Spatial resolution and optical sensitivity in the compound eyes of two common European wasps, Vespula germanica and Vespula vulgaris.

Vespula germanica and Vespula vulgaris are two common European wasps that have ecological and economic importance as a result of their artificial introduction into many different countries and environments. Their success has undoubtedly been aided by their capacity for visually guided hunting, foraging, learning and using visual cues in the context of homing and navigation. However, the visual systems of V. germanica and V. vulgaris have not received any deep attention. We used electrophysiology, together with optical and anatomical techniques, to measure the spatial resolution and optical sensitivity of the compound eyes of both species. We found that both wasps have high anatomical spatial resolution with narrow interommatidial angles (Δϕ between 1.0 and 1.5 deg) and a distinct acute zone in the fronto-ventral part of the eye. These narrow interommatidial angles are matched to photoreceptors having narrow angular sensitivities (acute zone acceptance angles Δρ below 1.3 deg), indicating eyes of high spatial resolution that are well suited to their ecological needs. Additionally, we found that both species possess an optical sensitivity that is typical of other day-flying hymenopterans.

Comparability of comparative toxicity: insect sensitivity to imidacloprid reveals huge variations across species but also within species.

Pesticides have been identified as major drivers of insect biodiversity loss. Thus, the study of their effects on non-pest insect species has attracted a lot of attention in recent decades. In general toxicology, the 'gold standard' to assess the toxicity of a substance is to measure mass-specific LD50 (i.e. median lethal dose per unit body mass). In entomology, reviews attempting to compare these data across all available studies are lacking. To fill this gap in knowledge, we performed a systematic review of the lethality of imidacloprid for adult insects. Imidacloprid is possibly the most extensively studied insecticide in recent times, yet we found that little is comparable across studies, owing to both methodological divergence and missing estimates of body mass. By accounting for body mass whenever possible, we show how imidacloprid sensitivity spans across an apparent range of approximately six orders of magnitude across insect species. Very high variability within species can also be observed owing to differences in exposure methods and observation time. We suggest that a more comparable and comprehensive approach has both biological and economic relevance. Ultimately, this would help to identify differences that could direct research towards preventing non-target species from being negatively affected.

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.

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.

Temporal and structural neural asymmetries in insects.

Neural asymmetries of the bilateral parts of the nervous system are found throughout the animal kingdom. The relative low complexity and experimental accessibility of the insect nervous system makes it well suited for studying the functions of neural asymmetries and their underlying mechanisms. Recent findings in insects reveal hardwired asymmetries in their peripheral and central nervous systems, which affect sensory perception, motor behaviours and cognitive-related tasks. Together, these findings underscore the tendency of the nervous system to segregate between the activities of its right and left sides either transiently or as permanent lateralized specializations.

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.

Ex vivo recordings reveal desert locust forelimb control is asymmetric.

Lateralized behaviours are widespread among the animals, including insects with their miniature brains, perhaps being a way of maximising neural capacity (reviewed in [1,2]). However, evidence for functional asymmetries in the neural circuitry itself is scarce. Here, using bilateral simultaneous recordings from the ex vivo nervous system of desert locusts, we show that the neural control of their forelimbs is asymmetric. This asymmetry was retained throughout the experimental period and either with or without the suboesophageal ganglion (SOG). These findings provide evidence for hard-wired neural sidedness and contribute to our understanding of the lateralization observed in in-vivo motor behaviours.

Lateralization of Sucrose Responsiveness and Non-associative Learning in Honeybees.

Lateralization is a fundamental property of the human brain that affects perceptual, motor, and cognitive processes. It is now acknowledged that left-right laterality is widespread across vertebrates and even some invertebrates such as fruit flies and bees. Honeybees, which learn to associate an odorant (the conditioned stimulus, CS) with sucrose solution (the unconditioned stimulus, US), recall this association better when trained using their right antenna than they do when using their left antenna. Correspondingly, olfactory sensilla are more abundant on the right antenna and odor encoding by projection neurons of the right antennal lobe results in better odor differentiation than those of the left one. Thus, lateralization arises from asymmetries both in the peripheral and central olfactory system, responsible for detecting the CS. Here, we focused on the US component and studied if lateralization exists in the gustatory system of Apis mellifera. We investigated whether sucrose sensitivity is lateralized both at the level of the antennae and the fore-tarsi in two independent groups of bees. Sucrose sensitivity was assessed by presenting bees with a series of increasing concentrations of sucrose solution delivered either to the left or the right antenna/tarsus and measuring the proboscis extension response to these stimuli. Bees experienced two series of stimulations, one on the left and the other on the right antenna/tarsus. We found that tarsal responsiveness was similar on both sides and that the order of testing affects sucrose responsiveness. On the contrary, antennal responsiveness to sucrose was higher on the right than on the left side, and this effect was independent of the order of antennal stimulation. Given this asymmetry, we also investigated antennal lateralization of habituation to sucrose. We found that the right antenna was more resistant to habituation, which is consistent with its higher sucrose sensitivity. Our results reveal that the gustatory system presents a peripheral lateralization that affects stimulus detection and non-associative learning. Contrary to the olfactory system, which is organized in two distinct brain hemispheres, gustatory receptor neurons converge into a single central region termed the subesophagic zone (SEZ). Whether the SEZ presents lateralized gustatory processing remains to be determined.

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.

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.

Asymmetric neural coding revealed by in vivo calcium imaging in the honey bee brain.

Left-right asymmetries are common properties of nervous systems. Although lateralized sensory processing has been well studied, information is lacking about how asymmetries are represented at the level of neural coding. Using in vivo functional imaging, we identified a population-level left-right asymmetry in the honey bee's primary olfactory centre, the antennal lobe (AL). When both antennae were stimulated via a frontal odour source, the inter-odour distances between neural response patterns were higher in the right than in the left AL. Behavioural data correlated with the brain imaging results: bees with only their right antenna were better in discriminating a target odour in a cross-adaptation paradigm. We hypothesize that the differences in neural odour representations in the two brain sides serve to increase coding capacity by parallel processing.

The Bee as a Model to Investigate Brain and Behavioural Asymmetries.

The honeybee Apis mellifera, with a brain of only 960,000 neurons and the ability to perform sophisticated cognitive tasks, has become an excellent model in life sciences and in particular in cognitive neurosciences. It has been used in our laboratories to investigate brain and behavioural asymmetries, i.e., the different functional specializations of the right and the left sides of the brain. It is well known that bees can learn to associate an odour stimulus with a sugar reward, as demonstrated by extension of the proboscis when presented with the trained odour in the so-called Proboscis Extension Reflex (PER) paradigm. Bees recall this association better when trained using their right antenna than they do when using their left antenna. They also retrieve short-term memory of this task better when using the right antenna. On the other hand, when tested for long-term memory recall, bees respond better when using their left antenna. Here we review a series of behavioural studies investigating bees' lateralization, integrated with electrophysiological measurements to study asymmetries of olfactory sensitivity, and discuss the possible evolutionary origins of these asymmetries. We also present morphological data obtained by scanning electron microscopy and two-photon microscopy. Finally, a behavioural study conducted in a social context is summarised, showing that honeybees control context-appropriate social interactions using their right antenna, rather than the left, thus suggesting that lateral biases in behaviour might be associated with requirements of social life.

A right antenna for social behaviour in honeybees.

Sophisticated cognitive abilities have been documented in honeybees, possibly an aspect of their complex sociality. In vertebrates brain asymmetry enhances cognition and directional biases of brain function are a putative adaptation to social behaviour. Here we show that honeybees display a strong lateral preference to use their right antenna in social interactions. Dyads of bees tested using only their right antennae (RA) contacted after shorter latency and were significantly more likely to interact positively (proboscis extension) than were dyads of bees using only their left antennae (LA). The latter were more likely to interact negatively (C-responses) even though they were from the same hive. In dyads from different hives C-responses were higher in RA than LA dyads. Hence, RA controls social behaviour appropriate to context. Therefore, in invertebrates, as well as vertebrates, lateral biases in behaviour appear to be associated with requirements of social life.

Spatial reorientation by geometry in bumblebees.

Human and non-human animals are capable of using basic geometric information to reorient in an environment. Geometric information includes metric properties associated with spatial surfaces (e.g., short vs. long wall) and left-right directionality or 'sense' (e.g. a long wall to the left of a short wall). However, it remains unclear whether geometric information is encoded by explicitly computing the layout of surface geometry or by matching images of the environment. View-based spatial encoding is generally thought to hold for insect navigation and, very recently, evidence for navigation by geometry has been reported in ants but only in a condition which does not allow the animals to use features located far from the goal. In this study we tested the spatial reorientation abilities of bumblebees (Bombus terrestris). After spatial disorientation, by passive rotation both clockwise and anticlockwise, bumblebees had to find one of the four exit holes located in the corners of a rectangular enclosure. Bumblebees systematically confused geometrically equivalent exit corners (i.e. corners with the same geometric arrangement of metric properties and sense, for example a short wall to the left of a long wall). However, when one wall of the enclosure was a different colour, bumblebees appeared to combine this featural information (either near or far from the goal) with geometric information to find the correct exit corner. Our results show that bumblebees are able to use both geometric and featural information to reorient themselves, even when features are located far from the goal.

A multimodal approach for tracing lateralisation along the olfactory pathway in the honeybee through electrophysiological recordings, morpho-functional imaging, and behavioural studies.

Recent studies have revealed asymmetries between the left and right sides of the brain in invertebrate species. Here we present a review of a series of recent studies from our laboratories, aimed at tracing asymmetries at different stages along the honeybee's (Apis mellifera) olfactory pathway. These include estimates of the number of sensilla present on the two antennae, obtained by scanning electron microscopy, as well as electroantennography recordings of the left and right antennal responses to odorants. We describe investigative studies of the antennal lobes, where multi-photon microscopy was used to search for possible morphological asymmetries between the two brain sides. Moreover, we report on recently published results obtained by two-photon calcium imaging for functional mapping of the antennal lobe aimed at comparing patterns of activity evoked by different odours. Finally, possible links to the results of behavioural tests, measuring asymmetries in single-sided olfactory memory recall, are discussed.

Lateralization in the invertebrate brain: left-right asymmetry of olfaction in bumble bee, Bombus terrestris.

Brain and behavioural lateralization at the population level has been recently hypothesized to have evolved under social selective pressures as a strategy to optimize coordination among asymmetrical individuals. Evidence for this hypothesis have been collected in Hymenoptera: eusocial honey bees showed olfactory lateralization at the population level, whereas solitary mason bees only showed individual-level olfactory lateralization. Here we investigated lateralization of odour detection and learning in the bumble bee, Bombus terrestris L., an annual eusocial species of Hymenoptera. By training bumble bees on the proboscis extension reflex paradigm with only one antenna in use, we provided the very first evidence of asymmetrical performance favouring the right antenna in responding to learned odours in this species. Electroantennographic responses did not reveal significant antennal asymmetries in odour detection, whereas morphological counting of olfactory sensilla showed a predominance in the number of olfactory sensilla trichodea type A in the right antenna. The occurrence of a population level asymmetry in olfactory learning of bumble bee provides new information on the relationship between social behaviour and the evolution of population-level asymmetries in animals.