Acclimatization and Foraging of Native Brazilian Stingless Bees in Arenas with Covering Materials of Different Spectral Properties.

The use of Meliponini for crop pollination in protected environments is practically non-existent. One of the reasons is the difficulty of acclimatizing Meliponini to the temperature and light conditions inside greenhouses. We investigated how covering materials used in greenhouses, which filter different intensities of ultraviolet (UV) light, affect the foraging behaviors, flight orientation, attraction to walls and ceilings, and mortality of Scaptotrigona cf. postica (Letreille), Frieseomelitta varia (Lepeletier), and Melipona quadrifasciata (Lepeletier). The experiments were conducted in 5.3 m3 arenas covered with four types of plastic films that do not polarize sunlight, with UV transmittance levels ranging from 0.1 to 54%, compared to a transparent glass control. The temperature inside the arenas varied between treatments, from 27 ± 3°C to 31 ± 2°C. All three species collected resources and returned to the colony, regardless of the covering material. However, the proportion of this behavior, the number of bees attracted to the ceiling and wall, and mortality varied among treatments and/or throughout the confinement days for each species. Melipona quadrifasciata and F. varia acclimatized better to the confined environments than S. cf. postica and showed consistent resource collection behavior throughout the confinement days in all tested materials, except for the one that filtered around 90% of UV. In all three species, the mortality gradually decreased throughout the confinement days. The results indicate that the choice of covering material, considering its optical characteristics, can be crucial to ensure greater effectiveness of the pollination services provided by stingless bees in protected systems.

A three-dimensional atlas of the honeybee central complex, associated neuropils and peptidergic layers of the central body.

The central complex (CX) in the brain of insects is a highly conserved group of midline-spanning neuropils consisting of the upper and lower division of the central body, the protocerebral bridge, and the paired noduli. These neuropils are the substrate for a number of behaviors, most prominently goal-oriented locomotion. Honeybees have been a model organism for sky-compass orientation for more than 70 years, but there is still very limited knowledge about the structure and function of their CX. To advance and facilitate research on this brain area, we created a high-resolution three-dimensional atlas of the honeybee's CX and associated neuropils, including the posterior optic tubercles, the bulbs, and the anterior optic tubercles. To this end, we developed a modified version of the iterative shape averaging technique, which allowed us to achieve high volumetric accuracy of the neuropil models. For a finer definition of spatial locations within the central body, we defined layers based on immunostaining against the neuropeptides locustatachykinin, FMRFamide, gastrin/cholecystokinin, and allatostatin and included them into the atlas by elastic registration. Our honeybee CX atlas provides a platform for future neuroanatomical work.

Bimodal Patterning Discrimination in Harnessed Honey Bees.

In natural environments, stimuli and events learned by animals usually occur in a combination of more than one sensory modality. An important problem in experimental psychology has been thus to understand how organisms learn about multimodal compounds and how they discriminate this compounds from their unimodal constituents. Here we tested the ability of honey bees to learn bimodal patterning discriminations in which a visual-olfactory compound (AB) should be differentiated from its visual (A) and olfactory (B) elements. We found that harnessed bees trained in classical conditioning of the proboscis extension reflex (PER) are able to solve bimodal positive and negative patterning (NP) tasks. In positive patterning (PP), bees learned to respond significantly more to a bimodal reinforced compound (AB+) than to non-reinforced presentations of single visual (A-) or olfactory (B-) elements. In NP, bees learned to suppress their responses to a non-reinforced compound (AB-) and increase their responses to reinforced presentations of visual (A+) or olfactory (B+) elements alone. We compared the effect of two different inter-trial intervals (ITI) in our conditioning approaches. Whereas an ITI of 8 min allowed solving both PP and NP, only PP could be solved with a shorter ITI of 3 min. In all successful cases of bimodal PP and NP, bees were still able to discriminate between reinforced and non-reinforced stimuli in memory tests performed one hour after conditioning. The analysis of individual performances in PP and NP revealed that different learning strategies emerged in distinct individuals. Both in PP and NP, high levels of generalization were found between elements and compound at the individual level, suggesting a similar difficulty for bees to solve these bimodal patterning tasks. We discuss our results in light of elemental and configural learning theories that may support the strategies adopted by honey bees to solve bimodal PP or NP discriminations.

Floral colour change in Byrsonima variabilis (Malpighiaceae) as a visual cue for pollen but not oil foraging by oil-collecting bees.

Pollinators search for multiple flora resources throughout their life cycle. Most studies, however, only assess how bees discriminate floral cues in the context of nectar foraging. In the present study, we sought to elucidate whether oil-collecting bees discriminate flowers of Byrsonima variabilis (Malpighiaceae) with petals of different colours when foraging for pollen or oil. As the colour of the standard petal changes during anthesis, we characterised the spectral reflectance patterns of flowers throughout anthesis and modelled chromatic perceptual space to determine how these colour patterns are perceived by bees. Through the quantification of flower pollen in the different phases, we found that the colour of the standard petal is an honest cue of the presence of pollen. Centridine bees preferentially visited flowers with a yellow (bee's green) colour when searching for pollen, but indiscriminately visited flowers with different petal colours when searching for floral oil. We suggest that standard petals, in the species studied and others of the genus, like nectar guides, act as pollen guides, which oil-collecting females use to detect pollen-rich flowers. Moreover, they use different floral clues during foraging for different resources in the same host plant.

Visual discrimination transfer and modulation by biogenic amines in honeybees.

For more than a century, visual learning and memory have been studied in the honeybee Apis mellifera using operant appetitive conditioning. Although honeybees show impressive visual learning capacities in this well-established protocol, operant training of free-flying animals cannot be combined with invasive protocols for studying the neurobiological basis of visual learning. In view of this, different attempts have been made to develop new classical conditioning protocols for studying visual learning in harnessed honeybees, though learning performance remains considerably poorer than that for free-flying animals. Here, we investigated the ability of honeybees to use visual information acquired during classical conditioning in a new operant context. We performed differential visual conditioning of the proboscis extension reflex (PER) followed by visual orientation tests in a Y-maze. Classical conditioning and Y-maze retention tests were performed using the same pair of perceptually isoluminant chromatic stimuli, to avoid the influence of phototaxis during free-flying orientation. Visual discrimination transfer was clearly observed, with pre-trained honeybees significantly orienting their flights towards the former positive conditioned stimulus (CS+), thus showing that visual memories acquired by honeybees are resistant to context changes between conditioning and the retention test. We combined this visual discrimination approach with selective pharmacological injections to evaluate the effect of dopamine and octopamine in appetitive visual learning. Both octopaminergic and dopaminergic antagonists impaired visual discrimination performance, suggesting that both these biogenic amines modulate appetitive visual learning in honeybees. Our study brings new insight into cognitive and neurobiological mechanisms underlying visual learning in honeybees.

Advances and limitations of visual conditioning protocols in harnessed bees.

Bees are excellent invertebrate models for studying visual learning and memory mechanisms, because of their sophisticated visual system and impressive cognitive capacities associated with a relatively simple brain. Visual learning in free-flying bees has been traditionally studied using an operant conditioning paradigm. This well-established protocol, however, can hardly be combined with invasive procedures for studying the neurobiological basis of visual learning. Different efforts have been made to develop protocols in which harnessed honey bees could associate visual cues with reinforcement, though learning performances remain poorer than those obtained with free-flying animals. Especially in the last decade, the intention of improving visual learning performances of harnessed bees led many authors to adopt distinct visual conditioning protocols, altering parameters like harnessing method, nature and duration of visual stimulation, number of trials, inter-trial intervals, among others. As a result, the literature provides data hardly comparable and sometimes contradictory. In the present review, we provide an extensive analysis of the literature available on visual conditioning of harnessed bees, with special emphasis on the comparison of diverse conditioning parameters adopted by different authors. Together with this comparative overview, we discuss how these diverse conditioning parameters could modulate visual learning performances of harnessed bees.

Synaptic Organization of Microglomerular Clusters in the Lateral and Medial Bulbs of the Honeybee Brain.

The honeybee Apis mellifera is an established model for the study of visual orientation. Yet, research on this topic has focused on behavioral aspects and has neglected the investigation of the underlying neural architectures in the bee brain. In other insects, the anterior optic tubercle (AOTU), the lateral (LX) and the central complex (CX) are important brain regions for visuospatial performances. In the central brain of the honeybee, a prominent group of neurons connecting the AOTU with conspicuous microglomerular synaptic structures in the LX has been recently identified, but their neural organization and ultrastructure have not been investigated. Here we characterized these microglomerular structures by means of immunohistochemical and ultrastructural analyses, in order to evaluate neurotransmission and synaptic organization. Three-dimensional reconstructions of the pre-synaptic and post-synaptic microglomerular regions were performed based on confocal microscopy. Each pre-synaptic region appears as a large cup-shaped profile that embraces numerous post-synaptic profiles of GABAergic tangential neurons connecting the LX to the CX. We also identified serotonergic broad field neurons that probably provide modulatory input from the CX to the synaptic microglomeruli in the LX. Two distinct clusters of microglomerular structures were identified in the lateral bulb (LBU) and medial bulb (MBU) of the LX. Although the ultrastructure of both clusters is very similar, we found differences in the number of microglomeruli and in the volume of the pre-synaptic profiles of each cluster. We discuss the possible role of these microglomerular clusters in the visuospatial behavior of honeybees and propose research avenues for studying their neural plasticity and synaptic function.

A multi-species bait for Chagas disease vectors.

BACKGROUND: Triatomine bugs are the insect vectors of Trypanosoma cruzi, the etiological agent of Chagas disease. These insects are known to aggregate inside shelters during daylight hours and it has been demonstrated that within shelters, the aggregation is induced by volatiles emitted from bug feces. These signals promote inter-species aggregation among most species studied, but the chemical composition is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In the present work, feces from larvae of the three species were obtained and volatile compounds were identified by solid phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS). We identified five compounds, all present in feces of all of the three species: Triatoma infestans, Panstrongylus megistus and Triatoma brasiliensis. These substances were tested for attractivity and ability to recruit insects into shelters. Behaviorally active doses of the five substances were obtained for all three triatomine species. The bugs were significantly attracted to shelters baited with blends of 160 ng or 1.6 µg of each substance. CONCLUSIONS/SIGNIFICANCE: Common compounds were found in the feces of vectors of Chagas disease that actively recruited insects into shelters, which suggests that this blend of compounds could be used for the development of baits for early detection of reinfestation with triatomine bugs.

Chromatic processing in the anterior optic tubercle of the honey bee brain.

Color vision in honey bees (Apis mellifera) has been extensively studied at the behavioral level and, to a lesser degree, at the physiological level by means of electrophysiological intracellular recordings of single neurons. Few visual neurons have been so far characterized in the lateral protocerebrum of bees. Therefore, the possible implication of this region in chromatic processing remains unknown. We performed in vivo calcium imaging of interneurons in the anterior optic tubercle (AOTu) of honey bees upon visual stimulation of the compound eye to analyze chromatic response properties. Stimulation with distinct monochromatic lights (ultraviolet [UV], blue, and green) matching the sensitivity of the three photoreceptor types of the bee retina induced different signal amplitudes, temporal dynamics, and spatial activity patterns in the AOTu intertubercle network, thus revealing intricate chromatic processing properties. Green light strongly activated both the dorsal and ventral lobes of the AOTu's major unit; blue light activated the dorsal lobe more while UV light activated the ventral lobe more. Eye stimulation with mixtures of blue and green light induced suppression phenomena in which responses to the mixture were lower than those to the color components, thus concurring with color-opponent processing. These data provide evidence for a spatial segregation of color processing in the AOTu, which may serve for navigation purposes.

Genetic basis of triatomine behavior: lessons from available insect genomes.

Triatomines have been important model organisms for behavioural research. Diverse reports about triatomine host search, pheromone communication in the sexual, shelter and alarm contexts, daily cycles of activity, refuge choice and behavioural plasticity have been published in the last two decades. In recent times, a variety of molecular genetics techniques has allowed researchers to investigate elaborate and complex questions about the genetic bases of the physiology of insects. This, together with the current characterisation of the genome sequence of Rhodnius prolixus allows the resurgence of this excellent insect physiology model in the omics era. In the present revision, we suggest that studying the molecular basis of behaviour and sensory ecology in triatomines will promote a deeper understanding of fundamental aspects of insect and, particularly, vector biology. This will allow uncovering unknown features of essential insect physiology questions for a hemimetabolous model organism, promoting more robust comparative studies of insect sensory function and cognition.

Genetic basis of triatomine behavior: lessons from available insect genomes

Triatomines have been important model organisms for behavioural research. Diverse reports about triatomine host search, pheromone communication in the sexual, shelter and alarm contexts, daily cycles of activity, refuge choice and behavioural plasticity have been published in the last two decades. In recent times, a variety of molecular genetics techniques has allowed researchers to investigate elaborate and complex questions about the genetic bases of the physiology of insects. This, together with the current characterisation of the genome sequence of Rhodnius prolixus allows the resurgence of this excellent insect physiology model in the omics era. In the present revision, we suggest that studying the molecular basis of behaviour and sensory ecology in triatomines will promote a deeper understanding of fundamental aspects of insect and, particularly, vector biology. This will allow uncovering unknown features of essential insect physiology questions for a hemimetabolous model organism, promoting more robust comparative studies of insect sensory function and cognition.

Lack of segregation between two species of Chagas disease vectors.

Triatoma infestans and Panstrongylus megistus are relevant Chagas disease vectors. An apparent segregation among these triatomine species inside human households was suggested to rely on mutual repellence between them. However, P. megistus and T. infestans show aggregation responses to chemical signals emitted by the other species. These findings do not rule out the possibility that stimuli other than chemical signals could mediate repellence when these species exploit shelters simultaneously. In the present study, we investigated how P. megistus and T. infestans exploit shelters in controlled laboratory conditions and how insect density and environmental illumination modulate this behavior. We evaluated whether these species aggregate inside shelters or mutually repel each other. Panstrongylus megistus and T. infestans show specific patterns of shelter exploitation, which are differentially affected by insect density and environment illumination. In particular, P. megistus is more sensitive to insect density than T. infestans, whereas T. infestans shows higher sensitivity to illumination than P. megistus. Nevertheless, these species exploit shelters randomly without any apparent repellence.

Visual conditioning of the sting extension reflex in harnessed honeybees.

Visual performances of honeybees have been extensively studied using free-flying individuals trained to choose visual stimuli paired with sucrose reward. By contrast, harnessed bees in the laboratory were not thought to be capable of learning a Pavlovian association between a visual stimulus (CS) and sucrose reward (US). For reasons as yet unknown, harnessed bees only learn visual cues in association with sucrose if their antennae are ablated. However, slow acquisition and low retention performances are obtained in this case. Here, we established a novel visual conditioning protocol, which allows studying visual learning and memory in intact harnessed bees in the laboratory. This protocol consists of conditioning the sting extension reflex (SER) by pairing a visual stimulus (CS+) with an electric shock punishment (US), and a different visual stimulus (CS-) with the absence of shock. Bees with intact antennae learned the discrimination between CS+ and CS- by using chromatic cues, achromatic cues or both. Antennae ablation was not only unnecessary for learning to occur but it even impaired visual SER conditioning because of a concomitant reduction of responsiveness to the electric shock. We thus established the first visual conditioning protocol on harnessed honeybees that does not require injuring the experimental subjects. This novel experimental approach opens new doors for accessing the neural correlates of visual learning and memory in honeybees.

Neural organization and visual processing in the anterior optic tubercle of the honeybee brain.

The honeybee Apis mellifera represents a valuable model for studying the neural segregation and integration of visual information. Vision in honeybees has been extensively studied at the behavioral level and, to a lesser degree, at the physiological level using intracellular electrophysiological recordings of single neurons. However, our knowledge of visual processing in honeybees is still limited by the lack of functional studies of visual processing at the circuit level. Here we contribute to filling this gap by providing a neuroanatomical and neurophysiological characterization at the circuit level of a practically unstudied visual area of the bee brain, the anterior optic tubercle (AOTu). First, we analyzed the internal organization and neuronal connections of the AOTu. Second, we established a novel protocol for performing optophysiological recordings of visual circuit activity in the honeybee brain and studied the responses of AOTu interneurons during stimulation of distinct eye regions. Our neuroanatomical data show an intricate compartmentalization and connectivity of the AOTu, revealing a dorsoventral segregation of the visual input to the AOTu. Light stimuli presented in different parts of the visual field (dorsal, lateral, or ventral) induce distinct patterns of activation in AOTu output interneurons, retaining to some extent the dorsoventral input segregation revealed by our neuroanatomical data. In particular, activity patterns evoked by dorsal and ventral eye stimulation are clearly segregated into distinct AOTu subunits. Our results therefore suggest an involvement of the AOTu in the processing of dorsoventrally segregated visual information in the honeybee brain.

Color modulates olfactory learning in honeybees by an occasion-setting mechanism.

A sophisticated form of nonelemental learning is provided by occasion setting. In this paradigm, animals learn to disambiguate an uncertain conditioned stimulus using alternative stimuli that do not enter into direct association with the unconditioned stimulus. For instance, animals may learn to discriminate odor rewarded from odor nonrewarded trials if these two situations are indicated by different colors that do not themselves become associated with the reward. Despite a growing interest in nonelemental learning in insects, no study has so far attempted to study occasion setting in restrained honeybees, although this would allow direct access to the neural basis of nonelemental learning. Here we asked whether colors can modulate olfactory conditioning of the proboscis extension reflex (PER) via an occasion-setting mechanism. We show that intact, harnessed bees are not capable of learning a direct association between color and sucrose. Despite this incapacity, bees solved an occasion-setting discrimination in which colors set the occasion for appropriate responding to an odor that was rewarded or nonrewarded depending on the color. We therefore provide the first controlled demonstration of bimodal (color-odor) occasion setting in harnessed honeybees, which opens the door for studying the neural basis of such bimodal, nonelemental discriminations in insects.

Multiple reversal olfactory learning in honeybees.

In multiple reversal learning, animals trained to discriminate a reinforced from a non-reinforced stimulus are subjected to various, successive reversals of stimulus contingencies (e.g. A+ vs. B-, A- vs. B+, A+ vs. B-). This protocol is useful to determine whether or not animals "learn to learn" and solve successive discriminations faster (or with fewer errors) with increasing reversal experience. Here we used the olfactory conditioning of proboscis extension reflex to study how honeybees Apis mellifera perform in a multiple reversal task. Our experiment contemplated four consecutive differential conditioning phases involving the same odors (A+ vs. B- to A- vs. B+ to A+ vs. B- to A- vs. B+). We show that bees in which the weight of reinforced or non-reinforced stimuli was similar mastered the multiple olfactory reversals. Bees which failed the task exhibited asymmetric responses to reinforced and non-reinforced stimuli, thus being unable to rapidly reverse stimulus contingencies. Efficient reversers did not improve their successive discriminations but rather tended to generalize their choice to both odors at the end of conditioning. As a consequence, both discrimination and reversal efficiency decreased along experimental phases. This result invalidates a learning-to-learn effect and indicates that bees do not only respond to the actual stimulus contingencies but rather combine these with an average of past experiences with the same stimuli.

Sinais de agregação em Panstrongylus megistus e a interação desta espécie com Triatoma infestans no interior de abrigos / Signals of aggregation in Panstrongylus megistus and the interaction of this species with Triatoma infestans in the interior of shelters

Pastrongylus megistus se destaca pelo seu alto potencial para transmissão da doença de Chagas. Estudos prévios descreveram compostos voláteis presentes nas fezes de P. megistus que provavelmente servem como marca química do abrigo. Alguns autores sugerem a existência de repelência mútua entre P. megistus e Triatoma infestans mediada pelas suas fezes, feromônios e pela sua própria presença. Entretanto, foi demonstrada uma reposta de agregação interespecífica a sinais químicos das fezes e da cutícula para T. infestans e P. megistus. [...] testamos a atração de P. megistus a cinco compostos identificados nas suas fezes e determinamos a relação dose-resposta comportamental para cada composto. Posteriormente foi desenvolvida uma mistura de substâncias voláteis capaz de recrutar P. megistus para o interior de abrigos. [...]. Finalmente avaliamos se existe agregação ou repelência interespecífica no interior de abrigos entre P. megistus e T. infestans. Houve atração de P. megistus pelas doses de 10 microgramas e 100 microgramas de ácidos acético, 1 nanograma e 10 microgramas de ácido hexanóico, 10 microgramas e 100 microgramas de acetamida e 100 nanogramas de ácido isovalérico. Por outro lado, observou-se uma resposta de rejeição à dose de 10 microgramas de 2,3-butanodiol. Uma mistura contendo 10 nanogramas de cada uma destas substâncias mostrou-se atraente. Abrigos impregnados com 160 nanogramas ou 1,6 microgramas de cada composto mostraram-se mais atrativos do que abrigos limpos. Demostramos que a densidade de insetos e o ciclo de iluminação interferem significativamente no uso do abrigo para ambas as espécies estudadas. Entretanto existem diferenças no padrão de comportamento das espécies, sendo P. megistus mais sensível ao efeito da densidade e T. infestans aparentemente mais sensível ao efeito da iluminação. Vimos que T. infestans e P. megistus se agregam aleatoriamente entre dois abrigos disponíveis. Além disso, as duas espécies se agregam de maneira conjunta n.

Sinais de agregação em Panstrongylus megistus e a interação desta espécie com Triatoma infestans no interior de abrigos

Pastrongylus megistus se destaca pelo seu alto potencial para transmissão da doença de Chagas. Estudos prévios descreveram compostos voláteis presentes nas fezes de P. megistus que provavelmente servem como marca química do abrigo. Alguns autores sugerem a existência de repelência mútua entre P. megistus e Triatoma infestans mediada pelas suas fezes, feromônios e pela sua própria presença. Entretanto, foi demonstrada uma reposta de agregação interespecífica a sinais químicos das fezes e da cutícula para T. infestans e P. megistus. [...] testamos a atração de P. megistus a cinco compostos identificados nas suas fezes e determinamos a relação dose-resposta comportamental para cada composto. Posteriormente foi desenvolvida uma mistura de substâncias voláteis capaz de recrutar P. megistus para o interior de abrigos. [...]. Finalmente avaliamos se existe agregação ou repelência interespecífica no interior de abrigos entre P. megistus e T. infestans. Houve atração de P. megistus pelas doses de 10 microgramas e 100 microgramas de ácidos acético, 1 nanograma e 10 microgramas de ácido hexanóico, 10 microgramas e 100 microgramas de acetamida e 100 nanogramas de ácido isovalérico. Por outro lado, observou-se uma resposta de rejeição à dose de 10 microgramas de 2,3-butanodiol. Uma mistura contendo 10 nanogramas de cada uma destas substâncias mostrou-se atraente. Abrigos impregnados com 160 nanogramas ou 1,6 microgramas de cada composto mostraram-se mais atrativos do que abrigos limpos. Demostramos que a densidade de insetos e o ciclo de iluminação interferem significativamente no uso do abrigo para ambas as espécies estudadas. Entretanto existem diferenças no padrão de comportamento das espécies, sendo P. megistus mais sensível ao efeito da densidade e T. infestans aparentemente mais sensível ao efeito da iluminação. Vimos que T. infestans e P. megistus se agregam aleatoriamente entre dois abrigos disponíveis. Além disso, as duas espécies se agregam de maneira conjunta n

Electrophysiological and behavioural characterization of gustatory responses to antennal 'bitter' taste in honeybees.

We combined behavioural and electrophysiological experiments to study whether bitter taste is perceived at the antennal level in honeybees, Apis mellifera. Our behavioural studies showed that neither quinine nor salicin delivered at one antenna at different concentrations induced a retraction of the proboscis once it was extended in response to 1 M sucrose solution delivered to the opposite antenna. Bees that extended massively their proboscis to 1 M sucrose responded only partially when stimulated with a mixture of 1 M sucrose and 100 mM quinine. The mixture of 1 m sucrose and 100 mM salicin had no such suppressive effect. No behavioural suppression was found for mixtures of salt solution and either bitter substance. Electrophysiological recordings of taste sensillae at the antennal tip revealed sensillae that responded specifically either to sucrose or salt solutions, but none responded to the bitter substances quinine and salicin at the different concentrations tested. The electrophysiological responses of sensillae to 15 mM sucrose solution were inhibited by a mixture of 15 mM sucrose and 0.1 mM quinine, but not by a mixture of 15 mM sucrose and 0.1 mM salicin. The responses of sensillae to 50 mM NaCl were reduced by a mixture of 50 mm NaCl and 1 mM quinine but not by a mixture of 50 mM NaCl and 1 mM salicin. We concluded that no receptor cells for the bitter substances tested, exist at the level of the antennal tip of the honeybee and that antennal bitter taste is not represented as a separate perceptual quality.