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.

Distribution of infectious and parasitic agents among three sentinel bee species across European agricultural landscapes.

Infectious and parasitic agents (IPAs) and their associated diseases are major environmental stressors that jeopardize bee health, both alone and in interaction with other stressors. Their impact on pollinator communities can be assessed by studying multiple sentinel bee species. Here, we analysed the field exposure of three sentinel managed bee species (Apis mellifera, Bombus terrestris and Osmia bicornis) to 11 IPAs (six RNA viruses, two bacteria, three microsporidia). The sentinel bees were deployed at 128 sites in eight European countries adjacent to either oilseed rape fields or apple orchards during crop bloom. Adult bees of each species were sampled before their placement and after crop bloom. The IPAs were detected and quantified using a harmonised, high-throughput and semi-automatized qPCR workflow. We describe differences among bee species in IPA profiles (richness, diversity, detection frequencies, loads and their change upon field exposure, and exposure risk), with no clear patterns related to the country or focal crop. Our results suggest that the most frequent IPAs in adult bees are more appropriate for assessing the bees' IPA exposure risk. We also report positive correlations of IPA loads supporting the potential IPA transmission among sentinels, suggesting careful consideration should be taken when introducing managed pollinators in ecologically sensitive environments.

Pesticide use negatively affects bumble bees across European landscapes.

Sustainable agriculture requires balancing crop yields with the effects of pesticides on non-target organisms, such as bees and other crop pollinators. Field studies demonstrated that agricultural use of neonicotinoid insecticides can negatively affect wild bee species1,2, leading to restrictions on these compounds3. However, besides neonicotinoids, field-based evidence of the effects of landscape pesticide exposure on wild bees is lacking. Bees encounter many pesticides in agricultural landscapes4-9 and the effects of this landscape exposure on colony growth and development of any bee species remains unknown. Here we show that the many pesticides found in bumble bee-collected pollen are associated with reduced colony performance during crop bloom, especially in simplified landscapes with intensive agricultural practices. Our results from 316 Bombus terrestris colonies at 106 agricultural sites across eight European countries confirm that the regulatory system fails to sufficiently prevent pesticide-related impacts on non-target organisms, even for a eusocial pollinator species in which colony size may buffer against such impacts10,11. These findings support the need for postapproval monitoring of both pesticide exposure and effects to confirm that the regulatory process is sufficiently protective in limiting the collateral environmental damage of agricultural pesticide use.

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.

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.

Green Leaf Volatile Confers Management of Late Blight Disease: A Green Vaccination in Potato.

Yield losses of crops due to plant pathogens are a major threat in all agricultural systems. In view of environmental issues and legislative limitations for chemical crop protection products, the need to design new environmentally friendly disease management strategies has gained interest. Despite the unique capability of green leaf volatiles (GLVs) to suppress a broad spectrum of plant pathogens, their capacity to control the potato late-blight-causing agent Phytophthora infestans has not been well studied. This study addresses the potential role of the GLV Z-3-hexenyl acetate (Z-3-HAC) in decreasing the severity of late blight and the underlying gene-based evidence leading to this effect. Nine-week-old potato plants (Solanum tuberosum L.) were exposed to Z-3-HAC before they were inoculated with P. infestans genotypes at different time points. These pre-exposed potato plants exhibited slower disease development after infection with the highly pathogenic genotype of P. infestans (EU-13-A2) over time. Qualitative assessment showed that the exposed, infected plants possessed significantly lower sporulation intensity and disease severity compared to the control plants. Hypersensitive response (HR)-like symptoms were observed on the treated leaves when inoculated with different pathogen genotypes. No HR-like lesions were detected on the untreated leaves after infection. It was shown that the transcript levels of several defense-related genes, especially those that are involved in reactive oxygen species (ROS) production pathways were significantly expressed in plants at 48 and 72 h postexposure to the Z-3-HAC. The current work provides evidence on the role of Z-3-HAC in the increased protection of potato plants against late blight through plant immunity and offers new opportunities for the sustainable control of potato diseases.

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.

Neural Code for Ambient Heat Detection in Elaterid Beetles.

Environmental thermal conditions play a major role at all levels of biological organization; however, there is little information on noxious high temperature sensation crucial in behavioral thermoregulation and survival of small ectothermic animals such as insects. So far, a capability to unambiguously encode heat has been demonstrated only for the sensory triad of the spike bursting thermo- and two bimodal hygro-thermoreceptor neurons located in the antennal dome-shaped sensilla (DSS) in a carabid beetle. We used extracellular single sensillum recording in the range of 20-45°C to demonstrate that a similar sensory triad in the elaterid Agriotes obscurus also produces high temperature-induced bursty spike trains. Several parameters of the bursts are temperature dependent, allowing the neurons in a certain order to encode different, but partly overlapping ranges of heat up to lethal levels in a graded manner. ISI in a burst is the most useful parameter out of six. Our findings consider spike bursting as a general, fundamental quality of the classical sensory triad of antennal thermo- and hygro-thermoreceptor neurons widespread in many insect groups, being a flexible and reliable mode of coding unfavorably high temperatures. The possible involvement of spike bursting in behavioral thermoregulation of the beetles is discussed. By contrast, the mean firing rate of the neurons in regular and bursty spike trains combined does not carry useful thermal information at the high end of noxious heat.

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.

Linking organismal growth, coping styles, stress reactivity, and metabolism via responses against a selective serotonin reuptake inhibitor in an insect.

Evidence suggests that brain serotonin (5-HT) is one of the central mediators of different types of animal personality. We tested this assumption in field crickets Gryllus integer using a selective serotonin reuptake inhibitor (SSRI). Crickets were selected for slow and rapid development and tested for their coping styles under non-stressful conditions (time spent exploring a novel object). Resting metabolic rate, maximum metabolic rate and latency to resume activity were measured under stressful conditions (stress reactivity). Measurements were taken (i) before and (ii) during the SSRI treatment. Before the SSRI treatment, a strong negative correlation was observed between coping style and stress reactivity, which suggests the existence of a behavioral syndrome. After the SSRI treatment, the syndrome was no longer evident. The results of this study show that 5-HT may be involved in regulating behavior not only along a stress reactivity gradient but also along a coping styles axis. The relationship between personality and the strength and direction of 5-HT treatment on observed behaviors indicates trait-like individual differences in 5-HT signaling. Overall, these findings do not support recent ideas arising from the pace-of-life syndrome (POLS) hypothesis, which predict higher exploration and metabolic rates in rapidly developing bold animals.

Dynamics of Supercooling Ability and Cold Tolerance of the Alder Beetle (Coleoptera: Chrysomelidae).

Agelastica alni L. (Coleoptera: Chrysomelidae) is a common beetle pest of alder trees (Alnus incana L.) in forests and parks across Estonia. The supercooling ability and capacity to survive low temperature exposure changes temporally. Relatively high unimodal supercooling point (SCP) levels (with a mean value of -6 to -8°C) were characteristic of the beetles in September during their diapause induction period, in April when the beetles had terminated their diapause development and in May when they started to reproduce. During their deep diapause period the SCPs of beetles had a bimodal distribution. Some beetles decreased their SCPs to -14 to -19°C while others retained a high SCP value of -6 to -8°C. Most vulnerable to low temperature were overwintered active beetles in May; after 1 h exposure their LTemp50 (median lethal temperature) was -6.3°C. In September with the onset of diapause tolerance of beetles started to increase, LTemp50 = -7.7°C. Beetles were most cold tolerant during their deep diapause period with LTemp50 < -12.0°C. A. alni beetles use one of two different strategies for overwintering, some are freeze-tolerant while others are freeze-avoidant. The freeze-avoidant beetles with low SCP (-12 to -15°C) acquired greater cold tolerance than those with higher SCP (-6 to -8°C), with LTemp50 = -13.7°C and LTemp50 = -11°C, respectively.

Bursty spike trains of antennal thermo- and bimodal hygro-thermoreceptor neurons encode noxious heat in elaterid beetles.

The main purpose of this study was to explain the internal fine structure of potential antennal thermo- and hygroreceptive sensilla, their innervation specifics, and responses of the sensory neurons to thermal and humidity stimuli in an elaterid beetle using focused ion beam scanning electron microscopy and electrophysiology, respectively. Several essential, high temperature induced turning points in the locomotion were determined using automated video tracking. Our results showed that the sensilla under study, morphologically, are identical to the dome-shaped sensilla (DSS) of carabids. A cold-hot neuron and two bimodal hygro-thermoreceptor neurons, the moist-hot and dry-hot neuron, innervate them. Above 25-30 °C, all the three neurons, at different threshold temperatures, switch from regular spiking to temperature dependent spike bursting. The percentage of bursty DSS neurons on the antenna increases with temperature increase suggesting that this parameter of the neurons may encode noxious heat in a graded manner. Thus, we show that besides carabid beetles, elaterids are another large group of insects with this ability. The threshold temperature of the beetles for onset of elevated locomotor activity (OELA) was lower by 11.9 °C compared to that of critical thermal maximum (39.4 °C). Total paralysis occurred at 41.8 °C. The threshold temperatures for spike bursting of the sensory neurons in DSS and OELA of the beetles coincide suggesting that probably the spike bursts are responsible for encoding noxious heat when confronted. In behavioural thermoregulation, spike bursting DSS neurons serve as a fast and firm three-fold early warning system for the beetles to avoid overheating and death.

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.

Metabolic rate associates with, but does not generate covariation between, behaviours in western stutter-trilling crickets, Gryllus integer.

The causes and consequences of among-individual variation and covariation in behaviours are of substantial interest to behavioural ecology, but the proximate mechanisms underpinning this (co)variation are still unclear. Previous research suggests metabolic rate as a potential proximate mechanism to explain behavioural covariation. We measured the resting metabolic rate (RMR), boldness and exploration in western stutter-trilling crickets, Gryllus integer, selected differentially for short and fast development over two generations. After applying mixed-effects models to reveal the sign of the covariation, we applied structural equation models to an individual-level covariance matrix to examine whether the RMR generates covariation between the measured behaviours. All traits showed among-individual variation and covariation: RMR and boldness were positively correlated, RMR and exploration were negatively correlated, and boldness and exploration were negatively correlated. However, the RMR was not a causal factor generating covariation between boldness and exploration. Instead, the covariation between all three traits was explained by another, unmeasured mechanism. The selection lines differed from each other in all measured traits and significantly affected the covariance matrix structure between the traits, suggesting that there is a genetic component in the trait integration. Our results emphasize that interpretations made solely from the correlation matrix might be misleading.