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.

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.

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.

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.

Honey bee exposure scenarios to selected residues through contaminated beeswax.

Twenty-two pesticides and veterinary drugs of which residues were detected in beeswax in Europe were selected according to different criteria. The risk to honey bee health posed by the presence of these residues in wax was assessed based on three exposure scenarios. The first one corresponds to the exposure of larvae following their close contact with wax constituting the cells in which they develop. The second one corresponds to the exposure of larvae following consumption of the larval food that was contaminated from contact with contaminated wax. The third one corresponds to the exposure of adult honey bees following wax chewing when building cells and based on a theoretical worst-case scenario (= intake of contaminants from wax). Following these three scenarios, maximum concentrations which should not be exceeded in beeswax in order to protect honey bee health were calculated for each selected substance. Based on these values, provisional action limits were proposed. Beeswax exceeding these limits should not be put on the market.

Reduced nest development of reared Bombus terrestris within apiary dense human-modified landscapes.

Wild bees are in decline on a local to global scale. The presence of managed honey bees can lead to competition for resources with wild bee species, which has not been investigated so far for human-modified landscapes. In this study we assess if managed honey bee hive density influence nest development (biomass) of bumble bees, an important trait affecting fitness. We hypothesize that domesticated honey bees can negatively affect Bombus terrestris nest development in human-modified landscapes. In Flanders, Belgium, where such landscapes are dominantly present, we selected 11 locations with landscape metrics ranging from urban to agricultural. The bee hive locations were mapped and each location contained one apiary dense (AD) and one apiary sparse (AS) study site (mean density of 7.6 ± 5.7 managed honey bee hives per km2 in AD sites). We assessed the effect of apiary density on the reproduction of reared B. terrestris nests. Reared B. terrestris nests had more biomass increase over 8 weeks in apiary sparse (AS) sites compared to nests located in apiary dense (AD) sites. This effect was mainly visible in urban locations, where nest in AS sites have 99.25 ± 60.99 g more biomass increase compared to nest in urban AD sites. Additionally, we found that managed bumble bee nests had higher biomass increase in urban locations. We conclude that the density of bee hives is a factor to consider in regard to interspecific competition between domesticated honey bees and bumble bees.

Metagenomic Approach with the NetoVIR Enrichment Protocol Reveals Virus Diversity within Ethiopian Honey Bees (Apis mellifera simensis).

Metagenomics studies have accelerated the discovery of novel or divergent viruses of the honey bee. However, most of these studies predominantly focused on RNA viruses, and many suffer from the relatively low abundance of viral nucleic acids in the samples (i.e., compared to that of the host). Here, we explored the virome of the Ethiopian honey bee, Apis mellifera simensis, using an unbiased metagenomic approach in which the next-generation sequencing step was preceded by an enrichment protocol for viral particles. Our study revealed five well-known bee viruses and 25 atypical virus species, most of which have never been found in A. mellifera before. The viruses belong to Iflaviridae, Dicistroviridae, Secoviridae, Partitiviridae, Parvoviridae, Potyviridae, and taxonomically unclassified families. Fifteen of these atypical viruses were most likely plant-specific, and the remaining ten were presumed to be insect-specific. Apis mellifera filamentous virus (AmFV) was found in one sampling site out of 10. Two samples contained high read counts of a virus similar to Diatraea saccharales densovirus (DsDNV), which is a virus that causes high mortality in the sugarcane borer. AmFV and the DsDNV-like virus were the only DNA viruses found. Three viruses that primarily infect Drosophila spp. were also discovered: La Jolla virus (LJV), Kilifi virus (KiV), and Thika virus. Our study suggests that phoretic varroa mites are involved in the transmission of LJV and KiV and that both viruses replicate in mites and adult bees. We also found an overwhelming dominance of the deformed wing virus type B variant, which fits well with the apparently harmless infestation by Varroa destructor. It was suggested that Ethiopian bees have developed tolerance against virus infections as the result of natural selection.

Heritability estimates of the novel trait 'suppressed in ovo virus infection' in honey bees (Apis mellifera).

Honey bees are under pressure due to abnormal high colony death rates, especially during the winter. The infestation by the Varroa destructor mite and the viruses that this ectoparasite transmits are generally considered as the bees' most important biological threats. Almost all efforts to remedy this dual infection have so far focused on the control of the Varroa mite alone and not on the viruses it transmits. In the present study, the sanitary control of breeding queens was conducted on eggs taken from drone brood for 4 consecutive years (2015-2018). The screening was performed on the sideline of an ongoing breeding program, which allowed us to estimate the heritabilities of the virus status of the eggs. We used the term 'suppressed in ovo virus infection' (SOV) for this novel trait and found moderate heritabilities for the presence of several viruses simultaneously and for the presence of single viral species. Colonies that expressed the SOV trait seemed to be more resilient to virus infections as a whole with fewer and less severe Deformed wing virus infections in most developmental stages, especially in the male caste. The implementation of this novel trait into breeding programs is recommended.

Nationwide Screening for Bee Viruses and Parasites in Belgian Honey Bees.

The health of honey bees is threatened by multiple factors, including viruses and parasites. We screened 557 honey bee (Apis mellifera) colonies from 155 beekeepers distributed all over Belgium to determine the prevalence of seven widespread viruses and two parasites (Varroa sp. and Nosema sp.). Deformed wing virus B (DWV-B), black queen cell virus (BQCV), and sacbrood virus (SBV) were highly prevalent and detected by real-time RT-PCR in more than 95% of the colonies. Acute bee paralysis virus (ABPV), chronic bee paralysis virus (CBPV) and deformed wing virus A (DWV-A) were prevalent to a lower extent (between 18 and 29%). Most viruses were only present at low or moderate viral loads. Nevertheless, about 50% of the colonies harbored at least one virus at high viral load (>107 genome copies/bee). Varroa mites and Nosema sp. were found in 81.5% and 59.7% of the honey bee colonies, respectively, and all Nosema were identified as Nosema ceranae by real time PCR. Interestingly, we found a significant correlation between the number of Varroa mites and DWV-B viral load. To determine the combined effect of these and other factors on honey bee health in Belgium, a follow up of colonies over multiple years is necessary.