Known and novel viruses in Belgian honey bees: yearly differences, spatial clustering, and associations with overwintering loss.

In recent years, managed honey bee colonies have been suffering from an increasing number of biotic and abiotic stressors, resulting in numerous losses of colonies worldwide. A pan-European study, EPILOBEE, estimated the colony loss in Belgium to be 32.4% in 2012 and 14.8% in 2013. In the current study, absolute viral loads of four known honey bee viruses (DWV-A, DWV-B, AmFV, and BMLV) and three novel putative honey bee viruses (Apis orthomyxovirus 1, apthili virus, and apparli virus) were determined in 300 Flemish honey bee samples, and associations with winter survival were determined. This revealed that, in addition to the known influence of DWV-A and DWV-B on colony health, one of the newly described viruses (apthili virus) shows a strong yearly difference and is also associated with winter survival. Furthermore, all scrutinized viruses revealed significant spatial clustering patterns, implying that despite the limited surface area of Flanders, local virus transmission is paramount. The vast majority of samples were positive for at least one of the seven investigated viruses, and up to 20% of samples were positive for at least one of the three novel viruses. One of those three, Apis orthomyxovirus 1, was shown to be a genuine honey bee-infecting virus, able to infect all developmental stages of the honey bee, as well as the Varroa destructor mite. These results shed light on the most prevalent viruses in Belgium and their roles in the winter survival of honey bee colonies. IMPORTANCE: The western honey bee (Apis mellifera) is a highly effective pollinator of flowering plants, including many crops, which gives honey bees an outstanding importance both ecologically and economically. Alarmingly high annual loss rates of managed honey bee colonies are a growing concern for beekeepers and scientists and have prompted a significant research effort toward bee health. Several detrimental factors have been identified, such as varroa mite infestation and disease from various bacterial and viral agents, but annual differences are often not elucidated. In this study, we utilize the viral metagenomic survey of the EPILOBEE project, a European research program for bee health, to elaborate on the most abundant bee viruses of Flanders. We complement the existing metagenomic data with absolute viral loads and their spatial and temporal distributions. Furthermore, we identify Apis orthomyxovirus 1 as a potentially emerging pathogen, as we find evidence for its active replication honey bees.

A field realistic model to assess the effects of pesticides residues and adulterants on honey bee gene expression.

While studies on the sublethal effects of chemical residues in beeswax on adult honey bees are increasing, the study protocols assessing the impacts on honey bee brood in realistic conditions still need to be investigated. Moreover, little is known about the residue's effect on gene expression in honey bee brood. This study reports the effects of chlorpyriphos-ethyl, acrinathrin and stearin worker pupae exposure through contaminated or adulterated beeswax on the gene expression of some key health indicators, using a novel in vivo realistic model. Larvae were reared in acrinathrin (12.5, 25, 10 and 100 ppb) and chlorpyriphos-ethyl (5, 10, 500 and 5000 ppb) contaminated or stearin adulterated beeswax (3, 4, 5, 6 and 9%) in newly formed colonies to reduce the influence of external factors. On day 11, mortality rates were assessed. Honey bee pupae were extracted from the comb after 19 days of rearing and were analysed for the gene expression profile of four genes involved in the immune response to pathogens and environmental stress factors (Imd, dorsal, domeless and defensin), and two genes involved in detoxifications mechanisms (CYP6AS14 and CYP9Q3). We found no linear relation between the increase in the pesticide concentrations and the brood mortality rates, unlike stearin where an increase in stearin percentage led to an exponential increase in brood mortality. The immune system of pupae raised in acrinathrin contaminated wax was triggered and the expression of CYP6AS14 was significantly upregulated (exposure to 12.5 and 25 ppb). Almost all expression levels of the tested immune and detoxification genes were down-regulated when pupae were exposed to chlorpyrifos-contaminated wax. The exposure to stearin triggered the immune system and detoxification system of the pupae. The identification of substance-specific response factors might ultimately serve to identify molecules that are safer for bees and the ecosystem's health.

Comprehensive Approach to Phenotype Varroa destructor Reproduction in Honey Bee Drone Brood and Its Correlation with Decreased Mite Reproduction (DMR).

The mechanisms of action behind decreased mite reproduction (DMR) are still unknown, but current hypotheses state that DMR is the result of brood-intrinsic and/or external disturbances in the V. destructor-honey bee pupa signal interactions. For accurate and precise DMR phenotyping, sufficient single infested honey bee brood cells are required (e.g., 35), which requires extensive labor and time and may exclude many samples not reaching the threshold. We defined a new comprehensive trait called the 'mean V. destructor reproduction rate' (mVR), which describes the mean number of offspring mites per infested cell in the sample while compensating for the reduced number of offspring with increasing multiple infested cells. We found a significant correlation between mVR and DMR, allowing for an estimation of DMR based on the mVR only. When the mVR was calculated with 10 infested cells, we found an average variation in mVR of 16.8%. For the same variation in DMR determination, 40 single infested cells are required. This broader look at V. destructor resistance phenotyping can improve the applicability and effectiveness of traits related to V. destructor reproduction in honey bee breeding programs.

Allele Frequencies of Genetic Variants Associated with Varroa Drone Brood Resistance (DBR) in Apis mellifera Subspecies across the European Continent.

Implementation of marker-assisted selection (MAS) in modern beekeeping would improve sustainability, especially in breeding programs aiming for resilience against the parasitic mite Varroa destructor. Selecting honey bee colonies for natural resistance traits, such as brood-intrinsic suppression of varroa mite reproduction, reduces the use of chemical acaricides while respecting local adaptation. In 2019, eight genomic variants associated with varroa non-reproduction in drone brood were discovered in a single colony from the Amsterdam Water Dune population in the Netherlands. Recently, a new study tested the applicability of these eight genetic variants for the same phenotype on a population-wide scale in Flanders, Belgium. As the properties of some variants varied between the two studies, one hypothesized that the difference in genetic ancestry of the sampled colonies may underly these contribution shifts. In order to frame this, we determined the allele frequencies of the eight genetic variants in more than 360 Apis mellifera colonies across the European continent and found that variant type allele frequencies of these variants are primarily related to the A. mellifera subspecies or phylogenetic honey bee lineage. Our results confirm that population-specific genetic markers should always be evaluated in a new population prior to using them in MAS programs.

Eggs sampling as an effective tool for identifying the incidence of viruses in honey bees involved in artificial queen rearing.

The Carniolan honey bee (Apis mellifera carnica) plays an essential role in crop pollination, environment diversity, and the production of honey bee products. However, the health of individual honey bees and their colonies is under pressure due to multiple stressors, including viruses as a significant threat to bees. Monitoring various virus infections could be a crucial selection tool during queen rearing. In the present study, samples from all developmental stages (eggs, larvae, pupae, and queens) were screened for the incidence of seven viruses during queen rearing in Slovenia. The screening of a total of 108 samples from five queen breeders was performed by the RT-qPCR assays. The results showed that the highest incidence was observed for black queen cell virus (BQCV), Lake Sinai virus 3 (LSV3), deformed wing virus B (DWV-B), and sacbrood virus (SBV). The highest viral load was detected in queens (6.07 log10 copies/queen) and larvae (5.50 log10 copies/larva) for BQCV, followed by SBV in larvae (5.47 log10 copies/larva). When comparing all the honey bee developmental stages, the eggs exhibited general screening for virus incidence and load in queen mother colonies. The results suggest that analyzing eggs is a good indicator of resilience to virus infection during queen development.

Population-wide modelling reveals prospects of marker-assisted selection for parasitic mite resistance in honey bees.

In 2019, a joint eight-variant model was published in which eight single nucleotide polymorphisms (SNPs) in seven Apis mellifera genes were associated with Varroa destructor drone brood resistance (DBR, i.e. mite non-reproduction in drone brood). As this model was derived from only one Darwinian Black Bee Box colony, it could not directly be applied on a population-overarching scale in the northern part of Belgium (Flanders), where beekeepers prefer the carnica subspecies. To determine whether these eight SNPs remained associated with the DBR trait on a Flemish colony-broad scope, we performed population-wide modelling through sampling of various A. mellifera carnica colonies, DBR scoring of Varroa-infested drone brood and variant genotyping. Novel eight-variant modelling was performed and the classification performance of the eight SNPs was evaluated. Besides, we built a reduced three-variant model retaining only three genetic variants and found that this model classified 76% of the phenotyped drones correctly. To examine the spread of beneficial alleles and predict the DBR probability distribution in Flanders, we determined the allelic frequencies of the three variants in 292 A. mellifera carnica queens. As such, this research reveals prospects of marker-assisted selection for Varroa drone brood resistance in honeybees.

Quantitative assessments of honeybee colony's response to an artificial vibrational pulse resulting in non-invasive measurements of colony's overall mobility and restfulness.

In this work we aim to provide a quantitative method allowing the probing of the physiological status of honeybee colonies by providing them with a gentle, short, external artificial vibrational shockwave, and recording their response. The knock is provided by an external electromagnetic shaker attached to the outer wall of a hive, driven by a computer with a 0.1 s long, monochromatic vibration at 340Hz set to an amplitude that occasionally yields a mild response from the bees, recorded by an accelerometer placed in the middle of the central frame of the colony. To avoid habituation, the stimulus is supplied at randomised times, approximately every hour. The method is pioneered with a pilot study on a single colony hosted indoors, then extended onto eight outdoors colonies. The results show that we can quantitatively sense the colony's overall mobility, independently from another physiological aspect, which is phenomenologically explored. Using this, a colony that is queenless is easily discriminated from the others.

System Mapping of the Production and Value Chain to Explore Beekeeping Potential in Southwest Ethiopia.

Ethiopia has a high potential for the production of honey and other apiary products due to its ideal agroecology. This potential is, however, not yet well utilized due to weak production and valorization systems. The study analyzed beekeeping systems and their honey value chain to detect the barriers and to explore ways to better exploit the existing potential. Descriptive statistics, a SWOT and PESTEL matrix, and system mapping were utilized for analysis. Ethiopian beekeeping is still dominated by traditional production systems, followed by modern and transitional systems, differing in types of beehives and the average amount of honey yield. The combined SWOT-PESTEL analysis revealed challenges like a limited supply and high cost of modern beehives, shortage of credit, absence of a honey marketing legal framework, pest and predator attacks, absconding, and uncontrolled application of agrochemicals. Opportunities include the globally increasing demand for honey, availability of good investment policy, conducive agroecology, and support from NGOs. The less productive techniques of smallholder beekeepers' crude honey production for local beverage making affected the good use of the potential and minimized its contribution to the local and national economy. On the contrary, strengthening private investors and cooperatives towards the production of fully and semi-processed honey impacted the utilization of the potential positively.

Shift in virus composition in honeybees (Apis mellifera) following worldwide invasion by the parasitic mite and virus vector Varroa destructor.

Invasive vectors can induce dramatic changes in disease epidemiology. While viral emergence following geographical range expansion of a vector is well known, the influence a vector can have at the level of the host's pathobiome is less well understood. Taking advantage of the formerly heterogeneous spatial distribution of the ectoparasitic mite Varroa destructor that acts as potent virus vector among honeybees Apis mellifera, we investigated the impact of its recent global spread on the viral community of honeybees in a retrospective study of historical samples. We hypothesized that the vector has had an effect on the epidemiology of several bee viruses, potentially altering their transmissibility and/or virulence, and consequently their prevalence, abundance, or both. To test this, we quantified the prevalence and loads of 14 viruses from honeybee samples collected in mite-free and mite-infested populations in four independent geographical regions. The presence of the mite dramatically increased the prevalence and load of deformed wing virus, a cause of unsustainably high colony losses. In addition, several other viruses became more prevalent or were found at higher load in mite-infested areas, including viruses not known to be actively varroa-transmitted, but which may increase opportunistically in varroa-parasitized bees.

Expression of Molecular Markers of Resilience against Varroa destructor and Bee Viruses in Ethiopian Honey Bees (Apis mellifera simensis) Focussing on Olfactory Sensing and the RNA Interference Machinery.

Varroa destructor mites and the viruses it vectors are two major factors leading to high losses of honey bees (Apis mellifera) colonies worldwide. However, honey bees in some African countries show resilience to varroa infestation and/or virus infections, although little is known about the mechanisms underlying this resilience. In this study, we investigated the expression profiles of some key molecular markers involved in olfactory sensing and RNA interference, as these processes may contribute to the bees' resilience to varroa infestation and virus infection, respectively. We found significantly higher gene expression of the odorant binding protein, OBP14, in the antennae of Ethiopian bees compared to Belgian bees. This result suggests the potential of OBP14 as a molecular marker of resilience to mite infestation. Scanning electron microscopy showed no significant differences in the antennal sensilla occurrence and distribution, suggesting that resilience arises from molecular processes rather than morphological adaptations. In addition, seven RNAi genes were upregulated in the Ethiopian honey bees and three of them-Dicer-Drosha, Argonaute 2, and TRBP2-were positively correlated with the viral load. We can conclude that the antiviral immune response was triggered when bees were experiencing severe viral infection and that this might contribute to the bees' resilience to viruses.

Beekeeping in Europe facing climate change: A mixed methods study on perceived impacts and the need to adapt according to stakeholders and beekeepers.

The beekeeping sector is suffering from the detrimental effects of climate change, both directly and indirectly. Despite numerous studies conducted on this subject, large-scale research incorporating stakeholders' and beekeepers' perspectives has remained elusive. This study aims to bridge this gap by assessing the extent to which stakeholders involved in the European beekeeping sector and European beekeepers perceive and experience the impacts of climate change on their operations, and whether they had to adapt their practices accordingly. To this end, a mixed-methods study including in-depth stakeholder interviews (n = 41) and a pan-European beekeeper survey (n = 844) was completed within the frame of the EU-funded H2020-project B-GOOD. The development of the beekeeper survey was informed by insights from literature and the stakeholder interviews. The results highlighted significant regional disparities in the perceived impacts of climate change, with beekeepers in Southern European regions expressing more negative outlooks, while Northern European beekeepers reported more favourable experiences. Furthermore, survey analysis revealed beekeepers who were classified as 'heavily impacted' by climate change. These beekeepers reported lower average honey yields, higher colony winter loss rates and a stronger perceived contribution of honey bees to pollination and biodiversity, underscoring climate change's detrimental impacts on the beekeeping sector. Multinomial logistic regression revealed determinants of the likelihood of beekeepers being classified as 'heavily impacted' by climate change. This analysis indicates that Southern European beekeepers experienced a 10-fold likelihood of being classified as heavily impacted by climate change compared to Northern European beekeepers. Other significant factors distinguishing 'winners' and 'losers' were self-reported level of professionalism as a beekeeper (ranging from pure hobbyist to fully professional, Odds Ratio (OR) = 1.31), number of years active in beekeeping (OR = 1.02), availability of floral resources throughout the bee season (OR = 0.78), beehives located in a forested environment (OR = 1.34), and the presence of local policy measures addressing climate change-related challenges (OR = 0.76).

Convivina is a specialised core gut symbiont of the invasive hornet Vespa velutina.

We provide a culturomics analysis of the cultivable bacterial communities of the crop, midgut and hindgut compartments, as well as the ovaries, of the invasive insect Vespa velutina, along with a cultivation-independent analysis of samples of the same nest through 16S rRNA amplicon sequencing. The Vespa velutina bacterial symbiont community was dominated by the genera Convivina, Fructobacillus, Lactiplantibacillus, Lactococcus, Sphingomonas and Spiroplasma. Lactococcus lactis and Lactiplantibacillus plantarum represented generalist core lactic acid bacteria (LAB) symbionts, while Convivina species and Fructobacillus fructosus represented highly specialised core LAB symbionts with strongly reduced genome sizes. Sphingomonas and Spiroplasma were the only non-LAB core symbionts but were not isolated. Convivina bacteria were particularly enriched in the hornet crop and included Convivina intestini, a species adapted towards amino acid metabolism, and Convivina praedatoris sp. nov. which was adapted towards carbohydrate metabolism.

Virus Prevalence in Egg Samples Collected from Naturally Selected and Traditionally Managed Honey Bee Colonies across Europe.

Monitoring virus infections can be an important selection tool in honey bee breeding. A recent study pointed towards an association between the virus-free status of eggs and an increased virus resistance to deformed wing virus (DWV) at the colony level. In this study, eggs from both naturally surviving and traditionally managed colonies from across Europe were screened for the prevalence of different viruses. Screenings were performed using the phenotyping protocol of the 'suppressed in ovo virus infection' trait but with qPCR instead of end-point PCR and a primer set that covers all DWV genotypes. Of the 213 screened samples, 109 were infected with DWV, 54 were infected with black queen cell virus (BQCV), 3 were infected with the sacbrood virus, and 2 were infected with the acute bee paralyses virus. It was demonstrated that incidences of the vertical transmission of DWV were more frequent in naturally surviving than in traditionally managed colonies, although the virus loads in the eggs remained the same. When comparing virus infections with queen age, older queens showed significantly lower infection loads of DWV in both traditionally managed and naturally surviving colonies, as well as reduced DWV infection frequencies in traditionally managed colonies. We determined that the detection frequencies of DWV and BQCV in honey bee eggs were lower in samples obtained in the spring than in those collected in the summer, indicating that vertical transmission may be lower in spring. Together, these patterns in vertical transmission show that honey bee queens have the potential to reduce the degree of vertical transmission over time.

Cold case: The disappearance of Egypt bee virus, a fourth distinct master strain of deformed wing virus linked to honeybee mortality in 1970's Egypt.

In 1977, a sample of diseased adult honeybees (Apis mellifera) from Egypt was found to contain large amounts of a previously unknown virus, Egypt bee virus, which was subsequently shown to be serologically related to deformed wing virus (DWV). By sequencing the original isolate, we demonstrate that Egypt bee virus is in fact a fourth unique, major variant of DWV (DWV-D): more closely related to DWV-C than to either DWV-A or DWV-B. DWV-A and DWV-B are the most common DWV variants worldwide due to their close relationship and transmission by Varroa destructor. However, we could not find any trace of DWV-D in several hundred RNA sequencing libraries from a worldwide selection of honeybee, varroa and bumblebee samples. This means that DWV-D has either become extinct, been replaced by other DWV variants better adapted to varroa-mediated transmission, or persists only in a narrow geographic or host range, isolated from common bee and beekeeping trade routes.

Beekeepers perception of risks affecting colony loss: A pilot survey.

Understanding amateur beekeepers' perception of risks affecting bee health and mortality is essential to analyse the reasons for adopting or rejecting good management practices. A perception survey on how beekeepers perceive and manage factors related to climate change, Varroa infestation, management practices, and pesticide exposure was designed and launched online. This unpreceded sociological survey involved 355 beekeepers spread all over Belgium. A two-sample t test with unequal variances comparing beekeepers with colony loss rates below or exceeding the acceptable level, that is <10% and ≥10%, indicates that beekeepers (N = 213) with colony loss rates <10% generally have greater average levels of perceived risks and the benefits of action that lead to increased motivation to act in better ways. The results of this survey highlight the importance of looking beyond socio-economic determinants in any risk mitigation strategy associated with bee mortality when dealing with amateur beekeepers.

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.

Pyrethroid target-site resistance mutations in populations of the honey bee parasite Varroa destructor (Acari: Varroidae) from Flanders, Belgium.

The honey bee ectoparasite Varroa destructor is considered the major threat to apiculture, as untreated colonies of Apis mellifera usually collapse within a few years. In order to control this mite, many beekeepers rely on a limited number of approved synthetic acaricides, including the pyrethroids tau-fluvalinate and flumethrin. Due to the intensive use of these products, resistance is now commonplace in many beekeeping regions across the world. In the present study, the occurrence of amino acid substitutions at residue L925 of the voltage-gate sodium channel-the pyrethroid target site-was studied in Varroa populations collected throughout Flanders, Belgium. Dose-response bioassays supported the involvement of the frequently observed L925V substitution in flumethrin resistance, resulting in a 12.64-fold increase of the LC50 in a Varroa population mostly consisting of homozygous 925 V/V mites. With the presence of L925 substitutions in about four out of 10 screened apiaries, the use of pyrethroid-based varroacides in Flanders, including the recently released PolyVar® Yellow, should be carefully considered.

Risk and protective indicators of beekeeping management practices.

Explaining the reasons for the high honey bee (Apis mellifera) colony loss rate in recent years has become a top global research priority in apicultural and agricultural sciences. Although there are indications of the role played by beekeeping management practices on honey bee health, very little information is currently available. Our study aimed to characterize the beekeeping management practices carried out in Belgium, and to determine the relationship between beekeeping management practices and colony losses. Variables obtained from face-to-face questioning of a representative randomized and stratified sample of Belgian beekeepers (n = 186) were integrated into a logistic regression model (univariate and multivariate) and correlated to the declared colony loss rates to identify risk and protective indicators. We used a classification tree analysis to validate the results. We present evidence of a relationship between poor beekeeping management practices and colony losses. The main factors protecting honey bee colonies are the aptitude of the beekeeper to change his management practices, the hive type, the equipment origin and hygiene, wintering in proper conditions (the use of divider boards, i.e. board blocks or space fillers off part of the hive body), the colony strength estimation before wintering, winter monitoring, and last but not least, appropriate integrated pest management. Proper estimation of the Varroa infestation level should be performed prior to treatment. The consequences of poor beekeeping practices on honey bee health can be addressed by proper training of beekeepers. An online tool was developed and published for beekeepers allowing them to evaluate the effect of their management practices on colony health.

Breeding for Virus Resistance and Its Effects on Deformed Wing Virus Infection Patterns in Honey Bee Queens.

Viruses, and in particular the deformed wing virus (DWV), are considered as one of the main antagonists of honey bee health. The 'suppressed in ovo virus infection' trait (SOV) described for the first time that control of a virus infection can be achieved from genetically inherited traits and that the virus state of the eggs is indicative for this. This research aims to explore the effect of the SOV trait on DWV infections in queens descending from both SOV-positive (QDS+) and SOV-negative (QDS-) queens. Twenty QDS+ and QDS- were reared from each time four queens in the same starter-finisher colony. From each queen the head, thorax, ovaries, spermatheca, guts and eviscerated abdomen were dissected and screened for the presence of the DWV-A and DWV-B genotype using qRT-PCR. Queens descending from SOV-positive queens showed significant lower infection loads for DWV-A and DWV-B as well as a lower number of infected tissues for DWV-A. Surprisingly, differences were less expressed in the reproductive tissues, the ovaries and spermatheca. These results confirm that selection on the SOV trait is associated with increased virus resistance across viral genotypes and that this selection drives DWV towards an increased tissue specificity for the reproductive tissues. Further research is needed to explore the mechanisms underlying the interaction between the antiviral response and DWV.

qPCR assays with dual-labeled probes for genotyping honey bee variants associated with varroa resistance.

BACKGROUND: The varroa mite is one of the main causes of honey bee mortality. An important mechanism by which honey bees increase their resistance against this mite is the expression of suppressed mite reproduction. This trait describes the physiological inability of mites to produce viable offspring and was found associated with eight genomic variants in previous research. RESULTS: This paper presents the development and validation of high-throughput qPCR assays with dual-labeled probes for discriminating these eight single-nucleotide variants. Amplicon sequences used for assay validation revealed additional variants in the primer/probe binding sites in four out of the eight assays. As for two of these the additional variants interfered with the genotyping outcome supplementary primers and/or probes were developed. Inclusion of these primers and probes in the assay mixes allowed for the correct genotyping of all eight variants of interest within our bee population. CONCLUSION: These outcomes underline the importance of checking for interfering variants in designing qPCR assays. Ultimately, the availability of this assay allows genotyping for the suppressed mite reproduction trait and paves the way for marker assisted selection in breeding programs.