Effect of bacterial metabolites on microsporidian Nosema ceranae and on its host Apis mellifera

Nosemosis, a disease caused by a microsporidian infection, is one of the most frequently observed parasitic pathologies affecting adult honeybees. Presently, Nosema ceranae seems to be the main microsporidian infection in Apis mellifera. The antibiotic fumagillin is the only compound available to treat Nosema diseases; however, it is no longer licensed in most EU member states; therefore, the need to identify new molecules/substances prevails. The intent of this paper is to test bacterial metabolites by Bacillus and Enterococcus strains, isolated from bee midgut and honey. The toxicity on bees and the antiparasitic activity on N. ceranae were assessed under laboratory conditions. Results did not yield toxicity for the administered surfactin or bacteriocin concentrations. Spores exposed to direct contact with a particular surfactin revealed a significant infectivity reduction when inoculated on bees. This surfactin, administered ad libitum from the individuals’ emergence, led to a significant reduction in parasitosis development when bees were infected with untreated spores 7 days postemergence. Based on the results obtained, one of the surfactins is herein postulated as a molecule capable of reducing N. ceranae development, acting either by direct exposure to purified spores or incorporated into the digestive tract of the bee.     

The microsporidian parasites Nosema ceranae and Nosema apis are widespread in honeybee (Apis mellifera) colonies across Scotland

Nosema ceranae is spreading into areas where Nosema apis already exists. N. ceranae has been reported to cause an asymptomatic infection that may lead, ultimately, to colony collapse. It is thought that there may be a temperature barrier to its infiltration into countries in colder climates. In this study, 71 colonies from Scottish Beekeeper’s Association members have been screened for the presence of N. apis and N. ceranae across Scotland. We find that only 11 of the 71 colonies tested positive for spores by microscopy. However, 70.4 % of colonies screened by PCR revealed the presence of both N. ceranae and N. apis, with only 4.2 or 7 % having either strain alone and 18.3 % being Nosema free. A range of geographically separated colonies testing positive for N. ceranae were sequenced to confirm their identity. All nine sequences confirmed the presence of N. ceranae and indicated the presence of a single new variant. Furthermore, two of the spore-containing colonies had only N. ceranae present, and these exhibited the presence of smaller spores that could be distinguished from N. apis by the analysis of average spore size. Differential quantification of the PCR product revealed N. ceranae to be the dominant species in all seven samples tested. In conclusion, N. ceranae is widespread in Scotland where it exists in combination with the endemic N. apis. A single variant, identical to that found in France (DQ374655) except for the addition of a single nucleotide polymorphism, is present in Scotland.     

Comparison of the energetic stress associated with experimental Nosema ceranae and Nosema apis infection of honeybees (Apis mellifera)

Nosema ceranae is a relatively new and widespread parasite of the western honeybee Apis mellifera that provokes a new form of nosemosis. In comparison to Nosema apis, which has been infecting the honeybee for much longer, N. ceranae seems to have co-evolved less with this host, causing a more virulent disease. Given that N. apis and N. ceranae are obligate intracellular microsporidian parasites, needing host energy to reproduce, energetic stress may be an important factor contributing to the increased virulence observed. Through feeding experiments on caged bees, we show that both mortality and sugar syrup consumption were higher in N. ceranae-infected bees than in N. apis-infected and control bees. The mortality and sugar syrup consumption are also higher in N. apis-infected bees than in controls, but are less than in N. ceranae-infected bees. With both microsporidia, mortality and sugar syrup consumption increased in function of the increasing spore counts administered for infection. The differences in energetic requirements between both Nosema spp. confirm that their metabolic patterns are not the same, which may depend critically on host–parasite interactions and, ultimately, on host pathology. The repercussions of this increased energetic stress may even explain the changes in host behavior due to starvation, lack of thermoregulatory capacity, or higher rates of trophallaxis, which might enhance transmission and bee death.     

Critical aspects of the Nosema spp. diagnostic sampling in honey bee (Apis mellifera L.) colonies

Nosemosis is one of the most widespread of the adult honey bee diseases and causes major economic losses to beekeepers. Two microsporidia have been described infecting honey bees worldwide, Nosema apis and Nosema ceranae, whose seasonality and pathology differ markedly. An increasing prevalence of microsporidian infections in honey bees has been observed worldwide during the last years. Because nosemosis has detrimental effects on both strength and productivity of the infected colonies, an accurate and reliable method to evaluate the presence of Nosema in honey bee colonies is needed. In this study a high degree of variability in the detection of microsporidia depending on the random subsample analyzed was found, suggesting that both sample size and the time of collection (month and day of sampling) notably affect the diagnosis.     

Senescence and learning in honeybee (Apis mellifera) workers

Foraging by honeybee workers was investigated from the moment of the first foraging flight until death. To minimise the influence of factors other than senescence the foragers were trained to collect food from an artificial flower close to their hive. During each foraging trip the workers repeatedly visited an artificial flower, collecting one microlitre of 50% sugar solution per visit. During the first 50 flights the mean time taken to collect one portion of food decreased significantly and the number of visits to the artificial flower per flight increased significantly. During flights following the 50th flight, the mean time taken to collect one portion of food increased significantly and the number of visits to the artificial flower per flight decreased significantly. The results confirm earlier observations that the foraging behaviour of honeybee workers is not only influenced by learning, but also by the effects of senescence.     

Computational analysis and identification of an emergent human adenovirus pathogen implicated in a respiratory fatality

Adenoviral infections are typically acute, self-limiting, and not associated with death. However, we present the genomic and bioinformatics analysis of a novel recombinant human adenovirus (HAdV-D56) isolated in France that caused a rare neonatal fatality, and keratoconjunctivitis in three health care workers who cared for the neonate. Whole genome alignments revealed the expected diversity in the penton base, hexon, E3, and fiber coding regions, and provided evidence for extensive recombination. Bootscan analysis confirmed recombination between HAdV-D9, HAdV-D26, HAdV-D15, and HAdV-D29 in the penton base and hexon proteins, centered around hypervariable loops within the putative proteins. Protein structure analysis of the fiber coding region revealed similarity with HAdV-D8, HAdV-D9, and HAdV-D53, possibly accounting for the ocular tropism of the virus. Based on these data, this virus appears to be a new HAdV-D type (HAdV-D56), underscoring the importance of recombination events in human adenovirus evolution and the emergence of new adenovirus pathogens.     

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.     

Enantioselectivity of odor perception in honeybees (Apis mellifera carnica).

The authors tested the ability of 60 free-flying honeybees (Apis mellifera carnica) to discriminate a conditioning odor from an array of 26 simultaneously presented substances. The stimuli included 10 pairs of enantiomers and 6 essential oils. The bees (a) significantly distinguished between 98% of the 540 odor pairs tested, thus showing an excellent overall discrimination performance, and (b) were able to discriminate between the optical isomers of limonene, alpha-pinene, beta-citronellol, menthol, and carvone but failed to distinguish between the (+)- and (-)-forms of alpha-terpineol, camphor, rose oxide, fenchone, and 2-butanol. The findings support the assumptions that enantioselective molecular odor receptors may exist only for some volatile enantiomers and that insects and mammals may share common principles of odor quality perception, irrespective of their completely differing repertoires of olfactory receptors.     

A new genotype of the Sacbrood virus in honeybee Apis mellifera

The sacbrood virus (SBV) causes the death of honeybee larvae. Until recently, three SBV genotypes were known: European, Asian, and South African [7, 9]. Serological assay, sequencing, and phylogenetic analysis of the SBV RNA polymerase gene fragment have revealed two genotypes of SBV circulating among honeybee Apis mellifera in the European region of the Russian Federation (RF); one of them forms an independent, not previously described genetic lineage (genotype 4), which occupies an intermediate position on the phylogenetic tree between the Asian and the South African virus genotypes. The strain 202/8 isolated in 2006 in Moscow oblast from honeybees previously imported from Uzbekistan and two variants (Tr5 and 6/A3) of the laboratory strain isolated in Kaluga oblast in 1986 belong to this group of viruses. The homology between them is 94.2%, while that with representatives of other groups does not exceed 80.6?C83.5%. Another group of SBVs comprise European genotype viruses circulating in Stavropol krai, Adygea, and Moscow oblast (isolates 207/4 and 212/3). The homology between them is 99%. The two SBV genotypes found in the RF were differentiated by PCR and RID (radial immunodiffusion test). As for RID, both genotypes react with antiserum no. 6(1), but only the fourth genotype reacts with antiserum no. 58.     

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).     

Comparative analysis of detection limits and specificity of molecular diagnostic markers for three pathogens (Microsporidia, Nosema spp.) in the key pollinators Apis mellifera and Bombus terrestris

Global pollinator decline has recently been discussed in the context of honey and bumble bee infections from various pathogens including viruses, bacteria, microsporidia and mites. The microsporidian pathogens Nosema apis, Nosema ceranae and Nosema bombi may in fact be major candidates contributing to this decline. Different molecular and non-molecular detection methods have been developed; however, a comparison, especially of the highly sensitive PCR based methods, is currently lacking. Here, we present the first comparative quantitative real-time PCR study of nine Nosema spp. primers within the framework of primer specificity and sensitivity. With the help of dilution series of defined numbers of spores, we reveal six primer pairs amplifying N. apis, six for N. bombi and four for N. ceranae. All appropriate primer pairs detected an amount of at least 10⁴ spores, the majority of which were even as sensitive to detect such low amounts as 10³ to ten spores. Species specificity of primers was observed for N. apis and N. bombi, but not for N. ceranae. Additionally, we did not find any significant correlation for the amplified fragments with PCR efficiency or the limit of detection. We discuss our findings on the background of false positive and negative results using quantitative real-time PCR. On the basis of these results, future research might be based on appropriate primer selection depending on the experimental needs. Primers may be selected on the basis of specificity or sensitivity. Pathogen species and load may be determined with higher precision enhancing all kinds of diagnostic studies.     

Behavioral development and olfactory learning in the honeybee (Apis mellifera)

Honeybees (Apis mellifera) can be readily conditioned in the laboratory to specific odors paired with a subsequent sucrose reward. A series of experiments are reported which demonstrate that the ability of bees to acquire and retain this learning is affected by the stage of behavioral development (caste). Results show rapid acquisition of the proboscis extension reflex (PER) conditioning in adult forager bees, however much slower acquisition and poorer retention of the same learning paradigm in younger adults, i.e., nurse bees and guard bees. Further, if nurse bees are made to forage precocially by manipulation of the hive population, these bees show excellent acquisition and retention of PER conditioning comparable to normal adult forager bees. Results are discussed in terms of olfactory learning requirements of bees performing caste‐specific behaviors and the maturation of the bee nervous system. © 1999 John Wiley & Sons, Inc. Dev Psychobiol 34: 21–27, 1999     

Canonical TTAGG-repeat telomeres and telomerase in the honey bee, Apis mellifera

The draft assembly of the honey bee Apis mellifera genome sequence reveals that the 17 centromeric-distal telomeres are of a simple, shared, and canonical structure, with 3-4 kb of a unique subtelomeric sequence, followed by several kilobases of TTAGG or variant telomeric repeats. This simple subtelomeric structure differs from the centromeric-proximal telomeres on the short arms of the 15 acrocentric chromosomes, which are apparently composed primarily of the 176-bp AluI tandem repeat. This dichotomy between the distal and proximal telomeres may involve differential participation of the telomeres of the 15 acrocentric chromosomes in the Rabl configuration after mitosis and the chromosome bouquet in meiotic prophase I. As expected from the presence of canonical TTAGG telomeric repeats, we identified a candidate telomerase gene in the bee, as well as the silkmoth Bombyx mori and the flour beetle Tribolium castaneum.     

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).     

Dopamine modulation of honey bee (Apis mellifera) antennal-lobe neurons

Primary olfactory centers [antennal lobes (ALs)] of the honey bee brain are invaded by dopamine (DA)-immunoreactive neurons early in development (pupal stage 3), immediately before a period of rapid growth and compartmentalization of the AL neuropil. Here we examine the modulatory actions of DA on honey bee AL neurons during this period. Voltage-clamp recordings in whole cell configuration were used to determine the effects of DA on ionic currents in AL neurons in vitro from pupal bees at stages 4-6 of the nine stages of metamorphic adult development. In approximately 45% of the neurons tested, DA (5-50 x 10(-5) M) reduced the amplitude of outward currents in the cells. In addition to a slowly activating, sustained outward current, DA reduced the amplitude of a rapidly activating, transient outward conductance in some cells. Both of the currents modulated by DA could be abolished by the removal of Ca2+ from the external medium or by treatment of cells with charybdotoxin (2 x 10(-8) M), a blocker of Ca2+-dependent K+ currents in the cells. Ca2+ currents were not affected by DA, nor were A-type K+ currents (I(A)). Results suggest that the delayed rectifier-like current (I(KV)) also remains intact in the presence of DA. Taken together, our data indicate that Ca2+-dependent K+ currents are targets of DA modulation in honey bee AL neurons. This study lends support to the hypothesis that DA plays a role in the developing brain of the bee.     

The ontogeny of kin discrimination cues in the honey bee,Apis mellifera

Cues used in the discrimination of relatives from nonrelatives by the honey bee reflect both genetic and environmental differences between groups. Discrimination is behaviorally expressed by acceptance into or agonistic rejection from the social group. We examine the development of these cues in field colonies and in controlled laboratory settings. Newly emerged worker honey bees are accepted by honey bee social groups at a high frequency. When bees are kept in a controlled laboratory environment for 5 days, acceptability into laboratory groups is determined largely by relatedness. Cues indicating relatedness develop in the laboratory within 12 h after the adult bee emerges. Bees older than 12 h are not accepted by field colonies regardless of relatedness. Bees maintained in a hive until 5 days after emergence are not accepted by related or unrelated laboratory groups (this is termed the hive effect). Bees maintained in hives for times as short as 5 h acquired the hive effect. In a cross-fostering experiment, the hive effect completely masked genetic differences.This work was supported by NSF Grants BNS 82-16787 and BNS 86-05604.     

Hemolectin expression reveals functional heterogeneity in honey bee (Apis mellifera) hemocytes

The identification of molecular markers considerably facilitated the classification and functional analysis of blood cell types. Apis mellifera hemocytes have been classified by morphological criteria and lectin binding properties; however, the use of molecular markers has been minimal. Here we describe a monoclonal antibody to a non-phagocytic subpopulation of A. mellifera hemocytes and to a constituent of the hemolymph clot. We demonstrate that the antibody identifies the A. mellifera hemolectin, a protein carrying human von Willebrand factor homology domains, characteristic of proteins involved in blood coagulation and platelet aggregation in mammals. Hemolectin expressing A. mellifera hemocytes contain the protein as cytoplasmic granules and contribute to the formation of a protein matrix, building up around foreign particles. Consequently, hemolectin as a marker molecule reveals a clear functional heterogeneity of hemocytes, allowing for the analytical separation of hemocyte classes, and could promote the molecular identification of hemocyte lineages in A. mellifera.     

Course of infection with the emergent pathogen Brucella microti in immunocompromised mice

A new Brucella species, Brucella microti, has been isolated from wild rodents and found to be pathogenic in mice. The biological relevance of this new mouse pathogen is clear, as it allows us to study Brucella infection in a species-specific model. The course of infection in wild-type (wt) and immunodeficient mice that lack B (Jh), T and B (SCID), or T, B, and NK (SCID.Beige) cells was analyzed over 3 weeks. wt mice completely cleared bacteria from the liver and spleen after that time. However, SCID mice showed a much higher bacterial load in the spleen and liver than wt and Jh mice after 1 week and maintained the same level during the next 2 weeks. All mice tested survived for the 3 weeks. In contrast, the bacterial levels in mice that lacked NK cell activity progressively increased and these mice succumbed to infection after 16 to 18 days. Histopathology analysis of infected mice showed extensive areas of necrotic tissue and thrombosis in liver after 1 week in all infected SCID.Beige mice but were not seen in either SCID or wt animals. These processes were dramatically increased after 21 days, corresponding with the death of SCID.Beige animals. Our results indicate that T and/or B cells are required for the control of infection with the mouse pathogen Brucella microti in liver and spleen but that NK cells are crucial for survival in the absence of B and T cells. In addition, they suggest that controlled granuloma formation is critical to clear this type of infection in wt mice.