Harmonic radar tracking reveals that honeybee drones navigate between multiple aerial leks.

Male honeybees (drones) are thought to congregate in large numbers in particular "drone congregation areas" to mate. We used harmonic radar to record the flight paths of individual drones and found that drones favored certain locations within the landscape which were stable over two years. Drones often visit multiple potential lekking sites within a single flight and take shared flight paths between them. Flights between such sites are relatively straight and begin as early as the drone's second flight, indicating familiarity with the sites acquired during initial learning flights. Arriving at congregation areas, drones display convoluted, looping flight patterns. We found a correlation between a drone's distance from the center of each area and its acceleration toward the center, a signature of collective behavior leading to congregation in these areas. Our study reveals the behavior of individual drones as they navigate between and within multiple aerial leks.

Evidence for facultative migratory flight behavior in Helicoverpa armigera (Noctuidae: Lepidoptera) in India.

Despite its deleterious impact on farming and agriculture, the physiology and energetics of insect migration is poorly understood due to our inability to track their individual movements in the field. Many insects, e.g. monarch butterflies, Danaus plexippus (L.), are facultative migrants. Hence, it is important to establish whether specific insect populations in particular areas migrate. The polyphagous insect, Helicoverpa armigera (Hübner), is especially interesting in this regard due to its impact on a variety of crops. Here, we used a laboratory-based flight mill assay to show that Helicoverpa armigera populations clearly demonstrate facultative migration in South India. Based on various flight parameters, we categorized male and female moths as long, medium or short distance fliers. A significant proportion of moths exhibited long-distance flight behavior covering more than 10 km in a single night, averaging about 8 flight hours constituting 61% flight time in the test period. The maximum and average flight speeds of these long fliers were greater than in the other categories. Flight activity across sexes also varied; male moths exhibited better performance than female moths. Wing morphometric parameters including forewing length, wing loading, and wing aspect ratio were key in influencing long-distance flight. Whereas forewing length positively correlated with flight distance and duration, wing loading was negatively correlated.

Odorant binding proteins promote flight activity in the migratory insect, Helicoverpa armigera.

Migratory insects are capable of actively sustaining powered flight for several hours. This extraordinary phenomenon requires a highly efficient transport system to cope with the energetic demands placed on the flight muscles. Here, we provide evidence that the role of the hydrophobic ligand binding of odorant binding proteins (OBPs) extends beyond their typical function in the olfactory system to support insect flight activity via lipid interactions. Transcriptomic and candidate gene analyses show that two phylogenetically clustered OBPs (OBP3/OBP6) are consistently over-expressed in adult moths of the migrant Old-World bollworm, Helicoverpa armigera, displaying sustained flight performance in flight activity bioassays. Tissue-specific over-expression of OBP6 was observed in the antennae, wings and thorax in long-fliers of H. armigera. Transgenic Drosophila flies over-expressing an H. armigera transcript of OBP6 (HarmOBP6) in the flight muscle attained higher flight speeds on a modified tethered flight system. Quantification of lipid molecules using mass spectrometry showed a depletion of triacylglyerol and phospholipids in flown moths. Protein homology models built from the crystal structure of a fatty acid carrier protein identified the binding site of OBP3 and OBP6 for hydrophobic ligand binding with both proteins exhibiting a stronger average binding affinity with triacylglycerols and phospholipids compared with other groups of ligands. We propose that HarmOBP3 and HarmOBP6 contribute to the flight capacity of a globally invasive and highly migratory noctuid moth, and in doing so, extend the function of this group of proteins beyond their typical role as chemosensory proteins in insects.

Do bumblebees have signatures? Demonstrating the existence of a speed-curvature power law in Bombus terrestris locomotion patterns.

We report the discovery that Bombus terrestris audax (Buff-tailed bumblebee) locomotor trajectories adhere to a speed-curvature power law relationship which has previously been found in humans, non-human primates and Drosophila larval trajectories. No previous study has reported such a finding in adult insect locomotion. We used behavioural tracking to study walking Bombus terrestris in an arena under different training environments. Trajectories analysed from this tracking show the speed-curvature power law holds robustly at the population level, displaying an exponent close to two-thirds. This exponent corroborates previous findings in human movement patterns, but differs from the three-quarter exponent reported for Drosophila larval locomotion. There are conflicting hypotheses for the principal origin of these speed-curvature laws, ranging from the role of central planning to kinematic and muscular skeletal constraints. Our findings substantiate the latter idea that dynamic power-law effects are robust, differing only through kinematic constraints due to locomotive method. Our research supports the notion that these laws are present in a greater range of species than previously thought, even in the bumblebee. Such power laws may provide optimal behavioural templates for organisms, delivering a potential analytical tool to study deviations from this template. Our results suggest that curvature and angular speed are constrained geometrically, and independently of the muscles and nerves of the performing body.

Mass Seasonal Migrations of Hoverflies Provide Extensive Pollination and Crop Protection Services.

Despite the fact that migratory insects dominate aerial bioflows in terms of diversity, abundance, and biomass [1-6], the migration patterns of most species, and the effects of their annual fluxes between high- and low-latitude regions, are poorly known. One important group of long-range migrants that remain understudied is a suite of highly beneficial species of hoverfly in the tribe Syrphini, which we collectively term "migrant hoverflies." Adults are key pollinators [7-10] and larvae are significant biocontrol agents of aphid crop pests [11], and thus, it is important to quantify the scale of their migrations and the crucial ecosystem services they provide with respect to energy, nutrient, and biomass transport; regulation of crop pests; and pollen transfer. Such assessments cannot be made by sporadic observations of mass arrivals at ground level, because hoverflies largely migrate unnoticed high above ground. We used insect-monitoring radars [12] to show that up to 4 billion hoverflies (80 tons of biomass) travel high above southern Britain each year in seasonally adaptive directions. The long-range migrations redistribute tons of essential nutrients (nitrogen [N] and phosphorus [P]) and transport billions of pollen grains between Britain and Europe, and locally produced populations consume 6 trillion aphids and make billions of flower visits. Migrant hoverfly abundance fluctuated greatly between years, but there was no evidence of a population trend during the 10-year study period. Considering that many beneficial insects are seriously declining [7, 10, 13-19], our results demonstrate that migrant hoverflies are key to maintaining essential ecosystem services.

The tethered flight technique as a tool for studying life-history strategies associated with migration in insects.

1. Every year billions of insects engage in long-distance, seasonal mass migrations which have major consequences for agriculture, ecosystem services and insect-vectored diseases. Tracking this movement in the field is difficult, with mass migrations often occurring at high altitudes and over large spatial scales. 2. As such, tethered flight provides a valuable tool for studying the flight behaviour of insects, giving insights into flight propensity (e.g. distance, duration and velocity) and orientation under controlled laboratory settings. By experimentally manipulating a variety of environmental and physiological traits, numerous studies have used this technology to study the flight behaviour of migratory insects ranging in size from aphids to butterflies. Advances in functional genomics promise to extend this to the identification of genetic factors associated with flight. Tethered flight techniques have been used to study migratory flight characteristics in insects for more than 50 years, but have never been reviewed. 3. This study summarises the key findings of this technology, which has been employed in studies of species from six Orders. By providing detailed descriptions of the tethered flight systems, the present study also aims to further the understanding of how tethered flight studies support field observations, the situations under which the technology is useful and how it might be used in future studies. 4. The aim is to contextualise the available tethered flight studies within the broader knowledge of insect migration and to describe the significant contribution these systems have made to the literature.

Higher flight activity in the offspring of migrants compared to residents in a migratory insect.

Migration has evolved among many animal taxa and migratory species are found across all major lineages. Insects are the most abundant and diverse terrestrial migrants, with trillions of animals migrating annually. Partial migration, where populations consist of resident and migratory individuals, is ubiquitous among many taxa. However, the underlying mechanisms are relatively poorly understood and may be driven by physiological, behavioural or genetic variation within populations. We investigated the differences in migratory tendency between migratory and resident phenotypes of the hoverfly, Episyrphus balteatus, using tethered flight mills. Further, to test whether migratory flight behaviour is heritable and to disentangle the effects of environment during development, we compared the flight behaviour of laboratory-reared offspring of migrating, overwintering and summer animals. Offspring of migrants initiated more flights than those of resident individuals. Interestingly, there were no differences among wild-caught phenotypes with regard to number of flights or total flight duration. Low activity in field-collected migrants might be explained by an energy-conserving state that migrants enter into when under laboratory conditions, or a lack of suitable environmental cues for triggering migration. Our results strongly suggest that flight behaviour is heritable and that genetic factors influence migratory tendency in E. balteatus These findings support the growing evidence that genetic factors play a role in partial migration and warrant careful further investigation.

Genome-Wide Characterization of DNA Methylation in an Invasive Lepidopteran Pest, the Cotton Bollworm Helicoverpa armigera.

The genes and genomes of insect pests are shaped by the wide array of selective forces encountered in their environments. While the molecular adaptations that evolve are beginning to be understood at the genomic and transcriptomic level, they have been less well characterized at an epigenetic level. Here, we present a genome-wide map of DNA methylation at single-nucleotide resolution for the cotton bollworm moth, Helicoverpa armigera, a globally invasive pest of agriculture. We show that methylation is almost identical in the larvae and adults of H. armigera and that, through whole-genome bisulfite sequencing (WGBS), at the most ∼0.9% of CpG sites in this species are methylated. We find that DNA methylation occurs primarily in exons, is positively correlated with gene expression, and that methylated genes are enriched for cellular "housekeeping" roles. H. armigera has an exceptional capacity for long-range migration. To explore the role of methylation in influencing the migratory phenotype of H. armigera, we performed targeted bisulfite sequencing on selected loci from 16 genes that were differentially expressed between adult moths exhibiting distinct flight performance in behavioral assays. While most CpG sites in these genes were not methylated between flight phenotypes, we identified hypermethylation in a demethylase (KDM4) that targets lysine-specific histone modifications, which are strongly associated with transcription and methylation. The H. armigera methylome provides new insights into the role of DNA methylation in a noctuid moth and is a valuable resource for further research into the epigenetic control of adaptive traits in this important pest.

Continuous Radar Tracking Illustrates the Development of Multi-destination Routes of Bumblebees.

Animals that visit multiple foraging sites face a problem, analogous to the Travelling Salesman Problem, of finding an efficient route. We explored bumblebees' route development on an array of five artificial flowers in which minimising travel distances between individual feeders conflicted with minimising overall distance. No previous study of bee spatial navigation has been able to follow animals' movement during learning; we tracked bumblebee foragers continuously, using harmonic radar, and examined the process of route formation in detail for a small number of selected individuals. On our array, bees did not settle on visit sequences that gave the shortest overall path, but prioritised movements to nearby feeders. Nonetheless, flight distance and duration reduced with experience. This increased efficiency was attributable mainly to experienced bees reducing exploration beyond the feeder array and flights becoming straighter with experience, rather than improvements in the sequence of feeder visits. Flight paths of all legs of a flight stabilised at similar rates, whereas the first few feeder visits became fixed early while bees continued to experiment with the order of later visits. Stabilising early sections of a route and prioritising travel between nearby destinations may reduce the search space, allowing rapid adoption of efficient routes.

Mass seasonal bioflows of high-flying insect migrants.

Migrating animals have an impact on ecosystems directly via influxes of predators, prey, and competitors and indirectly by vectoring nutrients, energy, and pathogens. Although linkages between vertebrate movements and ecosystem processes have been established, the effects of mass insect "bioflows" have not been described. We quantified biomass flux over the southern United Kingdom for high-flying (>150 meters) insects and show that ~3.5 trillion insects (3200 tons of biomass) migrate above the region annually. These flows are not randomly directed in insects larger than 10 milligrams, which exploit seasonally beneficial tailwinds. Large seasonal differences in the southward versus northward transfer of biomass occur in some years, although flows were balanced over the 10-year period. Our long-term study reveals a major transport process with implications for ecosystem services, processes, and biogeochemistry.

Optimal search patterns in honeybee orientation flights are robust against emerging infectious diseases.

Lévy flights are scale-free (fractal) search patterns found in a wide range of animals. They can be an advantageous strategy promoting high encounter rates with rare cues that may indicate prey items, mating partners or navigational landmarks. The robustness of this behavioural strategy to ubiquitous threats to animal performance, such as pathogens, remains poorly understood. Using honeybees radar-tracked during their orientation flights in a novel landscape, we assess for the first time how two emerging infectious diseases (Nosema sp. and the Varroa-associated Deformed wing virus (DWV)) affect bees' behavioural performance and search strategy. Nosema infection, unlike DWV, affected the spatial scale of orientation flights, causing significantly shorter and more compact flights. However, in stark contrast to disease-dependent temporal fractals, we find the same prevalence of optimal Lévy flight characteristics (µ ≈ 2) in both healthy and infected bees. We discuss the ecological and evolutionary implications of these surprising insights, arguing that Lévy search patterns are an emergent property of fundamental characteristics of neuronal and sensory components of the decision-making process, making them robust against diverse physiological effects of pathogen infection and possibly other stressors.

Life-Long Radar Tracking of Bumblebees.

Insect pollinators such as bumblebees play a vital role in many ecosystems, so it is important to understand their foraging movements on a landscape scale. We used harmonic radar to record the natural foraging behaviour of Bombus terrestris audax workers over their entire foraging career. Every flight ever made outside the nest by four foragers was recorded. Our data reveal where the bees flew and how their behaviour changed with experience, at an unprecedented level of detail. We identified how each bee's flights fit into two categories-which we named exploration and exploitation flights-examining the differences between the two types of flight and how their occurrence changed over the course of the bees' foraging careers. Exploitation of learned resources takes place during efficient, straight trips, usually to a single foraging location, and is seldom combined with exploration of other areas. Exploration of the landscape typically occurs in the first few flights made by each bee, but our data show that further exploration flights can be made throughout the bee's foraging career. Bees showed striking levels of variation in how they explored their environment, their fidelity to particular patches, ratio of exploration to exploitation, duration and frequency of their foraging bouts. One bee developed a straight route to a forage patch within four flights and followed this route exclusively for six days before abandoning it entirely for a closer location; this second location had not been visited since her first exploratory flight nine days prior. Another bee made only rare exploitation flights and continued to explore widely throughout its life; two other bees showed more frequent switches between exploration and exploitation. Our data shed light on the way bumblebees balance exploration of the environment with exploitation of resources and reveal extreme levels of variation between individuals.

Flight performance of actively foraging honey bees is reduced by a common pathogen.

Sudden and severe declines in honey bee (Apis mellifera) colony health in the US and Europe have been attributed, in part, to emergent microbial pathogens, however, the mechanisms behind the impact are unclear. Using roundabout flight mills, we measured the flight distance and duration of actively foraging, healthy-looking honey bees sampled from standard colonies, before quantifying the level of infection by Nosema ceranae and Deformed Wing Virus complex (DWV) for each bee. Neither the presence nor the quantity of N. ceranae were at low, natural levels of infection had any effect on flight distance or duration, but presence of DWV reduced flight distance by two thirds and duration by one half. Quantity of DWV was shown to have a significant, but weakly positive relation with flight distance and duration, however, the low amount of variation that was accounted for suggests further investigation by dose-response assays is required. We conclude that widespread, naturally occurring levels of infection by DWV weaken the flight ability of honey bees and high levels of within-colony prevalence are likely to reduce efficiency and increase the cost of resource acquisition. Predictions of implications of pathogens on colony health and function should take account of sublethal effects on flight performance.

Quantifying interspecific variation in dispersal ability of noctuid moths using an advanced tethered flight technique.

Dispersal plays a crucial role in many aspects of species' life histories, yet is often difficult to measure directly. This is particularly true for many insects, especially nocturnal species (e.g. moths) that cannot be easily observed under natural field conditions. Consequently, over the past five decades, laboratory tethered flight techniques have been developed as a means of measuring insect flight duration and speed. However, these previous designs have tended to focus on single species (typically migrant pests), and here we describe an improved apparatus that allows the study of flight ability in a wide range of insect body sizes and types. Obtaining dispersal information from a range of species is crucial for understanding insect population dynamics and range shifts. Our new laboratory tethered flight apparatus automatically records flight duration, speed, and distance of individual insects. The rotational tethered flight mill has very low friction and the arm to which flying insects are attached is extremely lightweight while remaining rigid and strong, permitting both small and large insects to be studied. The apparatus is compact and thus allows many individuals to be studied simultaneously under controlled laboratory conditions. We demonstrate the performance of the apparatus by using the mills to assess the flight capability of 24 species of British noctuid moths, ranging in size from 12-27 mm forewing length (~40-660 mg body mass). We validate the new technique by comparing our tethered flight data with existing information on dispersal ability of noctuids from the published literature and expert opinion. Values for tethered flight variables were in agreement with existing knowledge of dispersal ability in these species, supporting the use of this method to quantify dispersal in insects. Importantly, this new technology opens up the potential to investigate genetic and environmental factors affecting insect dispersal among a wide range of species.

Adaptive strategies in nocturnally migrating insects and songbirds: contrasting responses to wind.

Animals that use flight as their mode of transportation must cope with the fact that their migration and orientation performance is strongly affected by the flow of the medium they are moving in, that is by the winds. Different strategies can be used to mitigate the negative effects and benefit from the positive effects of a moving flow. The strategies an animal can use will be constrained by the relationship between the speed of the flow and the speed of the animal's own propulsion in relation to the surrounding air. Here we analyse entomological and ornithological radar data from north-western Europe to investigate how two different nocturnal migrant taxa, the noctuid moth Autographa gamma and songbirds, deal with wind by analysing variation in resulting flight directions in relation to the wind-dependent angle between the animal's heading and track direction. Our results, from fixed locations along the migratory journey, reveal different global strategies used by moths and songbirds during their migratory journeys. As expected, nocturnally migrating moths experienced a greater degree of wind drift than nocturnally migrating songbirds, but both groups were more affected by wind in autumn than in spring. The songbirds' strategies involve elements of both drift and compensation, providing some benefits from wind in combination with destination and time control. In contrast, moths expose themselves to a significantly higher degree of drift in order to obtain strong wind assistance, surpassing the songbirds in mean ground speed, at the cost of a comparatively lower spatiotemporal migratory precision. Moths and songbirds show contrasting but adaptive responses to migrating through a moving flow, which are fine-tuned to the respective flight capabilities of each group in relation to the wind currents they travel within.

Genomewide transcriptional signatures of migratory flight activity in a globally invasive insect pest.

Migration is a key life history strategy for many animals and requires a suite of behavioural, morphological and physiological adaptations which together form the 'migratory syndrome'. Genetic variation has been demonstrated for many traits that make up this syndrome, but the underlying genes involved remain elusive. Recent studies investigating migration-associated genes have focussed on sampling migratory and nonmigratory populations from different geographic locations but have seldom explored phenotypic variation in a migratory trait. Here, we use a novel combination of tethered flight and next-generation sequencing to determine transcriptomic differences associated with flight activity in a globally invasive moth pest, the cotton bollworm Helicoverpa armigera. By developing a state-of-the-art phenotyping platform, we show that field-collected H. armigera display continuous variation in flight performance with individuals capable of flying up to 40 km during a single night. Comparative transcriptomics of flight phenotypes drove a gene expression analysis to reveal a suite of expressed candidate genes which are clearly related to physiological adaptations required for long-distance flight. These include genes important to the mobilization of lipids as flight fuel, the development of flight muscle structure and the regulation of hormones that influence migratory physiology. We conclude that the ability to express this complex set of pathways underlines the remarkable flexibility of facultative insect migrants to respond to deteriorating conditions in the form of migratory flight and, more broadly, the results provide novel insights into the fundamental transcriptional changes required for migration in insects and other taxa.

Detection of flow direction in high-flying insect and songbird migrants.

Goal-oriented migrants travelling through the sea or air must cope with the effect of cross-flows during their journeys if they are to reach their destination. In order to counteract flow-induced drift from their preferred course, migrants must detect the mean flow direction, and integrate this information with output from their internal compass, to compensate for the deflection. Animals can potentially sense flow direction by two nonexclusive mechanisms: either indirectly, by visually assessing the effect of the current on their movement direction relative to the ground; or directly, via intrinsic properties of the current. Here, we report the first evidence that nocturnal compass-guided insect migrants use a turbulence-mediated mechanism for directly assessing the wind direction hundreds of metres above the ground. By comparison, we find that nocturnally-migrating songbirds do not use turbulence to detect the flow; instead they rely on visual assessment of wind-induced drift to indirectly infer the flow direction.

Evidence for a pervasive 'idling-mode' activity template in flying and pedestrian insects.

Understanding the complex movement patterns of animals in natural environments is a key objective of 'movement ecology'. Complexity results from behavioural responses to external stimuli but can also arise spontaneously in their absence. Drawing on theoretical arguments about decision-making circuitry, we predict that the spontaneous patterns will be scale-free and universal, being independent of taxon and mode of locomotion. To test this hypothesis, we examined the activity patterns of the European honeybee, and multiple species of noctuid moth, tethered to flight mills and exposed to minimal external cues. We also reanalysed pre-existing data for Drosophila flies walking in featureless environments. Across these species, we found evidence of common scale-invariant properties in their movement patterns; pause and movement durations were typically power law distributed over a range of scales and characterized by exponents close to 3/2. Our analyses are suggestive of the presence of a pervasive scale-invariant template for locomotion which, when acted on by environmental cues, produces the movements with characteristic scales observed in nature. Our results indicate that scale-finite complexity as embodied, for instance, in correlated random walk models, may be the result of environmental cues overriding innate behaviour, and that scale-free movements may be intrinsic and not limited to 'blind' foragers as previously thought.

So near and yet so far: harmonic radar reveals reduced homing ability of Nosema infected honeybees.

Pathogens may gain a fitness advantage through manipulation of the behaviour of their hosts. Likewise, host behavioural changes can be a defence mechanism, counteracting the impact of pathogens on host fitness. We apply harmonic radar technology to characterize the impact of an emerging pathogen--Nosema ceranae (Microsporidia)--on honeybee (Apis mellifera) flight and orientation performance in the field. Honeybees are the most important commercial pollinators. Emerging diseases have been proposed to play a prominent role in colony decline, partly through sub-lethal behavioural manipulation of their hosts. We found that homing success was significantly reduced in diseased (65.8%) versus healthy foragers (92.5%). Although lost bees had significantly reduced continuous flight times and prolonged resting times, other flight characteristics and navigational abilities showed no significant difference between infected and non-infected bees. Our results suggest that infected bees express normal flight characteristics but are constrained in their homing ability, potentially compromising the colony by reducing its resource inputs, but also counteracting the intra-colony spread of infection. We provide the first high-resolution analysis of sub-lethal effects of an emerging disease on insect flight behaviour. The potential causes and the implications for both host and parasite are discussed.

The ontogeny of bumblebee flight trajectories: from naïve explorers to experienced foragers.

Understanding strategies used by animals to explore their landscape is essential to predict how they exploit patchy resources, and consequently how they are likely to respond to changes in resource distribution. Social bees provide a good model for this and, whilst there are published descriptions of their behaviour on initial learning flights close to the colony, it is still unclear how bees find floral resources over hundreds of metres and how these flights become directed foraging trips. We investigated the spatial ecology of exploration by radar tracking bumblebees, and comparing the flight trajectories of bees with differing experience. The bees left the colony within a day or two of eclosion and flew in complex loops of ever-increasing size around the colony, exhibiting Lévy-flight characteristics constituting an optimal searching strategy. This mathematical pattern can be used to predict how animals exploring individually might exploit a patchy landscape. The bees' groundspeed, maximum displacement from the nest and total distance travelled on a trip increased significantly with experience. More experienced bees flew direct paths, predominantly flying upwind on their outward trips although forage was available in all directions. The flights differed from those of naïve honeybees: they occurred at an earlier age, showed more complex looping, and resulted in earlier returns of pollen to the colony. In summary bumblebees learn to find home and food rapidly, though phases of orientation, learning and searching were not easily separable, suggesting some multi-tasking.