Giant honeybees (Apis dorsata) trade off defensiveness against periodic mass flight activity.

The giant honeybee Apis dorsata (Fabricius, 1793) is an evolutionarily ancient species that builds its nests in the open. The nest consists of a single honeycomb covered with the bee curtain which are several layers of worker bees that remain almost motionless with their heads up and abdomens down on the nest surface, except for the mouth area, the hub between inner- and outer-nest activities. A colony may change this semi-quiescence several times a day, depending on its reproductive state and ambient temperature, to enter the state of mass flight activity (MFA), in which nest organisation is restructured and defense ability is likely to be suppressed (predicted by the mass-flight-suspend-defensiveness hypothesis). For this study, three episode of MFA (mfa1-3) of a selected experimental nest were analysed in a case study with sequences of >60 000 images at 50 Hz, each comprise a short pre-MFA session, the MFA and the post-MFA phase of further 10 min. To test colony defensiveness under normative conditions, a dummy wasp was cyclically presented with a standardised motion programme (Pd) with intervening sessions without such a presentation (nPd). Motion activity at five selected surveillance zones (sz1-5) on the nest were analysed. In contrast to mfa1,2, in mfa3 the experimental regime started with the cyclic presentation of the dummy wasp only after the MFA had subsided. As a result, the MFA intensity in mfa3 was significantly lower than in mfa1-2, suggesting that a colony is able to perceive external threats during the MFA. Characteristic ripples appear in the motion profiles, which can be interpreted as a start signal for the transition to MFA. Because they are strongest in the mouth zone and shift to higher frequencies on their way to the nest periphery, it can be concluded that MFA starts earlier in the mouth zone than in the peripheral zones, also suggesting that the mouth zone is a control centre for the scheduling of MFA. In Pd phases of pre- and postMFA, the histogram-based motion spectra are biphasic, suggesting two cohorts in the process, one remaining at quiescence and the other involved in shimmering. Under MFA, nPd and Pd spectra were typically Gaussian, suggesting that the nest mates with a uniform workload shifted to higher motion activity. At the end of the MFA, the spectra shift back to the lower motion activities and the Pd spectra form a biphasic again. This happens a few minutes earlier in the peripheral zones than in the mouth zone. Using time profiles of the skewness of the Pd motion spectra, the mass-flight-suspend-defensiveness hypothesis is confirmed, whereby the inhibition of defense ability was found to increase progressively during the MFA. These sawtooth-like time profiles of skewness during MFA show that defense capability is recovered again quite quickly at the end of MFA. Finally, with the help of the Pd motion spectra, clear indications can be obtained that the giant honeybees engage in a decision in the sense of a tradeoff between MFA and collective defensiveness, especially in the regions in the periphery to the mouth zone.

Automatic detection of spatio-temporal signaling patterns in cell collectives.

Increasing experimental evidence points to the physiological importance of space-time correlations in signaling of cell collectives. From wound healing to epithelial homeostasis to morphogenesis, coordinated activation of biomolecules between cells allows the collectives to perform more complex tasks and to better tackle environmental challenges. To capture this information exchange and to advance new theories of emergent phenomena, we created ARCOS, a computational method to detect and quantify collective signaling. We demonstrate ARCOS on cell and organism collectives with space-time correlations on different scales in 2D and 3D. We made a new observation that oncogenic mutations in the MAPK/ERK and PIK3CA/Akt pathways of MCF10A epithelial cells hyperstimulate intercellular ERK activity waves that are largely dependent on matrix metalloproteinase intercellular signaling. ARCOS is open-source and available as R and Python packages. It also includes a plugin for the napari image viewer to interactively quantify collective phenomena without prior programming experience.

Non-invasive diagnosis of viability in seeds and lichens by infrared thermography under controlled environmental conditions.

BACKGROUND: Non-invasive procedures for the diagnosis of viability of plant or fungal tissues would be valuable for scientific, industrial and biomonitoring purposes. Previous studies showed that infrared thermography (IRT) enables non-invasive assessment of the viability of individual "orthodox" (i.e. desiccation tolerant) seeds upon water uptake. However, this method was not tested for rehydrating tissues of other desiccation tolerant life forms. Furthermore, evaporative cooling could obscure the effects of metabolic processes that contribute to heating and cooling, but its effects on the shape of the "thermal fingerprints" have not been explored. Here, we further adapted this method using a purpose-built chamber to control relative humidity (RH) and gaseous atmosphere. This enabled us to test (i) the influence of relative humidity on the thermal fingerprints during the imbibition of Pisum sativum (Garden pea) seeds, (ii) whether thermal fingerprints can be correlated with viability in lichens, and (iii) to assess the potential influence of aerobic metabolism on thermal fingerprints by controlling the oxygen concentration in the gaseous atmosphere around the samples. Finally, we developed a method to artificially "age" lichens and validated the IRT-based method to assess lichen viability in three lichen species. RESULTS: Using either 30% or 100% RH during imbibition of pea seeds, we showed that "live" and "dead" seeds produced clearly discernible "thermal fingerprints", which significantly differed by > |0.15| °C in defined time windows, and that RH affected the shape of these thermal fingerprints. We demonstrated that IRT can also be used to assess the viability of the lichens Lobaria pulmonaria, Pseudevernia furfuracea and Peltigera leucophlebia. No clear relationship between aerobic metabolism and the shape of thermal fingerprints was found. CONCLUSIONS: Infrared thermography appears to be a promising method for the diagnosis of viability of desiccation-tolerant tissues at early stages of water uptake. For seeds, it is possible to diagnose viability within the first hours of rehydration, after which time they can still be re-dried and stored until further use. We envisage our work as a baseline study for the use of IR imaging techniques to investigate physiological heterogeneity of desiccation tolerant life forms such as lichens, which can be used for biomonitoring, and for sorting live and dead seeds, which is potentially useful for the seed trade.

Evidence for Ventilation through Collective Respiratory Movements in Giant Honeybee (Apis dorsata) Nests.

The Asian giant honeybees (Apis dorsata) build single-comb nests in the open, which makes this species particularly susceptible to environmental strains. Long-term infrared (IR) records documented cool nest regions (CNR) at the bee curtain (nCNR = 207, nnests > 20) distinguished by marked negative gradients (ΔTCNR/d < -3°C / 5 cm) at their margins. CNRs develop and recede within minutes, predominantly at higher ambient temperatures in the early afternoon. The differential size (ΔACNR) and temperature (ΔTCNR) values per time unit correlated mostly positively (RAT > 0) displaying the Venturi effect, which evidences funnel properties of CNRs. The air flows inwards through CNRs, which is verified by the negative spatial gradient ΔTCNR/d, by the positive grading of TCNR with Tamb and lastly by fanners which have directed their abdomens towards CNRs. Rare cases of RAT < 0 (< 3%) document closing processes (for ΔACNR/Δt < -0.4 cm2/s) but also suggest ventilation of the bee curtain (for ΔACNR/Δt > +0.4 cm2/s) displaying "inhalation" and "exhalation" cycling. "Inhalation" could be boosted by bees at the inner curtain layers, which stretch their extremities against the comb enlarging the inner nest lumen and thus causing a pressure fall which drives ambient air inwards through CNR funnels. The relaxing of the formerly "activated" bees could then trigger the "exhalation" process, which brings the bee curtain, passively by gravity, close to the comb again. That way, warm, CO2-enriched nest-borne air is pressed outwards through the leaking mesh of the bee curtain. This ventilation hypothesis is supported by IR imaging and laser vibrometry depicting CNRs in at least four aspects as low-resistance convection funnels for maintaining thermoregulation and restoring fresh air in the nest.

Giant honeybees (Apis dorsata) mob wasps away from the nest by directed visual patterns.

The open nesting behaviour of giant honeybees (Apis dorsata) accounts for the evolution of a series of defence strategies to protect the colonies from predation. In particular, the concerted action of shimmering behaviour is known to effectively confuse and repel predators. In shimmering, bees on the nest surface flip their abdomens in a highly coordinated manner to generate Mexican wave-like patterns. The paper documents a further-going capacity of this kind of collective defence: the visual patterns of shimmering waves align regarding their directional characteristics with the projected flight manoeuvres of the wasps when preying in front of the bees' nest. The honeybees take here advantage of a threefold asymmetry intrinsic to the prey-predator interaction: (a) the visual patterns of shimmering turn faster than the wasps on their flight path, (b) they "follow" the wasps more persistently (up to 100 ms) than the wasps "follow" the shimmering patterns (up to 40 ms) and (c) the shimmering patterns align with the wasps' flight in all directions at the same strength, whereas the wasps have some preference for horizontal correspondence. The findings give evidence that shimmering honeybees utilize directional alignment to enforce their repelling power against preying wasps. This phenomenon can be identified as predator driving which is generally associated with mobbing behaviour (particularly known in selfish herds of vertebrate species), which is, until now, not reported in insects.

Speeding up social waves. Propagation mechanisms of shimmering in giant honeybees.

Shimmering is a defence behaviour in giant honeybees (Apis dorsata), whereby bees on the nest surface flip their abdomen upwards in a Mexican wave-like process. However, information spreads faster than can be ascribed to bucket bridging, which is the transfer of information from one individual to an adjacent one. We identified a saltatoric process that speeds up shimmering by the generation of daughter waves, which subsequently merge with the parental wave, producing a new wave front. Motion patterns of individual "focus" bees (n = 10,894) and their shimmering-active neighbours (n = 459,558) were measured with high-resolution video recording and stereoscopic imaging. Three types of shimmering-active surface bees were distinguished by their communication status, termed "agents": "Bucket-bridging" agents comprised 74.98% of all agents, affected 88.17% of their neighbours, and transferred information at a velocity of v = 0.317±0.015 m/s. "Chain-tail" agents comprised 9.20% of the agents, were activated by 6.35% of their neighbours, but did not motivate others to participate in the wave. "Generator agents" comprised 15.82% of agents, showed abdominal flipping before the arrival of the main wave front, and initiated daughter waves. They affected 6.75% of their neighbourhood and speeded up the compound shimmering process compared to bucket bridging alone by 41.5% to v = 0.514±0.019 m/s. The main direction of shimmering was reinforced by 35.82% of agents, whereas the contribution of the complementing agents was fuzzy. We discuss that the saltatoric process could enable the bees to instantly recruit larger cohorts to participate in shimmering and to respond rapidly to changes in flight direction of preying wasps. A third, non-exclusive explanation is that at a distance of up to three metres from the nest the acceleration of shimmering could notably contribute to the startle response in mammals and birds.

Intraspecific Aggression in Giant Honey Bees (Apis dorsata).

We investigated intraspecific aggression in experimental nests (expN1, expN2) of the giant honey bee Apis dorsata in Chitwan (Nepal), focusing on interactions between surface bees and two other groups of bees approaching the nest: (1) homing "nestmate" foragers landing on the bee curtain remained unmolested by guards; and (2) supposed "non-nestmate" bees, which were identified by their erratic flight patterns in front of the nest, such as hovering or sideways scanning and splaying their legs from their body, and were promptly attacked by the surface bees after landing. These supposed non-nestmate bees only occurred immediately before and after migration swarms, which had arrived in close vicinity (and were most likely scouting for a nesting site). In total, 231 of the "nestmate" foragers (fb) and 102 approaches of such purported "non-nestmate" scouts (sc) were analysed (total observation time expN1: 5.43 min) regarding the evocation of shimmering waves (sh). During their landing the "nestmate" foragers provoked less shimmering waves (relnsh[fb] = 23/231 = 0.0996, relnsh[sc] = 75/102 = 0.7353; p <0.001, χ²-test) with shorter duration (Dsh[fb] = 197 ± 17 ms, Dsh[sc] = 488 ± 16 ms; p <0.001; t-test) than "non-nestmates". Moreover, after having landed on the nest surface, the "non-nestmates" were attacked by the surface bees (expN1, expN2: observation time >18 min) quite similarly to the defensive response against predatory wasps. Hence, the surface members of settled colonies respond differently to individual giant honey bees approaching the nest, depending on whether erratic flight patterns are displayed or not.

Social waves in giant honeybees (Apis dorsata) elicit nest vibrations.

Giant honeybees (Apis dorsata) nest in the open and have developed a wide array of strategies for colony defence, including the Mexican wave-like shimmering behaviour. In this collective response, the colony members perform upward flipping of their abdomens in coordinated cascades across the nest surface. The time-space properties of these emergent waves are response patterns which have become of adaptive significance for repelling enemies in the visual domain. We report for the first time that the mechanical impulse patterns provoked by these social waves and measured by laser Doppler vibrometry generate vibrations at the central comb of the nest at the basic (='natural') frequency of 2.156 ± 0.042 Hz which is more than double the average repetition rate of the driving shimmering waves. Analysis of the Fourier spectra of the comb vibrations under quiescence and arousal conditions provoked by mass flight activity and shimmering waves gives rise to the proposal of two possible models for the compound physical system of the bee nest: According to the elastic oscillatory plate model, the comb vibrations deliver supra-threshold cues preferentially to those colony members positioned close to the comb. The mechanical pendulum model predicts that the comb vibrations are sensed by the members of the bee curtain in general, enabling mechanoreceptive signalling across the nest, also through the comb itself. The findings show that weak and stochastic forces, such as general quiescence or diffuse mass flight activity, cause a harmonic frequency spectrum of the comb, driving the comb as an elastic plate. However, shimmering waves provide sufficiently strong forces to move the nest as a mechanical pendulum. This vibratory behaviour may support the colony-intrinsic information hypothesis herein that the mechanical vibrations of the comb provoked by shimmering do have the potential to facilitate immediate communication of the momentary defensive state of the honeybee nest to the majority of its members.

How to join a wave: decision-making processes in shimmering behavior of Giant honeybees (Apis dorsata).

Shimmering is a collective defence behaviour in Giant honeybees (Apis dorsata) whereby individual bees flip their abdomen upwards, producing Mexican wave-like patterns on the nest surface. Bucket bridging has been used to explain the spread of information in a chain of members including three testable concepts: first, linearity assumes that individual "agent bees" that participate in the wave will be affected preferentially from the side of wave origin. The directed-trigger hypothesis addresses the coincidence of the individual property of trigger direction with the collective property of wave direction. Second, continuity describes the transfer of information without being stopped, delayed or re-routed. The active-neighbours hypothesis assumes coincidence between the direction of the majority of shimmering-active neighbours and the trigger direction of the agents. Third, the graduality hypothesis refers to the interaction between an agent and her active neighbours, assuming a proportional relationship in the strength of abdomen flipping of the agent and her previously active neighbours. Shimmering waves provoked by dummy wasps were recorded with high-resolution video cameras. Individual bees were identified by 3D-image analysis, and their strength of abdominal flipping was assessed by pixel-based luminance changes in sequential frames. For each agent, the directedness of wave propagation was based on wave direction, trigger direction, and the direction of the majority of shimmering-active neighbours. The data supported the bucket bridging hypothesis, but only for a small proportion of agents: linearity was confirmed for 2.5%, continuity for 11.3% and graduality for 0.4% of surface bees (but in 2.6% of those agents with high wave-strength levels). The complimentary part of 90% of surface bees did not conform to bucket bridging. This fuzziness is discussed in terms of self-organisation and evolutionary adaptedness in Giant honeybee colonies to respond to rapidly changing threats such as predatory wasps scanning in front of the nest.

Training for Defense? From Stochastic Traits to Synchrony in Giant Honey Bees (Apis dorsata).

In Giant Honey Bees, abdomen flipping happens in a variety of contexts. It can be either synchronous or cascaded, such as in the collective defense traits of shimmering and rearing-up, or it can happen as single-agent behavior. Abdomen flipping is also involved in flickering behavior, which occurs regularly under quiescent colony state displaying singular or collective traits, with stochastic, and (semi-) synchronized properties. It presumably acts via visual, mechanoceptive, and pheromonal pathways and its goals are still unknown. This study questions whether flickering is preliminary to shimmering which is subject of the fs (flickering-shimmering)-transition hypothesis? We tested the respective prediction that trigger sites (ts) at the nest surface (where shimmering waves had been generated) show higher flickering activity than the alternative non-trigger sites (nts). We measured the flickering activity of ts- and nts-surface bees from two experimental nests, before and after the colony had been aroused by a dummy wasp. Arousal increased rate and intensity of the flickering activity of both ts- and nts cohorts (P < 0.05), whereby the flickering intensity of ts-bees were higher than that of nts-bees (P < 0.05). Under arousal, the colonies also increased the number of flickering-active ts- and nts-cohorts (P < 0.05). This provides evidence that cohorts which are specialist at launching shimmering waves are found across the quiescent nest zone. It also proves that arousal may reinforce the responsiveness of quiescent curtain bees for participating in shimmering, practically by recruiting additional trigger site bees for expanding repetition of rate and intensity of shimmering waves. This finding confirms the fs-transition hypothesis and constitutes evidence that flickering is part of a basal colony-intrinsic information system. Furthermore, the findings disprove that the muscle activity associated with flickering would heat up the surface bees. Hence, surface bees are not actively contributing to thermoregulation.

Stereoscopic motion analysis in densely packed clusters: 3D analysis of the shimmering behaviour in Giant honey bees.

BACKGROUND: The detailed interpretation of mass phenomena such as human escape panic or swarm behaviour in birds, fish and insects requires detailed analysis of the 3D movements of individual participants. Here, we describe the adaptation of a 3D stereoscopic imaging method to measure the positional coordinates of individual agents in densely packed clusters. The method was applied to study behavioural aspects of shimmering in Giant honeybees, a collective defence behaviour that deters predatory wasps by visual cues, whereby individual bees flip their abdomen upwards in a split second, producing Mexican wave-like patterns. RESULTS: Stereoscopic imaging provided non-invasive, automated, simultaneous, in-situ 3D measurements of hundreds of bees on the nest surface regarding their thoracic position and orientation of the body length axis. Segmentation was the basis for the stereo matching, which defined correspondences of individual bees in pairs of stereo images. Stereo-matched "agent bees" were re-identified in subsequent frames by the tracking procedure and triangulated into real-world coordinates. These algorithms were required to calculate the three spatial motion components (dx: horizontal, dy: vertical and dz: towards and from the comb) of individual bees over time. CONCLUSIONS: The method enables the assessment of the 3D positions of individual Giant honeybees, which is not possible with single-view cameras. The method can be applied to distinguish at the individual bee level active movements of the thoraces produced by abdominal flipping from passive motions generated by the moving bee curtain. The data provide evidence that the z-deflections of thoraces are potential cues for colony-intrinsic communication. The method helps to understand the phenomenon of collective decision-making through mechanoceptive synchronization and to associate shimmering with the principles of wave propagation. With further, minor modifications, the method could be used to study aspects of other mass phenomena that involve active and passive movements of individual agents in densely packed clusters.

Complex social waves of giant honeybees provoked by a dummy wasp support the special-agent hypothesis.

The social waves in giant honeybees termed as shimmering are more complex than mexican waves. it has been demonstrated1 that shimmering is triggered by special agents at the nest surface. in this paper, we have used a nest that originated by amalgamation of two previously separated nests and stimulated waves by a dummy wasp moved on a miniature cable car. we illustrate the plausibility of the special-agent hypothesis1 also for complex shimmering processes.

Noninvasive diagnosis of seed viability using infrared thermography.

Recent advances in the noninvasive analyses of plant metabolism include stress imaging techniques, mainly developed for vegetative tissues. We explored if infrared thermography can be used to predict whether a quiescent seed will germinate or die upon water uptake. Thermal profiles of viable, aged, and dead Pisum sativum seeds were recorded, and image analysis of 22,000 images per individual seed showed that infrared thermography can detect imbibition- and germination-associated biophysical and biochemical changes. These "thermal fingerprints" vary with viability in this species and in Triticum aestivum and Brassica napus seeds. Thermogenesis of the small individual B. napus seeds was at the limit of the technology. We developed a computer model of "virtual pea seeds," that uses Monte Carlo simulation, based on the heat production of major seed storage compounds to unravel physico-chemical processes of thermogenesis. The simulation suggests that the cooling that dominates the early thermal profiles results from the dissolution of low molecular-weight carbohydrates. Moreover, the kinetics of the production of such "cooling" compounds over the following 100 h is dependent on seed viability. We also developed a deterministic tool that predicts in the first 3 hours of water uptake, when seeds can be redried and stored again, whether or not a pea seed will germinate. We believe that the early separation of individual, ungerminated seeds (live, aged, or dead) before destructive germination assessment creates unique opportunities for integrative studies on cell death, differentiation, and development.

'Special agents' trigger social waves in giant honeybees (Apis dorsata).

Giant honeybees (Apis dorsata) nest in the open and have therefore evolved a variety of defence strategies. Against predatory wasps, they produce highly coordinated Mexican wavelike cascades termed 'shimmering', whereby hundreds of bees flip their abdomens upwards. Although it is well known that shimmering commences at distinct spots on the nest surface, it is still unclear how shimmering is generated. In this study, colonies were exposed to living tethered wasps that were moved in front of the experimental nest. Temporal and spatial patterns of shimmering were investigated in and after the presence of the wasp. The numbers and locations of bees that participated in the shimmering were assessed, and those bees that triggered the waves were identified. The findings reveal that the position of identified trigger cohorts did not reflect the experimental path of the tethered wasp. Instead, the trigger centres were primarily arranged in the close periphery of the mouth zone of the nest, around those parts where the main locomotory activity occurs. This favours the 'special-agents' hypothesis that suggest that groups of specialized bees initiate the shimmering.

Social waves in giant honeybees repel hornets.

Giant honeybees (Apis dorsata) nest in the open and have evolved a plethora of defence behaviors. Against predatory wasps, including hornets, they display highly coordinated Mexican wave-like cascades termed 'shimmering'. Shimmering starts at distinct spots on the nest surface and then spreads across the nest within a split second whereby hundreds of individual bees flip their abdomens upwards. However, so far it is not known whether prey and predator interact and if shimmering has anti-predatory significance. This article reports on the complex spatial and temporal patterns of interaction between Giant honeybee and hornet exemplified in 450 filmed episodes of two A. dorsata colonies and hornets (Vespa sp.). Detailed frame-by-frame analysis showed that shimmering elicits an avoidance response from the hornets showing a strong temporal correlation with the time course of shimmering. In turn, the strength and the rate of the bees' shimmering are modulated by the hornets' flight speed and proximity. The findings suggest that shimmering creates a 'shelter zone' of around 50 cm that prevents predatory wasps from foraging bees directly from the nest surface. Thus shimmering appears to be a key defence strategy that supports the Giant honeybees' open-nesting life-style.

Infrared imaging technology and biological applications.

Temperature is the most frequently measured physical quantity, second only to time. Infrared (IR) technology has been utilized successfully in astronomy (for a summary,see Hermans-Killam, 2002b) and in industrial and research settings (Gruner, 2002; Madding, 1982, 1989; Wolfe & Zissis, 1993) for decades. However, fairly recent innovations have reduced costs, increased reliability, and resulted in noncontact IR sensors offering mobile, smaller units of measurement (EOI, 2002; Flir, 2000, 2001,2002). The advantages of using IR imaging are (1) rapidity in the millisecond range, facilitating measurement of moving targets, (2) noncontact procedures, allowing measurements of hazardous or physically inaccessible objects, (3) no interference and no energy lost from the target, (4) no risk of contamination, and (5) no mechanical effect on the surface of the object. All these factors have led to IR technology's becoming an area of interest for new kinds of applications and users. In both manufacturing and quality control, temperature plays an important role as an indicator of the condition of a product or a piece of machinery (EOI, 2002; Flir, 2000, 2001, 2002; Raytek, 2002). In medical and veterinary applications, IR thermometry is increasingly used in organ diagnostics, in the evaluation of sports injuries and the progression of therapy, in disease evaluation (e.g, breast cancer, arthritis, and SARS; Flir, 2003), and in injury and inflammation examinations in horses, livestock (Tivey & Banhazi, 2002), and zoo animals (Hermans-Killam, 2002a; Thiesbrummel, 2002). Lastly, physiological expressions of life processes in animals (Kastberger, Winder, & Steindl, 2001; Stabentheiner, Kovac, & Hagmüller, 1995; Stabentheiner, Kovac, & Schmaranzer, 2002; Stabentheiner & Schmarnzer, 1987) and plants (Bermadinger-Stabentheiner & Stabentheiner, 1995) can be monitored. The most recent field in which IR technology has been applied is animal behavior. This article focuses on the practical options for noncontact IR thermometry--in particular, in biological applications.

5-Methyl-2-phenyl-2-hexenal in the scent gland secretion of male cave crickets, Troglophilus cavicola and T. neglectus (Ensifera: Rhaphidophoridae).

5-Methyl-2-phenyl-2-hexenal, a distinctly smelling compound, was identified as a constituent of the male scent gland secretion of two species of cave crickets, Troglophilus cavicola and T. neglectus. It is the first proof of a male specific exocrine compound in grasshoppers. A pheromonal function of the component could not be established; its possible biological role is discussed.