Claudia Groh

Behavioral performance in adult honey bees is influenced by the temperature experienced during their pupal development

To investigate the possible consequences of brood-temperature regulation in honey bee colonies on the quality of behavioral performance of adults, we placed honey bee pupae in incubators and allowed them to develop at temperatures held constant at 32°C, 34.5°C, and 36°C. This temperature range occurs naturally within hives. On emergence, the young adult bees were marked and introduced into foster colonies housed in normal and observation hives and allowed to live out their lives. No obvious difference in within-hive behavior was noted between the temperature-treated bees and the foster-colony bees. However, when the temperature-treated bees became foragers and were trained to visit a feeder 200 m from the hive, they exhibited clear differences in dance performance that could be correlated with the temperatures at which they had been raised: bees raised at 32°C completed only ≈20% of the dance circuits when compared with bees of the higher-temperature group. Also, the variance in the duration of the waggle phase is larger in 32°C-raised bees compared with 36°C-raised bees. All other parameters compared across all groups were not significantly different. One-trial learning and memory consolidation in the bees raised at different temperatures was investigated 1 and 10 min after conditioning the proboscis-extension reflex. Bees raised at 36°C performed as expected for bees typically classified as “good learners,” whereas bees raised at 32°C and 34.5°C performed significantly less well. We propose that the temperature at which pupae are raised will influence their behavioral performance as adults and may determine the tasks they carry out best inside and outside the hive.

Synaptic organization in the adult honey bee brain is influenced by brood-temperature control during pupal development

Recent studies have shown that the behavioral performance of adult honey bees is influenced by the temperature experienced during pupal development. Here we explore whether there are temperature-mediated effects on the brain. We raised pupae at different constant temperatures between 29 and 37°C and performed neuroanatomical analyses of the adult brains. Analyses focused on sensory-input regions in the mushroom bodies, brain areas associated with higher-order processing such as learning and memory. Distinct synaptic complexes [microglomeruli (MG)] within the mushroom body calyces were visualized by using fluorophore-conjugated phalloidin and an antibody to synapsin. The numbers of MG were different in bees that had been raised at different temperatures, and these differences persisted after the first week of adult life. In the olfactory-input region (lip), MG numbers were highest in bees raised at the temperature normally maintained in brood cells (34.5°C) and significantly decreased in bees raised at 1°C below and above this norm. Interestingly, in the neighboring visual-input region (collar), MG numbers were less affected by temperature. We conclude that thermoregulatory control of brood rearing can generate area- and modality-specific effects on synaptic neuropils in the adult brain. We propose that resulting differences in the synaptic circuitry may affect neuronal plasticity and may underlie temperature-mediated effects on multimodal communication and learning.

Environment- and Age-Dependent Plasticity of Synaptic Complexes in the Mushroom Bodies of Honeybee Queens

Diversity in behavior plays a crucial role for the division of labor in insect societies. Social insects such as honeybees provide excellent model systems to investigate neuronal principles underlying behavioral plasticity. The two female castes, queens and workers, differ substantially in anatomy, physiology, aging and behavior. The different phenotypes are induced by environmental factors rather than genetic differences. Here we investigated environment- and age-dependent effects on the synaptic organization within higher order neuropils of the honeybee brain. Synaptic complexes (microglomeruli) in sensory-input regions of the mushroom bodies, prominent higher sensory integration centers, were analyzed quantitatively using fluorescent markers and confocal microscopy. Pre- and postsynaptic compartments of individual microglomeruli were labeled by anti-synapsin immunolabeling and f-actin detection with phalloidin in dendritic spines of mushroom-body intrinsic neurons. The results demonstrate that in queens the numbers of microglomeruli in the olfactory and visual input regions of the mushroom-body calyx are significantly lower than in workers. In queens raised in incubators, microglomeruli were affected by differences in pupal rearing temperature within the range of naturally occurring temperatures (32–36°C). The highest numbers of microglomeruli developed at a lower temperature compared to workers (33.5 vs. 34.5°C). We found a striking adult plasticity of microglomeruli numbers throughout the extended life-span of queens. Whereas microglomeruli in the olfactory lip increased with age (∼55%), microglomeruli in the visual collar significantly decreased (∼35%). We propose that developmental and adult plasticity of the synaptic circuitry in the mushroom-body calyx might underlie caste- and age-specific adaptations in behavior.

Comparison of microglomerular structures in the mushroom body calyx of neopteran insects.

Mushroom bodies (MBs) are prominent neuropils in the insect brain involved in higher order processing such as sensory integration, learning and memory, and spatial orientation. The size and general morphology of MBs are diverse across insects. In this study we comparatively investigated the microstructure of synaptic complexes (microglomeruli) in major sensory input regions of the MBs, the calyces, across various neopteran insect species. Pre- and postsynaptic compartments of microglomeruli were analyzed using anti-synapsin immunocytochemistry, f-actin-phalloidin labeling and high-resolution confocal microscopy. Our results suggest that calycal microglomeruli are present across all investigated neopteran insect species, but differences are found in the distribution of synapsin and f-actin within their pre- and postsynaptic compartments. Hymenopteran MBs contain the highest number and packing density of microglomeruli compared to all other species from the different insect orders we investigated. We conclude that the evolution of high numbers of microglomeruli in Hymenoptera may reflect an increase in synaptic microcircuits, which could enhance the computational capacities of the MBs.

Caste-specific postembryonic development of primary and secondary olfactory centers in the female honeybee brain

Eusocial insects are characterized by division of labor among a sterile worker caste and a reproductive queen. In the honeybee both female castes are determined postembryonically by environmental factors, and queens develop substantially faster than workers. Since olfaction plays a crucial role in organizing honeybee behavior and social interactions, we compared the development of primary and secondary olfactory centers in the brain. Age-synchronized queen and worker pupae were raised in incubators at 34.5 degrees C, and their external morphology was characterized for all pupal stages. The development of olfactory synaptic neuropil was analyzed using anti-synapsin immunocytochemistry, f-actin-phalloidin labeling and confocal microscopy. In the antennal lobes of queens olfactory glomeruli formed approximately 4 days earlier than in workers. The adult number of olfactory glomeruli was in a similar range, but the total glomerular volume was slightly smaller in queens. Olfactory and visual subdivisions (lip, collar) of the mushroom-body calyx formed early, whereas the basal ring separated late. Synaptic microglomeruli in the olfactory lip were established approximately 3-4 days earlier in queens compared to workers. We propose that developmental heterochrony results in fewer synapses in olfactory centers (smaller glomeruli, fewer microglomeruli) in queens, which may result in poorer performance on olfactory learning tasks compared to workers.

Age‐related and light‐induced plasticity in opsin gene expression and in primary and secondary visual centers of the nectar‐feeding ant Camponotus rufipes

Camponotus rufipes workers are characterized by an age‐related polyethism. In the initial weeks of adult life, young workers perform tasks inside the nest before they switch to multimodal foraging tasks outside. We tested the hypothesis that this transition is accompanied by profound adaptations in the peripheral and central visual systems. Our results show that C. rufipes workers of all tested ages (between 1 and 42 days) express three genes encoding for ultraviolet (UV), blue (BL), and long‐wavelength (LW1) sensitive opsins in their retina, which are likely to provide the substrate for trichromatic color vision. Expression levels of all three opsin genes increased significantly within the first two weeks of adulthood and following light exposure. Interestingly, the volumes of all three optic neuropils (lamina, medulla, and lobula) showed corresponding volume increases. Tracing of connections to higher visual centers in the mushroom bodies (MBs) revealed only one optic pathway, the anterior superior optic tract, emerging from the medulla and sending segregated input to the MB‐calyx collar. The MB collar volumes and densities of synaptic complexes (microglomeruli, MGs) increased with age. Exposure to light for 4 days induced a decrease in MG densities followed by an increase after extended light exposure. This shows that plasticity in retinal opsin gene expression and structural neuroplasticity in primary and secondary visual centers comprise both “experience‐independent” and “experience‐dependent” elements. We conclude that both sources of plasticity in the visual system represent important components promoting optimal timing of the interior–forager transition and flexibility of age‐related division of labor.

Neuronal plasticity in the mushroom body calyx during adult maturation in the honeybee and possible pheromonal influences.

Honeybee workers express a pronounced age‐dependent polyethism switching from various indoor duties to foraging outside the hive. This transition is accompanied by tremendous changes in the sensory environment that sensory systems and higher brain centers have to cope with. Foraging and age have earlier been shown to be associated with volume changes in the mushroom bodies (MBs). Using age‐ and task‐controlled bees this study provides a detailed framework of neuronal maturation processes in the MB calyx during the course of natural behavioral maturation. We show that the MB calyx volume already increases during the first week of adult life. This process is mainly driven by broadening of the Kenyon cell dendritic branching pattern and then followed by pruning of projection neuron axonal boutons during the actual transition from indoor to outdoor duties. To further investigate the flexible regulation of division of labor and its neuronal correlates in a honeybee colony, we studied the modulation of the nurse‐forager transition via a chemical communication system, the primer pheromone ethyl oleate (EO). EO is found at high concentrations on foragers in contrast to nurse bees and was shown to delay the onset of foraging. In this study, EO effects on colony behavior were not as robust as expected, and we found no direct correlation between EO treatment and synaptic maturation in the MB calyx. In general, we assume that the primer pheromone EO rather acts in concert with other factors influencing the onset of foraging with its effect being highly adaptive.

Plasticity of Synaptic Microcircuits in the Mushroom-Body Calyx of the Honey Bee

Mushroom bodies (MBs) are prominent neuropils in the insect brain that have been implicated in higher order processing such as sensory integration, learning and memory, and spatial orientation. Hymenoptera, like the honey bee, possess particularly large MBs with doubled MB calyces (major sensory input structures of the MBs) that are divided into compartments. In this review we focus on characteristic modular synaptic complexes (microglomeruli, MG) in the honey bee MB calyx (CA). The main components of MG comprise a presynaptic bouton from projection neurons (PNs) (e.g. olfactory, visual), numerous dendritic spines from MB intrinsic neurons (Kenyon cells, KC), and processes from recurrent GABAergic neurons. Recent work has demonstrated a remarkable structural plasticity of MG associated with postembryonic brood care, age, sensory experience, and stable long-term memory. The mechanisms and functional significance of this neuronal plasticity are discussed and related to behavioral plasticity and social organization.

Wax perception in honeybees: contact is not necessary

In social insects, much progress has been made in identifying variations in the cuticular signatures of sexes, castes, kin and reproductive status. In contrast to this, we still do not know how the receivers perceive these recognition cues. This study was designed to investigate whether honeybees use contact-chemosensory or olfactory sensilla to perceive wax components. To answer this question in a behavioral assay, we combined classical conditioning of the proboscis extension reaction and a recently established method using zinc sulfate to selectively block antennal contact-chemosensory sensilla. Comparison of the responses to sucrose, wax and geraniol before and after antennal zinc sulfate treatment revealed that the sucrose response is lost after treatment but the responses to wax and geraniol are maintained. As sucrose is perceived by the contact-chemosensory sensilla, the retention of the wax response indicates that contact-chemosensory sensilla are not necessary for wax perception.

Density of mushroom body synaptic complexes limits intraspecies brain miniaturization in highly polymorphic leaf-cutting ant workers

Hymenoptera possess voluminous mushroom bodies (MBs), brain centres associated with sensory integration, learning and memory. The mushroom body input region (calyx) is organized in distinct synaptic complexes (microglomeruli, MG) that can be quantified to analyse body size-related phenotypic plasticity of synaptic microcircuits in these small brains. Leaf-cutting ant workers (Atta vollenweideri) exhibit an enormous size polymorphism, which makes them outstanding to investigate neuronal adaptations underlying division of labour and brain miniaturization. We particularly asked how size-related division of labour in polymorphic workers is reflected in volume and total numbers of MG in olfactory calyx subregions. Whole brains of mini, media and large workers were immunolabelled with anti-synapsin antibodies, and mushroom body volumes as well as densities and absolute numbers of MG were determined by confocal imaging and three-dimensional analyses. The total brain volume and absolute volumes of olfactory mushroom body subdivisions were positively correlated with head widths, but mini workers had significantly larger MB to total brain ratios. Interestingly, the density of olfactory MG was remarkably independent from worker size. Consequently, absolute numbers of olfactory MG still were approximately three times higher in large compared with mini workers. The results show that the maximum packing density of synaptic microcircuits may represent a species-specific limit to brain miniaturization.