Analysis of GABAergic and Non-GABAergic Neuron Activity in the Optic Lobes of the Forager and Re-Orienting Worker Honeybee (Apis mellifera L.)

Background

European honeybee (Apis mellifera L.) foragers have a highly developed visual system that is used for navigation. To clarify the neural basis underlying the highly sophisticated visual ability of foragers, we investigated the neural activity pattern of the optic lobes (OLs) in pollen-foragers and re-orienting bees, using the immediate early gene kakusei as a neural activity marker.

Methodology/Principal Findings

We performed double-in situ hybridization of kakusei and Amgad, the honeybee homolog of the GABA synthesizing enzyme GAD, to assess inhibitory neural activity. kakusei-related activity in GABAergic and non-GABAergic neurons was strongly upregulated in the OLs of the foragers and re-orienting bees, suggesting that both types of neurons are involved in visual information processing. GABAergic neuron activity was significantly higher than non-GABAergic neuron activity in a part of the OLs of only the forager, suggesting that unique information processing occurs in the OLs of foragers. In contrast, GABAergic neuron activity in the antennal lobe was significantly lower than that of GABAergic neurons in the OLs in the forager and re-orienting bees, suggesting that kakusei-related visual activity is dominant in the brains of these bees.

Conclusions/Significance

The present study provides the first evidence that GABAergic neurons are highly active in the OL neurons of free-moving honeybees and essential clue to reveal neural basis of the sophisticated visual ability that is equipped in the small and simple brain.     

Waggle dances in absconding colonies of the red dwarf honeybee, Apis florea

The directional information encoded in the waggle dances of absconding colonies of Apis florea shows how different sites are advertised during decision-making. Colonies of A. florea were observed from the inception of absconding until the swarm settled at a new nest site. The number of waggle dancers at the beginning of the absconding sequence was low, gradually increased and then declined shortly before liftoff. During the last 2 to 0.5?h before liftoff, the dances still indicated different directions. This significantly decreased in the last 0.5?h until only one or two dance directions were being advertised. All colonies reached a near consensus in the last 20 dances before liftoff. The swarm flight path is meandering so the actual distance flown is about twice that indicated by the dances. During the last 3?min the waggle dance in most colonies showed nest target angles that were closely clustered indicating that the final directions advertised were close to the chosen target site. In all absconding/migratory species of honeybees thus far studied, there is a special dance associated with absconding that appears not to select specific destinations but rather a particular direction in search of a new nesting area.     

The Brief Piping Signal of the Honey Bee: Begging Call or Stop Signal?

For over 40 yr, investigators have recognized that the brief piping signal plays a role in the foraging operation of a honey bee colony. The function of this signal, however, remains uncertain. The main objective of this study was to determine whether, under normal foraging conditions, bees following waggle dancers produce brief piping signals to beg nectar samples from the dancers. We made observations on waggle dancers and their followers in an undisturbed colony whose foragers gathered nectar and pollen from flowers. We found that waggle dancers do often receive brief piping signals, that the bees producing these signals are generally dance followers, and that these signals increase a waggle dancer's tendency to stop dancing. We also found, however, that the brief piping signal is clearly not a begging call; 0 of 41 waggle dancers that received a piping signal from a dance follower gave a nectar sample to the bee that produced the signal. Our results support the hypothesis that the brief piping signal is a stop signal; it serves to shut off waggle dancing. But why some dance followers pipe the dancer they are following, thereby inhibiting her dancing, remains unclear and warrants further investigation.     

Variations of brain biogenic amines in mature honeybees and induction of recruitment behavior

Mature honeybees (Apis mellifera L.) old enough to forage (>3 weeks) were segregated into three activity groups: waggle dancers (active foragers), followers of the dancers (potential recruits) and resting bees (not involved in foraging). Dopamine (DA) pathways in the brain of honeybees seemed to be involved in regulation of forager recruitment. Brain DA and N-β-alanyldopamine (NBAD) levels in the dancers were always higher than in followers, and an increased number of dancers was observed after feeding the colony dihydroxy-phenylalanine (DOPA). Dopamine is hypothesized to modulate the neural activity in the calyx of the mushroom bodies related to recruitment behavior. No consistant effect of octopamine (OA) or serotonin (5HT) on recruitment behavior was observed. Levels of all biogenic amines were strongly effected by season and day-to-day whether changes. Some diurnal changes were also observed.     

The scent of the waggle dance

The waggle dance of honey bee (Apis mellifera L.) foragers communicates to nest mates the location of a profitable food source. We used solid-phase microextraction and gas chromatography coupled with mass spectrometry to show that waggle-dancing bees produce and release two alkanes, tricosane and pentacosane, and two alkenes, Z-(9)-tricosene and Z-(9)-pentacosene, onto their abdomens and into the air. Nondancing foragers returning from the same food source produce these substances in only minute quantities. Injection of the scent significantly affects worker behavior by increasing the number of bees that exit the hive. The results of this study suggest that these compounds are semiochemicals involved in worker recruitment. By showing that honey bee waggle dancers produce and release behaviorally active chemicals, this study reveals a new dimension in the organization of honey bee foraging.     

A comparison of the dance language in<Emphasis Type="Italic"> Apis mellifera carnica</Emphasis> and<Emphasis Type="Italic"> Apis florea</Emphasis> reveals striking similarities

Honeybees have a dance language by which successful foragers inform nestmates about attractive food patches. The classical concept of dialects in the dance language of honeybees points to two differences in the dances by different species and races, firstly in the flight distance at which the dancers start performing waggle dances instead of round dances, and secondly in the circuit duration of the waggle dance performed for a given flight distance. However, recent findings have indicated that the dance language is influenced and affected by a number of parameters, both genetic and environmental. The current study was carried out to see whether the distance at which dancers change from round dances to waggle dances is statistically different in two different species, Apis mellifera carnica and A. florea and to develop a set of definitions for such comparative studies. Results show that the two species do not differ in the relative proportion of waggle dances and round dances performed at a given distance. Thus, this study points to the need of addressing the dialect question again.     

Dynamic temporal change of cerebral microbleeds: long-term follow-up MRI study

Background

Cerebral microbleeds (MBs) are understood as an important radiologic marker of intracerebral hemorrhage. We sought to investigate the temporal changes of MBs and clinical factors associated with the changes using long-term follow-up MRI.

Methods/Principal Findings

From October 2002 to July 2006, we prospectively enrolled patients with stroke or transient ischemic attack, and followed-up their brain MRIs with an interval >12 mo. We compared demographic factors, vascular risk factors, laboratory findings, and radiologic factors according to the presence or changes of MBs. A total of 224 patients successfully completed the follow-up examinations (mean, 27 months). Newly developed MBs were noted in 10 patients (6.8%) among those without MBs at baseline (n = 148), and in those with MBs at baseline (n = 76), the MB count had decreased in 11 patients (14.5%), and increased in 41 patients (53.9%). The estimated annual rate of change of MB numbers was 0.80 lesions per year in all patients, a value which became greater in those patients who exhibited MBs at baseline (MBs≥5, 5.43 lesions per year). Strokes due to small vessel occlusion and intracerebral hemorrhage, as well as white matter lesions were independently associated with an increased MB count, whereas the highest quartile of low-density lipoprotein (LDL) cholesterol was associated with a decreased MB count.

Conclusion

During the follow-up period, most of MBs showed dynamic temporal change. Symptomatic or asymptomatic small vessel diseases appear to act as risk factors while in contrast, a high level of LDL cholesterol may act as a protective factor against MB increase.     

Influence of flight time and flight environment on distance communication by dancing honey bees

Forager honey bees communicate the distance of food sources to nest mates through waggle dances, but how do bees measure these distances? Recent work suggests that bees measure distance flown in terms of the extent of image motion (integrated optic flow) that is experienced during flight. However, it is known that optic flow also regulates the speed of flight. Therefore, the duration of the flight to a destination is likely to co-vary with the optic flow that is experienced en route. This makes it difficult to tease apart the potential roles of flight duration and optic flow as cues in estimating distance flown. Here we examine whether flight duration alone can serve as an indicator of distance. We trained bees to visit feeders at two sites located in optically different environments, but positioned such that the flight durations to the two sites were similar. The distance estimates for the two sites, as reported in the waggle dance, were compared. We found that dances differed significantly between the two sites, even though flight times were similar. Flight time perse was a poor predictor of waggle dance behaviour. We conclude that foraging bees do not simply signal flight time as their measure of distance in the waggle dance; the environment through which they fly plays a dominant role. Received 11 April 2005; revised 16 May 2005; accepted 3 June 2005.     

Spatial foraging patterns of the African honey bee,Apis mellifera scutellata

Recruitment patterns were investigated for the African honey bee in the Okavango River Delta, Botswana. The waggle dances of two observation colonies maintained in the field were monitored and used to construct maps of daily recruitment activity. These maps revealed that the African colonies frequently adjusted the allocation of recruits among food patches, recruited for 16–17 different food sites/day over areas of 55–80 km ² ,and concentrated the majority of recruitment within 1 km of the hives (median foraging distances for the two colonies were 295 and 563 m). In both colonies pollen foragers were more abundant than nectar foragers, and pollen sources indicated by waggle dancers were significantly closer to the hives than nectar sources. Compared to the recruitment patterns of temperate climate colonies, the African colonies had smaller recruitment areas, smaller mean recruitment distances, and a greater emphasis on pollen foraging. These differences may be related to the contrasting survival strategies followed by tropical-versus temperate-climate honey bees.     

Genetic diversity within honeybee colonies increases signal production by waggle-dancing foragers

Recent work has demonstrated considerable benefits of intracolonial genetic diversity for the productivity of honeybee colonies: single-patriline colonies have depressed foraging rates, smaller food stores and slower weight gain relative to multiple-patriline colonies. We explored whether differences in the use of foraging-related communication behaviour (waggle dances and shaking signals) underlie differences in foraging effort of genetically diverse and genetically uniform colonies. We created three pairs of colonies; each pair had one colony headed by a multiply mated queen (inseminated by 15 drones) and one colony headed by a singly mated queen. For each pair, we monitored the production of foraging-related signals over the course of 3 days. Foragers in genetically diverse colonies had substantially more information available to them about food resources than foragers in uniform colonies. On average, in genetically diverse colonies compared with genetically uniform colonies, 36% more waggle dances were identified daily, dancers performed 62% more waggle runs per dance, foragers reported food discoveries that were farther from the nest and 91% more shaking signals were exchanged among workers each morning prior to foraging. Extreme polyandry by honeybee queens enhances the production of worker-worker communication signals that facilitate the swift discovery and exploitation of food resources.     

Inter‐individual variation in honey bee dance intensity correlates with expression of the foraging gene

Individual behavioural differences in responding to the same stimuli is an integral part of division of labour in eusocial insect colonies. Amongst honey bee nectar foragers, individuals strongly differ in their sucrose responsiveness, which correlates with strong differences in behavioural decisions. In this study, we explored whether the mechanisms underlying the regulation of foraging are linked to inter‐individual differences in the waggle dance activity of honey bee foragers. We first quantified the variation in dance activity amongst groups of foragers visiting an artificial feeder filled consecutively with different sucrose concentrations. We then determined, for these foragers, the sucrose responsiveness and the brain expression levels of three genes associated with food search and foraging; the foraging gene Amfor, octopamine receptor gene AmoctαR1 and insulin receptor AmInR‐2. As expected, foragers showed large inter‐individual differences in their dance activity, irrespective of the reward offered at the feeder. The sucrose responsiveness correlated positively with the intensity of the dance activity at the higher reward condition, with the more responsive foragers having a higher intensity of dancing. Out of the three genes tested, Amfor expression significantly correlated with dance activity, with more active dancers having lower expression levels. Our results show that dance and foraging behaviour in honey bees have similar mechanistic underpinnings and supports the hypothesis that the social communication behaviour of honey bees might have evolved by co‐opting behavioural modules involved in food search and foraging in solitary insects.     

Direct Visual Observation of Wing Movements during the Honey Bee Waggle Dance

Recruitment-related behaviours such as waggle dances enable honey bee foragers to inform their nestmates about the location of important resources. However, it is still not known how the information contained in a dance performed in the darkness of the nest is transferred to followers. Although, there are findings indicating that dancing honey bees produce airborne sounds which may convey the information, there has only been indirect evidence that moving wings are the source of these airborne sounds. In this study, honey bee dances were recorded using a high-speed camera in order to directly observe and precisely measure the frequency of wing beats and abdomen wags of dancers. Dancing bees moved their wings for 40.4% of the duration of a waggle run and for only 8.1% of the duration of a circle run. The episodes of wing movements consisted of one to five wing beats and were separated by intervals of motionless wings. The mean frequency of wing beats was 167.0 Hz and significantly differed depending on the number of wing beats in one episode (p < 0.001) and the position of the wings (p = 0.007). The mean frequency of abdomen wags was 14.6 Hz. The mean number of followers was 7.9 and significantly more of them gathered around the abdomens of dancers than around their heads and thoraxes (p = 0.001). The results of this study support the assumption that moving wings are the source of airborne sounds emitted during honey bee dances.     

Information needs at the beginning of foraging: grass-cutting ants trade off load size for a faster return to the nest

Background

Acquisition of information about food sources is essential for animals that forage collectively like social insects. Foragers deliver two commodities to the nest, food and information, and they may favor the delivery of one at the expenses of the other. We predict that information needs should be particularly high at the beginning of foraging: the decision to return faster to the nest will motivate a grass-cutting ant worker to reduce its loading time, and so to leave the source with a partial load.

Principal Findings

Field results showed that at the initial foraging phase, most grass-cutting ant foragers (Acromyrmex heyeri) returned unladen to the nest, and experienced head-on encounters with outgoing workers. Ant encounters were not simply collisions in a probabilistic sense: outgoing workers contacted in average 70% of the returning foragers at the initial foraging phase, and only 20% at the established phase. At the initial foraging phase, workers cut fragments that were shorter, narrower, lighter and tenderer than those harvested at the established one. Foragers walked at the initial phase significantly faster than expected for the observed temperatures, yet not at the established phase. Moreover, when controlling for differences in the fragment-size carried, workers still walked faster at the initial phase. Despite the higher speed, their individual transport rate of vegetable tissue was lower than that of similarly-sized workers foraging later at the same patch.

Conclusions/Significance

At the initial foraging phase, workers compromised their individual transport rates of material in order to return faster to the colony. We suggest that the observed flexible cutting rules and the selection of partial loads at the beginning of foraging are driven by the need of information transfer, crucial for the establishment and maintenance of a foraging process to monopolize a discovered resource.     

Does Foraging Behaviour Affect Female Mate Preferences and Pair Formation in Captive Zebra Finches?

Background

Successful foraging is essential for survival and reproductive success. In many bird species, foraging is a learned behaviour. To cope with environmental change and survive periods in which regular foods are scarce, the ability to solve novel foraging problems by learning new foraging techniques can be crucial. Although females have been shown to prefer more efficient foragers, the effect of males'' foraging techniques on female mate choice has never been studied. We tested whether females would prefer males showing the same learned foraging technique as they had been exposed to as juveniles, or whether females would prefer males that showed a complementary foraging technique.

Methodology/Principal Findings

We first trained juvenile male and female zebra finches (Taeniopygia guttata) to obtain a significant proportion of their food by one of two foraging techniques. We then tested whether females showed a preference for males with the same or the alternative technique. We found that neither a male''s foraging technique nor his foraging performance affected the time females spent in his proximity in the mate-choice apparatus. We then released flocks of these finches into an aviary to investigate whether assortative pairing would be facilitated by birds taught the same technique exploiting the same habitat. Zebra finches trained as juveniles in a specific foraging technique maintained their foraging specialisation in the aviary as adults. However, pair formation and nest location were random with regard to foraging technique.

Conclusions/Significance

Our findings show that zebra finches can be successfully trained to be foraging specialists. However, the robust negative results of the conditions tested here suggest that learned foraging specializations do not affect mate choice or pair formation in our experimental context.     

Reappraising Social Insect Behavior through Aversive Responsiveness and Learning

Background

The success of social insects can be in part attributed to their division of labor, which has been explained by a response threshold model. This model posits that individuals differ in their response thresholds to task-associated stimuli, so that individuals with lower thresholds specialize in this task. This model is at odds with findings on honeybee behavior as nectar and pollen foragers exhibit different responsiveness to sucrose, with nectar foragers having higher response thresholds to sucrose concentration. Moreover, it has been suggested that sucrose responsiveness correlates with responsiveness to most if not all other stimuli. If this is the case, explaining task specialization and the origins of division of labor on the basis of differences in response thresholds is difficult.

Methodology

To compare responsiveness to stimuli presenting clear-cut differences in hedonic value and behavioral contexts, we measured appetitive and aversive responsiveness in the same bees in the laboratory. We quantified proboscis extension responses to increasing sucrose concentrations and sting extension responses to electric shocks of increasing voltage. We analyzed the relationship between aversive responsiveness and aversive olfactory conditioning of the sting extension reflex, and determined how this relationship relates to division of labor.

Principal Findings

Sucrose and shock responsiveness measured in the same bees did not correlate, thus suggesting that they correspond to independent behavioral syndromes, a foraging and a defensive one. Bees which were more responsive to shock learned and memorized better aversive associations. Finally, guards were less responsive than nectar foragers to electric shocks, exhibiting higher tolerance to low voltage shocks. Consequently, foragers, which are more sensitive, were the ones learning and memorizing better in aversive conditioning.

Conclusions

Our results constitute the first integrative study on how aversive responsiveness affects learning, memory and social organization in honeybees. We suggest that parallel behavioral modules (e.g. appetitive, aversive) coexist within each individual bee and determine its tendency to adopt a given task. This conclusion, which is at odds with a simple threshold model, should open new opportunities for exploring the division of labor in social insects.     

Shearwater Foraging in the Southern Ocean: The Roles of Prey Availability and Winds

Background

Sooty (Puffinus griseus) and short-tailed (P. tenuirostris) shearwaters are abundant seabirds that range widely across global oceans. Understanding the foraging ecology of these species in the Southern Ocean is important for monitoring and ecosystem conservation and management.

Methodology/Principal Findings

Tracking data from sooty and short-tailed shearwaters from three regions of New Zealand and Australia were combined with at-sea observations of shearwaters in the Southern Ocean, physical oceanography, near-surface copepod distributions, pelagic trawl data, and synoptic near-surface winds. Shearwaters from all three regions foraged in the Polar Front zone, and showed particular overlap in the region around 140°E. Short-tailed shearwaters from South Australia also foraged in Antarctic waters south of the Polar Front. The spatial distribution of shearwater foraging effort in the Polar Front zone was matched by patterns in large-scale upwelling, primary production, and abundances of copepods and myctophid fish. Oceanic winds were found to be broad determinants of foraging distribution, and of the flight paths taken by the birds on long foraging trips to Antarctic waters.

Conclusions/Significance

The shearwaters displayed foraging site fidelity and overlap of foraging habitat between species and populations that may enhance their utility as indicators of Southern Ocean ecosystems. The results highlight the importance of upwellings due to interactions of the Antarctic Circumpolar Current with large-scale bottom topography, and the corresponding localised increases in the productivity of the Polar Front ecosystem.     

Adaptive Lévy Walks in Foraging Fallow Deer

Background

Lévy flights are random walks, the step lengths of which come from probability distributions with heavy power-law tails, such that clusters of short steps are connected by rare long steps. Lévy walks maximise search efficiency of mobile foragers. Recently, several studies raised some concerns about the reliability of the statistical analysis used in previous analyses. Further, it is unclear whether Lévy walks represent adaptive strategies or emergent properties determined by the interaction between foragers and resource distribution. Thus two fundamental questions still need to be addressed: the presence of Lévy walks in the wild and whether or not they represent a form of adaptive behaviour.

Methodology/Principal Findings

We studied 235 paths of solitary and clustered (i.e. foraging in group) fallow deer (Dama dama), exploiting the same pasture. We used maximum likelihood estimation for discriminating between a power-tailed distribution and the exponential alternative and rank/frequency plots to discriminate between Lévy walks and composite Brownian walks. We showed that solitary deer perform Lévy searches, while clustered animals did not adopt that strategy.

Conclusion/Significance

Our demonstration of the presence of Lévy walks is, at our knowledge, the first available which adopts up-to-date statistical methodologies in a terrestrial mammal. Comparing solitary and clustered deer, we concluded that the Lévy walks of solitary deer represent an adaptation maximising encounter rates with forage resources and not an epiphenomenon induced by a peculiar food distribution.     

Microbubble Mediated Thrombus Dissolution with Diagnostic Ultrasound for the Treatment of Chronic Venous Thrombi

Background

Central venous catheter (CVC) thrombi result in significant morbidity in children, and currently available treatments are associated with significant risk. We sought to investigate the therapeutic efficacy of microbubble (MB) enhanced sonothrombolysis for aged CVC associated thrombi in vivo.

Methods and Results

A model of chronic indwelling CVC in the low superior vena cava with thrombus in situ was established after feasibility and safety testing in 7 pigs; and subsequently applied for repeated, sonothrombolytic treatments in 9 pigs (total 24 treatments). Baseline intracardiac echocardiography (ICE, 10.5F, Siemens), fluoroscopy and saline flushing confirmed the absence of any pre-existing CVC thrombus. A thrombus was then allowed to form and age over 24 hours. The created thrombus was localized and measured by ICE, and transthoracic image guided high mechanical index (MI) two-dimensional US treatments (1.1–1.7 MI; iE33, Philips) applied intermittently whenever intravenously infused MBs (3% MRX-801; NuVox) were visualized near the thrombus (n = 10; Group A). Control pigs (n = 10; Group B) received US without MB. All treatments were randomized. Post-treatment thrombus area by ICE planimetry was compared with pre-treatment measurements. Thrombus area measurements before and after treatment were 0.22 and 0.10 cm² respectively in Group A; compared to 0.24 and 0.21 cm² in Group B (p  = 0.0003). Effectiveness of longer duration US and MB thrombolytic treatments were studied (n = 4), which suggested that near complete thrombus dissolution is possible. No pulmonary emboli, alterations in oxygen saturation, or hemodynamics occurred with either treatment.

Conclusions

Guided high MI diagnostic US+systemic MB facilitates reduction of aged CVC associated thrombi in vivo. MB enhanced sonothrombolytic therapy may be a non-invasive safe alternative to thrombolytic agents in treating thrombotic CVC occlusions.     

MiR-34a targeting of Notch ligand delta-like 1 impairs CD15+/CD133+ tumor-propagating cells and supports neural differentiation in medulloblastoma

Background

Through negative regulation of gene expression, microRNAs (miRNAs) can function as oncosuppressors in cancers, and can themselves show altered expression in various tumor types. Here, we have investigated medulloblastoma tumors (MBs), which arise from an early impairment of developmental processes in the cerebellum, where Notch signaling is involved in many of the cell-fate-determining stages. Notch regulates a subset of MB cells that have stem-cell-like properties and can promote tumor growth. On the basis of this evidence, we hypothesized that miRNAs targeting the Notch pathway can regulate these phenomena, and can be used in anti-cancer therapies.

Methodology/Principal Findings

In a screening of potential targets within Notch signaling, miR-34a was seen to be a regulator of the Notch pathway through its targeting of Notch ligand Delta-like 1 (Dll1). Down-regulation of Dll1 expression by miR-34a negatively regulates cell proliferation, and induces apoptosis and neural differentiation in MB cells. Using an inducible tetracycline on-off model of miR-34a expression, we show that in Daoy MB cells, Dll1 is the first target that is regulated in MB, as compared to the other targets analyzed here: Cyclin D1, cMyc and CDK4. MiR-34a expression negatively affects CD133⁺/CD15⁺ tumor-propagating cells, then we assay through reverse-phase proteomic arrays, Akt and Stat3 signaling hypo-phosphorylation. Adenoviruses carrying the precursor miR-34a induce neurogenesis of tumor spheres derived from a genetic animal model of MB (Patch1^+/- p53^-/-), thus providing further evidence that the miR-34a/Dll1 axis controls both autonomous and non autonomous signaling of Notch. In vivo, miR-34a overexpression carried by adenoviruses reduces tumor burden in cerebellum xenografts of athymic mice, thus demonstrating an anti-tumorigenic role of miR-34a in vivo.

Conclusions/Significance

Despite advances in our understanding of the pathogenesis of MB, one-third of patients with MB remain incurable. Here, we show that stable nucleic-acid-lipid particles carrying mature miR-34a can target Dll1 in vitro and show equal effects to those of adenovirus miR-34a cell infection. Thus, this technology forms the basis for their therapeutic use for the delivery of miR-34a in brain-tumor treatment, with no signs of toxicity described to date in non-human primate trials.