A blood test to monitor bee health across a European network of agricultural sites of different land-use by MALDI BeeTyping mass spectrometry.

There are substantial concerns about impaired honey bee health and colony losses due to several poorly understood factors. We used MALDI profiling (MALDI BeeTyping®) analysis to investigate how some environmental and management factors under field conditions across Europe affected the honey bee haemolymph peptidome (all peptides in the circulatory fluid), as a profile of molecular markers representing the immune status of Apis mellifera. Honey bees were exposed to a range of environmental stressors in 128 agricultural sites across eight European countries in four biogeographic zones, with each country contributing eight sites each for two different cropping systems: oilseed rape (OSR) and apple (APP). The full haemolymph peptide profiles, including the presence and levels of three key immunity markers, namely the antimicrobial peptides (AMPs) Apidaecin, Abaecin and Defensin-1, allowed the honey bee responses to environmental variables to be discriminated by country, crop type and site. When considering just the AMPs, it was not possible to distinguish between countries by the prevalence of each AMP in the samples. However, it was possible to discriminate between countries on the amounts of the AMPs, with the Swedish samples in particular expressing high amounts of all AMPs. A machine learning model was developed to discriminate the haemolymphs of bees from APP and OSR sites. The model was 90.6 % accurate in identifying the crop type from the samples used to build the model. Overall, MALDI BeeTyping® of bee haemolymph represents a promising and cost-effective "blood test" for simultaneously monitoring dozens of peptide markers affected by environmental stressors at the landscape scale, thus providing policymakers with new diagnostic and regulatory tools for monitoring bee health.

Novel indices reveal that pollinator exposure to pesticides varies across biological compartments and crop surroundings.

Declines in insect pollinators have been linked to a range of causative factors such as disease, loss of habitats, the quality and availability of food, and exposure to pesticides. Here, we analysed an extensive dataset generated from pesticide screening of foraging insects, pollen-nectar stores/beebread, pollen and ingested nectar across three species of bees collected at 128 European sites set in two types of crop. In this paper, we aimed to (i) derive a new index to summarise key aspects of complex pesticide exposure data and (ii) understand the links between pesticide exposures depicted by the different matrices, bee species and apple orchards versus oilseed rape crops. We found that summary indices were highly correlated with the number of pesticides detected in the related matrix but not with which pesticides were present. Matrices collected from apple orchards generally contained a higher number of pesticides (7.6 pesticides per site) than matrices from sites collected from oilseed rape crops (3.5 pesticides), with fungicides being highly represented in apple crops. A greater number of pesticides were found in pollen-nectar stores/beebread and pollen matrices compared with nectar and bee body matrices. Our results show that for a complete assessment of pollinator pesticide exposure, it is necessary to consider several different exposure routes and multiple species of bees across different agricultural systems.

Overlapping exposure effects of pathogen and dimethoate on honeybee (Apis mellifera Linnaeus) metabolic rate and longevity.

Introduction: Declines in honeybee abundance have been observed worldwide during last decades. This is partly due to plant protection agents used in intensive farming, landscaping and infrastructure maintenance. Another type of factors negatively affecting honeybees is the spread of diseases caused by different pathogens and pests. Lately, more focus has been paid to the interactions between different overlapping stressors affecting honeybee health, the combination of these often being more detrimental compared to individual stressors. The most widely used stress-evaluating methods take into account lethal- or motorial changes of the individuals or colonies. Comparatively little honeybee research has examined changes in initial recovery potential and physiological symptoms of toxification. The aim of this study was to examine the combined effect of Nosema apis and N. ceranae (according to a newer classification Vairimorpha apis and V. ceranae), the common causes of nosemosis in the honeybee Apis mellifera L., with the insecticide dimethoate. Methods: In this study, honeybee mortality and metabolic rate were used to assess the combined effects interactions of Nosema ssp. and dimethoate. Results: Our results showed that exposure to the low concentration of either dimethoate, either one or both species of Nosema ssp as single factors or in the combination had no significant effect on honeybee metabolic rate. The mortality increased with the two Nosema spp., as well as with infection by N. ceranae alone. The effect of dimethoate was observed only in combination with N. apis infection, which alone had no effect on individual honeybee mortality. Conclusion: This study demonstrates that the overlapping exposure to a non-lethal concentration of a pesticide and a pathogen can be hidden by stronger stressor but become observable with milder stressors.

Intra-specific variation in sensitivity of Bombus terrestris and Osmia bicornis to three pesticides.

There is growing evidence that pesticides may be among the causes of worldwide bee declines, which has resulted in repeated calls for their increased scrutiny in regulatory assessments. One recurring concern is that the current frameworks may be biased towards assessing risks to the honey bee. This paradigm requires extrapolating toxicity information across bee species. Most research effort has therefore focused on quantifying differences in sensitivity across species. However, our understanding of how responses to pesticides may vary within a species is still very poor. Here we take the first steps towards filling this knowledge gap by comparing acute, lethal hazards in sexes and castes of the eusocial bee Bombus terrestris and in sexes of the solitary bee Osmia bicornis after oral and contact exposure to the pesticides sulfoxaflor, Amistar (azoxystrobin) and glyphosate. We show that sensitivity towards pesticides varies significantly both within and across species. Bee weight was a meaningful predictor of pesticide susceptibility. However, weight could not fully explain the observed differences, which suggests the existence of unexplored mechanisms regulating pesticide sensitivity across bee sexes and castes. Our data show that intra-specific responses are an overlooked yet important aspect of the risk assessment of pesticides in bees.

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.

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.

Bumble Bee Foraged Pollen Analyses in Spring Time in Southern Estonia Shows Abundant Food Sources.

Agricultural landscapes usually provide higher quantities of single-source food, which are noticeably lacking in diversity and might thus have low nutrient value for bumble bee colony development. Here, in this study, we analysed the pollen foraging preferences over a large territory of a heterogeneous agricultural landscape: southern Estonia. We aimed to assess the botanical diversity of bumble bee food plants in the spring time there. We looked for preferences for some food plants or signs of food shortage that could be associated with any particular landscape features. For this purpose, we took Bombus terrestris commercial hives to the landscape, performed microscopy analyses and improved the results with the innovative DNA metabarcoding technique to determine the botanical origin of bumble bee-collected pollen. We found high variability of forage plants with no strong relationship with any particular landscape features. Based on the low number of plant species in single flights, we deduce that the availability of main forage plants is sufficient indicating rich forage availabilities. Despite specific limitations, we saw strong correlations between microscopy and DNA metabarcoding data usable for quantification analyses. As a conclusion, we saw that the spring-time vegetation in southern Estonia can support bumble bee colony development regardless of the detailed landscape structure. The absence of clearly dominating food preference by the tested generalist bumble bee species B. terrestris makes us suggest that other bumble bee species, at least food generalists, should also find plenty of forage in their early development phase.

Variation in the Distribution of Nosema Species in Honeybees (Apis mellifera Linnaeus) between the Neighboring Countries Estonia and Latvia.

The unicellular spore-forming parasites Nosema apis and Nosema ceranae are considered to be one of the causes of increased honey bee mortality in recent years. These pathogens attack their honey bee hosts through their gut, causing changes in behavioral stress responses and possibly resulting in decreased honey yield and increased honey bee mortality. The present study aimed to determine the prevalence of Nosema spp. (nosemosis) in Estonia and Latvia, as well as the persistence of the disease in previously infected hives. Currently, N. ceranae is considered the most virulent species and is predominant worldwide. However, in some regions, usually with colder climates, N. apis is still prevalent. To achieve better disease control, it is important to determine the species distribution. For this purpose, we selected 30 apiaries in Estonia and 60 in Latvia that were positive for Nosema spp. in the EPILOBEE (2012-2014) study, which was 5 years prior to the present study. The results show that, while both species are present in Estonia and Latvia, N. apis is dominant in Estonia (43%), and N. ceranae is dominant in Latvia (47%). We also found that the pathogens are very persistent, since 5 years later, only 33% of infected apiaries in Estonia and 20% of infected apiaries in Latvia, we could not detect any pathogens at the time of sampling.

Translocation of Tebuconazole between Bee Matrices and Its Potential Threat on Honey Bee (Apis mellifera Linnaeus) Queens.

Various pesticide residues can be found in different bee colony components. The queen larvae of honey bee (Apis mellifera L.) receive non-contaminated food from nurse bees. However, there is little knowledge about how pesticide residues affect developing bees. Additionally, little is known about the migration of lipophilic pesticides between bee matrices. While wax, royal jelly (RJ), and bee larvae are chemically distinct, they all contain lipids and we expected the lipophilic fungicide tebuconazole to be absorbed by different contacting materials. Our aim was to analyze the translocation of tebuconazole residues from queen cell wax to RJ, queen larvae, and newly emerged queens and to evaluate its potential risk to queens. We demonstrated the potential for the migration of tebuconazole from wax to RJ, with a strong dilution effect from the original contamination source. No residues were detected in queen bee larvae and newly emerged queens, indicating that the migration of tebuconazole probably did not directly endanger the queen bee, but there was some risk that tebuconazole might still affect the homeostasis of developing bees.

Pesticide residues in beehive matrices are dependent on collection time and matrix type but independent of proportion of foraged oilseed rape and agricultural land in foraging territory.

Pesticide residues in bee products is still a major issue. However, the relations to botanical source and land use characteristics are not clear. The large variability of residues detected questions the suitability of bee-collected- and other hive materials as indicators for environmental contamination. The aim of our study was to clarify whether different beehive matrices contain similar pesticide residues, and how these are correlated with forage preferences and land use types in foraging areas. We tested bee-collected pollen, beebread, honey, nurse bees and honey bee larvae for the presence of concurrently used agricultural pesticides in Estonia. Samples were collected at the end of May and mid-July to include the main crop in northern region - winter and spring oilseed rape (Brassica napus). We saw that different beehive matrices contained various types of pesticide residues in different proportions: pollen and beebread tended to contain more insecticides and fungicides, whereas herbicides represented the primary contaminant in honey. The variations were related to collection year and time but were not related to crops as basic forage resource nor the land use type. We found few positive correlations between amount of pesticides and proportion of pollen from any particular plant family. None of these correlations were related to any land-use type. We conclude that pesticide residues in different honey bee colony components vary largely in amount and composition. The occurrence rate of pesticide residues was not linked to any particular crop.

Potential of Colorado Potato Beetle (Coleoptera: Chrysomelidae) to Adapt to Alternative Host Plants.

Twelve selected cultivated or wild solanaceae (Solanum dulcamara, Solanum nigrum, Solanum villosum, Solanum lycopersicum, Solanum muricatum, Solanum melongena, Datura innoxia, Datura metel, Physalis spp., Capsicum annuum, Nicotiana alata, and Petunia spp.) (all species Solanales: Solanaceae) were tested as potential alternative host plants against Solanum tuberosum for a local population of the Colorado potato beetle Leptinotarsa decemlineata Say (CPB) in Estonia. Some CPB populations in America and southern Europe accept most of these plants. However, geographically isolated populations of beetles can differ in their acceptance of new host plants. Migrants from the southern Europe supplement Estonian beetle population regularly, so individuals may differ in their host plant preferences. S. melongena and S. dulcamara were well accepted by the beetles, Lycopersicum spp. (Solanales: Solanaceae) and N. alata were intermediate, and S. villosum and S. nigrum were least accepted. The beetles rejected S. muricatum, Physalis spp., C. annuum, Petunia spp., and D. metel. First-instar larvae completed a full life cycle only on S. dulcamara, N. alata, S. lycopesicum, S. melongena, although their development rate was slower, mortality was higher and emerging adults were underweight relative to those that fed on S. tuberosum. The fourth-instar larvae were less sensitive in relation to food. We found that S. dulcamara as a native plant could provide resources for CPB during early and late season. At the same time, the cultivated S. melongena would be suitable plant species for further testing as dead-end crop for integrated pest management.

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.

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.

Synergistic interactions between a variety of insecticides and an ergosterol biosynthesis inhibitor fungicide in dietary exposures of bumble bees (Bombus terrestris L.).

BACKGROUND: In recent years, concern has been raised over honey bee colony losses, and also among wild bees there is evidence for extinctions and range contractions in Europe and North America. Pesticides have been proposed as a potential cause of this decline. Bees are exposed simultaneously to a variety of agrochemicals, which may cause synergistically detrimental impacts, which are incompletely understood. We investigated the toxicity of the fungicide imazalil in mixture with four common insecticides: fipronil (phenylpyrazoid), cypermethrin (pyrethroid), thiamethoxam, and imidacloprid (neonicotinoids). Ergosterol biosynthesis inhibitor (EBI) fungicides like imazalil can inhibit P450 detoxification systems in insects and therefore fungicide - insecticide co-occurrence might produce synergistic toxicity in bees. We assessed the impact of dietary fungicide - insecticide mixtures on the mortality and feeding rates of laboratory bumble bees (Bombus terrestris L.). RESULTS: Regarding mortality, imazalil synergised the toxicity of fipronil, cypermethrin and thiamethoxam, but not imidacloprid. We found no synergistic effects on feeding rates. CONCLUSION: Our findings suggest that P450-based detoxification processes are differentially important in mitigating the toxicity of certain insecticides, even those of the same chemical class. Our evidence that cocktail effects can arise in bumble bees should extend concern about the potential impacts of agrochemical mixtures to include wild bee species in farmland. © 2017 Society of Chemical Industry.

Are pesticide residues in honey related to oilseed rape treatments?

Pesticide treatments before and during the flowering of honey bee forage crops may lead to residues in honey. In northern regions oilseed rape belongs to the main forage crops that is mostly cultivated by means of intensive agriculture, including several pesticide treatments. However, in addition to the focal forage crops, pesticides from non-forage crops can spread to wild flowers around fields, and thus the residues in honey would reflect the whole range of pesticides used in the agricultural landscape. The aim of our study was to clarify which currently used pesticides are present in honey gathered from heterogeneous agricultural landscapes after the end of flowering of oilseed crops. Honey samples (N = 33) were collected from beehives of Estonia during 2013 and 2014, and analysed for residues of 47 currently used agricultural pesticides using the multiresidue method with HPLC-MS/MS and GC-MS and a single residue method for glyphosate, aminopyralid and clopyralid. Residues of eight different active ingredients with representatives from all three basic pesticide classes were determined. Although no correlation was detected between the cumulative amount of pesticide residues and percent of oilseed crops in the foraging territory, most of the residues are those allowed for oilseed rape treatments. Among all pesticides, herbicide residues prevailed in 2013 but not in 2014. Despite the relatively small agricultural impact of Estonia, the detected levels of pesticide residues sometimes exceeded maximum residue level; however, these concentrations do not pose a health risk to consumers, also acute toxicity to honey bees would be very unlikely.

Reliability of the entomovector technology using Prestop-Mix and Bombus terrestris L. as a fungal disease biocontrol method in open field.

Botrytis cinerea Pers.:Fr. is a major plant pathogen, and a new approach is needed for its control in strawberry to minimise the increasing use of synthetic fungicides. The biofungicide Prestop-Mix, which contains Gliocladium catenulatum, is effective against Botrytis infections; however, the need for frequent applications increases the costs for farmers. Here, we demonstrate that bumble bees, Bombus terrestris L., effectively disseminate the preparation onto flowers in open field conditions. Over the course of three years, we found a highly significant decrease in the rate of Botrytis infection. Pathogen control was achieved with relatively low numbers of G. catenulatum spores per flower, even using flowers that are not highly attractive to bumble bees. An even distribution of spores was detected up to 100 m from the hives, either due to primary inoculation by bumble bees or secondary distribution by other flower visitors such as honey bees and solitary bees. We showed that the application of a biocontrol agent by bumble bees is reliable for the use of environmentally friendly pest control strategies in northern climatic conditions. This low cost technology is especially relevant for organic farming. This study provides valuable information for introducing this method into practice in open strawberry fields.

Effects of Fastac 50 EC on bumble bee Bombus terrestris L. respiration: DGE disappearance does not lead to increasing water loss.

Sublethal effects of pesticides in insects can be observed through physiological changes, which are commonly estimated by metabolic rate and respiratory patterns, more precisely by the patterns of discontinuous gas-exchange (DGE) cycles. The aim of the present research was to study the effect of some low concentrations of Fastac 50 EC on the cycles of CO(2) release and respiratory water loss rates (WLR) in bumble bee Bombus terrestris L. foragers. Bumble bees were dipped into 0.004% and 0.002% Fastac 50 EC solution. Flow-through respirometry was used to record the respiration and WLR 3h before and after the treatment. The respirometry was combined with infrared actography to enable simultaneous recording of abdominal movements. Our results show that Fastac 50 EC has an after-effect on bumble bee respiratory rhythms and muscle activity but does not affect WLR. Treatment with 0.004% Fastac 50 EC solution resulted in disappearance of the respiration cycles; also the lifespan of treated bumble bees was significantly shorter. Treatment with 0.002% Fastac 50 EC solution had no significant effect on respiration patterns or longevity. We found no evidence for the DGE cycles functioning as a water saving mechanism.

Impact of alpha-cypermethrin on honey bees foraging on spring oilseed rape (Brassica napus) flowers in field conditions.

BACKGROUND: Cruciferous oil-bearing crops have gained in importance worldwide. The expansion of the growing area of these crops has caused a proliferation of pests. Exposure to organophosphate, carbamate and pyrethroid insecticides has been associated with bee poisoning in food crops. This study examines the repellent effect of alpha-cypermethrin on the number of foraging honey bees, Apis mellifera L., on fields of spring oilseed rape, Brassica napus L. var. oleifera. RESULTS: The first experiment was conducted on differently sprayed 10 m(2) experimental plots where alpha-cypermethrin was applied at different times. Another experiment was conducted on a 4 ha seed production field divided into two parts: one part was treated with alpha-cypermethrin and the other was not treated with this insecticide. The results show that there was no difference in the number of honey bees between alpha-cypermethrin-treated and untreated patches. The result persisted through three observation years, regardless of varying flower and honey bee densities. CONCLUSION: No repellent effect of the insecticide on honey bees was found even 24 h after spraying. The density of oilseed rape flowers most likely played a major role in choosing the foraging area.