Two feedback mechanisms involved in the control of leaf fragment size in leaf-cutting ants.

Polymorphic leaf-cutting ants harvest leaf fragments that correlate in size with the workers' body size. When cutting, workers anchor their hind legs on the leaf edge and rotate, removing approximately semicircular fragments. Workers show behavioural plasticity and modify their leg extension while holding onto the leaf edge depending on, for instance, leaf toughness, cutting smaller fragments out of tough leaves. What sensory information workers use to control the cutting trajectory remains unknown. We investigated whether sensory information from both the leg contact with the leaf edge and from head movements underlies fragment size determination. In the laboratory, we recorded Atta sexdens workers cutting standardised ®Parafilm pseudoleaves of different thickness, and quantified cutting behaviour and body reach, i.e. the distance between the mandible and the anchored hind leg tarsus. Experimentally preventing contact with the leaf edge resulted in smaller fragments, evincing that workers control the cutting trajectory using information from the contact of the hind legs with the leaf edge. However, ants were able to cut fragments even when contact of all six legs with the edge was prevented, indicating the use of additional sensory information. Ablation of mechanosensory hairs at the neck joint alone did not influence fragment size determination, yet simultaneously preventing sensory feedback from both mechanosensory hairs and edge contact led to a loss of control over the cutting trajectory. Leaf-cutting ants, therefore, control their cutting trajectory using sensory information from both the leg contact with the leaf edge and the lateral bending of the head.

Symbiont demand guides resource supply: leaf-cutting ants preferentially deliver their harvested fragments to undernourished fungus gardens.

Leaf-cutting ants are highly successful herbivores in the Neotropics. They forage large amounts of fresh plant material to nourish a symbiotic fungus that sustains the colony. It is unknown how workers organize the intra-nest distribution of resources, and whether they respond to increasing demands in some fungus gardens by adjusting the amount of delivered resources accordingly. In laboratory experiments, we analyzed the spatial distribution of collected leaf fragments among nest chambers in Acromyrmex ambiguus leaf-cutting ants, and how it changed when one of the fungus gardens experienced undernourishment. Plant fragments were evenly distributed among nest chambers when the fungal symbiont was well nourished. That pattern changed when one of the fungus gardens was undernourished and had a higher leaf demand, resulting in more leaf discs delivered to the undernourished fungus garden over at least 2 days after deprivation. Some ants bypassed nourished gardens to directly deliver their resource to the chamber with higher nutritional demand. We hypothesize that cues arising from that chamber might be used for orientation and/or that informed individuals, presumably stemming from the undernourished chamber, may preferentially orient to them.

Carbon dioxide levels and ventilation in Acromyrmex nests: significance and evolution of architectural innovations in leaf-cutting ants.

Leaf-cutting ant colonies largely differ in size, yet all consume O2 and produce CO2 in large amounts because of their underground fungus gardens. We have shown that in the Acromyrmex genus, three basic nest morphologies occur, and investigated the effects of architectural innovations on nest ventilation. We recognized (i) serial nests, similar to the ancestral type of the sister genus Trachymyrmex, with chambers excavated along a vertical tunnel connecting to the outside via a single opening, (ii) shallow nests, with one/few chambers extending shallowly with multiple connections to the outside, and (iii) thatched nests, with an above-ground fungus garden covered with plant material. Ventilation in shallow and thatched nests, but not in serial nests, occurred via wind-induced flows and thermal convection. CO2 concentrations were below the values known to affect the respiration of the symbiotic fungus, indicating that shallow and thatched nests are not constrained by harmful CO2 levels. Serial nests may be constrained depending on the soil CO2 levels. We suggest that in Acromyrmex, selective pressures acting on temperature and humidity control led to nesting habits closer to or above the soil surface and to the evolution of architectural innovations that improved gas exchanges.

Selection and spatial arrangement of building materials during the construction of nest turrets by grass-cutting ants.

Ants build complex nest structures by reacting to simple, local stimuli. While underground nests result from the space generated by digging, some leaf- and grass-cutting ants also construct conspicuous aboveground turrets around nest openings. We investigated whether the selection of specific building materials occurs during turret construction in Acromyrmex fracticornis grass-cutting ants, and asked whether single building decisions at the beginning can modify the final turret architecture. To quantify workers' material selection, the original nest turret was removed and a choice between two artificial building materials, thin and thick sticks, was offered for rebuilding. Workers preferred thick sticks at the very beginning of turret construction, showed varying preferences thereafter, and changed to prefer thin sticks for the upper, final part of the turret, indicating that they selected different building materials over time to create a stable structure. The impact of a single building choice on turret architecture was evaluated by placing artificial beams that divided a colony's nest entrance at the beginning of turret rebuilding. Splitting the nest entrance led to the self-organized construction of turrets with branched galleries ending in multiple openings, showing that the spatial location of a single building material can strongly influence turret morphology.

Nest Turrets of Acromyrmex Grass-Cutting Ants: Micromorphology Reveals Building Techniques and Construction Dynamics.

Acromyrmex fracticornis grass-cutting ants construct conspicuous chimney-shaped nest turrets made of intermeshed grass fragments. We asked whether turrets are constructed by merely piling up nearby materials around the entrance, or whether ants incorporate different materials as the turret develops. By removing the original nest turrets and following their rebuilding process over three consecutive days, age-dependent changes in wall morphology and inner lining fabrics were characterized. Micromorphological descriptions based on thin sections of turret walls revealed the building behaviors involved. Ants started by collecting nearby twigs and dry grass fragments that are piled up around the nest entrance. Several large fragments held the structure like beams. As a net-like structure grew, soil pellets were placed in between the intermeshed plant fragments from the turret base to the top, reinforcing the structure. Concomitantly, the turret inner wall was lined with soil pellets, starting from the base. Therefore, the consolidation of the turret occurred both over time and from its base upwards. It is argued that nest turrets do not simply arise by the arbitrary deposition of nearby materials, and that workers selectively incorporate large materials at the beginning, and respond to the developing structure by reinforcing the intermeshed plant fragments over time.

Waste deposition in leaf-cutting ants is guided by olfactory cues from waste.

Social insects often use olfactory cues from their environment to coordinate colony tasks. We investigated whether leaf-cutting ants use volatiles as cues to guide the deposition of their copious amounts of colony refuse. In the laboratory, we quantified the relocation of a small pile of colony waste by workers of Atta laevigata towards volatiles offered at each side of the pile as a binary choice, consisting of either waste volatiles, fungus volatiles, or no volatiles. Fungus volatiles alone did not evoke relocation of waste. Waste volatiles alone, by contrast, led to a strong relocation of waste particles towards them. When fungus and waste volatiles were tested against each other, waste particles were also relocated towards waste volatiles, and in a high percentage of assays completely moved away from the source of fungus volatiles as compared to the previous series. We suggest that deposition and accumulation of large amounts of refuse in single external heaps or a few huge underground waste chambers of Atta nests is due to both olfactory preferences and stigmergic responses towards waste volatiles by waste-carrying workers.

Carbon dioxide sensing in the social context: Leaf-cutting ants prefer elevated CO2 levels to tend their brood.

Social insects show temperature and humidity preferences inside their nests to successfully rear brood. In underground nests, ants also encounter rising CO2 concentrations with increasing depth. It is an open question whether they use CO2 as a cue to decide where to place and tend the brood. Leaf-cutting ants do show CO2 preferences for the culturing of their symbiotic fungus. We evaluated their CO2 choices for brood placement in laboratory experiments. Workers of Acromyrmex lundii in the process of relocating brood were offered a binary choice consisting of two interconnected chambers with different CO2 concentrations. Values ranged from atmospheric to high concentrations of 4% CO2. The CO2 preferences shown by workers for themselves and for brood placement were assessed by quantifying the number of workers and relocated brood in each chamber. Ants showed clear CO2 preferences for brood placement. They avoided atmospheric levels, 1% and 4% CO2, and showed a preference for levels of 3%. This is the first report of CO2 preferences for the maintenance of brood in social insects. The observed preferences for brood location were independent of the workers' own CO2 preferences, since they showed no clear-cut pattern. Workers' CO2 preferences for brood maintenance were slightly higher than those reported for fungus culturing, although brood is reared in the same chambers as the fungus in leaf-cutting ant nests. Workers' choices for brood placement in natural nests are likely the result of competing preferences for other environmental factors more crucial for brood survival, aside from those for CO2.

Carbon dioxide sensing in an obligate insect-fungus symbiosis: CO2 preferences of leaf-cutting ants to rear their mutualistic fungus.

Defense against biotic or abiotic stresses is one of the benefits of living in symbiosis. Leaf-cutting ants, which live in an obligate mutualism with a fungus, attenuate thermal and desiccation stress of their partner through behavioral responses, by choosing suitable places for fungus-rearing across the soil profile. The underground environment also presents hypoxic (low oxygen) and hypercapnic (high carbon dioxide) conditions, which can negatively influence the symbiont. Here, we investigated whether workers of the leaf-cutting ant Acromyrmex lundii use the CO2 concentration as an orientation cue when selecting a place to locate their fungus garden, and whether they show preferences for specific CO2 concentrations. We also evaluated whether levels preferred by workers for fungus-rearing differ from those selected for themselves. In the laboratory, CO2 preferences were assessed in binary choices between chambers with different CO2 concentrations, by quantifying number of workers in each chamber and amount of relocated fungus. Leaf-cutting ants used the CO2 concentration as a spatial cue when selecting places for fungus-rearing. A. lundii preferred intermediate CO2 levels, between 1 and 3%, as they would encounter at soil depths where their nest chambers are located. In addition, workers avoided both atmospheric and high CO2 levels as they would occur outside the nest and at deeper soil layers, respectively. In order to prevent fungus desiccation, however, workers relocated fungus to high CO2 levels, which were otherwise avoided. Workers' CO2 preferences for themselves showed no clear-cut pattern. We suggest that workers avoid both atmospheric and high CO2 concentrations not because they are detrimental for themselves, but because of their consequences for the symbiotic partner. Whether the preferred CO2 concentrations are beneficial for symbiont growth remains to be investigated, as well as whether the observed preferences for fungus-rearing influences the ants' decisions where to excavate new chambers across the soil profile.

Nest enlargement in leaf-cutting ants: relocated brood and fungus trigger the excavation of new chambers.

During colony growth, leaf-cutting ants enlarge their nests by excavating tunnels and chambers housing their fungus gardens and brood. Workers are expected to excavate new nest chambers at locations across the soil profile that offer suitable environmental conditions for brood and fungus rearing. It is an open question whether new chambers are excavated in advance, or will emerge around brood or fungus initially relocated to a suitable site in a previously-excavated tunnel. In the laboratory, we investigated the mechanisms underlying the excavation of new nest chambers in the leaf-cutting ant Acromyrmex lundi. Specifically, we asked whether workers relocate brood and fungus to suitable nest locations, and to what extent the relocated items trigger the excavation of a nest chamber and influence its shape. When brood and fungus were exposed to unfavorable environmental conditions, either low temperatures or low humidity, both were relocated, but ants clearly preferred to relocate the brood first. Workers relocated fungus to places containing brood, demonstrating that subsequent fungus relocation spatially follows the brood deposition. In addition, more ants aggregated at sites containing brood. When presented with a choice between two otherwise identical digging sites, but one containing brood, ants' excavation activity was higher at this site, and the shape of the excavated cavity was more rounded and chamber-like. The presence of fungus also led to the excavation of rounder shapes, with higher excavation activity at the site that also contained brood. We argue that during colony growth, workers preferentially relocate brood to suitable locations along a tunnel, and that relocated brood spatially guides fungus relocation and leads to increased digging activity around them. We suggest that nest chambers are not excavated in advance, but emerge through a self-organized process resulting from the aggregation of workers and their density-dependent digging behavior around the relocated brood and fungus.

Vascular risk prediction in ischemic stroke patients undergoing in-patient rehabilitation - insights from the investigation of patients with ischemic stroke in neurologic rehabilitation (INSIGHT) registry.

BACKGROUND: In-patient rehabilitation following ischemic stroke offers a unique opportunity for risk factor and lifestyle modification. Quantification of risk in this setting may help to tailor therapy, increase physician awareness and patient compliance and thus to reduce recurrent vascular events. AIMS: To validate the predictive value of established secondary stroke risk scores. METHODS: One thousand one hundred sixty-three patients undergoing in-patient rehabilitation following recent ischemic stroke in 15 German neurologic rehabilitation centers were included 0·9 ± 0·5 months after the index event. Outcome information was available for 846 participants (72·7%) after a mean follow-up of 13 ± 2·3 months. RESULTS: Patients' mean age was 66·3 ± 12·3 years and 42·5% were women. The National Institutes of Health Stroke Scale (mean 4·0 ± 3·9), modified Rankin scale (median 2, range 0-5), and Barthel Index (median 90, range 0-100) indicated good functional status. A recurrent fatal or nonfatal stroke during follow-up occurred in 6·7% and combined vascular events (stroke, myocardial infarction, vascular death) in 10·9%. The predictive accuracy for recurrent stroke was slightly higher on the Essen Stroke Risk Score than on the Stroke Prognostis Instrument II (area under the curve 0·62 vs. 0·56), while both scores had a similar predictive accuracy for combined vascular events. CONCLUSIONS: Risk stratification on the Essen Stroke Risk Score and Stroke Prognostis Instrument II provides a moderate accuracy for the prediction of recurrent stroke and vascular events in patients undergoing neurologic in-patient rehabilitation. Although individual risk prediction may remain imprecise, the use of these scores should be encouraged.

Candidate polyanionic microbicides inhibit human T-cell lymphotropic virus type 1 receptor interactions, cell-free infection, and cell-cell spread.

The human T-cell lymphotropic virus type 1 (HTLV-1) is the cause of adult T-cell leukemia and inflammatory diseases including HTLV-1-associated myelopathy/tropical spastic paraparesis. HTLV-1 can be transmitted through sexual contact, mother-to-child transmission, and exposure to contaminated blood. Microbicides are agents that interfere with microbial infectivity at mucous membranes, and candidates are under development for use against sexually transmitted viruses such as human immunodeficiency virus type 1. We previously demonstrated that cell surface polyanionic heparan sulfate proteoglycans bind the HTLV-1 envelope glycoprotein surface subunit gp46, facilitating cell-cell and cell-free virus spread in vitro. We now show, using assays for Env-receptor binding inhibition, Env-induced cell-cell fusion, cell-cell virus spread, and pseudotype HTLV-1 infectivity, that the soluble polyanions PRO 2000 and dextran sulfate are potent inhibitors of HTLV-1 spread in vitro, with PRO 2000 being the more promising candidate. The results of these studies suggest that candidate topical microbicides may be of use in reducing HTLV-1 sexual transmission.