Brood Samples Research Articles

Molecular Characterization and Phylogenetic Analysis of Honeybee (Apis mellifera) Mite-Borne Pathogen DWV-A and DWV-B Isolated from Lithuania.

Deformed wing virus (DWV) is known as one of the main viruses that affect honeybees' health all around the world. The virus has two widespread genotypes, DWV-A and DWV-B (VDV-1), transmitted mainly by V. destructor mites. In this study, we collected honeycombs with covered broods from 73 apiaries in eight Lithuanian regions and initially investigated the prevalence of V. destructor mites. Mites were collected from May to the end of July in 2021 from 124 hives. The prevalence of V. destructor infestations in beehives reached 30% and 63% in investigated apiaries. The presence of DWV-A and DWV-B pathogens in mites and broods was examined by RT-qPCR targeting the CRPV-capsid region. The molecular characterization of the virus in mite samples was based on sequence analysis of the RNA-dependent RNA polymerase (RdRp) region. In addition, leader polypeptide (LP), structural protein (Vp3), Helicase, and RdRp genes were used for phylogenetic characterization of dual infection. The prevalences of DWV-B in mites and broods were 56.5% and 31.5%, respectively, while DWV-A was detected in 12.9% of mite samples and 24.7% of brood samples. Some of the examined mite samples harboured dual virus infections. Our findings showed that bee colonies from the same apiary were not always infected by the same viruses. Some bee colonies were virus-free, while others were highly infected. Phylogenetic analysis of 21 sequences demonstrated the presence of highly variable DWV-B and DWV-A genotypes in Lithuania and possible recombinant variants of the virus. This study represents the first molecular characterization of mite-borne pathogens hosted by honeybees (Apis mellifera) in Lithuania.

Screening of Bacterial Microflora from the Gastrointestinal tract of Apis mellifera (Apidae; Hymenoptera), its Brood and Soil samples from Prayagraj District: A Brief Study

Screening of Bacterial Microflora from the Gastrointestinal tract of Apis mellifera (Apidae; Hymenoptera), its Brood and Soil samples from Prayagraj District: A Brief Study

Occurrence and distribution of two bacterial brood diseases (American and European foulbrood) in US honey bee colonies and resistance to antibiotics from 2015 to 2022

Here, we present a retrospective study on honey bee brood diseases in the USA. The Bee Disease Diagnostic Laboratory (BDDL) in the USDA-ARS Beltsville Bee Research Laboratory received a total of 4790 brood samples between 2015 and 2022 from U.S. State Apiary Inspectors and beekeepers. Samples from 49 states were analyzed by microscopy for the presence and prevalence of two bacteria, Melissococcus plutonius and Paenibacillus larvae, causing European foulbrood (EFB) and American foulbrood (AFB) diseases, respectively. Samples that tested positive for AFB were cultured and subjected to antibiotic susceptibility tests via the agar disc diffusion method to determine their resistance to tetracycline (Terramycin®) and tylosin (Tylan®) antibiotics. A comprehensive data analysis was conducted at multiple levels, including state, month, year, nationwide, and climate region. Among the 49 states examined, EFB was identified in 44 states, while AFB was found in 31 states. Infection levels vary across states, ranging from 0% to 63.6% for EFB and from 0% to 54.8% for AFB. The national average of EFB (19.2%) was significantly (p < 0.001) higher than AFB (8.6%). Contrary to AFB, significant yearly increases (p < 0.001) were identified for EFB from 2015 to 2022. Furthermore, significant monthly variations were recorded for both brood diseases, with the highest occurrence of EFB observed from April to July. States with the lowest infection rates of both diseases were NV, ND, MS, AK, and AZ. The South U.S. climate region, which comprises six states, had the highest and lowest EFB and ABF infections, respectively. On a nationwide scale, the AFB resistance to tetracycline (38%) was significantly (p < 0.001) higher than the resistance to tylosin (27%).

Health status of honeybee colonies (Apis mellifera) and disease-associated risk factors in different agroecological zones of Southwest Ethiopia

A cross-sectional study design was conducted in different agroecological zones of southwest Ethiopia from October 2019 to October 2021. The study aimed to determine the prevalence and associated risk factors for honeybee diseases and pests, as well as the impact of these issues on honeybee colonies and their products. To identify potential risk factors for honeybee disease and pests, a multivariate random effects logistic regression analysis was used. Adult honeybee and brood samples from a total of 384 honeybee colonies were collected and tested using standard laboratory diagnostic methods. The highest prevalence (55.8%) of ants was recorded, followed by wax moths (22.5%) and hive beetles (23.3%). In the current study, the main honeybee diseases observed in the study areas were varroosis (36.5%), bee lice (5.2%), nosemosis (39.6%), amoeba (56%), and chalkbrood (4.5%). However, tracheal mites, sachbrood, and American and European foul brood, were not detected. The agroecological zone (OR = 5.2, 95% CI: 1.75–14.85), type of hive (OR = 2.9, 95% CI: 1.17–17.03), management system (OR = 4.3, 95% CI: 1.23–14.70), and the management of the colony (OR = 3.5, 95% CI: 1.31–9.14) were identified as risk factors for varroosis in these areas. The occurrence of nosemosis in colonies was also influenced by the agroecological zone (OR = 12.2, 95% CI: 3.06–48.54) and colony management (OR = 3.4, 95% CI: 1.59–7.23). The agroecological zone (OR = 10.5, 95% CI: 12.76–22.63) and hive type (OR = 3.0, 95% CI: 1.39–6.36) were the primary risk factors for the occurrence of amoeba in honeybee colonies. However, the occurrence of bee lice (OR = 34.7, 95% CI: 3.96–104.93) and chalkbrood (OR = 4.8, 95% CI: 1.44–13.16) in honeybee colonies was only influenced by the agroecological zone in the study areas. This study demonstrated that losses in honey production in the area are significantly attributed to honeybee disease and pests. Therefore, it is essential to increase public awareness of how honeybee diseases and pests affect honey production and to develop and implement appropriate control measures for these diseases and pests. Furthermore, more studies should be conducted to characterize and isolate other causes of honeybee diseases and pests in various locations.

Epidemiology, factors influencing prevalence and level of varroosis infestation (Varroa destructor) in honeybee (Apis mellifera) colonies in different agroecologies of Southwest Ethiopia

Little information is available on the epidemiology of varroosis caused by Varroa mite, Varroa destructor infestation in Ethiopia, although it is a devastating honeybee disease that results in significant economic losses in beekeeping. Therefore, between October 2021 and October 2022, a cross-sectional study was carried out in different agroecology zones in Southwest Ethiopia to determine the prevalence and associated risk factors for varroosis, as well as the effects of this disease on honeybee colonies and honey production. A multivariate logistic regression analysis was performed to identify possible risk factors for the prevalence of V. destructor. A total of 384 adult honeybee and worker or drone brood samples were collected from honeybee colonies and examined using standard diagnostic techniques in the laboratory. The result shows that the prevalence of V. destructor was found to be 39.3% (95% CI 34.44–44.21) and 43.2% (38.27–48.18) in adult honeybees and brood, respectively. The major risk factors for the prevalence of V. destructor in the study areas included agroecology (OR = 5.2, 95% CI 1.75–14.85), type of hive (OR = 2.9, 95% CI 1.17–17.03), management system (OR = 4.3, 95% CI 1.23–14.70), and colony management (OR = 3.5, 95% CI 1.31–9.14). The lower level of colony infestation in adult bees and brood was measured as 1.97 ± 0.14 and 3.19 ± 0.25, respectively. Season, colony status, colony management, and agroecology were among the determinant factors of the level of varroa mite infestation in adult bees and brood. The results of the study demonstrated that honey production losses are largely attributable to V. destructor infestation. Therefore, it is critical to inform the community about the effects of V. destructor on honey production and develop and implement effective management strategies for this disease. In addition, further research should be done to identify and isolate additional factors that contribute to varroosis in honeybees in different regions.

A Comparative Study of Healthy and American Foulbrood-Infected Bee Brood (Apis mellifera L.) through the Investigation of Volatile Compounds

American Foulbrood (AFB) is a major endemic disease affecting the bee brood and the absence of chemical therapeutic treatments leads beekeepers to develop alternative management plans, based mainly on the prevention and accurate diagnosis of symptoms. One of the main symptoms of the disease is the unpleasant odor caused by the rot of dead larvae. In the present comparative study, we analyzed the odor profile of bee larvae and the presence of characteristic volatile compounds (Gas Chromatography-Mass Spectometry), in an effort to discriminate healthy and AFB-infected brood. A greater number of volatile compounds was identified in the affected brood than the healthy. The presence of (Ε)-β-ocimene was prominent in healthy brood samples in percentages from 85.25 to 99.14%, a compound also detected in all samples of infected brood but in lower percentages (37%). The compounds toluene, xylene, 1,3-dimethylbenzene, 2-nonanone, dimethyl disulfide, and dimethyl trisulfide were detected in 100% of the diseased brood samples, with the latter three being absent from the healthy brood, while 2-undecanone was found in some samples of diseased brood (40.0%). Further investigation of volatile markers may contribute significantly to the successful diagnosis of the disease, aiming at its rapid treatment.

Ion chromatography coupled to Q-Orbitrap for the analysis of formic and oxalic acid in beehive matrices: a field study

There is an increasing concern about the use of synthetic acaricides to fight the ectoparasitic mite Varroa destructor. Natural products such as formic acid (FA) and oxalic acid (OA) have emerged as a possible alternative control strategy. However, given the difficulty of analysing these highly polar compounds and the lack of robust and reliable methods, there are very few studies of the concentration and distribution of these natural acaricides in the beehive compartments. We present a reliable and simple analytical methodology, based on sample extraction with modified quick polar pesticide (QuPPe) methods followed by ion chromatography coupled to a quadrupole Orbitrap mass analyser for the analysis of FA and OA in honeybees, honey, beeswax, and beebread. The developed methods have been used in a field study for the evaluation of the presence and distribution of FA and OA in the beehive products, as well as in adult bees and bee brood samples, before, during, and up to 3 months after the application of the treatments by the beekeeper. Beebread and honey samples presented the highest concentration levels of OA and FA, respectively, mainly due to their natural presence. As expected, the organic acids showed low persistence in wax after the treatments. The natural acaricides were found in adult and developing bees at concentration levels below the reported LD50 in all the cases; however, residue levels of OA in larvae during the treatment application were very close to the reported LD50.Graphical abstract

ERIC and WGS Typing of Paenibacillus larvae in Slovenia: Investigation of ERIC I Outbreaks.

Simple SummaryAmerican foulbrood is a serious disease of honeybees caused by Paenibacillus larvae. ERIC-PCR is a widely used method for typing of P. larvae that currently divides it into five ERIC types (ERIC I–V); these differ in certain phenotypic characteristics—most importantly, virulence. In the first part of the study, we assessed the distribution of ERIC types in Slovenia in the period 2017–2019 on a set of 506 P. larvae isolates. We identified ERIC II as the predominant type (70.2%), followed by ERIC I (29.8%). In the second part of the study, we typed 59 outbreak-related ERIC I isolates using whole-genome sequencing, which revealed seven ERIC I-ST2 outbreak clusters (≤35 allele differences). The transmission of the outbreak clone within a 3-km radius was observed in all seven clusters and could be explained by the activity of honeybees. The transmission of the outbreak clone between geographically distant apiaries was observed in three clusters and could be explained by migratory beekeeping and trading of bee colonies. The present findings highlight the importance of beekeeping activities in the transmission of P. larvae over large geographic distances. Paenibacillus larvae is the causative agent of American foulbrood (AFB), a fatal disease of honeybee brood. Here, we obtained 506 P. larvae isolates originating from honey or brood samples and from different geographic regions of Slovenia in the period 2017–2019. In the first part of the study, we conducted ERIC-PCR typing to assess the frequency of ERIC types in Slovenia. Capillary electrophoresis was used for the analysis of ERIC patterns, revealing good separation efficiency and enabling easy lane-to-lane comparisons. ERIC II was the predominant type (70.2%), followed by ERIC I (29.8%); two slightly altered ERIC I banding patterns were observed but were not considered relevant for the discrimination of ERIC types. No evident spatiotemporal clustering of ERIC types was observed. To assess the clonality of the outbreak-related P. larvae ERIC I isolates, 59 isolates of this type underwent whole-genome sequencing (WGS). Whole-genome multilocus sequence typing (wgMLST) revealed seven ERIC I-ST2 outbreak clusters (≤35 allele differences) with the median intra-outbreak diversity ranging from 7 to 27 allele differences. In all seven clusters, the transmission of P. larvae outbreak clone within a 3-km radius (AFB zone) was observed, which could be explained by the activity of honeybees. In three clusters, the transmission of the outbreak clone between geographically distant apiaries was revealed, which could be explained by the activities of beekeepers such as migratory beekeeping and trading of bee colonies. The present findings reinforce the importance of beekeeping activities in the transmission of P. larvae. WGS should be used as a reference typing method for the detection of P. larvae transmission clusters.

Assessment of risk to honey bees and honey consumers resulting from the insect exposure to captan, thiacloprid, penthiopyrad, and λ-cyhalothrin used in a commercial apple orchard.

Samples of leaves, flowers, soil, pollen, bee workers, bee brood, honey, and beeswax were collected to assess the possibility of a transfer of captan, thiacloprid, penthiopyrad, and λ-cyhalothrin from apple trees of Idared variety to honey bee (Apis mellifera) hives. Chemical analyses were performed using the Agilent 7890 Gas Chromatograph equipped with the Micro-cell Electron Capture Detector. It was found that significant amounts of penthiopyrad, the active ingredient of Fontelis 200 SC, were present in leaves, flowers, pollen, bee workers, and beeswax. Simultaneously, captan was present in the brood, worker bees, and honey samples. Significant levels of the captan residues were also detected on the soil surface. In honey samples, captan residue levels exceeded the acceptable standard, reaching 160% of its maximum residue level. However, in no case the amounts of captan, thiacloprid, penthiopyrad, and λ-cyhalothrin ingested with honey by an adult consumer exceeded the level of 0.02% of the acceptable daily intake. Despite the trace amounts of pesticide residues in honey samples collected during the field trial, bee honey consumption can be considered safe. An adult consumer can safely consume about 16kg of honey.

Identity and distribution of American foulbrood (Paenibacillus larvae) in South Africa

American foulbrood (AFB) is a serious and near-global disease of honey bees and was recently discovered in South Africa. The causative pathogen, Paenibacillus larvae, comprises various strains. It is currently unknown which of these are present in South Africa and what their distribution is. We address this issue by genotyping P. larvae isolates using repetitive element fingerprinting (rep-PCR) markers and microfabricated chip technology. A total of 71 brood, honey and wax samples were collected opportunistically in South Africa. Twenty-three samples tested positive for P. larvae (32.4%). Diagnostic rep-PCR fingerprinting using ERIC, BOX A1R and MBO REP1 primers found all P. larvae isolates in South Africa to correspond to the ERIC I genotype, with two subtypes revealed, Ab (13 samples) and ab (10 samples). These results are in agreement with the global prevalence of the ERIC I AFB genotype among P. larvae-infected honey bee colonies.

Diagnosis of Varroosis Based on Bee Brood Samples Testing with Use of Semiconductor Gas Sensors.

Varroosis is a dangerous and difficult to diagnose disease decimating bee colonies. The studies conducted sought answers on whether the electronic nose could become an effective tool for the efficient detection of this disease by examining sealed brood samples. The prototype of a multi-sensor recorder of gaseous sensor signals with a matrix of six semiconductor gas sensors TGS 823, TGS 826, TGS 832, TGS 2600, TGS 2602, and TGS 2603 from FIGARO was tested in this area. There were 42 objects belonging to 3 classes tested: 1st class—empty chamber (13 objects), 2nd class—fragments of combs containing brood sick with varroosis (19 objects), and 3rd class—fragments of combs containing healthy sealed brood (10 objects). The examination of a single object lasted 20 min, consisting of the exposure phase (10 min) and the sensor regeneration phase (10 min). The k-th nearest neighbors algorithm (kNN)—with default settings in RSES tool—was successfully used as the basic classifier. The basis of the analysis was the sensor reading value in 270 s with baseline correction. The multi-sensor MCA-8 gas sensor signal recorder has proved to be an effective tool in distinguishing between brood suffering from varroosis and healthy brood. The five-time cross-validation 2 test (5 × CV2 test) showed a global accuracy of 0.832 and a balanced accuracy of 0.834. Positive rate of the sick brood class was 0.92. In order to check the overall effectiveness of baseline correction in the examined context, we have carried out additional series of experiments—in multiple Monte Carlo Cross Validation model—using a set of classifiers with different metrics. We have tested a few variants of the kNN method, the Naïve Bayes classifier, and the weighted voting classifier. We have verified with statistical tests the thesis that the baseline correction significantly improves the level of classification. We also confirmed that it is enough to use the TGS2603 sensor in the examined context.

Ephemeral detection of Bonamia exitiosa (Haplosporida) in adult and larval European flat oysters Ostrea edulis in the Solent, United Kingdom

The haplosporidian parasite Bonamia exitiosa was detected using PCR in four adult and six larval brood samples of the European flat oyster Ostrea edulis from the Solent, UK. This represents the second reported detection of this parasite along the south coast of England. Adult oysters were collected and preserved from seabed populations or restoration broodstock cages between 2015 and 2018. The larvae within brooding adults sampled during 2017 and 2018 were also preserved. Molecular analysis of all samples was performed in 2019. The DNA of B. exitiosa was confirmed to be present within the gill tissue of one oyster within the Portsmouth wild fishery seabed population (n = 48), sampled in November 2015; the congeneric parasite Bonamia ostreae was not detected in this individual. This is the earliest record of B. exitiosa in the Solent. Concurrent presence of both B. ostreae and B. exitiosa, determined by DNA presence, was confirmed in the gill and heart tissue of three mature individuals from broodstock cages sampled in October 2017 (n = 99), two from a location on the River Hamble and one from the Camber Dock in Portsmouth Harbour. B. exitiosa was not detected in the November 2018 broodstock populations. A total of six larval broods were positive for B. exitiosa, with five also positive for B. ostreae. None of the brooding adults were positive for B. exitiosa suggesting that horizontal transmission from the surrounding environment to the brooding larvae is occurring. Further sampling of broodstock populations conducted by the Fish Health Inspectorate at the Centre for Environment, Fisheries and Aquaculture Science in June 2019 did not detect infection of O. edulis by B. exitiosa. These findings together suggest that the pathogen has not currently established in the area.

Distribution of chemical residues in the beehive compartments and their transfer to the honeybee brood

Honeybee (Apis mellifera) is one of the most important crop and wild plant pollinators, playing an essential role in the agricultural production and the natural ecosystems. However, the number of honeybee colonies is decreasing alarmingly, which has motivated extensive research on the factors affecting their development and survival in some regions. Honeybees' exposure to pesticides and other chemicals has been identified as one of the causes of their decline. The present study evaluates the distribution of plant protection products, veterinary treatments and environmental contaminants inside the beehive, their persistence and their migration to the bee brood. During the five-month sampling period, only amitraz was applied to the colonies. Samples of beeswax, beebread (processed pollen) and bee brood were extracted and analyzed using GC-MS/MS and LC-MS/MS with a multiresidue method. The results showed the presence of 31 chemical residues in the samples. The highest concentrations of residues were detected in the beeswax and corresponded to amitraz (expressed as the sum of DMF and DMPF), coumaphos and tau-fluvalinate, with total concentrations of up to 16,858, 7102 and 1775 μg kg−1, respectively. These and other veterinary treatments were found to accumulate in the beeswax and migrate to other beehive matrices such as beebread and bee brood. Plant protection products used in agriculture were also found in the beehive matrices, especially in the beebread. Five different chemical residues (acrinathrin, amitraz, coumaphos, cypermethrin and tau-fluvalinate) were found in bee brood samples at concentration levels ranging from 1 to 167 μg kg−1. These findings reveal that bee brood reared in field conditions is in fact exposed to plant protection products and veterinary residues through direct contact with contaminated wax and via beebread although they had not been applied to the beehive.

A Microbiomic Analysis of a Pasture-Raised Broiler Flock Elucidates Foodborne Pathogen Ecology Along the Farm-To-Fork Continuum.

While conventionally grown poultry continues to dominate the U. S. poultry industry, there is an increasing demand for locally-grown, “all natural” alternatives. The use of next generation sequencing allows for not only the gross (e.g., community structure) but also fine-scale (e.g., taxa abundances) examination of these complex microbial communities. This data provides a better understanding of how a pasture flock's microbiome changes throughout the production life cycle and how that change in microbial ecology changes foodborne pathogens in alternative poultry production systems. In order to understand this ecology better, pooled broiler samples were taken during the entire flock life cycle, from pre-hatch gastrointestinal samples (N = 12) to fecal samples from the brood (N = 5), and pasture (N = 10) periods. Additional samples were taken during processing, including skin and feather rinsates (N = 12), ceca (N = 12), and whole carcass rinses (N = 12), and finally whole carcasss rinsates of final products (N = 3). Genomic DNA was extracted, 16S rDNA microbiome sequencing was conducted (Illumina MiSeq), and microbiomes were analyzed and compared using QIIME 1.9.1 to determine how microbiomes shifted throughout production continuum, as well as what environmental factors may be influencing these shifts. Significant microbiome shifts occurred during the life cycle of the pasture broiler flock, with the brood and pasture fecal samples and cecal samples being very distinct from the other pre-hatch, processing, and final product samples. Throughout these varied microbiomes, there was a stable core microbiome containing 13 taxa. Within this core microbiome, five taxa represented known foodborne pathogens (Salmonella, Campylobacter) or potential/emerging pathogens (Pseudomonas, Enterococcus, Acinetobacter) whose relative abundances varied throughout the farm-to-fork continuum, although all were more prevalent in the fecal samples. Additionally, of the 25 physiochemical and nutrient variables measured from the fecal samples, the carbon to nitrogen ratio was one of the most significant variables to warrant further investigations because it impacted both general fecal microbial ecology and Campylobacter and Enterococcus taxa within the core fecal microbiomes. These findings demonstrate the need for further longitudinal, farm-to-fork studies to understand the ecology of the microbial ecology of pasture production flocks to improve animal, environmental, and public health.

The levels of natural Nosema spp. infection in Apis mellifera iberiensis brood stages

Nosema ceranae is the most prevalent endoparasite of Apis mellifera iberiensis and it is a major health problem for bees worldwide. The infective capacity of N. ceranae has been demonstrated experimentally in honey bee brood, however no data are available about its prevalence in brood under natural conditions. Thus, brood combs from 10 different hives were analyzed over two consecutive years, taking samples before and after winter. A total of 1433 larvae/pupae were analyzed individually and N. ceranae (3.53%) was the microsporidian most frequently detected, as opposed to Nosema apis (0.42%) which was more frequently detected in conjunction with N. ceranae (0.71%). The active multiplication of both microsporidians was confirmed by the expression (real-time-PCR) of the N. ceranae polar tube protein 3 gene and/or the N. apis RNA polymerase II gene in 24% of the brood samples positive for Nosema spp. Both genes are related to microsporidian multiplication. As such, N. ceranae multiplication was confirmed in 1.06% of the samples, while N. apis multiplication was only observed in co-infections with N. ceranae (0.07%). Brood cells were analyzed for the presence of Nosema spp., as those are the immediate environment where the brood stages develop. The brood samples infected by Nosema spp. were in brood cells in which that microsporidians were not detected, while brood cells positive for N. ceranae hosted brood stages that were not apparently infected, indicating that this is unlikely to be the main pathway of infection. Finally, the colonies with brood infected by N. ceranae showed higher levels (numbers) of infected adult bees, although the differences were not significant before (P = 0.260), during (P = 0.055) or after (P = 0.056) brood sampling. These results show that N. ceranae is a bee parasite ubiquitous to all members of the colony, irrespective of the age of the bee. It is also of veterinary interest and should be considered when studying the epidemiology of the disease.

Variation in nestling feather mercury concentrations at individual, brood, and breeding colony levels: Implications for sampling mercury in birds

Nestling feathers are often used to monitor and estimate Hg exposure in birds. Decision-making and results of studies on effects of exposure to Hg depend on adequate estimation of [Hg] at the studied level, which in turn is sensitive to variation in [Hg] in sampling units. However, there is little information on how feather [Hg] varies within nestlings, between nest mates, or between broods. We sampled feathers from sibling great egret nestlings in three breeding colonies of the Everglades where birds were exposed to variable Hg levels through diet. We compared the deviation of samples from average [Hg] within four levels of aggregation: individual (different scapular feathers of the same individual); brood (samples from siblings in the same nest), breeding colonies; and the entire wetland ecosystem. We found, on average, little intra-individual (<2%) and intra-brood (<6%) differences in [Hg], and no effects of chick hatch order in feather [Hg]. Further, intra-brood variability was not statistically different from intra-individual variability in [Hg]. In contrast, there was much higher variation between broods within colonies (>20%) and within the entire wetland (>30%) that could bias estimates of exposure to Hg obtained through small sample sizes. We simulated the influence of inter-brood variability in estimates of exposure to Hg under different sample sizes, and we found that uncertainty decreases markedly with samples sizes >6, and suggest a sample size of 10 individuals from different nests for monitoring programs and to estimate Hg contamination in areas where no previous knowledge is available.

Occurrence of honey bee (Apis mellifera L.) pathogens in commercial and traditional hives

This work was aimed at the detection of the differences in the occurrence of seven bee pathogens between bee colonies kept in commercial and traditional ways. The research was conducted on 120 apparently healthy, commercially kept colonies in DB hives and 24 traditionally kept colonies in primitive, so-called trmka hives on the Pester Plateau. Brood samples were taken from all colonies to assess the occurrence of bee brood disease agents (Paenibacillus larvae, Melissococcus plutonius, Ascosphaera apis and sacbrood virus – SBV) and adult bee pathogens (deformed wing virus – DWV, chronic bee paralysis virus – CBPV and acute bee paralysis virus – ABPV). PCR diagnostics was used in all cases, in compliance with the existing methods adopted by OIE. Concerning bee brood disease-causing agents, in commercial hives P. larvae (16.67% samples), A. apis (15.83%) and the SBV (96.67%) were confirmed, whilst in traditional hives, SBV was the only one detected (33.33%). M. plutonius was not found in any sample. As for adult bee diseases, in both commercial and traditional hives all of the three viruses were detected (DWV, ABPV, CBPV), but their occurrence in the former (100.00, 100.00 and 83.33%, respectively) was significantly (p<0.001) higher than in the latter (33.33% occurrence of each). No commercially kept colonies were free from all disease causes, while in the traditionally kept group there were 66.66% of such colonies. It can be concluded that the traditional way of beekeeping provides significantly better conditions for maintenance of bee health and their resistance to pathogens.

Transfer of plant protection products from raspberry crops of Laszka and Seedling varieties to beehives

Field studies were conducted to evaluate the transfer of active ingredients (AIs) of plant protection products (PPPs) to beehives. They were applied in two commodity red raspberry plantations of two varieties: Laszka (experiment 1) and Seedling (experiment 2). Samples of flowers, leaves, bees, brood, and honey were examined for the presence of chlorpyrifos, cypermethrin, difenoconazole, cyprodinil, and trifloxystrobin (experiment 1) and chlorpyrifos, boscalid, pyraclostrobin, cypermethrin, difenoconazole, and azoxystrobin (experiment 2). In experiment 1, the highest levels of trifloxystrobin were observed on the surface of flowers, (0.04 μg/flower) and for difenoconazole on the inside (0.023 μg/flower). Leaves contained only trace residues of cypermethrin and cyprodinil (0.001 μg/cm2 of leaves each) and trifloxystrobin (0.01 μg/cm2 of leaves) on the surface; inside the leaves, the highest levels of trifloxystrobin were observed (0.042 μg/cm2 of leaves). In experiment 2, boscalid was found on the surface and inside the flowers and leaves (0.063 and 0.018 μg/flower and 0.057 and 0.033 μg/cm2 of leaves, respectively). In bees, brood, and honey (experiment 1), chlorpyrifos was present in the highest quantity (7.3, 1.6, and 4.7 μg/kg, respectively). Additionally, cypermethrin and trifloxystrobin were found in bees, and trifloxystrobin was present in honey. Bees, brood, and honey from plantation 2 contained all studied AIs, with the highest levels of boscalid (28.6 μg/kg of bees, 37.0 μg/kg of brood, and 33.9 μg/kg of honey, respectively). In no case did the PPP residues in honey exceed acceptable maximum residue levels (MRLs)—from a formal and legal point of view, in terms of the used plant protection products, the analysed honey was fit for human consumption.

Risk factors associated with honey bee colony loss in apiaries in Galicia, NW Spain

A cross-sectional study was carried out in Galicia, NW Spain, in order to estimate the magnitude of honey bee colony losses and to identify potential risk factors involved. A total of 99 samples from 99 apiaries were collected in spring using simple random sampling. According to international guidelines, the apiaries were classified as affected by colony loss or asymptomatic. Each sample consisted of worker bees, brood and comb-stored pollen. All worker bees and brood samples were analysed individually in order to detect the main honey bee pathogens. Moreover, the presence of residues of the most prevalent agrotoxic insecticides and acaricides was assessed in comb-stored pollen. The general characteristics of the apiaries and sanitary information regarding previous years was evaluated through questionnaires, while the vegetation surrounding the apiaries sampled was assessed by palynological analysis of comb-stored pollen. The colony loss prevalence was 53.5% (CI95%=43.2-63.9) and Nosema ceranae was found to be the only risk factor strongly associated with colony loss. The decision tree also pointed out the impact of the Varroa mite presence while variables such as apiary size, the incorrect application of Varroa mite treatments, and the presence of Acarapis woodi and Kashmir bee virus (KBV) were identified as possible co-factors.

Coumaphos residues in honey, bee brood, and beeswax after Varroa treatment

Residues of acaricide coumaphos were assessed in honey, bee brood, and beeswax during a 2-year field experiment. Honey, bee brood, and beeswax samples were collected before and after routine use of coumaphos in the treatment of bee colonies against varroosis in two consecutive years. Determination of coumaphos in honey and bee brood was based on RP-HPLC with UV detection after a liquid-liquid extraction with hexane or ethyl acetate. Coumaphos in beeswax was identified and quantified by GC/MS. Results indicate the undetectable presence of coumaphos in honey. In bee brood, coumaphos was observed after the treatment. In beeswax, the accumulation of coumaphos was determined not only in hives where it was used but also in hives nearby in which coumaphos was not used. Results indicate the accumulation of coumaphos in bee brood and beeswax. Due to the coumaphos accumulation this drug should be used only in strongly affected bee colonies.