Leaf-cutting Ant Atta Sexdens Research Articles

Influence of the leaf-cutting ant Atta sexdens Linnaeus (1758) on the taxonomic and functional diversity of woody plants in Atlantic Forest fragments

Influence of the leaf-cutting ant Atta sexdens Linnaeus (1758) on the taxonomic and functional diversity of woody plants in Atlantic Forest fragments

Activity of a Recombinant Chitinase of the Atta sexdens Ant on Different Forms of Chitin and Its Fungicidal Effect against Lasiodiplodia theobromae.

This study evaluates the activity of a recombinant chitinase from the leaf-cutting ant Atta sexdens (AsChtII-C4B1) against colloidal and solid α- and β-chitin substrates. 1H NMR analyses of the reaction media showed the formation of N-acetylglucosamine (GlcNAc) as the hydrolysis product. Viscometry analyses revealed a reduction in the viscosity of chitin solutions, indicating that the enzyme decreases their molecular masses. Both solid state 13C NMR and XRD analyses showed minor differences in chitin crystallinity pre- and post-reaction, indicative of partial hydrolysis under the studied conditions, resulting in the formation of GlcNAc and a reduction in molecular mass. However, the enzyme was unable to completely degrade the chitin samples, as they retained most of their solid-state structure. It was also observed that the enzyme acts progressively and with a greater activity on α-chitin than on β-chitin. AsChtII-C4B1 significantly changed the hyphae of the phytopathogenic fungus Lasiodiplodia theobromae, hindering its growth in both solid and liquid media and reducing its dry biomass by approximately 61%. The results demonstrate that AsChtII-C4B1 could be applied as an agent for the bioproduction of chitin derivatives and as a potential antifungal agent.

Hemocyte morphology of worker subcastes of the leaf-cutting ant Atta sexdens rubropilosa (Hymenoptera: Formicidae)

Hemocytes are cells present in the hemolymph of insects that play a role in combating invasive pathogens, ensuring defense by the immune system in these organisms. While the types of hemocytes are well known in some insect representatives, data on these cells in Hymenoptera are limited to certain bees and wasps, with little information available for ants. Among ants, the genus Atta has environmental and economic importance, forming highly organized colonies consisting of the queen and workers, with the latter subdivided into subcastes: gardeners, waste removers, foragers, and soldiers, which are exposed to different pathogens. This study describes the morphology of hemocytes in the worker subcastes of Atta sexdens rubropilosa. Hemolymph samples from the ant were submitted to light, confocal, and scanning electron microscopy analyses. Five types of hemocytes were identified in the hemolymph of all ant subcastes, including prohemocytes, oenocytoids, spherulocytes, plasmatocytes, and granulocytes. They exhibited nuclei with a predominance of decondensed chromatin. The granulocytes were the most abundant cell type in the subcastes, followed by prohemocytes, plasmatocytes, oenocytoids, and spherulocytes. Phagocytosis assays reveal that plasmatocytes and granulocytes are the main phagocytic cells in all castes evaluated. This study fills an important gap in understanding the immune response in this ant species.

Identification, cloning, and characterization of a novel chitinase from leaf-cutting ant Atta sexdens: An enzyme with antifungal and insecticidal activity

Chitinases are enzymes that degrade chitin, a polysaccharide found in the exoskeleton of insects, fungi, yeast, and internal structures of other vertebrates. Although chitinases isolated from bacteria, fungi and plants have been reported to have antifungal or insecticide activities, chitinases from insects with these activities have been seldomly reported. In this study, a leaf-cutting ant Atta sexdens DNA fragment containing 1623 base pairs was amplified and cloned into a vector to express the protein (AsChtII-C4B1) in Pichia pastoris. AsChtII-C4B1, which contains one catalytic domain and one carbohydrate-binding module (CBM), was secreted to the extracellular medium and purified by ammonium sulfate precipitation followed by nickel column chromatography. AsChtII-C4B1 showed maximum activity at pH 5.0 and 55 °C when tested against colloidal chitin substrate and maintained >60% of its maximal activity in different temperatures during 48 h. AsChtII-C4B1 decreased the survival of Spodoptera frugiperda larvae fed with an artificial diet that contained AsChtII-C4B1. Our results have indicated that AsChtII-C4B1 has a higher effect on larva-pupa than larva-larva molts. AsChtII-C4B1 activity targets more specifically the growth of filamentous fungus than yeast. This work describes, for the first time, the obtaining a recombinant chitinase from ants and the characterization of its insecticidal and antifungal activities.

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.

Pathogenicity of Beauveria bassiana to Atta sexdens rubropilosa (Hymenoptera: Formicidae) in laboratory conditions

This research, under laboratory condition, evaluated the efficiency of different strains of entomopatogenic fungus Beauveria bassiana to control leaf-cutting ant Atta sexdens rubropilosa. Soldiers and workers were inoculated with suspensions containing 1.0x107, 1.0x108, 1.0x109 and 1.0x1010 con mL-1 and maintained in B.O.D, at 25±1°C, 70% RH and 12 hours of photophase, without food. The mortality was evaluated daily, with 15 days after inoculation. Experimental design was entirely random. The sporulation data were submitted to the analysis of variance and the LT50 values (in days) were obtained through the Probit analysis. All strains of B. bassiana were pathogenic, being workers more susceptible than soldiers. The strains IBCB 21 and IBCB 07 caused high percentages of confirmed mortality showing potential for use in the control of A. sexdens rubropilosa. Strain IBCB 21 was more virulent for workers, whereas isolated IBCB 07 was more virulent for soldiers. Both isolates presented no significant difference among tested concentrations.

Carbon dioxide levels in initial nests of the leaf-cutting ant Atta sexdens (Hymenoptera: Formicidae)

Claustral foundation of nests by Atta sexdens Forel (Hymenoptera: Formicidae) involves great effort by its queens, solely responsible for the cultivation of the fungus and care for her offspring at this stage. The minimum workers, after 4 months, open access to the external environment to foraging plants to cultivate the symbiotic fungus, which decomposes the plant fragments and produces gongilidea nodules as food for the individuals in the colony. Colony gas exchange and decomposition of organic matter in underground ant nests generate carbon dioxide (CO2) emitted into the atmosphere. We described the carbon dioxide concentration in colonies in the field. The objective was to evaluate the carbon dioxide concentration in initial A. sexdens colonies, in the field, and their development. The CO2 level was also measured in 4-month-old colonies in the field, using an open respirometric system fitted with an atmospheric air inlet. The CO2 level of the respirometric container was read by introducing a tube into the nest inlet hole and the air sucked by a peristaltic pump into the CO2 meter box. The CO2 concentration in the initial colony was also measured after 4 months of age, when the offspring production (number of eggs, larvae, pupae and adult workers) stabilized. Ten perforations (15 cm deep) was carried out in the adjacent soil, without a nest of ants nearby, to determine the concentration of CO2. The composition of the nests in the field was evaluated after excavating them using a gardening shovel and they were stored in 250 ml pots with 1 cm of moistened plaster at the bottom. The CO2 concentration was higher in field nest than in adjacent soil. The concentration of carbon dioxide in A. sexdens nests in the field is higher than in those in the soil, due to the production of CO2 by the fungus garden and colony.

Seasonal Parasitism of the Leaf-Cutting Ant Atta sexdens Linnaeus (Hymenoptera: Formicidae) by Phorid Flies (Diptera: Phoridae) in a Brazilian Cerrado-Atlantic Forest Ecotone

Phorid flies (Diptera: Phoridae) parasitize insects of many orders including leaf-cutting ants, which are pests of several crops. These parasitoids may have a significant impact on ant behavior by altering their foraging rhythm. Seasonal variations in climatic variables, such as temperature and humidity, influence the abundance, activity period, distribution, and reproduction of insects. Therefore, knowledge of parasitoid seasonal patterns is important for biological control success. In this study we evaluate the parasitism of the leaf-cutting ant Atta sexdens L. (Hymenoptera: Formicidae) by Apocephalus attophilus Borgmeier and Eibesfeldtphora tonhascai Brown (both Diptera: Phoridae) across seasons throughout a yr in a Brazilian Cerrado-Atlantic Forest ecotone, and compare their parasitism in a semi-deciduous seasonal forest fragment and urban area. The number of ants parasitized by A. attophilus and E. tonhascai was higher in the winter and spring, respectively, but similar in summer and fall. Apocephalus attophilus parasitism was negatively correlated with temperature but not with relative humidity, whereas ant parasitism by E. tonhascai presented no correlation with either parameter. The number of ants parasitized by A. attophilus and E. tonhascai in the semi-deciduous seasonal forest fragment was higher than in urban areas. Knowledge of biotic and abiotic factors influencing the population dynamics of phorid flies is important for rearing and releasing protocols for these parasitoids.

Analysis of the Gene Expression and RNAi-Mediated Knockdown of Chitin Synthase from Leaf-Cutting Ant Atta sexdens

Chitin synthase (CHS) is the enzyme specifically associated with chitin synthesis, an important component of diverse organisms including insects. Two alternative spliced transcripts of the CHS gene (AsCHS-A1 and AsCHS-A2) were identified by quantitative reverse-transcription polymerase chain reaction (RT-qPCR) during the development of the leaf-cutting ant Atta sexdens. Expression profiles of AsCHS-A transcripts increased from larva to pupae and decay in workers. Phylogenetic analysis showed both transcripts are classified within class A insect CHSs. AsCHS-A1 showed the highest expression level in larvae and pupae, while AsCHS-A2 is the main CHS transcript in workers. Our results suggest that these variants should be under regulation of different promoters. AsCHS-A1 has topology expected for insect CHSs, while the predicted AsCHS-A2 topology, with a missing A domain, is similar to some fungal CHSs. CHS-B (class B) was not identified in A. sexdens transcriptome. Ribonucleic acid interference (RNAi)-mediated gene silencing in pupae revealed that low reduction in CHS transcript levels (18%) was enough to cause morphological changes in the pupa exoskeleton impairing the process of cuticle sclerotization. To our knowledge, this work was the first to use and to show the feasibility of using RNA interference techniques on leaf-cutting ants.

INSECTICIDAL AND FUNGICIDAL ACTIVITY OF A MAGNESIUM COMPOUND CONTAINING ISOVANILLIC ACID AGAINST LEAF-CUTTING ANT AND ITS SYMBIOTIC FUNGUS

Pesticides offer a simple, cost-effective solution to ensure food supplies for a growing world population. However, their environmental performance needs improvement if pesticides are to continue contributing importantly. This study explores the insecticidal potential of a magnesium (II) metal complex containing 1,10-phenanthroline and isovanillic acid (MgPhenIso) against leaf-cutting ant (Atta sexdens rubropilosa) and its symbiotic fungus (Leucoagaricus gongylophorus). The MgPhenIso complex is easy-to-prepare, stable in solid state and in aqueous solution, and resistant to sunlight irradiation. The insecticidal activity of MgPhenIso-containing bait against A. sexdens rubropilosa and L. gongylophorus showed a delayed action mode at 2 mg g-1 (100% kill, 6th day, S50=2b) and suppressed fungus development and growth. These results, together with earlier investigations on MgPhenIso regarding its absence of toxicity to Zebrafish and Wistar male rats, emphasize the potential of this complex as an environmentally friendly insecticide candidate.

Bioactivity of Asclepias curassavica, Equisetum spp. and Rosmarinus officinalis Extracts Against Leaf-Cutting Ants

Chemical control of leaf-cutting ants is widely used, but alternative control with toxic plant extracts is promising. Substances with insecticidal potential extracted from plants have numerous ecological advantages. This study evaluated the insecticidal and/or fungicidal potential of the plants Asclepias curassavica (tropical milkweed), Rosmarinus officinalis L. (Lamiaceae) (rosemary) and Equisetum spp. (horsetail) for control of the leaf-cutting ant Atta sexdens rubropilosa Forel, 1908 (Hymenoptera: Formicidae). Forty laboratory-reared colonies of Atta sexdens rubropilosa were used. The plants were collected, dried out in a circulating air oven for 48 hours, ground, and macerated in 96o ethanol until exhaustion. After filtration, the products were evaporated under reduced pressure to obtain the ethanolic extracts. Acceptance of the reagent, topical application of the extracts, and application of baits containing 4% of the plant extracts were tested. The results showed that all plant extracts tested negatively influenced the development of the fungus garden. Baits produced with Asclepias curassavica caused the highest mortality of the colonies within 7 days. In conclusion, the ethanolic extracts of Asclepias curassavica, Rosmarinus officinalis and Equisetum spp. exhibit insecticidal (contact and ingestion) and fungicidal activity in colonies of the leaf-cutting ant Atta sexdens rubropilosa.

Parasitism by Phorids on Leaf Cutter Ants Atta sexdens (Linnaeus, 1758) (Hymenoptera: Formicidae) in Natural and Agricultural Environments.

The leaf cutter ant Atta sexdens (L.) (Hymenoptera:Formicidae) causes huge economic losses to agriculture in the Neotropics. In natural environments, parasitic flies of the Phoridae family are natural enemies of leaf cutter ants. Habitat modification is considered one of the main causes of species decline. In this study, we compare the occurrence of parasitic phorids on A. sexdens and the parasitism that they cause on colonies located in agricultural and natural habitats. Phorid flies were collected from trails, nest entrances, and cutting sites when they were hovering over workers of A. sexdens nests from natural vegetation, farmland, and Eucalyptus plantation areas. Simultaneously, workers from A. sexdens nests were collected from these environments, and the parasitism rates of phorids were determined. Ants were parasitized by Apocephalus attophilus, A. vicosae, Eibesfeldtphora bragancai, E. tonhascai, and Myrmosicarius grandicornis. The highest parasitism rate (3.54 ± 0.49%) was registered for nests from the Eucalyptus plantations. The rate of parasitism of nest from natural vegetation was 2.42 ± 0.40% and in the farmland was 1.91 ± 0.39%. The parasitism of each phorid genus varied according to habitat and month. Apocephalus attophilus had the highest parasitism in the three habitats and displayed biological characteristics that give it great potential as a biological control agent for A. sexdens. Eibesfeldtphora spp. had higher parasitism in a natural environment and M. grandicornis in agricultural environments. Apocephalus attophilus and M. grandicornis seemed able to occur in a wide variety of habitats that their hosts occupy, but the rate of parasitism by Eibesfeldtphora spp. was significantly lower in agricultural environments.

Inhibition of symbiote fungus of the leaf cutter ant Atta sexdens by secondary metabolites from the bacterium Xenorhabdus szentirmaii associated with entomopathogenic nematodes

ABSTRACT: Leaf-cutter ants (Hymenoptera: Formicidae) have evolved as dominant herbivores on the American continent. These social insects remove the leaves of economically important plant species to maintain their colony’s food reserves, the symbiotic fungus Leucocoprinus gongylophorus, a basidiomycete. Such fungus can be used for applications of fungicide molecules from metabolites generated by symbiont bacteria (Xenorhabdus and Photorhabdus) from entomopathogenic nematodes (Steinernema and Heterorhabditis). Through isolation and multiplication in tryptic soy broth (TSB) medium of the bacteria Xenorhabdus szentirmaii isolated PAM 25, we conducted laboratorial tests using treatments with 10, 25, and 50% of the metabolites obtained in the sixth day of cultivation. The treatments were centrifuged and filtered to generate a supernatant, which was diluted in potato + dextrose + agar (PDA), to verify the consequences of exposure to the fungus L. gongylophorus in Petri dishes. To confirm metabolite efficiency, the control treatments in PDA only and mixed (PDA+TSB) media were conducted simultaneously for 14 days. We observed total inhibition of the symbiont fungus in both the 25 and 50% dilutions during the first days of the tests. Our results support that these metabolites have inhibitory effect on the development of symbiont fungus of leaf-cutter ants.

Simarouba versicolor (Simaroubaceae) Dispersal by the Leaf-Cutter Ant Atta sexdens

The importance of Simarouba versicolor St. Hil. fruit dispersal by the leaf-cutting ant Atta sexdens (L.) was studied in the Cerrado, Tocantins, Brazil. The trees and nests were located between a forest area and a Brachiaria decumbens Stapf pasture. Seeds were collected in October 2015 along foraging trails and on the anthill of an A. sexdens colony. Germination of three groups of seeds was tested: (1) seeds with the tegument removed by the ants; (2) seeds without tegument, cleaned manually, and (3) seeds with tegument. The germination rates for the three treatments were similar; however, it was verified that the seeds cleaned by ants germinated faster. In addition, it was verified that the ants dispersed the seeds by at least 20 meters in the study area. Simarouba versicolor is a plant studied for its insecticidal properties, and this is the first study to our knowledge reporting its dispersal by ants.

Social Carrying and Defensive Behavior During Colony Emigration in the Leaf-cutting Ant Atta sexdens

In this work, we describe for the first time and under laboratory conditions, the behaviors related to social carrying and defensive strategies during colony emigration in the leaf-cutting ant Atta sexdens. Once colonies were laid on a tray under suboptimal conditions, groups of workers aggregated all over the body surface of the queen and brood, with mandibles half open and legs widely open in a ‘entangle’ formation. Queens were the first caste to be reallocated, followed respectively by the transportation of brood, newly-emerged workers, and pieces of fungus garden to the new nesting site. Contrary to what have been reported to the Myrmicinae species, adult transport followed a stereotyped sequence of acts involving approach, seize and transportation of newly-emerged workers to new target areas. Our results suggest that, in front of rapid unfavorable changes, leaf-cutting ants are capable of reorganize the nest in order to protect their members and resources.

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

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.

Susceptibility of Atta sexdens worker ants treated with the immunosuppressant Sandimmun Neoral to Metarhizium anisopliae

Abstract: The objective of this work was to evaluate if the immunosuppressant Sandimmun Neoral enhances the activity of Metarhizium anisopliae against the leaf-cutting ant Atta sexdens. The vulnerability to the pathogen was measured by comparing the mortality rate of worker ants subjected to the following treatments: immunosuppressant+control, immunosuppressant+M. anisopliae, excipient+control, excipient+M. anisopliae, control+M. anisopliae, and control. Worker ants treated with immunosuppressant+M. anisopliae showed the highest mortality rate in comparison with those subjected to all other treatments. The use of the immunosuppressant together with entomopathogenic fungus controlled leaf-cutting ants in laboratory conditions.

Lipid Mobilization and Acyl-Coenzyme A Dehydrogenase Abundance in the Postpharyngeal Gland of a Leaf-Cutting Ant

The postpharyngeal gland (PPG) in ants has a central role in lipidaccumulation. Our objective was to investigate whether the PPG ofworkers of the leaf-cutting ant Atta sexdens rubropilosa (F.) performslipid mobilization to the hemolymph, and to verify whether lipidsin gland cells are in part oxidized to acetyl-CoA. Increased levelsof fatty acids were observed in both the PPG and the hemolymphafter 48 h of lipid supplementation. In contrast, after 120 h of lipidsupplementation, fatty acid levels were reduced in the PPG andsignificantly increased in the hemolymph. The gland cells of the PPGof workers supplemented with lipids exhibited a high amount of Acyl-CoA dehydrogenase, and the lipids were in part β-oxidized to Acetyl-CoA. These findings suggest that the PPG of A. sexdens rubropilosaworkers mobilize fatty acids from their gland cells to the hemolymph.Further work will be needed to elucidate lipid transport from thehemolymph to other organs in this organism.

Mandibles of Leaf-Cutting Ants: Morphology Related to Food Preference

Feed adaptation is crucial for the ecological success of animals, which explore specific or varied resources according to the suitability of the selected feature. We hypothesized that the mandibles of leaf-cutting ants exhibit a greater degree of specialization because of the specific food preference of some species. The objective of this study was to highlight possible morphological differences between the mandibles of leaf-cutting ants of the genus Atta related to the food preference of the species, i.e., to investigate the morphofunctionality of mandibles in conjunction with a study of the leaf anatomy of plants selected. A detailed description of the mandibles of workers of the grass-cutting ant Atta bisphaerica and of the leaf-cutting ant Atta sexdens rubropilosa was provided, and the anatomy and chemical composition of the plant material foraged by these species were investigated. For morphometric analysis, 50 individuals of each of the four size classes were dissected for removal of the right mandible and one sample of each caste was processed for scanning electron microscopy. The Atta species had a similar mandibular morphology and differences were only observed between species and among castes, demonstrating the clear specialization of workers. Together with the anatomical study of the foraged plant material, the results indicate a high abundance of lignified cells in grasses, permitting to infer that the foraging and processing of these plants can lead to differences in behavioral acts during their preparation and incorporation in the fungus garden. We accept the hypothesis of morphofunctionality in view of the mandibular morphology observed.

Initial development and production of CO2 in colonies of the leaf-cutting ant Atta sexdens during the claustral foundation

Queens in the genus Atta are solely responsible for fungus cultivation and for care of herself and her offspring. Only few studies have investigated their nests in the claustral foundation and it is unknown the production rate of expelled carbon dioxide and/or of oxygen consumption in the initial colonies of leaf-cutting ants. Thus, we have studied the development of 50 initial colonies of Atta sexdens, and production of expelled carbon dioxide under laboratory conditions. The number of eggs was counted one week after nest foundation on the seventh day, the larvae counted on day 28, and the pupae between days 42 and 49. The workers emerged on the 63rd and 70th day. The CO2 concentration increased steeply in the 42nd days (20.60 ± 8.36%) and 49th days (15,37 ± 13,11 %), at 42nd days, and subsequently returned to lower values, for example, 3.35±2.84% at week seven. The present study is the first to present CO2 emission data in initial nests, in their claustral foundation under laboratory conditions.