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A liquid chromatographic tandem mass spectrometric method for the determination of sulfa drugs in beeswax was developed. When performing residue control on beeswax intended for the fabrication of wax foundations, residues of sulfonamides were found. A migration test was set up to study whether sulfonamide-containing beeswax could lead to the contamination of honey. The higher the concentration of sulfamethazine doped in the wax, the higher was the concentration of sulfamethazine found in the honey. The maximum transfer was 15.6, 56.9, and 29.5% of the initial amount spiked in the wax foundation. In a second experiment, the percentage of sulfamethazine migrating from medicated winter feed to beeswax in relation to the concentration in the syrup and the contact time was studied. The maximum transfer of sulfamethazine from medicated sucrose syrup to beeswax was 3.1%.

We report on the first proficiency test in the context of honey bee disease testing on a broad international scale. Honey samples were distributed to 12 participating laboratories for isolation and identification of spores of the etiological agent of American foulbrood, Paenibacillus larvae. Of the 11 laboratories responding to this proficiency testing event, only 6 (54%) provided data that were in full agreement with the previously determined status of the honey samples; eight (72%) laboratories fell within the threshold for qualification as defined a priori in this proficiency test (=level of agreement of at least 90%). Some lessons can now be drawn from the organizational point of view and will certainly improve new initiatives to be taken.

In this study an important and often neglected aspect of gene expression studies in insects, the validation of appropriate reference genes with stable expression levels between sample groups, is addressed. Although in this paper the reference gene selection for the honeybee, Apis mellifera L. (Hymenoptera: Apidae) head was tested in the context of bacterial challenge with Escherichia coli, this work can serve as a resource to help select and screen insect reference genes for gene expression studies in any tissue and under any experimental manipulation. Since it is recommended to use multiple reference genes for accurate normalization, we analyzed the expression of eleven candidate reference genes in the honeybee head, for their potential use in the analysis of differential gene expression following bacterial challenge. Three software programs, BestKeeper, Normfinder and geNorm, were used to assess candidate reference genes. GeNorm recommended the use of four reference genes. Both geNorm and Normfinder identified the genes GAPDH, RPS18, actin and RPL13a as the most stable ones, only differing in their ranking order. BestKeeper identified RPS18 as being the reference gene with the least overall variation, but also actin and GAPDH were found to be the second and third most stable expressed gene. By a combination of three software programs the genes actin, RPS18 and GAPDH were found suitable reference genes in the honeybee head in the context of bacterial infection.

Bidirectional interactions between the immune and nervous systems are well established in vertebrates. Insects show similar neuro-immune-behavioral interactions to those seen in vertebrates. Using quantitative real-time PCR, we present evidence that gene expression in the honeybee head is influenced by activation of the immune system 8h after a bacterial challenge with Escherichia coli. Seven genes were selected for quantitative analysis in order to cover both typical functions of the head such as exocrine secretion (mrjp3 and mrjp4) and olfactory processes (obp17) as well as more general processes such as structural functions (mlc2 and paramyosin), stress response (ERp60) and energy housekeeping (enolase). In this way, we show at the molecular level that the immune system functions as a sensory organ in insects -- as it does in vertebrates -- which signals to the head that a bacterial infection is present, and leads to regulation of expression of several genes in the head by a yet unidentified mechanism.

The study evaluated the technical feasibility of distinguishing the different honeybee blood cells by flow cytometric analysis with and without staining by 3 lectins: soybean agglutinin (SBA), concanavalin A (ConA) and wheat germ agglutinin (WGA). Flow cytometric analysis of unstained cells provided a forward scatter-side scatter dotplot with no distinct haemocyte population. Examination of blood samples stained with FITC-labelled ConA or WGA revealed a notable population of fluorescently marked cells on FL1-histo- grams. Microscopic analysis, run parallel, demonstrated low fluorescence of the granular cells, strong fluo- rescence of the plasmatocytes, with P1's stained all over their surfaces and P2's with a rather dotted appearence. Prohaemocytes were not stained at all. SBA-FITC did not stain honeybee haemocytes as demon- strated by both the flow cytometric and the microscopic examinations honeybee / haemocytes / flow cytometry / lectin staining

The aim of this investigation was to establish whether Varroa destructor can play a role in the transmission of Paenibacillus larvae larvae spores from infected to healthy bee colonies. Mites, collected from an Apis mellifera carnica colony heavily infected with American foulbrood and treated with Apistan®, were suspended in distilled water and treated in three different ways: homogenizing, shaking and stirring, or sonication. The resulting fluid samples were transferred onto selective agar medium. All culture plates showed colonies that could be identified as P. l. larvae. In view of the numbers of spores they can carry, it is concluded that mites may transmit American foulbrood from infected to healthy bee colonies.

(2010). Honey bee colony losses in Belgium during the 2008–9 winter. Journal of Apicultural Research: Vol. 49, No. 4, pp. 337-339.

The early infection of the honeybee midgut by the microsporidian parasite Nosema apis was examined with both light and electron microscopy. Newly ingested spores were found closer to the apex of the epithelium as they moved toward the distal end. The initial infection of the epithelial cells was restricted mainly to a very narrow area, at the posterior end of the midgut. Neither released sporoplasms in the midgut lumen, nor were polar tubes penetrating the host cell plasmalemma detected. The differences in the fine structure between the first and second meronts were minor.

Nosema apis spore hatching was examined in different salt solutions. Best results were obtained in 0.5 M NaCl + 0.5 M NaHCO3 at pH 6. No differences could be detected between sodium and potassium salts. The differences in efficacy between monovalent anions were more obvios, and a rank order could be formed: Cl- > Br- > F- > I-. The carbohydrate content of ungerminated and germinated spores was measured by GC-MS analysis. Only a very small shift in the ratio αα-trehalose/ d -glucose was found. Scanning electron microscopy of extruded spores was done. No convincing images of released sporoplasms were obtained.

Honey bee workers use venom for the defence of the colony and themselves when they are exposed to dangers and predators. It is produced by a long thin, convoluted, and bifurcated gland, and consists of several toxic proteins and peptides. The present study was undertaken in order to identify the mechanisms that protect the venom gland secretory cells against these harmful components. Samples of whole venom glands, including the interconnected reservoirs, were separated by two-dimensional gel electrophoresis and the most abundant protein spots were subjected to mass spectrometric identification using MALDI TOF/TOF-MS and LC MS/MS. This proteomic study revealed four antioxidant enzymes: CuZn superoxide dismutase (SOD1), glutathione-S-transferase sigma 1 isoform A (GSTS1), peroxiredoxin 2540 (PXR2540) and thioredoxin peroxidase 1 isoform A (TPX1). Although glutathione-S-transferase (GST) has also been associated with xenobiotic detoxification, the protein we found belongs to the GST Sigma class which is known to protect against oxidative stress only. Moreover, we could demonstrate that the GST and SOD activity of the venom gland was low and moderate, respectively, when compared to other tissues from the adult honey bee. Several proteins involved in other forms of stress were likewise found but it remains uncertain what their function is in the venom gland. In addition to major royal jelly protein 9 (MRJP9), already found in a previous proteomic study, we identified MRJP8 as second member of the MRJP protein family to be associated with the venom gland. Transcripts of both MRJPs were amplified and sequenced. Two endocuticular structural proteins were abundantly present in the 2D-gel and most probably represent a structural component of the epicuticular lining that protects the secretory cells from the toxins they produce.

The 1045bp full-length cDNA sequence of a new bee venom component was obtained by rapid amplification of cDNA ends. The 672bp coding sequence corresponds to a protein with a signal peptide and multiple carbohydrate binding sites, and it was named icarapin. It has the new consensus sequence N-[TS]-T-S-[TV]-x-K-[VI](2)-[DN]-G-H-x-V-x-I-N-[ED]-T-x-Y-x-[DHK]-x(2,6)- [STA]-[VLFI]-x-[KR]-V-R-[VLI]-[IV]-[DN]-V-x-P. At least two transcript variants were found. Recombinant icarapin was tested for recognition by IgE antibodies and gave a positive dot blot with sera from 4 out of 5 bee venom allergic patients, all beekeepers. Indirect immunofluorescent staining localized the protein in the cuticular lining of the venom duct.

We provide the link between population dynamics and the dynamics of Darwinian evolution via studying the joint population dynamics of similar populations. Similarity implies that the relative dynamics of the populations is slow compared to, and decoupled from, their aggregated dynamics. The relative dynamics is simple, and captured by a Taylor expansion in the difference between the populations. The emerging evolution is directional, except at the singular points of the evolutionary state space. Here ‘‘evolutionary branching’’ may occur. The diversification of life forms thus is demonstrated to be a natural consequence of the Darwinian process.

The aim of this investigation was to establish whether Varroa destructor can play a role in the transmission of Paenibacillus larvae larvae spores from infected to healthy bee colonies. Mites, collected from an Apis mellifera carnica colony heavily infected with American foulbrood and treated with Apistan, were suspended in distilled water and treated in three different ways:homogenizing, shaking and stirring, or sonication. The resulting fluid samples were transferred onto selective agar medium. All culture plates showed colonies that could be identified as P.l. larvae. In view of the numbers of spores they can carry, it is concluded that mites may transmit American foulbrood from infected to healthy bee colonies.

A survey based on the bacteriological analysis of 1328 honey samples from the harvest in the summer of 1999 yielded 146 samples contaminated with P. larvae spores (11.0%), ranging from the detection of only a single colony of P. larvae to completely overgrown plates. Risk factor analy- sis by logistic regression modelling revealed that honey samples collected in the proximity of apiaries with AFB cases from 1998 were at 3-times higher risk of being contaminated compared to other geographic locations (odds ratio = 3.04; P < 0.001). Comb replacement was found to lower the risk (odds ratio = 0.46; P = 0.033) and should be encouraged in beekeeping management. There was also a relation between the positive honey samples collected in 1999 and the cases of AFB in the year 2000 (odds ratio = 2.64; P = 0.009), supporting the value of honey analysis for sanitary control of bee colonies. Paenibacillus larvae larvae / American foulbrood / honey / risk factor analysis / prevalence

SummaryA comparative analysis of fatty acids and sterols in colony- and laboratory-stored pollen from the same source revealed some important differences. The sterol ester concentration was 56% higher in pollen stored in the colony; there was also a much lower concentration of the essential fatty acids linoleic (C18: 2) and linolenic (C18: 3), and more of the saturated C16 and C18 acids.

SummaryThe most important changes in lipid composition during larval development in the honeybee, Apis mellifera, were noted in the total weight of the fatty acid classes. Palmitic acid and a C18:1 acid, presumably oleic, were the most abundant fatty acids in the different lipid classes during larval development, together accounting for more than 70% of the total. The proportions of the fatty acids changed very little during development. Of the sterols, 24-methylene cholesterol was most abundant in both the free and esterified forms except on the first and second days when cholesterol ester was more abundant. Cholesterol-ester concentration decreased in such a way that on the fifth larval day the percentage composition of the sterol esters was almost identical to that of the free sterols. The composition of the free sterols remained practically constant during development.

SummaryCaged workers fed colony-stored pollen had a lower mortality than those fed laboratory-stored pollen. For Nosema-infected bees the difference was even greater.

International audience

International audience