Identification of chemosensory genes in the stingless bee Tetragonisca fiebrigi.

Reception of chemical information from the environment is crucial for insects' survival and reproduction. The chemosensory reception mainly occurs by the antennae and mouth parts of the insect, when the stimulus contacts the chemoreceptors located within the sensilla. Chemosensory receptor genes have been well-studied in some social hymenopterans such as ants, honeybees, and wasps. However, although stingless bees are the most representative group of eusocial bees, little is known about their odorant, gustatory, and ionotropic receptor genes. Here, we analyze the transcriptome of the proboscis and antennae of the stingless bee Tetragonisca fiebrigi. We identified and annotated 9 gustatory and 15 ionotropic receptors. Regarding the odorant receptors, we identified 204, and we were able to annotate 161 of them. In addition, we compared the chemosensory receptor genes of T. fiebrigi with those annotated for other species of Hymenoptera. We found that T. fiebrigi showed the largest number of odorant receptors compared with other bees. Genetic expansions were identified in the subfamilies 9-exon, which was also expanded in ants and paper wasps; in G02A, including receptors potentially mediating social behavior; and in GUnC, which has been related to pollen and nectar scent detection. Our study provides the first report of chemosensory receptor genes in T. fiebrigi and represents a resource for future molecular and physiological research in this and other stingless bee species.

In search of behavioral and brain processes involved in honey bee dance communication.

Honey bees represent an iconic model animal for studying the underlying mechanisms affecting advanced sensory and cognitive abilities during communication among colony mates. After von Frisch discovered the functional value of the waggle dance, this complex motor pattern led ethologists and neuroscientists to study its neural mechanism, behavioral significance, and implications for a collective organization. Recent studies have revealed some of the mechanisms involved in this symbolic form of communication by using conventional behavioral and pharmacological assays, neurobiological studies, comprehensive molecular and connectome analyses, and computational models. This review summarizes several critical behavioral and brain processes and mechanisms involved in waggle dance communication. We focus on the role of neuromodulators in the dancer and the recruited follower, the interneurons and their related processing in the first mechano-processing, and the computational navigation centers of insect brains.

Glyphosate affects larval gut microbiota and metamorphosis of honey bees with differences between rearing procedures.

The honey bee Apis mellifera is a sentinel species of the pollinator community which is exposed to a wide variety of pesticides. In the last half-century, the pesticide most applied worldwide has been the herbicide glyphosate (GLY) used for weed control and with microbiocide effects. After its application in crops, the GLY residues have been detected in flowers visited by honey bees as well as in the stored food of their hives. Therefore, the honey bee brood can ingest the herbicide during larval development. Recent studies proved that GLY has detrimental effects on adult honey bees and other insects associated with the disturbance of their gut microbiota. GLY induces changes in the growth, metabolism and survival of honey bees and stingless bees reared in vitro. However, the effect of GLY on larval microbiota is unknown so far and there are few studies with an in-hive exposure to GLY. For these reasons, this study aims to determine whether GLY induces dysbiosis in honey bee larvae and affects their metamorphosis during the exposure period (pre-defecation) and the post-exposure period. Furthermore, we assessed this herbicide in vitro and in the hive to compare its effects on different rearing procedures. Finally, we tested the pigment BLUE1 as an indirect exposure marker to detect and estimate the in-hive intake concentration of GLY. Our results indicate that the intake of field-relevant concentrations of GLY induced a slowdown in growth with dysbiosis in the larval gut microbiota followed by late effects on their metamorphosis such as teratogenesis and mortality of newly emerged bees. Nevertheless, brood from the same colonies expressed different signs of toxicity depending on the rearing procedure and in a dose-dependent manner.

In-hive learning of specific mimic odours as a tool to enhance honey bee foraging and pollination activities in pear and apple crops.

The areas devoted to agriculture that depend on pollinators have been sharply increased in the last decades with a concomitant growing global demand for pollination services. This forces to consider new strategies in pollinators' management to improve their efficiency. To promote a precision pollination towards a specific crop, we developed two simple synthetic odorant mixtures that honey bees generalized with their respective natural floral scents of the crop. We chose two commercial crops for fruit production that often coexist in agricultural settings, the apple (Malus domesticus) and the pear trees (Pyrus communis). Feeding colonies with sucrose solution scented with the apple mimic (AM) or the pear mimic (PM) odour enabled the establishment of olfactory memories that can bias bees towards the flowers of these trees. Encompassing different experimental approaches, our results support the offering of scented food to improve foraging and pollination activities of honey bees. The circulation of AM-scented sucrose solution inside the hive promoted higher colony activity, probably associated with greater activity of nectar foragers. The offering of PM-scented sucrose solution did not increase colony activity but led to greater pollen collection, which is consistent with pear flowers offering mainly pollen as resources for the bees. Results obtained from apple and pear crops suggest that the offering of AM- and PM-scented sucrose solution increased fruit yields. This preliminary study highlights the role of in-hive olfactory learning to bias foraging preferences within pome fruit crops.

Learning of a mimic odor combined with nectar nonsugar compounds enhances honeybee pollination of a commercial crop.

The increasing demand on pollination services leads food industry to consider new strategies for management of pollinators to improve their efficiency in agroecosystems. Recently, it was demonstrated that feeding beehives food scented with an odorant mixture mimicking the floral scent of a crop (sunflower mimic, SM) enhanced foraging activity and improved recruitment to the target inflorescences, which led to higher density of bees on the crop and significantly increased yields. Besides, the oral administration of nonsugar compounds (NSC) naturally found in nectars (caffeine and arginine) improved short and long-term olfactory memory retention in conditioned bees under laboratory conditions. To test the effect of offering of SM-scented food supplemented with NSC on honeybees pollinating sunflower for hybrid seed production, in a commercial plantation we fed colonies SM-scented food (control), and SM-scented food supplemented with either caffeine, arginine, or a mixture of both, in field realistic concentrations. Their foraging activity was assessed at the hive and on the crop up to 90 h after treatment, and sunflower yield was estimated prior to harvest. Our field results show that SM + Mix-treated colonies exhibited the highest incoming rates and densities on the crop. Additionally, overall seed mass was significantly higher by 20% on inflorescences close to these colonies than control colonies. Such results suggest that combined NSC potentiate olfactory learning of a mimic floral odor inside the hive, promoting faster colony-level foraging responses and increasing crop production.

Managed honeybees and South American bumblebees exhibit complementary foraging patterns in highbush blueberry.

Despite Apis mellifera being the most widely managed pollinator to enhance crop production, they are not the most suitable species for highbush blueberries, which possess restrictive floral morphology and require buzz-pollination. Thus, the South American bumblebee Bombus pauloensis is increasingly managed as an alternative species in this crop alongside honeybees. Herein, we evaluated the foraging patterns of the two species, concerning the potential pollen transfer between two blueberry co-blooming cultivars grown under open high tunnels during two seasons considering different colony densities. Both managed pollinators showed different foraging patterns, influenced by the cultivar identity which varied in their floral morphology and nectar production. Our results demonstrate that both species are efficient foragers on highbush blueberry and further suggest that they contribute positively to its pollination in complementary ways: while bumblebees were more effective at the individual level (visited more flowers and carried more pollen), the greater densities of honeybee foragers overcame the difficulties imposed by the flower morphology, irrespective of the stocking rate. This study supports the addition of managed native bumblebees alongside honeybees to enhance pollination services and emphasizes the importance of examining behavioural aspects to optimize management practices in pollinator-dependent crops.

Evaluating the Impact of Post-Emergence Weed Control in Honeybee Colonies Located in Different Agricultural Surroundings.

The honeybee Apis mellifera is exposed to agricultural intensification, which leads to an improved reliance upon pesticide use and the reduction of floral diversity. In the present study, we assess the changes in the colony activity and the expression profile of genes involved in xenobiotic detoxification in larvae and adult honeybees from three apiaries located in agricultural environments that differ in their proportion of the crop/wild flora. We evaluated these variables before and after the administration of a mixture of three herbicides during the summer season. The expression of several cytochrome P450 monooxygenases decreased significantly in larvae after post-emergence weed control and showed significant differences between apiaries in the case of honeybee workers. Principal component analysis (PCA) revealed that colonies located in the plot near to a wetland area exhibited a different relative gene expression profile after herbicide application compared with the other plots. Moreover, we found significant positive correlations between pollen collection and the pesticide detoxification genes that discriminated between plots in the PCA. Our results suggest that nutrition may modify herbicide impact on honeybees and that larvae are more harmed than adults in agroecosystems, a factor that will alter the colonies' population growth at the end of the blooming period.

Combined secondary compounds naturally found in nectars enhance honeybee cognition and survival.

The alkaloid caffeine and the amino acid arginine are present as secondary compounds in nectars of some flower species visited by pollinators. Each of these compounds affects honeybee appetitive behaviours by improving foraging activity and learning. While caffeine potentiates responses of mushroom body neurons involved in honeybee learning processes, arginine acts as precursor of nitric oxide, enhancing the protein synthesis involved in memory formation. Despite existing evidence on how these compounds affect honeybee cognitive ability individually, their combined effect on this is still unknown. We evaluated acquisition and memory retention in a classical olfactory conditioning procedure, in which the reward (sucrose solution) contained traces of caffeine, arginine or a mixture of the two. The results indicate that the presence of the single compounds and their most concentrated mixture increases bees' learning performance. However, memory retention, measured in the short and long term, increases significantly only in those treatments offering combinations of the two compounds in the reward. Additionally, the most concentrated mixture triggers a significant survival rate in the conditioned bees. Thus, some nectar compounds, when combined, show synergistic effects on cognitive ability and survival in an insect.

Learning of a Mimic Odor within Beehives Improves Pollination Service Efficiency in a Commercial Crop.

The growing global demand for pollination services leads producers to consider new strategies in pollinator management to improve its efficiency in agroecosystems [1-3]. Central place foragers, like honeybees, learn floral cues not only in the field but also inside the nest, where resource cues introduced into the hive improve foraging by guiding bees toward the learned stimuli [4]. In this regard, attempts to condition bees with crop-odor-scented food produced ambiguous results and lacked yield measurements [5-7]. To deepen our understanding of the use of odors as part of a precision pollination strategy, we developed a simple synthetic odorant mixture that bees generalized with the natural floral scent of sunflower for hybrid seed production, an economically important and highly pollinator-dependent crop [8]. Encompassing different experimental approaches, our results show that feeding colonies food scented with the sunflower mimic (SM) odor enabled the establishment of olfactory memories that biased bees to the sunflower crop. The offering of a rewarded odor mimicking the sunflower floral fragrance promoted higher foraging activity, increased the proportion of dances advertising the target inflorescences and reduced delays in dance onset, positively affected the density of bees on the crop, and increased yields from 29% to 57% in different sunflower hybrids. This study highlights the role of olfactory learning within the social context of the hive to bias foraging preferences in a novel agricultural environment and suggest that improvements in the tested parameters were due to bees anticipated response to the sunflower scent.

Sleep in honey bees is affected by the herbicide glyphosate.

Sleep plays an essential role in both neural and energetic homeostasis of animals. Honey bees (Apis mellifera) manifest the sleep state as a reduction in muscle tone and antennal movements, which is susceptible to physical or chemical disturbances. This social insect is one of the most important pollinators in agricultural ecosystems, being exposed to a great variety of agrochemicals, which might affect its sleep behaviour. The intake of glyphosate (GLY), the herbicide most widely used worldwide, impairs learning, gustatory responsiveness and navigation in honey bees. In general, these cognitive abilities are linked with the amount and quality of sleep. Furthermore, it has been reported that animals exposed to sleep disturbances show impairments in both metabolism and memory consolidation. Consequently, we assessed the sleep pattern of bees fed with a sugar solution containing GLY (0, 25, 50 and 100 ng) by quantifying their antennal activity during the scotophase. We found that the ingestion of 50 ng of GLY decreased both antennal activity and sleep bout frequency. This sleep deepening after GLY intake could be explained as a consequence of the regenerative function of sleep and the metabolic stress induced by the herbicide.

Chemosensory reception in the stingless bee Tetragonisca angustula.

In stingless bees, unlike honey bees, the relationship between chemosensory abilities and colony labor division has been poorly studied. Here we examined odor reception and gustatory responsiveness of the stingless bee Tetragonisca angustula focusing on workers, whose are involved in different tasks. Using the proboscis extension response, we studied sucrose response thresholds (SRTs) of foragers and guards. Peripheral responses to odors at the antennae were recorded by electroantennography (EAG). Additionally, we quantified and described the number and type of sensilla present on the antennae using scanning electron microscopy. Foragers' SRTs changed according to the resource collected: nonpollen foragers showed higher SRTs than pollen foragers and guards, that showed similar sucrose responsiveness. EAG signal strength of both foragers and guards increased with increasing odor concentration. Interestingly, guard bees showed the highest response to citral, an odor that triggers defensive behavior in T. angustula. Type and number of sensilla present in the antennae of guards and foragers were similar. Our results suggest that differences found in chemosensory responses among worker subcastes are task dependent.

Chronic exposure to glyphosate induces transcriptional changes in honey bee larva: A toxicogenomic study.

The honey bee Apis mellifera is the most abundant managed pollinator in diverse crops worldwide. Consequently, it is exposed to a plethora of environmental stressors, among which are the agrochemicals. In agroecosystems, the herbicide glyphosate (GLY) is one of the most applied. In laboratory assessments, GLY affects the honey bee larval development by delaying its moulting, among other negative effects. However, it is still unknown how GLY affects larval physiology when there are no observable signs of toxicity. We carried out a longitudinal experimental design using the in vitro rearing procedure. Larvae were fed with food containing or not a sub-lethal dose of GLY in chronic exposure (120 h). Individuals without observable signs of toxicity were sampled and their gene expression profile was analyzed with a transcriptomic approach to compare between treatments. Even though 29% of larvae were asymptomatic in the exposed group, they showed transcriptional changes in several genes after the GLY chronic intake. A total of 19 transcripts were found to be differentially expressed in the RNA-Seq experiment, mainly linked with defensive response and intermediary metabolism processes. Furthermore, the enriched functional categories in the transcriptome of the exposed asymptomatic larvae were linked with enzymes with catalytic and redox activity. Our results suggest an enhanced catabolism and oxidative metabolism in honey bee larvae as a consequence of the sub-lethal exposure to GLY, even in the absence of observable symptoms.

Effects of the Herbicide Glyphosate on Honey Bee Sensory and Cognitive Abilities: Individual Impairments with Implications for the Hive.

The honeybee Apis mellifera is an important pollinator in both undisturbed and agricultural ecosystems. Its great versatility as an experimental model makes it an excellent proxy to evaluate the environmental impact of agrochemicals using current methodologies and procedures in environmental toxicology. The increase in agrochemical use, including those that do not target insects directly, can have deleterious effects if carried out indiscriminately. This seems to be the case of the herbicide glyphosate (GLY), the most widely used agrochemical worldwide. Its presence in honey has been reported in samples obtained from different environments. Hence, to understand its current and potential risks for this pollinator it has become essential to not only study the effects on honeybee colonies located in agricultural settings, but also its effects under laboratory conditions. Subtle deleterious effects can be detected using experimental approaches. GLY negatively affects associative learning processes of foragers, cognitive and sensory abilities of young hive bees and promotes delays in brood development. An integrated approach that considers behavior, physiology, and development allows not only to determine the effects of this agrochemical on this eusocial insect from an experimental perspective, but also to infer putative effects in disturbed environments where it is omnipresent.

Honey bee workers generate low-frequency vibrations that are reliable indicators of their activity level.

In social insects, the tuning of activity levels among different worker task groups, which constitutes a fundamental basis of colony organization, relies on the exchange of reliable information on the activity level of individuals. The underlying stimuli, however, have remained largely unexplored so far. In the present study, we describe low-frequency thoracic vibrations generated by honey bee workers (Apis mellifera) within the colony, whose velocity amplitudes and main frequency components significantly increased with the level of an individual's activity. The characteristics of these vibrations segregated three main activity level-groups: foragers, active hive bees, and inactive hive bees. Nectar foragers, moreover, modulated their low-frequency vibrations during trophallactic food unloading to nestmates according to the quality of the collected food. Owing to their clear association with the activity level of an individual and their potential perceptibility during direct contacts, these low-frequency thoracic vibrations are candidate stimuli for providing unambiguous local information on the motivational status of honey bee workers.

Glyphosate affects the larval development of honey bees depending on the susceptibility of colonies.

As the main agricultural insect pollinator, the honey bee (Apis mellifera) is exposed to a number of agrochemicals, including glyphosate (GLY), the most widely used herbicide. Actually, GLY has been detected in honey and bee pollen baskets. However, its impact on the honey bee brood is poorly explored. Therefore, we assessed the effects of GLY on larval development under chronic exposure during in vitro rearing. Even though this procedure does not account for social compensatory mechanisms such as brood care by adult workers, it allows us to control the herbicide dose, homogenize nutrition and minimize environmental stress. Our results show that brood fed with food containing GLY traces (1.25-5.0 mg per litre of food) had a higher proportion of larvae with delayed moulting and reduced weight. Our assessment also indicates a non-monotonic dose-response and variability in the effects among colonies. Differences in genetic diversity could explain the variation in susceptibility to GLY. Accordingly, the transcription of immune/detoxifying genes in the guts of larvae exposed to GLY was variably regulated among the colonies studied. Consequently, under laboratory conditions, the response of honey bees to GLY indicates that it is a stressor that affects larval development depending on individual and colony susceptibility.

Assessment of Appetitive Behavior in Honey Bee Dance Followers.

Honey bees transfer different informational components of the discovered feeding source to their nestmates during the waggle dance. To decode the multicomponent information of this complex behavior, dance followers have to attend to the most relevant signal elements while filtering out less relevant ones. To achieve that, dance followers should present improved abilities to acquire information compared with those bees not engaged in this behavior. Through proboscis extension response assays, sensory and cognitive abilities were tested in follower and non-follower bees. Individuals were captured within the hive, immediately after following waggle runs or a bit further from the dancer. Both behavioral categories present low and similar spontaneous odor responses (SORs). However, followers exhibit differences in responsiveness to sucrose and odor discrimination: followers showed increased gustatory responsiveness and, after olfactory differential conditioning, better memory retention than non-followers. Thus, the abilities of the dance followers related to appetitive behavior would allow them to improve the acquisition of the dance surrounding information.

Odor Learning and Its Experience-Dependent Modulation in the South American Native Bumblebee Bombus atratus (Hymenoptera: Apidae).

Learning about olfactory stimuli is essential in bumblebees' life since it is involved in orientation, recognition of nest sites, foraging efficiency and food yield for the colony as a whole. To evaluate associative learning abilities in bees under controlled environmental conditions, the proboscis extension response (PER) assay is a well-established method used in honey bees, stingless bees and successfully adapted to bumblebees of the genus Bombus. However, studies on the learning capacity of Bombus atratus (Hymenoptera: Apidae), one of the most abundant native species in South America, are non-existent. In this study, we examined the cognitive abilities of worker bees of this species, carrying out an olfactory PER conditioning experiment. Bumblebees were able to learn a pure odor when it was presented in paired association with sugared reward, but not when odor and reward were presented in an unpaired manner. Furthermore, if the bees were preexposed to the conditioned odor, the results differed depending on the presence of the scent either as a volatile in the rearing environment or diluted in the food. A decrement in learning performance results from the non-reinforced pre-exposure to the to-be-conditioned odor, showing a latent inhibition phenomenon. However, if the conditioned odor has been previously offered diluted in sugared reward, the food odor acts as a stimulus that improves the learning performance during PER conditioning. The native bumblebee B. atratus is thus a new hymenopteran species capable of being trained under controlled experimental conditions. Since it is an insect increasingly reared for pollination service, this knowledge could be useful in its management in crops.

Impaired associative learning after chronic exposure to pesticides in young adult honey bees.

Neonicotinoids are the most widespread insecticides in agriculture, preferred for their low toxicity to mammals and their systemic nature. Nevertheless, there have been increasing concerns regarding their impact on non-target organisms. Glyphosate is also widely used in crops and, therefore, traces of this pesticide are likely to be found together with neonicotinoids. Although glyphosate is considered a herbicide, adverse effects have been found on animal species, including honey bees. Apis mellifera is one of the most important pollinators in agroecosystems and is exposed to both these pesticides. Traces can be found in nectar and pollen of flowers that honey bees visit, but also in honey stores inside the hive. Young workers, which perform in-hive tasks that are crucial for colony maintenance, are potentially exposed to both these contaminated resources. These workers present high plasticity and are susceptible to stimuli that can modulate their behaviour and impact on colony state. Therefore, by performing standardised assays to study sublethal effects of these pesticides, these bees can be used as bioindicators. We studied the effect of chronic joint exposure to field-realistic concentrations of the neonicotinoid imidacloprid and glyphosate on gustatory perception and olfactory learning. Both pesticides reduced sucrose responsiveness and had a negative effect on olfactory learning. Glyphosate also reduced food uptake during rearing. The results indicate differential susceptibility according to honey bee age. The two agrochemicals had adverse effects on different aspects of honey bee appetitive behaviour, which could have repercussions for food distribution, propagation of olfactory information and task coordination within the nest.

An Early Sensitive Period Induces Long-Lasting Plasticity in the Honeybee Nervous System.

The effect of early experiences on the brain during a sensitive period exerts a long-lasting influence on the mature individual. Despite behavioral and neural plasticity caused by early experiences having been reported in the honeybee Apis mellifera, the presence of a sensitive period in which associative experiences lead to pronounced modifications in the adult nervous system is still unclear. Laboratory-reared bees were fed with scented food within specific temporal windows and were assessed for memory retention, in the regulation of gene expression related to the synaptic formation and in the olfactory perception of their antennae at 17 days of age. Bees were able to retain a food-odor association acquired 5-8 days after emergence, but not before, and showed better retention than those exposed to an odor at 9-12 days. In the brain, the odor-rewarded experiences that occurred at 5-8 days of age boosted the expression levels of the cell adhesion proteins neurexin 1 (Nrx1) and neuroligin 2 (Nlg2) involved in synaptic strength. At the antennae, the experiences increased the electrical response to a novel odor but not to the one experienced. Therefore, a sensitive period that induces long-lasting behavioral, functional and structural changes is found in adult honeybees.