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.

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.

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.

Learning through the waste: olfactory cues from the colony refuse influence plant preferences in foraging leaf-cutting ants.

Leaf-cutting ants learn to avoid plants initially harvested if they prove to be harmful for their symbiotic fungus once incorporated into the nest. At this point, waste particles removed from the fungus garden are likely to contain cues originating from both the unsuitable plant and the damaged fungus. We investigated whether leaf-cutting ant foragers learn to avoid unsuitable plants solely through the colony waste. We fed subcolonies of Acromymex ambiguus privet leaves treated with a fungicide undetectable to the ants, then collected the produced waste, and placed it into the fungus chamber of naive subcolonies. In individual choice tests, naive foragers preferred privet leaves before waste was put into the fungus chamber, but avoided them afterwards. Evidence on the influence of olfactory cues from the waste on decision making by foragers was obtained by scenting and transferring waste particles from subcolonies that had been fed either fungicide-treated or untreated leaves. In choice experiments, foragers from subcolonies given scented waste originating from fungicide-treated leaves collected fewer sugared paper discs with that scent compared with foragers from subcolonies given scented waste from untreated leaves. The results indicate that foragers learn to avoid plants unsuitable for the fungus by associating plant odours and cues from the damaged fungus that are present in waste particles. It is argued that waste particles may contribute to spread information about noxious plants for the fungus within the colony.

Foraging leaf-cutting ants learn to reject Vitis vinifera ssp. vinifera plants that emit herbivore-induced volatiles.

Leaf-cutting ants (LCAs) are dominant herbivores of the Neotropics, as well as economically important pests. Their foraging ecology and patterns/mechanisms of food selection have received considerable attention. Recently, it has been documented that LCAs exhibit a delayed rejection of previously accepted food plants following treatment with a fungicide that makes the plants unsuitable as substrate for their symbiotic fungus. Here, we investigated whether LCAs similarly reject plants with induced chemical defenses, by combining analysis of volatile emissions with dual-choice bioassays that used LCA subcolonies (Atta sexdens L.). On seven consecutive days, foraging ants were given the choice between leaf disks from untreated control plants and test plants of Vitis vinifera ssp. vinifera L. treated with the phytohormone jasmonic acid (JA) to mimic herbivore attack. Chemical analysis revealed the emission of a characteristic set of herbivore-induced volatile organic compounds (VOC) from JA-induced plants. Dual-choice experiments indicated that workers did not show any preference initially, but that they avoided JA-treated plants from day five onwards. Our finding that A. sexdens foragers learn to avoid VOC-emitting plants, which are likely detrimental to their symbiotic fungus, represents the first evidence for avoidance learning in attine ants toward plants with induced defenses.

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.

The determination of nest depth in founding queens of leaf-cutting ants (Atta vollenweideri): idiothetic and temporal control.

Leaf-cutting ant queens excavate a founding nest consisting of a vertical tunnel and a final horizontal chamber. Nest foundation is very time consuming, and colony success depends on the excavated depth. Although shallow nests may be energetically cheaper to dig, queens may be more exposed to the changing environment. Deeper chambers, in contrast, may be climatically more stable, but are more expensive to dig. We investigated the mechanisms underlying the control of nest depth in queens of the leaf-cutting ant Atta vollenweideri. We focused on the use of internal information for the control of nest depth, and therefore maintained the soil and environmental conditions invariant during the different laboratory experiments. We compared the tunnel lengths excavated by queens that were able to complete their nests earlier, faster or slower than under standard conditions. An earlier and faster nest completion was obtained by offering queens either pre-excavated tunnels of different lengths, soils at different temperatures, or soft sandy soils. A slower nest excavation was induced by offering queens harder dry soils, and by delaying the start of digging several days after the nuptial flight. Results indicate that the determination of nest depth was a regulated process involving the use of internal references: queens excavated their tunnels either until a particular depth was reached or for some predetermined length of time. Queens appear to monitor their movements while walking up und down the tunnel, and to compare this sensory information with a motor command that represents a preset tunnel length to be excavated before switching to chamber digging. In addition to this form of idiothetic control, results indicate that the elapsed digging time also feeds back onto the control system. It is argued that the determination of nest depth, i.e. the transition from tunnel to chamber digging, is initiated either after a preset tunnel length is reached, or as soon as a maximal time interval has elapsed, irrespective of the excavated tunnel length. A control system using both idiothetic and temporal information, as demonstrated in the present study, allows queens to flexibly react to different soil conditions, and therefore avoid excessive time and energy investments. Possible mechanisms underlying the control of chamber size are also discussed.

The energetics of running stability: costs of transport in grass-cutting ants depend on fragment shape.

Grass-cutting ants (Atta vollenweideri) carry fragments that can be many times heavier and longer than the ants themselves and it is important for them to avoid falling over during load transport. To investigate whether the energetic costs of transport are affected by the need to maintain stability, the rate of CO(2) production was measured in both unladen workers and workers carrying standardized paper fragments of different size and shape. We tested: (1) the effect of mass by comparing workers carrying either light or heavy fragments of the same size, and (2) the effect of shape by comparing short and long fragments of the same mass. Consistent with previous studies, metabolic rate increased but running speed remained constant when ants carried heavier fragments. The net cost of transport (normalized to the total mass of ant and fragment) was the same for heavy and light fragments, and did not differ from the costs of carrying a unit body mass. Ants carrying long fragments showed similar metabolic rates but ran significantly slower than ants carrying short fragments. As a consequence, net cost of transport was significantly higher for long fragments than for short ones, and higher than the costs of carrying a unit body mass. The observed reduction in running speed is likely a result of the ants' need to maintain stability. When the absolute costs of transport were compared, smaller ants required more energy to carry heavier and longer fragments than larger workers, but the opposite was found for lighter and shorter fragments. The absolute costs of transport per unit fragment mass suggest that it is energetically advantageous for a colony to allocate smaller workers for the transport of small fragments and larger workers for large fragments. The present results underline the importance of biomechanical factors for the understanding of leaf-cutting ant foraging strategies.

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.

Detection of minute temperature transients by thermosensitive neurons in ants.

The antennae of leaf-cutting ants are equipped with sensilla coeloconica that house three receptor neurons, one of which is thermosensitive. Using convective heat (air at different temperatures), we investigated the physiological characteristics of the thermosensitive neuron associated with the sensilla coeloconica in the leaf-cutting ant Atta vollenweideri. The thermosensitive neuron very quickly responds to a drop in temperature with a brief phasic increase (50 ms) in spike rate and thus classifies as cold receptor (ambient temperature = 24°C). The short latency and the brief phasic response enable the thermosensitive neuron to follow temperature transients up to an estimated frequency of around 5 Hz. Although the neuron responds as a cold receptor, it is extremely sensitive to warm stimuli. A temperature increase of only 0.005°C already leads to a pronounced decrease in the resting activity of the thermosensitive neuron. Through sensory adaptation, the sensitivity to temperature transients is maintained over a wide range of ambient temperatures (18-30°C). We conclude that the thermosensitive neuron of the sensilla coeloconica is adapted to detect minute temperature transients, providing the ants with thermal information of their microenvironment, which they may use for orientation.

Foraging grass-cutting ants (Atta vollenweideri) maintain stability by balancing their loads with controlled head movements.

Grass-cutting ants (Atta vollenweideri) carry leaf fragments several times heavier and longer than the workers themselves over considerable distances back to their nest. Workers transport fragments in an upright, slightly backwards-tilted position. To investigate how they maintain stability and control the carried fragment's position, we measured head and fragment positions from video recordings. Load-transporting ants often fell over, demonstrating the biomechanical difficulty of this behavior. Long fragments were carried at a significantly steeper angle than short fragments of the same mass. Workers did not hold fragments differently between the mandibles, but performed controlled up and down head movements at the neck joint. By attaching additional mass at the fragment's tip to load-carrying ants, we demonstrated that they are able to adjust the fragment angle. When we forced ants to transport loads across inclines, workers walking uphill carried fragments at a significantly steeper angle, and downhill at a shallower angle than ants walking horizontally. However, we observed similar head movements in unladen workers, indicating a generalized reaction to slopes that may have other functions in addition to maintaining stability. Our results underline the importance of proximate, biomechanical factors for the understanding of the foraging process in leaf-cutting ants.

The thermoregulatory function of thatched nests in the South American grass-cutting ant, Acromyrmex heyeri.

The construction of mound-shaped nests by ants is considered as a behavioral adaptation to low environmental temperatures, i.e., colonies achieve higher and more stables temperatures than those of the environment. Besides the well-known nests of boreal Formica wood-ants, several species of South American leaf-cutting ants of the genus Acromyrmex construct thatched nests. Acromyrmex workers import plant fragments as building material, and arrange them so as to form a thatch covering a central chamber, where the fungus garden is located. Thus, the degree of thermoregulation attained by the fungus garden inside the thatched nest largely depends on how the thatch affects the thermal relations between the fungus and the environment. This work was aimed at studying the thermoregulatory function of the thatched nests built by the grass-cutting ant Acromyrmex heyeri Forel (Hymenoptera: Formicidae: Myrmicinae). Nest and environmental temperatures were measured as a function of solar radiation on the long-term. The thermal diffusivity of the nest thatch was measured and compared to that of the surrounding soil, in order to assess the influence of the building material on the nest's thermoregulatory ability. The results showed that the average core temperature of thatched nests was higher than that of the environment, but remained below values harmful for the fungus. This thermoregulation was brought about by the low thermal diffusivity of the nest thatch built by workers with plant fragments, instead of the readily-available soil particles that have a higher thermal diffusivity. The thatch prevented diurnal nest overheating by the incoming solar radiation, and avoided losses of the accumulated daily heat into the cold air during the night. The adaptive value of thatching behavior in Acromyrmex leaf-cutting ants occurring in the southernmost distribution range is discussed.

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.