The Importance of Time and Place: Nutrient Composition and Utilization of Seasonal Pollens by European Honey Bees (Apis mellifera L.).

Honey bee colonies have a yearly cycle that is supported nutritionally by the seasonal progression of flowering plants. In the spring, colonies grow by rearing brood, but in the fall, brood rearing declines in preparation for overwintering. Depending on where colonies are located, the yearly cycle can differ especially in overwintering activities. In temperate climates of Europe and North America, colonies reduce or end brood rearing in the fall while in warmer climates bees can rear brood and forage throughout the year. To test the hypothesis that nutrients available in seasonal pollens and honey bee responses to them can differ we analyzed pollen in the spring and fall collected by colonies in environments where brood rearing either stops in the fall (Iowa) or continues through the winter (Arizona). We fed both types of pollen to worker offspring of queens that emerged and open mated in each type of environment. We measured physiological responses to test if they differed depending on the location and season when the pollen was collected and the queen line of the workers that consumed it. Specifically, we measured pollen and protein consumption, gene expression levels (hex 70, hex 110, and vg) and hypopharyngeal gland (HPG) development. We found differences in macronutrient content and amino and fatty acids between spring and fall pollens from the same location and differences in nutrient content between locations during the same season. We also detected queen type and seasonal effects in HPG size and differences in gene expression between bees consuming spring vs. fall pollen with larger HPG and higher gene expression levels in those consuming spring pollen. The effects might have emerged from the seasonal differences in nutritional content of the pollens and genetic factors associated with the queen lines we used.

Can supplementary pollen feeding reduce varroa mite and virus levels and improve honey bee colony survival?

Varroa destructor is an ectoparasitic mite of immature and adult honey bees that can transmit several single-stranded RNA viruses to its host. Varroa reproduce in brood cells, and mite populations increase as colonies produce brood in spring and summer. Mite numbers also can sharply rise, particularly in the fall, by the migration of varroa into hives on foragers. Colonies with high levels of varroa and viruses often die over the winter. Feeding colonies pollen might keep virus levels low and improve survival because of the positive effects of pollen on immunity and colony growth. We compared varroa and virus levels and overwinter survival in colonies with (fed) and without (unfed) supplemental pollen. We also measured the frequency of capturing foragers with mites (FWM) at colony entrances to determine its relationship to varroa and virus levels. Colonies fed supplemental pollen were larger than unfed colonies and survived longer. Varroa populations and levels of Deformed wing virus (DWV) rose throughout the season, and were similar between fed and unfed colonies. The growth of varroa populations was correlated with FWM in fed and unfed colonies, and significantly affected DWV levels. Increasing frequencies of FWM and the effects on varroa populations might reduce the positive influence of supplemental pollen on immune function. However, pollen feeding can stimulate colony growth and this can improve colony survival.

Pollen Alters Amino Acid Levels in the Honey Bee Brain and This Relationship Changes With Age and Parasitic Stress.

Pollen nutrition is necessary for proper growth and development of adult honey bees. Yet, it is unclear how pollen affects the honey bee brain and behavior. We investigated whether pollen affects amino acids in the brains of caged, nurse-aged bees, and what the behavioral consequences might be. We also tested whether parasitic stress altered this relationship by analyzing bees infected with prevalent stressor, Nosema ceranae. Levels of 18 amino acids in individual honey bee brains were measured using Gas Chromatography - Mass Spectrometry at two different ages (Day 7 and Day 11). We then employed the proboscis extension reflex to test odor learning and memory. We found that the honey bee brain was highly responsive to pollen. Many amino acids in the brain were elevated and were present at higher concentration with age. The majority of these amino acids were non-essential. Without pollen, levels of amino acids remained consistent, or declined. Nosema-infected bees showed a different profile. Infection altered amino acid levels in a pollen-dependent manner. The majority of amino acids were lower when pollen was given, but higher when pollen was deprived. Odor learning and memory was not affected by feeding pollen to uninfected bees; but pollen did improve performance in Nosema-infected bees. These results suggest that pollen in early adulthood continues to shape amino acid levels in the brain with age, which may affect neural circuitry and behavior over time. Parasitic stress by N. ceranae modifies this relationship revealing an interaction between infection, pollen nutrition, and behavior.

Smoke Conditions Affect the Release of the Venom Droplet Accompanying Sting Extension in Honey Bees (Hymenoptera: Apidae).

Honey bees (Apis mellifera) (Hymenoptera: Apidae) are social insects that have evolved a coordinated defensive response to ensure colony survival. Their nests may contain valuable resources such as pollen and nectar that are attractive to a range of insect and mammalian intruders and need protecting. With sufficient provocation, honey bees will mobilize and sting intruders, who are likely to incur additional stings. To inspect and manage their colonies, beekeepers apply smoke to decrease the likelihood of being stung. The use of smoke is a ubiquitous beekeeping practice, but the reasons behind its efficacy remain unknown. In this study, we examined the effects of smoke on honey bee defensive behavior by assessing individual sting extension responses under smoke conditions. We applied a brief voltage to the bee, ranging from a mild to a strong perturbation, and assessed four components of the sting extension reflex using two types of smoke. We found that smoke did not influence the probability of sting extension, but it did affect whether a venom droplet was released with the stinger. The venom droplet was more likely to be released at higher voltage levels, but this effect was significantly reduced under smoke conditions. Based on these results, we propose that the venom droplet coincides with greater agitation in individual bees; and smoke reduces the probability of its release. We speculate that the venom droplet serves to amplify the sting alarm pheromone, and smoke, in its ability to reduce droplet formation, may indicate that less alarm pheromone is released.

Connecting the nutrient composition of seasonal pollens with changing nutritional needs of honey bee (Apis mellifera L.) colonies.

Free-ranging herbivores have yearly life cycles that generate dynamic resource needs. Honey bee colonies also have a yearly life cycle that might generate nutritional requirements that differ between times of brood rearing and colony expansion in the spring and population contraction and preparation for overwintering in the fall. To test this, we analyzed polyfloral mixes of spring and fall pollens to determine if the nutrient composition differed with season. Next, we fed both types of seasonal pollens to bees reared in spring and fall. We compared the development of brood food glands (i.e., hypopharyngeal glands - HPG), and the expression of genes in the fat body between bees fed pollen from the same (in-season) or different season (out-of-season) when they were reared. Because pathogen challenges often heighten the effects of nutritional stress, we infected a subset of bees with Nosema to determine if bees responded differently to the infection depending on the seasonal pollen they consumed. We found that spring and fall pollens were similar in total protein and lipid concentrations, but spring pollens had higher concentrations of amino and fatty acids that support HPG growth and brood production. Bees responded differently when fed in vs. out of season pollen. The HPG of both uninfected and Nosema-infected spring bees were larger when they were fed spring (in-season) compared to fall pollen. Spring bees differentially regulated more than 200 genes when fed in- vs. out-of-season pollen. When infected with Nosema, approximately 400 genes showed different infection-induced expression patterns in spring bees depending on pollen type. In contrast, HPG size in fall bees was not affected by pollen type, though HPG were smaller in those infected with Nosema. Very few genes were differentially expressed with pollen type in uninfected (4 genes) and infected fall bees (5 genes). Pollen type did not affect patterns of infection-induced expression in fall bees. Our data suggest that physiological responses to seasonal pollens differ between bees reared in the spring and fall with spring bees being significantly more sensitive to pollen type especially when infected with Nosema. This study provides evidence that seasonal pollens may provide levels of nutrients that align with the activities of honey bees during their yearly colony cycle. The findings are important for the planning and establishment of forage plantings to sustain honey bees, and in the development of seasonal nutritional supplements fed to colonies when pollen is unavailable.

Nosema ceranae parasitism impacts olfactory learning and memory and neurochemistry in honey bees (Apis mellifera).

Nosema sp. is an internal parasite of the honey bee, Apis mellifera, and one of the leading contributors to colony losses worldwide. This parasite is found in the honey bee midgut and has profound consequences for the host's physiology. Nosema sp. impairs foraging performance in honey bees, yet, it is unclear whether this parasite affects the bee's neurobiology. In this study, we examined whether Nosema sp. affects odor learning and memory and whether the brains of parasitized bees show differences in amino acids and biogenic amines. We took newly emerged bees and fed them with Nosema ceranae At approximate nurse and forager ages, we employed an odor-associative conditioning assay using the proboscis extension reflex and two bioanalytical techniques to measure changes in brain chemistry. We found that nurse-aged bees infected with N. ceranae significantly outperformed controls in odor learning and memory, suggestive of precocious foraging, but by forager age, infected bees showed deficits in learning and memory. We also detected significant differences in amino acid concentrations, some of which were age specific, as well as altered serotonin, octopamine, dopamine and l-dopa concentrations in the brains of parasitized bees. These findings suggest that N. ceranae infection affects honey bee neurobiology and may compromise behavioral tasks. These results yield new insight into the host-parasite dynamic of honey bees and N. ceranae, as well as the neurochemistry of odor learning and memory under normal and parasitic conditions.

The role of nitric oxide in memory is modulated by diurnal time.

Nitric oxide (NO) is thought to play an important neuromodulatory role in the olfactory system. This modulation has been suggested to be particularly important for olfactory learning and memory in the antennal lobe (the primary olfactory network in invertebrates). We are using the hawkmoth, Manduca sexta, to further investigate the role of NO in olfactory memory. Recent findings suggest that NO affects short-term memory traces and that NO concentration fluctuates with the light cycle. This gives rise to the hypothesis that NO may be involved in the connection between memory and circadian rhythms. In this study, we explore the role of diurnal time and NO in memory by altering the time of day when associative-olfactory conditioning is performed. We find a strong effect of NO on short-term memory, and two surprising effects of diurnal time. We find that (1) at certain time points, NO affects longer traces of memory in addition to short-term memory; and (2) when conditioning is performed close to the light cycle switches-both from light to dark and dark to light-NO does not significantly affect memory at all. These findings suggest an intriguing functional role for NO in olfactory conditioning that is modulated as a function of diurnal time.

Nitric oxide affects short-term olfactory memory in the antennal lobe of Manduca sexta.

Nitric oxide (NO) is thought to play an important neuromodulatory role in olfaction. We are using the hawkmoth Manduca sexta to investigate the function of NO signaling in the antennal lobe (AL; the primary olfactory network in invertebrates). We have found previously that NO is present at baseline levels, dramatically increases in response to odor stimulation, and alters the electrophysiology of AL neurons. It is unclear, however, how these effects contribute to common features of olfactory systems such as olfactory learning and memory, odor detection and odor discrimination. In this study, we used chemical detection and a behavioral approach to further examine the function of NO in the AL. We found that basal levels of NO fluctuate with the daily light cycle, being higher during the nocturnal active period. NO also appears to be necessary for short-term olfactory memory. NO does not appear to affect odor detection, odor discrimination between dissimilar odorants, or learning acquisition. These findings suggest a modulatory role for NO in the timing of olfactory-guided behaviors.

Olfactory modulation by dopamine in the context of aversive learning.

The need to detect and process sensory cues varies in different behavioral contexts. Plasticity in sensory coding can be achieved by the context-specific release of neuromodulators in restricted brain areas. The context of aversion triggers the release of dopamine in the insect brain, yet the effects of dopamine on sensory coding are unknown. In this study, we characterize the morphology of dopaminergic neurons that innervate each of the antennal lobes (ALs; the first synaptic neuropils of the olfactory system) of the moth Manduca sexta and demonstrate with electrophysiology that dopamine enhances odor-evoked responses of the majority of AL neurons while reducing the responses of a small minority. Because dopamine release in higher brain areas mediates aversive learning we developed a naturalistic, ecologically inspired aversive learning paradigm in which an innately appetitive host plant floral odor is paired with a mimic of the aversive nectar of herbivorized host plants. This pairing resulted in a decrease in feeding behavior that was blocked when dopamine receptor antagonists were injected directly into the ALs. These results suggest that a transient dopaminergic enhancement of sensory output from the AL contributes to the formation of aversive memories. We propose a model of olfactory modulation in which specific contexts trigger the release of different neuromodulators in the AL to increase olfactory output to downstream areas of processing.

Contrast enhancement of stimulus intermittency in a primary olfactory network and its behavioral significance.

BACKGROUND: An animal navigating to an unseen odor source must accurately resolve the spatiotemporal distribution of that stimulus in order to express appropriate upwind flight behavior. Intermittency of natural odor plumes, caused by air turbulence, is critically important for many insects, including the hawkmoth, Manduca sexta, for odor-modulated search behavior to an odor source. When a moth's antennae receive intermittent odor stimulation, the projection neurons (PNs) in the primary olfactory centers (the antennal lobes), which are analogous to the olfactory bulbs of vertebrates, generate discrete bursts of action potentials separated by periods of inhibition, suggesting that the PNs may use the binary burst/non-burst neural patterns to resolve and enhance the intermittency of the stimulus encountered in the odor plume. RESULTS: We tested this hypothesis first by establishing that bicuculline methiodide reliably and reversibly disrupted the ability of PNs to produce bursting response patterns. Behavioral studies, in turn, demonstrated that after injecting this drug into the antennal lobe at the effective concentration used in the physiological experiments animals could no longer efficiently locate the odor source, even though they had detected the odor signal. CONCLUSIONS: Our results establish a direct link between the bursting response pattern of PNs and the odor-tracking behavior of the moth, demonstrating the behavioral significance of resolving the dynamics of a natural odor stimulus in antennal lobe circuits.