and peripheral effects of 5-HT.

Amino Acid Sequence↗

Modification of potassium movement through the retina of the drone (Apis mellifera male) by glial uptake.

Intracellular recordings were made in photoreceptors and glial cells (outer pigment cells) of the superfused cut head of the honey-bee drone (Apis mellifera male). When the [K+] in the superfusate was abruptly increased from 3.2 mM to 17.9 mM both photoreceptors and glial cells depolarized. The time course of the depolarization of the photoreceptors was slower with increasing depth from the surface. Half time of depolarization was plotted against depth: this graph was compatible with the arrival of K+ being exclusively by diffusion through the extracellular clefts. However, as we then showed, this interpretation is inadequate. The time course of depolarization of the glial cells was almost the same at all depths. This indicates that they are electrically coupled. Consequently, current-mediated K+ flux (spatial buffering) through glial cells will contribute to the transport of K+ through the tissue: K+ ions enter the glial syncytium in the region of high external potassium concentration, [K+]0, and an equivalent quantity of K+ ions leave in regions of low [K+]0. Intracellular K+ activity (aiK) was measured with double-barrelled K+-sensitive micro-electrodes in slices of retina superfused on both faces. When [K+] in the superfusate was increased from 7.5 mM to 17.9 mM an increase in aiK was observed in glial cells at all depths in the slice (initial rate 1.7 mM min-1, S.E. of the mean = 0.2 mM min-1), but there was little increase in the photoreceptors (0.3 +/- 0.2 mM min-1). The increase in aiK in glial cells near the centre of the slice could not have been caused by spatial buffering; it presumably resulted from net uptake. We conclude that when [K+] is increased at the surface of this tissue, the build up of K+ in the extracellular clefts depends on extracellular diffusion, spatial buffering and net uptake. The latter two processes, which have opposing effects, involve about 10 times as much K+ as the first. This is in rough agreement with less direct experiments on mammalian brain (Gardner-Medwin, 1977, 1983b).

Animals↗

Thrice out of Africa: ancient and recent expansions of the honey bee, Apis mellifera.

We characterized Apis mellifera in both native and introduced ranges using 1136 single-nucleotide polymorphisms genotyped in 341 individuals. Our results indicate that A. mellifera originated in Africa and expanded into Eurasia at least twice, resulting in populations in eastern and western Europe that are geographically close but genetically distant. A third expansion in the New World has involved the near-replacement of previously introduced "European" honey bees by descendants of more recently introduced A. m. scutellata ("African" or "killer" bees). Our analyses of spatial transects and temporal series in the New World revealed differential replacement of alleles derived from eastern versus western Europe, with admixture evident in all individuals.

Africa↗

Use of the API rapid NFT system for identifying nonfermentative and fermentative marine bacteria.

Thirty-five American Type Culture Collection type strains of marine bacteria were used to evaluate the Rapid NFT system (API Analab Products, Plainview, N.Y.) for use in identifying heterotrophic marine bacteria. The 21 biochemical and assimilation tests on the Rapid NFT test strips were treated according to the manufacturer's protocol, which included use of AUX medium (provided with the Rapid NFT system) for preparing assimilation tests, and by substituting phenol red broth base (BBL Microbiology Systems, Cockeysville, Md.) with and without an oil overlay for the AUX medium. A seven-digit numerical profile was obtained for each NFT test strip from each of the three procedures and matched to its corresponding number in the Rapid NFT identification codebook. Also, all biochemical and assimilation test results were analyzed with SASTAXAN and SAS/GRAPH programs (SAS Institute, Inc., Cary, N.C.); similarity matrices were computed for all 35 strains. For comparison purposes, bacterial strains were grouped at a similarity level of 70%. The results indicated a low efficacy of identification for all three procedures. In addition, similarity matrix analysis showed more cohesive grouping based on results of phenol red broth base-treated strains than for the AUX medium provided by the manufacturer. However, none of the three treatments provided exclusive grouping of type strains at the genus level. Thus, the reliability of the data obtained from the NFT system and modifications thereof should be evaluated carefully when environmental isolates are characterized.

Bacteria↗