While the ability of honeybees to navigate relying on sky-compass information has been investigated in a large number of behavioral studies, the underlying neuronal system has so far received less attention. The sky-compass pathway has recently been described from its input region, the dorsal rim area (DRA) of the compound eye, to the anterior optic tubercle (AOTU). The aim of this study is to reveal the connection from the AOTU to the central complex (CX). For this purpose, we investigated the anatomy of large microglomerular synaptic complexes in the medial and lateral bulbs (MBUs/LBUs) of the lateral complex (LX). The synaptic complexes are formed by tubercle-lateral accessory lobe neuron 1 (TuLAL1) neurons of the AOTU and GABAergic tangential neurons of the central body’s (CB) lower division (TL neurons). Both TuLAL1 and TL neurons strongly resemble neurons forming these complexes in other insect species. We further investigated the ultrastructure of these synaptic complexes using transmission electron microscopy. We found that single large presynaptic terminals of TuLAL1 neurons enclose many small profiles (SPs) of TL neurons. The synaptic connections between these neurons are established by two types of synapses: divergent dyads and divergent tetrads. Our data support the assumption that these complexes are a highly conserved feature in the insect brain and play an important role in reliable signal transmission within the sky-compass pathway.

Honeybees show a remarkable behavioral plasticity at the transition from nursing inside the hive to foraging for nectar and/or pollen outside. This plasticity is important for age-related division of labor in honeybee colonies. The behavioral transition is associated with significant volume and synaptic changes in the mushroom bodies (MBs), brain centers for sensory integration, learning, and memory. We tested whether precocious sensory exposure to light leads to changes in the density of synaptic complexes [microglomeruli (MG)] in the MBs. The results show that exposure to light pulses over 3 days induces a significant decrease in the MG density in visual subregions (collar) of the MB. Earlier studies had shown that foragers have increased levels of juvenile hormone (JH) co-occurring with a decrease of vitellogenin (Vg). Previous work further established that RNAi-mediated knockdown of vg and ultraspiracle (usp) induced an upregulation of JH levels, which can lead to precocious foraging. By disturbing both Vg and JH pathways using gene knockdown of vg and usp, we tested whether the changes in the hormonal system directly affect MG densities. Our study shows that MG numbers remained unchanged when Vg and JH pathways were perturbed, suggesting no direct hormonal influences on MG densities. However, mass spectrometry detection of JH revealed that precocious light exposure triggered an increase in JH levels in the hemolymph (HL) of young bees. This suggests a dual effect following light exposure via direct effects on MG reorganization in the MB calyx and a possible positive feedback on HL JH levels.

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.

Honeybees are able to solve complex learning tasks and memorize learned information for long time periods. The molecular mechanisms mediating long-term memory (LTM) in the honeybee Apis mellifera are, to a large part, still unknown. We approached this question by investigating the potential function of the calcium/calmodulindependent protein kinase II (CaMKII), an enzyme known as a 'molecular memory switch' in vertebrates. CaMKII is able to switch to a calcium-independent constitutively active state, providing a mechanism for a molecular memory and has further been shown to play an essential role in structural synaptic plasticity. Using a combination of knockdown by RNA interference and pharmacological manipulation, we disrupted the function of CaMKII during olfactory learning and memory formation. We found that learning, memory acquisition and mid-term memory were not affected, but all manipulations consistently resulted in an impaired LTM. Both early LTM (24 h after learning) and late LTM (72 h after learning) were significantly disrupted, indicating the necessity of CaMKII in two successive stages of LTM formation in the honeybee.

Tyramine is an important neurotransmitter, neuromodulator, and neurohormone in insects. In honeybees, it is assumed to have functions in modulating sensory responsiveness and controlling motor behavior. Tyramine can bind to two characterized receptors in honeybees, both of which are coupled to intracellular cAMP pathways. How tyramine acts on neuronal, cellular and circuit levels is unclear. We investigated the spatial brain expression of the tyramine receptor AmTAR1 using a specific antibody. This antibody detects a membrane protein of the expected molecular weight in western blot analysis. In honeybee brains, it labels different structures which process sensory information. Labeling along the antennal nerve, in projections of the dorsal lobe and in the gnathal ganglion suggest that tyramine receptors are involved in modulating gustatory and tactile perception. Furthermore, the ellipsoid body of the central complex and giant synapses in the lateral complex show AmTAR1-like immunoreactivity (AmTAR1-IR), suggesting a role of this receptor in modulating sky-compass information and/or higher sensor-motor control. Additionally, intense signals derive from the mushroom bodies, higher-order integration centers for olfactory, visual, gustatory and tactile information. To investigate whether AmTAR1-expressing brain structures are in vicinity to tyramine releasing sites, a specific tyramine antibody was applied. Tyramine-like labeling was observed in AmTAR1-IR positive structures, although it was sometimes weak and we did not always find a direct match of ligand and receptor. Moreover, tyramine-like immunoreactivity was also found in brain regions without AmTAR1-IR (optic lobes, antennal lobes), indicating that other tyramine-specific receptors may be expressed there.

Specific experimental protocols necessitate transportation, a potentially stressful event that could confound results. We determined adrenocortical activity by measuring fecal corticosterone metabolites (FCMs), as a stress marker, in prepuberal (three-week old) female C57BL/6J, C57BL/6NCrl, FVB/NCrl, Crl:CD1(ICR), and BALB/cAnCrl mice. On each transport day, five female cage mates per genetic background were weaned and transported in stable groups via truck from the breeding to the research facility. Fecal pellets were collected on Days 0, 1, and 4. Mice were superovulated for embryo production to determine if repeated fecal collection impacts this procedure. The average duration of transportation over 600 km and from packing to unpacking of mice was 7.24 and 22.62 h, respectively. FCM levels increased from Day 0 to Day 1 and decreased on Day 4 in all genetic backgrounds except in FVB/NCrl, but only B6N showed significantly higher FCM levels on Day 1. Furthermore, embryo production was not affected by repeated feces collection. The results show that weaning and immediate transport of prepuberal mice from the breeding to the research facility led to temporal and genetic background-dependent increases of adrenocortical activity in four of the five genetic backgrounds investigated, which returned to baseline levels within four days.

Tyramine is an important neurotransmitter, neuromodulator, and neurohormone in insects. In honeybees, it is assumed to have functions in modulating sensory responsiveness and controlling motor behavior. Tyramine can bind to two characterized receptors in honeybees, both of which are coupled to intracellular cAMP pathways. How tyramine acts on neuronal, cellular and circuit levels is unclear. We investigated the spatial brain expression of the tyramine receptor AmTAR1 using a specific antibody. This antibody detects a membrane protein of the expected molecular weight in western blot analysis. In honeybee brains, it labels different structures which process sensory information. Labeling along the antennal nerve, in projections of the dorsal lobe and in the gnathal ganglion suggest that tyramine receptors are involved in modulating gustatory and tactile perception. Furthermore, the ellipsoid body of the central complex and giant synapses in the lateral complex show AmTAR1‐like immunoreactivity (AmTAR1‐IR), suggesting a role of this receptor in modulating sky‐compass information and/or higher sensor‐motor control. Additionally, intense signals derive from the mushroom bodies, higher‐order integration centers for olfactory, visual, gustatory and tactile information. To investigate whether AmTAR1‐expressing brain structures are in vicinity to tyramine releasing sites, a specific tyramine antibody was applied. Tyramine‐like labeling was observed in AmTAR1‐IR positive structures, although it was sometimes weak and we did not always find a direct match of ligand and receptor. Moreover, tyramine‐like immunoreactivity was also found in brain regions without AmTAR1‐IR (optic lobes, antennal lobes), indicating that other tyramine‐specific receptors may be expressed there.