Hymenopteran-specific TRPA channel from the Texas leaf cutter ant (Atta texana) is heat and cold activated and expression correlates with environmental temperature.

Leaf cutting ants of the genus Atta cultivate fungal gardens, carefully modifying environmental conditions to maintain optimal temperature for fungal growth. Antennal nerves from Atta are highly temperature sensitive, but the underlying molecular sensor is unknown. Here, we utilize Atta texana (Texas leaf cutter ant) to investigate the molecular basis of ant temperature sensation and how it might have evolved as the range expanded northeast across Texas from ancestral populations in Mexico. We focus on transient receptor potential (TRP) channel genes, the best characterized temperature sensor proteins in animals. Atta texana antennae express 6 of 13 Hymenopteran TRP channel genes and sequences are under a mix of relaxed and intensified selection. In a behavioral assay, we find A. texana workers prefer 24 °C (range 21-26 °C) for fungal growth. There was no evidence of regulatory evolution across a temperature transect in Texas, but instead Hymenoptera-specific TRPA (HsTRPA) expression highly correlated with ambient temperature. When expressed in vitro, HsTRPA from A. texana is temperature activated with Q10 values exceeding 100 on initial exposure to temperatures above 33 °C. Surprisingly, HsTRPA also appears to be activated by cooling, and therefore to our knowledge, the first non-TRPA1 ortholog to be described with dual heat/cold activation and the first in any invertebrate.

Leaf-cutter ants - mycorrhizal fungi: observations and research questions from an unexpected mutualism.

Leaf-cutter ants (LCAs) are widely distributed and alter the physical and biotic architecture above and below ground. In neotropical rainforests, they create aboveground and belowground disturbance gaps that facilitate oxygen and carbon dioxide exchange. Within the hyperdiverse neotropical rainforests, arbuscular mycorrhizal (AM) fungi occupy nearly all of the forest floor. Nearly every cubic centimeter of soil contains a network of hyphae of Glomeromycotina, fungi that form arbuscular mycorrhizae. Our broad question is as follows: how can alternative mycorrhizae, which are-especially ectomycorrhizae-essential for the survival of some plant species, become established? Specifically, is there an ant-mycorrhizal fungus interaction that facilitates their establishment in these hyperdiverse ecosystems? In one lowland Costa Rican rainforest, nests of the LCA Atta cephalotes cover approximately 1.2% of the land surface that is broadly scattered throughout the forest. On sequencing the DNA from soil organisms, we found the inocula of many AM fungi in their nests, but the nests also contained the inocula of ectomycorrhizal, orchid mycorrhizal, and ericoid mycorrhizal fungi, including Scleroderma sinnamariense, a fungus critical to Gnetum leyboldii, an obligate ectomycorrhizal plant. When the nests were abandoned, new root growth into the nest offered opportunities for new mycorrhizal associations to develop. Thus, the patches created by LCAs appear to be crucial sites for the establishment and survival of shifting mycorrhizal plant-fungal associations, in turn facilitating the high diversity of these communities. A better understanding of the interactions of organisms, including cross-kingdom and ant-mycorrhizal fungal interactions, would improve our understanding of how these ecosystems might tolerate environmental change.

Collaborative Use of Sensor Networks and Cyberinfrastructure to Understand Complex Ecosystem Interactions in a Tropical Rainforest: Challenges and Lessons Learned.

Collaborations between ecosystem ecologists and engineers have led to impressive progress in developing complex models of biogeochemical fluxes in response to global climate change. Ecology and engineering iteratively inform and transform each other in these efforts. Nested data streams from local sources, adjacent networks, and remote sensing sources together magnify the capacity of ecosystem ecologists to observe systems in near real-time and address questions at temporal and spatial scales that were previously unobtainable. We describe our research experiences working in a Costa Rican rainforest ecosystem with the challenges presented by constant high humidity, 4300 mm of annual rainfall, flooding, small invertebrates entering the tiniest openings, stinging insects, and venomous snakes. Over the past two decades, we faced multiple challenges and learned from our mistakes to develop a broad program of ecosystem research at multiple levels of integration. This program involved integrated networks of diverse sensors on a series of canopy towers linked to multiple belowground soil sensor arrays that could transport sensor data streams from the forest directly to an off-site location via a fiber optic cable. In our commentary, we highlight three components of our work: (1) the eddy flux measurements using canopy towers; (2) the soil sensor arrays for measuring the spatial and temporal patterns of CO2 and O2 fluxes at the soil-atmosphere interface; and (3) focused investigations of the ecosystem impact of leaf-cutter ants as "ecosystem engineers" on carbon fluxes.

From Plants to Ants: Fungal Modification of Leaf Lipids for Nutrition and Communication in the Leaf-Cutter Ant Fungal Garden Ecosystem.

Lipids are essential to all living organisms, as an energy source, as an important cellular structural component, and as a communication tool. In this study, we used global lipidomic methods to evaluate the lipids in leaf-cutter ant fungal gardens. Leaf-cutter ants and their coevolved fungal cultivar, Leucoagaricus gongylophorus, are a model mutualistic system. The fungus enzymatically digests fresh plant material that the ants cut and deliver, converting energy and nutrients from plants and providing them to the ants through specialized hyphal swellings called gongylidia. Using combined liquid chromatography, ion mobility spectrometry, and tandem mass spectrometry, we evaluated differences between the molecular species of lipids in the leaf-cutter ant fungal garden ecosystem. This lipidomic study characterized leaves that are fed to the gardens, gongylidia that are produced by the fungus to feed the ants, and spatially resolved regions of the fungal garden through stages of leaf degradation. Lipids containing alpha-linolenic acid (18:3) were enriched in leaves and the top of the gardens but not dominant in the middle or bottom regions. Gongylidia were dominated by lipids containing linoleic acid (18:2). To evaluate the communicative potential of the lipids in fungal gardens, we conducted a behavioral experiment that showed Atta leaf-cutter ants responded differently to 18:3 and 18:2 fatty acids, with aggression toward 18:3 and attraction for 18:2. This work demonstrates the role of lipids in both the transfer of energy and as an interkingdom communication tool in leaf-cutter ant fungal gardens.IMPORTANCE In this work, we examined the role of lipids in the mutualism between leaf-cutter ants and fungus. These ants cut fresh leaf material, which they provide to their fungal cultivar, that converts energy and nutrients from the plants and provides it to the ants in specialized hyphal swellings called gongylidia. This work constitutes the first example of a global lipidomics study of a symbiotic system and provides insights as to how the fungus modifies plant lipids into a usable source for the ants. Through a behavioral experiment, this work also demonstrates how lipids can be used as an interkingdom communication tool, in this case, as an attractant rather than as a repellant, which is more often seen.

Insecticidal and Cholinesterase Activity of Dichloromethane Extracts of Tithonia diversifolia on Atta cephalotes Worker Ants (Formicidae: Myrmicinae).

Leaf-cutter ants are agricultural and urban pests that defy chemical control methods. Laboratory and field studies have revealed repellent and insecticidal activity by the extracts of Tithonia diversifolia (Asteraceae), known as Mexican sunflower, as a promising alternative for the control of the leaf-cutter ant Atta cephalotes. This study evaluated the effects of different extracts (non-polar and polar) of T. diversifolia dry leaves on worker ants from laboratory colonies of A. cephalotes through ingestion and contact. In addition, the biological activity of the extracts as inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) was evaluated. A dichloromethane extract at 1000 ppm presented the highest insecticidal activity through ingestion, causing 70% and 90% worker ant mortality after five and seven days of treatment, respectively. The acetylcholinesterase inhibition values showed that the dichloromethane presented the best AChE concentration of inhibition (IC50) at 73.9 ± 11.06 µg/mL, compared to its fractions, which demonstrates that its activity is potentiated when the crude extract is used. Our results can be attributed to the existence of terpenes and sesquiterpene lactones, which are likely inhibitors of AChE, in T. diversifolia.

Do Workers from Subspecies Acromyrmex subterraneus Prepare Leaves and Toxic Baits in Similar Ways for Their Fungus Garden?

Toxic baits are the most efficient method to control leaf-cutter ants in eucalyptus forests for paper and cellulose production. For the proper use of these baits, insecticide compounds must reach workers and contaminate them. Thus, understanding how these baits are processed inside the nests is vital for a successful control, especially when it comes to genus Acromyrmex. Lack of information on toxic baits and on contamination of Acromyrmex workers raises the question: do workers from subspecies Acromyrmex subterraneus (Forel) prepare leaves and toxic baits in similar ways for their fungus garden? To answer it, this study described and analyzed the behavioral repertoire executed by A. subterraneus workers during the preparation of leaf disks and baits and their incorporation into the fungus garden. Results show that the act of licking the substrate was the most frequently executed behavior, regardless of subspecies or size categories. Moreover, additional behaviors have been observed when workers processed the baits, such as licking and scraping their jaws on the surface of the bait pellet, as well as licking and biting fragments of bait pellets, moistening them. Thus, it is concluded that the preparation of baits is different from that of leaves; baits are more processed and can therefore contribute to contaminating workers via insecticides.

Large ants do not carry their fair share: maximal load-carrying performance of leaf-cutter ants (Atta cephalotes).

Although ants are lauded for their strength, little is known about the limits of their load-carrying abilities. We determined the maximal load-carrying capacity of leaf-cutter ants by incrementally adding mass to the leaves they carried. Maximal load-carrying ability scaled isometrically with body size, indicating that larger ants had the capacity to lift the same proportion of their body mass as smaller ants (8.78 times body mass). However, larger ants were captured carrying leaf fragments that represented a lower proportion of their body mass compared with their smaller counterparts. Therefore, when selecting leaves, larger ants retained a higher proportion of their load-carrying capacity in reserve. This suggests that either larger ants require greater power reserves to overcome challenges they encounter along the trail or leaf-cutter ants do not select loads that maximize the overall leaf transport rate of the colony.

Aflatoxins produced by Aspergillus nomius ASR3, a pathogen isolated from the leaf-cutter ant Atta sexdens rubropilosa

ABSTRACT Aspergillus spp. cause economic impacts due to aflatoxins production. Although the toxicity of aflatoxins is already known, little information about their ecological roles is available. Here we investigated the compounds produced by Aspergillus nomius ASR3 directly from a dead leaf-cutter queen ant Atta sexdens rubropilosa and the fungal axenic culture. Chemical analyses were carried out by high-resolution mass spectrometry and tandem mass spectrometry techniques. Aflatoxins B1 and G1 were detected in both the axenic culture and in the dead leaf-cutter queen ant. The presence of these mycotoxins in the dead leaf-cutter queen ant suggests that these compounds can be related to the insect pathogenicity of A. nomius against A. sexdens rubropilosa.

Pathogenic nature of Syncephalastrum in Atta sexdens rubropilosa fungus gardens.

BACKGROUND: Leaf-cutter ants are considered to be a major herbivore and agricultural pest in the Neotropics. They are often controlled by environmentally persistent insecticides. Biological control using pathogenic fungi is regarded as an alternative for the management of these insects. Here, we assess whether the filamentous fungus Syncephalastrum sp. is a pathogenic microorganism responsible for a characteristic disease in fungus gardens. We also characterise the damage caused by this fungus by evaluating physiological and behavioural responses of Atta sexdens rubropilosa subcolonies infected with Syncephalastrum sp. RESULTS: Syncephalastrum sp. fulfils Koch's postulates characterising it as a pathogenic microorganism. Ant workers recognise the infection and remove contaminated fragments from the fungus garden. Syncephalastrum sp. infection causes an interruption of foraging activity, an increase in ant mortality, subcolony deterioration and an increase in the amount of waste generated, all resulting in subcolony death. Syncephalastrum sp. also inhibits the ant fungal cultivar in vitro. The pathogenic effect of Syncephalastrum sp. does not depend on host morbidity or stress (e.g. worker mortality caused by an entomopathogenic fungus). CONCLUSION: Syncephalastrum sp. treatment resulted in progressive damage in subcolonies. The interactions among Syncephalastrum sp., fungus garden and ants offer new opportunities in integrated pest management of leaf-cutter ants. © 2016 Society of Chemical Industry.

The fungal cultivar of leaf-cutter ants produces specific enzymes in response to different plant substrates.

Herbivores use symbiotic microbes to help derive energy and nutrients from plant material. Leaf-cutter ants are a paradigmatic example, cultivating their mutualistic fungus Leucoagaricus gongylophorus on plant biomass that workers forage from a diverse collection of plant species. Here, we investigate the metabolic flexibility of the ants' fungal cultivar for utilizing different plant biomass. Using feeding experiments and a novel approach in metaproteomics, we examine the enzymatic response of L. gongylophorus to leaves, flowers, oats or a mixture of all three. Across all treatments, our analysis identified and quantified 1766 different fungal proteins, including 161 putative biomass-degrading enzymes. We found significant differences in the protein profiles in the fungus gardens of subcolonies fed different plant substrates. When provided with leaves or flowers, which contain the majority of their energy as recalcitrant plant polymers, the fungus gardens produced more proteins predicted to break down cellulose: endoglucanase, exoglucanase and ß-glucosidase. Further, the complete metaproteomes for the leaves and flowers treatments were very similar, while the mixed substrate treatment closely resembled the treatment with oats alone. This indicates that when provided a mixture of plant substrates, fungus gardens preferentially break down the simpler, more digestible substrates. This flexible, substrate-specific enzymatic response of the fungal cultivar allows leaf-cutter ants to derive energy from a wide range of substrates, which likely contributes to their ability to be dominant generalist herbivores.

Genetic variability and social structure of colonies in Acromyrmex heyeri and A. striatus (Hymenoptera: Formicidae)

The breeding structure of both colony and population of social insects can be examined by genetic analysis. Colonies of the leaf-cutting ants Acromyrmex heyeri and A. striatus (Myrmicinae, Attini) were thus analyzed for isoenzyme systems MDH, a-GPDH, and AMY to describe genotype variability and social structure. A total of five loci were investigated (three for amylase and one for each other system). Ninety-seven colonies of A. heyeri and 103 of A. striatus were sampled in different localities in Southern Brazil (State of Rio Grande do Sul). The genotypes found show the occurrence of monogyny and polygyny associated or not with polyandry, which indicates that the social organization is colony-specific. The polygyny and polyandry observed are likely to be responsible for the great genotypic diversity of the colonies. The average inbreeding coefficient per colony was higher in A. striatus than in A. heyeri, which may reflect the different patterns of production of sexual individuals and nuptial flight of those two species.

A estrutura de cruzamento de colônias e populações de insetos sociais pode ser observada por análise genética. Assim, colônias de formigas cortadeiras Acromyrmex heyeri e A. striatus (Myrmicinae, Attini) foram analisadas para os sistemas isoenzimáticos MDH, a-GPDH e AMY, a fim de descrever sua variabilidade genotípica e estrutura social. Foram investigados cinco locos (três para amilase e um para cada outro sistema), em 97 colônias de A. heyeri e 103 de A. striatus, amostradas em diversas localidades do Rio Grande do Sul. Os genótipos encontrados indicaram a ocorrência de monoginia e poliginia associadas ou não à poliandria, indicando que a organização social é colônia específica. Tanto a poliginia quanto a poliandria são responsáveis pela grande diversidade genotípica das colônias. O coeficiente de endocruzamento médio por colônia foi mais alto em A. striatus do que em A. heyeri e pode refletir os diferentes padrões de produção dos indivíduos sexuados e de vôo nupcial das duas espécies.

The effects of light on foliar chemistry, growth and susceptibility of seedlings of a canopy tree to an attine ant.

Seedlings of Inga oerstediana Benth. (Mimosaceae) growing in three different light environments (the understory, tree-fall gaps and full sun) were tested for differences in chemistry (nutrients and tannins), wound-induced increases in tannins, growth, and susceptibility to leaf-cutter ants, Atta cephalotes (L.) (Formicidae: Attini). I hypothesized that seedlings of I. oerstediana would contain higher concentrations of tannins when growing in high light conditions and, therefore, would be less susceptible to leaf-cutter ants.Foliar concentrations of condensed tannins were much higher in plants growing in full sun compared to those growing in the understory. The concentrations of condensed tannins did not increase following damage. Despite higher concentrations of condensed tannins in sun foliage, leaf-cutter ants found these leaves more acceptable. The preference for sun leaves was consistent with higher concentrations of foliar nutrients. I suggest that the magnitude of the increase in condensed tannins was not great enough to override the benefits of increased concentrations of foliar nutrients. Finally, based on these results and those of others, I suggest that foraging by leaf-cutter ants may be an important factor determining patterns of succession in early successional habitats.