Large deformation diffeomorphic mapping of 3D shape variation reveals two distinct mandible and head capsule morphs in Atta vollenweideri leaf-cutter worker ants.

Ants are crucial ecosystem engineers, and their ecological success is facilitated by a division of labour among sterile "workers". In some ant lineages, workers have undergone further morphological differentiation, resulting in differences in body size, shape, or both. Distinguishing between changes in size and shape is not trivial. Traditional approaches based on allometry reduce complex 3D shapes into simple linear, areal, or volume metrics; modern approaches using geometric morphometrics typically rely on landmarks, introducing observer bias and a trade-off between effort and accuracy. Here, we use a landmark-free method based on large deformation diffeomorphic metric mapping (LDDMM) to assess the co-variation of size and 3D shape in the mandibles and head capsules of Atta vollenweideri leaf-cutter ants, a species exhibiting extreme worker size-variation. Body mass varied by more than two orders of magnitude, but a shape atlas created via LDDMM on µ-CT-derived 3D mesh files revealed only two distinct head capsule and mandibles shapes-one for the minims (body mass < 1 mg) and one for all other workers. We discuss the functional significance of the identified 3D shape variation, and its implications for the evolution of extreme polymorphism in Atta.

Developmental biomechanics and age polyethism in leaf-cutter ants.

Many social insects display age polyethism: young workers stay inside the nest, and only older workers forage. This behavioural transition is accompanied by genetic and physiological changes, but the mechanistic origin of it remains unclear. To investigate if the mechanical demands on the musculoskeletal system effectively prevent young workers from foraging, we studied the biomechanical development of the bite apparatus in Atta vollenweideri leaf-cutter ants. Fully matured foragers generated peak in vivo bite forces of around 100 mN, more than one order of magnitude in excess of those measured for freshly eclosed callows of the same size. This change in bite force was accompanied by a sixfold increase in the volume of the mandible closer muscle, and by a substantial increase of the flexural rigidity of the head capsule, driven by a significant increase in both average thickness and indentation modulus of the head capsule cuticle. Consequently, callows lack the muscle force capacity required for leaf-cutting, and their head capsule is so compliant that large muscle forces would be likely to cause damaging deformations. On the basis of these results, we speculate that continued biomechanical development post eclosion may be a key factor underlying age polyethism, wherever foraging is associated with substantial mechanical demands.

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.

Strong positive allometry of bite force in leaf-cutter ants increases the range of cuttable plant tissues.

Atta leaf-cutter ants are the prime herbivore in the Neotropics: differently sized foragers harvest plant material to grow a fungus as a crop. Efficient foraging involves complex interactions between worker size, task preferences and plant-fungus suitability; it is, however, ultimately constrained by the ability of differently sized workers to generate forces large enough to cut vegetation. In order to quantify this ability, we measured bite forces of Atta vollenweideri leaf-cutter ants spanning more than one order of magnitude in body mass. Maximum bite force scaled almost in direct proportion to mass; the largest workers generated peak bite forces 2.5 times higher than expected from isometry. This remarkable positive allometry can be explained via a biomechanical model that links bite forces with substantial size-specific changes in the morphology of the musculoskeletal bite apparatus. In addition to these morphological changes, we show that bite forces of smaller ants peak at larger mandibular opening angles, suggesting a size-dependent physiological adaptation, probably reflecting the need to cut leaves with a thickness that corresponds to a larger fraction of the maximum possible gape. Via direct comparison of maximum bite forces with leaf mechanical properties, we demonstrate (i) that bite forces in leaf-cutter ants need to be exceptionally large compared with body mass to enable them to cut leaves; and (ii), that the positive allometry enables colonies to forage on a wider range of plant species without the need for extreme investment in even larger workers. Our results thus provide strong quantitative arguments for the adaptive value of a positively allometric bite force.

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.

Morphological determinants of bite force capacity in insects: a biomechanical analysis of polymorphic leaf-cutter ants.

The extraordinary success of social insects is partially based on division of labour, i.e. individuals exclusively or preferentially perform specific tasks. Task preference may correlate with morphological adaptations so implying task specialization, but the extent of such specialization can be difficult to determine. Here, we demonstrate how the physical foundation of some tasks can be leveraged to quantitatively link morphology and performance. We study the allometry of bite force capacity in Atta vollenweideri leaf-cutter ants, polymorphic insects in which the mechanical processing of plant material is a key aspect of the behavioural portfolio. Through a morphometric analysis of tomographic scans, we show that the bite force capacity of the heaviest colony workers is twice as large as predicted by isometry. This disproportionate 'boost' is predominantly achieved through increased investment in muscle volume; geometrical parameters such as mechanical advantage, fibre length or pennation angle are likely constrained by the need to maintain a constant mandibular opening range. We analyse this preference for an increase in size-specific muscle volume and the adaptations in internal and external head anatomy required to accommodate it with simple geometric and physical models, so providing a quantitative understanding of the functional anatomy of the musculoskeletal bite apparatus in insects.

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.

Landscape genomics of an obligate mutualism: Concordant and discordant population structures between the leafcutter ant Atta texana and its two main fungal symbiont types.

To explore landscape genomics at the range limit of an obligate mutualism, we use genotyping-by-sequencing (ddRADseq) to quantify population structure and the effect of host-symbiont interactions between the northernmost fungus-farming leafcutter ant Atta texana and its two main types of cultivated fungus. Genome-wide differentiation between ants associated with either of the two fungal types is of the same order of magnitude as differentiation associated with temperature and precipitation across the ant's entire range, suggesting that specific ant-fungus genome-genome combinations may have been favoured by selection. For the ant hosts, we found a broad cline of genetic structure across the range, and a reduction of genetic diversity along the axis of range expansion towards the range margin. This population-genetic structure was concordant between the ants and one cultivar type (M-fungi, concordant clines) but discordant for the other cultivar type (T-fungi). Discordance in population-genetic structures between ant hosts and a fungal symbiont is surprising because the ant farmers codisperse with their vertically transmitted fungal symbionts. Discordance implies that (a) the fungi disperse also through between-nest horizontal transfer or other unknown mechanisms, and (b) genetic drift and gene flow can differ in magnitude between each partner and between different ant-fungus combinations. Together, these findings imply that variation in the strength of drift and gene flow experienced by each mutualistic partner affects adaptation to environmental stress at the range margin, and genome-genome interactions between host and symbiont influence adaptive genetic differentiation of the host during range evolution in this obligate mutualism.

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.

Appetitive and aversive learning of plants odors inside different nest compartments by foraging leaf-cutting ants.

Cues inside the nest provide social insect foragers with information about resources currently exploited that may influence their decisions outside. Leaf-cutting ants harvest leaf fragments that are either further processed as substrate for their symbiotic fungus, or disposed of if unsuitable. We investigated whether Acromyrmex ambiguus foragers develop learned preferences for olfactory cues they experienced either in the fungus or in the waste chamber of the nest. Foragers' olfactory preferences were quantified as a choice between sugared papers disks scented with a novel odor and with the odor experienced in one of the nest compartments, before and after odor addition. Odors incorporated in the fungus chamber led to preferences towards paper disks smelling of them. Conversely, odors experienced in the waste chambers led to avoidance of similarly-scented disks. To investigate context-specificity of responses, we quantified learned preferences towards an odor that occurred first in the fungus chamber, and 14 h later in the waste chamber. Foragers initially developed a preference for the odor added in the fungus chamber that turned into avoidance when the same odor solely occurred later in the waste chamber. Avoidance of plants could also be induced in a more natural context, when fresh leaf disks of novel plants, privet or firethorn, were presented in the waste chamber. We conclude that learned acceptance or rejection of suitable plants by foragers depend on the learning context: smells can lead to appetitive learning when present in the fungus garden, or to avoidance learning when they occur at the dump.

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.

The Circadian Clock of the Ant Camponotus floridanus Is Localized in Dorsal and Lateral Neurons of the Brain.

The circadian clock of social insects has become a focal point of interest for research, as social insects show complex forms of timed behavior and organization within their colonies. These behaviors include brood care, nest maintenance, foraging, swarming, defense, and many other tasks, of which several require social synchronization and accurate timing. Ants of the genus Camponotus have been shown to display a variety of daily timed behaviors such as the emergence of males from the nest, foraging, and relocation of brood. Nevertheless, circadian rhythms of isolated individuals have been studied in few ant species, and the circadian clock network in the brain that governs such behaviors remains completely uncharacterized. Here we show that isolated minor workers of Camponotus floridanus exhibit temperature overcompensated free-running locomotor activity rhythms under constant darkness. Under light-dark cycles, most animals are active during day and night, with a slight preference for the night. On the neurobiological level, we show that distinct cell groups in the lateral and dorsal brain of minor workers of C. floridanus are immunostained with an antibody against the clock protein Period (PER) and a lateral group additionally with an antibody against the neuropeptide pigment-dispersing factor (PDF). PER abundance oscillates in a daily manner, and PDF-positive neurites invade most parts of the brain, suggesting that the PER/PDF-positive neurons are bona fide clock neurons that transfer rhythmic signals into the relevant brain areas controlling rhythmic behavior.

The construction of ventilation turrets in Atta vollenweideri leaf-cutting ants: Carbon dioxide levels in the nest tunnels, but not airflow or air humidity, influence turret structure.

Nest ventilation in the leaf-cutting ant Atta vollenweideri is driven via a wind-induced mechanism. On their nests, workers construct small turrets that are expected to facilitate nest ventilation. We hypothesized that the construction and structural features of the turrets would depend on the colony's current demands for ventilation and thus might be influenced by the prevailing environmental conditions inside the nest. Therefore, we tested whether climate-related parameters, namely airflow, air humidity and CO2 levels in the outflowing nest air influenced turret construction in Atta vollenweideri. In the laboratory, we simulated a semi-natural nest arrangement with fungus chambers, a central ventilation tunnel providing outflow of air and an aboveground building arena for turret construction. In independent series, different climatic conditions inside the ventilation tunnel were experimentally generated, and after 24 hours, several features of the built turret were quantified, i.e., mass, height, number and surface area (aperture) of turret openings. Turret mass and height were similar in all experiments even when no airflow was provided in the ventilation tunnel. However, elevated CO2 levels led to the construction of a turret with several minor openings and a larger total aperture. This effect was statistically significant at higher CO2 levels of 5% and 10% but not at 1% CO2. The construction of a turret with several minor openings did not depend on the strong differences in CO2 levels between the outflowing and the outside air, since workers also built permeated turrets even when the CO2 levels inside and outside were both similarly high. We propose that the construction of turrets with several openings and larger opening surface area might facilitate the removal of CO2 from the underground nest structure and could therefore be involved in the control of nest climate in leaf-cutting ants.

Biogeography of mutualistic fungi cultivated by leafcutter ants.

Leafcutter ants propagate co-evolving fungi for food. The nearly 50 species of leafcutter ants (Atta, Acromyrmex) range from Argentina to the United States, with the greatest species diversity in southern South America. We elucidate the biogeography of fungi cultivated by leafcutter ants using DNA sequence and microsatellite-marker analyses of 474 cultivars collected across the leafcutter range. Fungal cultivars belong to two clades (Clade-A and Clade-B). The dominant and widespread Clade-A cultivars form three genotype clusters, with their relative prevalence corresponding to southern South America, northern South America, Central and North America. Admixture between Clade-A populations supports genetic exchange within a single species, Leucocoprinus gongylophorus. Some leafcutter species that cut grass as fungicultural substrate are specialized to cultivate Clade-B fungi, whereas leafcutters preferring dicot plants appear specialized on Clade-A fungi. Cultivar sharing between sympatric leafcutter species occurs frequently such that cultivars of Atta are not distinct from those of Acromyrmex. Leafcutters specialized on Clade-B fungi occur only in South America. Diversity of Clade-A fungi is greatest in South America, but minimal in Central and North America. Maximum cultivar diversity in South America is predicted by the Kusnezov-Fowler hypothesis that leafcutter ants originated in subtropical South America and only dicot-specialized leafcutter ants migrated out of South America, but the cultivar diversity becomes also compatible with a recently proposed hypothesis of a Central American origin by postulating that leafcutter ants acquired novel cultivars many times from other nonleafcutter fungus-growing ants during their migrations from Central America across South America. We evaluate these biogeographic hypotheses in the light of estimated dates for the origins of leafcutter ants and their cultivars.

Underground anemotactic orientation in leaf-cutting ants: perception of airflow and experience-dependent choice of airflow direction during digging.

Air exchange between the large nests of Atta vollenweideri leaf-cutting ants and the environment strongly relies on a passive, wind-induced ventilation mechanism. Air moves through nest tunnels and airflow direction depends on the location of the tunnel openings on the nest mound. We hypothesized that ants might use the direction of airflow along nest tunnels as orientation cue in the context of climate control, as digging workers might prefer to broaden or to close tunnels with inflowing or outflowing air in order to regulate nest ventilation. To investigate anemotactic orientation in Atta vollenweideri, we first tested the ants' ability to perceive air movements by confronting single workers with airflow stimuli in the range 0 to 20 cm/s. Workers responded to airflow velocities ≥ 2 cm/s, and the number of ants reacting to the stimulus increased with increasing airflow speed. Second, we asked whether digging workers use airflow direction as an orientation cue. Workers were exposed to either inflow or outflow of air while digging in the nest and could subsequently choose between two digging sites providing either inflow or outflow of air, respectively. Workers significantly chose the side with the same airflow direction they experienced before. When no airflow was present during initial digging, workers showed no preference for airflow directions. Workers developed preferences for airflow direction only after previous exposure to a given airflow direction. We suggest that experience-modified anemotaxis might help leaf-cutting ants spatially organize their digging activity inside the nest during tasks related to climate control.

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.

Avoidance of plants unsuitable for the symbiotic fungus in leaf-cutting ants: Learning can take place entirely at the colony dump.

Plants initially accepted by foraging leaf-cutting ants are later avoided if they prove unsuitable for their symbiotic fungus. Plant avoidance is mediated by the waste produced in the fungus garden soon after the incorporation of the unsuitable leaves, as foragers can learn plant odors and cues from the damaged fungus that are both present in the recently produced waste particles. We asked whether avoidance learning of plants unsuitable for the symbiotic fungus can take place entirely at the colony dump. In order to investigate whether cues available in the waste chamber induce plant avoidance in naïve subcolonies, we exchanged the waste produced by subcolonies fed either fungicide-treated privet leaves or untreated leaves and measured the acceptance of untreated privet leaves before and after the exchange of waste. Second, we evaluated whether foragers could perceive the avoidance cues directly at the dump by quantifying the visits of labeled foragers to the waste chamber. Finally, we asked whether foragers learn to specifically avoid untreated leaves of a plant after a confinement over 3 hours in the dump of subcolonies that were previously fed fungicide-treated leaves of that species. After the exchange of the waste chambers, workers from subcolonies that had access to waste from fungicide-treated privet leaves learned to avoid that plant. One-third of the labeled foragers visited the dump. Furthermore, naïve foragers learned to avoid a specific, previously unsuitable plant if exposed solely to cues of the dump during confinement. We suggest that cues at the dump enable foragers to predict the unsuitable effects of plants even if they had never been experienced in the fungus garden.