Ocurrence of the antibiotic producing bacterium Burkholderia sp. in colonies of the leaf-cutting ant Atta sexdens rubropilosa

Fungus garden material from recently established Atta sexdens rubropilosa colonies (6-12 months old) was sampled to detect antibiotic producing microorganisms that inhibited the growth of pathogens of insects and of the fungus gardens but did not affect their mutualistic fungus. A bacterium with activity against the entomopathogenic fungus Beauveria bassiana was isolated from 56% of the gardens tested (n=57) and identified from its biochemical profile and from 16S and 23S ribosomal DNA sequences as a member of the genus Burkholderia. The ant-associated Burkholderia isolates secreted a potent, anti-fungal agent that inhibited germination of conidia of the entomopathogenic fungi B. bassiana, Metarhizium anisopliae, of the saprophytic Verticillium lecanii, and also of a specialist fungus garden Escovopsis weberi. Growth of the ant's mutualist fungus was unaffected.     

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.

     

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.     

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.     

Low intraspecific aggression level in the polydomous and facultative polygynous ant Ectatomma tuberculatum

Nestmate recognition is a key feature of social insects, as it preserves colony integrity. However, discrimination of non‐nestmates and nestmate recognition mechanisms are highly variable according to species and social systems. Here, we investigated the intraspecific level of aggression in the facultative polygynous and polydomous ant, Ectatomma tuberculatum Olivier (Hymenoptera: Formicidae: Ectatomminae), in a population with a strong genetic structure. We found that the intraspecific level of aggression was generally low in this population of E. tuberculatum. However, the level of aggression was significantly correlated with the geographical distance, suggesting that both genetic and environmental cues could be involved in nestmate recognition and discrimination mechanisms. Moreover, polydomy was confirmed by the absence of aggression between workers from nests at a distance of 3 m, while the level of aggression was significantly higher between workers from nests separated by a distance of 10 or 1300 m. Field experiments showed that the low level of aggression between neighbouring colonies was associated with shared foraging areas, which could suggest that familiarization processes may occur in this species. We propose that the particular social organization of this species, with secondary polygyny, polydomy, and budding, may have favoured a high acceptance threshold, because of the low probability of interactions with unrelated conspecifics, the high cost of erroneously rejecting nestmates, and the low cost of accepting non‐nestmate workers. The resulting open recognition system can thus allow privileged relationships between neighbouring colonies and promote the ecological dominance of E. tuberculatum in the mosaic of arboreal ants.     

Variation in ant aggression and kin discrimination ability within and between colonies

Significant variation in aggressiveness and kin discrimination ability occurs between different laboratory colonies of the ant Rhytidoponera confusa.Different colonies show consistently high (or low) levels of aggression toward nonnestmates over 4–19 weeks. Earlier studies excluded colony size and the natural presence or absence of the queen in colonies and differences in hunger as possible sources of variation. The present study excluded the number of larvae in colonies and the time of the light period of the light cycle when recognition tests were carried out. Highly significant variation occurs between the kin discrimination ability of individual workers in any particular colony. Approximately 28% of the workers in colonies of R. confusashowed very poor kin discrimination. Much of the colony's kin discrimination is carried out by a small number of highly aggressive workers.     

Temporal variation in foraging activity and efficiency and the role of hitchhiking behaviour in the leaf-cutting ant, Atta cephalotes

Two phenomena are integral to the foraging behaviour of leaf‐cutting ants in the genus Atta: hitchhiking (where small ants ride on leaf fragments carried by larger workers) and rhythmic foraging (where foraging activity shows marked fluctuations over time). While parasitism by phorid flies has been implicated in eliciting both behaviours, recent research suggests fungal contaminants and the need to procure sap also play a key role in eliciting hitchhiking. For wild colonies of Atta cephalotes L. (Hymenoptera: Formicidae), we investigated the extent to which hitchhiking frequency varied in space and time and the foraging performance of day‐time and night‐time workers. Day‐time foragers were considerably smaller than nocturnal foragers, a trend previously described as a response to diurnal phorids. Despite their smaller size, day‐time foragers had higher foraging performance, perhaps as a consequence of decreased trail congestion. Larger leaf‐carriers were more likely to carry hitchhikers and hitchhiking frequency was higher at night, an observation that conflicts with the parasitoid defence hypothesis, but not with the leaf sap and fungal defence hypotheses. Hitchhikers constitute a major proportion (typically 12%) of the loads carried by workers, and have three times the effect of leaf fragment mass on forager velocity. However, they reduced energetic efficiency by only 2.6% and provisioning rate by 5.9%. Our results provide partial support for the parasitoid defence hypothesis, but suggest that both the risks of parasitism and the opportunity‐cost to foraging associated with carrying hitchhikers may be low.     

Courtship with two spoons—Anatomy and presumed function of the bizarre antennae of Cardiocondyla zoserka ant males

Mating in ants often occurs on the wing during nuptial flights or on the ground when scattered female sexuals attract males by pheromones. In both scenarios, there is little opportunity for males to engage in prolonged aggressive competition or elaborate courtship displays. Male morphology is therefore adapted to locating female sexuals and mating, and it lacks specific weapons or other traits associated with courtship. In contrast, sexuals of the ant genus Cardiocondyla typically mate in their natal nests. As a consequence, in many species winged males have been replaced by wingless fighter or territorial males, which kill or expel rival males with their strong mandibles and show complex mating behavior. However, no wingless males are known from Cardiocondyla zoserka from West Africa, and instead, winged males have evolved a bizarre secondary sexual trait: uniquely shaped antennae with spoon‐like tips that show heavily sculptured ventral surfaces with numerous invaginations. We here report on the courtship behavior of C. zoserka males and describe antennal glands with class 3 gland cells, which presumably secrete a close range sex pheromone. Antennal glands have not yet been found in males of other ant species, including a close relative of C. zoserka, suggesting that in ants with intranidal mating sexual selection can rapidly lead to highly divergent adaptations and the evolution of novel structures.     

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.

     

Structure of ground‐dwelling ant communities in burned and unburned areas in Brazilian subtropical grasslands

Fire is frequently used in the management of pastures in southern Brazil, but its effects on ground‐dwelling ant communities in Brazilian subtropical grasslands is still poorly understood. Here, we compared ant species richness and composition between periodically burned and unburned areas in native grasslands of the Atlantic Forest biome. In total, we found 35 epigeic ant species in burned and unburned areas. There was slightly higher species richness in burned than in unburned areas, independent of the sampling period (season). There was a significant difference in richness over the sampling period (season effect). Species composition varied significantly between the areas, in which nine species (26%) occurred only in burned areas, eight (23%) occurred only in unburned areas, and 18 (51%) occurred in both. Four species showed a significant preference for burned sites (Camponotus crassus, Linepithema humile and two undetermined species of Pheidole and Solenopsis). Although this study did not separate fire effects on ground‐dwelling ant communities (due to sampling design), it provides new information regarding subtropical native grasslands that can be used as a baseline for future studies.     

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.     

Memory and chemical communication in the orientation of two mass-recruiting ant species

Summary The relative contribution of visual and chemical components in the orientation ofLasius niger andIridomyrmex humilis (Argentine ant) workers during mass recruitment to newly discovered food sources is analyzed over short time intervals. While both species orient in response to the trail pheromone, a large number ofL. niger foragers rapidly switch to a more individual orientation, based on their memory of environmental cues.I. humilis workers, on the other hand, predominantly use collective chemical cues. The effect of the number of reinforcements on visual learning and its interference with chemical communication show that olfactory cues always prevail in the Argentine ant. InL. niger, the proportion of ants orienting to visual cues is independent of the trail concentration. Detailed observations of the trail-laying behavior of individually marked foragers show that nearly all theI. humilis workers initially lay a trail, whereas only half theL. niger foragers do so. This proportion decreases considerably with the number of trips performed byL. niger workers, while remaining constant for the Argentine ants. These results are interpreted with respect to the species' behavioral ecology.     

Reformation process of the neuronal template for nestmate-recognition cues in the carpenter ant <Emphasis Type="Italic">Camponotus floridanus</Emphasis>

Ants use cuticular hydrocarbons (CHC-profiles) as multicomponent recognition cues to identify colony members (nestmates). Recognition cues (label) are thought to be perceived during ant–ant encounters and compared to a neuronal template that represents the colony label. Over time, the CHC-profile may change, and the template is adjusted accordingly. A phenotype mismatch between label and template, as happens with CHC-profiles of foreign workers (non-nestmates), frequently leads to aggressive behavior. We investigated the template reformation in workers of the carpenter ant Camponotus floridanus by masking their antennae with postpharyngeal gland (PPG) extracts from nestmates or non-nestmates. The behavioral response of manipulated workers encountering unmanipulated workers was measured independently after 2 and after 15 h. After 2 h of incubation, workers treated with either of the two PPG-extracts showed low aggression towards nestmates and high aggression towards non-nestmates. In contrast, after 15 h of incubation, workers treated with non-nestmate PPG-extract showed low aggression towards both nestmates and non-nestmates. The slow (>2 h) adjustment of the template indicates a reformation localized in the central nervous system rather than in chemosensory neurons. In addition, our data show that template adjustment to a new CHC-profile does not impair the assessment of the old CHC-profile as nestmate label.     

Nestmate recognition in burying beetles: the "breeder's badge" as a cue used by females to distinguish their mates from male intruders

Burying beetles use small vertebrate carcasses as food for theirlarvae and defend these carcasses against intra- and interspecificcompetitors. Breeding associations on carcasses can consistof single females, heterosexual pairs, or various combinationsof males and females. When a heterosexual pair collaborate ina breeding attempt, they do not typically exhibit aggressivebehavior toward each other, but do attack newly arrived conspecificsthat attempt to usurp the carcass. We investigated the cuesinvolved in discrimination between breeding partners and intrudersby female burying beetles. We found that resident females toleratemales that have cared for a brood, as well as males that havenot cared for a brood but have been on a carcass for a day ortwo. Males that have had no prior contact with a carcass areattacked. Females appear to use a chemical cue, the "breeder'sbadge," an apolar substance on the male's cuticle that can beremoved by washing with pentane. This cue is reliably correlatedwith recent male experience with a carcass that is suitablefor reproduction. The breeder's badge develops as a result ofprolonged contact with such a carcass, and disappears on removalfrom the carcass; its presence does not require contact witha female or with larvae. Female recognition of their male partnersin burying beetles thus does not involve individual recognition,but rather recognition of reproductive condition.     

Digging effort in leaf-cutting ant queens (<Emphasis Type="Italic">Atta sexdens rubropilosa</Emphasis>) and its effects on survival and colony growth during the claustral phase

Nest foundation in the leaf-cutting ant Atta sexdens is claustral, and the single queen completely relies on its body reserves throughout, approximately, 9 weeks until the first workers emerge and initiate foraging. Nest digging is much time- and energy-consuming, and it is an open question how queens decide on the length of the tunnel they dig and therefore the depth of the initial chamber. Shallow founding nests may be energetically cheaper to dig, but queens may be more exposed to changing environmental variables. Deeper nests, on the other hand, may be climatically more stable and suitable, but more expensive to dig. We hypothesized that the maximal nest depth excavated by Atta founding queens may represent the outcome of an evolutionary trade-off between maximizing nest depth and minimizing energy expenditure during digging, so as to save energy for the long claustral phase. We tested this hypothesis by comparing the fitness consequences of increased digging effort in queens that were experimentally stimulated to excavate a complete founding nest either once, twice or three times consecutively compared to control queens that did not dig. Fitness was quantified as mortality rates, rates of egg-laying and offspring production, and size of the fungus garden until the emergence of the first workers. Results showed that, in contrast with the initial expectations, fungus growth, egg-laying rates and offspring production were not affected by the increased digging effort in the experimentally induced successive excavations. However, a significant higher mortality was observed in queens with increased digging effort, i.e., those that dug two or three nests consecutively. It is argued that in queens a behavioral mechanism for the control of nest depth has evolutionary been selected for as a trade-off between maximizing nest depth, to favor protection of the queen against unsuitable environmental variables, and minimizing energy expenditure during digging, which significantly affects survival.     

Evolution of cuticular hydrocarbon diversity in ants

The cuticular hydrocarbons (CHCs) of ants provide important cues for nest-mate and caste recognition. There is enormous diversity in the composition of these CHCs, but the manner in which this diversity has evolved is poorly understood. We gathered data on CHC profiles for 56 ant species, relating this information to their phylogeny. We deduced the mode of evolution of CHC profiles by reconstructing character evolution and then relating the number of changes in CHC components along each branch of the phylogeny to the length of the branch. There was a strong correlation between branch length and number of component changes, with fewer changes occurring on short branches. Our analysis thereby indicated a gradual mode of evolution. Different ant species tend to use specific CHC structural types that are exclusive of other structural types, indicating that species differences may be generated in part by switching particular biosynthetic pathways on or off in different lineages. We found limited, and contradictory, evidence for abiotic factors (temperature and rainfall) driving change in CHC profiles.