Leaf-cutting Ant Workers Research Articles

Contamination and mortality of leaf-cutting ant workers by the quinone inside inhibitor fungicide after social interactions

Leaf-cutting ants of the genera Atta and Acromyrmex (Hymenoptera: Formicidae) are the most important pests in forest and agricultural plantations and livestock. Toxic baits are the main method to manage these insects. The objective was to determine whether the behavior of allogrooming, touch, and self-grooming among Atta sexdens rubropilosa Forel, 1908 (Hymenoptera: Formicidae) workers disperse the fungicide quinone inside inhibitor and whether this product is toxic to them. This fungicide was applied, topically, in groups of workers and the social interactions between them and their mortality with and without the fungicide were evaluated. The interactions and the quinone inside inhibitor fungicide contamination increased with the number of leaf-cutting ant workers per group. Excessive touches, with subsequent allogrooming, and self-grooming among the ant workers dispersed the quinone inside the inhibitor fungicide causing 100% mortality and indicating its toxicity to this insect. The hypothesis that social interactions contaminated ant colony mates and the toxicity of the fungicide quinone inside inhibitor to workers of the leaf-cutting ant A. sexdens rubropilosa was proven.

Division of labor in work shifts by leaf-cutting ants

Foraging rhythms in eusocial insects are determined by the colony´s overall pattern. However, in leaf-cutting ant workers, individual rhythms are not fully synchronized with the colonies’ rhythm. The colony as a whole is nocturnal, since most worker activity takes place at night; however some workers forage during the day. Previous studies in individualized ants suggest nocturnal and diurnal workers coexistence. Here observations within the colony, in leaf-cutting ants, showed that workers have differential foraging time preference, which interestingly is associated to body size and differential leaf transportation engagement. Nocturnal ants are smaller and less engaged in leaf transportation whereas diurnal ants are bigger and more engaged in leaf carriage. Mechanisms underlying division of labor in work shifts in ants are still unknown but much can be extrapolated from honeybees; another social system bearing a similar pattern. A collective organization like this favors constant exploitation of food sources while preserving natural individual rhythm patterns, which arise from individual differences, and thermal tolerance, given by the size polymorphism presented by this species.

Queen loss increases worker survival in leaf-cutting ants under paraquat-induced oxidative stress.

Longevity is traded off with fecundity in most solitary species, but the two traits are positively linked in social insects. In ants, the most fecund individuals (queens and kings) live longer than the non-reproductive individuals, the workers. In many species, workers may become fertile following queen loss, and recent evidence suggests that worker fecundity extends worker lifespan. We postulated that this effect is in part owing to improved resilience to oxidative stress, and tested this hypothesis in three Myrmicine ants: Temnothorax rugatulus, and the leaf-cutting ants Atta colombica and Acromyrmex echinatior. We removed the queen from colonies to induce worker reproduction and subjected workers to oxidative stress. Oxidative stress drastically reduced survival, but this effect was less pronounced in leaf-cutting ant workers from queenless nests. We also found that, irrespective of oxidative stress, outside workers died earlier than inside workers did, likely because they were older. Since At. colombica workers cannot produce fertile offspring, our results indicate that direct reproduction is not necessary to extend the lives of queenless workers. Our findings suggest that workers are less resilient to oxidative stress in the presence of the queen, and raise questions on the proximate and ultimate mechanisms underlying socially mediated variation in worker lifespan. This article is part of the theme issue 'Ageing and sociality: why, when and how does sociality change ageing patterns?'

Screening of Fungi Isolated from the Brazilian Restinga for Insecticidal Activity

Research on microorganisms for the control of pests and pathogens is increasing. Such organisms display antagonistic effects on pests and pathogens, at the same time, they do not interfere with the sustainable development processes and are environmentally safe for human populations. Thus, bioprospection of fungi from restinga ecosystems is of interest as a novel source of microorganism and a yet unexplored source of chemical structures. This study selected endophytic fungi and fungi from the restinga soil samples to investigate their biological activity against insects. Fifty-three fungal isolates were used in screening bioassays against Atta sexdens rubropilosa leaf-cutting ant workers via direct contact of the insects with sporulating fungi cultures. This assay indicated that Trichoderma caused the highest mortality. Extracts from Trichoderma were then assessed for biological activity via ingestion, contact or exposure to fungal volatiles. Results showed that one Trichoderma sp. isolate (TR1) caused 50% mortality in 2, 1.5 and 4 days when ingested, sprayed onto the ants or by exposing ants to volatiles, respectively. Although this fungus is not known to be entomopathogenic, it could have potential use as an additional tool for pest control as it produces metabolites with antagonistic effects.

Avoiding traffic jams: Hitchhiking behavior as a strategy to reduce ant workers’ traffic on the foraging trail

During foraging, thousands of leaf-cutting ant workers travel along high traffic foraging trails which, when narrow, reduce the leaf delivery rate due to the reduction in workers’ travel-speed. On the other hand, high worker traffic promotes head-on encounters which are supposed to mediate worker task allocation and so could constitute a cue which induces traffic reduction. Very small workers along trails, for example, could change their task between marking the trail chemically to hitchhiking. Since they assume the hitchhiker function even in the absence of phorid parasitoids, one can suppose that hitchhiker behavior could be a strategy mediated by head-on encounters to avoid the high density of workers. Thus, we studied how the variation of worker density on the trail influences the hitchhiker frequency, testing the hypothesis that very small workers climb on the transported leaves to reduce trail traffic. Therefore, five Acromyrmex subterraneus colonies were linked to a foraging area by trails of different width (1.5 or 3 cm). We counted the number of hitchhikers and the outbound worker flow. The frequency of hitchhikers increased along narrow trails, and also due to outbound workers in both trail widths. Regardless of outbound foraging flow being comparable in both trail widths, the narrower ones had high density of workers leading to a presumed increase in head-on encounters. Head-on encounter rates cause a reduction in travel speed and, furthermore, are regulatory factors of task-allocation. Thus, high density trails lead to an increase in the rate of head-on encounters which could constitute as a stimulus to task-allocation of very small workers to the function of hitchhiker to avoid traffic jams.

Communication or Toxicity: What Is the Effect of Cycloheximide on Leaf-Cutting Ant Workers?

Leaf-cutting ants are insects that use plant material to grow fungus from which they feed. These fungus-growing ants perform various behavioral activities to establish an environment conducive to the fungus. Among these behaviors are activities that can serve to detect materials harmful to the colony, such as licking, scraping, chopping, etc. However, there are substances that may not be detected as harmful to the fungus on first contact. Cycloheximide (CHX) is one such substance, described as a fungicide that inhibits the synthesis of proteins in eukaryotes, although its mechanism of action remains unclear. The present study aimed at evaluating the behavioral changes of worker ants, time carrying orange pellets, waste deposition and mortality, when subjected to seven days of CHX-incorporated pellets and another seven days of CHX-free pellets. The fungicide adversely and structurally affected the leaf-cutter ant colonies. Their behavior went through changes, such as an increase in pellet-licking frequencies and cleaning among the ants from the third day onward. Moreover, there was an increase in time carrying orange pellets, as well as in the mortality of workers during CHX incorporation.

Allogrooming, Self-Grooming, and Touching Behavior: Contamination Routes of Leaf-Cutting Ant Workers Using a Fat-Soluble Tracer Dye.

The aim of this study was to determine whether worker self-grooming, allogrooming, and direct contact promotes the dispersal of substances among members of the colony. For this purpose, a tracer (Sudan III dye) was applied topically to a worker ant and the social interactions between the worker with the tracer and workers without the tracer were studied. Additionally, the worker heads were dissected to visualize whether or not the post-pharyngeal gland was stained. The post-pharyngeal glands from 50% to 70% of workers were stained depending on the size of the group. With the increase in the experimental group size, the frequency of interactions between workers increased, with touching being the most frequent behavior. The tracer dye was probably passed on by direct contact between workers, followed by self-grooming and allogrooming. These behaviors are responsible for the rapid dispersal of substances among colony members as observed in our experiment. The results therefore support the hypothesis that contact with substances promotes the contamination of nestmates, even in the absence of feeding, serving as a model for further studies on the contamination of workers with the active ingredients of insecticides.

The trade-off between the transmission of chemical cues and parasites: behavioral interactions between leaf-cutting ant workers of different age classes

Social animals are faced with an intriguing dilemma. On the one hand, interactions between individuals are essential to exchange information and to promote cohesion, while on the other hand such interactions carry with them the risk of catching and transmitting parasites. This trade-off is particularly significant for social insects because low within-colony genetic diversity makes their colonies potentially vulnerable to parasites while frequent interactions are essential to the development of the colonial odor profile necessary for nestmate recognition. Here we investigate whether social interactions between young and old leaf-cutting ant workers show evidence of this trade-off. We find that old workers engage in more selfgrooming and mandibular scraping than young workers, both in keeping with old workers having been more exposed to parasites. In contrast, we find that young workers engaged in more allogrooming than old workers, which seems likely to have a different motivation possibly the transfer of recognition cues. Furthermore, young workers tended to engage in allogrooming with other young workers, although it was the old workers that were most active and with whom allogrooming would seem likely to optimize information or chemicals transfer. This suggests that young workers may be attempting to minimize the risk of parasite transmission during their social interactions. Although limited to behavioral data, these results hint that ant workers may be sensitive to the trade-off between the transmission of recognition cues and disease, and adjust their social interactions accordingly.

A Convenient Trip: an Analysis of the Impact of Hitchhiker Ants on Forager Transport Rates

During foraging, leaf-cutting ant workers of different size classes perform various tasks along foraging trails. Commonly, small workers called hitchhikers climb on leaf fragments imposing an extra transport cost, so their presence is thought to reduce the individual foraging performance. There are four main hypotheses which may explain the occurrence of hitchhikers and a different behavioral act related to their role can be predicted for each. Hitchhiker behavior was observed considering these hypotheses and the effect of the hitchhikers on the walking speed and transport rate of foragers was evaluated. The behavioral registers were obtained from 1371 hitchhikers on foraging trails of Acromyrmex subterraneus subterraneus nests in the field. To verify the influence of hitchhikers on walking speed and transport rate, 239 foragers with hitchhikers and 250 foragers transporting only leaf fragments were analyzed. The walking speed, burden and transport rate of each forager were calculated. Data indicated not only that hitchhikers are vigilant but that they remain motionless on the leaf fragment probably in order to reduce the impact of their presence for the loaded forager. The impact of their presence is verified through walking speed reduction but as they ride preferentially on larger workers who transport larger leaf fragments, there are no losses in the individual transport rate. The transporter selection made by the hitchhiker ensures at the same time enhanced protection against phorid parasitoids and the maintenance of the leaf transport rate.

Use of Seeds as Fungus Garden Substrate Changes the Organization of Labor Among Leaf-Cutting Ant Workers.

Seeds of different plant species constitute an alternative but also significant substrate that leaf-cutting ants use to cultivate their fungus garden. However, how they are processed inside the nest and if their use implies differential allocation of worker size classes are still poorly known. Using laboratory colonies of Acromyrmex subterraneus (Forel) as a model, the behaviors related to the processing of three different seeds (sesame, guava, and grape) as fungus substrate were listed. At the same time, we measured how each worker size class contributed to the execution of these behaviors by registering their respective frequency. It was found that medium-sized (1.2 > head width < 1.6mm) and minimum-sized (head width <1.1mm) workers assumed the role of incorporation for sesame and grape seeds, respectively. Major-sized workers (head width >1.7mm) were concentrated on licking and holding guava seeds. Tegument removal was the only task observed that differs between treatment of seeds and treatment of leaves before their incorporation, as described in the literature. It was verified that different species of seeds imply a differential allocation of worker size classes and the inclusion or exclusion of some tasks from the behavioral repertoire. Regardless of the substrate type, leaf-cutting ant workers follow a coordinated and specialized procedure to cultivate the fungus garden but always maintain a high degree of cooperation.

Variation in nestmate recognition ability among polymorphic leaf-cutting ant workers

A key feature for the success of social insects is division of labour, allowing colony members to specialize on different tasks. Nest defence is a defining task for social insects since it is crucial for colony integrity. A particularly impressive and well-known case of worker specialization in complex hymenopteran societies is found in leaf-cutting ants of the genera Atta and Acromyrmex. We hypothesized that three morphological worker castes of Acromyrmex echinatior differ in their likelihood to attack intruders, and show that major workers are more aggressive towards non-nestmate workers than medium and minor workers. Moreover, minors do not discriminate between nestmate and non-nestmate brood, while larger workers do. We further show that A. echinatior ants use cuticular chemical compounds for nestmate recognition. We took advantage of the natural variation in the cuticular compounds between colonies to investigate the proximate factors that may have led to the observed caste differences in aggression. We infer that major workers differ from medium workers in their general propensity to attack intruders (the “action component” of the nestmate recognition system), while minors seem to be less sensitive to foreign odours (“perception component”). Our results highlight the importance of proximate mechanisms underlying social insect behaviour, and encourage an appreciation of intra-colony variation when analysing colony-level traits such as nest defence.

Density of mushroom body synaptic complexes limits intraspecies brain miniaturization in highly polymorphic leaf-cutting ant workers.

Hymenoptera possess voluminous mushroom bodies (MBs), brain centres associated with sensory integration, learning and memory. The mushroom body input region (calyx) is organized in distinct synaptic complexes (microglomeruli, MG) that can be quantified to analyse body size-related phenotypic plasticity of synaptic microcircuits in these small brains. Leaf-cutting ant workers (Atta vollenweideri) exhibit an enormous size polymorphism, which makes them outstanding to investigate neuronal adaptations underlying division of labour and brain miniaturization. We particularly asked how size-related division of labour in polymorphic workers is reflected in volume and total numbers of MG in olfactory calyx subregions. Whole brains of mini, media and large workers were immunolabelled with anti-synapsin antibodies, and mushroom body volumes as well as densities and absolute numbers of MG were determined by confocal imaging and three-dimensional analyses. The total brain volume and absolute volumes of olfactory mushroom body subdivisions were positively correlated with head widths, but mini workers had significantly larger MB to total brain ratios. Interestingly, the density of olfactory MG was remarkably independent from worker size. Consequently, absolute numbers of olfactory MG still were approximately three times higher in large compared with mini workers. The results show that the maximum packing density of synaptic microcircuits may represent a species-specific limit to brain miniaturization.

Energy substrate used by workers of leaf-cutting ants during nest excavation

Energy substrate used by workers of leaf-cutting ants during nest excavation. In this study we aimed to ascertain whether leaf-cutting ant workers lose body reserves (fat or sugars) as a function of nest excavation. For each treatment, we isolated 10 workers of Atta sexdens into two experimental groups, Control (C- without excavation) and Soil (S- with excavation), which were kept for different time intervals (0, 24, 48 or 72 hours), totaling 700 tested workers. We then determined the concentration of soluble carbohydrates and total lipid content in them. The total carbohydrates were determined colorimetrically, based on the reaction between carbohydrates and sulfuric acid-phenol. For determination of lipids, the insects were immersed in organic solvent until they reached a constant weight. Our results showed that carbohydrates are consumed during nest excavation activities. In the experimental groups S24, S48 and S72, there was an average reduction of 5.82 (20.42%), 14.31 (44.96%) and 13.27 (43.96%) µ.mg-1 in soluble sugar when compared with the experimental groups that did not excavate. Furthermore, the lipids were not used during this activity. With respect to dry mass of the workers, their values were C0 = 8%, C24 = 10.4%, C48 = 9.2%, C72 = 10%, S24 = 9.2%, S48 = 8.7% and S72 = 8.5%. Our results show experimentally that the source of energy for nest excavation is carbohydrates, whereas lipids are conserved for other activities.

Energetic cost of digging behavior in workers of the leaf-cutting ant Atta sexdens (Fabricius)

Energetic cost of digging behavior in workers of the leaf-cutting ant Atta sexdens (Fabricius). During nest excavation, leaf-cutting ant workers undergo reduction in their body reserve, particularly carbohydrates. In order to estimate the energetic cost of digging, groups of 30 workers of the leaf-cutting ant Atta sexdens were sealed in a hermetic chamber for 24, 48 and 72 hours, with and without soil for digging, and had the CO2 concentration measured using respirometric chambers as well as volume of soil excavated (g). As expected, the worker groups that carried out soil excavation expelled more carbon dioxide than the groups that did not excavate. Therefore, a worker with body mass of 9.65 ± 1.50 mg dug in average 0.85 ± 0.27 g of soil for 24 hours, consuming ca. 0.58 ± 0.23 J. In this study, we calculate that the energetic cost of excavation per worker per day in the experimental set-up was ca. 0.58 J.

Nest Digging by Leaf-Cutting Ants: Effect of Group Size and Functional Structures

Leaf-cutting ant workers dig underground chambers, for housing their symbiotic fungus, interconnected by a vast quantity of tunnels whose function is to permit the entrance of food (leaves), gaseous exchanges, and movement of workers, offspring, and the queen. Digging is a task executed by a group of workers, but little is known about the group effect and group-constructed functional structures. Thus, we analyzed the structures formed by worker groups (5, 10, 20, and 40 individuals) of the leaf-cutting ant,Atta sexdens rubropilosa, for 2 days of excavation. The digging arena was the same for the 4 groups, with each group corresponding to a different density. Our results verified a pattern of tunneling by the workers, but no chamber was constructed. The group effect is well known, since the 40-worker group dug significantly more than the groups of 5, 10, and 20. These groups did not differ statistically from each other. Analysis of load/worker verified that workers of the smallest group carried the greatest load. Our paper demonstrates the group effect on the digging of nests, namely, that excavation is proportional to group size, but without emergence of a functional structure such as a chamber.

Polymorphism and Division of Labor During Foraging Cycles in the Leaf-cutting Ant Acromyrmex octospinosus (Formicidae; Attini)

While division of labor within leaf-cutting ant nests has been well-characterized in the context of the collection and processing of leaf material, environmental factors such as day-night cycles and heavy rainfall limit the time during which leaf-cutting ant workers leave the nest to gather forage. Using a novel “flat panel” nest design, we studied how patterns of within-nest task performance changed when a colony of the leaf-cutting ant Acromyrmex octospinosus was and was not provided access to forage. We conducted scan samples of individuals working within the nest under both conditions and compared task allocation patterns across provisioning regimes and between workers of different sizes. When labor was compared between worker size groups, “minor” workers (head width ≤2.0 mm) and “major” workers (head width >2.0 mm) showed significantly different task performance patterns when forage was available: minors performed mostly brood-care and garden maintenance, while majors were mostly involved in the handling of freshly-cut leaf fragments. In contrast, when the colony was deprived of forage, the task performance patterns of minor and major workers converged and did not significantly differ. Marked major workers known to be foragers tended to remain idle within the nest when the colony was deprived of forage, while non-foragers of similar head width engaged in a variety of within-nest tasks, suggesting polyethism in majors may be based on factors other than size.

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.

Phenotypic plasticity in number of glomeruli and sensory innervation of the antennal lobe in leaf‐cutting ant workers (A. vollenweideri)

In the leaf-cutting ant Atta vollenweideri, the worker caste exhibits a pronounced size-polymorphism, and division of labor is dependent on worker size (alloethism). Behavior is largely guided by olfaction, and the olfactory system is highly developed. In a recent study, two different phenotypes of the antennal lobe of Atta vollenweideri workers were found: MG- and RG-phenotype (with/without a macroglomerulus). Here we ask whether the glomerular numbers are related to worker size. We found that the antennal lobes of small workers contain approximately 390 glomeruli (low-number; LN-phenotype), and in large workers we found a substantially higher number of approximately 440 glomeruli (high-number; HN-phenotype). All LN-phenotype workers and some small HN-phenotype workers do not possess an MG (LN-RG-phenotype and HN-RG-phenotype), and the remaining majority of HN-phenotype workers do possess an MG (HN-MG-phenotype). Using mass-staining of antennal olfactory receptor neurons we found that the sensory tracts divide the antennal lobe into six clusters of glomeruli (T1-T6). In LN-phenotype workers, approximately 50 glomeruli are missing in the T4-cluster. Selective staining of single sensilla and their associated receptor neurons revealed that T4-glomeruli are innervated by receptor neurons from the main type of olfactory sensilla, the Sensilla trichodea curvata. The other type of olfactory sensilla (Sensilla basiconica) exclusively innervates T6-glomeruli. Quantitative analyses of differently sized workers revealed that the volume of T6 glomeruli scales with the power of 2.54 to the number of Sensilla basiconica. The results suggest that developmental plasticity leading to antennal-lobe phenotypes promotes differences in olfactory-guided behavior and may underlie task specialization within ant colonies.

Information transfer in head-on encounters between leaf-cutting ant workers: food, trail condition or orientation cues?

Living in large societies involves costs associated with high density of individuals, but being near others includes the benefit of access to conspecifics' information. High densities of workers in ant colonies impose traffic congestion costs on foraging trails. It has been postulated that crowding also increases foraging efficiency by facilitating information transfer between workers in head-on encounters. However, this hypothesis remains untested. Here we assessed, in 24 field nests of the leaf-cutting ant Atta cephalotes, whether head-on encounters between workers facilitate information transfer about trail condition, orientation and food. Several experimental manipulations failed to fit predictions of certain types of communication. (1) Trail disturbance (and thus potential need for information transfer) did not affect the rate of head-on encounters, (2) head-on encounters did not decrease the time required for laden ants to properly orient when entering a trail, and (3) ants that had been experimentally disoriented did not increase the number of head-on encounters when they returned to the trail. Nevertheless, one experiment strongly suggested information acquisition: (4) outbound ants were more likely to find and collect food after a head-on encounter with an ant carrying the same kind of food. These results do not support the hypotheses that workers exchange information about trail condition and orientation in head-on encounters, but suggest that workers acquire food information. The information transferred in head-on encounters could thus increase foraging efficiency under crowded conditions. The cost of the reduced speed due to worker collisions might be outweighed by the benefits of information acquisition, and could explain why leaf-cutting ants do not form distinct lanes of outbound and returning workers. Our results reinforce the key role of information use in the adaptive behaviour of animals and in the maintenance of group living.

Does genetic diversity hinder parasite evolution in social insect colonies?

Polyandry is often difficult to explain because benefits of the behaviour have proved elusive. In social insects, polyandry increases the genetic diversity of workers within a colony and this has been suggested to improve the resistance of the colony to disease. Here we examine the possible impact of host genetic diversity on parasite evolution by carrying out serial passages of a virulent fungal pathogen through leaf-cutting ant workers of known genotypes. Parasite virulence increased over the nine-generation span of the experiment while spore production decreased. The effect of host relatedness upon virulence appeared limited. However, parasites cycled through more genetically diverse hosts were more likely to go extinct during the experiment and parasites cycled through more genetically similar hosts had greater spore production. These results indicate that host genetic diversity may indeed hinder the ability of parasites to adapt while cycling within social insect colonies.