Acute and chronic toxicity of imidacloprid in the pollinator fly, Eristalis tenax L., assessed using a novel oral bioassay.

Imidacloprid is a neonicotinoid neurotoxin that remains widely used worldwide and persists in the environment, resulting in chronic exposure to non-target insects. To accurately map dose-dependent effects of such exposure across taxa, toxicological assays need to assess relevant modes of exposure across indicator species. However, due to the difficulty of these experiments, contact bioassays are most frequently used to quantify dose. Here, we developed a novel naturalistic feeding bioassay to precisely measure imidacloprid ingestion and its toxicity for acute and chronic exposure in a dipteran, Eristalis tenax L., an important member of an under-represented pollinator group. Flies which ingested imidacloprid dosages lower than 12.1 ng/mg all showed consistent intake volumes and learned improved feeding efficiency over successive feeding sessions. In contrast, at doses of 12.1 ng/mg and higher flies showed a rapid onset of severe locomotive impairment which prevented them from completing the feeding task. Neither probability of survival nor severe locomotive impairment were significantly higher than the control group until doses of 1.43 ng/mg or higher were reached. We were unable to measure a median lethal dose for acute exposure (72 h) due to flies possessing a relatively high tolerance for imidacloprid. However, with chronic exposure (18 days), mortality went up and an LD50 of 0.41 ng/mg was estimated. Severe locomotive impairment (immobilisation) tended to occur earlier and at lower dosages than lethality, with ED50s of 7.82 ng/mg and 0.17 ng/mg for acute and chronic exposure, respectively. We conclude that adult Eristalis possess a much higher tolerance to this toxin than the honeybees that they mimic. The similarity of the LD50 to other dipterans such as the fruitfly and the housefly suggests that there may be a phylogenetic component to pesticide tolerance that merits further investigation. The absence of obvious adverse effects at sublethal dosages also underscores a need to develop better tools for quantifying animal behaviour to evaluate the impact of insecticides on foraging efficiency in economically important species.

Preattentive facilitation of target trajectories in a dragonfly visual neuron.

The ability to pursue targets in visually cluttered and distraction-rich environments is critical for predators such as dragonflies. Previously, we identified Centrifugal Small-Target Motion Detector 1 (CSTMD1), a dragonfly visual neuron likely involved in such target-tracking behaviour. CSTMD1 exhibits facilitated responses to targets moving along a continuous trajectory. Moreover, CSTMD1 competitively selects a single target out of a pair. Here, we conducted in vivo, intracellular recordings from CSTMD1 to examine the interplay between facilitation and selection, in response to the presentation of paired targets. We find that neuronal responses to both individual trajectories of simultaneous, paired targets are facilitated, rather than being constrained to the single, selected target. Additionally, switches in selection elicit suppression which is likely an important attribute underlying target pursuit. However, binocular experiments reveal these results are constrained to paired targets within the same visual hemifield, while selection of a target in one visual hemifield establishes ocular dominance that prevents facilitation or response to contralaterally presented targets. These results reveal that the dragonfly brain preattentively represents more than one target trajectory, to balance between attentional flexibility and resistance against distraction.

A new, fluorescence-based method for visualizing the pseudopupil and assessing optical acuity in the dark compound eyes of honeybees and other insects.

Recent interest in applying novel imaging techniques to infer optical resolution in compound eyes underscores the difficulty of obtaining direct measures of acuity. A widely used technique exploits the principal pseudopupil, a dark spot on the eye surface representing the ommatidial gaze direction and the number of detector units (ommatidia) viewing that gaze direction. However, dark-pigmented eyes, like those of honeybees, lack a visible pseudopupil. Attempts over almost a century to estimate optical acuity in this species are still debated. Here, we developed a method to visualize a stable, reliable pseudopupil by staining the photoreceptors with fluorescent dyes. We validated this method in several species and found it to outperform the dark pseudopupil for this purpose, even in pale eyes, allowing more precise location of the gaze centre. We then applied this method to estimate the sampling resolution in the frontal part of the eye of the honeybee forager. We found a broad frontal acute zone with interommatidial angles below 2° and a minimum interommatidial angle of 1.3°, a broader, sharper frontal acute zone than previously reported. Our study provides a new method to directly measure the sampling resolution in most compound eyes of living animals.

Modeling Nonlinear Dendritic Processing of Facilitation in a Dragonfly Target-Tracking Neuron.

Dragonflies are highly skilled and successful aerial predators that are even capable of selectively attending to one target within a swarm. Detection and tracking of prey is likely to be driven by small target motion detector (STMD) neurons identified from several insect groups. Prior work has shown that dragonfly STMD responses are facilitated by targets moving on a continuous path, enhancing the response gain at the present and predicted future location of targets. In this study, we combined detailed morphological data with computational modeling to test whether a combination of dendritic morphology and nonlinear properties of NMDA receptors could explain these observations. We developed a hybrid computational model of neurons within the dragonfly optic lobe, which integrates numerical and morphological components. The model was able to generate potent facilitation for targets moving on continuous trajectories, including a localized spotlight of maximal sensitivity close to the last seen target location, as also measured during in vivo recordings. The model did not, however, include a mechanism capable of producing a traveling or spreading wave of facilitation. Our data support a strong role for the high dendritic density seen in the dragonfly neuron in enhancing non-linear facilitation. An alternative model based on the morphology of an unrelated type of motion processing neuron from a dipteran fly required more than three times higher synaptic gain in order to elicit similar levels of facilitation, despite having only 20% fewer synapses. Our data support a potential role for NMDA receptors in target tracking and also demonstrate the feasibility of combining biologically plausible dendritic computations with more abstract computational models for basic processing as used in earlier studies.

Acute Application of Imidacloprid Alters the Sensitivity of Direction Selective Motion Detecting Neurons in an Insect Pollinator.

Cholinergic pesticides, such as the neonicotinoid imidacloprid, are the most important insecticides used for plant protection worldwide. In recent decades, concerns have been raised about side effects on non-target insect species, including altered foraging behavior and navigation. Although pollinators rely on visual cues to forage and navigate their environment, the effects of neonicotinoids on visual processing have been largely overlooked. To test the effect of acute treatment with imidacloprid at known concentrations in the brain, we developed a modified electrophysiological setup that allows recordings of visually evoked responses while perfusing the brain in vivo. We obtained long-lasting recordings from direction selective wide-field, motion sensitive neurons of the hoverfly pollinator, Eristalis tenax. Neurons were treated with imidacloprid (3.9 µM, 0.39 µM or a sham control treatment using the solvent (dimethylsulfoxide) only. Exposure to a high, yet sub-lethal concentration of imidacloprid significantly alters their physiological response to motion stimuli. We observed a general effect of imidacloprid (3.9 µM) increasing spontaneous activity, reducing contrast sensitivity and giving weaker directional tuning to wide-field moving stimuli, with likely implications for errors in flight control, hovering and routing. Our electrophysiological approach reveals the robustness of the fly visual pathway against cholinergic perturbance (i.e., at 0.39 µM) but also potential threatening effects of cholinergic pesticides (i.e., evident at 3.9 µM) for the visual motion detecting system of an important pollinator.

Comparison of Transparency and Shrinkage During Clearing of Insect Brains Using Media With Tunable Refractive Index.

Improvement of imaging quality has the potential to visualize previously unseen building blocks of the brain and is therefore one of the great challenges in neuroscience. Rapid development of new tissue clearing techniques in recent years have attempted to solve imaging compromises in thick brain samples, particularly for high resolution optical microscopy, where the clearing medium needs to match the high refractive index of the objective immersion medium. These problems are exacerbated in insect tissue, where numerous (initially air-filled) tracheal tubes branching throughout the brain increase the scattering of light. To date, surprisingly few studies have systematically quantified the benefits of such clearing methods using objective transparency and tissue shrinkage measurements. In this study we compare a traditional and widely used insect clearing medium, methyl salicylate combined with permanent mounting in Permount ("MS/P") with several more recently applied clearing media that offer tunable refractive index (n): 2,2'-thiodiethanol (TDE), "SeeDB2" (in variants SeeDB2S and SeeDB2G matched to oil and glycerol immersion, n = 1.52 and 1.47, respectively) and Rapiclear (also with n = 1.52 and 1.47). We measured transparency and tissue shrinkage by comparing freshly dissected brains with cleared brains from dipteran flies, with or without addition of vacuum or ethanol pre-treatments (dehydration and rehydration) to evacuate air from the tracheal system. The results show that ethanol pre-treatment is very effective for improving transparency, regardless of the subsequent clearing medium, while vacuum treatment offers little measurable benefit. Ethanol pre-treated SeeDB2G and Rapiclear brains show much less shrinkage than using the traditional MS/P method. Furthermore, at lower refractive index, closer to that of glycerol immersion, these recently developed media offer outstanding transparency compared to TDE and MS/P. Rapiclear protocols were less laborious compared to SeeDB2, but both offer sufficient transparency and refractive index tunability to permit super-resolution imaging of local volumes in whole mount brains from large insects, and even light-sheet microscopy. Although long-term permanency of Rapiclear stored samples remains to be established, our samples still showed good preservation of fluorescence after storage for more than a year at room temperature.

A Target-Detecting Visual Neuron in the Dragonfly Locks on to Selectively Attended Targets.

The visual world projects a complex and rapidly changing image onto the retina of many animal species. This presents computational challenges for those animals reliant on visual processing to provide an accurate representation of the world. One such challenge is parsing a visual scene for the most salient targets, such as the selection of prey amid a swarm. The ability to selectively prioritize processing of some stimuli over others is known as 'selective attention'. We recently identified a dragonfly visual neuron called 'Centrifugal Small Target Motion Detector 1' (CSTMD1) that exhibits selective attention when presented with multiple, equally salient targets. Here we conducted in vivo, electrophysiological recordings from CSTMD1 in wild-caught male dragonflies (Hemicordulia tau), while presenting visual stimuli on an LCD monitor. To identify the target selected in any given trial, we uniquely modulated the intensity of the moving targets (frequency tagging). We found that the frequency information of the selected target is preserved in the neuronal response, while the distracter is completely ignored. We also show that the competitive system that underlies selection in this neuron can be biased by the presentation of a preceding target on the same trajectory, even when it is of lower contrast than an abrupt, novel distracter. With this improved method for identifying and biasing target selection in CSTMD1, the dragonfly provides an ideal animal model system to probe the neuronal mechanisms underlying selective attention.SIGNIFICANCE STATEMENT We present the first application of frequency tagging to intracellular neuronal recordings, demonstrating that the frequency component of a stimulus is encoded in the spiking response of an individual neuron. Using this technique as an identifier, we demonstrate that CSTMD1 'locks on' to a selected target and encodes the absolute strength of this target, even in the presence of abruptly appearing, high-contrast distracters. The underlying mechanism also permits the selection mechanism to switch between targets mid-trial, even among equivalent targets. Together, these results demonstrate greater complexity in this selective attention system than would be expected in a winner-takes-all network. These results are in contrast to typical findings in the primate and avian brain, but display intriguing resemblance to observations in human psychophysics.

Differential Tuning to Visual Motion Allows Robust Encoding of Optic Flow in the Dragonfly.

Visual cues provide an important means for aerial creatures to ascertain their self-motion through the environment. In many insects, including flies, moths, and bees, wide-field motion-sensitive neurons in the third optic ganglion are thought to underlie such motion encoding; however, these neurons can only respond robustly over limited speed ranges. The task is more complicated for some species of dragonflies that switch between extended periods of hovering flight and fast-moving pursuit of prey and conspecifics, requiring motion detection over a broad range of velocities. Since little is known about motion processing in these insects, we performed intracellular recordings from hawking, emerald dragonflies (Hemicordulia spp.) and identified a diverse group of motion-sensitive neurons that we named lobula tangential cells (LTCs). Following prolonged visual stimulation with drifting gratings, we observed significant differences in both temporal and spatial tuning of LTCs. Cluster analysis of these changes confirmed several groups of LTCs with distinctive spatiotemporal tuning. These differences were associated with variation in velocity tuning in response to translated, natural scenes. LTCs with differences in velocity tuning ranges and optima may underlie how a broad range of motion velocities are encoded. In the hawking dragonfly, changes in LTC tuning over time are therefore likely to support their extensive range of behaviors, from hovering to fast-speed pursuits.SIGNIFICANCE STATEMENT Understanding how animals navigate the world is an inherently difficult and interesting problem. Insects are useful models for understanding neuronal mechanisms underlying these activities, with neurons that encode wide-field motion previously identified in insects, such as flies, hawkmoths, and butterflies. Like some Dipteran flies, dragonflies exhibit complex aerobatic behaviors, such as hovering, patrolling, and aerial combat. However, dragonflies lack halteres that support such diverse behavior in flies. To understand how dragonflies might address this problem using only visual cues, we recorded from their wide-field motion-sensitive neurons. We found these differ strongly in the ways they respond to sustained motion, allowing them collectively to encode the very broad range of velocities experienced during diverse behavior.

Properties of predictive gain modulation in a dragonfly visual neuron.

Dragonflies pursue and capture tiny prey and conspecifics with extremely high success rates. These moving targets represent a small visual signal on the retina and successful chases require accurate detection and amplification by downstream neuronal circuits. This amplification has been observed in a population of neurons called small target motion detectors (STMDs), through a mechanism we term predictive gain modulation. As targets drift through the neuron's receptive field, spike frequency builds slowly over time. This increased likelihood of spiking or gain is modulated across the receptive field, enhancing sensitivity just ahead of the target's path, with suppression of activity in the remaining surround. Whilst some properties of this mechanism have been described, it is not yet known which stimulus parameters modulate the amount of response gain. Previous work suggested that the strength of gain enhancement was predominantly determined by the duration of the target's prior path. Here, we show that predictive gain modulation is more than a slow build-up of responses over time. Rather, the strength of gain is dependent on the velocity of a prior stimulus combined with the current stimulus attributes (e.g. angular size). We also describe response variability as a major challenge of target-detecting neurons and propose that the role of predictive gain modulation is to drive neurons towards response saturation, thus minimising neuronal variability despite noisy visual input signals.

Photoreceptor signalling is sufficient to explain the detectability threshold of insect aerial pursuers.

An essential biological task for many flying insects is the detection of small, moving targets, such as when pursuing prey or conspecifics. Neural pathways underlying such 'target-detecting' behaviours have been investigated for their sensitivity and tuning properties (size, velocity). However, which stage of neuronal processing limits target detection is not yet known. Here, we investigated several skilled, aerial pursuers (males of four insect species), measuring the target-detection limit (signal-to-noise ratio) of light-adapted photoreceptors. We recorded intracellular responses to moving targets of varying size, extended well below the nominal resolution of single ommatidia. We found that the signal detection limit (2× photoreceptor noise) matches physiological or behavioural target-detection thresholds observed in each species. Thus, across a diverse range of flying insects, individual photoreceptor responses to changes in light intensity establish the sensitivity of the feature detection pathway, indicating later stages of processing are dedicated to feature tuning, tracking and selection.

An autonomous robot inspired by insect neurophysiology pursues moving features in natural environments.

OBJECTIVE: Many computer vision and robotic applications require the implementation of robust and efficient target-tracking algorithms on a moving platform. However, deployment of a real-time system is challenging, even with the computational power of modern hardware. Lightweight and low-powered flying insects, such as dragonflies, track prey or conspecifics within cluttered natural environments, illustrating an efficient biological solution to the target-tracking problem. APPROACH: We used our recent recordings from 'small target motion detector' neurons in the dragonfly brain to inspire the development of a closed-loop target detection and tracking algorithm. This model exploits facilitation, a slow build-up of response to targets which move along long, continuous trajectories, as seen in our electrophysiological data. To test performance in real-world conditions, we implemented this model on a robotic platform that uses active pursuit strategies based on insect behaviour. MAIN RESULTS: Our robot performs robustly in closed-loop pursuit of targets, despite a range of challenging conditions used in our experiments; low contrast targets, heavily cluttered environments and the presence of distracters. We show that the facilitation stage boosts responses to targets moving along continuous trajectories, improving contrast sensitivity and detection of small moving targets against textured backgrounds. Moreover, the temporal properties of facilitation play a useful role in handling vibration of the robotic platform. We also show that the adoption of feed-forward models which predict the sensory consequences of self-movement can significantly improve target detection during saccadic movements. SIGNIFICANCE: Our results provide insight into the neuronal mechanisms that underlie biological target detection and selection (from a moving platform), as well as highlight the effectiveness of our bio-inspired algorithm in an artificial visual system.

A predictive focus of gain modulation encodes target trajectories in insect vision.

When a human catches a ball, they estimate future target location based on the current trajectory. How animals, small and large, encode such predictive processes at the single neuron level is unknown. Here we describe small target-selective neurons in predatory dragonflies that exhibit localized enhanced sensitivity for targets displaced to new locations just ahead of the prior path, with suppression elsewhere in the surround. This focused region of gain modulation is driven by predictive mechanisms, with the direction tuning shifting selectively to match the target's prior path. It involves a large local increase in contrast gain which spreads forward after a delay (e.g. an occlusion) and can even transfer between brain hemispheres, predicting trajectories moved towards the visual midline from the other eye. The tractable nature of dragonflies for physiological experiments makes this a useful model for studying the neuronal mechanisms underlying the brain's remarkable ability to anticipate moving stimuli.

Visual acuity of the honey bee retina and the limits for feature detection.

Visual abilities of the honey bee have been studied for more than 100 years, recently revealing unexpectedly sophisticated cognitive skills rivalling those of vertebrates. However, the physiological limits of the honey bee eye have been largely unaddressed and only studied in an unnatural, dark state. Using a bright display and intracellular recordings, we here systematically investigated the angular sensitivity across the light adapted eye of honey bee foragers. Angular sensitivity is a measure of photoreceptor receptive field size and thus small values indicate higher visual acuity. Our recordings reveal a fronto-ventral acute zone in which angular sensitivity falls below 1.9°, some 30% smaller than previously reported. By measuring receptor noise and responses to moving dark objects, we also obtained direct measures of the smallest features detectable by the retina. In the frontal eye, single photoreceptors respond to objects as small as 0.6° × 0.6°, with >99% reliability. This indicates that honey bee foragers possess significantly better resolution than previously reported or estimated behaviourally, and commonly assumed in modelling of bee acuity.

Performance of an insect-inspired target tracker in natural conditions.

Robust and efficient target-tracking algorithms embedded on moving platforms, are a requirement for many computer vision and robotic applications. However, deployment of a real-time system is challenging, even with the computational power of modern hardware. As inspiration, we look to biological lightweight solutions-lightweight and low-powered flying insects. For example, dragonflies pursue prey and mates within cluttered, natural environments, deftly selecting their target amidst swarms. In our laboratory, we study the physiology and morphology of dragonfly 'small target motion detector' neurons likely to underlie this pursuit behaviour. Here we describe our insect-inspired tracking model derived from these data and compare its efficacy and efficiency with state-of-the-art engineering models. For model inputs, we use both publicly available video sequences, as well as our own task-specific dataset (small targets embedded within natural scenes). In the context of the tracking problem, we describe differences in object statistics within the video sequences. For the general dataset, our model often locks on to small components of larger objects, tracking these moving features. When input imagery includes small moving targets, for which our highly nonlinear filtering is matched, the robustness outperforms state-of-the-art trackers. In all scenarios, our insect-inspired tracker runs at least twice the speed of the comparison algorithms.

Properties of neuronal facilitation that improve target tracking in natural pursuit simulations.

Although flying insects have limited visual acuity (approx. 1°) and relatively small brains, many species pursue tiny targets against cluttered backgrounds with high success. Our previous computational model, inspired by electrophysiological recordings from insect 'small target motion detector' (STMD) neurons, did not account for several key properties described from the biological system. These include the recent observations of response 'facilitation' (a slow build-up of response to targets that move on long, continuous trajectories) and 'selective attention', a competitive mechanism that selects one target from alternatives. Here, we present an elaborated STMD-inspired model, implemented in a closed loop target-tracking system that uses an active saccadic gaze fixation strategy inspired by insect pursuit. We test this system against heavily cluttered natural scenes. Inclusion of facilitation not only substantially improves success for even short-duration pursuits, but it also enhances the ability to 'attend' to one target in the presence of distracters. Our model predicts optimal facilitation parameters that are static in space and dynamic in time, changing with respect to the amount of background clutter and the intended purpose of the pursuit. Our results provide insights into insect neurophysiology and show the potential of this algorithm for implementation in artificial visual systems and robotic applications.

Multisensory perception: pinpointing visual enhancement by appropriate odors.

Multisensory contributions to perception are well studied, but their underlying brain mechanisms are poorly understood. A new study has exploited advances in fly optogenetics to pinpoint mechanisms that enhance responses to visual motion in the presence of ecologically relevant odors.

Neurogenic potential of dental pulp stem cells isolated from murine incisors.

INTRODUCTION: Interest in the use of dental pulp stem cells (DPSC) to enhance neurological recovery following stroke and traumatic injury is increasing following successful pre-clinical studies. A murine model of autologous neural stem cell transplantation would be useful for further pre-clinical investigation of the underlying mechanisms. However, while human-derived DPSC have been well characterised, the neurogenic potential of murine DPSC (mDPSC) has been largely neglected. In this study we demonstrate neuronal differentiation of DPSC from murine incisors in vitro. METHODS: mDPSC were cultured under neuroinductive conditions and assessed for neuronal and glial markers and electrophysiological functional maturation. RESULTS: mDPSC developed a neuronal morphology and high expression of neural markers nestin, ßIII-tubulin and GFAP. Neurofilament M and S100 were found in lower abundance. Differentiated cells also expressed protein markers for cholinergic, GABAergic and glutaminergic neurons, indicating a mixture of central and peripheral nervous system cell types. Intracellular electrophysiological analysis revealed the presence of voltage-gated L-type Ca2+ channels in a majority of cells with neuronal morphology. No voltage-gated Na+ or K+ currents were found and the cultures did not support spontaneous action potentials. Neuronal-like networks expressed the gap junction protein, connexin 43 but this was not associated with dye coupling between adjacent cells after injection of the low-molecular weight tracers Lucifer yellow or Neurobiotin. This indicated that the connexin proteins were not forming traditional gap junction channels. CONCLUSIONS: The data presented support the differentiation of mDPSC into immature neuronal-like networks.

Contrast sensitivity and the detection of moving patterns and features.

Theories based on optimal sampling by the retina have been widely applied to visual ecology at the level of the optics of the eye, supported by visual behaviour. This leads to speculation about the additional processing that must lie in between-in the brain itself. But fewer studies have adopted a quantitative approach to evaluating the detectability of specific features in these neural pathways. We briefly review this approach with a focus on contrast sensitivity of two parallel pathways for motion processing in insects, one used for analysis of wide-field optic flow, the other for detection of small features. We further use a combination of optical modelling of image blur and physiological recording from both photoreceptors and higher-order small target motion detector neurons sensitive to small targets to show that such neurons operate right at the limits imposed by the optics of the eye and the noise level of single photoreceptors. Despite this, and the limitation of only being able to use information from adjacent receptors to detect target motion, they achieve a contrast sensitivity that rivals that of wide-field motion sensitive pathways in either insects or vertebrates-among the highest in absolute terms seen in any animal.