Sensory reception of the primer pheromone ethyl oleate

Social work force distribution in honeybee colonies critically depends on subtle adjustments of an age-related polyethism. Pheromones play a crucial role in adjusting physiological and behavioral maturation of nurse bees to foragers. In addition to primer effects of brood pheromone and queen mandibular pheromone—both were shown to influence onset of foraging—direct worker–worker interactions influence adult behavioral maturation. These interactions were narrowed down to the primer pheromone ethyl oleate, which is present at high concentrations in foragers, almost absent in young bees and was shown to delay the onset of foraging. Based on chemical analyses, physiological recordings from the antenna (electroantennograms) and the antennal lobe (calcium imaging), and behavioral assays (associative conditioning of the proboscis extension response), we present evidence that ethyl oleate is most abundant on the cuticle, received by olfactory receptors on the antenna, processed in glomeruli of the antennal lobe, and learned in olfactory centers of the brain. The results are highly suggestive that the primer pheromone ethyl oleate is transmitted and perceived between individuals via olfaction at close range.     

The role of tyramine and octopamine in the regulation of reproduction in queenless worker honeybees

In honeybees, workers under queenless condition compete for reproduction and establish reproductive dominance hierarchy. Ovary activation is generally accompanied by the expression of queen-like pheromones. Biogenic amines (BAs), in particular dopamine, are believed to be involved in this process by regulating ovarian development. However, the role of BAs in establishing reproductive dominance or their effect on queen-like pheromone production was not investigated. Here, we explored the effect of octopamine (OA) and tyramine (TA) oral treatments on the propensity of treated bees to become reproductively dominant and produce queen-like pheromones in Dufour’s and mandibular glands. One bee in a pair was treated with either OA or TA while the other was fed sugar solution. TA was found to enhance ovary development and the production of esters in the Dufour’s gland and 9HDA (queen component) in the mandibular glands, thus facilitating worker reproductive dominance. OA, on the other hand, did not enhance ovarian development or ester production, but increased the production of 10HDA (worker major component) in the mandibular glands of their sugar-paired mates. OA is known to induce foraging behavior by workers, while increased production of 10HDA characterizes nursing workers. Therefore, we suggest that TA induces reproductive division of labor, while OA treatment results in caste differentiation of workers to foragers and nurses.     

Uncoupling primer and releaser responses to pheromone in honey bees

Pheromones produce dramatic behavioral and physiological responses in a wide variety of species. Releaser pheromones elicit rapid responses within seconds or minutes, while primer pheromones produce long-term changes which may take days to manifest. Honeybee queen mandibular pheromone (QMP) elicits multiple distinct behavioral and physiological responses in worker bees, as both a releaser and primer, and thus produces responses on vastly different time scales. In this study, we demonstrate that releaser and primer responses to QMP can be uncoupled. First, treatment with the juvenile hormone analog methoprene leaves a releaser response (attraction to QMP) intact, but modulates QMP’s primer effects on sucrose responsiveness. Secondly, two components of QMP (9-ODA and 9-HDA) do not elicit a releaser response (attraction) but are as effective as QMP at modulating a primer response, downregulation of foraging-related brain gene expression. These results suggest that different responses to a single pheromone may be produced via distinct pathways.     

Pheromone-modulated behavioral suites influence colony growth in the honey bee (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

The success of a species depends on its ability to assess its environment and to decide accordingly which behaviors are most appropriate. Many animal species, from bacteria to mammals, are able to communicate using interspecies chemicals called pheromones. In addition to exerting physiological effects on individuals, for social species, pheromones communicate group social structure. Communication of social structure is important to social insects for the allocation of its working members into coordinated suites of behaviors. We tested effects of long-term treatment with brood pheromone on suites of honey bee brood rearing and foraging behaviors. Pheromone-treated colonies reared significantly greater brood areas and more adults than controls, while amounts of stored pollen and honey remained statistically similar. Brood pheromone increased the number of pollen foragers and the pollen load weights they returned. It appeared that the pheromone-induced increase in pollen intake was directly canalized into more brood rearing. A two-way pheromone priming effect was observed, such that some workers from the same age cohorts showed an increased and extended capacity to rear larvae, while others were recruited at significantly younger ages into pollen-specific foraging. Brood pheromone affected suites of nursing and foraging behaviors allocating worker and pollen resources associated with an important fitness trait, colony growth.     

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. David Santos Marco Antonio and Karina Rosa Guidugli-Lazzarini contributed equally to the present study.     

Foraging scent marks of bumblebees: footprint cues rather than pheromone signals

In their natural habitat foraging bumblebees refuse to land on and probe flowers that have been recently visited (and depleted) by themselves, conspecifics or other bees, which increases their overall rate of nectar intake. This avoidance is often based on recognition of scent marks deposited by previous visitors. While the term 'scent mark' implies active labelling, it is an open question whether the repellent chemicals are pheromones actively and specifically released during flower visits, or mere footprints deposited unspecifically wherever bees walk. To distinguish between the two possibilities, we presented worker bumblebees (Bombus terrestris) with three types of feeders in a laboratory experiment: unvisited control feeders, passive feeders with a corolla that the bee had walked over on its way from the nest (with unspecific footprints), and active feeders, which the bee had just visited and depleted, but which were immediately refilled with sugar-water (potentially with specific scent marks). Bumblebees rejected both active and passive feeders more frequently than unvisited controls. The rate of rejection of passive feeders was only slightly lower than that of active feeders, and this difference vanished completely when passive corollas were walked over repeatedly on the way from the nest. Thus, mere footprints were sufficient to emulate the repellent effect of an actual feeder visit. In confirmation, glass slides on which bumblebees had walked on near the nest entrance accumulated hydrocarbons (alkanes and alkenes, C23 to C31), which had previously been shown to elicit repellency in flower choice experiments. We conclude that repellent scent marks are mere footprints, which foraging bees avoid when they encounter them in a foraging context.     

Age-related changes in biogenic amines in individual brains of the ant Pheidole dentata

The behavioral development of minor workers of the ant Pheidole dentata involves a progression of tasks beginning with brood care and culminating in foraging as individuals age. To understand the role of brain neurochemistry in age-related division of labor, we measured the levels of serotonin, dopamine and octopamine in individual brains of minor workers of different age. Serotonin and dopamine levels were significantly correlated with worker age: both increased as minor workers matured, and serotonin rose significantly in the oldest ants. In addition, the serotonin:dopamine ratio was significantly higher in the oldest workers. Octopamine levels did not change with age, although the ratios of octopamine:serotonin and octopamine:dopamine were significantly higher in the youngest workers. These age-associated changes in biogenic amine levels suggest an involvement of neuromodulators in minor worker behavioral ontogeny and temporal polyethism in P. dentata.     

Pheromonal regulation of starvation resistance in honey bee workers (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Most animals can modulate nutrient storage pathways according to changing environmental conditions, but in honey bees nutrient storage is also modulated according to changing behavioral tasks within a colony. Specifically, bees involved in brood care (nurses) have higher lipid stores in their abdominal fat bodies than forager bees. Pheromone communication plays an important role in regulating honey bee behavior and physiology. In particular, queen mandibular pheromone (QMP) slows the transition from nursing to foraging. We tested the effects of QMP exposure on starvation resistance, lipid storage, and gene expression in the fat bodies of worker bees. We found that indeed QMP-treated bees survived much longer compared to control bees when starved and also had higher lipid levels. Expression of vitellogenin RNA, which encodes a yolk protein that is found at higher levels in nurses than foragers, was also higher in the fat bodies of QMP-treated bees. No differences were observed in expression of genes involved in insulin signaling pathways, which are associated with nutrient storage and metabolism in a variety of species; thus, other mechanisms may be involved in increasing the lipid stores. These studies demonstrate that pheromone exposure can modify nutrient storage pathways and fat body gene expression in honey bees and suggest that chemical communication and social interactions play an important role in altering metabolic pathways.     

Colony size affects division of labour in the ponerine ant<Emphasis Type="Italic"> Rhytidoponera metallica</Emphasis>

In theory, larger colonies of social insects should have greater colony organisation. While inter-specific comparative studies provide support for this idea, there is little direct intra-specific evidence. We investigated differences in task specialisation between large (>450 workers) and small (<80 workers) colonies of the ponerine ant Rhytidoponera metallica. Observations of individually marked young or old workers revealed greater task specialisation in large colonies. Age polyethism was detected in large but not small colonies. In large colonies, old workers spent significantly more time foraging than young workers did, while young workers spent more time caring for brood. In small colonies, young and old workers spent a similar amount of time foraging and caring for brood. This difference in task allocation patterns in large and small colonies was associated with a difference in contact rates between workers. Workers in small colonies have a lower contact rate between nestmates and a greater variability in time between contacts than workers from large colonies.     

The effect of queen pheromones on worker honey bee ovary development

We report results that address a long-standing controversy in honey bee biology, the identity of the queen-produced compounds that inhibit worker honey bee ovary development. As the honey bee is the only organism for which identities have been proposed for any pheromone that regulates reproduction, the resolution of its identity is of broad significance. We examined the effects of synthetic honey bee queen mandibular pheromone (QMP), four newly identified queen retinue pheromone components, and whole-queen extracts on the ovary development of caged worker bees. The newly identified compounds did not inhibit worker ovary development alone, nor did they improve the efficacy of QMP when applied in combination. QMP was as effective as queen extracts at ovary regulation. Caged workers in the QMP and queen extract treatments had better developed ovaries than did workers remaining in queenright colonies. We conclude that QMP is responsible for the ovary-regulating pheromonal capability of queens from European-derived Apis mellifera subspecies.     

Queen regulates biogenic amine level and nestmate recognition in workers of the fire ant, <Emphasis Type="Italic">Solenopsis invicta</Emphasis>

Nestmate recognition is a critical element in social insect organization, providing a means to maintain territoriality and close the colony to parasites and predators. Ants detect the colony chemical label via their antennae and respond to the label mismatch of an intruder with aggressive behavior. In the fire ant, Solenopsis invicta, worker ability to recognize conspecific nonnestmates decreases if the colony queen is removed, such that they do not recognize conspecific nonnestmates as different. Here, we tested the hypothesis that the presence of the colony queen influences the concentration of octopamine, a neuromodulator, in worker ants, which in turn has an effect on nestmate recognition acuity in workers. We demonstrate that queenless workers exhibit reduced brain octopamine levels and reduced discriminatory acuteness; however, feeding queenless workers octopamine restored both. Dopamine levels are influenced by honeybee queen pheromones; however, levels of this biogenic amine were unchanged in our experiments. This is the first demonstration of a link between the presence of the colony queen, a worker biogenic amine, and conspecific nestmate recognition, a powerful expression of colony cohesion and territoriality.     

Lethal fighting between honeybee queens and parasitic workers (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Pheromonal signals associated with queen and worker policing prevent worker reproduction and have been identified as important factors for establishing harmony in the honeybee (Apis mellifera) colony. However, "anarchic workers", which can evade both mechanisms, have been detected at low frequency in several honeybee populations. Worker bees of the Cape honeybee, Apis mellifera capensis, also show this anarchistic trait but to an extreme degree. They can develop into so called "pseudoqueens", which release a pheromonal bouquet very similar to that of queens. They prime and release very similar reactions in sterile workers to those of true queens (e.g. suppress ovary activation; release retinue behavior). Here we show in an experimental bioassay that lethal fights between these parasitic workers and the queen (similar to queen–queen fights) occur, resulting in the death of either queen or worker. Although it is usually the queen that attacks the parasitic workers and kills many of them, in a few cases the workers succeeded in killing the queen. If this also occurs in a parasitized colony where the queen encounters many parasitic workers, she may eventually be killed in one of the repeated fights she engages in.     

Variations in circulating hemocytes are affected by age and caste in the stingless bee <Emphasis Type="Italic">Melipona quadrifasciata</Emphasis>

The insect immune system faces various challenges; particularly in social bees, caste system and age polyethism expose individuals to numerous environmental and working conditions. However, little is known about how cellular defenses in social bees may be organized to respond to a variety of immune challenges. Here, we describe the morphological features and the total and differential counts of hemocytes in different female classes (newly emerged workers, nurses, foragers, and virgin queens) of the eusocial stingless bee Melipona quadrifasciata. Granulocytes and prohemocytes were, respectively, the most and the least abundant cells among all classes of females. Furthermore, there were more prohemocytes in virgin queens than in foragers. The total number of hemocytes was smaller in foragers, whereas the largest number was observed in nurse workers. This reduced amount of hemocytes in foragers might allow energy savings to perform colony activities such as foraging and defense. Foragers also had the biggest hemocytes (either prohemocytes, granulocytes, or plasmatocytes) in comparison to the other classes of females, which might have arisen as a compensation for the reduction in number of these cells during aging. These results suggest that profiles of hemocytes of M. quadrifasciata vary according to the caste and age of this eusocial bee.     

Heat-balling wasps by honeybees

Defensiveness of honeybee colonies of Apis cerana and Apis mellifera (actively balling the wasps but reduction of foraging) against predatory wasps, Vespa velutina, and false wasps was assessed. There were significantly more worker bees in balls of the former than latter. Core temperatures in a ball around a live wasp of A. cerana were significantly higher than those of A. mellifera, and also significantly more when exposed to false wasps. Core temperatures of bee balls exposed to false wasps were significantly lower than those exposed to V. velutina for both A. cerana and for A. mellifera. The lethal thermal limits for V. velutina, A. cerana and A. mellifera were significantly different, so that both species of honeybees have a thermal safety factor in heat-killing such wasp predators. During wasps attacks at the hives measured at 3, 6 and 12 min, the numbers of Apis cerana cerana and Apis cerana indica bees continuing to forage were significantly reduced with increased wasp attack time. Tropical lowland A. c. indica reduced foraging rates significantly more than the highland A. c. cerana bees; but, there was no significant effect on foraging by A. mellifera. The latency to recovery of honeybee foraging was significantly greater the longer the duration of wasp attacks. The results show remarkable thermal fine-tuning in a co-evolving predator–prey relationship.     

Signaling hunger through aggression—the regulation of foraging in a primitively eusocial wasp

Primitively eusocial wasps are generally headed by behaviorally dominant queens who use their aggression to suppress worker reproduction. In contrast, queens in the primitively eusocial wasp Ropalidia marginata are strikingly docile and non-aggressive. However, workers exhibit dominance-subordinate interactions among themselves. These interactions do not appear to reflect reproductive competition because there is no correlation between the relative position of an individual in the dominance hierarchy of the colony and the likelihood that she will succeed a lost/removed queen. Based on the observation that foraging continues unaltered in the absence of the queen and the correlation between dominance behavior and foraging, we have previously suggested that dominance-subordinate interactions among workers in R. marginata have been co-opted to serve the function of decentralized, self-organized regulation of foraging. This idea has been supported by an earlier experimental study where it was found that a reduced demand for food led to a significant decrease in dominance behavior. In this study, we perform the converse experiment, demonstrate that dominance behavior increases under conditions of starvation, and thus provide further evidence in support of the hypothesis that intranidal workers signal hunger through aggression.     

Aggressive reproductive competition among hopelessly queenless honeybee workers triggered by pheromone signaling

In the honeybee, Apis mellifera, the queen monopolizes reproduction, while the sterile workers cooperate harmoniously in nest maintenance. However, under queenless (QL) conditions, cooperation collapses and reproductive competition among workers ensues. This is mediated through aggression and worker oviposition, as well as shifts in pheromones, from worker to queen-like composition. Many studies suggest a dichotomy between conflict resolution through aggression or through pheromonal signaling. In this paper, we demonstrate that both phenomena comprise essential components of reproductive competition and that pheromone signaling actually triggers the onset of aggression. We kept workers as QL groups until first aggression was observed and subsequently determined the contestants' reproductive status and content of the mandibular (MG) and Dufour's glands (DG). In groups in which aggression occurred early, the attacked bee had consistently more queen-like pheromone in both the MG and DG, although both contestants had undeveloped ovaries. In groups with late aggression, the attacked bee had consistently larger oocytes and more queen-like pheromone in the DG, but not the MG. We suggest that at early stages of competition, the MG secretion is utilized to establish dominance and that the DG provides an honest fertility signal. We further argue that it is the higher amount of DG pheromone that triggers aggression.     

Coming out of the woods: do termites need a specialized worker caste to search for new food sources?

Most small-colony termites live confined within a single piece of wood on which they feed and do not possess permanent workers: Tasks are done by developmentally flexible immatures (pseudergates). By contrast, large-colony termites possess a specialized (true) worker caste and forage outside their nest for food. To shed light on possible transitional steps between these contrasting patterns of social organization, we studied an atypical Rhinotermitidae, Prorhinotermes inopinatus. In this species, despite the absence of a true worker caste, soldiers, pseudergates, and neotenic reproductives may leave the nest and explore their surroundings. Although evidence presented in this study indicates that termites recognize unknown areas, there is no directional recruitment toward them. The discovery of a food source, i.e., a piece of wood, is followed by the establishment of a long-lasting trail between the nest and the food source. A large fraction of the colony, including neotenic reproductives, ultimately migrates into the piece of wood. Our results thus demonstrate that multiple features of external foraging behavior can evolve independently of the existence of a true worker caste in termites. We suggest that large colonies with true workers, like those of most Rhinotermitidae, may easily have evolved from a Prorhinotermes-like pattern if submitted to increasing selective pressures for worker efficiency in a stable environment.     

Beeping and piping: characterization of two mechano-acoustic signals used by honey bees in swarming

Of the many signals used by honey bees during the process of swarming, two of them??the stop signal and the worker piping signal??are not easily distinguished for both are mechano-acoustic signals produced by scout bees who press their bodies against other bees while vibrating their wing muscles. To clarify the acoustic differences between these two signals, we recorded both signals from the same swarm and at the same time, and compared them in terms of signal duration, fundamental frequency, and frequency modulation. Stop signals and worker piping signals differ in all three variables: duration, 174?±?64 vs. 602?±?377?ms; fundamental frequency, 407 vs. 451?Hz; and frequency modulation, absent vs. present. While it remains unclear which differences the bees use to distinguish the two signals, it is clear that they do so for the signals have opposite effects. Stop signals cause inhibition of actively dancing scout bees whereas piping signals cause excitation of quietly resting non-scout bees.     

Pollen foraging: learning a complex motor skill by bumblebees (<Emphasis Type="Italic">Bombus terrestris</Emphasis>)

To investigate how bumblebees (Bombus terrestris) learn the complex motor skills involved in pollen foraging, we observed naïve workers foraging on arrays of nectarless poppy flowers (Papaver rhoeas) in a greenhouse. Foraging skills were quantified by measuring the pollen load collected during each foraging bout and relating this to the number of flowers visited and bout duration on two consecutive days. The pollen standing crop (PSC) in each flower decreased drastically from 0530 to 0900 hours. Therefore, we related foraging performance to the changing levels of pollen available (per flower) and found that collection rate increased over the course of four consecutive foraging bouts (comprising between 277 and 354 individual flower visits), suggesting that learning to forage for pollen represents a substantial time investment for individual foragers. The pollen collection rate and size of pollen loads collected at the start of day 2 were markedly lower than at the end of day 1, suggesting that components of pollen foraging behaviour could be subject to imperfect overnight retention. Our results suggest that learning the necessary motor skills to collect pollen effectively from morphologically simple flowers takes three times as many visits as learning how to handle the most morphologically complex flowers to extract nectar, potentially explaining why bees are more specialised in their choice of pollen flowers.     

Individual lifetime pollen and nectar foraging preferences in bumble bees

Foraging specialization plays an important role in the ability of social insects to efficiently allocate labor. However, relatively little is known about the degree to which individual bumble bees specialize on collecting nectar or pollen, when such preferences manifest, and if individuals can alter their foraging preferences in response to changes in the colony workforce. Using Bombus impatiens, we monitored all foraging visits made by every bee in multiple colonies and showed that individual foragers exhibit consistent lifetime foraging preferences. Based upon the distribution of foraging preferences, we defined three forager types (pollen specialists, nectar specialists, and generalists). In unmanipulated colonies, 16–36?% of individuals specialized (≥90?% of visits) on nectar or pollen only. On its first day of foraging, an individual’s foraging choices (nectar only, pollen only, or nectar and pollen) significantly predicted its lifetime foraging preferences. Foragers that only collected pollen on their first day of foraging made 1.61- to 1.67-fold more lifetime pollen foraging visits (as a proportion of total trips) than foragers that only collected nectar on their first foraging day. Foragers were significantly larger than bees that stayed only in the nest. We also determined the effect of removing pollen specialists at early (brood present) or later (brood absent) stages in colony life. These results suggest that generalists can alter their foraging preferences in response to the loss of a small subset of foragers. Thus, bumble bees exhibit individual lifetime foraging preferences that are established early in life, but generalists may be able to adapt to colony needs.