Using individual-based trait frequency distributions to forecast plant-pollinator network responses to environmental change.

Determining how and why organisms interact is fundamental to understanding ecosystem responses to future environmental change. To assess the impact on plant-pollinator interactions, recent studies have examined how the effects of environmental change on individual interactions accumulate to generate species-level responses. Here, we review recent developments in using plant-pollinator networks of interacting individuals along with their functional traits, where individuals are nested within species nodes. We highlight how these individual-level, trait-based networks connect intraspecific trait variation (as frequency distributions of multiple traits) with dynamic responses within plant-pollinator communities. This approach can better explain interaction plasticity, and changes to interaction probabilities and network structure over spatiotemporal or other environmental gradients. We argue that only through appreciating such trait-based interaction plasticity can we accurately forecast the potential vulnerability of interactions to future environmental change. We follow this with general guidance on how future studies can collect and analyse high-resolution interaction and trait data, with the hope of improving predictions of future plant-pollinator network responses for targeted and effective conservation.

Genomic variation in montane bumblebees in Scandinavia: High levels of intraspecific diversity despite population vulnerability.

Populations of many bumblebee species are declining, with distributions shifting northwards to track suitable climates. Climate change is considered a major contributing factor. Arctic species are particularly vulnerable as they cannot shift further north, making assessment of their population viability important. Analysis of levels of whole-genome variation is a powerful way to analyse population declines and fragmentation. Here, we use genome sequencing to analyse genetic variation in seven species of bumblebee from the Scandinavian mountains, including two classified as vulnerable. We sequenced 333 samples from across the ranges of these species in Sweden. Estimates of effective population size (NE ) vary from ~55,000 for species with restricted high alpine distributions to 220,000 for more widespread species. Population fragmentation is generally very low or undetectable over large distances in the mountains, suggesting an absence of barriers to gene flow. The relatively high NE and low population structure indicate that none of the species are at immediate risk of negative genetic effects caused by high levels of genetic drift. However, reconstruction of historical fluctuations in NE indicates that the arctic specialist species Bombus hyperboreus has experienced population declines since the last ice age and we detected one highly inbred diploid male of this species close to the southern limit of its range, potentially indicating elevated genetic load. Although the levels of genetic variation in montane bumblebee populations are currently relatively high, their ranges are predicted to shrink drastically due to the effects of climate change and monitoring is essential to detect future population declines.

Genomic Signatures of Recent Adaptation in a Wild Bumblebee.

Environmental changes threaten insect pollinators, creating risks for agriculture and ecosystem stability. Despite their importance, we know little about how wild insects respond to environmental pressures. To understand the genomic bases of adaptation in an ecologically important pollinator, we analyzed genomes of Bombus terrestris bumblebees collected across Great Britain. We reveal extensive genetic diversity within this population, and strong signatures of recent adaptation throughout the genome affecting key processes including neurobiology and wing development. We also discover unusual features of the genome, including a region containing 53 genes that lacks genetic diversity in many bee species, and a horizontal gene transfer from a Wolbachia bacteria. Overall, the genetic diversity we observe and how it is distributed throughout the genome and the population should support the resilience of this important pollinator species to ongoing and future selective pressures. Applying our approach to more species should help understand how they can differ in their adaptive potential, and to develop conservation strategies for those most at risk.

Toxic temperatures: Bee behaviours exhibit divergent pesticide toxicity relationships with warming.

Climate change and agricultural intensification are exposing insect pollinators to temperature extremes and increasing pesticide usage. Yet, we lack good quantification of how temperature modulates the sublethal effects of pesticides on behaviours vital for fitness and pollination performance. Consequently, we are uncertain if warming decreases or increases the severity of different pesticide impacts, and whether separate behaviours vary in the direction of response. Quantifying these interactive effects is vital in forecasting pesticide risk across climate regions and informing pesticide application strategies and pollinator conservation. This multi-stressor study investigated the responses of six functional behaviours of bumblebees when exposed to either a neonicotinoid (imidacloprid) or a sulfoximine (sulfoxaflor) across a standardised low, mid, and high temperature. We found the neonicotinoid had a significant effect on five of the six behaviours, with a greater effect at the lower temperature(s) when measuring responsiveness, the likelihood of movement, walking rate, and food consumption rate. In contrast, the neonicotinoid had a greater impact on flight distance at the higher temperature. Our findings show that different organismal functions can exhibit divergent thermal responses, with some pesticide-affected behaviours showing greater impact as temperatures dropped, and others as temperatures rose. We must therefore account for environmental context when determining pesticide risk. Moreover, we found evidence of synergistic effects, with just a 3°C increase causing a sudden drop in flight performance, despite seeing no effect of pesticide at the two lower temperatures. Our findings highlight the importance of multi-stressor studies to quantify threats to insects, which will help to improve dynamic evaluations of population tipping points and spatiotemporal risks to biodiversity across different climate regions.

Signatures of increasing environmental stress in bumblebee wings over the past century: Insights from museum specimens.

Determining when animal populations have experienced stress in the past is fundamental to understanding how risk factors drive contemporary and future species' responses to environmental change. For insects, quantifying stress and associating it with environmental factors has been challenging due to a paucity of time-series data and because detectable population-level responses can show varying lag effects. One solution is to leverage historic entomological specimens to detect morphological proxies of stress experienced at the time stressors emerged, allowing us to more accurately determine population responses. Here we studied specimens of four bumblebee species, an invaluable group of insect pollinators, from five museums collected across Britain over the 20th century. We calculated the degree of fluctuating asymmetry (FA; random deviations from bilateral symmetry) between the right and left forewings as a potential proxy of developmental stress. We: (a) investigated whether baseline FA levels vary between species, and how this compares between the first and second half of the century; (b) determined the extent of FA change over the century in the four bumblebee species, and whether this followed a linear or nonlinear trend; (c) tested which annual climatic conditions correlated with increased FA in bumblebees. Species differed in their baseline FA, with FA being higher in the two species that have recently expanded their ranges in Britain. Overall, FA significantly increased over the century but followed a nonlinear trend, with the increase starting c. 1925. We found relatively warm and wet years were associated with higher FA. Collectively our findings show that FA in bumblebees increased over the 20th century and under weather conditions that will likely increase in frequency with climate change. By plotting FA trends and quantifying the contribution of annual climate conditions on past populations, we provide an important step towards improving our understanding of how environmental factors could impact future populations of wild beneficial insects.

How competition between overlapping generations can influence optimal egg-laying strategies in annual social insects.

Annual social insects are an integral functional group of organisms, particularly in temperate environments. An emblematic part of their annual cycle is the social phase, during which the colony-founding queen rears workers that later assist her in rearing sexual progeny (gynes and drones). In many annual social insects, such as species of bees, wasps, and other groups, developing larvae are provisioned gradually as they develop (progressive provisioning) leading to multiple larval generations being reared simultaneously. We present a model for how the queen in such cases should optimize her egg-laying rate throughout the social phase depending on number-size trade-offs, colony age-structure, and energy balance. Complementing previous theory on optimal allocation between workers vs. sexuals in annual social insects and on temporal egg-laying patterns in solitary insects, we elucidate how resource competition among overlapping larval generations can influence optimal egg-laying strategies. With model parameters informed by knowledge of a common bumblebee species, the optimal egg-laying schedule consists of two temporally separated early broods followed by a more continuous rearing phase, matching empirical observations. However, eggs should initially be laid continuously at a gradually increasing rate when resources are scarce or mortality risks high and in cases where larvae are fully supplied with resources at the egg-laying stage (mass-provisioning). These factors, alongside sexual:worker body size ratios, further determine the overall trend in egg-laying rates over the colony cycle. Our analysis provides an inroad to study and mechanistically understand variation in colony development strategies within and across species of annual social insects.

A micro-CT-based standard brain atlas of the bumblebee.

In recent years, bumblebees have become a prominent insect model organism for a variety of biological disciplines, particularly to investigate learning behaviors as well as visual performance. Understanding these behaviors and their underlying neurobiological principles requires a clear understanding of brain anatomy. Furthermore, to be able to compare neuronal branching patterns across individuals, a common framework is required, which has led to the development of 3D standard brain atlases in most of the neurobiological insect model species. Yet, no bumblebee 3D standard brain atlas has been generated. Here we present a brain atlas for the buff-tailed bumblebee Bombus terrestris using micro-computed tomography (micro-CT) scans as a source for the raw data sets, rather than traditional confocal microscopy, to produce the first ever micro-CT-based insect brain atlas. We illustrate the advantages of the micro-CT technique, namely, identical native resolution in the three cardinal planes and 3D structure being better preserved. Our Bombus terrestris brain atlas consists of 30 neuropils reconstructed from ten individual worker bees, with micro-CT allowing us to segment neuropils completely intact, including the lamina, which is a tissue structure often damaged when dissecting for immunolabeling. Our brain atlas can serve as a platform to facilitate future neuroscience studies in bumblebees and illustrates the advantages of micro-CT for specific applications in insect neuroanatomy.

Insecticide exposure during brood or early-adult development reduces brain growth and impairs adult learning in bumblebees.

For social bees, an understudied step in evaluating pesticide risk is how contaminated food entering colonies affects residing offspring development and maturation. For instance, neurotoxic insecticide compounds in food could affect central nervous system development predisposing individuals to become poorer task performers later-in-life. Studying bumblebee colonies provisioned with neonicotinoid spiked nectar substitute, we measured brain volume and learning behaviour of 3 or 12-day old adults that had experienced in-hive exposure during brood and/or early-stage adult development. Micro-computed tomography scanning and segmentation of multiple brain neuropils showed exposure during either of the developmental stages caused reduced mushroom body calycal growth relative to unexposed workers. Associated with this was a lower probability of responding to a sucrose reward and lower learning performance in an olfactory conditioning test. While calycal volume of control workers positively correlated with learning score, this relationship was absent for exposed workers indicating neuropil functional impairment. Comparison of 3- and 12-day adults exposed during brood development showed a similar degree of reduced calycal volume and impaired behaviour highlighting lasting and irrecoverable effects from exposure despite no adult exposure. Our findings help explain how the onset of pesticide exposure to whole colonies can lead to lag-effects on growth and resultant dysfunction.

Caste- and pesticide-specific effects of neonicotinoid pesticide exposure on gene expression in bumblebees.

Social bees are important insect pollinators of wildflowers and agricultural crops, making their reported declines a global concern. A major factor implicated in these declines is the widespread use of neonicotinoid pesticides. Indeed, recent research has demonstrated that exposure to low doses of these neurotoxic pesticides impairs bee behaviours important for colony function and survival. However, our understanding of the molecular-genetic pathways that lead to such effects is limited, as is our knowledge of how effects may differ between colony members. To understand what genes and pathways are affected by exposure of bumblebee workers and queens to neonicotinoid pesticides, we implemented a transcriptome-wide gene expression study. We chronically exposed Bombus terrestriscolonies to either clothianidin or imidacloprid at field-realistic concentrations while controlling for factors including colony social environment and worker age. We reveal that genes involved in important biological processes including mitochondrial function are differentially expressed in response to neonicotinoid exposure. Additionally, clothianidin exposure had stronger effects on gene expression amplitude and alternative splicing than imidacloprid. Finally, exposure affected workers more strongly than queens. Our work demonstrates how RNA-Seq transcriptome profiling can provide detailed novel insight on the mechanisms mediating pesticide toxicity to a key insect pollinator.

Lower bumblebee colony reproductive success in agricultural compared with urban environments.

Urbanization represents a rapidly growing driver of land-use change. While it is clear that urbanization impacts species abundance and diversity, direct effects of urban land use on animal reproductive success are rarely documented. Here, we show that urban land use is linked to long-term colony reproductive output in a key pollinator. We reared colonies from wild-caught bumblebee (Bombus terrestris) queens, placed them at sites characterized by varying degrees of urbanization from inner city to rural farmland and monitored the production of sexual offspring across the entire colony cycle. Our land-use cluster analysis identified three site categories, and this categorization was a strong predictor of colony performance. Crucially, colonies in the two clusters characterized by urban development produced more sexual offspring than those in the cluster dominated by agricultural land. These colonies also reached higher peak size, had more food stores, encountered fewer parasite invasions and survived for longer. Our results show a link between urbanization and bumblebee colony reproductive success, supporting the theory that urban areas provide a refuge for pollinator populations in an otherwise barren agricultural landscape.

Foraging bumblebees acquire a preference for neonicotinoid-treated food with prolonged exposure.

Social bees represent an important group of pollinating insects that can be exposed to potentially harmful pesticides when foraging on treated or contaminated flowering plants. To investigate if such exposure is detrimental to bees, many studies have exclusively fed individuals with pesticide-spiked food, informing us about the hazard but not necessarily the risk of exposure. While such studies are important to establish the physiological and behavioural effects on individuals, they do not consider the possibility that the risk of exposure may change over time. For example, many pesticide assays exclude potential behavioural adaptations to novel toxins, such as rejection of harmful compounds by choosing to feed on an uncontaminated food source, thus behaviourally lowering the risk of exposure. In this paper, we conducted an experiment over 10 days in which bumblebees could forage on an array of sucrose feeders containing 0, 2 and 11 parts per billion of the neonicotinoid pesticide thiamethoxam. This more closely mimics pesticide exposure in the wild by allowing foraging bees to (i) experience a field realistic range of pesticide concentrations across a chronic exposure period, (ii) have repeated interactions with the pesticide in their environment, and (iii) retain the social cues associated with foraging by using whole colonies. We found that the proportion of visits to pesticide-laced feeders increased over time, resulting in greater consumption of pesticide-laced sucrose relative to untreated sucrose. After changing the spatial position of each feeder, foragers continued to preferentially visit the pesticide-laced feeders which indicates that workers can detect thiamethoxam and alter their behaviour to continue feeding on it. The increasing preference for consuming the neonicotinoid-treated food therefore increases the risk of exposure for the colony during prolonged pesticide exposure. Our results highlight the need to incorporate attractiveness of pesticides to foraging bees (and potentially other insect pollinators) in addition to simply considering the proportion of pesticide-contaminated floral resources within the foraging landscape.

Combined pesticide exposure severely affects individual- and colony-level traits in bees.

Reported widespread declines of wild and managed insect pollinators have serious consequences for global ecosystem services and agricultural production. Bees contribute approximately 80% of insect pollination, so it is important to understand and mitigate the causes of current declines in bee populations . Recent studies have implicated the role of pesticides in these declines, as exposure to these chemicals has been associated with changes in bee behaviour and reductions in colony queen production. However, the key link between changes in individual behaviour and the consequent impact at the colony level has not been shown. Social bee colonies depend on the collective performance of many individual workers. Thus, although field-level pesticide concentrations can have subtle or sublethal effects at the individual level, it is not known whether bee societies can buffer such effects or whether it results in a severe cumulative effect at the colony level. Furthermore, widespread agricultural intensification means that bees are exposed to numerous pesticides when foraging, yet the possible combinatorial effects of pesticide exposure have rarely been investigated. Here we show that chronic exposure of bumblebees to two pesticides (neonicotinoid and pyrethroid) at concentrations that could approximate field-level exposure impairs natural foraging behaviour and increases worker mortality leading to significant reductions in brood development and colony success. We found that worker foraging performance, particularly pollen collecting efficiency, was significantly reduced with observed knock-on effects for forager recruitment, worker losses and overall worker productivity. Moreover, we provide evidence that combinatorial exposure to pesticides increases the propensity of colonies to fail.

A genotyping array for the globally invasive vector mosquito, Aedes albopictus.

BACKGROUND: Although whole-genome sequencing (WGS) is the preferred genotyping method for most genomic analyses, limitations are often experienced when studying genomes characterized by a high percentage of repetitive elements, high linkage, and recombination deserts. The Asian tiger mosquito (Aedes albopictus), for example, has a genome comprising up to 72% repetitive elements, and therefore we set out to develop a single-nucleotide polymorphism (SNP) chip to be more cost-effective. Aedes albopictus is an invasive species originating from Southeast Asia that has recently spread around the world and is a vector for many human diseases. Developing an accessible genotyping platform is essential in advancing biological control methods and understanding the population dynamics of this pest species, with significant implications for public health. METHODS: We designed a SNP chip for Ae. albopictus (Aealbo chip) based on approximately 2.7 million SNPs identified using WGS data from 819 worldwide samples. We validated the chip using laboratory single-pair crosses, comparing technical replicates, and comparing genotypes of samples genotyped by WGS and the SNP chip. We then used the chip for a population genomic analysis of 237 samples from 28 sites in the native range to evaluate its usefulness in describing patterns of genomic variation and tracing the origins of invasions. RESULTS: Probes on the Aealbo chip targeted 175,396 SNPs in coding and non-coding regions across all three chromosomes, with a density of 102 SNPs per 1 Mb window, and at least one SNP in each of the 17,461 protein-coding genes. Overall, 70% of the probes captured the genetic variation. Segregation analysis found that 98% of the SNPs followed expectations of single-copy Mendelian genes. Comparisons with WGS indicated that sites with genotype disagreements were mostly heterozygotes at loci with WGS read depth < 20, while there was near complete agreement with WGS read depths > 20, indicating that the chip more accurately detects heterozygotes than low-coverage WGS. Sample sizes did not affect the accuracy of the SNP chip genotype calls. Ancestry analyses identified four to five genetic clusters in the native range with various levels of admixture. CONCLUSIONS: The Aealbo chip is highly accurate, is concordant with genotypes from WGS with high sequence coverage, and may be more accurate than low-coverage WGS.

Chronic sublethal stress causes bee colony failure.

Current bee population declines and colony failures are well documented yet poorly understood and no single factor has been identified as a leading cause. The evidence is equivocal and puzzling: for instance, many pathogens and parasites can be found in both failing and surviving colonies and field pesticide exposure is typically sublethal. Here, we investigate how these results can be due to sublethal stress impairing colony function. We mathematically modelled stress on individual bees which impairs colony function and found how positive density dependence can cause multiple dynamic outcomes: some colonies fail while others thrive. We then exposed bumblebee colonies to sublethal levels of a neonicotinoid pesticide. The dynamics of colony failure, which we observed, were most accurately described by our model. We argue that our model can explain the enigmatic aspects of bee colony failures, highlighting an important role for sublethal stress in colony declines.

The threat of pesticide and disease co-exposure to managed and wild bee larvae.

Brood diseases and pesticides can reduce the survival of bee larvae, reduce bee populations, and negatively influence ecosystem biodiversity. However, major gaps persist in our knowledge regarding the routes and implications of co-exposure to these stressors in managed and wild bee brood. In this review, we evaluate the likelihood for co-exposure to brood pathogen and pesticide stressors by examining the routes of potential co-exposure and the possibility for pollen and nectar contaminated with pathogens and pesticides to become integrated into brood food. Furthermore, we highlight ways in which pesticides may increase brood disease morbidity directly, through manipulating host immunity, and indirectly through disrupting microbial communities in the guts of larvae, or compromising brood care provided by adult bees. Lastly, we quantify the brood research bias towards Apis species and discuss the implications the bias has on brood disease and pesticide risk assessment in wild bee communities. We advise that future studies should place a higher emphasis on evaluating bee brood afflictions and their interactions with commonly encountered stressors, especially in wild bee species.

Workers influence royal reproduction.

Understanding which parties regulate reproduction is fundamental to understanding conflict resolution in animal societies. In social insects, workers can influence male production and sex ratio. Surprisingly, few studies have investigated worker influence over which queen(s) reproduce(s) in multiple queen (MQ) colonies (skew), despite skew determining worker-brood relatedness and so worker fitness. We provide evidence for worker influence over skew in a functionally monogynous population of the ant Leptothorax acervorum. Observations of MQ colonies leading up to egg laying showed worker aggressive and non-aggressive behaviour towards queens and predicted which queen monopolized reproduction. In contrast, among-queen interactions were rare and did not predict queen reproduction. Furthermore, parentage analysis showed workers favoured their mother when present, ensuring closely related fullsibs (average r = 0.5) were reared instead of less related offspring of other resident queens (r ≤ 0.375). Discrimination among queens using relatedness-based cues, however, seems unlikely as workers also biased their behaviour in colonies without a mother queen. In other polygynous populations of this species, workers are not aggressive towards queens and MQs reproduce, showing the outcome of social conflicts varies within species. In conclusion, this study supports non-reproductive parties having the power and information to influence skew within cooperative breeding groups.

No evidence of volatile chemicals regulating reproduction in a multiple queen ant.

Efficient cooperation in eusocial insect colonies requires effective communication, and there is abundant evidence of non-volatile chemicals playing a role in regulating reproduction within colonies. In contrast, there have been fewer studies investigating the role of volatile chemicals. This study investigated the potential role of volatile chemicals in regulating queen reproduction either by directly inhibiting queen reproduction or by honestly signalling queen fecundity to workers. We tested this using multiple queen colonies of the ant (Leptothorax acervorum) from a functionally monogynous population where one queen monopolizes all reproduction. Nine colonies, each with an established laying queen, were split to produce two colony fragments-one containing the reproducing queen (group 1) and one containing only previously non-reproducing queens (group 2). Each group was separated by a fine wire mesh preventing physical contact, but allowing volatile chemical contact. In each group 2 fragment, we found that a single formerly non-reproductive queen commenced reproduction and that the rate of egg laying and maximum number of eggs recorded did not significantly differ between groups 1 and 2, results that do not support volatile chemicals as playing a role in regulating queen reproduction. Instead, our findings suggest that physical contact is necessary to maintain functional monogyny.

Pollen Source Richness May Be a Poor Predictor of Bumblebee (Bombus terrestris) Colony Growth.

Agricultural intensification has drastically altered foraging landscapes for bees, with large-scale crop monocultures associated with floral diversity loss. Research on bumblebees and honeybees has shown individuals feeding on pollen from a low richness of floral sources can experience negative impacts on health and longevity relative to higher pollen source richness of similar protein concentrations. Florally rich landscapes are thus generally assumed to better support social bees. Yet, little is known about whether the effects of reduced pollen source richness can be mitigated by feeding on pollen with higher crude protein concentration, and importantly how variation in diet affects whole colony growth, rearing decisions and sexual production. Studying queen-right bumblebee (Bombus terrestris) colonies, we monitored colony development under a polyfloral pollen diet or a monofloral pollen diet with 1.5-1.8 times higher crude protein concentration. Over 6 weeks, we found monofloral colonies performed better for all measures, with no apparent long-term effects on colony mass or worker production, and a higher number of pupae in monofloral colonies at the end of the experiment. Unexpectedly, polyfloral colonies showed higher mortality, and little evidence of any strategy to counteract the effects of reduced protein; with fewer and lower mass workers being reared, and males showing a similar trend. Our findings (i) provide well-needed daily growth dynamics of queenright colonies under varied diets, and (ii) support the view that pollen protein content in the foraging landscape rather than floral species richness per se is likely a key driver of colony health and success.

Urbanisation is associated with reduced Nosema sp. infection, higher colony strength and higher richness of foraged pollen in honeybees.

Bees are vital pollinators, but are faced with numerous threats that include loss of floral resources and emerging parasites amongst others. Urbanisation is a rapidly expanding driver of land-use change that may interact with these two major threats to bees. Here we investigated effects of urbanisation on food store quality and colony health in honeybees (Apis mellifera) by sampling 51 hives in four different land-use categories: urban, suburban, rural open and rural wooded during two seasons (spring and autumn). We found positive effects of urban land use on colony strength and richness of stored pollen morphotypes, alongside lower late-season Nosema sp. infection in urban and suburban colonies. Our results reveal that honeybees exhibit lower colony performance in strength in rural areas, adding to the growing evidence that modern agricultural landscapes can constitute poor habitat for insect pollinators.