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.

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.

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.

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.

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.

Dynamics of cell and tissue genesis in the male reproductive system of ticks (Acari: Ixodidae) Amblyomma cajennense [corrected] (Fabricius, 1787) and Amblyomma aureolatum (Pallas, 1772): a comparative analysis.

Ticks are classified into three families: Argasidae, Ixodidae, and Nutalliellidae. The taxonomy and phylogeny within Ixodidae are still discussed by the specialists, thus requiring further studies. Amblyomma cajennese and Amblyomma aureolatum (Brazil) belong to two species complexes known as "cajennese" and "ovale", respectively, and are directly related to the transmission of the Brazilian spotted fever. This confirms the medical and veterinary significance of these species, as well as the need for further morphological studies that will bring a better understanding of their taxonomy, phylogeny, and control. In this context, the present study aimed to characterize the morphology of the male reproductive system of A. cajennese and A. aureolatum when unfed and after 4 days of feeding, thereby seeking to: (a) distinguish the two species or "complexes", and (b) study an internal system which has the potential to be targeted by acaricides. Therefore, males from both species (unfed and after 4 days of feeding) were cold-anesthetized, dissected, and had their reproductive systems removed for histological analysis. The results showed that the morphology of the male reproductive system is generally similar between both species, like in other Ixodidae ticks, exhibiting a multilobed accessory gland complex related to seminal fluid secretion, a pair of vasa deferentia and a pair of testes housing germ cells (spermatocytes) in different stages. The main differences were found in the development of the accessory gland complex cells and germ cells, showing that the maturation of the male reproductive system starts later in A. aureolatum, when compared to A. cajennese. However, during the blood meal, A. aureolatum development is increased, thus making germ cell maturation and gland complex activity higher than in A. cajennese. This study shows the differences in the development of the male reproductive systems of both species, while providing information that can assist in the establishment of new control methods.

On the morphology of the digestive system of two Monomorium ant species.

The digestive system of adults and mature larvae of two ant species of Monomorium Mayr (Hymoneptera: Formicidae) were described with the aid of light and scanning electron microscopy, as there is a lack of studies in this area. These two ant species are recurrently found in urban habitats and are known as 'tramp species,' as they cause problems in households, businesses, and hospitals. The most interesting finds of the present study include the existence of spinules in the crop of adults, and the number of Malpighian tubules and rectal pads was constant among different castes, ages, and species.

Interaction between Linepithema micans (Hymenoptera: Formicidae) and Eurhizococcus brasiliensis (Hemiptera: Margarodidae) in vineyards.

Eurhizococcus brasiliensis (Wille) (Hemiptera: Margarodidae) is a soil scale that is considered the main pest of vineyards in Brazil. The ant Linepithema micans (Forel) (Hymenoptera: Formicidae) is frequently found associated with this species of scale in infested areas. The effect of the presence of L. micans on the infestation and dispersal capacity of E. brasiliensis on vine roots was measured in a greenhouse, using Paulsen 1103 rootstock seedlings planted in simple and double "Gallotti Cages." Treatments measured were: infestation of roots with E. brasiliensis or L. micans, and infestation with both species together. In the experiment using simple Gallotti Cages, with E. brasiliensis associated with L. micans, higher mean numbers of cysts and ants per plant were recorded, a result significantly different from that found for infestation with scale only. When double Gallotti Cages were used, first-instar nymphs were transported between the cages. The results showed that L. micans transports and aids in the attachment of E. brasiliensis to vine plants.

A metabolic pathway assembled by enzyme selection may support herbivory of leaf-cutter ants on plant starch.

Mutualistic associations shape the evolution in different organism groups. The association between the leaf-cutter ant Atta sexdens and the basidiomycete fungus Leucoagaricus gongylophorus has enabled them to degrade starch from plant material generating glucose, which is a major food source for both mutualists. Starch degradation is promoted by enzymes contained in the fecal fluid that ants deposit on the fungus culture in cut leaves inside the nests. To understand the dynamics of starch degradation in ant nests, we purified and characterized starch degrading enzymes from the ant fecal fluid and from laboratory cultures of L. gongylophorus and found that the ants intestine positively selects fungal α-amylase and a maltase likely produced by the ants, as a negative selection is imposed to fungal maltase and ant α-amylases. Selected enzymes are more resistant to catabolic repression by glucose and proposed to structure a metabolic pathway in which the fungal α-amylase initiates starch catalysis to generate byproducts which are sequentially degraded by the maltase to produce glucose. The pathway is responsible for effective degradation of starch and proposed to represent a major evolutionary innovation enabling efficient starch assimilation from plant material by leaf-cutters.