Mechanisms and rules of social learning in crickets.

A new study on insect social learning shows that crickets learn to prefer a rewarded odorant by observing the choice of a conspecific and without experiencing the reward themselves. The mere perception of the conspecific activates octopaminergic reward neurons in the brain of the observer, thus facilitating odorant learning.

The Neural Signature of Visual Learning Under Restrictive Virtual-Reality Conditions.

Honey bees are reputed for their remarkable visual learning and navigation capabilities. These capacities can be studied in virtual reality (VR) environments, which allow studying performances of tethered animals in stationary flight or walk under full control of the sensory environment. Here, we used a 2D VR setup in which a tethered bee walking stationary under restrictive closed-loop conditions learned to discriminate vertical rectangles differing in color and reinforcing outcome. Closed-loop conditions restricted stimulus control to lateral displacements. Consistently with prior VR analyses, bees learned to discriminate the trained stimuli. Ex vivo analyses on the brains of learners and non-learners showed that successful learning led to a downregulation of three immediate early genes in the main regions of the visual circuit, the optic lobes (OLs) and the calyces of the mushroom bodies (MBs). While Egr1 was downregulated in the OLs, Hr38 and kakusei were coincidently downregulated in the calyces of the MBs. Our work thus reveals that color discrimination learning induced a neural signature distributed along the sequential pathway of color processing that is consistent with an inhibitory trace. This trace may relate to the motor patterns required to solve the discrimination task, which are different from those underlying pathfinding in 3D VR scenarios allowing for navigation and exploratory learning and which lead to IEG upregulation.

Visual learning in a virtual reality environment upregulates immediate early gene expression in the mushroom bodies of honey bees.

Free-flying bees learn efficiently to solve numerous visual tasks. Yet, the neural underpinnings of this capacity remain unexplored. We used a 3D virtual reality (VR) environment to study visual learning and determine if it leads to changes in immediate early gene (IEG) expression in specific areas of the bee brain. We focused on kakusei, Hr38 and Egr1, three IEGs that have been related to bee foraging and orientation, and compared their relative expression in the calyces of the mushroom bodies, the optic lobes and the rest of the brain after color discrimination learning. Bees learned to discriminate virtual stimuli displaying different colors and retained the information learned. Successful learners exhibited Egr1 upregulation only in the calyces of the mushroom bodies, thus uncovering a privileged involvement of these brain regions in associative color learning and the usefulness of Egr1 as a marker of neural activity induced by this phenomenon.

Inhibitory learning of phototaxis by honeybees in a passive-avoidance task.

Honeybees are a standard model for the study of appetitive learning and memory. Yet, fewer attempts have been performed to characterize aversive learning and memory in this insect and uncover its molecular underpinnings. Here, we took advantage of the positive phototactic behavior of bees kept away from the hive in a dark environment and established a passive-avoidance task in which they had to suppress positive phototaxis. Bees placed in a two-compartment box learned to inhibit spontaneous attraction to a compartment illuminated with blue light by associating and entering into that chamber with shock delivery. Inhibitory learning resulted in an avoidance memory that could be retrieved 24 h after training and that was specific to the punished blue light. The memory was mainly operant but involved a Pavlovian component linking the blue light and the shock. Coupling conditioning with transcriptional analyses in key areas of the brain showed that inhibitory learning of phototaxis leads to an up-regulation of the dopaminergic receptor gene Amdop1 in the calyces of the mushroom bodies, consistently with the role of dopamine signaling in different forms of aversive learning in insects. Our results thus introduce new perspectives for uncovering further cellular and molecular underpinnings of aversive learning and memory in bees. Overall, they represent an important step toward comparative learning studies between the appetitive and the aversive frameworks.

A Tyrosine-Hydroxylase Characterization of Dopaminergic Neurons in the Honey Bee Brain.

Dopamine (DA) plays a fundamental role in insect behavior as it acts both as a general modulator of behavior and as a value system in associative learning where it mediates the reinforcing properties of unconditioned stimuli (US). Here we aimed at characterizing the dopaminergic neurons in the central nervous system of the honey bee, an insect that serves as an established model for the study of learning and memory. We used tyrosine hydroxylase (TH) immunoreactivity (ir) to ensure that the neurons detected synthesize DA endogenously. We found three main dopaminergic clusters, C1-C3, which had been previously described; the C1 cluster is located in a small region adjacent to the esophagus (ES) and the antennal lobe (AL); the C2 cluster is situated above the C1 cluster, between the AL and the vertical lobe (VL) of the mushroom body (MB); the C3 cluster is located below the calyces (CA) of the MB. In addition, we found a novel dopaminergic cluster, C4, located above the dorsomedial border of the lobula, which innervates the visual neuropils of the bee brain. Additional smaller processes and clusters were found and are described. The profuse dopaminergic innervation of the entire bee brain and the specific connectivity of DA neurons, with visual, olfactory and gustatory circuits, provide a foundation for a deeper understanding of how these sensory modules are modulated by DA, and the DA-dependent value-based associations that occur during associative learning.

Monoterpenoid-based preparations in beehives affect learning, memory, and gene expression in the bee brain.

Bees are exposed in their environment to contaminants that can weaken the colony and contribute to bee declines. Monoterpenoid-based preparations can be introduced into hives to control the parasitic mite Varroa destructor. The long-term effects of monoterpenoids are poorly investigated. Olfactory conditioning of the proboscis extension reflex (PER) has been used to evaluate the impact of stressors on cognitive functions of the honeybee such as learning and memory. The authors tested the PER to odorants on bees after exposure to monoterpenoids in hives. Octopamine receptors, transient receptor potential-like (TRPL), and γ-aminobutyric acid channels are thought to play a critical role in the memory of food experience. Gene expression levels of Amoa1, Rdl, and trpl were evaluated in parallel in the bee brain because these genes code for the cellular targets of monoterpenoids and some pesticides and neural circuits of memory require their expression. The miticide impaired the PER to odors in the 3 wk following treatment. Short-term and long-term olfactory memories were improved months after introduction of the monoterpenoids into the beehives. Chronic exposure to the miticide had significant effects on Amoa1, Rdl, and trpl gene expressions and modified seasonal changes in the expression of these genes in the brain. The decrease of expression of these genes in winter could partly explain the improvement of memory. The present study has led to new insights into alternative treatments, especially on their effects on memory and expression of selected genes involved in this cognitive function. Environ Toxicol Chem 2017;36:337-345. © 2016 SETAC.

Prolonged effects of in-hive monoterpenoids on the honey bee Apis mellifera.

Honey bees are exposed in their environment to contaminants but also to biological stressors such as Varroa destructor that can weaken the colony. Preparations containing monoterpenoids that are essential oil components, can be introduced into hives to control Varroa. The long-term sublethal effects of monoterpenoids used as miticides have been poorly investigated. Analysis of behavior of free-moving bees in the laboratory is useful to evaluate the impact of chemical stressors on their cognitive functions such as vision function. Here, the walking behavior was quantified under a 200-lux light intensity. Weeks and months after introduction of the miticide (74 % thymol) into the hives, decreases of phototaxis was observed with both summer and winter bees. Curiously, in spring, bees collected in treated hives were less attracted by light in the morning than control bees. The survival of bees collected in spring was increased by treatment. After a 1-year period of observation, the colony losses were identical in treated and non-treated groups. Colony loss started earlier in the non-treated group. In public opinion, natural substances as essential oils are safer and more environmentally friendly. We demonstrated that a monoterpenoid-based treatment affects bee responses to light. The latter results have notable implications regarding the evaluation of miticides in beekeeping.

Honey Bee Allatostatins Target Galanin/Somatostatin-Like Receptors and Modulate Learning: A Conserved Function?

Sequencing of the honeybee genome revealed many neuropeptides and putative neuropeptide receptors, yet functional characterization of these peptidic systems is scarce. In this study, we focus on allatostatins, which were first identified as inhibitors of juvenile hormone synthesis, but whose role in the adult honey bee (Apis mellifera) brain remains to be determined. We characterize the bee allatostatin system, represented by two families: allatostatin A (Apime-ASTA) and its receptor (Apime-ASTA-R); and C-type allatostatins (Apime-ASTC and Apime-ASTCC) and their common receptor (Apime-ASTC-R). Apime-ASTA-R and Apime-ASTC-R are the receptors in bees most closely related to vertebrate galanin and somatostatin receptors, respectively. We examine the functional properties of the two honeybee receptors and show that they are transcriptionally expressed in the adult brain, including in brain centers known to be important for learning and memory processes. Thus we investigated the effects of exogenously applied allatostatins on appetitive olfactory learning in the bee. Our results show that allatostatins modulate learning in this insect, and provide important insights into the evolution of somatostatin/allatostatin signaling.

Fasting increases survival to cold in FOXO, DIF, autophagy mutants and in other genotypes of Drosophila melanogaster.

Fasting increases survival to a severe cold stress in young and middle-aged wild-type flies, this effect being lowered or absent at old age. As an attempt to determine the mechanisms of this effect, genes involved in metabolism (dFOXO), autophagy (Atg7), innate immunity (Dif (1) ), and resistance to cold (Frost) were studied. The 12 mutant, RNAi and control lines tested in this study displayed an increased survival to cold after fasting. This shows that fasting has a robust effect on survival to cold in many genotypes, but the mechanism of this effect remains unknown. This mechanism does not seem to be linked to metabolic pathways often considered to play a critical role in ageing and longevity determinations (insulin/insulin-like growth factor-1 pathway and autophagy).

Differential involvement of glutamate-gated chloride channel splice variants in the olfactory memory processes of the honeybee Apis mellifera.

Glutamate-gated chloride channels (GluCl) belong to the cys-loop ligand-gated ion channel superfamily and their expression had been described in several invertebrate nervous systems. In the honeybee, a unique gene amel_glucl encodes two alternatively spliced subunits, Amel_GluCl A and Amel_GluCl B. The expression and differential localization of those variants in the honeybee brain had been previously reported. Here we characterized the involvement of each variant in olfactory learning and memory processes, using specific small-interfering RNA (siRNA) targeting each variant. Firstly, the efficacy of the two siRNAs to decrease their targets' expression was tested, both at mRNA and protein levels. The two proteins showed a decrease of their respective expression 24h after injection. Secondly, each siRNA was injected into the brain to test whether or not it affected olfactory memory by using a classical paradigm of conditioning the proboscis extension reflex (PER). Amel_GluCl A was found to be involved only in retrieval of 1-nonanol, whereas Amel_GluCl B was involved in the PER response to 2-hexanol used as a conditioned stimulus or as new odorant. Here for the first time, a differential behavioral involvement of two highly similar GluCl subunits has been characterized in an invertebrate species.

The NF-κB-like factor DIF could explain some positive effects of a mild stress on longevity, behavioral aging, and resistance to strong stresses in Drosophila melanogaster.

A mild cold stress can have positive effects on longevity, aging and resistance to severe stresses in flies (heat, cold, fungal infection), but the causes of these effects remain elusive. In order to know whether these effects could be explained by the DIF transcription factor (a NF-κB-like factor in the Toll innate immunity pathway), the Dif ( 1 ) mutant and its control cn bw strain were subjected to a pretreatment by cold. The DIF factor seems to be involved in the response to fungal infection after a mild cold stress and in the resistance to heat. However, DIF seems to have no role in the increased longevity of non-infected flies and resistance to a severe cold shock, because the cold pretreatment slightly increased longevity in females, mainly in Dif ( 1 ) ones, and resistance to a long cold shock in both sexes of these strains.

Cold stress increases resistance to fungal infection throughout life in Drosophila melanogaster.

Flies were subjected to one of three mild stresses known to have positive effects on longevity (heat, hypergravity, cold), prior to an infection with the entomopathogenic fungus Beauveria bassiana. Flies subjected to cold survived longer to infection, while the other mild stresses had no positive effect. These positive effects of a cold stress on resistance to infection were observed mainly in males and throughout life, i.e., a long time after the cold stress was applied. It was confirmed that cold and hypergravity stresses increased longevity of non-infected flies, but no positive effect of heat shocks were however observed.

In situ hybridization assays for localization of the chronic bee paralysis virus in the honey bee (Apis mellifera) brain.

Chronic bee paralysis virus (CBPV) is a common single-stranded RNA virus which may cause significant losses in honey bee colonies. As this virus seems to exhibit neurotropism, an in situ hybridization based method was developed to localize the genomic and antigenomic CBPV RNAs in infected honey bee brains. Double-stranded cDNA probes as well as genomic and antigenomic-specific single-stranded cDNA probes were prepared, using the polymerase chain reaction in presence of labelled d-UTP with non-radioactive digoxigenin. Both genomic and antigenomic RNAs were detected the brain of honey bee infected naturally or artificially. Hybridization signals were obtained in some somata and neuropile regions of the brain. In particular, high signals were observed at the level of the mushroom bodies and central complex, regions that are known to be engaged in higher neuronal functions and in the optic and antennal lobes that are sensorial neuropiles. Thus, the presence of virus at these levels may explain the nervous symptoms observed in infected bees. The in situ hybridization procedure proved to be a useful tool to localize specifically CBPV and may be helpful for understanding the observed symptoms.