The Influence of Selected Insecticides on the Oxidative Response of Atta sexdens (Myrmicinae, Attini) Workers.

Reactive oxygen species (ROS) are generated as products of normal cellular metabolic activities; however, the use of pesticides to control leafcutter ants leads to unbalanced ROS production. We evaluated the effects of two insecticides (fipronil, sulfluramid) and metallic insecticide complex (magnesium complex [Mg(hesp)2(phen)] (1)) on the superoxide dismutase (SOD), glutathione (GSH) and the overall antioxidant capacity using two different methodologies: total radical-trapping potential (TRAP) and oxygen radical absorbance capacity (ORAC). Media workers of Atta sexdens (C. Linnaeus) were exposed to the insecticides for 24 h, 48 h, 72 h and 96 h before their fat bodies were dissected for analysis. The results showed that although the sulfluramid may cause the production of ROS, its slow action in the organism does not lead to oxidative stress. There is a rise in oxidative stress in workers of leafcutter ants treated with fipronil because SOD significantly increased when compared to the control group. On the other hand, Mg1-complex suppressed both GSH and SOD, indicating that the immune system may be affected by Mg1-complex, which has a delayed activity ideal for its use in chemical pest control. Both TRAP and ORAC evaluated total antioxidant capacities; however, ORAC proved to be a more sensitive method. In conclusion, the Mg1-complex is a new compound that should be further investigated as a potential replacement for fipronil and sulfluramid in pest control.

Collection and Long-Term Maintenance of Leaf-Cutting Ants (Atta) in Laboratory Conditions.

Ants are one of the most biodiverse groups of animals on the planet and inhabit different environments. The maintenance of ant colonies in controlled environments enables an enriched comprehension of their biology that can contribute to applied research. This practice is usually employed in population control studies of species that cause economic loss, such as Atta ants. To cultivate their mutualistic fungus, these leaf-cutting ants collect leaves and for this are considered agricultural pests widely distributed throughout the American continent. They are highly socially organized and inhabit elaborated underground nests composed of a variety of chambers. Their maintenance in a controlled environment depends on a daily routine of several procedures and frequent care that are described here. It starts with the collection of queens during the reproductive season (i.e., nuptial flight), which are then individually transferred to plastic containers. Due to the high mortality rate of queens, a second collection can be carried out about 6 months after the nuptial flight, when incipient nests with developed fungus wad are excavated, hand-picked, and placed in plastic containers. In the laboratory, leaves are daily provided to established colonies, and ant-produced waste is weekly removed along with remaining dry plant material. As the fungus garden keeps growing, colonies are transferred to different types of containers according to the experimental purpose. Leaf-cutting ant colonies are placed in interconnected containers, representing the organizational system with functional chambers built by those insects in nature. This setup is ideal to monitor factors such as waste amount, fungus garden health, and the behavior of workers and queen. Facilitated data collection and more detailed observations are considered the greatest advantage of keeping ant colonies in controlled conditions.

Exploring the diversity and potential interactions of bacterial and fungal endophytes associated with different cultivars of olive (Olea europaea) in Brazil.

The olive crop has expanded in the southeastern region of South America, particularly in Brazil. Thus, the objectives of this study were to identify the diversity of endophytic microorganisms associated with olive leaves with culture-dependent and culture-independent methods, to explore which factors influence the composition and abundance of this microbial community, to identify the trophic mode of these fungi by FunGuild and, to verify type associations between bacterial and fungal communities. Leaf samples were collected from 93 plants in nine locations in the Brazilian states of São Paulo and Minas Gerais. Leaves were first superficially disinfected before fungal isolation and next-generation metabarcoding sequencing was completed targeting the 16S rRNA regions for bacteria and ITS1 for fungi. In total, 800 isolates were obtained, which were grouped into 191 morphotypes and molecularly identified, resulting in 38 genera, 32 of which were recorded for the first time in cultivated olive trees in Brazil. For the isolated fungi, the most abundant trophic level was pathotrophic and for the culture-independent method was unidentified followed by symbiotrophic. The metabarcoding results revealed that factors such as plant age, altitudinal gradient, and geographic location can influence the microbial community of commercial olive plants, while the specific cultivar did not.

Proteomic characterization of the fibroin-based silk fibers produced by weaver ant Camponotus textor.

The fibroin-based silk fibers of weaver ants are an alternative biomaterial to be investigated and explored for potential biomedical applications. In this context, the silk fibers from the nest of the weaver ant Camponotus textor was solubilized and fractionated by gel permeation. The different fractions were collected, pooled and submitted to analysis with a series of biochemical methods, nuclear magnetic resonance (NMR) spectroscopy, analytical proteomic strategies, and data treatment with bioinformatic tools to perform the structural characterization of the fibroin-based silk fibers produced by the ant. Our data demonstrated the identification of one fibroin proteoform in the ant silk fibers. The protein chracterized as a glycoprotein with MW around 40 kDa and presenting 66% (w/w) of total sugars attached to it through O-linked carbohydrates. The 3D of protein was modeled, revealing a structure predominantly constituted of coiled-coil secondary units in the whole model, featuring at least four superhelices (arrangement with multiple α-helices). The scientific outcomes reported herein may be relevant for the development of novel approaches for the synthetic or recombinant production of novel silk-based polymers for biomedical applications. BIOLOGICAL SIGNIFICANCE: The present investigation significantly expanded knowledge regarding to the fibroin-based silk fibers from weaver ants, contributing to improvements in our understanding of the properties and characteristics of these silk fibers. For example, as reported here, carbohydrates were detected in the ants' silk for the first time presenting the fibroin as a glycoprotein. Moreover, the 3D structure provided new insights into the secondary structures considering the whole model of the protein.

Machaerium opacum Vogel (Fabaceae): phytochemical study and toxicity to Atta sexdens Linnaeus (Hymenoptera: Formicidae).

In this work was described the phytochemical investigation of Machaerium opacum Vogel (Fabaceae) leaves as well as the insecticidal activity of its crude extract and fractions against Atta sexdens Linnaeus (Hymenoptera: Formicidae). The phytochemical study led to the identification of α-amyrin, ß-amyrin, lupeol, phytol, isomucronulatol and rutin, described for the first time in M. opacum and mucronulatol. Insecticidal activity was assessed by the ingestion of the crude extract and fractions incorporated into an artificial diet at three different concentrations (0.2, 1.0 and 2.0 mg mL-1). Statistical analysis revealed that all the samples of M. opacum at all concentrations tested showed significant results when compared to the pure diet control.

What Can the Bacterial Community of Atta sexdens (Linnaeus, 1758) Tell Us about the Habitats in Which This Ant Species Evolves?

Studies of bacterial communities can reveal the evolutionary significance of symbiotic interactions between hosts and their associated bacteria, as well as identify environmental factors that may influence host biology. Atta sexdens is an ant species native to Brazil that can act as an agricultural pest due to its intense behavior of cutting plants. Despite being extensively studied, certain aspects of the general biology of this species remain unclear, such as the evolutionary implications of the symbiotic relationships it forms with bacteria. Using high-throughput amplicon sequencing of 16S rRNA genes, we compared for the first time the bacterial community of A. sexdens (whole ant workers) populations according to the habitat (natural versus agricultural) and geographical location. Our results revealed that the bacterial community associated with A. sexdens is mainly influenced by the geographical location, and secondarily by the differences in habitat. Also, the bacterial community associated with citrus differed significantly from the other communities due to the presence of Tsukamurella. In conclusion, our study suggests that environmental shifts may influence the bacterial diversity found in A. sexdens.

Histopatology and HSP70 analysis of the midgut of Rhinocricus padbergi (Diplopoda) in the evaluation of the toxicity of two new metallic-insecticides.

Millipedes are organisms of the edaphic fauna and have been used as bioindicators for the evaluation of pollutants in the environment, as they are in constant contact with the soil. This study used the millipede Rhinocricus padbergi as surrogate to evaluate the toxicity of two metallic-insecticides that has been developed for leaf-cutting ants management. Millipedes were exposed in terrariums containing different concentrations of the metallic-insecticides and, after periods of 21 and 90 days, three individuals from each terrarium were dissected in order to remove the midgut, the organ where absorption of nutrients and, consequently, toxic substances occurs. The toxic action of the metallic-insecticides was analyzed through qualitative and semi-quantitative analysis of morphophysiological alterations and by quantitative analysis of the HSP70 stress protein. The results showed that the metallic-insecticides may increase HSP70 labeling, although not at all concentrations and periods of exposure. Histopathological alterations were not significant at any concentration, indicating that the cytoprotective action of HSP70 is able to prevent severe damage to the midgut. It is therefore suggested that the metallic-insecticides are not toxic to the species studied here as no toxicity was observed under the conditions tested. In addition, stress protein localization in midgut helps understand how morphophysiological processes can potentially be affected by pesticide exposure.

Transovarian Transmission of Blochmannia and Wolbachia Endosymbionts in the Neotropical Weaver Ant Camponotus textor (Hymenoptera, Formicidae).

Camponotus is a hyper-diverse ant genus that is associated with the obligate endosymbiont Blochmannia, and often also with Wolbachia, but morphological studies on the location of these bacteria in the queen's ovaries during oogenesis remain limited. In the present study, we used the Neotropical weaver ant Camponotus textor to characterize the ovary using histology (HE) techniques, and to document the location of Blochmannia and Wolbachia during oogenesis through fluorescence in situ hybridization (FISH). This is the first morphological report of these two bacteria in the same host with polytrophic meroistic ovaries and reveals that Blochmannia is found inside late-stage oocytes and Wolbachia is associated with the nuclei of the nurse cells. Our results provide insights into the developmental sequence of when these bacteria reach the egg, with Blochmannia establishing itself in the egg first, and Wolbachia only reaching the egg shortly before completing egg development. Studies such as this provide understanding about the mechanisms and timing of the establishment of these endosymbionts in the host.

Species-specific signatures of the microbiome from Camponotus and Colobopsis ants across developmental stages.

Symbiotic relationships between hosts and bacteria are common in nature, and these may be responsible for the evolutionary success of various groups of animals. Among ants, these associations have been well studied in some genera of the Camponotini, but several questions remain regarding the generality of the previous findings across all the members of this ant tribe and if bacterial communities change across development in these hosts. This study is the first to characterize the bacterial community associated with a colony of the recently recognized genus Colobopsis and three colonies of Camponotus (two distinct species) and show how different the composition of the bacterial community is when compared across the different genera. Our data reveal that Colobopsis (species: Co. riehlii) and Camponotus (species: Ca. floridanus and Ca. planatus) have distinct microbiota, and we were able to verify that the identity of the species contributes more to the bacterial diversity. We also demonstrated that there were no significant differences between colonies of the same species (Camponotus planatus), and between stages of development from different colonies. We did find that some developmental stages have distinct bacteria, confirming that each stage of development could have a specific microbiota. Our results show species are one of the factors that shape the bacterial community in these Camponotini ants. Additional studies of the intra-colonial microbiome of other hosts and across development may reveal additional clues about the function and importance of bacteria in colony recognition, individual and colony health, and nutritional upgrading.

Microbial composition of spiny ants (Hymenoptera: Formicidae: Polyrhachis) across their geographic range.

BACKGROUND: Symbiotic relationships between insects and bacteria are found across almost all insect orders, including Hymenoptera. However there are still many remaining questions about these associations including what factors drive host-associated bacterial composition. To better understand the evolutionary significance of this association in nature, further studies addressing a diversity of hosts across locations and evolutionary history are necessary. Ants of the genus Polyrhachis (spiny ants) are distributed across the Old World and exhibit generalist diets and habits. Using Next Generation Sequencing (NGS) and bioinformatics tools, this study explores the microbial community of >80 species of Polyrhachis distributed across the Old World and compares the microbiota of samples and related hosts across different biogeographic locations and in the context of their phylogenetic history. RESULTS: The predominant bacteria across samples were Enterobacteriaceae (Blochmannia - with likely many new strains), followed by Wolbachia (with multiple strains), Lactobacillus, Thiotrichaceae, Acinetobacter, Nocardia, Sodalis, and others. We recovered some exclusive strains of Enterobacteriaceae as specific to some subgenera of Polyrhachis, corroborating the idea of coevolution between host and bacteria for this bacterial group. Our correlation results (partial mantel and mantel tests) found that host phylogeny can influence the overall bacterial community, but that geographic location had no effect. CONCLUSIONS: Our work is revealing important aspects of the biology of hosts in structuring the diversity and abundance of these host-associated bacterial communities including the role of host phylogeny and shared evolutionary history.

Genotoxicity evaluation of two metallic-insecticides using Allium cepa and Tradescantia pallida: A new alternative against leaf-cutting ants.

In order to combat leaf-cutting ants, the pesticide sulfluramid used to be the most widely utilized active ingredient. However, its use was banned in 2009 by the Stockholm Convention, although some countries were allowed to continue using it. As an effective alternative to its replacement, researchers developed a metallic-insecticide system, which is a natural product linked to metal complexes. Thus, the aim of this study was to evaluate the ability of these new metallic-insecticides in change the genetic material of non-target organisms. The tests were performed utilizing chromosomal aberrations and micronucleus tests in the Allium cepa test system and the Trad-MCN test in Tradescantia pallida. To better understand the results, one of the components of the formula, 5-methyl-phenanthroline, was also analyzed according to the same parameters. To A. cepa, the results showed that one of the metallic insecticides induced cytotoxicity and genotoxicity at different concentrations, while the other metallic-insecticide showed chromosomal instability only at the highest concentration. The analysis of 5-methyl-phenanthroline revealed that it can be related with the positive results, since genotoxic effects were induced. In the Trad-MCN test, none of the metallic-insecticides showed genotoxic activity, although one of them induced more micronucleus formation.

Control of Linepithema micans (Hymenoptera: Formicidae) and Eurhizococcus brasiliensis (Hemiptera: Margarodidae) in Vineyards Using Toxic Baits.

Linepithema micans (Forel) (Hymenoptera: Formicidae) is the main ant species responsible for dispersal of Eurhizococcus brasiliensis (Wille) (Hemiptera: Margarodidae), a root scale that damages grapevines in southern Brazil. The effects of different formulations of toxic baits based on boric acid and hydramethylnon to control L. micans and E. brasiliensis were evaluated. Toxic baits with boric acid (1.0%) mixed in different concentrations of inverted sugar (20%, 30%, and 40%), and hydramethylnon, mixed with sardines (paste), cassava flour and peanut, brown sugar (sucrose), or sardine oil-based gel, were evaluated in a greenhouse and in the field. In the greenhouse experiment, the number of foraging ants was significantly reduced in the pots where the hydramethylnon in sardine paste (Solid S), sardine oil-brown sugar-based gel (GEL SAM), and peanut oil-brown-sugar gel (GEL AM) formulations were applied. The GEL SAM toxic bait effectively reduced the infestation of L. micans, and could be used for indirect control of E. brasiliensis on young grapevines.

The Postpharyngeal Gland: Specialized Organ for Lipid Nutrition in Leaf-Cutting Ants.

There are several hypotheses about the possible functions of the postpharyngeal gland (PPG) in ants. The proposed functions include roles as cephalic or gastric caeca and diverticulum of the digestive tract, mixing of hydrocarbons, nestmate recognition, feeding larvae, and the accumulation of lipids inside this gland, whose origin is contradictory. The current study aimed to investigate the functions of these glands by examining the protein expression profile of the PPGs of Atta sexdens rubropilosa (Hymenoptera, Formicidae). Mated females received lipid supplementation and their glands were extracted and analyzed using a proteomic approach. The protocol used combined two-dimensional electrophoresis and shotgun strategies, followed by mass spectrometry. We also detected lipid ß-oxidation by immunofluorescent marking of acyl-CoA dehydrogenase. Supplying ants with lipids elicited responses in the glandular cells of the PPG; these included increased expression of proteins related to defense mechanisms and signal transduction and reorganization of the cytoskeleton due to cell expansion. In addition, some proteins in PPG were overexpressed, especially those involved in lipid and energy metabolism. Part of the lipids may be reduced, used for the synthesis of fatty alcohol, transported to the hemolymph, or may be used as substrate for the synthesis of acetyl-CoA, which is oxidized to form molecules that drive oxidative phosphorylation and produce energy for cellular metabolic processes. These findings suggest that this organ is specialized for lipid nutrition of adult leaf-cutting ants and characterized like a of diverticulum foregut, with the ability to absorb, store, metabolize, and mobilize lipids to the hemolymph. However, we do not rule out that the PPG may have other functions in other species of ants.

Comparative analysis of spermatids of Rhipicephalus sanguineus sensu lato (Ixodidae) and Ornithodoros rostratus ticks (Argasidae): morphophysiology aimed at systematics.

The phylogenetic relationships among tick species (Acari: Ixodida) have been revisited by several researchers over the last decades. Two subfamilies, Rhipicephalinae (Ixodidae) and Ornithodorinae (Argasidae), deserve special attention. The male reproductive system morphology, as well as the ultrastructure of the germ cells, may provide important information for phylogeny and systematics of metazoan groups, with spermatozoa exhibiting characters that can be used for this purpose. With that information in mind, this study aimed at evaluating, through a comparative analysis, the morphology of the male reproductive systems and germ cells of ticks species Rhipicephalus sanguineus and Ornithodoros rostratus. In order to do that, histology and scanning electron microscopy techniques were used. The results have shown that despite the similarities in the general morphology of the male reproductive system among studied Ixodida so far, there are morphological differences among the species studied herein, mainly the U-shaped testis (ancestral character) in O. rostratus and the pair testes (derived character) in R. sanguineus, and the general morphology of germ cells (spermatids V). Besides that, the morphological changes observed during the spermiogenesis appear to be different between the species studied here, probably characterizing the two families considered. The data generated in this study showed the importance of comparative internal morphology studies, mainly in regard to spermatology, despite the morphological data obtained herein not being enough to product a cladogram (sperm cladistics), it was already possible to observe clear differences among families Argasidae and Ixodidae in regard to the organization of their male reproductive systems and concerning the external morphology of spermatids. Data yet to be obtained through transmission electron microscopy techniques will allow the application of spermiocladistics and spermiotaxonomy as tools for tick systematics.

The ant fauna of hospitals: advancements in public health and research priorities in Brazil

Ants inhabit several types of natural and urban habitats, where they successfully nest. In urban environments, the hospitals should be considered priority for studies, as ants pose risks to human health due to their pathogen carrying potential. We aimed at surveying the literature about studies on ants in hospital settings in Brazil in the past 20 years. We found 40 papers in 22 journals, the first one published in 1993. Among them, 26 papers assessed pathogenic microorganisms on ants. We recorded 59 ant species, being Tapinoma melanocephalum the most common. The Minas Gerais and São Paulo states had the largest number of published papers. Mato Grosso do Sul and Rio Grande do Sul showed the highest number of species. Exotic ant species were recorded in all states, except Goiás. Considering the potential to carry microorganisms and the importance of thorough studies on the ecology of ant species, our results can support and guide further research in Brazil.

Allium cepa and Tradescantia pallida bioassays to evaluate effects of the insecticide imidacloprid.

The indiscriminate use of pesticides has become a serious environmental concern. Of them, imidacloprid (IMI) is one of the most widely used worldwide. In 2010 in Brazil, 1.934 tonnes of IMI were sold and mainly used for sugarcane crops. Several studies have examined the toxicity of IMI as well as its possible ecological effects. However, few studies have examined its toxicity at the genetic level. This is one of the biggest challenges for the scientific community, which is concerned about the impacts of these contaminants on the environment and human health. In this study, we evaluated the effects of IMI above the genetic material in Allium cepa and Tradescantia pallida following exposure to different concentrations of this insecticide. The results demonstrated that the concentrations tested induced chromosomal alterations and increased the frequency of micronuclei. Therefore, IMI in these concentrations was genotoxic to the tested organisms. These factors should be taken into account when applying this pesticide.

Mitochondrial and peroxisomal population in post-pharyngeal glands of leaf-cutting ants after lipid supplementation.

The post-pharyngeal gland (PPG) occurs in ants and some Sphecidae wasps. Among its several roles is the storage of lipids from food. In order to investigate the effect of lipids on the cell, especially on mitochondria and peroxisomes, the present study was aimed at examining the peroxisomal and mitochondrial population in the PPG of Atta sexdens rubropilosa after lipid supplementation by confocal laser scanning microscopy and transmission electron microscopy. Soybean oil provided as lipid supplement was not toxic for A. sexdens rubropilosa workers for the first 48 h and 120 h. However, the ultrastructural cytochemical analysis revealed an accumulation of lipid droplets in the PPGs of ants after lipid supplementation at 48 h and 120 h, and smaller lipid droplets in the basal membrane of the PPG epithelium, showing lipid mobilization from the PPG to the hemolymph. The lipid supplementation reduces the life expectancy of medium workers, probably due to the high lipid metabolism. Most importantly, the PPGs of medium workers of leaf-cutting A. sexdens rubropilosa is probably a specialized gland in the lipid metabolism, due to the increased mitochondrial and peroxisomal population inside cells after lipid supplementation; participation of peroxisomal population in the ß-oxidation of long chain fatty acids into smaller chains and participation of mitochondrial population in the ß-oxidation of fatty acids for energy, or mobilization of lipid derivatives from the PPG to hemolymph, a process that requires energy. However, the hypothesis that the PPGs convert lipids from food in aldehydes and/or hydrocarbons must be better investigated.

Molecular characterization of fire ants, Solenopsis spp., from Brazil based on analysis of mtDNA gene cytochrome oxidase I.

Species from the Solenopsis saevissima (Smith) (Hymenoptera: Formicidae) species group are native to South America and have a cosmopolitan distribution because they have been accidentally introduced in many countries around the world. In Brazil, they have a wide distribution, including urban areas. The present study was conducted to investigate the characterization of Solenopsis genus populations associated with urban/human interference sites in Brazil by analyzing the mitochondrial gene cytochrome oxidase I and estimating the degree of relatedness of these populations to make inferences about their phylogeny and also observe the patterns of mitochondrial haplotype (mitotype) distribution across their range. The results revealed complete geographical coherence and polyphyly for the Solenopsis invicta Buren and Solenopsis saevissima species groups, which confirms the diversity of the genera. It also suggests the possibility that reproductively-isolated populations occur, resulting in the evolutionary process of speciation. No predominant haplotype was found in the populations analyzed, but some were more prevalent.

Ants (Hymenoptera: Formicidae) in vineyards that are infested or uninfested with Eurhizococcus brasiliensis (Hemiptera: Margarodidae) in Southeastern Brazil.

The association between ants and mealybugs can result in damage to agriculture, including vineyards. In southern Brazil, the ant Linepithema micans F. contributes to the dispersal of Eurhizococcus brasiliensis (Wille) (ground pearl), a root mealybug that can lead to economic losses. In this study, the ant communities in vineyards that were infested or uninfested with ground pearls were evaluated in the primary municipalities that produce the Niágara Rosada variety of grapes in southeastern Brazil. The hypothesis of this study was that the composition of the ant community differs between vineyards with and without E. brasiliensis. The ants were collected using subterranean traps in 10 vineyards infested with this mealybug and 10 uninfested vineyards. There was no significant association between ground pearls and the composition or richness of the ant species. Solenopsis invicta (Buren) (Hymenoptera: Formicidae) was the most frequently observed, and Pheidole aberrans (Mayr), Pheidole subarmata (Mayr), and Brachymyrmex incisus F. were common, especially in the rainy season when ground-pearl nymphs were prevalent in the state of São Paulo. Species from preserved or specialized environments were recorded in the vineyards, even with the use of conventional management techniques.

Foraging activity and seasonal food preference of Linepithema micans (Hymenoptera: Formicidae), a species associated with the spread of Eurhizococcus brasiliensis (Hemiptera: Margarodidae).

Linepithema micans (Forel) (Hymenoptera: Formicidae) is the main ant species responsible for the spread of Eurhizococcus brasiliensis (Wille) (Hemiptera: Margarodidae), a soil scale that damages vine plants in southern Brazil. The daily foraging activity of L. micans and its seasonal preference for protein- and carbohydrate-based foods were evaluated. The study was carried out in a greenhouse using seedlings of the Paulsen 1103 rootstock (Vitis berlandieri x Vitis rupestris) planted individually in pots and infested with colonies of L. micans. To determine the daily foraging activity and seasonal preference, a cricket (Gryllus sp.) and a 70% solution of inverted sugar and water were offered once a month for 12 mo. The ants foraging on each food source were counted hourly for 24 h. L. micans foraged from dusk until the end of the next morning, with higher intensity in the spring and summer. Workers of L. micans showed changes in food preference during the year, with a predominance of protein-based food during winter and spring and carbohydrate-based food during autumn. The implications of this behavior for control of the species with the use of toxic baits are discussed.