collection-long-term-maintenance-leaf-cutting-ants-atta-laboratory

Collection and Long-Term Maintenance of Leaf-Cutting Ants (<em>Atta</em>) 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 ...

São Paulo State University

Some ant species of the genus Monomorium Mayr occur worldwide and are considered important urban pests. The larvae of only a few species of this genus have been described, and these descriptions are either superficial or incomplete. This study aimed to determine the number of larval instars and describe the immature stages of the ant Monomorium floricola Jerdon (Formicidae: Myrmicinae). Specimens were analyzed and measured using light and scanning electron microscopy. Three larval instars were found, and all larvae had pheidoloid bodies with ectatommoid mandibles, consistent with other Monomorium species described previously. Five types of body hairs were described, and their distribution was instar-specific. Body and mandible dimensions of the larvae also were constant for each instar. Like other Myrmicinae, the larvae did not create a cocoon. Some of differences among the hair types and sensilla were observed by comparing the samples with larvae of other species in the genus, and these differences may have taxonomic utility.

Morphological description of the immatures of the ant, Monomorium floricola.

Linepithema micans Forel is a poorly studied species that is now considered a pest in vineyards in South Brazil. Larval characters have been used in a few phylogenetic studies, and their importance greatly depends in the amount of available information on different species. This study presents a complete panorama on the external morphology of the immatures of L. micans based on observations by light and scanning electron microscopy. The number of larval instars was estimated as three based on the frequency distribution of head widths of 965 larvae. Larvae of L. micans were similar to other Linepithema Mayr in the general format of body and mandibles, presenting a dorsal abdominal protuberance, nine pairs of spiracle, and unbranched hairs. On the other hand, L. micans was unique for having shorter hairs, predominantly denticulate, intraspecific variation in the number of antennal sensilla and in the types of sensilla on the labial palps were reported.

Description of the immatures of workers of the ant Linepithema micans Forel (Hymenoptera: Formicidae).

The Neotropical ant genus Myrmelachista Roger comprises 69 described species and subspecies, and still is a poorly studied group. Larvae play a paramount role in colony nutrition in social hymenopterans and bear considerable value in the reconstruction of group phylogenies, however, they are generally neglected. Larvae of different instars of Myrmelachista catharinae Mayr (Hymenoptera: Formicidae) are herein described in detail by light and scanning electron microscopy. The number of larval instars was estimated as three based on the frequency distribution of maximum head capsule widths. The described larvae confirmed some traits typical of the genus: general shape of body and mandibles, general aspect and distribution of body hairs, and the number of sensilla on the palps and galea. Differently from other Myrmelachista larvae previously described, M. catharinae presented two distinct kinds of second instars, some additional types of body hairs, different number of antennal sensilla, and a distinct labrum shape. M. catharinae presented ten pairs of spiracles, which is the first record for this genus.

Description of the immatures of the ant, Myrmelachista catharinae.

The Argentine ant Linepithema humile is an important invasive species because of the levels of infestation that it can reach; however, there is little information about its presence, histological organization, and function of the dorsal protuberance, which is found exclusively in their larvae. The objective of this study was to describe it in L. humile through scanning electron microscopy and transmission electron microscopy, bringing information about this structure. The epidermis of these larvae have cuticles covering the whole body, and is formed by a sequence of overlapping lamellas where the inner ones were thicker and presented lower electron density, whereas the outer ones were thinner and highly electron dense. Pores or pore-like channels were not observed. A thick and acellular region composed of granular material was found under the cuticular layer. Out of this region, the flattened epidermic cells formed an epithelial layer. For the dorsal protuberance region, these cells become prismatic, and similarly to the cuticle, presents significant thickening. These cells presented extended microvilli, as well as a great amount of lamellar rough endoplasmic reticulum. Under this epithelium was observed a concentration of fat body cells, more numerous in the dorsal protuberance region. This study indicated that the dorsal protuberance present in the first segment of L. humile larvae has apparently no secretory function because no pores were found. This fact allowed to conclude that in L. humile larvae the dorsal protuberance would have the function to make it easier for the worker ants to carry them within the colony.

Morphological organization of the dorsal protuberance of Linepithema humile (Mayr, 1868) ant's larvae (Hymenoptera, Formicidae).

Leafcutter ants of the genus Atta Fabricius are serious agricultural pests. Morphological studies of immature stages within this group are few, and the data provided for species of considerable importance are usually incomplete. In this study, the immatures of Atta sexdens Linnaeus are described and compared using light and scanning electron microscopy. Only specimens from founding stage colonies (i.e., lacking adult workers) were used. The existence of four larval instars was estimated by a frequency plot of maximum head widths, and the larvae of different instars differed from each other mainly by their bodily dimensions. Worker larvae belonged to two distinct morphological castes: (1) gardeners and nurses and (2) within-nest generalists. The worker larvae described in this study differed from a previous description of the same species by the following traits: the existence of a genal lobe, the number of clypeal hairs, the presence of two hairs on the ninth abdominal somite, the presence of hairs on the anterior surface of the labrum, and the shape of the maxillary palpus. This study provides a comparative analysis of immature stages of A. sexdens that may be relevant to future morphological and biological studies of the Attini.

On the morphology of the worker immatures of the leafcutter ant Atta sexdens Linnaeus (Hymenoptera: Formicidae).

The immatures of males of two species of Camponotus ants (Hymenoptera: Formicidae) are described and compared by light and electron microscopy. The numbers of larval instars were determined: Camponotus rufipes Fabricius (Hymenoptera: Formicidae) have four instars; and Camponotus vittatus Forel have three. Male larvae of the two species are similar to previously described Camponotus larvae, sharing the following traits: basic shape of body and mandible, presence of 'chiloscleres', 'praesaepium' (some specimens), labial pseudopalps, and ten pairs of spiracles. However, larvae of the two species can be separated by bodily dimensions and based on their hair number and types. Worker larvae of C. vittatus previously described are extensively similar to male larvae, with only a few inconspicuous differences that may result from intraspecific variation or sexual differences.

Compared morphology of the immatures of males of two urban ant species of Camponotus.

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.

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

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.

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

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. 

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

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.

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

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.

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

Leaf-cutting ants of the genera Atta and Acromyrmex are at constant risk of epizootics due to their dense living conditions and frequent social interactions between genetically related individuals. To help mitigate the risk of epizootics, these ants display individual and collective immune responses, including associations with symbiotic bacteria that can enhance their resistance to pathogenic infections. For example, Acromyrmex spp. harbor actinobacteria that control infection by Escovopsis in their fungal gardens. Although Atta spp. do not maintain symbiosis with protective actinobacteria, the evidence suggests that these insects are colonized by bacterial microbiota that may play a role in their defense against pathogens. The potential role of the bacterial microbiome of Atta workers in enhancing host immunity remains unexplored. We evaluated multiple parameters of the individual immunity of Atta cephalotes (Linnaeus, 1758) workers, including hemocyte count, encapsulation response, and the antimicrobial activity of the hemolymph in the presence or absence of bacterial microbiota. Experiments were performed on ants reared under standard conditions as well as on ants previously exposed to the entomopathogenic fungus Metharrizium anisopliae. Furthermore, the effects of the presence/absence of bacteria on the survival of workers exposed to M. anisopliae were evaluated. The bacterial microbiota associated with A. cephalotes workers does not modulate the number of hemocytes under control conditions or under conditions of exposure to the fungal pathogen. In addition, infection by M. anisopliae, but not microbiota, increases the encapsulation response. Similarly, the exposure of workers to this fungus led to increased hemolymph antimicrobial activity. Conversely, the removal of bacterial microbiota did not have a significant impact on the survival of workers with M. anisopliae. Our results suggest that the bacterial microbiota associated with the cuticle of A. cephalotes workers does not play a role as a modulator of innate immunity, either at baseline or after exposure to the entomopathogen M. anisopliae. Further, upon infection, workers rely on mechanisms of humoral immunity to respond to this threat. Overall, our findings indicate that the bacterial microbiota associated with A. cephalotes workers does not play a defensive role.

Immunity and survival response of Atta cephalotes (Hymenoptera: Myrmicinae) workers to Metarhizium anisopliae infection: Potential role of their associated microbiota.

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.

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

Article	Year
Morphological description of the immatures of the ant, Monomorium floricola. Journal of insect science (Online)\| PubMed ID: 20575746	2010
Description of the immatures of workers of the ant Linepithema micans Forel (Hymenoptera: Formicidae). Microscopy research and technique\| PubMed ID: 20734410	2011
Description of the immatures of the ant, Myrmelachista catharinae. Journal of insect science (Online)\| PubMed ID: 21529152	2011
Morphological organization of the dorsal protuberance of Linepithema humile (Mayr, 1868) ant's larvae (Hymenoptera, Formicidae). Microscopy research and technique\| PubMed ID: 21604325	2011
On the morphology of the worker immatures of the leafcutter ant Atta sexdens Linnaeus (Hymenoptera: Formicidae). Microscopy research and technique\| PubMed ID: 22419653	2012
Compared morphology of the immatures of males of two urban ant species of Camponotus. Journal of insect science (Online)\| PubMed ID: 22934715	2012
On the morphology of the digestive system of two Monomorium ant species. Journal of insect science (Online)\| PubMed ID: 24224520	2013
Allium cepa and Tradescantia pallida bioassays to evaluate effects of the insecticide imidacloprid. Chemosphere\| PubMed ID: 25225953	2015
Ants (Hymenoptera: Formicidae) in vineyards that are infested or uninfested with Eurhizococcus brasiliensis (Hemiptera: Margarodidae) in Southeastern Brazil. Journal of insect science (Online)\| PubMed ID: 25347833	2014
Transovarian Transmission of Blochmannia and Wolbachia Endosymbionts in the Neotropical Weaver Ant Camponotus textor (Hymenoptera, Formicidae). Current microbiology\| PubMed ID: 29468305	2018
Histopatology and HSP70 analysis of the midgut of Rhinocricus padbergi (Diplopoda) in the evaluation of the toxicity of two new metallic-insecticides. Environmental science and pollution research international\| PubMed ID: 31838689	2020
Immunity and survival response of Atta cephalotes (Hymenoptera: Myrmicinae) workers to Metarhizium anisopliae infection: Potential role of their associated microbiota. PloS one\| PubMed ID: 33626077	2021
Exploring the diversity and potential interactions of bacterial and fungal endophytes associated with different cultivars of olive (Olea europaea) in Brazil. Microbiological research\| PubMed ID: 35868260	2022

Odair Correa Bueno

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