Honeybees are well known for their complex division of labor. Each bee sequentially performs a series of social tasks during its life. The changes in social task performance are linked to gross differences in behavior and physiology. We tested whether honeybees performing different social tasks (nursing versus foraging) would differ in their gustatory responsiveness and associative learning behavior in addition to their daily tasks in the colony. Further, we investigated the role of the biogenic amine tyramine and its receptors in the behavior of nurse bees and foragers. Tyramine is an important insect neurotransmitter, which has long been neglected in behavioral studies as it was believed to only act as the metabolic precursor of the better-known amine octopamine. With the increasing number of characterized tyramine receptors in diverse insects, we need to understand the functions of tyramine on its own account. Our findings suggest an important role for tyramine and its two receptors in regulating honeybee gustatory responsiveness, social organization and learning behavior. Foragers, which were more responsive to gustatory stimuli than nurse bees and performed better in appetitive learning, also differed from nurse bees in their tyramine brain titers and in the mRNA expression of a tyramine receptor in the brain. Pharmacological activation of tyramine receptors increased gustatory responsiveness of nurse bees and foragers and improved appetitive learning in nurse bees. These data suggest that a large part of the behavioral differences between honeybees may be directly linked to tyramine signaling in the brain.

The honey bee is an excellent visual learner, but we know little about how and why it performs so well, or how visual information is learned by the bee brain. Here we examined the different roles of two key integrative regions of the brain in visual learning: the mushroom bodies and the central complex. We tested bees' learning performance in a new assay of color learning that used electric shock as punishment. In this assay a light field was paired with electric shock. The other half of the conditioning chamber was illuminated with light of a different wavelength and not paired with shocks. The unrestrained bee could run away from the light stimulus and thereby associate one wavelength with punishment, and the other with safety. We compared learning performance of bees in which either the central complex or mushroom bodies had been transiently inactivated by microinjection of the reversible anesthetic procaine. Control bees learned to escape the shock-paired light field and to spend more time in the safe light field after a few trials. When ventral lobe neurons of the mushroom bodies were silenced, bees were no longer able to associate one light field with shock. By contrast, silencing of one collar region of the mushroom body calyx did not alter behavior in the learning assay in comparison to control treatment. Bees with silenced central complex neurons did not leave the shock-paired light field in the middle trials of training, even after a few seconds of being shocked. We discussed how mushroom bodies and the central complex both contribute to aversive visual learning with an operant component.

Division of labor is a hallmark of social insects. In the honeybee (Apis mellifera) each sterile female worker performs a series of social tasks. The most drastic changes in behavior occur when a nurse bee, who takes care of the brood and the queen in the hive, transitions to foraging behavior. Foragers provision the colony with pollen, nectar or water. Nurse bees and foragers differ in numerous behaviors, including responsiveness to gustatory stimuli. Differences in gustatory responsiveness, in turn, might be involved in regulating division of labor through differential sensory response thresholds. Biogenic amines are important modulators of behavior. Tyramine and octopamine have been shown to increase gustatory responsiveness in honeybees when injected into the thorax, thereby possibly triggering social organization. So far, most of the experiments investigating the role of amines on gustatory responsiveness have focused on the brain. The potential role of the fat body in regulating sensory responsiveness and division of labor has large been neglected. We here investigated the role of the fat body in modulating gustatory responsiveness through tyramine signaling in different social roles of honeybees. We quantified levels of tyramine, tyramine receptor gene expression and the effect of elevating fat body tyramine titers on gustatory responsiveness in both nurse bees and foragers. Our data suggest that elevating the tyramine titer in the fat body pharmacologically increases gustatory responsiveness in foragers, but not in nurse bees. This differential effect of tyramine on gustatory responsiveness correlates with a higher natural gustatory responsiveness of foragers, with a higher tyramine receptor (Amtar1) mRNA expression in fat bodies of foragers and with lower baseline tyramine titers in fat bodies of foragers compared to those of nurse bees. We suggest that differential tyramine signaling in the fat body has an important role in the plasticity of division of labor through changing gustatory responsiveness.

Conventional definitions of drug addiction are focused on characterizing the neurophysiological and behavioral responses of mammals. Although mammalian models have been invaluable in studying specific and complex aspects of addiction, invertebrate systems have proven advantageous in investigating how drugs of abuse corrupt the most basic motivational and neurochemical systems. It has recently been shown that invertebrates and mammals have remarkable similarities in their behavioral and neurochemical responses to drugs of abuse. However, until now only mammals have demonstrated drug seeking and selfadministration without the concurrent presence of a natural reward, e.g. sucrose. Using a sucrose-fading paradigm, followed by a twodish choice test, we establish ants as an invertebrate model of opioid addiction. The ant species Camponotus floridanus actively seeks and self-administers morphine even in the absence of caloric value or additional natural reward. Using HPLC equipped with electrochemical detection, the neurochemicals serotonin, octopamine and dopamine were identified and subsequently quantified, establishing the concurrent neurochemical response to the opioid morphine within the invertebrate brain. With this study, we demonstrate dopamine to be governing opioid addiction in the brains of ants. Thus, this study establishes ants as the first non-mammalian model of selfadministration that is truly analogous to mammals.