Identification and characterization of a recombinant cysteine peptidase (AsCathL) from leaf-cutting ant Atta sexdens Linnaeus, 1758 (Hymenoptera, Formicidae).

Cysteine peptidases are involved in physiological processes of insect development and have been considered as potential targets for the development of insect control strategies. In this study, we obtained a recombinant cysteine cathepsin L (AsCathL) from leaf-cutting ant (Atta sexdens), a species from the order Hymenoptera who causes enormous damage to crops, natural forests and reforested areas. RT-qPCR showed AsCathL expression throughout insect development and in all body parts of the adult insect analysed, suggesting its role as a lysosomal cathepsin. AsCathL encodes a protein of 320 amino acid residues consisting of a pro-peptide and the mature with amino acids sequence over 67% similarity with lysosomal cathepsin L of species from Lepidoptera and Diptera. Phylogenetic tree revealed that AsCathL is very similar to predicted cathepsins found in other ants. Recombinant AsCathL was expressed in insoluble form by Escherichia coli Arctic Express (DE3) RIL, purified under denaturing conditions and refolded. The enzyme showed hydrolytic activity in vitro towards synthetic substrate Z-Phe-Arg-AMC at acidic pH. Synthetic inhibitor E-64 acted against peptidase activity and a study regarding the interaction between E-64 and AsCathL using nuclear magnetic resonance (NMR) revealed that 83.18% from all E-64 molecules are irreversibly bound to AsCathL. In addition, the proteolytic activity of AsCathL was strongly inhibited by recombinant sugarcane cystatins with Ki ranging from 0.6 nM to 2.95 nM. To the best of our knowledge this is the first report characterizing a cysteine peptidase from leaf-cutting ants, which may contribute to future studies of ants' cathepsins.

Host Size Preference of the Leafcutter Ant Parasitoid Eibesfeldtphora tonhascai (Diptera: Phoridae).

Eibesfeldtphora tonhascai Brown (Diptera: Phoridae) is a natural enemy of Atta sexdens Linnaeus and Atta laevigata Smith and is considered a promising candidate for the biological control of these ants. The aim of this study was to verify if E. tonhascai has a preference for specific sizes of A. sexdens foragers and whether this parasitoid demonstrated host species preference when comparing parasitism of A. sexdens and A. laevigata. Worker ants were classified into four different size groups and introduced into an attack arena with one E. tonhascai female. To verify host species preference, both A. sexdens and A. laevigata workers were placed in the attack arena together with one E. tonhascai female. A no-choice test was also performed, when each ant species was offered to a phorid female at different times. Eibesfeldtphora tonhascai performed the highest number of inspection flights, attempted attacks, and actual attacks against A. sexdens workers with a cephalic capsule size of 3-5 mm. When comparing attacks on A. sexdens and A. laevigata workers, they were equally inspected, attacked, and the total parasitism rates were similar. However, the phorid adult emergence rate was significantly higher in the A. sexdens parasitized workers. We conclude that E. tonhascai females express preference for parasitizing larger hosts (cephalic capsule width of 3-5 mm), but do not display a preference for host species when comparing A. sexdens versus A. laevigata, and thus can similarly contribute to both leafcutter ants' natural biological control.

Allogrooming, Self-grooming, and Touching Behavior as a Mechanism to Disperse Insecticides Inside Colonies of a Leaf-Cutting Ant.

Toxic baits, containing the active ingredients sulfluramid or fipronil, are the main methods to control leaf-cutting ants of the genera Atta Fabricius, 1805, and Acromyrmex Mayr, 1865 (Hymenoptera: Formicidae). However, the insecticide dispersion among members of the colony during the control needs further studies. We studied whether the behaviors of allogrooming, self-grooming, and contact among individuals spread the insecticides among those of the colony. The insecticides sulfluramid and fipronil (0.1% and 1.0% (w/w)) were applied topically in groups of workers of Atta sexdens (Linnaeus, 1758), and the social interactions among them with or without insecticide were studied. In addition, toxic baits (sulfluramid or fipronil) were provided to colonies and their behavioral acts were observed. At the end of the experiment, colony mortality, number and mass of dead workers, and mass of wet waste were compared between ant nests receiving baits and ants with topical application. In the topical application, behavioral analysis showed higher interaction between ants in the colonies and touch and allogrooming behaviors as the most frequent in those that received the concentrations of sulfluramid. In the baits, the behavior of licking the pellet and allogrooming was more frequent. Colony mortality was faster for those that received topical application, especially with the insecticide fipronil (0.1%). However, the number and mass of dead workers was similar between topical application and toxic baits. In the toxic baits, the licking behavior of the bait pellets and subsequent allogrooming probably dispersed the insecticides. In the topical application, the route of the insecticide occurred by excessive touches among workers, with subsequent allogrooming. Thus, allogrooming, self-grooming, and touching among workers increased the dispersion of insecticides among members of the ant colonies.

Low Virulence of the Fungi Escovopsis and Escovopsioides to a Leaf-Cutting Ant-Fungus Symbiosis.

Eusocial insects interact with a diversity of parasites that can threaten their survival and reproduction. The amount of harm these parasites cause to their hosts (i.e., their virulence) can be influenced by numerous factors, such as the ecological context in which the parasite and its host are inserted. Leaf-cutting ants (genera Atta, Acromyrmex and Amoimyrmex, Attini: Formicidae) are an example of a eusocial insect whose colonies are constantly threatened by parasites. The fungi Escovopsis and Escovopsioides (Ascomycota: Hypocreales) are considered a highly virulent parasite and an antagonist, respectively, to the leaf-cutting ants' fungal cultivar, Leucoagaricus gongylophorus (Basidiomycota: Agaricales). Since Escovopsis and Escovopsioides are common inhabitants of healthy colonies that can live for years, we expect them to have low levels of virulence. However, this virulence could vary depending on ecological context. We therefore tested two hypotheses: (i) Escovopsis and Escovopsioides are of low virulence to colonies; (ii) virulence increases as colony complexity decreases. For this, we used three levels of complexity: queenright colonies (fungus garden with queen and workers), queenless colonies (fungus garden and workers, without queen) and fungus gardens (without any ants). Each was inoculated with extremely high concentrations of conidia of Escovopsis moelleri, Escovopsioides nivea, the mycoparasitic fungus Trichoderma longibrachiatum or a blank control. We found that these fungi were of low virulence to queenright colonies. The survival of queenless colonies was decreased by E. moelleri and fungus gardens were suppressed by all treatments. Moreover, E. nivea and T. longibrachiatum seemed to be less aggressive than E. moelleri, observed both in vivo and in vitro. The results highlight the importance of each element (queen, workers and fungus garden) in the leaf-cutting ant-fungus symbiosis. Most importantly, we showed that Escovopsis may not be virulent to healthy colonies, despite commonly being described as such, with the reported virulence of Escovopsis being due to poor colony conditions in the field or in laboratory experiments.

Contamination routes and mortality of the leaf-cutting ant Atta sexdens (Hymenoptera: Formicidae) by the insecticides fipronil and sulfluramid through social interactions.

BACKGROUND: Leaf-cutting ants (LCAs) of the genera Atta and Acromyrmex (Hymenoptera: Formicidae) are important pests of forest plantations, agriculture and livestock. Toxic baits containing the active ingredients fipronil or sulfluramid are the main method used to control LCAs. Insecticide dispersion among members of an LCA colony during control with toxic bait is not well understood. The objective of the study was to determine whether self-grooming, allogrooming or touching behavior among Atta sexdens (Hymenoptera: Formicidae) workers disperses the insecticides fipronil and sulfluramid among members of the colony. The insecticides were topically applied on groups of A. sexdens workers and social interactions between ants with and without insecticide, and group mortality were evaluated. RESULTS: Behavioral analysis showed an increase in interaction among LCA workers as the numbers of individuals increased, with touches between workers being the most frequent behavior. The frequency of observed behaviors was higher in groups treated with sulfluramid compared with fipronil. The mortality of groups treated with fipronil was almost twice as high compared with ants treated with sulfluramid. The insecticides are probably dispersed by excessive touching among workers and subsequent self-grooming and allogrooming. CONCLUSION: These behaviors were responsible for the rapid dispersion of insecticides among members of the colony. Corroboration of the hypothesis that social interactions contaminate nestmates is a model for future studies on contamination of ant workers with active insecticide ingredients. © 2021 Society of Chemical Industry.

Growth of Leucoagaricus gongylophorus Möller (Singer) and production of key enzymes in submerged and solid-state cultures with lignocellulosic substrates.

The aim of this study was to characterize the growth of the fungus Leucoagaricus gongylophorus LEU18496, isolated from the fungus garden of the nest of leaf cutter ants Atta mexicana. The fungus garden was cultivated in an artificial laboratory nest and the fungus further grown in submerged (SmC) and solid state (SSC) cultures with sugarcane bagasse, grass or model substrates containing CM-cellulose, xylan or lignin. The CO2 production rate with grass in SmC (Vmax 34.76 mg CO2 Lgas-1 day- 1) was almost four times than SSC (Vmax 9.49 mg CO2 Lgas-1 day- 1), while the production rate obtained in sugarcane bagasse in SmC (Vmax 16.02 mg CO2 Lgas-1 day- 1) was almost three times than that for SSC (Vmax 5.42 mg CO2 Lgas-1 day- 1). In addition, the fungus grew with defined carbon substrates mixtures in SmC, but at different rates, first xylan, followed by CM-cellulose and lignin. Endoglucanase and xylanase activities (U mgprotein-1) were detected in all cultures, the specific activity was higher in the fungus-garden, 5.2 and 1.8; followed by SSC-grass, 1.5 and 0.8, and SSC-bagasse, 0.9 and 0.8, respectively. Laccase activity in the fungus-garden was 44.8 U L- 1 and 10.9 U L- 1 in the SSC-grass. The gongylidia structures observed by environmental scanning electron microscopy were ca. 40 µm and the hyphae width ca. 5 µm. The results show that L. gongylophorus from A. mexicana have promising applications for the treatment of plant residues to release fermentable sugars and the production of high value lignocellulolytic enzymes such as endoglucanase, xylanase or laccases.

Mortality of leaf-cutting ants with salicylic acid / Mortalidade de formigas-cortadeiras com ácido salicílico

Strategies for the control of leaf-cutting ants have mainly involved granular baits based on fipronil and sulfluramid as active ingredients, which are commonly coated with attractive citrus-based substances. Their constant use and the lack of alternatives in the market may favor the perception of these substances by ants, causing rejection for foraging and consequent difficulty in their control. In this respect, this study examined the mortality of leaf-cutting ants of the genera Atta and Acromyrmex subjected to direct application with dry powders, in laboratory conditions. As a preliminary treatment, a commercial antiseptic talc powder (C. A. P. T.) was used, followed by isolated treatments that corresponded to its components with potential insecticidal action, namely, salicylic acid, sulfur, boric acid, zinc oxide, in addition to an inert talc powder (Quimidrol®) as the control. For each treatment, 40 (worker) ants, whose activity was reduced due to remaining in a refrigerator prior to the treatment, were placed in transparent crystal polystyrene ('Gerbox' type) and were sprinkled with a salt shaker. The ants were kept at 25 ± 2 °C, under a 12-h photophase, and cumulative mortality was recorded every 24 h, considering dead ants as those that were unable to maintain the natural position of their body, i.e., even dying ants were considered dead when they exhibited no reaction when touched by a paintbrush. When sprinkled on ants, C. A. P. T. causes 40% mortality in both species 24 h after application. Subsequent studies of the components of this commercial product have found that when sprinkled on both Atta sexdens rubropilosa and Acromyrmex crassispinus, salicylic acid causes 100% mortality of workers in the first 24 h. This result provides a new prospect of control through a low environmental-impact product, representing an alternative for control in nests in the field that can contribute to the integrated control of ants.(AU)

As estratégias de controle de formigas-cortadeiras têm sido realizadas principalmente com iscas granuladas, à base dos princípios ativos fipronil e sulfluramida, que são comumente revestidas por substâncias atrativas cítricas. O uso constante das mesmas e a falta de alternativas no mercado podem favorecer a percepção de tais substâncias pelas formigas, causando rejeição para forrageamento e consequente dificuldade do seu controle. Neste sentido, o objetivo do trabalho foi avaliar a mortalidade de formigas-cortadeiras dos gêneros Atta e Acromyrmex, submetidas a aplicação direta com pós secos, em laboratório. Como tratamento preliminar, utilizou-se: um talco antisséptico comercial e, posteriormente seus componentes, com potencial ação inseticida, isoladamente, sendo: ácido salicílico, enxofre, ácido bórico, óxido de zinco, além de um talco inerte (Quimidrol®), como testemunha. Para cada tratamento foram utilizadas 40 formigas (operárias) acondicionadas em poliestireno cristal transparente (Tipo Gerbox), que foram polvilhadas com auxílio de um saleiro, estando com atividade reduzida, devido a permanência em geladeira, previamente ao tratamento. As formigas foram mantidas em 25 ± 2°C, fotofase de 12 horas e a mortalidade acumulada foi observada a cada 24 horas, considerando-se formigas mortas aquelas que não conseguiam manter a posição natural do corpo, ou seja, mesmo formigas moribundas foram consideradas mortas, quando não exibiam reação, ao serem tocadas por um pincel. O talco antisséptico comercial, quando polvilhado sobre as formigas, causa mortalidade de 40%, em ambas as espécies, 24 horas após as aplicações. Estudos subsequentes dos componentes do referido produto comercial detectaram que o ácido salicílico, quando polvilhado tanto para Atta sexdens rubropilosa quanto para Acromyrmex crassispinus, causa mortalidade de 100% das operárias nas primeiras 24 horas. Esse resultado abre uma nova perspectiva de controle, com produto de baixo impacto ambiental, sinalizando uma alternativa de controle em ninhos a campo, podendo contribuir para o controle integrado de formigas.(AU)

Dillapiole in Piper holtonii as an Inhibitor of the Symbiotic Fungus Leucoagaricus gongylophorus of Leaf-Cutting Ants.

Plants of the Piperaceae family are studied for their diverse secondary metabolism with a vast array of compounds that act as chemical defense agents against herbivores. Of all the agricultural pests, the management of insects is a highly significant challenge in the Neotropics, and ants of the Attini tribe pose a major problem. Due to their symbiotic association with the fungus Leucoagaricus gongylophorus (Möller) Singer (Agaricaceae), the species of Atta and Acromyrmex have exhaustive foraging activity which has intensified as deforestation and monoculture farming have increased. The control of leaf-cutting ants is still carried out with synthetic products with negative consequences to the environment and human health. In search for natural and sustainable alternatives to synthetic pesticides, Piper holtonii C. DC. was selected among other plant species after field observations of the foraging activity of Atta cephalotes, which revealed that P. holtonii was never chosen by ants. In vitro evaluation of an ethanol extract of the leaves of P. holtonii resulted in promising inhibitory activity (IC50 102 ppm) against L. gongylophorus. Subsequently, bioassay-guided fractionation led to the isolation of the phenylpropanoid dillapiole, which was also detected in the essential oil. This compound demonstrated inhibition of the fungus with an IC50 of 38 ppm. Considering the symbiotic relationship between the Attini ants and L. gongylophorus, the negative effect on the survival of one of the organisms will affect the survival of the other, so dillapiole or standardized essential oil extracts of P. holtonii containing this active principle could be a unique and useful source as a control agent for leaf cutting-ants.

Artifacts Caused by Leaf-Cutting Ants of the Genus Atta (Hymenoptera: Formicidae): Postmortem Bite Injuries and the Tearing of Clothes.

Ants are one of the first insects to find an exposed cadaver and can be present during all stages of decomposition. Although these organisms are not commonly used in postmortem interval estimates, they are to be taken into account on criminal investigations involving human corpses, since they can leave bite marks that can be mistaken for antemortem or perimortem injuries, which could be misleading when ascertaining the occurrence of abuse or physical altercation during a crime. A few studies report the action of ants on human cadavers and even though leaf-cutting ants of the genus Atta are frequently encountered in succession studies that use animal carcasses, there are no records of these fungus-growing species on human corpses. Atta is a genus restricted to the New World, ranging from northern Argentina to southern United States and acts as one of the most conspicuous neotropical herbivores. In this study, we report three cases of violent death that illustrate the impact of ants, especially those of the genus Atta, in a forensic setting. We compare the patterns displayed by postmortem bite injuries caused by leaf-cutter ants and other common species with less robust mandibles. We also present the capability of Atta ants to create artifacts by cutting victim's clothes in a crime scene, contributing to the knowledge of ant-mediated confounding factors in crime scene investigation.

Diel pattern driven by free convection controls leaf-cutter ant nest ventilation and greenhouse gas emissions in a Neotropical rain forest.

Leaf-cutter ant nests are biogeochemical hot spots where ants live and import vegetation to grow fungus. Metabolic activity and (in wet tropical forests) soil gas flux to the nest may result in high nest CO2 concentrations if not adequately ventilated. Wind-driven ventilation mitigates high CO2 concentrations in grasslands, but little is known about exchange for forest species faced with prolonged windless conditions. We studied Atta cephalotes nests located under dense canopy (leaf area index > 5) in a wet tropical rainforest in Costa Rica, where wind events are infrequent. We instrumented nests with thermocouples and flow-through CO2 sensing chambers. The results showed that CO2 concentrations exiting leaf-cutter ant nests follow a diel pattern with higher values at night. We developed an efflux model based on pressure differences that evaluated the observed CO2 diel pattern in terms of ventilation by (1) free convection (warm, less dense air rises out the nest more prominently at night) and (2) episodic wind-forced convection events providing occasional supplemental ventilation during daytime. Average greenhouse gas emissions were estimated through nest vents at about 78 kg CO2eq nest-1 year-1. At the ecosystem level, leaf-cutter ant nest vents accounted for 0.2% to 1% of total rainforest soil emissions. In wet, clayey tropical soils, leaf-cutter ant nests act as free convection-driven conduits for exporting CO2 and other greenhouse gases produced within the nest (fungus and ant respiration, refuse decay), and by roots and soil microbes surrounding the nest. This allows A. cephalotes nests to be ventilated without reliable wind conditions.

Disentangling the effects of foliar vs. floral herbivory of leaf-cutting ants on the plant reproductive success of Miconia nervosa (Smith) Triana (Family Melastomataceae).

Flower and leaf herbivory might cause relevant and negative impacts on plant fitness. While flower removal or damage by florivores produces direct negative effects on plant fitness, folivores affect plant fitness by reducing resource allocation to reproduction. In this study, we examine the effects of both flower and leaf herbivory by leaf-cutting ants on the reproductive success of the shrub species Miconia nervosa (Smith) Triana (Family Melastomataceae) in a fragment of Atlantic Forest in Northeast Brazil. We conducted a randomized block-designed field experiment with nine replicates (blocks), in which three plants per block were assigned to one of the three following treatments: undamaged plants (ant exclusion), leaf-damaged plants (ant exclusion from reproductive organs, but not from leaves), and flower + leaf-damaged plants (no exclusion of ants). We then measured flower production, fruit set, and fruit production. Our results showed that flower + leaf-damaged plants reduced flower production nearly twofold in relation to undamaged plants, while flower set in leaf-damaged plants remained constant. The number of flowers that turned into fruits (i.e., fruit set), however, increased by 15% in flower + leaf-damaged plants, while it slightly decreased in leaf-damaged compared to undamaged plants. Contrastingly, fruit production was similar between all treatments. Taken together, our results suggest a prominent role of ant floral herbivory across different stages of the reproductive cycle in M. nervosa, with no consequences on final fruit production. The tolerance of M. nervosa to leaf-cutting ant herbivory might explain its high abundance in human-modified landscapes where leaf-cutting ants are hyper-abundant.

Fat-Soluble Substance Flow During Symbiotic Fungus Cultivation by Leaf-Cutter Ants.

Leaf-cutter ants perform a series of specialized behaviors in preparing plant substrates for their symbiotic fungus. This process may be related to contamination of workers by substances such as insecticides, leading us to hypothesize that substances are spread among workers through behaviors they perform to grow the fungus. To test this hypothesis, we analyzed the behavioral acts of workers during the processing of the pellets by using a fat-soluble tracing dye, since the active ingredient that composes toxic baits, used for control of leaf-cutter ants, is fat-soluble. The frequencies of performed behaviors were recorded and the number of dyed workers was assessed after fungus cultivation. The most frequent behavior is allogrooming and corresponds to 45.87% of the contamination process in workers, followed by holding, licking, and cutting pellets, which account for 40.22% of the process. After pellet processing, the workers had their external and internal morphological structures marked by the tracing dye-93.75% and 79.25%, respectively. These results confirm that behaviors performed during fungus cultivation contribute to dispersing substances such as insecticides, causing the contamination of workers.

Nest Architecture Development of Grass-Cutting Ants, Atta capiguara (Hymenoptera: Formicidae)

ABSTRACT Atta capiguara grass-cutting ants are commonly found in the Cerrado biome, in open fields. Although grass-cutting ants build giant nests, little has been elucidated about this building pattern and when chambers and tunnels emerge. The present study describes the nest architecture development of A. capiguara grass-cutting ants from data on 31 cement-molded nests. A. capiguara nests grow with increases in the number of fungus chambers and emergence and increase of waste chambers and foraging tunnels. The structural growth of A. capiguara nests in the first year and a half of age (18 months) is vertical, with the building of the first chambers in the soil profile. After 18 months, the nests grow sideways with the addition of chambers and tunnels, and the first waste chambers appear. Between 18 and 54 months, the number of fungus chambers increases from 1-3 to 21-32, and the chambers are concentrated at the soil surface, although they can be found more than 3 m deep. In addition, the total volume of the waste chambers increases with the increment in the fungus chambers volume. Thus, this study contributes to understanding the nest architecture development of A. capiguara grass-cutting ants and demonstrates that the total volume of waste chambers is proportional to the total volume of fungus chambers suitable for the colony.

Caracterización genética de la hormiga Atta cephalotes (Hymenoptera: Formicidae) en Isla Gorgona, Colombia

La variación genética entre las poblaciones insulares y continentales de las hormigas cortadoras de hojas ha sido poco estudiada, a pesar de su importancia para comprender su capacidad de dispersión, estructura regional y distribución de una de las especies más abundantes en el Neotrópico. Objetivo: Analizar la variación genética de una población insular de Atta cephalotes en un contexto regional más amplio para América Central y del Sur. Métodos: Se secuenció el gen de la Citocromo oxidasa I de A. cephalotes para las poblaciones de la isla Gorgona y el continente para realizar un análisis de la varianza molecular, flujo de genes, así como para calcular el tiempo de divergencia de los haplogrupos que se encuentran en la isla. Se realizó una prueba de asignación de individuos para estimar la conectividad genética espacial y la distribución de esta hormiga en el Pacífico Oriental Tropical. Resultados: Un total de 33 muestras de la isla y el continente colombiano se compararon con 389 secuencias de A. cephalotes registradas en Genbank para América Central y Suramérica. Esta hormiga cortadora de hojas en la Isla Gorgona está estrechamente relacionada con el continente colombiano a pesar de que la separación entre la isla y el continente probablemente constituya una barrera para el flujo de genes. Es probable que dos de los haplogrupos más comunes en el continente llegaran a la isla recientemente. Por lo tanto, no se puede excluir la influencia antropogénica en el proceso de colonización de la isla por parte de esta hormiga cortadora de hojas. Un mapa de conectividad muestra que esta población tiene límites geográficos que se superponen en gran medida con una región biogeográfica dentro del Pacífico Oriental Tropical. Esta población trans-andina acumula el 23 % de la diversidad de herencia matrilínea de A. cephalotes. Conclusiones: Esta hormiga cortadora de hojas en la isla Gorgona y el Pacífico colombiano está estrechamente relacionada con la población centroamericana y toda esta región alberga un segmento significativo de la diversidad genética de A. cephalotes de Suramérica.

Introduction: The genetic variation between island and mainland populations of leaf-cutter ants have been poorly studied, despite its importance to understand their local dispersion, regional structure, and the distribution of one of the most abundant species in the neotropics. Objective: To analyze the genetic variation of an insular population of Atta cephalotes in a larger regional context in Central and South America. Methods: The Cytochrome Oxidase I gene of A. cephalotes was sequenced for the Gorgona Island and mainland populations in order to conduct an analysis of molecular variance, gene flow, and to calculate divergence time of haplogroups occurring in the island. An individual assignment test was conducted to estimate the spatial genetic connectivity and the distribution for this ant in the Eastern Tropical Pacific. Results: A total of 33 samples from the island and the Colombian mainland were compared to 389 Genebank accessions ofA. cephalotes from Central and South America. This leaf-cutter ant in the Gorgona Island is closely related to the Colombian mainland, despite that the separation between the island and the mainland likely constitute a barrier to gene flow. It is likely that two of the most common haplogroups on the continent arrived on the island recently. Therefore, the anthropogenic influence in the process of colonization of the island by this leaf-cutting ant cannot be excluded. A map of connectivity shows that this population has geographic limits that largely overlap with a biogeographic region within the Eastern Tropical Pacific. This trans-andean population accumulates 23 % of the maternally inherited diversity of theA. cephalotes. Conclusions: This leaf-cutter ant in the Gorgona Island and the Colombian Pacific is closely related to the Central American population and the whole region harbor a significant segment of A. cephalotes genetic diversity in South America.

Chemical control of leaf-cutting ants: how do workers disperse toxic bait fragments onto fungus garden?

ABSTRACT Leaf-cutting ants are controlled with toxic baits. For the method's greater efficiency, the baits must be distributed and processed by workers during fungus cultivation. To test hypotheses whether the mode of action of the active ingredients, which blocks the sodium channels and interrupts the production of ATP in the mitochondria, interferes with fragment distribution in nests; the dispersion of pellets fragments during fungus cultivation by workers was assessed, spatially referencing the fragment onto the fungus garden. Pellets fragments were randomly distributed, but the amount pellets fragments was influenced by the way that the active ingredient acts in the colony.

Leaf-cutter ants engineer large nitrous oxide hot spots in tropical forests.

Though tropical forest ecosystems are among the largest natural sources of the potent greenhouse gas nitrous oxide (N2O), the spatial distribution of emissions across landscapes is often poorly resolved. Leaf cutter ants (LCA; Atta and Acromyrmex, Myrmicinae) are dominant herbivores throughout Central and South America, and influence multiple aspects of forest structure and function. In particular, their foraging creates spatial heterogeneity by concentrating large quantities of organic matter (including nitrogen, N) from the surrounding canopy into their colonies, and ultimately into colony refuse dumps. Here, we demonstrate that refuse piles created by LCA species Atta colombica in tropical rainforests of Costa Rica provide ideal conditions for extremely high rates of N2O production (high microbial biomass, potential denitrification enzyme activity, N content and anoxia) and may represent an unappreciated source of heterogeneity in tropical forest N2O emissions. Average instantaneous refuse pile N2O fluxes surpassed background emissions by more than three orders of magnitude (in some cases exceeding 80 000 µg N2O-N m-2 h-1) and generating fluxes comparable to or greater than those produced by engineered systems such as wastewater treatment tanks. Refuse-concentrating Atta species are ubiquitous in tropical forests, pastures and production ecosystems, and increase density strongly in response to disturbance. As such, LCA colonies may represent an unrecognized greenhouse gas point source throughout the Neotropics.

Cryptic Diversity in Colombian Edible Leaf-Cutting Ants (Hymenoptera: Formicidae).

Leaf-cutting ants are often considered agricultural pests, but they can also benefit local people and serve important roles in ecosystems. Throughout their distribution, winged reproductive queens of leaf-cutting ants in the genus Atta Fabricius, 1804 are consumed as a protein-rich food source and sometimes used for medical purposes. Little is known, however, about the species identity of collected ants and the accuracy of identification when ants are sold, ambiguities that may impact the conservation status of Atta species as well as the nutritional value that they provide to consumers. Here, 21 samples of fried ants bought in San Gil, Colombia, were identified to species level using Cytochrome Oxidase I (COI) barcoding sequences. DNA was extracted from these fried samples using standard Chelex extraction methods, followed by phylogenetic analyses with an additional 52 new sequences from wild ant colonies collected in Panama and 251 publicly available sequences. Most analysed samples corresponded to Atta laevigata (Smith, 1858), even though one sample was identified as Atta colombica Guérin-Méneville, 1844 and another one formed a distinct branch on its own, more closely related to Atta texana (Buckley, 1860) and Atta mexicana (Smith, 1858). Analyses further confirm paraphyly within Atta sexdens (Linnaeus, 1758) and A. laevigata clades. Further research is needed to assess the nutritional value of the different species.

Do an ecosystem engineer and environmental gradient act independently or in concert to shape juvenile plant communities? Tests with the leaf-cutter ant Atta laevigata in a Neotropical savanna.

BACKGROUND: Ecosystem engineers are species that transform habitats in ways that influence other species.While the impacts of many engineers have been well described, our understanding of how their impact varies along environmental gradients remains limited. Although disentangling the effects of gradients and engineers on biodiversity is complicated-the gradients themselves can be altered by engineers-doing so is necessary to advance conceptual and mathematical models of ecosystem engineering. We used leaf-cutter ants (Atta spp.) to investigate the relative influence of gradients and environmental engineers on the abundance and species richness of woody plants. METHODS: We conducted our research in South America's Cerrado. With a survey of plant recruits along a canopy cover gradient, and data on environmental conditions that influence plant recruitment, we fit statistical models that addressed the following questions: (1) Does A. laevigata modify the gradient in canopy cover found in our Cerrado site? (2) Do environmental conditions that influence woody plant establishment in the Cerrado vary with canopy cover or proximity to A. laevigata nests? (3) Do A. laevigata and canopy cover act independently or in concert to influence recruit abundance and species richness? RESULTS: We found that environmental conditions previously shown to influence plant establishment in the Cerrado varied in concert with canopy cover, but that ants are not modifying the cover gradient or cover over nests. However, ants are modifying other local environmental conditions, and the magnitude and spatial extent of these changes are consistent across the gradient. In contrast to prior studies, we found that ant-related factors (e.g., proximity to nests, ant changes in surface conditions), rather than canopy cover, had the strongest effect on the abundance of plant recruits. However, the diversity of plants was influenced by both the engineer and the canopy cover gradient. DISCUSSION: Atta laevigata in the Cerrado modify local conditions in ways that have strong but spatially restricted consequences for plant communities. We hypothesize that ants indirectly reduce seedling establishment by clearing litter and reducing soil moisture, which leads to seed and seedling desiccation. Altering soil nutrients could also reduce juvenile growth and survivorship; if so these indirect negative effects of engineering could exacerbate their direct effects of harvesting plants. The effects of Atta appear restricted to nest mounds, but they could be long-lasting because mounds persist long after a colony has died or migrated. Our results support the hypothesis that leaf-cutter ants play a dominant role in Cerrado plant demography. We suggest the ecological and economic footprint of these engineers may increase dramatically in coming decades due to the transformation of the Cerrado by human activities.

Lack of correlation between micro fungi species and chemical control method of Atta treated with toxic baits / Ausência de correlação entre espécies de microfungos e método de controle químico de Atta tratadas com iscas tóxicas

Atta sexdens rubropilosa (leaf-cutter ants) has a symbiotic association with a fungus and has a negative interaction with other fungi due to parasitism of the fungus cultivated by ants; also, there are several other fungi with no exact known role occurring in their cultivated fungus garden. In the present study, we use the ITS region (internal transcribed spacer) to identify fungi in colonies treated with toxic baits. Experiments using two toxic baits were carried out: 0.75g of sulfluramid [0.3%] and 0.75g fipronil [0.003%]. Samples of fungi were collected and cultured in Czapek medium for seven days to allow fungal growth and subsequent identification. Total DNA was isolated from 100-150 mg of mycelium using the CTAB method and using PCR, with the universal primers (ITS4 and ITS5), to amplify the ITS region. Sequencing was performed using the Sanger method. Sequences were subjected to BLAST, allowing the identification of nine different species of the orders Agaricales, Eurotiales, Hypocreales, Pleosporales, Saccharomycetales and Tremellales showing a variation in identity of 96-100%. Using "The Automatic Barcode Gap Discovery" analysis, nine groups were identified, corresponding to species described in NCBI. The K2P distances were used to generate a tree using Neighbour-joining, demonstrating that the species were grouped according to phylogenetic groups. We concluded that leaf-cutter ant colonies exhibited a wide variety of fungi and this study suggested that there is no correlation between the species of fungi isolated with the control method used on the ant nest.(AU)

Atta sexdens rubropilosa (cortadeira de folha) possui associação simbiótica com fungos e interação negativa com outros fungos devido ao parasitismo do fungo cultivado pelas formigas. Quando colônias da formiga cortadeira de folhas são submetidas ao tratamento com iscas tóxicas, diversas espécies de fungos surgem dentro da colônia, podendo contribuir com a morte ou sobrevivência da colônia. Para entender os relacionamentos ecológicos em colônias de formigas, a identificação de espécie de fungos se torna muito importante e, o uso de DNA barcoding tem sido um método rápido e eficiente para identificação de espécies usando métodos moleculares. No presente trabalho, usamos a região ITS (internal transcribed spacer) para identificar fungos em colônias tratadas com iscas tóxicas. Dois experimentos com iscas tóxicas foram aplicados: 0.75g de Fipronil [0.003%] e 0.75g de Sulfluramid [0.3%]. As amostras, contendo os possíveis fungos, foram coletadas e cultivadas em meio Czaped durante sete dias para o crescimento do fungo e posterior identificação. O DNA total foi isolado de 100-150mg de micélio usando o método CTAB, usado para amplificar a região ITS por PCR empregando primers universais (ITS5 e ITS4). O sequenciamento foi realizado utilizando o método de Sanger. As sequências foram submetidas ao BLAST, permitindo identificar nove diferentes espécies das ordens Agaricales, Eurotiales, Hypocreales, Pleosporales, Saccharomycetales e Tremellales, mostrando variação 96-100% de identidade. Empregando a análise "The Automatic Barcode Gap Discovery", identificou-se nove grupos, correspondendo as espécies descritas no NCBI. As distâncias K2P foram usadas para gerar uma árvore usando Neighbour-Joining, apresentando que as espécies foram agrupadas de acordo com as filogenias dos grupos. Conclui-se que as colônias de formigas cortadeira de folhas apresentam grande diversidade de fungos e que DNA barcoding é eficiente para identificação destes.(AU)

Lack of correlation between micro fungi species and chemical control method of Atta treated with toxic baits / Ausência de correlação entre espécies de microfungos e método de controle químico de Atta tratadas com iscas tóxicas

ABSTRACT: Atta sexdens rubropilosa (leaf-cutter ants) has a symbiotic association with a fungus and has a negative interaction with other fungi due to parasitism of the fungus cultivated by ants; also, there are several other fungi with no exact known role occurring in their cultivated fungus garden. In the present study, we use the ITS region (internal transcribed spacer) to identify fungi in colonies treated with toxic baits. Experiments using two toxic baits were carried out: 0.75g of sulfluramid [0.3%] and 0.75g fipronil [0.003%]. Samples of fungi were collected and cultured in Czapek medium for seven days to allow fungal growth and subsequent identification. Total DNA was isolated from 100-150 mg of mycelium using the CTAB method and using PCR, with the universal primers (ITS4 and ITS5), to amplify the ITS region. Sequencing was performed using the Sanger method. Sequences were subjected to BLAST, allowing the identification of nine different species of the orders Agaricales, Eurotiales, Hypocreales, Pleosporales, Saccharomycetales and Tremellales showing a variation in identity of 96-100%. Using "The Automatic Barcode Gap Discovery" analysis, nine groups were identified, corresponding to species described in NCBI. The K2P distances were used to generate a tree using Neighbour-joining, demonstrating that the species were grouped according to phylogenetic groups. We concluded that leaf-cutter ant colonies exhibited a wide variety of fungi and this study suggested that there is no correlation between the species of fungi isolated with the control method used on the ant nest.

RESUMO: Atta sexdens rubropilosa (cortadeira de folha) possui associação simbiótica com fungos e interação negativa com outros fungos devido ao parasitismo do fungo cultivado pelas formigas. Quando colônias da formiga cortadeira de folhas são submetidas ao tratamento com iscas tóxicas, diversas espécies de fungos surgem dentro da colônia, podendo contribuir com a morte ou sobrevivência da colônia. Para entender os relacionamentos ecológicos em colônias de formigas, a identificação de espécie de fungos se torna muito importante e, o uso de DNA barcoding tem sido um método rápido e eficiente para identificação de espécies usando métodos moleculares. No presente trabalho, usamos a região ITS (internal transcribed spacer) para identificar fungos em colônias tratadas com iscas tóxicas. Dois experimentos com iscas tóxicas foram aplicados: 0.75g de Fipronil [0.003%] e 0.75g de Sulfluramid [0.3%]. As amostras, contendo os possíveis fungos, foram coletadas e cultivadas em meio Czaped durante sete dias para o crescimento do fungo e posterior identificação. O DNA total foi isolado de 100-150mg de micélio usando o método CTAB, usado para amplificar a região ITS por PCR empregando primers universais (ITS5 e ITS4). O sequenciamento foi realizado utilizando o método de Sanger. As sequências foram submetidas ao BLAST, permitindo identificar nove diferentes espécies das ordens Agaricales, Eurotiales, Hypocreales, Pleosporales, Saccharomycetales e Tremellales, mostrando variação 96-100% de identidade. Empregando a análise "The Automatic Barcode Gap Discovery", identificou-se nove grupos, correspondendo as espécies descritas no NCBI. As distâncias K2P foram usadas para gerar uma árvore usando Neighbour-Joining, apresentando que as espécies foram agrupadas de acordo com as filogenias dos grupos. Conclui-se que as colônias de formigas cortadeira de folhas apresentam grande diversidade de fungos e que DNA barcoding é eficiente para identificação destes.