The epizootiology of a Metarhizium infection in mini-nests of the leaf-cutting ant Atta sexdens rubropilosa

There is growing interest in the use of entomopathogenic organisms to control leaf-cutting ants (Hymenoptera: Formicidae: Attini). However, the way leaf-cutting ants react as a colony to biohazards is poorly understood. We investigated the effects of Metarhizium anisopliae (Metschnikoff) (Deuteromycotina: Hyphomycetes) applied to the foraging arenas of mini-nests (queenless sub-colonies) of the leaf-cutting ant Atta sexdens rubropilosa (Forel). Dry spores were applied either alone or mixed with citrus powder, at 0.5 g or 0.05 g per mini-nest. The spores were removed four days after application, and all dead ants removed every three days. Ant numbers near the Metarhizium increased as the ants attempted to clean up the biohazard. The ants attempted to place the spores in piles, which they then covered over with other material. They were able to deal with the low doses in this way, but the high doses overwhelmed them. All treated mini-nests suffered increased ant mortality during the first ten days after application. This mortality was particularly high in the media worker caste which had played the major role in attempting to clean up the spores. Foraging activity decreased, as did the health of the fungus gardens. The mini-nests exposed to the low dose of spores mixed with citrus powder then recovered fully. The health of the other treated mini-nests declined gradually until around 26 days after application, when they began deteriorating sharply. However, the decline of these mini-nests after day 26 was not due directly to the pathogenic action of the Metarhizium, nor to the initial ant mortality it had caused. The results suggest that the social stress caused by even such a short-lived Metarhizium epizootic was sufficient to cause the decline and ultimate death of the mini-nests. This has important implications for the control of leaf-cutting ants. It also demonstrates how important the social homeostasis of the colony is to leaf-cutting ants.     

Synthetic amides toxic to the leaf-cutting ant Atta sexdens rubropilosa L. and its symbiotic fungus

• 1 Nine synthetic amides similar to natural N‐piperidine‐3‐(4,5‐methylenedioxyphenyl)‐2‐(E)‐propenamide and N‐pyrrolidine‐3‐(4,5‐methylenedyoxiphenyl)‐2‐(E)‐propenamide were synthesized and identified by their spectroscopic data.
• 2 The toxicity of these synthetic amides to the Atta sexdens rubropilosa workers and the antifungal activity against Leucoagaricus gongylophorus, the symbiotic fungus of the leaf‐cutting ants, were determined.
• 3 Workers ants that were fed daily on an artificial diet to which these compounds were added had a higher mortality rate than the controls for N‐pyrrolidine‐3‐(3′,4′‐methylenedioxyphenyl)‐2‐(E)‐propenamide and N‐benzyl‐3‐(3′,4′‐methylenedioxyphenyl)‐2‐(E)‐propenamide at a concentration of 100 µg/mL.
• 4 The completely inhibition (100%) of the fungal growth was observed with N‐piperidine‐3‐(3′,4′‐methylenedioxyphenyl)‐2‐(E)‐propenamide and N,N‐diethyl‐3‐(3′,4′‐methylenedioxyphenyl)‐2‐(E)‐propenamide at concentrations of 50 and 100 µg/mL and N‐pirrolidine‐3‐(3′,4′‐methylenedioxyphenyl)‐2‐(E)‐propenamide at a concentration of 100 µg/mL.
• 5 The possibility of controlling these insects in the future using synthetic piperamides that can simultaneously target both organisms is discussed.

     

Polyethism and nestmate recognition in the alarm reaction of Atta leaf-cutting ants

Abstract.  Ethological studies are conducted under laboratory conditions using workers of Atta sexdens sexdens and Atta opaciceps collected from field colonies to investigate the mechanisms involved in the alarm response and intra-specific recognition in leaf-cutting ants. Hexane extracts from the heads of gardeners and generalist workers elicit higher levels of alarm response in foragers from the same colony than do mandibular gland extracts from foragers and soldiers, indicating that gardeners, generalists and foragers are primarily responsible for the production of alarm pheromone. Foragers subjected to extracts from non-nestmates exhibit significantly greater alarm responses than are induced by similar extracts derived from nestmate workers, suggesting that the alarm pheromone may have a role in nestmate recognition.     

The role of leaf-cutting ant workers (Hymenoptera: Formicidae) in fungus garden maintenance

Abstract.

• 1 We studied the role of leaf-cutting ant workers (Atta sexdens (L.) in fungus garden maintenance, by temporarily excluding workers from the garden. This increased its subsequent attractiveness, as expressed by an increase in the numbers of workers licking it.
• 2 The length of free mycelia on areas of the garden from which workers were excluded increased but was reduced again when workers were returned. Workers therefore removed hyphae from the garden surface.
• 3 The maximum‘isolation effect’was obtained by preventing ant access for 2–3 days, after which the effect declined. Removing staphylae from portions of garden kept ant-free for 4 and 6 days restored the effect, as the ants were not distracted by harvesting staphylae. Portions of garden kept ant-free for longer than this were no more attractive than non-isolated control garden.
• 4 Workers were highly efficient in detecting and removing contaminants from their fungus garden. Samples of garden could be isolated from workers for up to 12 days before major growth of contaminants occurred, and this contrasted with the maximum of 6 days for the isolation effect on licking. The isolation effect was therefore not a response to contaminant growths on the garden.
• 5 Workers on the garden surface may remove hyphae for nutritional reasons, or to‘prune’their fungus and stimulate its growth. In either case, the result is a regulation of fungal growth.

     

Communication between the fungus garden and workers of the leaf-cutting ant, Atta sexdens rubropilosa, regarding choice of substrate for the fungus

Bait made from orange peel, containing the fungicide cycloheximide, was initially harvested by workers of Atta sexdens rubropilosa (Forel) and incorporated into the fungus garden as substrate for the fungus. The bait was subsequently rejected by the worker ants days later. Exposure of the fungus to cycloheximide, in laboratory sub-colonies, resulted in the fungus being ‘stressed’. By interchanging normal fungus garden with ‘stressed’ fungus garden, a change in the foraging behaviour of the workers was evident. –Two hypotheses to explain the behavioural changes were tested: a volatile semiochemical is produced by the fungus which affects the foragers directly, or contact between workers (and fungus garden) is necessary for information regarding fungal substrate to be transmitted through the worker force. When pairs of sub-colonies were connected (one colony of each pair exposed to cycloheximide in the bait) and workers were initially prevented from passing from one colony to the other, one colony continued to forage on orange bait while the other did not. When both colonies were allowed to make full contact then both colonies failed to accept orange bait. This discounted the first hypothesis, but supported the second, as a highly volatile chemical should be able to diffuse between colonies. When large foragers were prevented from making contact with the second colony, the information may be communicated by smaller workers.     

Waste management in the leaf-cutting ant Atta colombica

Unlike most leaf-cutting ants, which have underground wastedumps, the leaf-cutting ant Atta colombica dumps waste in aheap outside the nest. Waste is hazardous, as it is contaminatedwith pathogens. We investigated the organization of the workforceinvolved in outside-nest tasks (foraging, waste disposal) andquantified task switching and heap location to test hypothesesthat these tasks are organized to minimize contact between the heap and foraging entrances and trails. Waste management isan important task: 11% of externally working ants were eithertransporting waste or manipulating waste on the heap, and theother 89% were foragers. There is strict division of laborbetween foragers and waste workers, with no task switching.Waste management also has division of labor and is undertakenby transporters that carry waste to the heap margins and heapworkers that manage the heap. Waste heaps are always locateddownhill from nest entrances. The distance to the waste heapis positively related to colony size and negatively relatedto slope. Foraging trails avoid the heap, with 92% of trailsgoing away from the heap. This avoidance behavior is costly,increasing foraging trail length by at least 6%. Waste managementin A. colombica is a sophisticated system that encompassesboth work and spatial organization. This organization is probablyadaptive in reducing disease transmission. Division of labor separates waste management from foraging, reducing the likelihoodof foragers becoming contaminated with waste. The downhilllocation of heaps reduces waste entering entrances during rain.The orientation of foraging trails reduces the possibilityof foragers becoming accidentally contaminated with waste.     

Influence of physical and chemical factors during foraging and culture of the symbiont fungus in Atta sexdens rubropilosa （Hymenoptera： Formicidae）

The aim of this paper is to investigate the influence of physical and chemical factors on transport and use of substrate for Atta sexdens rubropilosa workers. Three types of rectangular fragments were used to study the physical influence factors： filter paper with paraffin, filter paper without paraffin and polyester film. To study the chemical factors, some fragments were impregnated with organic extract of orange albedo, others were soaked with soybean oil and for the remaining ones nothing was applied. The fonowing parameters were evaluated： （i） attractiveness of substrate for transport and number of loading workers per treatment; （ii） foraged material incorporation; （iii） rejection by numbers of fragments deposited in the garbage or beside the fungus garden. All the polyester film fragments carried out to the fungus garden were subsequently rejected. We verified that chemical factors of the substrate were more quickly detected by the workers, whereas physical factors were used as a criterion in the decision-making to reject or accept the substrate collected.     

Agonistic Behavior of the Leaf-Cutting Ant Atta sexdens rubropilosa Elicited by Caryophyllene

Caryophyllene is shown to evoke agonistic behavior among nestmates of the leaf-cutting ant Atta sexdens rubropilosa. Laboratory colonies were presented with caryophyllene on an acceptable bait. The behavioral responses observed included repellency, alarm, self-grooming, and arrestant behavior, followed by fighting. When caryophyllene was presented to the workers on inert filter paper fighting did not take place, but the other behaviors were observed. Individual workers smeared with caryophyllene produce an alarm response in nestmates. Caryophyllene produces the strongest reaction in workers that were within 2.5 cm of the source and a variation in sensitivity to caryophyllene appears to exist in different subcastes, with soldiers being the least sensitive. The behavior of the workers to caryophyllene was quantified from video recordings.     

Behavioural and olfactory variations in the leek moth, Acrolepiopsis assectella, after several generations of rearing under diverse conditions

Females of the parasitic phorid Neodohrniphora sp. were collected in the field and released singly inside an observation chamber placed between a laboratory colony of Atta sexdens (L.) and its foraging arena. The number and speed of loaded and unloaded ants returning to the nest, the weight of foragers and their loads, the number of leaf fragments abandoned by ants, and the number of small workers 'hitchhiking' on leaf fragments were measured before phorids were released, while they were in the observation chamber, and after they were removed. Relatively few ants were attacked by Neodohrniphora sp., but the presence of flies prompted outbound ants to return to the nest and caused a significant reduction on the number and mass of foragers. Additionally, the weight of leaf fragments transported by ants was reduced and the number of abandoned fragments increased in response to Neodohrniphora sp. Presence of the parasitoid caused no significant changes in the number of hitchhiking ants. The regular ants' traffic was resumed after phorids were removed, but foraging activity remained below normal for up to three hours. In the field A. sexdens forages mostly at night, but colonies undergo periods of diurnal foraging during which ants are subject to parasitism from several species of phorid flies. Considering that daytime foraging may be necessary for nutritional or metabolical needs, phorids may have a significant impact on their hosts by altering their foraging behavior regardless of the numerical values of parasitism.     

Cryptococcus haglerorum,sp. nov., an anamorphic basidiomycetous yeast isolated from nests of the leaf-cutting ant Atta sexdens

A yeast strain (CBS 8902) was isolated from the nest of a leaf-cutting ant and was shown to be related to Cryptococcus humicola. Sequencing of the D1/D2 region of the 26S ribosomal DNA and physiological characterization revealed a separate taxonomic position. A novel species named Cryptococcus haglerorum is proposed to accommodate strain CBS 8902 that assimilates n-hexadecane and several benzene compounds. Physiological characteristics distinguishing the novel species from some other members of the C. humicola complex are presented. The phylogenetic relationship of these strains to species of the genus Trichosporon Behrend is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cellulose degradation byLeucocoprinus gongylophorus,the fungus cultured by the leaf-cutting antAtta sexdens rubropilosa

Leucocoprinus gongylophorus, the fungus cultured by the leaf-cutting antAtta sexdens rubropilosa, is able to degrade efficiently cellulose, microcrystaline cellulose, carboximethylcellulose, and cellobiose. Analysis of the degradation products indicate that the fungus produce extracellular -glucosidase, exo- and endo-glucanase. The importance of cellulose degradation to the association of fungus and ant is discussed.     

Effect of constituents of the foliar wax of Didymopanax vinosum on the foraging activity of the leaf-cutting ant Atta sexdens rubropilosa

Leaf epicuticular waxes may affect substrate selection by leaf-cutting ants, and host recognition by several phytophagous insects. The influence of the crude epicuticular wax of Didymopanax vinosum E. March. (Araliaceae), and its major constituents lupeol and free primary alcohols, on substrate selection by the leaf-cutting ant Atta sexdens rubropilosa Forel, was investigated. Two ant colonies were used in laboratory bioassays. Corn flakes (zea) were impregnated with chloroform solutions of the substances being tested and fed to the adults. One of the colonies gave results, in most of the tests, not significantly different from the controls. The other colony, however, discriminated against the crude wax, a strong deterrent effect being observed from the first of a series of eight trials. The same colony was able to discriminate against lupeol after the second trial. The fraction of primary n-alcohols (22% C₂₈, 66% C₃₀ and 12% C₃₂) deterred feeding only after the fifth trial. The results demonstrate that some constituents of epicuticular waxes may deter the foraging activity of A. sexdens rubropilosa, depending, however, on the colony under observation. It is suggested that lupeol, of the foliar wax of D. vinosum, is an important deterrent to leaf-cutting ants, although with less pronounced effects than those observed in tests with the crude wax.     

Effects of cycloheximide on the mortality of Atta sexdens leaf-cutting worker ants

Leaf-cutting ants live symbiotically with a fungus that they cultivate on the plant leaves that they cut. The innumerous studies on the plant selection mechanism used by leaf-cutting ants show the researchers’ interest in this issue. Many classical studies propose that plants are selected according to the fungus garden nutritional needs and the absence of potentially harmful substances. This hypothesis is corroborated by behavioral experiments using cycloheximide (fungicide) with citric pulp or forage plants greatly accepted by leaf-cutting ants. According to this hypothesis, under the action of a fungicide, the fungus emits an allomone that informs worker ants that some food is inadequate to its growth. Although some authors state that the cycloheximide “fungicide” used is specific and non toxic to ants, our findings are distinct. In our study, various concentrations of cycloheximide were administered orally to leaf-cutting worker ants in a citric pulp paste diet. After the ingestion period, the ants were isolated and offered the symbiotic fungus for 21 days and the mortality rate was evaluated. As expected, the treatment with 0.01% cycloheximide showed a low mortality rate (8.86%). At 0.1%, the mortality rate was mild (27.85%), and treatment with 1% cycloheximide resulted in moderate mortality (45.57%). In contrast, the positive control with 0.1% sulfluramid showed a high mortality rate (91.14%). Therefore, we concluded that the ingestion of high concentrations of cycloheximide results in a moderate mortality rate in leaf-cutting worker ants.     

Biology and oviposition behavior of the phorid Apocephalus attophilus and the response of its host, the leaf-cutting ant Atta laevigata

Atta laevigata (Smith) (Hymenoptera: Formicidae) foragers collected in the field and parasitized by Apocephalus attophilus Borgmeier (Diptera: Phoridae) (1.2% of sampled ants) were larger and had lower survival rates than similar non-parasitized foragers. Moreover, the size of parasitized ants was significantly correlated with the number of A. attophilus puparia and percentage of adult emergence. These results suggest that host size is important for the reproductive success of A. attophilus. Different from most ant-parasitizing phorids, A. attophilus exhibits a pre-oviposition behavior that involves walking towards the host and inspecting it, and this careful approach may be responsible for a relatively low percentage of parasitoid detection by A. laevigata. When an exotic resource (Acalypha sp. leaves) was placed on ants' foraging trails, more foragers were recruited, which resulted in the attraction of A. attophilus. The number and recruiting rates of small workers (minima) were significantly higher on leaves visited by A. attophilus, but larger foragers showed no response to phorids. These results demonstrated that minima react to the presence of A. attophilus and suggest a defensive role of these ants against phorid parasitism.     

Influence of leaf epicuticular waxes from cerrado species on substrate selection byAtta sexdens rubropilosa

The influence of leaf epicuticular waxes of nine woody species from cerrado (a savanna ecosystem of Brazil) on the foraging behavior of the leaf-cutting antAtta sexdens rubropilosa Forel, 1908 (Formicidae, Attini) was investigated. Some waxes showed neither inhibitory nor attractive effect over a series of tests, while a second group caused a delayed rejection. Waxes of two species were shown to be strongly deterrent to the ants. Since different waxes caused different responses, it is concluded that their chemical composition, rather than hydrophobicity or mechanical constraints, accounts for the observed effects. Plant species with leaves rejected by the ants did not necessarily have repellent waxes. On the other hand, no species with acceptable leaves yielded waxes with deterrent effects.     

The effect of wilting on the selection of leaves by the leaf-cutting ant Atta laevigata

Using field assays of leaf preference, we tested the hypothesis that wilting affects the selection of leaves by the leaf-cutting ant Atta laevigata (Fr. Smith). Detached leaves were left to air-dry until noticeably wilted. The area removed by the ants from wilted leaves was significantly greater than the area removed from fresh leaves, this effect being observed in several plant species, in leaves of different age, and in assays with different ant colonies. Leaves collected from water-stressed plants were also preferred to leaves from non-stressed plants. A. laevigata was found to employ a two-stage, size-related, strategy when cutting plants. Larger workers climbed the plant stem and dropped whole leaves to the ground by severing their petioles; smaller workers cut the lamina of the dropped leaves. The ants frequently left dropped leaves on the ground, until the next foraging day or even later, when they were harvested in a wilted condition in preference to newly-dropped leaves.It is possible that during wilting some repellent substances evaporate or become less effective, thus enhancing leaf palatability. Alternatively or in addition, changes in nutrient and water content may have rendered wilted leaves more palatable to leaf-cutting ants.     

Forager polymorphism, size-matching, and load delivery in the leaf-cutting ant, Atta cephalotes

Abstract.

• 1 This study examined the importance of forager polymorphism and division of labour among foragers of different size for the economics of load delivery in a leaf-cutting ant, Atta cephalotes (L.). I collected A.cephalotes foragers coming down trees carrying leaf fragments to evaluate the degree of match between forager mass and the density (mass per unit area) of leaves being cut, and to quantify how this match affects whether the mass of leaf fragments cut by the ants are within the range which maximizes the rate and efficiency of load delivery.
• 2 Foragers ranged 23-fold in mass (1.4–32.1 mg). On average, larger workers cut at denser leaf sources. Leaf fragment area increased with ant mass, but relative area (fragment area/ant mass) decreased with ant mass. The density of a leaf type had little or no effect on the area cut by ants of a given size. As a result, ants of a given mass cut heavier fragments from the denser leaves. The effect of leaf density, however, was partly counteracted at the colony level by recruitment of larger ants, which cut smaller area fragments relative to their body mass, to cut at denser leaf sources.
• 3 Despite a fairly high variance in the relationship between ant mass and fragment mass, overall 87% of the laden ants (74–100% for different trees) carried leaf fragments in the 1.5–6 times body mass range. Earlier studies indicate that loads in this range yield the highest biomass transport rate and transport efficiency. Thus, the variance falls within bounds such that it has little effect on load transport efficiency. Having a broad range in optimal load mass may be considered an adaptation to the expected variability in load masses.
• 4 If there were no correlation between ant mass and leaf density, mismatches between ant mass and load mass would be more common than observed. Thus, size-matching of larger workers to cut denser leaves increases the rate and ergonomic efficiency of load delivery.

     

Starch metabolism in Leucoagaricus gongylophorus, the symbiotic fungus of leaf-cutting ants

Leucoagaricus gongylophorus, the symbiotic fungus of the leaf-cutting ants, degrades starch, this degradation being supposed to occur in the plant material which leafcutters forage to the nests, generating most of the glucose which the ants utilize for food. In the present investigation, we show that laboratory cultures of L. gongylophorus produce extracellular -amylase and maltase which degrade starch to glucose, reinforcing that the ants can obtain glucose from starch through the symbiotic fungus. Glucose was found to repress -amylase and, more severely, maltase activity, thus repressing starch degradation by L. gongylophorus, so that we hypothesize that: (1) glucose down-regulation of starch degradation also occurs in the Atta sexdens fungus garden; (2) glucose consumption from the fungus garden by A. sexdens stimulates degradation of starch from plant material by L. gongylophorus, which may represent a mechanism by which leafcutters can control enzyme production by the symbiotic fungus. Since glucose is found in the fungus garden inside the nests, down-regulation of starch degradation by glucose is supposed to occur in the nest and play a part in the control of fungal enzyme production by leafcutters.