Leaf-cutting ant workers (Acromyrmex heyeri) trade off nest thermoregulation for humidity control

This study investigated whether workers of the thatching grass-cutting ant Acromyrmex heyeri respond to the competing demands of temperature and humidity control by modifying the architecture of the nest thatch. First, we evaluated whether the opening and closing of nest apertures are thermoregulatory responses. Second, we explored whether the control of nest humidity is compromised by the thermoregulatory responses, and to what extent workers trade off the control of one variable for the other. At temperatures ranging from 20–30°C, workers created more openings in the nest thatch, the higher the internal nest temperature. When the air surrounding the nest was experimentally desiccated at constant temperature, workers were observed to close nest openings at temperatures that previously triggered the opposite response, i.e., opening of apertures. This demonstrates that A. heyeri workers trade off a response related to thermoregulation for the maintenance of internal nest humidity.     

Larval isolation and brood care in Acromyrmex leaf-cutting ants

The larvae of leaf-cutting ants are maintained within the fungus gardens of their colonies and are fed pieces of fungus by the adult workers. However, little else is known about the nature of the worker-larva interaction in these ecologically important ants. To examine whether workers can gauge the needs of individual larvae, we isolated larvae without adult workers for different lengths of time. We then placed workers with the larvae and recorded the type and frequency of the subsequent behaviours of the workers. Workers scraped the mouthparts of larvae, ingested their faecal fluid, fed them with fungal hyphae, transported them around the fungus garden and, most frequently, licked their bodies. The workers were also observed to ‘plant’ fungal hyphae on the bodies of larvae. Workers interacted more frequently with larvae that had been isolated without workers than with those that had not, but there was no effect of the length of isolation. The results suggest that the interactions are complex, involving a number of behaviours that probably serve different functions, and that workers are to some extent able to assess the individual needs of larvae. Received 8 November 2004; revised 31 March 2005; accepted 22 April 2005.     

Oral trophallaxis in adult leaf-cutting ants Acromyrmex subterraneus subterraneus (Hymenoptera, Formicidae)

Adult leaf-cutting ants of the subspecies Acromyrmex subterraneus subterraneus were fed with an Evans Blue dye solution, which allowed the investigation of subsequent exchange of liquids between ants by oral trophallaxis. Trophallactic behavior was filmed and the antennation patterns of donor and recipient ants were described. The ants’ crop capacity was measured following ad libitum feeding on dye solution. Ants previously fed on the dye solution (donors) were placed individually with unfed ants of the same caste (recipients) and the amount of dye solution passed from the donor to the recipient by oral trophallaxis was measured after one hour. The volume received was 0.26 ± 0.15μL (mean ± SD) and the residual volume of dye in the crop of the donors was 0.49 ± 0.23μL. There were 38 trophallactic events recorded for 50 pairs of ants. Trophallaxis was observed from 2.3 min to 21.5 min after initial exposure, with a mean latency of 8.4 ± 5.6 min. The mean duration of a trophallatic event was 2.3 ± 1.3 min. As far as we know, this is the first time that trophallaxis in leaf-cutting ants has been described and quantified. Received 2 December 2005; revised 6 April 2006; accepted 10 April 2006.     

Wingless virgin queens assume helper roles in Acromyrmex leaf-cutting ants

Division of labour is the hallmark of advanced societies, because specialization carries major efficiency benefits in spite of costs owing to reduced individual flexibility [1]. The trade-off between efficiency and flexibility is expressed throughout the social insects, where facultative social species have small colonies and reversible caste roles and advanced eusocial species have permanently fixed queen and worker castes. This usually implies that queens irreversibly specialize on reproductive tasks [2]. Here, we report an exception to this rule by showing that virgin queens (gynes) of the advanced eusocial leaf-cutting ant Acromyrmex echinatior switch to carrying out worker tasks such as brood care and colony defence when they fail to mate and disperse. These behaviours allow them to obtain indirect fitness benefits (through assisting the reproduction of their mother) after their direct fitness options (their own reproduction) have become moot. We hypothesize that this flexibility could (re-)evolve secondarily because these ants only feed on fungal mycelium and thus could not benefit from cannibalising redundant gynes, and because queens have retained behavioural repertoires for foraging, nursing, and defense, which they naturally express during colony founding.     

Soil temperature, digging behaviour, and the adaptive value of nest depth in South American species of Acromyrmex leaf-cutting ants

In leaf-cutting ants, workers are expected to excavate the nest at a soil depth that provides suitable temperatures, since the symbiotic fungus cultivated inside nest chambers is highly dependent on temperature for proper growth. We hypothesize that the different nesting habits observed in Acromyrmex leaf-cutting ants in the South American continent, i.e. superficial and subterranean nests, depend on the occurrence, across the soil profile, of the temperature range preferred by workers for digging. To test this hypothesis, we first explored whether the nesting habits in the genus Acromyrmex are correlated with the prevailing soil temperature regimes at the reported nest locations. Second, we experimentally investigated whether Acromyrmex workers engaged in digging use soil temperature as a cue to decide where to excavate the nest. A bibliographic survey of nesting habits of 21 South American Acromyrmex species indicated that nesting habits are correlated with the soil temperature regimes: the warmer the soil at the nesting site, the higher the number of species inhabiting subterranean nests, as compared to superficial nests. For those species showing nesting plasticity, subterranean nests occurred in hot soils, and superficial nests in cold ones. Experimental results indicated that Acromyrmex lundi workers use soil temperature as an orientation cue to decide where to start digging, and respond to rising and falling soil temperatures by moving to alternative digging places, or by stopping digging, respectively. The soil temperature range preferred for digging, between 20°C and maximally 30.6°C, matched the range at which colony growth would be maximized. It is suggested that temperature-sensitive digging guides digging workers towards their preferred range of soil temperature. Workers’ thermopreferences lead to a concentration of digging activity at the soil layers where the preferred range occurs, and therefore, to the construction of superficial nests in cold soils, and subterranean ones in hot soils. The adaptive value of the temperature-related nesting habits, and the temperature-sensitive digging, is further discussed.     

Dynamic Wolbachia prevalence in Acromyrmex leaf-cutting ants: potential for a nutritional symbiosis

Wolbachia are renowned as reproductive parasites, but their phenotypic effects in eusocial insects are not well understood. We used a combination of qrt-PCR, fluorescence in situ hybridization and laser scanning confocal microscopy to evaluate the dynamics of Wolbachia infections in the leaf-cutting ant Acromyrmex octospinosus across developmental stages of sterile workers. We confirm that workers are infected with one or two widespread wsp genotypes of Wolbachia, show that colony prevalence is always 100% and characterize two rare recombinant genotypes. One dominant genotype is always present and most abundant, whereas another only proliferates in adult workers of some colonies and is barely detectable in larvae and pupae. An explanation may be that Wolbachia genotypes compete for host resources in immature stages while adult tissues provide substantially more niche space. Tissue-specific prevalence of the two genotypes differs, with the rarer genotype being over-represented in the adult foregut and thorax muscles. Both genotypes occur extracellularly in the foregut, suggesting an unknown mutualistic function in worker ant nutrition. Both genotypes are also abundant in the faecal fluid of the ants, suggesting that they may have extended functional phenotypes in the fungus garden that the ants manure with their own faeces.     

Forager size and ecology of Acromyrmex coronatus and other leaf-cutting ants in Costa Rica

I compare forager size and foraging ecology of the leaf-cutting ant Acromyrmex coronatus (Fabricius) with published data on three other leaf-cutter species in Costa Rica, Atta cephalotes (L.), Acromyrmex octospinosus (Reich), and Acromyrmex volcanus Wheeler. Intra-and interspecific differences in forager size in these leaf-cutting ants appear to reflect the economics of harvesting different preferred resources. Ac. coronatus colonies have relatively small foragers (mean mass=3.4±1.4 mg) that cut almost exclusively the thin, soft leaves and other parts of small herbaceous plants. Similarly, small A. cephalotes colonies have small foragers (3.3±1.0 mg) that attack the leaves of small herbaceous plants. In contrast, mature A. cephalotes colonies have a wider sizerange of foragers (7.3±4.1 mg) that primarily attack the leaves of trees, with larger foragers cutting thicker, tougher leaves. In A. cephalotes, the match of forager size to leaf type (both ontogenetically and behaviorally) increases foraging efficiency. Extreme forager polymorphism in mature A. cephalotes colonies appears to broaden the diversity of tree species that they can exploit efficiently. Ac. octospinosus and Ac. volcanus both have large, relatively monomorphic foragers (13.3±4.2 mg and 30.6±4.3 mg, respectively) that typically scavenge for pieces of fallen vegetation, such as dead leaves, fruit, and flowers, in addition to cutting herbs. The large foragers of Ac. octospinosus and Ac. volcanus appear to be well suited as generalist foragers, able to cut or collect any desirable vegetation encountered. Ac. coronatus is similar to A. cephalotes in other ways. Both Ac. coronatus and A. cephalotes establish and maintain cleared trunk trails for foraging, and both have minima workers that hitchhike on the loads carried by foragers, apparently serving to protect the larger foragers from attack by phorid flies. Trunk trails and hitchhikers are not known for Ac. octospinosus and Ac. volcanus. That A. coronatus and A. cephalotes show little overlap in geographic distribution within Costa Rica may relate both to differences in habitat requirements and to interspecific competition.     

Male induction and function of workers' excitability during swarming in leaf-cutting ants (Atta and Acromyrmex) (Hymenoptera,Formicidae)

Worker excitation in species of Atta and Acromyrmex prior to and during the swarming of reproductives is initiated by secretions from the male mandibular glands. During this phase, workers are highly aggressive, attacking all foreign objects. Field data suggest that this behavior is not only advantageous in protecting sexuals as they swarm, but also to prime workers to execute queens trying to initiate colonies near mature colonies following nuptial flights.     

Acromyrmex ameliae sp. n. （Hymenoptera： Formicidae）： A new social parasite of leaf-cutting ants in Brazil

The fungus-growing ants （Tribe Attini） are a New World group of〉 200 species, all obligate symbionts with a fungus they use for food. Four attine taxa are known to be social parasites of other attines. Acromyrmex （ Pseudoatta） argentina argentina and Acromyrmex （Pseudoatta） argentina platensis （parasites of Acromyrmex lundi）, and Acromyrmex sp. （a parasite of Acromyrmex rugosus） produce no worker caste. In contrast, the recently discovered Acromyrmex insinuator （a parasite of Acromyrmex echinatior） does produce workers. Here, we describe a new species, Acromyrmex ameliae, a social parasite of Acromyrmex subterraneus subterraneus and Acromyrmex subterraneus brunneus in Minas Gerais, Brasil. Like A. insinuator, it produces workers and appears to be closely related to its hosts. Similar social parasites may be fairly common in the fungus-growing ants, but overlooked due to the close resemblance between parasite and host workers.     

Isolation of the symbiotic fungus of Acromyrmex pubescens and phylogeny of Leucoagaricus gongylophorus from leaf-cutting ants

Leaf-cutting ants live in an obligate symbiosis with a Leucoagaricus species, a basidiomycete that serves as a food source to the larvae and queen. The aim of this work was to isolate, identify and complete the phylogenetic study of Leucoagaricus gongylophorus species of Acromyrmex pubescens. Macroscopic and microscopic features were used to identify the fungal symbiont of the ants. The ITS1-5.8S-ITS2 region was used as molecular marker for the molecular identification and to evaluate the phylogeny within the Leucoagaricus genus. One fungal symbiont associated with A. pubescens was isolated and identified as L. gongylophorus. The phylogeny of Leucoagaricus obtained using the ITS molecular marker revealed three well established monophyletic groups. It was possible to recognize one clade of Leucoagaricus associated with phylogenetically derived leaf-cutting ants (Acromyrmex and Atta). A second clade of free living forms of Leucoagaricus (non-cultivated), and a third clade of Leucoagaricus associated with phylogenetically basal genera of ants were also recognized. The clades corresponded to traditional taxonomic groups, and were differentiated by ecological habitats of different species.     

Somatic incompatibility and genetic structure of fungal crops in sympatric Atta colombica and Acromyrmex echinatior leaf-cutting ants

Obligate mutualistic symbioses rely on mechanisms that secure host-symbiont commitments to maximize host benefits and prevent symbiont cheating. Previous studies showed that somatic incompatibilities correlate with neutral-marker-based genetic distances between fungal symbionts of Panamanian Acromyrmex leaf-cutting ants, but the extent to which this relationship applies more generally remained unclear. Here we showed that genetic distances accurately predicted somatic incompatibility for Acromyrmex echinatior symbionts irrespective of whether neutral microsatellites or AFLP markers were used, but that such correlations were weaker or absent in sympatric Atta colombica colonies. Further analysis showed that the symbiont clades maintained by A. echinatior and A. colombica were likely to represent separate gene pools, so that neutral markers were unlikely to be similarly correlated with incompatibility loci that have experienced different selection regimes. We suggest that evolutionarily derived claustral colony founding by Atta queens may have removed selection for strong incompatibility in Atta fungi, as this condition makes the likelihood of symbiont swaps much lower than in Acromyrmex, where incipient nests stay open because queens have to forage until the first workers emerge.     

Specificity of the mutualistic association between actinomycete bacteria and two sympatric species of Acromyrmex leaf-cutting ants

Acromyrmex leaf-cutting ants maintain two highly specialized, vertically transmitted mutualistic ectosymbionts: basidiomycete fungi that are cultivated for food in underground gardens and actinomycete Pseudonocardia bacteria that are reared on the cuticle to produce antibiotics that suppress the growth of Escovopsis parasites of the fungus garden. Mutualism stability has been hypothesized to benefit from genetic uniformity of symbionts, as multiple coexisting strains are expected to compete and, thus, reduce the benefit of the symbiosis. However, the Pseudonocardia symbionts are likely to be involved in Red-Queen-like antagonistic co-evolution with Escovopsis so that multiple strains per host might be favoured by selection provided the cost of competition between bacterial strains is low. We examined the genetic uniformity of the Pseudonocardia symbionts of two sympatric species of Acromyrmex ants by comparing partial sequences of the nuclear Elongation Factor-Tu gene. We find no genetic variation in Pseudonocardia symbionts among nest mate workers, neither in Acromyrmex octospinosus, where colonies are founded by a single queen, nor in Acromyrmex echinatior, where mixing of bacterial lineages might happen when unrelated queens cofound a colony. We further show that the two ant species maintain the same pool of Pseudonocardia symbionts, indicating that horizontal transmission occasionally occurs, and that this pool consists of two distinct clades of closely related Pseudonocardia strains. Our finding that individual colonies cultivate a single actinomycete strain is in agreement with predictions from evolutionary theory on host-symbiont conflict over symbiont mixing, but indicates that there may be constraints on the effectiveness of the bacterial symbionts on an evolutionary timescale.     

Excavated substrate modulates growth instability during nest building in ants

In social insects, the nests of the same species can show a large difference in size and shape. Despite these large variations, the nests share the same substructures, some appearing during nest growth. In ants, the interplay between nest size and digging activity leads to two successive morphological transitions from circular to branched shapes (budding along the perimeter of the circular cavity and tunnelling of the galleries). Like several other self-organized collective behaviours, this phenomenon, as well as the entire nest-digging process, is thought to be modulated by environmental properties. The present study investigates the effect of excavated substrate on the nest morphogenesis and the morphological transitions by using two materials with different cohesions. Here, we show that the two morphological transitions occur more frequently with a cohesive substrate than with a granular one: 96 per cent of cohesive experiments showed both transitions, whereas only 50 per cent did in granular experiments. We found that transitions and excavation cessation follow area–response thresholds: the shape transitions take place and the digging activity stops when the dug area reaches the corresponding threshold values. The shape transition thresholds are lower with the cohesive substrate and that of stopping digging is independent of nest shape and material. According to simulations, the experimental frequencies of transitions found their origin in the competition between transitions and activity cessation and in the difference between the transition threshold values of each substrate. Our results demonstrate how the substrate properties modulate the collective response and lead to various patterns. Considering the non-specific mechanisms at work, such effects of substrate coarseness have their counterparts in various collective behaviours, generating alternative patterns to colonize and exploit the environment.     

The thermo-sensitive sensilla coeloconica of leaf-cutting ants (Atta vollenweideri)

Social insects show a variety of temperature-guided behaviors. Depending on whether heat reaches the sensillum via air movements (convective heat) or as radiant heat, specific adaptations of thermo-sensitive sensilla are expected. In the present study the morphology and the physiology of thermo-sensitive peg-in-pit sensilla (S. coeloconica) of the leaf-cutting ant Atta vollenweideri were investigated. S. coeloconica are located predominantly in a single cluster on the apical antennomere, and connect to the outside through a small aperture. The sensory peg is double-walled, embedded in a chamber and innervated by three unbranched dendrites. Using tungsten electrodes, activity of the sensory neurons was measured. In most cases, the neuron with the largest spike amplitude responds to changes in air temperature (convective heat) as well as to radiant heat. In response to a drop in air temperature, the neuron shows a phasic-tonic response followed by a complete adaptation within 1 min (cold-sensitive neuron). Based on their morphology and physiology, it is suggested that the S. coeloconica are involved in the recently described thermal orientation behavior of A. vollenweideri leaf-cutting ants.     

Digestive enzymes of leaf-cutting ants, Acromyrmex subterraneus (Hymenoptera: Formicidae: Attini): distribution in the gut of adult workers and partial characterization

Enzyme activities associated with the labial glands, midgut and rectum of adult Acromyrmex subterraneus were investigated in order to understand their role in digestion of plant and fungal material. High chitinolytic activity was detected in the labial glands, indicating a possible role in the degradation of fungus ingested by the ants. Chitinolytic activity seen in other compartments of the alimentary canal probably originated in the labial glands. The highest activity detected in the midgut was for alpha-glucosidase, which was considered to be of insect origin due to its association with midgut epithelium and it is probably involved in glucose assimilation from nutrient sources such as maltose and sucrose present in plant material. A large range of enzyme activities were detected in the rectal lumen contents, and as in the midgut the highest values were for alpha-glucosidase activity. The absence of activity associated with the epithelium, in the particulate fraction, indicates that the rectal epithelium does not have a secretory function. The detection of enzymes in the rectal lumen contents, which were not detected in the midgut lumen contents, indicates that the rectum acts as a reservoir, accumulating enzymes. The major digestive enzymes were partially characterized using hydrophobic interaction chromatography, gel filtration and SDS-PAGE. The pH of the adult intestinal tract and flow rate of dye through the tract was investigated. A gradual acidification of the intestinal tract was noted commencing with the crop (pH 6-8.2) and terminating with the rectum (pH 3-5). The flow of dye through the different compartments of the tract showed a rapid fill time for all the gut compartments and a short residence time in the crop. In all other compartments, the dye remained detectable for 10 days or longer.     

Queen adoption in colonies of the leaf-cutting ant Acromyrmex subterraneus molestans (Hymenoptera: Formicidae)

Bioassays were conducted in both laboratory and the field to determine if monogynous colonies of Acromyrmex subterraneus molestans (Myrmicinae, Attini) adopt queens from other colonies of the same subspecies. The results suggest that the adoption of fertilized queens is a possible mechanism to explain the occurrence of colonies with multiple queens in this subspecies. Only minor workers were able to discriminate queens from other colonies and were aggressive toward them. Therefore, queen recognition differs among subcastes.