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.

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.

The insula mediates the effects of glucocorticoids in anxiety.

Increased activity in the insula has been consistently reported to be associated with anxiety and anxiety-related disorders. However, little is known on how the insula regulates anxiety. The present study aims at determining the role of the insula on the effects of glucocorticoids in anxiety. A combination of pharmacological manipulations, including blockade of adrenal GC synthesis by metyrapone and intra-insular microinjections of corticosterone, corticosterone-BSA, mineralocorticoid receptor (MR) antagonist spironolactone and glucocorticoid receptor (GR) antagonist mifepristone, were used to assess the short-term (5 min) effects of intra-insular corticosterone in two anxiety-like behaviors in male Sprague-Dawley rats. The elevated plus maze (EPM) and Novelty Suppressed Feeding (hyponeophagia) were utilized. We found that corticosterone in the insula is sufficient to prevent the anxiolytic effects corticosterone synthesis blockade in anxiety, and that intra-insular corticosterone has anxiolytic or anxiogenic effects depending on the amount of corticosterone microinjected and the arousal associated to the test, without affecting the HPA axis. Glucocorticoid anxiolytic effects in the insula are mediated by MRs, while its anxiogenic effects are dependent on a mifepristone-sensitive membrane-bound mechanism. Anxiety appears to be modulated at the insula through a competition between fast MR-dependent anxiolytic and membrane-associated anxiogenic signaling pathways that orchestrate the behavioral response to stress and determines the resulting level of anxiety.

The endoplasmic reticulum stress sensor IRE1 regulates collagen secretion through the enforcement of the proteostasis factor P4HB/PDIA1 contributing to liver damage and fibrosis.

Collagen is one the most abundant proteins and the main cargo of the secretory pathway, contributing to hepatic fibrosis and cirrhosis due to excessive deposition of extracellular matrix. Here we investigated the possible contribution of the unfolded protein response, the main adaptive pathway that monitors and adjusts the protein production capacity at the endoplasmic reticulum, to collagen biogenesis and liver disease. Genetic ablation of the ER stress sensor IRE1 reduced liver damage and diminished collagen deposition in models of liver fibrosis triggered by carbon tetrachloride (CCl 4 ) administration or by high fat diet. Proteomic and transcriptomic profiling identified the prolyl 4-hydroxylase (P4HB, also known as PDIA1), which is known to be critical for collagen maturation, as a major IRE1-induced gene. Cell culture studies demonstrated that IRE1 deficiency results in collagen retention at the ER and altered secretion, a phenotype rescued by P4HB overexpression. Taken together, our results collectively establish a role of the IRE1/P4HB axis in the regulation of collagen production and its significance in the pathogenesis of various disease states.

The unfolded protein response transcription factor XBP1s ameliorates Alzheimer's disease by improving synaptic function and proteostasis.

Alteration in the buffering capacity of the proteostasis network is an emerging feature of Alzheimer's disease (AD), highlighting the occurrence of endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) is the main adaptive pathway to cope with protein folding stress at the ER. Inositol-requiring enzyme-1 (IRE1) operates as a central ER stress sensor, enabling the establishment of adaptive and repair programs through the control of the expression of the transcription factor X-box binding protein 1 (XBP1). To artificially enforce the adaptive capacity of the UPR in the AD brain, we developed strategies to express the active form of XBP1 in the brain. Overexpression of XBP1 in the nervous system using transgenic mice reduced the load of amyloid deposits and preserved synaptic and cognitive function. Moreover, local delivery of XBP1 into the hippocampus of an 5xFAD mice using adeno-associated vectors improved different AD features. XBP1 expression corrected a large proportion of the proteomic alterations observed in the AD model, restoring the levels of several synaptic proteins and factors involved in actin cytoskeleton regulation and axonal growth. Our results illustrate the therapeutic potential of targeting UPR-dependent gene expression programs as a strategy to ameliorate AD features and sustain synaptic function.

Nutritional stress exacerbates impact of a novel insecticide on solitary bees' behaviour, reproduction and survival.

Pesticide exposure and food stress are major threats to bees, but their potential synergistic impacts under field-realistic conditions remain poorly understood and are not considered in current pesticide risk assessments. We conducted a semi-field experiment to examine the single and interactive effects of the novel insecticide flupyradifurone (FPF) and nutritional stress on fitness proxies in the solitary bee Osmia bicornis. Individually marked bees were released into flight cages with monocultures of buckwheat, wild mustard or purple tansy, which were assigned to an insecticide treatment (FPF or control) in a crossed design. Nutritional stress, which was high in bees foraging on buckwheat, intermediate on wild mustard and low on purple tansy, modulated the impact of insecticide exposure. Within the first day after application of FPF, mortality of bees feeding on buckwheat was 29 times higher compared with control treatments, while mortality of FPF exposed and control bees was similar in the other two plant species. Moreover, we found negative synergistic impacts of FPF and nutritional stress on offspring production, flight activity, flight duration and flower visitation frequency. These results reveal that environmental policies and risk assessment schemes that ignore interactions among anthropogenic stressors will fail to adequately protect bees and the pollination services they provide.

Unfolded protein response IRE1/XBP1 signaling is required for healthy mammalian brain aging.

Aging is a major risk factor to develop neurodegenerative diseases and is associated with decreased buffering capacity of the proteostasis network. We investigated the significance of the unfolded protein response (UPR), a major signaling pathway activated to cope with endoplasmic reticulum (ER) stress, in the functional deterioration of the mammalian brain during aging. We report that genetic disruption of the ER stress sensor IRE1 accelerated age-related cognitive decline. In mouse models, overexpressing an active form of the UPR transcription factor XBP1 restored synaptic and cognitive function, in addition to reducing cell senescence. Proteomic profiling of hippocampal tissue showed that XBP1 expression significantly restore changes associated with aging, including factors involved in synaptic function and pathways linked to neurodegenerative diseases. The genes modified by XBP1 in the aged hippocampus where also altered. Collectively, our results demonstrate that strategies to manipulate the UPR in mammals may help sustain healthy brain aging.

No evidence for impaired solitary bee fitness following pre-flowering sulfoxaflor application alone or in combination with a common fungicide in a semi-field experiment.

Pesticide exposure is considered a major driver of pollinator decline and the use of neonicotinoid insecticides has been restricted by regulatory authorities due to their risks for pollinators. Impacts of new alternative sulfoximine-based compounds on solitary bees and their potential interactive effects with other commonly applied pesticides in agriculture remain unclear. Here, we conducted a highly replicated full-factorial semi-field experiment with the solitary bee Osmia bicornis, an important pollinator of crops and wild plants in Europe, and Phacelia tanacetifolia as a model crop. We show that spray applications of the insecticide sulfoxaflor (product Closer) and the fungicide azoxystrobin (product Amistar), both alone and combined, had no significant negative impacts on adult female survival or the production, mortality, sex ratio and body size of offspring when sulfoxaflor was applied five days before crop flowering. Our results indicate that for O. bicornis (1) the risk of adverse impacts of sulfoxaflor (Closer) on fitness is small when applied at least five days before crop flowering and (2) that azoxystrobin (Amistar) has a low potential of exacerbating sulfoxaflor effects under field-realistic conditions.

Flowering resources modulate the sensitivity of bumblebees to a common fungicide.

Bees are exposed to various stressors, including pesticides and lack of flowering resources. Despite potential interactions between these stressors, the impacts of pesticides on bees are generally assumed to be consistent across bee-attractive crops, and regulatory risk assessments of pesticides neglect interactions with flowering resources. Furthermore, impacts of fungicides on bees are rarely examined in peer-reviewed studies, although these are often the pesticides that bees are most exposed to. In a full-factorial semi-field experiment with 39 large flight cages, we assessed the single and combined impacts of the globally used azoxystrobin-based fungicide Amistar® and three types of flowering resources (Phacelia, buckwheat, and a floral mix) on Bombus terrestris colonies. Although Amistar is classified as bee-safe, Amistar exposure through Phacelia monocultures reduced adult worker body mass and colony growth (including a 55% decline in workers and an 88% decline in males), while the fungicide had no impact on colonies in buckwheat or the floral mix cages. Furthermore, buckwheat monocultures hampered survival and fecundity irrespective of fungicide exposure. This shows that bumblebees require access to complementary flowering species to gain both fitness and fungicide tolerance and that Amistar impacts are flowering resource-dependent. Our findings call for further research on how different flowering plants affect bees and their pesticide tolerance to improve guidelines for regulatory pesticide risk assessments and inform the choice of plants that are cultivated to safeguard pollinators.

Astroglial gliotransmitters released via Cx43 hemichannels regulate NMDAR-dependent transmission and short-term fear memory in the basolateral amygdala.

Astrocytes release gliotransmitters via connexin 43 (Cx43) hemichannels into neighboring synapses, which can modulate synaptic activity and are necessary for fear memory consolidation. However, the gliotransmitters released, and their mechanisms of action remain elusive. Here, we report that fear conditioning training elevated Cx43 hemichannel activity in astrocytes from the basolateral amygdala (BLA). The selective blockade of Cx43 hemichannels by microinfusion of TAT-Cx43L2 peptide into the BLA induced memory deficits 1 and 24 h after training, without affecting learning. The memory impairments were prevented by the co-injection of glutamate and D-serine, but not by the injection of either alone, suggesting a role for NMDA receptors (NMDAR). The incubation with TAT-Cx43L2 decreased NMDAR-mediated currents in BLA slices, effect that was also prevented by the addition of glutamate and D-serine. NMDARs in primary neuronal cultures were unaffected by TAT-Cx43L2, ruling out direct effects of the peptide on NMDARs. Finally, we show that D-serine permeates through purified Cx43 hemichannels reconstituted in liposomes. We propose that the release of glutamate and D-serine from astrocytes through Cx43 hemichannels is necessary for the activation of post-synaptic NMDARs during training, to allow for the formation of short-term and subsequent long-term memory, but not for learning per se.

The first annotated checklist of Pentatomoidea (Hemiptera: Heteroptera) fauna of Alta Murgia National Park (Apulia region, Southern Italy).

Information regarding Pentatomoidea is scarce in Southern Italy, and in particular concering the Apulia region (Sourthern Italy), an area of high biodiversity potential. We conducted an extensive survey of Pentatomoidea fauna in the Alta Murgia National Park, a Site of Community Importance, and neighbouring areas located in central part of Apulia region. The survey was carried out in 2016, 2017 and 2020, sampling different habitat types. We found 55 species, with five new records for Apulia: Solenosthedium bilunatum, Acrosternum millierei, Sciocoris homalonotus, Cyphostethus tristriatus and Holcogaster fibulata. The polyphagous pest Halyomorpha halys was found only in urban areas, whereas Ventocoris falcatus, Ellipsocoris kalashiani and Tholagmus flavolineatus, considered rare species, were collected in dry grasslands, confirming the crucial role of natural habitat for biodiversity conservation.

Fungicide and insecticide exposure adversely impacts bumblebees and pollination services under semi-field conditions.

Sulfoximines, the next generation systemic insecticides developed to replace neonicotinoids, have been shown to negatively impact pollinator development and reproduction. However, field-realistic studies on sulfoximines are few and consequences on pollination services unexplored. Moreover, the impacts of other agrochemicals such as fungicides, and their combined effects with insecticides remain poorly investigated. Here, we show in a full factorial semi-field experiment that spray applications of both the product Closer containing the insecticide sulfoxaflor and the product Amistar containing the fungicide azoxystrobin, negatively affected the individual foraging performance of bumblebees (Bombus terrestris). Insecticide exposure further reduced colony growth and size whereas fungicide exposure decreased pollen deposition. We found indications for resource limitation that might have exacerbated pesticide effects on bumblebee colonies. Our work demonstrates that field-realistic exposure to sulfoxaflor can adversely impact bumblebees and that applications before bloom may be insufficient as a mitigation measure to prevent its negative impacts on pollinators. Moreover, fungicide use during bloom could reduce bumblebee foraging performance and pollination services.

Sulfoxaflor insecticide and azoxystrobin fungicide have no major impact on honeybees in a realistic-exposure semi-field experiment.

Exposure to pesticides is considered a major threat to bees and several neonicotinoid insecticides were recently banned in cropland within the European Union in light of evidence of their potential detrimental effects. Nonetheless, bees remain exposed to many pesticides whose effects are poorly understood. Recent evidence suggests that one of the most prominent replacements of the banned neonicotinoids - the insecticide sulfoxaflor - harms bees and that fungicides may have been overlooked as a driver of bee declines. Realistic-exposure studies are, however, lacking. Here, we assess the impact of the insecticide Closer (active ingredient: sulfoxaflor) and the widely used fungicide Amistar (a.i.: azoxystrobin) on honeybees in a semi-field study (10 flight cages containing a honeybee colony, for each of three treatments: Closer, Amistar, control). The products were applied according to label instructions either before (Closer) or during (Amistar) the bloom of purple tansy. We found no significant effects of Closer or Amistar on honeybee colony development or foraging activity. Our study suggests that these pesticides pose no notable risk to honeybees when applied in isolation, following stringent label instructions. The findings on Closer indicate that a safety-period of 5-6 days between application and bloom, which is only prescribed in a few EU member states, may prevent its impacts on honeybees. However, to conclude whether Closer and Amistar can safely be applied, further realistic-exposure studies should examine their effects in combination with other chemical or biological stressors on various pollinator species.

Agricultural diversification promotes multiple ecosystem services without compromising yield.

Enhancing biodiversity in cropping systems is suggested to promote ecosystem services, thereby reducing dependency on agronomic inputs while maintaining high crop yields. We assess the impact of several diversification practices in cropping systems on above- and belowground biodiversity and ecosystem services by reviewing 98 meta-analyses and performing a second-order meta-analysis based on 5160 original studies comprising 41,946 comparisons between diversified and simplified practices. Overall, diversification enhances biodiversity, pollination, pest control, nutrient cycling, soil fertility, and water regulation without compromising crop yields. Practices targeting aboveground biodiversity boosted pest control and water regulation, while those targeting belowground biodiversity enhanced nutrient cycling, soil fertility, and water regulation. Most often, diversification practices resulted in win-win support of services and crop yields. Variability in responses and occurrence of trade-offs highlight the context dependency of outcomes. Widespread adoption of diversification practices shows promise to contribute to biodiversity conservation and food security from local to global scales.

Species traits elucidate crop pest response to landscape composition: a global analysis.

Recent synthesis studies have shown inconsistent responses of crop pests to landscape composition, imposing a fundamental limit to our capacity to design sustainable crop protection strategies to reduce yield losses caused by insect pests. Using a global dataset composed of 5242 observations encompassing 48 agricultural pest species and 26 crop species, we tested the role of pest traits (exotic status, host breadth and habitat breadth) and environmental context (crop type, range in landscape gradient and climate) in modifying the pest response to increasing semi-natural habitats in the surrounding landscape. For natives, increasing semi-natural habitats decreased the abundance of pests that exploit only crop habitats or that are highly polyphagous. On the contrary, populations of exotic pests increased with an increasing cover of semi-natural habitats. These effects might be related to changes in host plants and other resources across the landscapes and/or to modified top-down control by natural enemies. The range of the landscape gradient explored and climate did not affect pests, while crop type modified the response of pests to landscape composition. Although species traits and environmental context helped in explaining some of the variability in pest response to landscape composition, the observed large interspecific differences suggest that a portfolio of strategies must be considered and implemented for the effective control of rapidly changing communities of crop pests in agroecosystems.

The Role of the Rodent Insula in Anxiety.

The human insula has been consistently reported to be overactivated in all anxiety disorders, activation which has been suggested to be proportional to the level of anxiety and shown to decrease with effective anxiolytic treatment. Nonetheless, studies evaluating the direct role of the insula in anxiety are lacking. Here, we set out to investigate the role of the rodent insula in anxiety by either inactivating different insular regions via microinjections of glutamatergic AMPA receptor antagonist CNQX or activating them by microinjection of GABA receptor antagonist bicuculline in rats, before measuring anxiety-like behavior using the elevated plus maze. Inactivation of caudal and medial insular regions induced anxiogenic effects, while their activation induced anxiolytic effects. In contrast, inactivation of more rostral areas induced anxiolytic effects and their activation, anxiogenic effects. These results suggest that the insula in the rat has a role in the modulation of anxiety-like behavior in rats, showing regional differences; rostral regions have an anxiogenic role, while medial and caudal regions have an anxiolytic role, with a transition area around bregma +0.5. The present study suggests that the insula has a direct role in anxiety.

Pollination benefits are maximized at intermediate nutrient levels.

Yield production in flowering crops depends on both nutrient availability and pollination, but their relative roles and potential interactions are poorly understood. We measured pollination benefits to yield in sunflower, combining a gradient in insect pollination (0, 25, 50, 100%) with a continuous gradient in nitrogen (N) fertilization (from 0 to 150 kg N ha-1) in an experiment under realistic soil field conditions. We found that pollination benefits to yield were maximized at intermediate levels of N availability, bolstering yield by approximately 25% compared with complete pollinator exclusion. Interestingly, we found little decrease in yield when insect visits were reduced by 50%, indicating that the incremental contribution of pollination by insects to yield is greater when the baseline pollination service provision is very low. Our findings provide strong evidence for interactive, nonlinear effects of pollination and resource availability on seed production. Our results support ecological intensification as a promising strategy for sustainable management of agroecosystems. In particular, we found optimal level of pollination to potentially compensate for lower N applications.

Soil pathogen-aphid interactions under differences in soil organic matter and mineral fertilizer.

There is increasing evidence showing that microbes can influence plant-insect interactions. In addition, various studies have shown that aboveground pathogens can alter the interactions between plants and insects. However, little is known about the role of soil-borne pathogens in plant-insect interactions. It is also not known how environmental conditions, that steer the performance of soil-borne pathogens, might influence these microbe-plant-insect interactions. Here, we studied effects of the soil-borne pathogen Rhizoctonia solani on aphids (Sitobion avenae) using wheat (Triticum aestivum) as a host. In a greenhouse experiment, we tested how different levels of soil organic matter (SOM) and fertilizer addition influence the interactions between plants and aphids. To examine the influence of the existing soil microbiome on the pathogen effects, we used both unsterilized field soil and sterilized field soil. In unsterilized soil with low SOM content, R. solani addition had a negative effect on aphid biomass, whereas it enhanced aphid biomass in soil with high SOM content. In sterilized soil, however, aphid biomass was enhanced by R. solani addition and by high SOM content. Plant biomass was enhanced by fertilizer addition, but only when SOM content was low, or in the absence of R. solani. We conclude that belowground pathogens influence aphid performance and that the effect of soil pathogens on aphids can be more positive in the absence of a soil microbiome. This implies that experiments studying the effect of pathogens under sterile conditions might not represent realistic interactions. Moreover, pathogen-plant-aphid interactions can be more positive for aphids under high SOM conditions. We recommend that soil conditions should be taken into account in the study of microbe-plant-insect interactions.