Precocious Foragers Research Articles

Honeybees age faster but bring back fattier pollen after consuming pesticide

Fat can be a blessing and a curse, sustaining creatures through times of scarcity but also causing disease when carried in excess. However, the loss of fat carried by honeybees in the fat body triggers the insects’ transition from life as hive-bound nurses to adventurous foragers around 21 days of age, ready to embark on a search for nectar and pollen to feed the hive. The premature loss of fat caused by stress can also accelerate this ageing process, leading nurse bees to initiate the transformation sooner. Megan Deeter from the University of Arizona, USA, wondered whether these precocious foragers may also shift their choice of diet to fattier pollen, in an attempt to compensate for their loss. Joining forces with Lucy Snyder, Charlotte Meador and Vanessa Corby Harris from the Carl Hayden Bee Research Center, USA, Deeter decided to find out.‘We were interested in a group of pesticides, insect growth regulators, and their potential to affect bee ageing and behaviour’, says Deeter, who selected two pesticides, pyriproxyfen and spirodiclofen, which target different aspects of the ageing process, to discover first how dining on the chemicals affected the bees’ survival. Collecting recently emerged young adults from a hive, the team gently fed the youngsters a single 2 μl sip of sugar water laced with either pyriproxyfen or spirodiclofen, ranging from 21 to 84 ng. The researchers then monitored the bees, keeping track of those that perished, and discovered that increasing the dose of pyriproxyfen had a dramatic effect: 50% of the insects survived the weakest dose for 25 days, falling to 17 days after the strongest dose. In contrast, the size of the spirodiclofen dose seemed to have little effect on the bees’ survival.Next, the team tagged 900 bees, administered a dose of pesticide to each and monitored when the insects departed on their maiden flights – noting the insects’ ages, when they left and returned, the duration of their flight and whether they were carrying nectar or pollen when they came back, which is indicative of foraging rather than simply getting their bearings – to find out whether the pesticides affected the bees’ ageing process and behaviour. Comparing the novices’ departure ages, it was clear that all of the young adults embarked on their orientation flights around similar ages, from 5 to 15 days, regardless of the pesticide they had consumed. However, the pyriproxyfen-fed bees began foraging sooner than the insects that had been fed spirodiclofen, although when the team checked whether the insects were returning with pollen or nectar, it seemed that the pesticides did not skew the bees’ choice of diet. But were the stressful pesticides affecting the quality of the pollen that the bees returned with and the amount of fat carried by the insects in their fat bodies?Collecting returning foragers with their corbiculae (pollen baskets) crammed with pollen, the team recorded the amount of pollen they were transporting and its fat content, in addition to the mass of the insects’ fat bodies, and found that the fat stores of the spirodiclofen-fed bees dwindled significantly, causing them to age faster and collect less pollen, but the pollen they collected was fattier.So, stressed forager bees lose their fat reserves faster than unstressed bees, especially when fed spirodiclofen, which prevents the insects from producing fat, but the insects compensate for their loss by bringing back fattier pollen, whereas bees that have consumed pyriproxyfen begin ageing faster and foraging sooner. Deeter suggests that beekeepers provide hives with high calorie supplements to maintain the bees’ fat stores once the flowering season has passed.

Changes in gut microbiota and metabolism associated with phenotypic plasticity in the honey bee Apis mellifera.

Honey bees exhibit an elaborate social structure based in part on an age-related division of labor. Young workers perform tasks inside the hive, while older workers forage outside the hive, tasks associated with distinct diets and metabolism. Critical to colony fitness, the work force can respond rapidly to changes in the environment or colony demography and assume emergency tasks, resulting in young foragers or old nurses. We hypothesized that both task and age affect the gut microbiota consistent with changes to host diet and physiology. We performed two experiments inducing precocious foragers and reverted nurses, then quantified tissue-specific gut microbiota and host metabolic state associated with nutrition, immunity and oxidative stress. In the precocious forager experiment, both age and ontogeny explained differences in midgut and ileum microbiota, but host gene expression was best explained by an interaction of these factors. Precocious foragers were nutritionally deficient, and incurred higher levels of oxidative damage relative to age-matched nurses. In the oldest workers, reverted nurses, the oxidative damage associated with age and past foraging was compensated by high Vitellogenin expression, which exceeded that of young nurses. Host-microbial interactions were evident throughout the dataset, highlighted by an age-based increase of Gilliamella abundance and diversity concurrent with increased carbonyl accumulation and CuZnSOD expression. The results in general contribute to an understanding of ecological succession of the worker gut microbiota, defining the species-level transition from nurse to forager.

A Machine Learning Approach to Study Demographic Alterations in Honeybee Colonies Using SDS-PAGE Fingerprinting.

Simple SummaryHoneybees are vital pollinators for the human food chain. Colony depopulation is a serious threat to Apis mellifera populations and unfortunately it is also one of the most elusive and difficult to study. This research deals with the problem at its foundation: population imbalances. The proposed method allows to discriminate, with remarkably good performances, precocious foragers from proper aged ones using SDS-PAGE patterns of haemolymph proteins. Implications and future perspectives are discussed.Honeybees, as social insects, live in highly organised colonies where tasks reflect the age of individuals. As is widely known, in this context, emergent properties arise from interactions between them. The accelerated maturation of nurses into foragers, stimulated by many negative factors, may disrupt this complex equilibrium. This complexity needs a paradigm shift: from the study of a single stressor to the study of the effects exerted by multiple stressors on colony homeostasis. The aim of this research is, therefore, to study colony population disturbances by discriminating overaged nurses from proper aged nurses and precocious foragers from proper aged foragers using SDS-PAGE patterns of haemolymph proteins and a machine-learning algorithm. The KNN (K Nearest Neighbours) model fitted on the forager dataset showed remarkably good performances (accuracy 0.93, sensitivity 0.88, specificity 1.00) in discriminating precocious foragers from proper aged ones. The main strength of this innovative approach lies in the possibility of it being deployed as a preventive tool. Depopulation is an elusive syndrome in bee pathology and early detection with the method described could shed more light on the phenomenon. In addition, it enables countermeasures to revert this vicious circle.

Transcriptional identification of differentially expressed genes associated with division of labor in Apis cerana cerana

Workers of Apis cerana cerana undergo an in-hive nursing to outdoor foraging transition, but the genes underlying this age-related transition remain largely unknown. Here, we sequenced the head transcriptomes of its 7-day-old normal nurses, 18- and 22-day-old normal foragers, 7-day-old precocious foragers and 22-day-old over-aged nurses to unravel the genes associated with this transition. Mapping of the sequence reads to Apis mellifera genome showed that the three types of foragers had a greater percentage of reads from annotated exons and intergenic regions, whereas the two types of nurses had a greater percentage of reads from introns. Pair- and group-wise comparisons of the five transcriptomes revealed 59 uniquely expressed genes (18 in nurses and 41 in foragers) and 14 nurse- and 15 forager-upregulated genes. The uniquely expressed genes are usually low-abundance long noncoding RNAs, transcription factors, transcription coactivators, RNA-binding proteins, kinases or phosphatases that are involved in signaling and/or regulation, whereas the nurse- or forager-upregulated genes are often high-abundance downstream genes that directly perform the tasks of nurses or foragers. Taken together, these results suggest that the nurse-forager transition is coordinated by a social signal-triggered epigenetic shift from introns to exons/intergenic regions and the resulting transcriptional shift between the nurse- and forager-associated genes.

Methoprene does not affect juvenile hormone titers in honey bee (Apis mellifera) workers.

Methoprene, a juvenile hormone (JH) analog, is a widely used insecticide that also accelerates behavioral development in honey bees (Apis mellifera). JH regulates the transition from nursing to foraging in adult worker bees, and treatment with JH or methoprene have both been shown to induce precocious foraging. To determine how methoprene changes honey bee behavior, we compared JH titers of methoprene-treated and untreated bees. Behavioral observations confirmed that methoprene treatment significantly increased the number of precocious foragers in 3 out of 4 colonies. In only 1 out of 4 colonies, however, was there a significant difference in JH titers between the methoprene-treated and control bees. Further, in all 4 colonies, there was no significant differences in JH titers between precocious and normal-aged foragers. These results suggest that methoprene did not directly affect the endogenous JH secreted by corpora allata. Because methoprene caused early foraging without changing workers' JH titers, we conclude that methoprene most likely acts directly on the JH receptors as a substitute for JH.

Preparation of Single-cohort Colonies and Hormone Treatment of Worker Honeybees to Analyze Physiology Associated with Role and/or Endocrine System.

Honeybee workers are engaged in various tasks related to maintaining colony activity. The tasks of the workers change according to their age (age-related division of labor). Young workers are engaged in nursing the brood (nurse bees), while older workers are engaged in foraging for nectar and pollen (foragers). The physiology of the workers changes in association with this role shift. For example, the main function of the hypopharyngeal glands (HPGs) changes from the secretion of major royal jelly proteins (MRJPs) to the secretion of carbohydrate-metabolizing enzymes. Because worker tasks change as the workers age in typical colonies, it is difficult to discriminate the physiological changes that occur with aging from those that occur with the role shift. To study the physiological changes in worker tissues, including the HPGs, in association with the role shift, it would be useful to manipulate the honeybee colony population by preparing single-cohort colonies in which workers of almost the same age perform different tasks. Here we describe a detailed protocol for preparing single-cohort colonies for this analysis. Six to eight days after single-cohort colony preparation, precocious foragers that perform foraging tasks earlier than usual appear in the colony. Representative results indicated role-associated changes in HPG gene expression, suggesting role-associated HPG function. In addition to manipulating the colony population, analysis of the endocrine system is important for investigating role-associated physiology. Here, we also describe a detailed protocol for treating workers with 20-hydroxyecdysone (20E), an active form of ecdysone, and methoprene, a juvenile hormone analogue. The survival rate of treated bees was sufficient to examine gene expression in the HPGs. Gene expression changes were observed in response to 20E- and/or methoprene-treatment, suggesting that hormone treatments induce physiological changes of the HPGs. The protocol for hormone treatment described here is appropriate for examining hormonal effects on worker physiology.

Pteridine levels and head weights are correlated with age and colony task in the honey bee, Apis mellifera.

Background. The age of an insect strongly influences many aspects of behavior and reproduction. The interaction of age and behavior is epitomized in the temporal polyethism of honey bees in which young adult bees perform nurse and maintenance duties within the colony, while older bees forage for nectar and pollen. Task transition is dynamic and driven by colony needs. However, an abundance of precocious foragers or overage nurses may have detrimental effects on the colony. Additionally, honey bee age affects insecticide sensitivity. Therefore, determining the age of a set of individual honey bees would be an important measurement of colony health. Pteridines are purine-based pigment molecules found in many insect body parts. Pteridine levels correlate well with age, and wild caught insects may be accurately aged by measuring pteridine levels. The relationship between pteridines and age varies with a number of internal and external factors among many species. Thus far, no studies have investigated the relationship of pteridines with age in honey bees.Methods. We established single-cohort colonies to obtain age-matched nurse and forager bees. Bees of known ages were also sampled from colonies with normal demographics. Nurses and foragers were collected every 3–5 days for up to 42 days. Heads were removed and weighed before pteridines were purified and analyzed using previously established fluorometric methods.Results. Our analysis showed that pteridine levels significantly increased with age in a linear manner in both single cohort colonies and colonies with normal demography. Pteridine levels were higher in foragers than nurses of the same age in bees from single cohort colonies. Head weight significantly increased with age until approximately 28-days of age and then declined for both nurse and forager bees in single cohort colonies. A similar pattern of head weight in bees from colonies with normal demography was observed but head weight was highest in 8-day old nurse bees and there was no relationship of head weight with age of foragers.Discussion. Although the relationship between pteridine levels and age was significant, variation in the data yielded a +4-day range in age estimation. This allows an unambiguous method to determine whether a bee may be a young nurse or old forager in colonies with altered demographics as in the case of single cohort colonies. Pteridine levels in bees do not correlate with age as well as in other insects. However, most studies used insects reared under tightly controlled laboratory conditions, while we used free-living bees. The dynamics of head weight change with age is likely to be due to growth and atrophy of the hypopharyngeal glands. Taken together, these methods represent a useful tool for assessing the age of an insect. Future studies utilizing these methods will provide a more holistic view of colony health.

Rapid behavioral maturation accelerates failure of stressed honey bee colonies

Many complex factors have been linked to the recent marked increase in honey bee colony failure, including pests and pathogens, agrochemicals, and nutritional stressors. It remains unclear, however, why colonies frequently react to stressors by losing almost their entire adult bee population in a short time, resulting in a colony population collapse. Here we examine the social dynamics underlying such dramatic colony failure. Bees respond to many stressors by foraging earlier in life. We manipulated the demography of experimental colonies to induce precocious foraging in bees and used radio tag tracking to examine the consequences of precocious foraging for their performance. Precocious foragers completed far fewer foraging trips in their life, and had a higher risk of death in their first flights. We constructed a demographic model to explore how this individual reaction of bees to stress might impact colony performance. In the model, when forager death rates were chronically elevated, an increasingly younger forager force caused a positive feedback that dramatically accelerated terminal population decline in the colony. This resulted in a breakdown in division of labor and loss of the adult population, leaving only brood, food, and few adults in the hive. This study explains the social processes that drive rapid depopulation of a colony, and we explore possible strategies to prevent colony failure. Understanding the process of colony failure helps identify the most effective strategies to improve colony resilience.

Accelerated behavioural development changes fine-scale search behaviour and spatial memory in honey bees (Apis mellifera L.).

Normally, worker honey bees (Apis mellifera) begin foraging when more than 2weeks old as adults, but if individual bees or the colony is stressed, bees often begin foraging precociously. Here, we examined whether bees that accelerated their behavioural development to begin foraging precociously differed from normal-aged foragers in cognitive performance. We used a social manipulation to generate precocious foragers from small experimental colonies and tested their performance in a free-flight visual reversal learning task, and a test of spatial memory. To assess spatial memory, bees were trained to learn the location of a small sucrose feeder within an array of three landmarks. In tests, the feeder and one landmark were removed and the search behaviour of the bees was recorded. Performance of precocious and normal-aged foragers did not differ in a visual reversal learning task, but the two groups showed a clear difference in spatial memory. Flight behaviour suggested normal-aged foragers were better able to infer the position of the removed landmark and feeder relative to the remaining landmarks than precocious foragers. Previous studies have documented the cognitive decline of old foragers, but this is the first suggestion of a cognitive deficit in young foragers. These data imply that worker honey bees continue their cognitive development during the adult stage. These findings may also help to explain why precocious foragers perform quite poorly as foragers and have a higher than normal loss rate.

The effects of age and behavioral development on honey bee (Apis mellifera) flight performance

A critical but seldom-studied component of life history theory is how behavior and age affect whole-organism performance. To address this issue we compared the flight performance of honey bees (whose behavioral development and age can be assessed independently via simple manipulations of colony demographics) between distinct behavioral castes (in-hive nurse bees vs out-of-hive foragers) and across lifespan. Variable-density gases and high-speed video were used to determine the maximum hovering flight capacity and wing kinematics of age-matched nurse bees and foragers sampled from a single-cohort colony over a period of 34 days. The transition from hive work to foraging was accompanied by a 42% decrease in body mass and a proportional increase in flight capacity (defined as the minimum gas density allowing hovering flight). The lower flight capacity of hive bees was primarily due to the fact that in air they were functioning at a near-maximal wing angular velocity due to their high body masses. Foragers were lighter and when hovering in air required a much lower wing angular velocity, which they were able to increase by 32% during maximal flight performance. Flight performance of hive bees was independent of age, but in foragers the maximal wingbeat frequency and maximal average angular velocity were lowest in precocious (7-14 day old) foragers, highest in normal-aged (15-28 day old) foragers and intermediate in foragers older than 29 days. This pattern coincides with previously described age-dependent biochemical and metabolic properties of honey bee flight muscle.

Expression of the dopamine transporter in the brain of the honeybee, Apis mellifera L. (Hymenoptera: Apidae)

The dopamine transporter (DAT) is located on the presynaptic membrane of dopaminergic terminals and recovers the released dopamine from synaptic clefts. Therefore, its expression level is expected to reflect the activity of dopaminergic neurons. We identified the honeybee ortholog of dat (Amdat) and measured its expression levels in honeybee brain by reverse transcription real-time quantitative polymerase chain reaction. Worker honeybees change their tasks from nursing to foraging in an age-dependent manner, and Amdat transcript levels in their brains were found to increase with age. Furthermore, the expression level was significantly higher in precocious foragers than in nurses of the same age, suggesting that Amdat expression is related to task shifts. In queens, Amdat expression levels decreased after a mating flight. Amdat expression in drone honeybees increased with age. Our data suggest that dopamine pathway activity is correlated with the behavioral activity of honeybee.

RNAi-mediated silencing of vitellogenin gene function turns honeybee (Apis mellifera) workers into extremely precocious foragers

The switch from within-hive activities to foraging behavior is a major transition in the life cycle of a honeybee (Apis mellifera) worker. A prominent regulatory role in this switch has long been attributed to juvenile hormone (JH), but recent evidence also points to the yolk precursor protein vitellogenin as a major player in behavioral development. In the present study, we injected vitellogenin double-stranded RNA (dsVg) into newly emerged worker bees of Africanized genetic origin and introduced them together with controls into observation hives to record flight behavior. RNA interference-mediated silencing of vitellogenin gene function shifted the onset of long-duration flights (>10 min) to earlier in life (by 3-4 days) when compared with sham and untreated control bees. In fact, dsVg bees were observed conducting such flights extremely precociously, when only 3 days old. Short-duration flights (<10 min), which bees usually perform for orientation and cleaning, were not affected. Additionally, we found that the JH titer in dsVg bees collected after 7 days was not significantly different from the controls. The finding that depletion of the vitellogenin titer can drive young bees to become extremely precocious foragers could imply that vitellogenin is the primary switch signal. At this young age, downregulation of vitellogenin gene activity apparently had little effect on the JH titer. As this unexpected finding stands in contrast with previous results on the vitellogenin/JH interaction at a later age, when bees normally become foragers, we propose a three-step sequence in the constellation of physiological parameters underlying behavioral development.

Hard working nurses rather than over-aged nurses permit Ropalidia marginata to respond to the loss of young individuals

The primitively eusocial wasp Ropalidia marginata exhibits a remarkably well-developed honey bee like age polyethism. Individuals perform different tasks sequentially as they age, starting with intranidal tasks (nursing and building in that order) and ending with extranidal tasks (foraging for pulp and food in that order). As in the case of honey bees such age polyethism is rather flexible; in the absence of old individuals (in young-cohort colonies), precocious foragers forage at abnormally young ages. Here we demonstrate that the absence of young individuals (in old-cohort colonies) does not result in over-aged nurses nursing at abnormally old ages, as seen in the case of honey bees. Instead it results in hard working nurses who nurse at abnormally high rates. The possible reasons for the absence of over-aged nurses and the presence of hard working nurses are discussed.

Satiation differentially affects performance in a learning assay by nurse and forager honey bees.

When not satiated prior to training, there were no differences between foragers and nurse honey bees in the acquisition of an appetitively based conditioned response in an olfactory associative learning assay, but when satiated foragers showed faster acquisition than did nurses. Satiation-related differences between foragers and nurses were more a function of behavioral state than age, because satiated precocious foragers also showed faster acquisition rates than did satiated nurse bees, despite their similar ages. Tests of sucrose responsiveness and retention of conditioned responses indicate that the observed performance differences between nurses and foragers were more likely due to differential sensitivity of sensory and motor processes related to satiation rather than differences in cognitive ability.

Octopamine influences division of labor in honey bee colonies.

Forager honey bees have higher brain levels of octopamine than do bees tending larvae in the hive. To test the hypothesis that octopamine influences honey bee division of labor we treated bees orally with octopamine or its immediate precursor tyramine and determined whether these treatments increased the probability of initiating foraging. Octopamine treatment significantly elevated levels of octopamine in the brain and caused a significant dose-dependent increase in the number of new foragers. This effect was seen for precocious foragers in single-cohort colonies and foragers in larger colonies with more typical age demographies. Tyramine treatment did not increase the number of new foragers, suggesting that octopamine was exerting a specific effect. Octopamine treatment was effective only when given to bees old enough to forage, i.e., older than 4 days of age. Treatment when bees were 1-3 days of age did not cause a significant increase in the number of new foragers when the bees reached the minimal foraging age. These results demonstrate that octopamine influences division of labor in honey bee colonies. We speculate that octopamine is acting in this context as a neuromodulator.

Differences in performance on a reversal learning test and division of labor in honey bee colonies

We studied the association between honey bee (Apis mellifera) division of labor and performance on an olfactory reversal-learning test. Manipulations of colony age structure and flight experience were used to test whether differences in performance are associated with age, current behavioral state, or flight experience. Nurse bees showed significantly faster rates of extinction to a learned odor than did foragers. This difference was associated primarily with differences in behavioral state, rather than age; it was seen when comparing nurses and foragers from typical colonies and normal-age nurses and precocious foragers from single-cohort colonies. Differences in extinction rate were not related to differences in flight experience; there was no difference between foragers and foraging-age bees denied flight experience. These results suggest that changes in learning and memory occur in association with division of labor. We speculate on the possible functional significance of the difference in extinction rate between nurses and foragers.

Biogenic amines and division of labor in honey bee colonies

Brain levels of dopamine, serotonin, and octopamine were measured in relation to both age-related division of labor and inter-individual differences in task specialization independent of age in honey bee colonies. The only differences among similarly aged bees performing different tasks were significantly lower levels of dopamine in food storers than comb builders and significantly lower levels of octopamine in soldiers than foragers, but soldiers also were slightly younger than foragers. Differences associated with age-related division of labor were stronger. Older bees, notably foragers, had significantly higher levels of all three amines than did younger bees working in the hive. Using social manipulations to unlink chronological age and behavioral status, octopamine was found to exhibit the most robust association between behavior and amine level, independent of age. Octopamine levels were significantly lower in normal-age nurses versus precocious foragers and overage nurses versus normal-age foragers, but not different in reverted nurses versus reversion colony foragers. Dopamine levels were significantly lower in normal-age nurses versus precocious foragers, but higher in reverted nurses versus reversion colony foragers. Serotonin levels did not differ in any of these comparisons. These correlative results suggest that octopamine is involved in the regulation of age-related division of labor in honey bees.

Flexible division of labor mediated by social interactions in an insect colony-a simulation model

The primitively eusocial wasp Ropalidia marginata shows an age-based division of labor in which workers allocate tasks according to their relative ages (age ranks). This age polyethism seems quite flexible because in colonies devoid of old workers, young individuals can perform the tasks normally performed by older workers. Social interactions appear to be a plausible mechanism by which workers can assess their relative ages. To explore possible proximate mechanisms that can potentially generate such a flexible, age-based task allocation, the activator-inhibitor model was adapted to the social biology of R. marginata and tested using computer simulations. The model generated a clear age polyethism including the phenomena of precocious foragers in colonies with only young individuals and reverted nurses in colonies consisting of only old individuals. A simple extension of the model to allow the brood:adult ratio to modulate the rates of social interactions, shows how increasing task demands can be met by a decrease in the ages of first performance of, and an increase in the proportions of individuals engaged in, various tasks. These results show how a pattern of division of labor based on relative age can be generated and modulated by social interactions. Copyright 1999 Academic Press.

Effects of social environment and worker mandibular glands on endocrine-mediated behavioral development in honey bees.

Previous studies suggest that older honey bee workers possess an inhibitory signal that regulates behavioral development in younger bees. To study how this inhibitor is transmitted, bees were reared for 7 days in double-screen cages, single-screen cages, or unrestricted in a typical colony (control bees). Double-screen cages prevented physical contact with colony members while single-screen cages allowed only antennation and food exchange. Bees reared in double-screen cages showed accelerated endocrine and behavioral development; they had significantly higher rates of juvenile hormone biosynthesis and juvenile hormone titers than did control bees and also were more likely to become precocious foragers. Relative to the other two groups, bees reared in single-screen cages showed intermediate juvenile hormone biosynthesis rates and titers, and intermediate rates of behavioral development. These results indicate that physical contact is required for total inhibition. We also began to test the hypothesis that worker mandibular glands are the sources of an inhibitory signal. Old bees with mandibular glands removed were significantly less inhibitory towards young bees than were sham-operated and unoperated bees. These results suggest that an inhibitor is produced by the worker mandibular glands.

Effects of colony food shortage on behavioral development in honey bees

Three experiments were conducted to explore the effects of severe food shortage on the control of two important and interrelated aspects of temporal division of labor in colonies of the honey bee (Apis mellifera): the size and age distribution of a colony's foraging force. The experiments were conducted with single-cohort colonies, composed entirely of young bees, allowing us to quickly distinguish the development of new (precocious) foragers from increases in activity of bees already competent to forage. In experiment 1, colony food shortage caused an acceleration of behavioral development; a significantly greater proportion of bees from starved colonies than from fed colonies became precocious foragers, and at significantly younger ages. Temporal aspects of this starvation effect were further explored in experiment 2 by feeding colonies that we initially starved, and starving colonies that we initially fed. There was a significant decrease in the number of new foragers in starved colonies that were fed, detected 1 day after feeding. There also was a significant increase in the number of new foragers in fed colonies that were starved, but only after a 2-day lag. These results suggest that colony nutritional status does affect long-term behavioral development, rather than only modulate the activity of bees already competent to forage. In experiment 3, we uncoupled the nutritional status of a colony from that of the individual colony members. The behavior of fed individuals in starved colonies was indistinguishable from that of bees in fed colonies, but significantly different from that of bees in starved colonies, in terms of both the number and age distribution of foragers. These results demonstrate that effects of starvation on temporal polyethism are not mediated by the most obvious possible worker-nest interaction: a direct interaction with colony food stores. This is consistent with previous findings suggesting the importance of worker-worker interactions in the regulation of temporal polyethism in honey bees as well as other social insects.