A simulated natural heatwave perturbs bumblebee immunity and resistance to infection.

As a consequence of ongoing climate change, heatwaves are predicted to increase in frequency, intensity, and duration in many regions. Such extreme events can shift organisms from thermal optima for physiology and behaviour, with the thermal stress hypothesis predicting reduced performance at temperatures where the maintenance of biological functions is energetically costly. Performance includes the ability to resist biotic stressors, including infectious diseases, with increased exposure to extreme temperatures having the potential to synergise with parasite infection. Climate change is a proposed threat to native bee pollinators, directly and through indirect effects on floral resources, but the thermal stress hypothesis, particularly pertaining to infectious disease resistance, has received limited attention. We exposed adult Bombus impatiens bumblebee workers to simulated, ecologically relevant heatwave or control thermal regimes and assessed longevity, immunity, and resistance to concurrent or future parasite infections. We demonstrate that survival and induced antibacterial immunity are reduced following heatwaves. Supporting that heatwave exposure compromised immunity, the cost of immune activation was thermal regime dependent, with survival costs in control but not heatwave exposed bees. However, in the face of real infections, an inability to mount an optimal immune response will be detrimental, which was reflected by increased trypanosomatid parasite infections following heatwave exposure. These results demonstrate interactions between heatwave exposure and bumblebee performance, including immune and infection outcomes. Thus, the health of bumblebee pollinator populations may be affected through altered interactions with parasites and pathogens, in addition to other effects of extreme manifestations of climate change.

Quantitative trait loci mapping for survival of virus infection and virus levels in honey bees.

Israeli acute paralysis virus (IAPV) is a highly virulent, Varroa-vectored virus that is of global concern for honey bee health. Little is known about the genetic basis of honey bees to withstand infection with IAPV or other viruses. We set up and analyzed a backcross between preselected honey bee colonies of low and high IAPV susceptibility to identify quantitative trait loci (QTL) associated with IAPV susceptibility. Experimentally inoculated adult worker bees were surveyed for survival and selectively sampled for QTL analysis based on SNPs identified by whole-genome resequencing and composite interval mapping. Additionally, natural titers of other viruses were quantified in the abdomen of these workers via qPCR and also used for QTL mapping. In addition to the full dataset, we analyzed distinct subpopulations of susceptible and non-susceptible workers separately. These subpopulations are distinguished by a single, suggestive QTL on chromosome 6, but we identified numerous other QTL for different abdominal virus titers, particularly in the subpopulation that was not susceptible to IAPV. The pronounced QTL differences between the susceptible and non-susceptible subpopulations indicate either an interaction between IAPV infection and the bees' interaction with other viruses or heterogeneity among workers of a single cohort that manifests itself as IAPV susceptibility and results in distinct subgroups that differ in their interaction with other viruses. Furthermore, our results indicate that low susceptibility of honey bees to viruses can be caused by both, virus tolerance and virus resistance. QTL were partially overlapping among different viruses, indicating a mixture of shared and specific processes that control viruses. Some functional candidate genes are located in the QTL intervals, but their genomic co-localization with numerous genes of unknown function delegates any definite characterization of the underlying molecular mechanisms to future studies.

Land-use-associated stressors interact to reduce bumblebee health at the individual and colony level.

In agricultural landscapes, bees face a variety of stressors, including insecticides and poor-quality food. Although both stressors individually have been shown to affect bumblebee health negatively, few studies have focused on stressor interactions, a scenario expected in intensively used agricultural landscapes. Using the bumblebee Bombus terrestris, a key pollinator in agricultural landscapes, we conducted a fully factorial laboratory experiment starting at nest initiation. We assessed the effects of food quality and insecticides, alone and in interaction, on health traits at various levels, some of which have been rarely studied. Pollen with a diluted nutrient content (low quality) reduced ovary size and delayed colony development. Wing asymmetry, indicating developmental stress, was increased during insecticide exposure and interactions with poor food, whereas both stressors reduced body size. Both stressors and their interaction changed the workers' chemical profile and reduced worker interactions and the immune response. Our findings suggest that insecticides combined with nutritional stress reduce bumblebee health at the individual and colony levels, thus possibly affecting colony performance, such as development and reproduction, and the stability of plant-pollinator networks. The synergistic effects highlight the need of combining stressors in risk assessments and when studying the complex effects of anthropogenic stressors on health outcomes.

The impact of heavy metal pollution on wild bee communities in smallholder farmlands.

Wild bees provide important pollination services, but they face numerous stressors that threaten them and their ecosystem services. Wild bees can be exposed to heavy metal pollution through the consumption of nectar, pollen, and water, which might cause bee decline. While some studies have measured heavy metal concentrations in honeybees, few studies have monitored heavy metal concentrations in wild bees or explored their potential effects on wild bee communities. To investigate the impact of heavy metal pollution on wild bee communities, heavy metal concentrations, including vanadium (V), chromium (Cr), nickel (Ni), cadmium (Cd), Zinc (Zn) and lead (Pb) in multiple wild bee species were measured. Multiple wild bee species, including: Xylocopa tranquabaroroum, Eucera floralia, Apis cerana, and small bee mixtures (representing multiple small wild bee species) were sampled from 18 sites in Quzhou, Zhejiang Province, China. The findings demonstrated that there were significant differences in heavy metal concentrations among different bee species. The concentrations of V, Zn, Cd, and Pb in X. tranquabaroroum, the largest bee species in this study, were lower than that in the other three sample groups. Furthermore, there were significant negative correlations between heavy metal pollution and wild bee diversity and species richness, but not with abundance. Particularly, there was no significant relationship between heavy metal pollution and the abundance of small bees. Given these worrying findings, monitoring multiple heavy metals in wild bees should be conducted for protecting wild bee diversity and securing their pollination services.

Environmental Effects on Bee Microbiota.

Anthropogenic activities and increased land use, which include industrialization, agriculture and urbanization, directly affect pollinators by changing habitats and floral availability, and indirectly by influencing their microbial composition and diversity. Bees form vital symbioses with their microbiota, relying on microorganisms to perform physiological functions and aid in immunity. As altered environments and climate threaten bees and their microbiota, characterizing the microbiome and its complex relationships with its host offers insights into understanding bee health. This review summarizes the role of sociality in microbiota establishment, as well as examines if such factors result in increased susceptibility to altered microbiota due to environmental changes. We characterize the role of geographic distribution, temperature, precipitation, floral resources, agriculture, and urbanization on bee microbiota. Bee microbiota are affected by altered surroundings regardless of sociality. Solitary bees that predominantly acquire their microbiota through the environment are particularly sensitive to such effects. However, the microbiota of obligately eusocial bees are also impacted by environmental changes despite typically well conserved and socially inherited microbiota. We provide an overview of the role of microbiota in plant-pollinator relationships and how bee microbiota play a larger role in urban ecology, offering microbial connections between animals, humans, and the environment. Understanding bee microbiota presents opportunities for sustainable land use restoration and aiding in wildlife conservation.

Sunflower plantings reduce a common gut pathogen and increase queen production in common eastern bumblebee colonies.

Community diversity can reduce the prevalence and spread of disease, but certain species may play a disproportionate role in diluting or amplifying pathogens. Flowers act as both sources of nutrition and sites of pathogen transmission, but the effects of specific plant species in shaping bee disease dynamics are not well understood. We evaluated whether plantings of sunflower (Helianthus annuus), whose pollen reduces infection by some pathogens when fed to bees in captivity, lowered pathogen levels and increased reproduction in free-foraging bumblebee colonies (Bombus impatiens). Sunflower abundance reduced the prevalence of a common gut pathogen, Crithidia bombi, and reduced infection intensity, with an order of magnitude lower infection intensity at high sunflower sites compared with sites with little to no sunflower. Sunflower abundance was also positively associated with greater queen production in colonies. Sunflower did not affect prevalence of other detected pathogens. This work demonstrates that a single plant species can drive disease dynamics in foraging B. impatiens, and that sunflower plantings can be used as a tool for mitigating a prevalent pathogen while also increasing reproduction of an agriculturally important bee species.

Signatures of Adaptation, Constraints, and Potential Redundancy in the Canonical Immune Genes of a Key Pollinator.

All organisms require an immune system to recognize, differentiate, and defend against pathogens. From an evolutionary perspective, immune systems evolve under strong selective pressures exerted by fast-evolving pathogens. However, the functional diversity of the immune system means that different immune components and their associated genes may evolve under varying forms of selection. Insect pollinators, which provide essential ecosystem services, are an important system in which to understand how selection has shaped immune gene evolution as their populations are experiencing declines with pathogens highlighted as a potential contributing factor. To improve our understanding of the genetic variation found in the immune genes of an essential pollinator, we performed whole-genome resequencing of wild-caught Bombus terrestris males. We first assessed nucleotide diversity and extended haplotype homozygosity for canonical immune genes finding the strongest signatures of positive selection acting on genes involved in pathogen recognition and antiviral defense, possibly driven by growing pathogen spread in wild populations. We also identified immune genes evolving under strong purifying selection, highlighting potential constraints on the bumblebee immune system. Lastly, we highlight the potential loss of function alleles present in the immune genes of wild-caught haploid males, suggesting that such genes are potentially less essential for development and survival and represent redundancy in the gene repertoire of the bumblebee immune system. Collectively, our analysis provides novel insights into the recent evolutionary history of the immune system of a key pollinator, highlighting targets of selection, constraints to adaptation, and potential redundancy.

Expression of subunits of an insecticide target receptor varies across tissues, life stages, castes, and species of social bees.

Global losses of insects jeopardize ecosystem stability and crop pollination. Robust evidence indicates that insecticides have contributed to these losses. Notably, insecticides targeting nicotinic acetylcholine receptors (nAChRs) have neurotoxic effects on beneficial insects. Because each nAChR consists of five subunits, the alternative arrangements of subunits could create a multitude of receptors differing in structure and function. Therefore, understanding whether the use of subunits varies is essential for evaluating and predicting the effects of insecticides targeting such receptors. To better understand how the use and composition of nAChRs differ within and between insect pollinators, we analysed RNA-seq gene expression data from tissues and castes of Apis mellifera honey bees and life stages and castes of the Bombus terrestris bumble bees. We reveal that all analysed tissues express nAChRs and that relative expression levels of nAChR subunits vary widely across almost all comparisons. Our work thus shows fine-tuned spatial and temporal expression of nAChRs. Given that coexpression of subunits underpins the compositional diversity of functional receptors and that the affinities of insecticides depend on nAChR composition, our findings provide a likely mechanism for the various damaging effects of nAChR-targeting insecticides on insects. Furthermore, our results indicate that the appraisal of insecticide risks should carefully consider variation in molecular targets.

Investigating the influence of diet diversity on infection outcomes in a bumble bee (Bombus impatiens) and microsporidian (Nosema bombi) host-pathogen system.

Diet can have an array of both direct and indirect effects on an organism's health and fitness, which can influence the outcomes of host-pathogen interactions. Land use changes, which could impact diet quantity and quality, have imposed foraging stress on important natural and agricultural pollinators. Diet related stress could exacerbate existing negative impacts of pathogen infection. Accounting for most of its nutritional intake in terms of protein and many micronutrients, pollen can influence bee health through changes in immunity, infection, and various aspects of individual and colony fitness. We investigate how adult pollen consumption, pollen type, and pollen diversity influence bumble bee Bombus impatiens survival and infection outcomes for a microsporidian pathogen Nosema (Vairimorpha) bombi. Experimental pathogen exposures of larvae occurred in microcolonies and newly emerged adult workers were given one of three predominantly monofloral, polyfloral, or no pollen diets. Workers were assessed for size, pollen consumption, infection 8-days following adult-eclosion, survival, and the presence of extracellular microsporidian spores at death. Pollen diet treatment, specifically absence of pollen, and infection independently reduced survival, but we saw no effects of pollen, pollen type, or pollen diet diversity on infection outcomes. The latter suggests infection outcomes were likely already set, prior to differential diets. Although infection outcomes were not altered by pollen diet in our study, it highlights both pathogen infection and pollen availability as important for bumble bee health, and these factors may interact at different stages of bumble bee development, at the colony level, or under different dietary regimes.

Mutualism has its limits: consequences of asymmetric interactions between a well-defended plant and its herbivorous pollinator.

Concern for pollinator health often focuses on social bees and their agricultural importance at the expense of other pollinators and their ecosystem services. When pollinating herbivores use the same plants as nectar sources and larval hosts, ecological conflicts emerge for both parties, as the pollinator's services are mitigated by herbivory and its larvae are harmed by plant defences. We tracked individual-level metrics of pollinator health-growth, survivorship, fecundity-across the life cycle of a pollinating herbivore, the common hawkmoth, Hyles lineata, interacting with a rare plant, Oenothera harringtonii, that is polymorphic for the common floral volatile (R)-(-)-linalool. Linalool had no impact on floral attraction, but its experimental addition suppressed oviposition on plants lacking linalool. Plants showed robust resistance against herbivory from leaf-disc to whole-plant scales, through poor larval growth and survivorship. Higher larval performance on other Oenothera species indicates that constitutive herbivore resistance by O. harringtonii is not a genus-wide trait. Leaf volatiles differed among populations of O. harringtonii but were not induced by larval herbivory. Similarly, elagitannins and other phenolics varied among plant tissues but were not herbivore-induced. Our findings highlight asymmetric plant-pollinator interactions and the importance of third parties, including alternative larval host plants, in maintaining pollinator health. This article is part of the theme issue 'Natural processes influencing pollinator health: from chemistry to landscapes'.

Potential effects of nectar microbes on pollinator health.

Floral nectar is prone to colonization by nectar-adapted yeasts and bacteria via air-, rain-, and animal-mediated dispersal. Upon colonization, microbes can modify nectar chemical constituents that are plant-provisioned or impart their own through secretion of metabolic by-products or antibiotics into the nectar environment. Such modifications can have consequences for pollinator perception of nectar quality, as microbial metabolism can leave a distinct imprint on olfactory and gustatory cues that inform foraging decisions. Furthermore, direct interactions between pollinators and nectar microbes, as well as consumption of modified nectar, have the potential to affect pollinator health both positively and negatively. Here, we discuss and integrate recent findings from research on plant-microbe-pollinator interactions and their consequences for pollinator health. We then explore future avenues of research that could shed light on the myriad ways in which nectar microbes can affect pollinator health, including the taxonomic diversity of vertebrate and invertebrate pollinators that rely on this reward. This article is part of the theme issue 'Natural processes influencing pollinator health: from chemistry to landscapes'.

Natural processes influencing pollinator health.

Evidence from the last few decades indicates that pollinator abundance and diversity are at risk, with many species in decline. Anthropogenic impacts have been the focus of much recent work on the causes of these declines. However, natural processes, from plant chemistry, nutrition and microbial associations to landscape and habitat change, can also profoundly influence pollinator health. Here, we argue that these natural processes require greater attention and may even provide solutions to the deteriorating outlook for pollinators. Existing studies also focus on the decline of individuals and colonies and only occasionally at population levels. In the light of this we redefine pollinator health and argue that a top-down approach is required focusing at the ecological level of communities. We use examples from the primary research, opinion and review articles published in this special issue to illustrate how natural processes influence pollinator health, from community to individuals, and highlight where some of these processes could mitigate the challenges of anthropogenic and natural drivers of change. This article is part of the theme issue 'Natural processes influencing pollinator health: from chemistry to landscapes'.

Engineering insects from the endosymbiont out.

Insects are an incredibly diverse group of animals with species that benefit and harm natural ecosystems, agriculture, and human health. Many insects have consequential associations with microbes: bacterial symbionts may be embedded in different insect tissues and cell types, inherited across insect generations, and required for insect survival and reproduction. Genetically engineering insect symbionts is key to understanding and harnessing these associations. We summarize different types of insect-bacteria relationships and review methods used to genetically modify endosymbiont and gut symbiont species. Finally, we discuss recent studies that use this approach to study symbioses, manipulate insect-microbe interactions, and influence insect biology. Further progress in insect symbiont engineering promises to solve societal challenges, ranging from controlling pests to protecting pollinator health.

Parasite Prevalence May Drive the Biotic Impoverishment of New England (USA) Bumble Bee Communities.

Numerous studies have reported a diversity of stressors that may explain continental-scale declines in populations of native pollinators, particularly those in the genus Bombus. However, there has been little focus on the identification of the local-scale dynamics that may structure currently impoverished Bombus communities. For example, the historically diverse coastal-zone communities of New England (USA) now comprise only a few species and are primarily dominated by a single species, B. impatiens. To better understand the local-scale factors that might be influencing this change in community structure, we examined differences in the presence of parasites in different species of Bombus collected in coastal-zone communities. Our results indicate that Bombus species that are in decline in this region were more likely to harbor parasites than are B. impatiens populations, which were more likely to be parasite-free and to harbor fewer intense infections or co-infections. The contrasting parasite burden between co-occurring winners and losers in this community may impact the endgame of asymmetric contests among species competing for dwindling resources. We suggest that under changing climate and landscape conditions, increasing domination of communities by healthy, synanthropic Bombus species (such as B. impatiens) may be another factor hastening the further erosion of bumble bee diversity.

Supplemental forage ameliorates the negative impact of insecticides on bumblebees in a pollinator-dependent crop.

Insecticide use and insufficient forage are two of the leading stressors to pollinators in agroecosystems. While these factors have been well studied individually, the experimental designs do not reflect real-world conditions where insecticide exposure and lack of forage occur simultaneously and could interactively suppress pollinator health. Using outdoor enclosures, we tested the effects of insecticides (imidacloprid + lambda-cyhalothrin) and non-crop forage (clover) in a factorial design, measuring the survival, behaviour and performance of bumblebees (Bombus impatiens), as well as pollination of the focal crop, watermelon. Colony survival was synergistically reduced to 17% in watermelon alone + insecticides (survival was 100% in all other treatments). However, behavioural shifts in foraging were mainly owing to insecticides (e.g. 95% reduced visitation rate to watermelon flowers), while impacts on hive performance were primarily driven by clover presence (e.g. 374% increase in the number of live eggs). Insecticide-mediated reductions in foraging decreased crop pollination (fruit set) by 32%. Altogether, these data indicate that both insecticides and non-crop forage play integral roles in shaping pollinator health in agricultural landscapes, but the relative importance and interaction of these two factors depend on which aspect of 'health' is being considered.

Field-Level Exposure of Bumble Bees to Fungicides Applied to a Commercial Cherry Orchard.

Bumble bees, Bombus spp. (Apidae), are important native pollinators; however, populations of some species are declining in North America and agricultural chemicals are a potential cause. Fungicides are generally not highly toxic to bees, but little is known about sublethal or synergistic effects. This study evaluates bumble bee exposure to fungicides by quantifying concentrations of boscalid and pyraclostrobin in nectar and pollen collected by colonies of Bombus huntii Greene, 1860 (Hunt bumble bee) deployed in a commercial cherry Prunus avium L. orchard in the spring of 2016. Seven colonies were placed adjacent to an orchard block that was sprayed with a fungicide mixture of boscalid and pyraclostrobin and a control group of seven colonies was placed next to an unsprayed block of orchard 400 m away from the treated block. Nectar and pollen were collected daily, beginning 1 d before spray application and continuing for a total of 12 d, and analyzed for both fungicides. Fungicide concentrations varied spatially by colony and temporally by day. The highest concentrations in nectar occurred 1 and 3 d after spraying: up to 440 ng/g boscalid and 240 ng/g pyraclostrobin. Six days after application, pollen from cherry flowers contained the highest concentrations of the fungicides: up to 60,500 ng/g boscalid and 32,000 ng/g pyraclostrobin. These data can help to determine field-level fungicide concentrations in nectar and pollen and direct future work on understanding the effects of these compounds, including their interactions with important bumble bee pathogenic and beneficial symbionts.

Supporting Bees in Cities: How Bees Are Influenced by Local and Landscape Features.

Urbanization is a major anthropogenic driver of decline for ecologically and economically important taxa including bees. Despite their generally negative impact on pollinators, cities can display a surprising degree of biodiversity compared to other landscapes. The pollinating communities found within these environments, however, tend to be filtered by interacting local and landscape features that comprise the urban matrix. Landscape and local features exert variable influence on pollinators within and across taxa, which ultimately affects community composition in such a way that contributes to functional trait homogenization and reduced phylogenetic diversity. Although previous results are not easily generalizable, bees and pollinators displaying functional trait characteristics such as polylectic diet, cavity-nesting behavior, and later emergence appear most abundant across different examined cities. To preserve particularly vulnerable species, most notably specialists that have become underrepresented within city communities, green spaces like parks and urban gardens have been examined as potential refuges. Such spaces are scattered across the urban matrix and vary in pollinator resource availability. Therefore, ensuring such spaces are optimized for pollinators is imperative. This review examines how urban features affect pollinators in addition to ways these green spaces can be manipulated to promote greater pollinator abundance and diversity.

Comparing Survival of Israeli Acute Paralysis Virus Infection among Stocks of U.S. Honey Bees.

Among numerous viruses that infect honey bees (Apis mellifera), Israeli acute paralysis virus (IAPV) can be linked to severe honey bee health problems. Breeding for virus resistance may improve honey bee health. To evaluate the potential for this approach, we compared the survival of IAPV infection among stocks from the U.S. We complemented the survival analysis with a survey of existing viruses in these stocks and assessing constitutive and induced expression of immune genes. Worker offspring from selected queens in a common apiary were inoculated with IAPV by topical applications after emergence to assess subsequent survival. Differences among stocks were small compared to variation within stocks, indicating the potential for improving honey bee survival of virus infections in all stocks. A positive relation between worker survival and virus load among stocks further suggested that honey bees may be able to adapt to better cope with viruses, while our molecular studies indicate that toll-6 may be related to survival differences among virus-infected worker bees. Together, these findings highlight the importance of viruses in queen breeding operations and provide a promising starting point for the quest to improve honey bee health by selectively breeding stock to be better able to survive virus infections.

Crithidia bombi can infect two solitary bee species while host survivorship depends on diet.

Pathogens and lack of floral resources interactively impair global pollinator health. However, epidemiological and nutritional studies aimed at understanding bee declines have historically focused on social species, with limited evaluations of solitary bees. Here, we asked whether Crithidia bombi, a trypanosomatid gut pathogen known to infect bumble bees, could infect the solitary bees Osmia lignaria (females) and Megachile rotundata (males), and whether nutritional stress influenced infection patterns and bee survival. We found that C. bombi was able to infect both solitary bee species, with 59% of O. lignaria and 29% of M. rotundata bees experiencing pathogen replication 5­11 days following inoculation. Moreover, access to pollen resulted in O. lignaria living longer, although it did not influence M. rotundata survival. Access to pollen did not affect infection probability or resulting pathogen load in either species. Similarly, inoculating with the pathogen did not drive survival patterns in either species during the 5­11-day laboratory assays. Our results demonstrate that solitary bees can be hosts of a known bumble bee pathogen, and that access to pollen is an important contributing factor for bee survival, thus expanding our understanding of factors contributing to solitary bee health.

Temporal changes in the microbiome of stingless bee foragers following colony relocation.

Maintaining beneficial interactions with microbial symbionts is vital for animal health. Yet, for social insects, the stability of microbial associations within and between cohorts is largely unknown. We investigated temporal changes in the microbiomes of nine stingless bee (Tetragonula carbonaria) colonies at seven timepoints across a 10-month period when moved between two climatically and florally different sites. Bacterial 16S rRNA gene and fungal ITS amplicon sequencing confirmed that microbiomes varied considerably between colonies initially at site one. However, following relocation, considerable changes occurred in bacterial community composition within each colony, and the microbiome composition became more similar across colonies. Notably, Snodgrassella disappeared and Zymobacter appeared as relatively abundant taxa. Remarkably, bacterial communities within colonies continued to shift over time but remained similar across colonies, becoming dominated by Acinetobacter six months after returning to the original site. Our results indicate that the stingless bee microbiome can undergo major changes in response to the environment, and that these changes can be long-lasting. Such legacy effects have not been reported for corbiculate bees. Further understanding the microbial ecology of stingless bees will aid future management of colonies used in agricultural production.