Lack of patriline-specific differences in chemical composition of the metapleural gland secretion in <Emphasis Type="Italic">Acromyrmex octospinosus</Emphasis>

Summary. Multiple queen-mating (polyandry) in social insects increases the genetic variability among worker offspring, which may enhance colony survival, social productivity and defence against parasites. The unique and complex symbiosis of leaf-cutting ants with a clonal mutualistic fungus makes this social system particularly vulnerable to contamination by pathogenic and unwanted saprophytic fungi and bacteria. Proper defence against such threats requires effective and flexible chemical defence mechanisms. A prime candidate for providing such defences is the metapleural gland secretion, which is known to have broad antibiotic properties. Here we use the leaf-cutting ant Acromyrmex octospinosus to specifically test the hypothesis that genetically more diverse worker-offspring produce a more variable spectrum of metapleural gland compounds. We used DNA microsatellite markers to assign workers from two colonies to the six most common patrilines in each colony, and have analysed the degree to which the observed variance in the quantitative chemical composition of the metapleural gland secretion can be explained by genetic differences among patrilines. We found a marginally significant patriline-effect on the overall variability of metapleural gland compounds in one colony, but could not detect such effect in the other colony. We discuss a number of possible reasons why the genetic variance component for quantitative variation in metapleural gland secretion may be low.     

Nest-founding in <Emphasis Type="Italic">Acromyrmex octospinosus</Emphasis> (Hymenoptera,Formicidae, Attini): demography and putative prophylactic behaviors

Summary Foundresses of the leaf-cutting ant Acromyrmex octospinosus in central Panamá forage for leaves as garden substrate (semi-claustral foundation). The fungal pellet and substrate usually are attached to rootlets, which are used as a platform for the garden. This arrangement keeps the garden suspended away from the earthen chamber of the underground nest during early colony growth, and we hypothesize that it serves to minimize contact between the garden and contaminants. A. octospinosus foundresses produce from 3 to 7 workers in 2.7 months after founding the nest, but workers do not forage for substrate at this time. Incipient nests died or were abandoned at a monthly rate of ca. 50%. We show that ants routinely clean their legs before manipulating the garden substrate. We also describe how foundresses use their fore-legs to rub the surface of the metapleural gland (MPG), and they then use typical grooming behaviors to pass the forelegs through the mouthparts, after which the ant then licks the garden substrate. Similarly, ants apparently use their mouths to transfer fecal droplets to their legs. We briefly discuss the functional significance of these grooming behaviors, and hypothesize that they are prophylactic behaviors that may help the foundress maintain a hygienic garden.     

Examination of the immune responses of males and workers of the leaf-cutting ant Acromyrmex echinatior and the effect of infection

Summary. Parasites represent significant challenges to social insects. The high density, interaction rate and relatedness of individuals within colonies are all predicted to make social insect colonies particularly vulnerable to parasites. To cope with this pressure, social insects have evolved a number of defence mechanisms. These include the immune response, which, aside from in bumblebees, has been relatively little studied in social insects. Here we compare the immune responses of males and workers of the leaf-cutting ant Acromyrmex echinatior and examine the effect upon immunocompetence of prior exposure to a virulent parasite. Males have a far lower immune response than workers, suggesting either haploid susceptibility or reduced investment in immunity by the short-lived males. There was also significantly less variation in the immune response of males than of workers, which may be due to leaf-cutting ant workers being more variable in age or more genetically diverse within colonies. When exposed to the entomopathogenic fungus Metarhizium, workers expressed a substantially reduced immune response 96 h after infection, suggesting that the immune system was either depleted by having to respond to the Metarhizium infection or was depressed by the parasite. The results suggest that the immune response is a costly and limited process, but further experiments are needed to distinguish between the alternative explanations for the effects observed.Received 3 August 2004; revised 3 February 2005; accepted 2 March 2005.     

Plasticity of grooming behavior against entomopathogenic fungus Metarhizium anisopliae in the ant Lasius japonicus

Social insects employ many types of defense mechanisms against parasites and pathogens because they face high risks from infections due to crowded living conditions with closely related nestmates. Grooming behavior, including self-grooming and allogrooming, can remove fungal spores on the cuticles of social insects and may be a behavioral defense mechanism to improve survivorship. Allogrooming between nestmates has been predicted to be especially important as a defense against ectoparasites. However, little is known about the plasticity of grooming behavior in susceptible environments. Here, we show that the ant Lasius japonicus increases the frequency of allogrooming rather than self-grooming to improve survivorship against the entomopathogenic fungus Metarhizium anisopliae. We found that increasing fungal dosage and ant group size influenced the plasticity of grooming behavior. Additionally, the survival rate of the ants over 30 days improved with increased group size. Our results suggest that social insects opt for altruistic behavior to prevent the spread of diseases. This study illustrates how ants maintain colonies through grooming behavior in the presence of fungal spores.     

Arboreality and the evolution of disease resistance in ants

1. Parasites are an important selective force for almost all organisms and drive the evolution by hosts of defence mechanisms that are energetically costly. The strength of parasitism will vary between host species according to their specific ecology and life history, and so the optimal investment in costly resistance mechanisms is also likely to vary between host species with differing ecologies. 2. Parasites are particularly important for social species such as ants, but very little is known about the strength of selection in different species. It has been suggested that, because arboreality reduces exposure to soil‐borne fungal pathogens, arboreal ant species may invest less in disease resistance. However, testing hypotheses such as this requires data on disease resistance in multiple species, and such studies have not previously been attempted. 3. Here we examine the arboreality hypothesis by comparing the disease resistance of seven Neotropical ant species with different degrees of arboreality. We exposed ants to controlled doses of the generalist, virulent fungal parasite, Metarhizium anisopliae (Metchnikoff) Sorokin. We then monitored survival, parasite sporulation, and the anti‐fungal grooming response of the ants. 4. Contrary to the hypothesis, we found that arboreal species were not less resistant to M. anisopliae than species that were ground‐dwelling, and that the species that inhabited both arboreal and ground habitats had the greatest resistance. Surprisingly, the most resistant species was one that lacked the antibiotic‐producing metapleural gland, previously considered the lynchpin of disease resistance in ants. 5. The results suggest that it may be the diversity of parasites encountered that is the greatest selection pressure. Further experimental studies with other parasites are needed to confirm the generality of the results, and similar comparative studies of other taxa are needed to understand the relationship between host ecology and the evolution of disease resistance.     

Functional role of phenylacetic acid from metapleural gland secretions in controlling fungal pathogens in evolutionarily derived leaf-cutting ants

Fungus-farming ant colonies vary four to five orders of magnitude in size. They employ compounds from actinomycete bacteria and exocrine glands as antimicrobial agents. Atta colonies have millions of ants and are particularly relevant for understanding hygienic strategies as they have abandoned their ancestors'' prime dependence on antibiotic-based biological control in favour of using metapleural gland (MG) chemical secretions. Atta MGs are unique in synthesizing large quantities of phenylacetic acid (PAA), a known but little investigated antimicrobial agent. We show that particularly the smallest workers greatly reduce germination rates of Escovopsis and Metarhizium spores after actively applying PAA to experimental infection targets in garden fragments and transferring the spores to the ants'' infrabuccal cavities. In vitro assays further indicated that Escovopsis strains isolated from evolutionarily derived leaf-cutting ants are less sensitive to PAA than strains from phylogenetically more basal fungus-farming ants, consistent with the dynamics of an evolutionary arms race between virulence and control for Escovopsis, but not Metarhizium. Atta ants form larger colonies with more extreme caste differentiation relative to other attines, in societies characterized by an almost complete absence of reproductive conflicts. We hypothesize that these changes are associated with unique evolutionary innovations in chemical pest management that appear robust against selection pressure for resistance by specialized mycopathogens.     

Bacterial symbiont sharing in Megalomyrmex social parasites and their fungus‐growing ant hosts

Bacterial symbionts are important fitness determinants of insects. Some hosts have independently acquired taxonomically related microbes to meet similar challenges, but whether distantly related hosts that live in tight symbiosis can maintain similar microbial communities has not been investigated. Varying degrees of nest sharing between Megalomyrmex social parasites (Solenopsidini) and their fungus‐growing ant hosts (Attini) from the genera Cyphomyrmex, Trachymyrmex and Sericomyrmex allowed us to address this question, as both ant lineages rely on the same fungal diet, interact in varying intensities and are distantly related. We used tag‐encoded FLX 454 pyrosequencing and diagnostic PCR to map bacterial symbiont diversity across the Megalomyrmex phylogenetic tree, which also contains free‐living generalist predators. We show that social parasites and hosts share a subset of bacterial symbionts, primarily consisting of Entomoplasmatales, Bartonellaceae, Acinetobacter, Wolbachia and Pseudonocardia and that Entomoplasmatales and Bartonellaceae can co‐infect specifically associated combinations of hosts and social parasites with identical 16S rRNA genotypes. We reconstructed in more detail the population‐level infection dynamics for Entomoplasmatales and Bartonellaceae in Megalomyrmex symmetochus guest ants and their Sericomyrmex amabilis hosts. We further assessed the stability of the bacterial communities through a diet manipulation experiment and evaluated possible transmission modes in shared nests such as consumption of the same fungus garden food, eating of host brood by social parasites, trophallaxis and grooming interactions between the ants, or parallel acquisition from the same nest environment. Our results imply that cohabiting ant social parasites and hosts may obtain functional benefits from bacterial symbiont transfer even when they are not closely related.     

Ant Colonies Prefer Infected over Uninfected Nest Sites

During colony relocation, the selection of a new nest involves exploration and assessment of potential sites followed by colony movement on the basis of a collective decision making process. Hygiene and pathogen load of the potential nest sites are factors worker scouts might evaluate, given the high risk of epidemics in group-living animals. Choosing nest sites free of pathogens is hypothesized to be highly efficient in invasive ants as each of their introduced populations is often an open network of nests exchanging individuals (unicolonial) with frequent relocation into new nest sites and low genetic diversity, likely making these species particularly vulnerable to parasites and diseases. We investigated the nest site preference of the invasive pharaoh ant, Monomorium pharaonis, through binary choice tests between three nest types: nests containing dead nestmates overgrown with sporulating mycelium of the entomopathogenic fungus Metarhizium brunneum (infected nests), nests containing nestmates killed by freezing (uninfected nests), and empty nests. In contrast to the expectation pharaoh ant colonies preferentially (84%) moved into the infected nest when presented with the choice of an infected and an uninfected nest. The ants had an intermediate preference for empty nests. Pharaoh ants display an overall preference for infected nests during colony relocation. While we cannot rule out that the ants are actually manipulated by the pathogen, we propose that this preference might be an adaptive strategy by the host to “immunize” the colony against future exposure to the same pathogenic fungus.     

Microfungal “Weeds” in the Leafcutter Ant Symbiosis

Leafcutter ants (Formicidae: tribe Attini) are well-known insects that cultivate basidiomycete fungi (Agaricales: Lepiotaceae) as their principal food. Fungus gardens are monocultures of a single cultivar strain, but they also harbor a diverse assemblage of additional microbes with largely unknown roles in the symbiosis. Cultivar-attacking microfungi in the genus Escovopsis are specialized parasites found only in association with attine gardens. Evolutionary theory predicts that the low genetic diversity in monocultures should render ant gardens susceptible to a wide range of diseases, and additional parasites with roles similar to that of Escovopsis are expected to exist. We profiled the diversity of cultivable microfungi found in 37 nests from ten Acromyrmex species from Southern Brazil and compared this diversity to published surveys. Our study revealed a total of 85 microfungal strains. Fusarium oxysporum and Escovopsis were the predominant species in the surveyed gardens, infecting 40.5% and 27% of the nests, respectively. No specific relationship existed regarding microfungal species and ant-host species, ant substrate preference (dicot versus grass) or nesting habit. Molecular data indicated high genetic diversity among Escovopsis isolates. In contrast to the garden parasite, F. oxysporum strains are not specific parasites of the cultivated fungus because strains isolated from attine gardens have similar counterparts found in the environment. Overall, the survey indicates that saprophytic microfungi are prevalent in South American leafcutter ants. We discuss the antagonistic potential of these microorganisms as “weeds” in the ant–fungus symbiosis.     

Asian weaver ants,Oecophylla smaragdina,and their repelling of pollinators

The Asian weaver ant, Oecophylla smaragdina, is known to have outstanding predatory power. This ant can protect the host plants from attacks of phytophagous insects and therefore has been used for biological control in the tropics. We present evidence for a possible negative effect of Oecophylla on the performance of host plants. Our observation in a fruit orchard of rambutan in Sumatra suggested that the presence of Oecohylla nests on the trees statistically significantly lowered the flower-visiting rate of flying insects, involving the major pollinator Trigona minangkabau. The visiting rate of Oecophylla workers to each flower shoot of rambutan significantly negatively correlated with the visiting rate of flying insects. Empirical evidence of such an inhibitory effect on flower-visiting of pollinators cased by aggressive ants has been scarce so far.     

Differential resistance and the importance of antibiotic production in Acromyrmex echinatior leaf-cutting ant castes towards the entomopathogenic fungus Aspergillus nomius

Paired exocrine metapleural glands are present in almost all ants and produce compounds with antibiotic properties towards a variety of pathogenic fungi and bacteria. In Acromyrmex leaf-cutting ants, small workers have relatively large metapleural glands compared to large workers, and thus harbour approximately half the number of gland cells of large workers, despite being only one-fifteenth their body mass. Here we present results showing that when the two worker castes of A. echinatior are treated with spores of the pathogenic fungus Aspergillus nomius in doses that correspond to the difference in metapleural gland cell numbers they do not differ in survival. However, we also show, for the first time, that small workers survive significantly longer than large workers when both are challenged with a dose of spores that corresponds to their difference in body mass. Furthermore, the time until Aspergillus nomius hyphae and spores appear on the cadavers of workers dead from infection, is significantly increased in the small worker caste. In addition to supporting previous findings that the metapleural glands have an important defence function, the results of this study indicate that the relatively large glands in small workers makes this caste particularly well adapted to preventing pathogenic microorganisms from entering the colony. Received 23 January 2006; revised 7 April 2006; accepted 11 April 2006.     

Urea in Weaver Ant Feces: Quantification and Investigation of the Uptake and Translocation of Urea in <Emphasis Type="Italic">Coffea arabica</Emphasis>

Weaver ants are tropical insects that nest in tree canopies, and for centuries these ants have been used for pest control in tropical orchards. Trees hosting weaver ants might benefit not only from the pest protective properties of these insects but also an additional supply of nutrients from ant feces deposited on the leaves. In a recent study, we demonstrated that Coffea arabica plants hosting Oecophylla smaragdina weaver ants under laboratory conditions experienced enhanced nitrogen availability compared with plants grown without ants. Therefore, the aim of the present study was to further investigate the interactions of weaver ants with the host plants with respect to plant nutrition. Here, we report the identification and quantification of urea, a highly effective foliar nutrient present in the fecal depositions of O. smaragdina. Feces samples obtained from six O. smaragdina colonies were analyzed, and urea concentrations ranging from 1.98 to 31.05 μg/mg ant feces were detected. Subsequently, we investigated the uptake and translocation of ¹⁵N₂-urea in amounts corresponding to the estimated urea contribution via feces depositions on single host plant leaves under laboratory conditions. The results clearly demonstrated that fecal urea was not only assimilated but also translocated within the plant. This evidence strongly supports the hypothesis that the fecal urea of weaver ants is a source of nitrogen for the host trees. Thus, weaver ant feces likely contribute to an improved nutritional status of ant-hosting trees in tropical orchards, thereby adding value to the use of weaver ants for the biocontrol of insect pests.     

An evaluation of the possible adaptive function of fungal brood covering by Attine ants

Fungus-growing ants (Myrmicinae: Attini) live in an obligate symbiotic relationship with a fungus that they rear for food, but they can also use the fungal mycelium to cover their brood. We surveyed colonies from 20 species of fungus-growing ants and show that brood-covering behavior occurs in most species, but to varying degrees, and appears to have evolved shortly after the origin of fungus farming, but was partly or entirely abandoned in some genera. To understand the evolution of the trait we used quantitative phylogenetic analyses to test whether brood-covering behavior covaries among attine ant clades and with two hygienic traits that reduce risk of disease: mycelial brood cover did not correlate with mutualistic bacteria that the ants culture on their cuticles for their antibiotics, but there was a negative relationship between metapleural gland grooming and mycelial cover. A broader comparative survey showed that the pupae of many ant species have protective cocoons but that those in the subfamily Myrmicinae do not. We therefore evaluated the previously proposed hypothesis that mycelial covering of attine ant brood evolved to provide cocoon-like protection for the brood.     

Pseudocopulation of an orchid by male ants: a test of two hypotheses accounting for the rarity of ant pollination

Summary The orchid Leporella fimbriata is pollinated by pseudocopulation with winged males of the ant Myrmecia urens. This recently studied interaction provides a unique opportunity to examine the two current hypotheses concerning the apparent rarity of ant pollination systems worldwide. The first hypothesis requires a series of specialized growth forms and floral characteristics regarded as adaptations to ant pollination. L. fimbriata does not possess them. The second considers the pollenicidal effects of secretions from the metapleural gland of ants. These glands are absent in M. urens males and it may be that the occurrence of ant pollination requires the absence of metapleural glands in the vector.     

Foraging Ants as Scavengers on Entomopathogenic Nematode-Killed Insects

Ants were the most apparent invertebrate scavengers observed foraging on entomopathogenic nematode-killed insects (i.e., insect cadavers containing entomopathogenic nematodes and their symbiotic bacteria) in the present study. Workers of the Argentine ant,Linepithema humile(Mayr), scavenged nematode-killed insects on the surface and those buried 2 cm below the soil surface. Ant workers scavenged significantly more steinernematid-killed (60–85%) than heterorhabditid-killed (10–20%) insects. More 4-day-postinfected cadavers (hosts died within 48 h after exposure to nematodes) were scavenged than 10-day-postinfected cadavers. Ten-day-postinfected hosts contained live infective juvenile nematodes therefore ants may serve as phoretic agents. Other ant species, includingVeromessor andrei(Mayr),Pheidole vistanaForel,Formica pacificaFrancoeur, andMonomoriom ergatogynaWheeler, also scavenged nematode-killed insects. These ant species removed or destroyed about 45% of the steinernematid-killed insects. These results suggest that survival of steinernematid nematodes may be more significantly impacted by invertebrate scavengers, especially ants, than that of heterorhabditid nematodes, and placement of steinernematid-killed insects in the field for biological control may be an ineffective release strategy. Because entomopathogenic nematodes kill insects with the help of symbiotic bacteria, we tested the role of these bacterial species in deterring invertebrate scavengers by injecting bacteria (without nematodes) into insects and placing the cadavers in the field. None of the insects killed by the symbiotic bacterium,Photorhabdus luminescens(Thomas and Poinar) fromHeterorhabditis bacteriophoraPoinar, were scavanged, whereas 70% of the insects killed by the symbiotic bacterium,Xenorhabdus nematophilus(Poinar and Thomas) fromSteinernema carpocapsae(Weiser), and 90% of the insects killed byBacillus thuringiensisBerliner were scavenged by the Argentine ant. We conclude thatP. luminescensis responsible for preventing ants from foraging on heterorhabditid-killed hosts.     

Trade-offs in group living: transmission and disease resistance in leaf-cutting ants

Sociality can be associated with significant costs due to the increased risk of disease transmission. However, in some organisms the costs may be offset by benefits due to improvements in defences against parasites. To examine this possible trade-off between infection risk and disease resistance, we used Acromyrmex leaf-cutting ants and the entomopathogenic fungus Metarhizium anisopliae as the model system. Ants exposed to the parasite were found to have substantially improved survival when they were kept with nest-mates, while the cost of being in a group in terms of increased disease transmission was very low. The efficiency of transmission is described by the transmission parameter, which decreased with increasing host density showing that transmission rates are inversely density dependent. Both grooming and antibiotic secretions appeared to be important in resistance against the parasite, with the defences of small workers being particularly effective. The results indicate that leaf-cutting ant colonies may have much greater resistance to disease than would be predicted from the high densities of host individuals within them. Unlike most organisms, group living in these ants may actually be associated with a net benefit in terms of disease dynamics.     

Selective isolation of dematiaceous fungi from the workers of <Emphasis Type="Italic">Atta laevigata</Emphasis> (Formicidae: Attini)

Leaf-cutting ants (Formicidae: Attini) are considered pests in agriculture for their impact in human crops, as they utilize leaf fragments to raise their fungal mutualist (Agaricales: Lepiotaceae). Basically, the basidiomycetous fungus is cultivated to supply food to adult workers and broads; in return, the ants protect it against natural enemies. However, recent studies have claimed that other microorganisms are associated to ant nests where a wide range of interactions may take place. To investigate the occurrence of dematiaceous fungi on the cuticle of Atta laevigata ants, 30 workers were sampled from an adult nest located in the surroundings of the Center for the Studies of Social Insects, UNESP-Rio Claro, SP, Brazil. The use of selective techniques to avoid high-sporulation fungi has been recommended and was tested in this study. To favor the isolation of the desired fungi, heads and cuticle scrapings of ant bodies were inoculated on Mycosel agar and incubated for 3 weeks at 35°C. Morphological and molecular methods were used to identify the filamentous fungi recovered. From 56 isolates, 19 were hyaline filamentous species, and among the remaining 37, some are mentioned as phyto-associated fungi like Alternaria arborescens, Bipolaris sorokiniana, Bipolaris eleusines, Bipolaris zeae, Curvularia trifolii, and Paraphaeosphaeria michotii. These species are reported from A. laevigata bodies for the first time. None of the isolation trials revealed the presence of the parasite Escovopsis or entomopathogenic fungi. The possible spread of the fungi in nature by the ants is discussed.     

Diversity,prevalence and virulence of fungal entomopathogens in colonies of the ant <Emphasis Type="Italic">Formica selysi</Emphasis>

The richness of the parasitic community associated with social insect colonies has rarely been investigated. Moreover, understanding how hosts and pathogens interact in nature is important to interpret results from laboratory experiments. Here, we assessed the diversity, prevalence and virulence of fungal entomopathogens present around and within colonies of the ant Formica selysi. We detected eight fungal species known to be entomopathogenic in soil sampled from the habitat of ants. Six of these entomopathogens were found in active nests, abandoned nests, and corpses from dump piles or live ants. A systematic search for the presence of three generalist fungal entomopathogens in ant colonies revealed a large variation in their prevalence. The most common of the three pathogens, Paecilomyces lilacinus, was detected in 44% of the colonies. Beauveria bassiana occurred in 17% of the colonies, often in association with P. lilacinus, whereas we did not detect Metarhizium brunneum (formerly M. anisopliae) in active colonies. The three fungal species caused significant mortality to experimentally challenged ants, but varied in their degree of virulence. There was a high level of genetic diversity within B. bassiana isolates, which delineated three genetic strains that also differed significantly in their virulence. Overall, our study indicates that the ants encounter a diversity of fungal entomopathogens in their natural habitat. Moreover, some generalist pathogens vary greatly in their virulence and prevalence in ant colonies, which calls for further studies on the specificity of the interactions between the ant hosts and their fungal pathogens.