Honey Bee Samples Research Articles

Genome Characterization, Prevalence and Distribution of a Macula-Like Virus from Apis mellifera and Varroa destructor.

Around 14 distinct virus species-complexes have been detected in honeybees, each with one or more strains or sub-species. Here we present the initial characterization of an entirely new virus species-complex discovered in honeybee (Apis mellifera L.) and varroa mite (Varroa destructor) samples from Europe and the USA. The virus has a naturally poly-adenylated RNA genome of about 6500 nucleotides with a genome organization and sequence similar to the Tymoviridae (Tymovirales; Tymoviridae), a predominantly plant-infecting virus family. Literature and laboratory analyses indicated that the virus had not previously been described. The virus is very common in French apiaries, mirroring the results from an extensive Belgian survey, but could not be detected in equally-extensive Swedish and Norwegian bee disease surveys. The virus appears to be closely linked to varroa, with the highest prevalence found in varroa samples and a clear seasonal distribution peaking in autumn, coinciding with the natural varroa population development. Sub-genomic RNA analyses show that bees are definite hosts, while varroa is a possible host and likely vector. The tentative name of Bee Macula-like virus (BeeMLV) is therefore proposed. A second, distantly related Tymoviridae-like virus was also discovered in varroa transcriptomes, tentatively named Varroa Tymo-like virus (VTLV).

Differential diagnosis of the honey bee trypanosomatids Crithidia mellificae and Lotmaria passim

Trypanosomatids infecting honey bees have been poorly studied with molecular methods until recently. After the description of Crithidia mellificae (Langridge and McGhee, 1967) it took about forty years until molecular data for honey bee trypanosomatids became available and were used to identify and describe a new trypanosomatid species from honey bees, Lotmaria passim (Evans and Schwarz, 2014). However, an easy method to distinguish them without sequencing is not yet available. Research on the related bumble bee parasites Crithidia bombi and Crithidia expoeki revealed a fragment length polymorphism in the internal transcribed spacer 1 (ITS1), which enabled species discrimination. In search of fragment length polymorphisms for differential diagnostics in honey bee trypanosomatids, we studied honey bee trypanosomatid cell cultures of C. mellificae and L. passim. This research resulted in the identification of fragment length polymorphisms in ITS1 and ITS1-2 markers, which enabled us to develop a diagnostic method to differentiate both honey bee trypanosomatid species without the need for sequencing. However, the amplification success of the ITS1 marker depends probably on the trypanosomatid infection level. Further investigation confirmed that L. passim is the dominant species in Belgium, Japan and Switzerland. We found C. mellificae only rarely in Belgian honey bee samples, but not in honey bee samples from other countries. C. mellificae was also detected in mason bees (Osmia bicornis and Osmia cornuta) besides in honey bees. Further, the characterization and comparison of additional markers from L. passim strain SF (published as C. mellificae strain SF) and a Belgian honey bee sample revealed very low divergence in the 18S rRNA, ITS1-2, 28S rRNA and cytochrome b sequences. Nevertheless, a variable stretch was observed in the gp63 virulence factor.

Determination of neonicotinoid insecticides and their metabolites in honey bee and honey by liquid chromatography tandem mass spectrometry.

The original analytical method for the simultaneous determination and confirmation of neonicotinoids insecticides (imidacloprid, clothianidin, acetamiprid, thiametoxam, thiacloprid, nitenpyram, dinotefuran) and some of their metabolites (imidacloprid guanidine, imidacloprid olefin, imidacloprid urea, desnitro-imidacloprid hydrochloride, thiacloprid-amid and acetamiprid-N-desmethyl) in honey bee and honey was developed. Preparation of honey bee samples involves the extraction with mixture of acetonitrile and ethyl acetate followed by cleaned up using the Sep-Pak Alumina N Plus Long cartridges. Honey samples were dissolved in 1% mixture of acetonitrile and ethyl acetate with addition of TEA, then extracts were cleaned up with Strata X-CW cartridges. The identity of analytes was confirmed using liquid chromatography tandem mass spectrometry. All compounds were separated on a Luna C18 column with gradient elution. The whole procedure was validated according to the requirements of SANCO 12571/2013. The average recoveries of the analytes ranged from 85.3% to 112.0%, repeatabilities were in the range of 2.8-11.2%, within-laboratory reproducibility was in the range of 3.3-14.6%, the limits of quantitation were in the range of 0.1-0.5μgkg(-1), depending of analyte and matrices. The validated method was successfully applied for the determination of clothianidin, imidacloprid and imidacloprid urea in real incurred honey bee samples and clothianidin in honey.

Comparison of acute effects of heroin and Kerack on sensory and motor activity of honey bees (Apis mellifera).

Previous studies demonstrated a functional similarity between vertebrate and honey bee nervous systems. The aim of the present study was to compare the effects of heroin and Iranian street Kerack, a combination of heroin and caffeine, on sensory threshold and locomotor activity in honey bees. All drugs were given orally to honey bees 30 min before each experiment. The levels of these drugs and their metabolites in brain samples of honey bees were determined by GC/MS. The sucrose sensitivity test was used for evaluation of changes in honey bees' sensory threshold. Following the administration of both drugs, the honey bees' locomotor activity changes were evaluated in open fields. 6-acetylmorphine had a higher concentration in comparison with other heroin metabolites in honey bees' brains. Concentration of the compound in the brain was directly proportional to the amount ingested. Heroin reduced the sensory threshold of honey bees, but Kerack increased it in the same doses. Locomotor activity of honey bee in open field was enhanced after the administration of both drugs. However, immobility time of honey bees was only affected by high doses of heroin. Acute effects of heroin andKerack on the sensory and motor functions of honey bees were different. Findings of this research suggest that these differences originated from the activation of different neurotransmitter systems by caffeine together with activation of opioid receptors by heroin.

Development of a Gas Chromatography - Tandem Mass Spectrometry Procedure for Determination of Pesticide Residues in Honey and Honeybee Samples

A new analytical procedure of determination of multiresidue of pesticides based on application of the modified QuEChERS approach at the step of sample preparation and technique GC-MS/MS has been developed and applied for studies the real honey and honeybee samples. Samples were collected from northern Poland (Pomerania). The proposed procedure enables for the determination of 34 and 30 pesticide residues respectively in honey and honeybee samples. The recovery of analytes was between 70 and 120% with relative standard deviation ≤ 20%. The limits of quantification were in the range of 2.3-27 ng/g and 2.2-29 ng/g respectively for honey and honeybee samples. The proposed procedure was proven to be a powerful, highly sensitive, and environmentally friendly analytical tool that requires minimal sample preparation.

Microsatellite DNA polymorphism in selectively controlled Apis mellifera carnica and Apis mellifera caucasica populations from Poland

Genetic polymorphism in selectively controlled honeybee populations of A. m. carnica and A. m. caucasica in Poland, was characterized by microsatellite DNA analysis. All honeybee samples were analyzed for nine microsatellite loci: Ac011; A024; A043; A088; Ap226; Ap238; Ap243; Ap249 and Ap256, which were found to be polymorphic in both populations. The mean number of alleles per locus was 6.222 for A. m. carnica and 4.556 for A. m. caucasica. Average observed and expected heterozygosity values were calculated as 0.976 and 0.734 in A. m. carnica and as 0.933 and 0.603 in A. m. caucasica, respectively. For the nine microsatellite loci, a total of 76 alleles were found in both populations. Thirty-five private alleles were observed in A. m. carnica and 20 in A. m. caucasica. Information about allele frequencies, FST values and genotypic differentiation is given. Nei?s genetic distance between studied populations of A. m. carnica and A. m. caucasica was calculated as 0.384.

Use of geometric morphometrics to differentiate selected lines of Carniolan honeybees (Apis mellifera carnica) in Serbia and Montenegro

In a selection of honeybees from autochthonous ecotypes, different lines must be identified. Honeybee lineages are usually distinguished by classical morphometrics and molecular markers, but these approaches are both costly and time-consuming to implement. Recognition of the purity of races is very important for regional and country regulations to allow a sustainable conservation of the huge variety of local honeybees. A geometric morphometric approach has been frequently used. In this work, honeybee samples were collected from stationary apiaries (belonging to the centers for honeybee queen selection) from two different Serbian areas: Vrsac (northeastern Serbia, mostly flatland) and Vranje (southern Serbia, mostly mountainous), and two different Montenegrin areas: Bijelo Polje (northern Montenegro, mountainous region) and Sutomore (southern Montenegro, coastal region). Each sample consisted of 150 honeybee workers, collected from 10 hives (15 specimens each). On the honeybee left forewings, a total of 19 vein intersections were used to determine the differences among the honeybees using MorphoJ 1.4a software. Canonical variate analysis (CVA) slightly separated the honeybee lines into one overlapping cloud of specimens at the individual level. The first canonical variable (60.57% of the total variability) discriminated mainly between Bijelo Polje and Sutomore honeybee lines. Therefore, on the colony level, CVA separated all four groups of breeding honeybee lines. The results show that geometric morphometrics are reliable in the discrimination of honeybee lines within subspecies only at the colony level.

Error correction and diversity analysis of population mixtures determined by NGS

The impetus for this work was the need to analyse nucleotide diversity in a viral mix taken from honeybees. The paper has two findings. First, a method for correction of next generation sequencing error in the distribution of nucleotides at a site is developed. Second, a package of methods for assessment of nucleotide diversity is assembled. The error correction method is statistically based and works at the level of the nucleotide distribution rather than the level of individual nucleotides. The method relies on an error model and a sample of known viral genotypes that is used for model calibration. A compendium of existing and new diversity analysis tools is also presented, allowing hypotheses about diversity and mean diversity to be tested and associated confidence intervals to be calculated. The methods are illustrated using honeybee viral samples. Software in both Excel and Matlab and a guide are available at http://www2.warwick.ac.uk/fac/sci/systemsbiology/research/software/, the Warwick University Systems Biology Centre software download site.

Biologic Activities of Honeybee Products Obtained From Different Phytogeographical Regions of Turkey

Honeybee products are rich in phenolic compounds, which effect as natural antioxidants. These compounds may be attached as indicators in studies into the floral and geographical origin of the natural bee products. In this study, we aimed to determine average total antioxidant capacity, average total oxidant capacity and average oxidative stress index of natural bee products obtained from different regions of Turkey. Collected honeybee samples were kept at +4o C until extracted. Natural bee products were extracted with specific methods and antioxidant capacities were defined with in vitro analyses and data were compared. As a result, the highest average total antioxidant capacities were observed in propolis and pollen samples. Total antioxidant capacities of honeybee products collected from various regions demonstrated differences (P

Molecular characterization of Paenibacillus larvae larvae, for early diagnosis of american foulbrood of honeybees in Egypt

Article History A reliable procedure for early detection of Paenibacillus larvae subsp. larvae (P. l. larvae), the causal agent of American Foulbrood disease (AFB) of honeybees (Apis mellifera L.) based on the polymerase chain reaction (PCR) and subspecies - specific KAT primers. A PCR amplicon of the expected size 550 bp only found in P. l. larvae strains was used for positive AFB. This PCR assay provides a specific detection for P. l. larvae from week 1 post infection even if there is no clinical symptoms appeared in a colony. The technique can be directly used to detect presence or absence of P. l. larvae spores in honeybee samples and contaminated honeys.

Pesticide residues in honeybees, honey and bee pollen by LC–MS/MS screening: Reported death incidents in honeybees

The aim of this study was to investigate reported cases of honeybee death incidents with regard to the potential interrelation to the exposure to pesticides. Thus honeybee, bee pollen and honey samples from different areas of Greece were analyzed for the presence of pesticide residues. In this context an LC–ESI-MS/MS multiresidue method of total 115 analytes of different chemical classes such as neonicotinoids, organophosphates, triazoles, carbamates, dicarboximides and dinitroanilines in honeybee bodies, honey and bee pollen was developed and validated. The method presents good linearity over the ranges assayed with correlation coefficient values r2≥0.99, recoveries ranging for all matrices from 59 to 117% and precision (RSD%) values ranging from 4 to 27%. LOD and LOQ values ranged – for honeybees, honey and bee pollen – from 0.03 to 23.3ng/g matrix weight and 0.1 up to 78ng/g matrix weight, respectively. Therefore this method is sufficient to act as a monitoring tool for the determination of pesticide residues in cases of suspected honeybee poisoning incidents. From the analysis of the samples the presence of 14 active substances was observed in all matrices with concentrations ranging for honeybees from 0.3 to 81.5ng/g, for bee pollen from 6.1 to 1273ng/g and for honey one sample was positive to carbendazim at 1.6ng/g. The latter confirmed the presence of such type of compounds in honeybee body and apicultural products.

Determination of Pesticide Residues in Honeybees using Modified QUEChERS Sample Work-Up and Liquid Chromatography-Tandem Mass Spectrometry

Increasing emissions of chemical compounds to the environment, especially of pesticides, is one of factors that may explain present honeybee colony losses. In this work, an analytical method employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) was optimized for the simultaneous screening of 19 pesticides which have not been yet determined in honeybee samples from northern Poland (Pomerania). The sample preparation, based on the QuEChERS method combining salting-out liquid-liquid extraction to acetonitrile and a dispersive-SPE clean-up, was adjusted to honeybee samples by adding a small amount of hexane to eliminate beeswax. The recovery of analytes ranged from 70% to 120% with relative standard deviation ≤20%. The limits of detection were in the range of 0.91–25 ng/g. A total of 19 samples of honeybees from suspected pesticide poisoning incidents were analyzed, in which 19 different pesticides were determined.

Israeli acute paralysis virus not detected in Apis mellifera in New Zealand in a national survey

SummaryA survey was carried out aimed at determining whether Israeli acute paralysis virus (IAPV) was present in honey bees (Apis mellifera) in New Zealand. There were two parts to the survey. The first, carried out during autumn 2010, comprised sampling of bees from a risk-based purposive selection of apiaries in areas from the North Island where an incursion of IAPV was considered most likely. From each selected apiary, pooled samples of approximately ten bees were tested. The bee sample was a composite sample made up of bees from all or a proportion of colonies present at the site. The second part of the survey was carried out approximately one year later during autumn 2011. It involved selecting apiaries to be sampled using generalised random tessellation, a selection technique designed to maximise spatial coverage over areas of New Zealand where beekeeping occurs. For each selected apiary a pooled sample of approximately twenty bees from each of three colonies were tested. In total, 1050 tests were carried out on honey bee samples from 499 apiaries. Israeli acture paralysis virus was not detected in any of the bee samples. The probability of absence of IAPV in the North Island of New Zealand was determined to be 96% from those colonies in the North Island associated with risk pathways and 92% for New Zealand as a whole from the subsequent spatially balanced survey. The survey assumed that IAPV if present occurred in 1% of apiaries and 65% of colonies within an affected apiary. This is the first published study estimating the probability of country absence from a bee virus.

Transcriptome Sequencing Analysis Reveals the Regulation of the Hypopharyngeal Glands in the Honey Bee, Apis mellifera carnica Pollmann

Transcriptome sequencing has become the main methodology for analyzing the relationship between genes and characteristics of interests, particularly those associated with diseases and economic traits. Because of its role of functional food for humans, commercial royal jelly (RJ) and its production are major research focuses in the field of apiculture. Multiple lines of evidence have demonstrated that many factors affect RJ output by activating or inhibiting various target genes and signaling pathways. Available coding sequences from the Honey Bee Genome Sequencing Consortium have permitted a pathway-based approach for investigating the development of the hypopharyngeal glands (HGs). In the present study, 3573941, 3562730, 3551541, 3524453, and 3615558 clean reads were obtained from the HGs of five full-sister honey bee samples using Solexa RNA sequencing technology. These reads were then assembled into 18378, 17785, 17065, 17105, and 17995 unigenes, respectively, and aligned to the DFCI Honey Bee Gene Index database. The differentially expressed genes (DEGs) data were also correlated with detailed morphological data for HGs acini.

Honeybees and honey as monitors for heavy metal contamination near thermal power plants in Mugla, Turkey

In the present work, 6 honeydew samples of known geographical and botanical origins and 11 honeybee samples were analyzed to detect possible contamination by the thermoelectric power plants in Mugla, Turkey. The contents of trace elements were determined by atomic absorption spectrometry after application of microwave digestion. The samples from the thermal power plants, which were 10-22 km away from the hives, that did not cause pollution in honeydew honeys were also analyzed. The levels of copper, cadmium (Cd), lead (Pb), zinc, manganese, iron, chromium, nickel, and aluminum were similar to the values found in other recent studies in literature. However, it was found that the contamination levels of the toxic elements such as Pb and Cd in honeybee samples measured relatively higher than that of honey samples. The study concludes that honeybees may be better bioindicators of heavy metal pollution than honey.

COMPARISON OF METHODS FOR DETECTION OF MICROSPORIDIA SPECIES OF THE GENUS NOSEMA IN HONEY BEES (APIS MELLIFERA)

Two microsporidia species of the Nosema genus cause nosemosis in the adult honeybee: N. apis and N. ceranae. For diagnostic purposes and the determination of infection level various microscopic and molecular biological methods are used. Th e aim of this research was to compare the reliability of the traditional microscopic assessment and two PCR techniques: simplex- and duplex-PCR. Honey bee samples were taken from 150 colonies. Microscopic examination, performed according to the recommendations of the OIE, revealed Nosema spores in 68.67% samples analysed, whilst with the simplex-PCR method all samples (100.0%) proved positive. On the other hand, duplex-PCR method used for the identifi cation of Nosema species resulted in 84.0% positive samples, all of which were N. ceranae. Our recommendation of the simplex-PCR method for the monitoring of honey bees in fi eld conditions is based on its higher reliability than the microscopic assessment in the detection of low-level infections, as well as its potential for the detection of vegetative Nosema sp. stages; thus the early detection and timely prevention of Nosema infection would be possible. Nosema species identifi cation is simplest and most cost-eff ective if performed with the duplex-PCR analysis. However, the simplex-PCR is more reliable, thus, it is suggested that samples that were negative when assessed with microscopy and duplex-PCR analysis undergo simplex-PCR.

The prevalence of the honeybee brood pathogens Ascosphaera apis, Paenibacillus larvae and Melissococcus plutonius in Spanish apiaries determined with a new multiplex PCR assay

The microorganisms Ascosphaera apis, Paenibacillus larvae and Melissococcus plutonius are the three most important pathogens that affect honeybee brood. The aim of the present study was to evaluate the prevalence of these pathogens in honeybee colonies and to elucidate their role in the honeybee colony losses in Spain. In order to get it, a multiplex polymerase chain reaction (PCR) assay was developed to simultaneously amplify the16S ribosomal ribonucleic acid (rRNA) gene of P. larvae and M. plutonius, and the 5.8S rRNA gene of A. apis. The multiplex PCR assay provides a quick and specific tool that successfully detected the three infectious pathogens (P. larvae, M. plutonius and A. apis) in brood and adult honeybee samples without the need for microbiological culture. This technique was then used to evaluate the prevalence of these pathogens in Spanish honeybee colonies in 2006 and 2007, revealing our results a low prevalence of these pathogens in most of the geographic areas studied.

The development, validation and application of a GC-dual detector (NPD-ECD) multi-pesticide residue method for monitoring bee poisoning incidents

A simple multiresidue method based on matrix solid phase dispersion (MSPD) combined with clean-up has been developed for the simultaneous screening of 153 pesticides in honeybees suspected of suffering from pesticide poisoning during field spraying. Extraction and clean-up were carried out in a glass column containing anhydrous sulphate, 2.0g of octadecyl (C18) and a 2.0-g sample of bees (23 insects on average) macerated with 4.0g of Florisil. An additional layer of anhydrous sodium sulphate was added, and acetonitrile was used as the elution solvent. This combination of clean-up steps ensured an efficient purification. A gas chromatograph with dual selective detectors for electron capture and nitrogen–phosphorous was used. The results presented in this paper demonstrate that matrix solid-phase dispersion (MSPD) with the one-step clean-up procedure is the most effective extraction technique. MSPD method recoveries ranged from 70 to 118%, with precision values expressed as a relative standard of <20%, except for 10 pesticides that had recoveries of 50–70% and two with 120–130%. Low limits of detection (0.003–0.04μg/g) and quantification (0.005–0.05μg/g) were readily achieved with this method for all tested pesticides. A “top down” empirical model was used to estimate the expanded uncertainty at 28% on average (coverage factor k=2, confidence level 95%). The MSPD method was successfully used on real bee samples to analyse four acaricides, 55 fungicides, 16 herbicides and 78 insecticides from various regions of Poland. A total of 33 honeybee samples from suspected pesticide poisoning incidents were analysed, in which 17 different pesticides were determined (14 insecticides and three fungicides). The pesticides most often found in honeybees were cypermethrin (in 51% of the samples, 0.008–0.563µg/bee), chlorpyrifos (27%, 0.001–51.5µg/bee) and biphentin (21%, 0.002–0.012µg/bee).

Biomonitoring with Honeybees of Heavy Metals and Pesticides in Nature Reserves of the Marche Region (Italy)

The aim of this study was to carry out biomonitoring with honeybees (Apis mellifera L.) to assess the presence of pesticides and heavy metals (cadmium, chromium, nickel, lead) in all of the ten nature reserves of the Marche Region (central–eastern Italy). The study was carried out during the spring and summer seasons when the honeybees were active, over 3 years (2008–2010). Twenty-two colonies of honeybees bred in hives were used. Samples of live and dead honeybees and of honey were collected from 11 sampling stations from May to October in each year. No pesticide pollution was found. Significant differences in heavy metal concentrations were found among years, months and sites, and in particular situations. The analysis reveals that high heavy-metal concentrations occurred exclusively in live honeybees. For the seasonal averages, the most detected heavy metal was chromium, which exceeded the threshold more often than for the other elements, followed by cadmium and lead; nickel never exceeded the threshold. The data are discussed with an evaluation of the natural and anthropic sources taken from the literature and from local situations that were likely to involve heavy metal pollution.

Widespread occurrence of chemical residues in beehive matrices from apiaries located in different landscapes of Western France.

BackgroundThe honey bee, Apis mellifera, is frequently used as a sentinel to monitor environmental pollution. In parallel, general weakening and unprecedented colony losses have been reported in Europe and the USA, and many factors are suspected to play a central role in these problems, including infection by pathogens, nutritional stress and pesticide poisoning. Honey bee, honey and pollen samples collected from eighteen apiaries of western France from four different landscape contexts during four different periods in 2008 and in 2009 were analyzed to evaluate the presence of pesticides and veterinary drug residues.Methodology/FindingsA multi-residue analysis of 80 compounds was performed using a modified QuEChERS method, followed by GC-ToF and LC−MS/MS. The analysis revealed that 95.7%, 72.3% and 58.6% of the honey, honey bee and pollen samples, respectively, were contaminated by at least one compound. The frequency of detection was higher in the honey samples (n = 28) than in the pollen (n = 23) or honey bee (n = 20) samples, but the highest concentrations were found in pollen. Although most compounds were rarely found, some of the contaminants reached high concentrations that might lead to adverse effects on bee health. The three most frequent residues were the widely used fungicide carbendazim and two acaricides, amitraz and coumaphos, that are used by beekeepers to control Varroa destructor. Apiaries in rural-cultivated landscapes were more contaminated than those in other landscape contexts, but the differences were not significant. The contamination of the different matrices was shown to be higher in early spring than in all other periods.Conclusions/SignificanceHoney bees, honeys and pollens are appropriate sentinels for monitoring pesticide and veterinary drug environmental pollution. This study revealed the widespread occurrence of multiple residues in beehive matrices and suggests a potential issue with the effects of these residues alone or in combination on honey bee health.