Authoritative subspecies diagnosis tool for European honey bees based on ancestry informative SNPs.

BACKGROUND: With numerous endemic subspecies representing four of its five evolutionary lineages, Europe holds a large fraction of Apis mellifera genetic diversity. This diversity and the natural distribution range have been altered by anthropogenic factors. The conservation of this natural heritage relies on the availability of accurate tools for subspecies diagnosis. Based on pool-sequence data from 2145 worker bees representing 22 populations sampled across Europe, we employed two highly discriminative approaches (PCA and FST) to select the most informative SNPs for ancestry inference. RESULTS: Using a supervised machine learning (ML) approach and a set of 3896 genotyped individuals, we could show that the 4094 selected single nucleotide polymorphisms (SNPs) provide an accurate prediction of ancestry inference in European honey bees. The best ML model was Linear Support Vector Classifier (Linear SVC) which correctly assigned most individuals to one of the 14 subspecies or different genetic origins with a mean accuracy of 96.2% ± 0.8 SD. A total of 3.8% of test individuals were misclassified, most probably due to limited differentiation between the subspecies caused by close geographical proximity, or human interference of genetic integrity of reference subspecies, or a combination thereof. CONCLUSIONS: The diagnostic tool presented here will contribute to a sustainable conservation and support breeding activities in order to preserve the genetic heritage of European honey bees.

How stressors disrupt honey bee biological traits and overwintering mechanisms.

High winter mortality of honey bees (Apis mellifera) has been observed in temperate regions over the past 30 years. Several biotic and abiotic stressors associated with winter colony losses have been identified, but the mechanisms and interactions underlying their effects remain unclear. We reviewed the effects of stressors on key overwintering biological traits, distinguishing between individual and colony traits. We found that disturbances at the level of individual traits can be amplified when transmitted to colony traits. By analyzing these cascading effects, we propose a concept of a feedback loop mechanism of winter mortality. We found that population size, social thermoregulation and honey reserve are integrative traits and can predict overwintering failure. Furthermore, we identified social thermoregulation as a good candidate for an early warning indicator. We therefore discuss existing tools for monitoring hive temperature to help mitigate the current high winter mortality of honey bees and support the sustainability of beekeeping.

Environmental assessment of PAHs through honey bee colonies - A matrix selection study.

The steady conditions of temperature, humidity and air flux within beehives make them a valuable location for conducting environmental monitoring of pollutants such as PAHs. In this context, the selection of an appropriate apicultural matrix plays a key role in these monitoring studies, as it maximizes the information that will be obtained in the analyses while minimizing the inaccurate results. In the present study, three apicultural matrices (honey bees, pollen and propolis) and two passive samplers (APIStrips and silicone wristbands) are compared in terms of the number and total load of PAHs detected in them. Samplings took place in a total of 11 apiaries scattered in Austria, Denmark, and Greece, with analyses performed by GC-MS/MS. Up to 14 different PAHs were identified in silicone wristbands and pollen, whereas the remaining matrices contained a maximum of five contaminants. Naphthalene, 1-methylnaphthalene, 2-methylnaphthalene, and pyrene were found to be the most prevalent substances in the environment. Recovery studies were also performed; these suggested that the chemical structure of APIStrips is likely to produce very strong interactions with PAHs, thus hindering the adequate desorption of these substances from their surface. Overall, silicone wristbands placed inside the beehives proved the most suitable matrix for PAH monitoring through honey bee colonies.

Semi-automated sequence curation for reliable reference datasets in ITS2 vascular plant DNA (meta-)barcoding.

One of the most critical steps for accurate taxonomic identification in DNA (meta)-barcoding is to have an accurate DNA reference sequence dataset for the marker of choice. Therefore, developing such a dataset has been a long-term ambition, especially in the Viridiplantae kingdom. Typically, reference datasets are constructed with sequences downloaded from general public databases, which can carry taxonomic and other relevant errors. Herein, we constructed a curated (i) global dataset, (ii) European crop dataset, and (iii) 27 datasets for the EU countries for the ITS2 barcoding marker of vascular plants. To that end, we first developed a pipeline script that entails (i) an automated curation stage comprising five filters, (ii) manual taxonomic correction for misclassified taxa, and (iii) manual addition of newly sequenced species. The pipeline allows easy updating of the curated datasets. With this approach, 13% of the sequences, corresponding to 7% of species originally imported from GenBank, were discarded. Further, 259 sequences were manually added to the curated global dataset, which now comprises 307,977 sequences of 111,382 plant species.

More insight into the chemical composition of Greek propolis; differences and similarities with Turkish propolis.

We aimed to determine the differences and similarities between Greek and Turkish propolis with respect to their chemical composition given that the two countries have many similarities in floral biodiversity. We observed that: a) Greek propolis is different from the European-type propolis, having a high terpene content; therefore we can definitely characterize it as a Mediterranean type; b) the Turkish propolis collected along the coast line of the Aegean Sea is similiar to the examined Greek propolis; c) the remaining Turkish samples, originating from the European part of Turkey, were found to be similiar to the European-type propolis, having a high flavonoid content. Finally, especially two compounds, beta-elemene and totarol, were found in Greek samples in quite high amounts that are thought to have important biological properties.

Four-Year Overview of Winter Colony Losses in Greece: Citizen Science Evidence That Transitioning to Organic Beekeeping Practices Reduces Colony Losses.

The honey bee is one of the most important pollinators with a close relationship to humans. The questionnaire from the non-governmental association "COLOSS", answered by beekeepers around the world, is a valuable tool for monitoring and analyzing factors involved in overwintering losses, as well as for understanding the evolution of the beekeeping sector over the years. Between 2018-2021, Greece's participation in this survey involved collecting data from 752 beekeepers and 81,903 hives, from almost the whole country, with a stable balance between professional/non-professional participants and hives, providing a solid representation of the beekeeping practices and winter losses during this period. The results of this study identify a transition towards more natural beekeeping practices concomitant with a significant decrease in winter losses (average losses in 2018: 22.3% and 2019: 24%, dropped in 2020: 14.4% and 2021: 15.3%). Indeed, some factors, such as the increased use of natural landscapes for honey production (from 66.7% usage in 2018 to 76.3% in 2021) and the reduction in the exclusive use of synthetic acaricides (from 24.7% usage in 2018 to 6.7% in 2021) seem to have a significant impact on hive survival. Although these correlations remain to be confirmed experimentally, our study shows that Greek beekeepers follow recommendations and policies toward more sustainable practices. In the future, these trends could be further analyzed and integrated into training programs to strengthen the cooperation and information exchange between citizens and science.

Enhancing the environmental monitoring of pesticide residues through Apis mellifera colonies: Honey bees versus passive sampling.

The use of apicultural matrices for the environmental monitoring of pesticides is a widely employed approach that facilitates to a great extent the sampling procedures. Honey bees are one of the most commonly employed matrices in these studies due to their abundance in the colonies and their direct contact with the beehive and the environment. However, the analysis of this matrix is associated to a lack of representativity of the contaminants accumulated within the beehive, due mainly to the limited number of honey bees that are sampled and analyzed compared to the population in a hive. This small proportion of organisms which are sampled from the colony may lead to underestimations or overestimations of the total pesticide load, depending on the specific individuals that are included in the analysis. In the present work, the passive, non-invasive APIStrip-based sampling approach is compared to active bee sampling with a total of 240 samples taken from 15 apiaries from Austria, Denmark and Greece over a two-month period in 2022. The APIStrips have been found to provide a more comprehensive image of the pesticide residues accumulated in the beehive in terms of number of identified residues and robustness of the results. A total of 74 different pesticide residues were detected: the use of APIStrips allowed to detect 66 pesticides in the three countries, compared to 38 residues in honey bees. The use of APIStrips also resulted in a higher percentage of positive samples (containing at least one pesticide residue). The results provided by the passive sampling approach were also more consistent among the replicates and over time, which reveals an increased sampling robustness.

Honey bee (Apis mellifera) wing images: a tool for identification and conservation.

BACKGROUND: The honey bee (Apis mellifera) is an ecologically and economically important species that provides pollination services to natural and agricultural systems. The biodiversity of the honey bee in parts of its native range is endangered by migratory beekeeping and commercial breeding. In consequence, some honey bee populations that are well adapted to the local environment are threatened with extinction. A crucial step for the protection of honey bee biodiversity is reliable differentiation between native and nonnative bees. One of the methods that can be used for this is the geometric morphometrics of wings. This method is fast, is low cost, and does not require expensive equipment. Therefore, it can be easily used by both scientists and beekeepers. However, wing geometric morphometrics is challenging due to the lack of reference data that can be reliably used for comparisons between different geographic regions. FINDINGS: Here, we provide an unprecedented collection of 26,481 honey bee wing images representing 1,725 samples from 13 European countries. The wing images are accompanied by the coordinates of 19 landmarks and the geographic coordinates of the sampling locations. We present an R script that describes the workflow for analyzing the data and identifying an unknown sample. We compared the data with available reference samples for lineage and found general agreement with them. CONCLUSIONS: The extensive collection of wing images available on the Zenodo website can be used to identify the geographic origin of unknown samples and therefore assist in the monitoring and conservation of honey bee biodiversity in Europe.

Honey vs. Mite-A Trade-Off Strategy by Applying Summer Brood Interruption for Varroa destructor Control in the Mediterranean Region.

In this study, we investigated the effect of queen caging on honey bee colonies' post-treatment development and the optimal timing of method application on honey production during the main summer nectar flow. We conducted the study in nine apiaries (N = 9) across six Mediterranean countries, with a total of 178 colonies. The colonies were divided into three test groups: QC1, QC2, and C. The QC1 group involved queens caged for a total of 28 days before the expected harvesting day. In the QC2 group, queens were caged for 28 days, but only 14 days before the expected harvesting day. The C group consisted of queens that were not caged, and the colonies received common local treatments. In both the QC1 and QC2 groups, the colonies were treated with a 4.2% oxalic acid (OA) solution by trickling after the queen release. Our findings revealed no significant adverse effects (p > 0.05) on colony strength at the end of the study resulting from queen caging. However, significantly lower amounts of honey were extracted from the QC1 group compared to both the QC2 group (p = 0.001) and the C group (p = 0.009). Although there were no initial differences in Varroa destructor infestation between the groups, ten weeks later, a significantly higher infestation was detected in the C group compared to both the QC1 group (p < 0.01) and the QC2 group (p = 0.003). Overall, our study demonstrates that queen caging, in combination with the use of OA, is an effective treatment for controlling V. destructor. However, the timing of caging plays a crucial role in honey production outcomes.

Emerging threats and opportunities to managed bee species in European agricultural systems: a horizon scan.

Managed bee species provide essential pollination services that contribute to food security worldwide. However, managed bees face a diverse array of threats and anticipating these, and potential opportunities to reduce risks, is essential for the sustainable management of pollination services. We conducted a horizon scanning exercise with 20 experts from across Europe to identify emerging threats and opportunities for managed bees in European agricultural systems. An initial 63 issues were identified, and this was shortlisted to 21 issues through the horizon scanning process. These ranged from local landscape-level management to geopolitical issues on a continental and global scale across seven broad themes-Pesticides & pollutants, Technology, Management practices, Predators & parasites, Environmental stressors, Crop modification, and Political & trade influences. While we conducted this horizon scan within a European context, the opportunities and threats identified will likely be relevant to other regions. A renewed research and policy focus, especially on the highest-ranking issues, is required to maximise the value of these opportunities and mitigate threats to maintain sustainable and healthy managed bee pollinators within agricultural systems.

Polar tube protein gene diversity among Nosema ceranae strains derived from a Greek honey bee health study.

Honey bee samples from 54 apiaries originating from 37 geographic locations of Greece were screened for Nosema apis and Nosema ceranae. Furthermore 15 samples coming from 12 geographic locations were screened also for Paenibacilluslarvae and Melissococcus plutonius and seven honey bee virus species, for the first time on a nation-wide level. There was a tendency in finding proportionally higher spore counts in samples from apiaries that suffered important colony losses. P. larvae bacteria were identified in two samples and each of the tested bee viruses could be detected in at least one of the examined samples, with IAPV, CBPV and SBV being the least abundant and BQCV and DWV being the most abundant. In the study we focused on polymorphism of a N. ceranae gene encoding a polar tube protein (PTP) as similar genes were proven to be highly polymorphic in the microsporidian parasites Encephalitozoon cuniculi and Encephalitozoon hellem. The polymorphism observed in the PTP gene sequences from a single sample (bee hive) was unexpected and can thus be considered to be a major obstacle for genotyping.

Genetic structure of Marchalina hellenica (Hemiptera: Margarodidae) populations from Turkey: preliminary mtDNA sequencing data.

The scale insect Marchalina hellenica (Gennadius) (Hemiptera: Margarodidae) contributes to the production of pine honey in Turkey and Greece via the honeydew excreted when it feeds on pine trees. Although it is an insect of prime economic importance, there is no information on its genetic structure. Preliminary data were obtained based on sequencing analysis of 12s rDNA and COI mtDNA gene segments from samples from four areas of Turkey. Sequences of the 12s rDNA gene segment from Greek samples available in GenBank were also included. No variability was detected concerning the COI mtDNA gene segment analysis, although 13 haplotypes were revealed based on the 12s rDNA gene segment. The most distant population was from Mudanya-Bursa Province (Turkey). Further research is necessary on the genetic structure and variability of M. hellenica populations from the two neighboring countries.

Biological Properties of Bee Bread Collected from Apiaries Located across Greece.

Bee bread is the only fermented product of the beehive. It constitutes the main source of proteins, lipids, vitamins, and macro- and microelements in honeybee nutrition and it exerts antioxidant and antimicrobial properties, though research on these aspects has been limited so far. In this study 18 samples of Greek bee bread, two of which were monofloral, were collected during different seasons from diverse locations such as Crete and Mount Athos and were tested for their bioactivity. Samples were analyzed for their antibacterial properties, antioxidant activity, total phenolic content (TPC), and total flavonoid content (TFC). The antimicrobial activity of each sample was tested against Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Salmonella typhimurium. Our data demonstrate that all samples exert inhibitory and most of them bactericidal activity against at least two pathogens. Furthermore, all samples exert significant antioxidant activity, where the monofloral Castanea Sativa sample demonstrated superior antioxidant activity. Nevertheless, the antioxidant and antimicrobial activity were not strongly correlated. Furthermore, machine learning methods demonstrated that the palynological composition of the samples is a good predictor of their TPC and ABTS activity. This is the first study that focuses on the biological properties of Greek bee bread and demonstrates that bee bread can be considered a functional food and a possible source of novel antimicrobial compounds.

CSI Pollen: Diversity of Honey Bee Collected Pollen Studied by Citizen Scientists.

A diverse supply of pollen is an important factor for honey bee health, but information about the pollen diversity available to colonies at the landscape scale is largely missing. In this COLOSS study, beekeeper citizen scientists sampled and analyzed the diversity of pollen collected by honey bee colonies. As a simple measure of diversity, beekeepers determined the number of colors found in pollen samples that were collected in a coordinated and standardized way. Altogether, 750 beekeepers from 28 different regions from 24 countries participated in the two-year study and collected and analyzed almost 18,000 pollen samples. Pollen samples contained approximately six different colors in total throughout the sampling period, of which four colors were abundant. We ran generalized linear mixed models to test for possible effects of diverse factors such as collection, i.e., whether a minimum amount of pollen was collected or not, and habitat type on the number of colors found in pollen samples. To identify habitat effects on pollen diversity, beekeepers' descriptions of the surrounding landscape and CORINE land cover classes were investigated in two different models, which both showed that both the total number and the rare number of colors in pollen samples were positively affected by 'urban' habitats or 'artificial surfaces', respectively. This citizen science study underlines the importance of the habitat for pollen diversity for bees and suggests higher diversity in urban areas.

A Pesticide Residues Insight on Honeybees, Bumblebees and Olive Oil after Pesticidal Applications against the Olive Fruit Fly Bactrocera oleae (Diptera: Tephritidae).

In 2017 and 2018, a field survey was initiated on Greek olive orchards to investigate the attractiveness of bait spray applications and the impact of cover and bait sprays applied against the olive fruit fly Bactrocera oleae (Diptera: Tephritidae), on the honeybee, Apis mellifera L. and bumblebees Bombus terrestris, by investigating the pesticides' residual prevalence. Bee colonies were evenly distributed in three sites located on coastal areas of Western Crete and visited almost weekly between July and October. Samples collected, were analyzed using existing or developed-optimized liquid and gas chromatographic methods. In bee samples, concentrations varied from 0.0013 to 2.3 mg/kg for dimethoate, from 0.0013-0.059 mg/kg for its metabolite omethoate, and from 0.0035 to 0.63 mg/kg regarding the pyrethroids, ß-cyfluthrin and λ-cyhalothrin. In one bee sample dimethoate concentration exceeded both acute oral and contact median lethal dose (LD50). Residue findings in bees, along with verified olive oil residues corroborated that those insecticides had been applied in the olive orchards and transferred to bees. The possibility of non-target effects of the bait sprays to the bees, as well as the impact of the contaminated olive to the bees are discussed.

Evaluation of Suppressed Mite Reproduction (SMR) Reveals Potential for Varroa Resistance in European Honey Bees (Apis mellifera L.).

In the fight against the Varroa destructor mite, selective breeding of honey bee (Apis mellifera L.) populations that are resistant to the parasitic mite stands as a sustainable solution. Selection initiatives indicate that using the suppressed mite reproduction (SMR) trait as a selection criterion is a suitable tool to breed such resistant bee populations. We conducted a large European experiment to evaluate the SMR trait in different populations of honey bees spread over 13 different countries, and representing different honey bee genotypes with their local mite parasites. The first goal was to standardize and validate the SMR evaluation method, and then to compare the SMR trait between the different populations. Simulation results indicate that it is necessary to examine at least 35 single-infested cells to reliably estimate the SMR score of any given colony. Several colonies from our dataset display high SMR scores indicating that this trait is present within the European honey bee populations. The trait is highly variable between colonies and some countries, but no major differences could be identified between countries for a given genotype, or between genotypes in different countries. This study shows the potential to increase selective breeding efforts of V. destructor resistant populations.

Stress indicator gene expression profiles, colony dynamics and tissue development of honey bees exposed to sub-lethal doses of imidacloprid in laboratory and field experiments.

In this study, different context-dependent effects of imidacloprid exposure on the honey bee response were studied. Honey bees were exposed to different concentrations of imidacloprid during a time period of 40 days. Next to these variables, a laboratory-field comparison was conducted. The influence of the chronic exposure on gene expression levels was determined using an in-house developed microarray targeting different immunity-related and detoxification genes to determine stress-related gene expression changes. Increased levels of the detoxification genes encoding, CYP9Q3 and CYT P450, were detected in imidacloprid-exposed honey bees. The different context-dependent effects of imidacloprid exposure on honey bees were confirmed physiologically by decreased hypopharyngeal gland sizes. Honey bees exposed to imidacloprid in laboratory cages showed a general immunosuppression and no detoxification mechanisms were triggered significantly, while honey bees in-field showed a resilient response with an immune stimulation at later time points. However, the treated colonies had a brood and population decline tendency after the first brood cycle in the field. In conclusion, this study highlighted the different context-dependent effects of imidacloprid exposure on the honey bee response. These findings warn for possible pitfalls concerning the generalization of results based on specific experiments with short exposure times. The increased levels of CYT P450 and CYP9Q3 combined with an immune response reaction can be used as markers for bees which are exposed to pesticides in the field.

Widespread dispersal of the microsporidian Nosema ceranae, an emergent pathogen of the western honey bee, Apis mellifera.

The economically most important honey bee species, Apis mellifera, was formerly considered to be parasitized by one microsporidian, Nosema apis. Recently, [Higes, M., Martín, R., Meana, A., 2006. Nosema ceranae, a new microsporidian parasite in honeybees in Europe, J. Invertebr. Pathol. 92, 93-95] and [Huang, W.-F., Jiang, J.-H., Chen, Y.-W., Wang, C.-H., 2007. A Nosema ceranae isolate from the honeybee Apis mellifera. Apidologie 38, 30-37] used 16S (SSU) rRNA gene sequences to demonstrate the presence of Nosema ceranae in A. mellifera from Spain and Taiwan, respectively. We developed a rapid method to differentiate between N. apis and N. ceranae based on PCR-RFLPs of partial SSU rRNA. The reliability of the method was confirmed by sequencing 29 isolates from across the world (N =9 isolates gave N. apis RFLPs and sequences, N =20 isolates gave N. ceranae RFLPs and sequences; 100% correct classification). We then employed the method to analyze N =115 isolates from across the world. Our data, combined with N =36 additional published sequences demonstrate that (i) N. ceranae most likely jumped host to A. mellifera, probably within the last decade, (ii) that host colonies and individuals may be co-infected by both microsporidia species, and that (iii) N. ceranae is now a parasite of A. mellifera across most of the world. The rapid, long-distance dispersal of N. ceranae is likely due to transport of infected honey bees by commercial or hobbyist beekeepers. We discuss the implications of this emergent pathogen for worldwide beekeeping.