The wild solitary bees Andrena vaga, Anthophora plumipes, Colletes cunicularius, and Osmia cornuta microbiota are host specific and dominated by endosymbionts and environmental microorganisms.

We characterized the microbial communities of the crop, midgut, hindgut, and ovaries of the wild solitary bees Andrena vaga, Anthophora plumipes, Colletes cunicularius, and Osmia cornuta through 16S rRNA gene and ITS2 amplicon sequencing and a large-scale isolation campaign. The bacterial communities of these bees were dominated by endosymbionts of the genera Wolbachia and Spiroplasma. Bacterial and yeast genera representing the remaining predominant taxa were linked to an environmental origin. While only a single sampling site was examined for Andrena vaga, Anthophora plumipes, and Colletes cunicularius, and two sampling sites for Osmia cornuta, the microbiota appeared to be host specific: bacterial, but not fungal, communities generally differed between the analyzed bee species, gut compartments and ovaries. This may suggest a selective process determined by floral and host traits. Many of the gut symbionts identified in the present study are characterized by metabolic versatility. Whether they exert similar functionalities within the bee gut and thus functional redundancy remains to be elucidated.

Signals of adaptation to agricultural stress in the genomes of two European bumblebees.

Human-induced environmental impacts on wildlife are widespread, causing major biodiversity losses. One major threat is agricultural intensification, typically characterised by large areas of monoculture, mechanical tillage, and the use of agrochemicals. Intensification leads to the fragmentation and loss of natural habitats, native vegetation, and nesting and breeding sites. Understanding the adaptability of insects to these changing environmental conditions is critical to predicting their survival. Bumblebees, key pollinators of wild and cultivated plants, are used as model species to assess insect adaptation to anthropogenic stressors. We investigated the effects of agricultural pressures on two common European bumblebees, Bombus pascuorum and B. lapidarius. Restriction-site Associated DNA Sequencing was used to identify loci under selective pressure across agricultural-natural gradients over 97 locations in Europe. 191 unique loci in B. pascuorum and 260 in B. lapidarius were identified as under selective pressure, and associated with agricultural stressors. Further investigation suggested several candidate proteins including several neurodevelopment, muscle, and detoxification proteins, but these have yet to be validated. These results provide insights into agriculture as a stressor for bumblebees, and signal for conservation action in light of ongoing anthropogenic changes.

Linking remote sensing data to the estimation of pollination services in agroecosystems.

Wild bees are key providers of pollination services in agroecosystems. The abundance of these pollinators and the ecosystem services they provide rely on supporting resources in the landscape. Spatially explicit models that quantify wild bee abundance and pollination services in food crops are built on the foundations of foraging and nesting resources. This dependence limits model implementation as land-cover maps and pollination experts capable of evaluating habitat resource quality are scarce. This study presents a novel approach to assessing crop pollination services using remote sensing data (RSD) as an alternative to the more conventional use of land-cover data and local expertise on spatially explicit models. We used landscape characteristics derived from remote sensors to qualify nesting resources in the landscape and to evaluate the delivery of pollination services by mining bees (Andrena spp.) in 30 fruit orchards located in the Flemish region of Belgium. For this study, we selected mining bees for their importance as local pollinators and underground nesting behavior. We compared the estimated pollination services derived from RSD with those derived from the conventional qualification of nesting resources. We did not observe significant differences (p = 0.68) in the variation in mining bee activity predicted by the two spatial models. Estimated pollination services derived from RSD and conventional characterizations explained 69% and 72% of the total variation, respectively. These results confirmed that RSD can deliver nesting suitability characterizations sufficient for estimating pollination services. This research also illustrates the importance of nesting resources and landscape characteristics when estimating pollination services delivered by insects like mining bees. Our results support the development of holistic agroenvironmental policies that rely on modern tools like remote sensors and promote pollinators by considering nesting resources.

Honey bees and climate explain viral prevalence in wild bee communities on a continental scale.

Viruses are omnipresent, yet the knowledge on drivers of viral prevalence in wild host populations is often limited. Biotic factors, such as sympatric managed host species, as well as abiotic factors, such as climatic variables, are likely to impact viral prevalence. Managed and wild bees, which harbor several multi-host viruses with a mostly fecal-oral between-species transmission route, provide an excellent system with which to test for the impact of biotic and abiotic factors on viral prevalence in wild host populations. Here we show on a continental scale that the prevalence of three broad host viruses: the AKI-complex (Acute bee paralysis virus, Kashmir bee virus and Israeli acute paralysis virus), Deformed wing virus, and Slow bee paralysis virus in wild bee populations (bumble bees and solitary bees) is positively related to viral prevalence of sympatric honey bees as well as being impacted by climatic variables. The former highlights the need for good beekeeping practices, including Varroa destructor management to reduce honey bee viral infection and hive placement. Furthermore, we found that viral prevalence in wild bees is at its lowest at the extreme ends of both temperature and precipitation ranges. Under predicted climate change, the frequency of extremes in precipitation and temperature will continue to increase and may hence impact viral prevalence in wild bee communities.

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.

More is less: mass-flowering fruit tree crops dilute parasite transmission between bees.

Parasites influence wild bee population dynamics and are regarded as one of the main drivers of wild bee decline. Most of these parasites are mainly transmitted between bee species via the use of shared floral resources. Disturbance of the plant-pollinator network at a location can hence disturb the transmission of these parasites. Expansion and intensification of agriculture, another major driver of wild bee decline, often disturbs local plant-pollinator networks by altering the availability and diversity of floral resources. Mass-flowering crops are an extreme example as they provide an abundance of floral resources for a short period of time, substantially altering the present plant-pollinator network. This likely has repercussions on parasite transmission in the pollinator community. Using the bloom of mass-flowering crops we tested the hypothesis that an increase in floral resources can dilute parasite transmission in the pollinator community. To test this, we analysed the presence of parasites in the pollen of the brood cell provisions of Osmia spp., collected from trap nests placed in apple and sweet cherry orchards. We collected pollen at several time intervals during and after mass bloom, and found that pollen collected during mass bloom had significantly lower parasite prevalence compared with pollen collected after mass bloom. Furthermore, using pollen barcoding data we found that the presence of MFCs in pollen was a good predictor for lower parasite prevalence. Taken together, our results indicate that an increase in flower availability can reduce parasite transmission between bees.

The Bee Hemolymph Metabolome: A Window into the Impact of Viruses on Bumble Bees.

State-of-the-art virus detection technology has advanced a lot, yet technology to evaluate the impacts of viruses on bee physiology and health is basically lacking. However, such technology is sorely needed to understand how multi-host viruses can impact the composition of the bee community. Here, we evaluated the potential of hemolymph metabolites as biomarkers to identify the viral infection status in bees. A metabolomics strategy based on ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry was implemented. First, we constructed a predictive model for standardized bumble bees, in which non-infected bees were metabolically differentiated from an overt Israeli acute paralysis virus (IAPV) infection (R2Y = 0.993; Q2 = 0.906), as well as a covert slow bee paralysis virus (SBPV) infection (R2Y = 0.999; Q2 = 0.875). Second, two sets of potential biomarkers were identified, being descriptors for the metabolomic changes in the bee's hemolymph following viral infection. Third, the biomarker sets were evaluated in a new dataset only containing wild bees and successfully discriminated virus infection versus non-virus infection with an AUC of 0.985. We concluded that screening hemolymph metabolite markers can underpin physiological changes linked to virus infection dynamics, opening promising avenues to identify, monitor, and predict the effects of virus infection in a bee community within a specific environment.

Managed bumble bees acquire parasites from their foraging environment: A case study on parasite spillback.

The use of commercially reared bumble bees in agricultural environments has been recognized as a potential threat to wild pollinators due to competition, genetic contamination, and most notably, disease transmission. Higher parasite prevalence near greenhouses where managed bumble bees are used has been linked to parasite spillover from managed to wild bees. However, pathogen transmission is not unidirectional, and can also flow from wild to managed bees. These newly infected managed bees can subsequently re-infect (other) wild bees, in a process known as spillback, which is an alternative explanation for the increased parasite prevalence near greenhouses. Reducing parasite prevalence in managed bees is key to controlling host-parasite dynamics in cases of spillover; in spillback, producing managed bees that are resilient to infection is important. Here we establish that the managed bumble bee Bombus terrestris can acquire parasites from their foraging environment, which is the major infection route for Apicystis spp. and Crithidia spp., but not for Nosema spp.. Managed B. terrestris were found to have a higher prevalence of Crithdia and a higher load of Apicystis than local wild conspecifics, showing that for these parasites, spillback is a possible risk scenario.

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.

Network Centrality as an Indicator for Pollinator Parasite Transmission via Flowers.

Parasites are important actors within ecosystems. However, a key aspect to unraveling parasite epidemiology is understanding transmission. The bee pollinator community harbors several multihost parasites, which have been shown to be able to spread between species via flowers. Hence the plant-pollinator network can provide valuable information on the transmission of these parasites between species. Although several controlled experiments have shown that flowers function as a transmission hub for parasites, the link with the plant-pollinator network has rarely been addressed in the field. Here, one can hypothesize that the most central flowers in the network are more likely to enable parasite transmission between species. In this study, we test this hypothesis in three local plant-pollinator networks and show that the centrality of a plant in a weighted plant-pollinator network is a good predictor of the presence of multihost pollinator parasites on the plant's flowers.

A Growers' Perspective on Crop Pollination and Measures to Manage the Pollination Service of Wild Pollinators in Sweet Cherry Cultivation.

Recent declines in insect pollinators and the increasing dependence on insect pollination in agriculture present major challenges to ensuring future food production. As part of the effort to deal with this challenge, there is a pressing need to understand growers' perceptions with regard to pollinator diversity and crop pollination management. At present, many growers are dependent on domesticated honey bees (Apis mellifera), however, targeted management strategies involving naturally occurring pollinator species might be necessary to ensure future crop pollination. In this study we used semi-structured interviews to explore growers' knowledge about crop pollination and current practices to manage insect pollination in sweet cherry cultivation. Our findings suggest that growers have a clear understanding of the importance of pollination and its determining factors. However, with respect to their current pollination management, growers depend mainly on honey bees and only apply measures to enhance wild pollinator communities to a limited extent. Our study highlights the gap between the growers' perception of the contribution of wild pollinators to crop pollination, and their efforts to manage these species. We conclude that this is due to a lack of communication of recent scientific findings on the contribution of pollinating insects to crop pollination through the information channels that are being used by growers today. It is therefore crucial that scientists, government and other stakeholders work together with growers and communicate scientific evidence as well as practical guidelines to growers.

Metabolomic Analysis of Cricket paralysisvirus Infection in Drosophila S2 Cells Reveals Divergent Effects on Central Carbon Metabolism as Compared with Silkworm Bm5 Cells.

High-throughput approaches have opened new opportunities for understanding biological processes such as persistent virus infections, which are widespread. However, the potential of persistent infections to develop towards pathogenesis remains to be investigated, particularly with respect to the role of host metabolism. To explore the interactions between cellular metabolism and persistent/pathogenic virus infection, we performed untargeted and targeted metabolomic analysis to examine the effects of Cricket paralysis virus (CrPV, Dicistroviridae) in persistently infected silkworm Bm5 cells and acutely infected Drosophila S2 cells. Our previous study (Viruses 2019, 11, 861) established that both glucose and glutamine levels significantly increased during the persistent period of CrPV infection of Bm5 cells, while they decreased steeply during the pathogenic stages. Strikingly, in this study, an almost opposite pattern in change of metabolites was observed during different stages of acute infection of S2 cells. More specifically, a significant decrease in amino acids and carbohydrates was observed prior to pathogenesis, while their abundance significantly increased again during pathogenesis. Our study illustrates the occurrence of diametrically opposite changes in central carbon mechanisms during CrPV infection of S2 and Bm5 cells that is possibly related to the type of infection (acute or persistent) that is triggered by the virus.

A Metabolomics Approach to Unravel Cricket Paralysis Virus Infection in Silkworm Bm5 Cells.

How a host metabolism responds to infection with insect viruses and how it relates to pathogenesis, is little investigated. Our previous study observed that Cricket paralysis virus (CrPV, Dicistroviridae) causes short term persistence in silkworm Bm5 cells before proceeding to acute infection. In this study, a metabolomics approach based on high resolution mass spectrometry was applied to investigate how a host metabolism is altered during the course of CrPV infection in Bm5 cells and which changes are characteristic for the transition from persistence to pathogenicity. We observed that CrPV infection led to significant and stage-specific metabolic changes in Bm5 cells. Differential metabolites abundance and pathway analysis further identified specific metabolic features at different stages in the viral life cycle. Notably, both glucose and glutamine levels significantly increased during CrPV persistent infection followed by a steep decrease during the pathogenic stages, suggesting that the central carbon metabolism was significantly modified during CrPV infection in Bm5 cells. In addition, dynamic changes in levels of polyamines were detected. Taken together, this study characterized for the first time the metabolic dynamics of CrPV infection in insect cells, proposing a central role for the regulation of both amino acid and carbohydrate metabolism during the period of persistent infection of CrPV in Bm5 cells.

Metabolomics-based biomarker discovery for bee health monitoring: A proof of concept study concerning nutritional stress in Bombus terrestris.

Bee pollinators are exposed to multiple natural and anthropogenic stressors. Understanding the effects of a single stressor in the complex environmental context of antagonistic/synergistic interactions is critical to pollinator monitoring and may serve as early warning system before a pollination crisis. This study aimed to methodically improve the diagnosis of bee stressors using a simultaneous untargeted and targeted metabolomics-based approach. Analysis of 84 Bombus terrestris hemolymph samples found 8 metabolites retained as potential biomarkers that showed excellent discrimination for nutritional stress. In parallel, 8 significantly altered metabolites, as revealed by targeted profiling, were also assigned as candidate biomarkers. Furthermore, machine learning algorithms were applied to the above-described two biomarker sets, whereby the untargeted eight components showed the best classification performance with sensitivity and specificity up to 99% and 100%, respectively. Based on pathway and biochemistry analysis, we propose that gluconeogenesis contributed significantly to blood sugar stability in bumblebees maintained on a low carbohydrate diet. Taken together, this study demonstrates that metabolomics-based biomarker discovery holds promising potential for improving bee health monitoring and to identify stressor related to energy intake and other environmental stressors.

Pattern of population structuring between Belgian and Estonian bumblebees.

Several population genetic studies investigated the extent of gene flow and population connectivity in bumblebees. In general, no restriction in gene flow is considered for mainland populations of common bumblebee species. Whether this assumption holds true for all species is not known. An assessment of bumblebee genetic structure in the context of their geographic distribution is needed to prioritize conservation and management needs. Here, we conducted a genetic study on seven bumblebee species occurring in Belgium and Estonia. Using 16 microsatellite markers, we investigated genetic diversity and population structuring in each species. This is the first study investigating population structuring of both declining and stable bumblebee species on both small and large geographic scales. Our results showed no or only low population structuring between the populations of the restricted and declining bumblebee species on both scales, while significant structuring was found for populations of the common species on the larger scale. The latter result, which may be due to human or environmental changes in the landscape, implies the need for the conservation of also widespread bumblebee species. Conservation strategies to improve gene flow and connectivity of populations could avoid the isolation and future losses of populations of these important species.

Honey bee-collected pollen is a potential source of Ascosphaera apis infection in managed bumble bees.

The trade of bumble bees started in the early nineties for pollinator-dependent greenhouse plants. Nowadays, its rearing and transport have received public attention, since managed bees can transfer pathogens to wild bee populations. Therefore, guaranteeing pathogen-free bumble bees is fundamental. The major protein source used in rearing facilities is honey bee-collected pollen. This can carry pathogens, however to date, solid data on the risk of this food source to the health of bumble bees is lacking. Here we performed a large pathogen screening of non-irradiated honey bee-collected pollen to discover particles infective to Bombus terrestris. We identified seven parasites (Apicystis bombi, Ascosphaera apis, Crithidia mellificae, Nosema ceranae, Paenibacillus larvae and two parasites resembling Nosema thomsoni and Microsporidium sp. Oise) and four viruses (CBPV, DWV, IAPV and SBV) in 17 pollen batches from two major European pollen source regions (Spain and Romania). Ascosphaera apis was capable of infecting bumble bees; the larvae showed similar symptoms to chalkbrood disease reported in honey bees. Bumble bee breeding facilities need to be cautious about the potential presence of this disease, which was originally reported in honey bees. Thorough diagnostic and control methods are needed, as risk of spillover to wild bee species is possible.

Double-stranded RNA reduces growth rates of the gut parasite Crithidia mellificae.

Parasites of managed bees can disrupt the colony success of the host, but also influence local bee-parasite dynamics, which is regarded as a threat for wild bees. Therapeutic measures have been suggested to improve the health of managed bees, for instance, exploiting the bees' RNA interference (RNAi) pathway to treat against viral pathogens. Gut trypanosomes are an important group of bee parasites in at least two common managed bee species, i.e., managed Apis mellifera and reared Bombus terrestris. In several trypanosomes, RNAi activity is present, while in other associated genes of RNAi, such as Dicer-like (DCL) and Argonaute (AGO), it is lost. Up to date, the ability to exploit the RNAi of gut trypanosomes of bees has remained unexplored. Here, we screened parasite genomes of two honey bee protozoa (Crithidia mellificae and Lotmaria passim) and two bumble bee protozoa (Crithidia bombi and Crithidia expoeki) for the presence of DCL and AGO proteins. For C. mellificae, we constructed a double-stranded RNA (dsRNA) targeting kinetoplastid membrane protein-11 (KMP-11) to test the RNAi potential to kill this parasite. Transfection with KMP-11 dsRNA, but also adding it to the growth medium resulted in small growth reduction of the trypanosome C. mellificae, thereby showing the limited potential to apply dsRNA therapeutics to control trypanosome infection in managed honey bee species. Within bumble bees, there seems to be no application potentials against C. bombi, as we could only retrieve non-functional DCL- and AGO-related genes within the genome of this bumble bee parasite.

Establishment of wildflower fields in poor quality landscapes enhances micro-parasite prevalence in wild bumble bees.

The current worldwide pollinator decline is caused by the interplay of different drivers. Several strategies have been undertaken to counteract or halt this decline, one of which is the implementation of wildflower fields. These supplementary flowers provide extra food resources and have proven their success in increasing pollinator biodiversity and abundance. Yet such landscape alterations could also alter the host-pathogen dynamics of pollinators, which could affect the populations. In this study, we investigated the influence of sown wildflower fields on the prevalence of micro-parasites and viruses in the wild bumble bee Bombus pascuorum, one of the most abundant bumble bee species in Europe and the Netherlands. We found that the effect of sown wildflower fields on micro-parasite prevalence is affected by the composition of the surrounding landscape and the size of the flower field. The prevalence of micro-parasites increases with increasing size of sown wildflower fields in landscapes with few semi-natural landscape elements. This effect was not observed in landscapes with a high amount of semi-natural landscape elements. We elaborate on two mechanisms which can support these findings: (1) "transmission hot spots" within the altered flower-networks, which could negatively impact hosts experiencing an increased exposure; (2) improved tolerance of the hosts, withstanding higher parasite populations.

Influence of microbiota in the susceptibility of parasitic wasps to abamectin insecticide: deep sequencing, esterase and toxicity tests.

BACKGROUND: The parasitic wasps Eretmocerus mundus, Eretmocerus eremicus and Encarsia formosa are important natural enemies of whiteflies. A broad understanding of their biology, ecology and behavior has been achieved, but the composition and role of their microbiota is not fully determined. The knowledge of the bacteria present in insects might be useful to manage species of human concern such as natural enemies or pests. Here, we performed a residual contact test to study a possible change in the susceptibility of E. mundus adults to abamectin insecticide after antibiotic treatment. Moreover, we assessed the microbiota present in adults of E. eremicus, E. formosa and two strains of E. mundus by MiSeq 16S rRNA amplicon sequencing. Finally, enzymatic tests were done to determine the influence of Arthrobacter species in the susceptibility of E. mundus to pesticides. RESULTS: The assays showed that when E. mundus adults were pretreated with antibiotic, the toxicity of abamectin was significantly higher. Among the different bacteria associated with parasitic wasps, Arthrobacter has been shown to be involved in the degradation of several kinds of pesticides. Four Arthrobacter species were detected in all the studied insects and the presence of esterases in this bacterial species was confirmed. CONCLUSIONS: The results suggest that the microbiota can modify the susceptibility of E. mundus to pesticides, which in turn supports the importance of the microbial community in natural enemies that it should be considered as a factor in risk assessment tests of pesticides. © 2018 Society of Chemical Industry.

Level of Genetic Diversity in European Bumblebees is Not Determined by Local Species Abundance.

Bumblebee species with declining population trends tend to show lower genetic diversity levels than stable species. The observed difference might be explained by abundance differences, with declining bumblebee species having lower genetic diversity levels simply due to their lower local species abundances. However, whether this holds true is not known. Here, we investigated whether bumblebee local abundances determines population genetic diversity levels. Therefore, local species abundances were measured for bumblebee species at four locations in Belgium and two locations in Estonia during bumblebee foraging years 2013-2017. These locations and countries were chosen to ensure the greatest possible variance in both local abundances and population trends for these species. Hence, genetic diversity levels were obtained for seven species by genotyping collected specimens with 16 microsatellites. Our results showed that the observed patterns in genetic diversity did not depend on local species abundance. So, although declining bumblebee species can become locally abundant, they will still show lower genetic diversity levels than stable species. This result implies that measuring bumblebees' local abundance cannot be used to directly determine the health status of a population. Furthermore, this result has also major impact on future conservation strategies as increasing the genetic diversity levels of declining species will be very difficult, and habitat quality should be high to maintain their populations, otherwise these species are doomed to disappear first.