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.

Two feedback mechanisms involved in the control of leaf fragment size in leaf-cutting ants.

Polymorphic leaf-cutting ants harvest leaf fragments that correlate in size with the workers' body size. When cutting, workers anchor their hind legs on the leaf edge and rotate, removing approximately semicircular fragments. Workers show behavioural plasticity and modify their leg extension while holding onto the leaf edge depending on, for instance, leaf toughness, cutting smaller fragments out of tough leaves. What sensory information workers use to control the cutting trajectory remains unknown. We investigated whether sensory information from both the leg contact with the leaf edge and from head movements underlies fragment size determination. In the laboratory, we recorded Atta sexdens workers cutting standardised ®Parafilm pseudoleaves of different thickness, and quantified cutting behaviour and body reach, i.e. the distance between the mandible and the anchored hind leg tarsus. Experimentally preventing contact with the leaf edge resulted in smaller fragments, evincing that workers control the cutting trajectory using information from the contact of the hind legs with the leaf edge. However, ants were able to cut fragments even when contact of all six legs with the edge was prevented, indicating the use of additional sensory information. Ablation of mechanosensory hairs at the neck joint alone did not influence fragment size determination, yet simultaneously preventing sensory feedback from both mechanosensory hairs and edge contact led to a loss of control over the cutting trajectory. Leaf-cutting ants, therefore, control their cutting trajectory using sensory information from both the leg contact with the leaf edge and the lateral bending of the head.

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

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

Distinct and enhanced hygienic responses of a leaf-cutting ant toward repeated fungi exposures.

Leaf-cutting ants and their fungal crops are a textbook example of a long-term obligatory mutualism. Many microbes continuously enter their nest containing the fungal cultivars, destabilizing the symbiosis and, in some cases, outcompeting the mutualistic partners. Preferably, the ant workers should distinguish between different microorganisms to respond according to their threat level and recurrence in the colony. To address these assumptions, we investigated how workers of Atta sexdens sanitize their fungal crop toward five different fungi commonly isolated from the fungus gardens: Escovopsis sp., Fusarium oxysporum, Metarhizium anisopliae, Trichoderma spirale, and Syncephalastrum sp. Also, to investigate the plasticity of these responses toward recurrences of these fungi, we exposed the colonies with each fungus three times fourteen days apart. As expected, intensities in sanitization differed according to the fungal species. Ants significantly groom their fungal crop more toward F. oxysporum, M. anisopliae, and Syncephalastrum sp. than toward Escovopsis sp. and T. spirale. Weeding, self-, and allogrooming were observed in less frequency than fungus grooming in all cases. Moreover, we detected a significant increase in the overall responses after repeated exposures for each fungus, except for Escovopsis sp. Our results indicate that A. sexdens workers are able to distinguish between different fungi and apply distinct responses to remove these from the fungus gardens. Our findings also suggest that successive exposures to the same antagonist increase hygiene, indicating plasticity of ant colonies' defenses to previously encountered pathogens.

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

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

Comparison of radial growth rate of the mutualistic fungus of Atta sexdens rubropilosa forel in two culture media.

In vitro culture of the mutualistic fungus of leaf-cutting ants is troublesome due to its low growth rate, which leads to storage problems and contaminants accumulation. This paper aims at comparing the radial growth rate of the mutualistic fungus of Atta sexdens rubropilosa Forel in two different culture media (Pagnocca B and MEA LP). Although total MEA LP radial growth was greater all along the bioassay, no significant difference was detected between growth efficiencies of the two media. Previous evidences of low growth rate for this fungus were confirmed. Since these data cannot point greater efficiency of one culture medium over the other, MEA LP medium is indicated for in vitro studies with this mutualistic fungus due its simpler composition and translucent color, making the analysis easier.

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

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

Allogrooming, Self-Grooming, and Touching Behavior: Contamination Routes of Leaf-Cutting Ant Workers Using a Fat-Soluble Tracer Dye.

The aim of this study was to determine whether worker self-grooming, allogrooming, and direct contact promotes the dispersal of substances among members of the colony. For this purpose, a tracer (Sudan III dye) was applied topically to a worker ant and the social interactions between the worker with the tracer and workers without the tracer were studied. Additionally, the worker heads were dissected to visualize whether or not the post-pharyngeal gland was stained. The post-pharyngeal glands from 50% to 70% of workers were stained depending on the size of the group. With the increase in the experimental group size, the frequency of interactions between workers increased, with touching being the most frequent behavior. The tracer dye was probably passed on by direct contact between workers, followed by self-grooming and allogrooming. These behaviors are responsible for the rapid dispersal of substances among colony members as observed in our experiment. The results therefore support the hypothesis that contact with substances promotes the contamination of nestmates, even in the absence of feeding, serving as a model for further studies on the contamination of workers with the active ingredients of insecticides.

Foliar Substrate Affects Cuticular Hydrocarbon Profiles and Intraspecific Aggression in the Leafcutter Ant Atta sexdens.

Cuticular hydrocarbons (CHCs) are traditionally considered to be one of the most important chemical cues used in the nestmate recognition process of social hymenopterans. However, it has been suggested that in the leafcutter ant genus Atta, it is not the CHCs, but the alarm pheromone that is involved in the nestmate recognition process. In this study we used a laboratory population of Atta sexdens to explore the association between their CHC profile variation and intraspecific aggression. In the first part of the experiment, four colonies were divided into two groups with distinct diets to stimulate differentiation of their CHC profiles. In the second part of the experiment, all colonies received the same diet to examine resemblance of chemical profiles. At the end of each part of the experiment we extracted the CHCs from workers. The results demonstrated that colonies that shared the same food resource had similar cuticular hydrocarbon profiles. Furthermore, colonies were significantly more aggressive towards conspecifics that used a different foliar substrate and consequently had greater differences in their cuticular chemical composition. This study suggests that the CHC profiles of A. sexdens can be affected by the foliar substrates used, and that the CHCs are used in the nestmate recognition process of this species.

Dealing with water deficit in Atta ant colonies: large ants scout for water while small ants transport it.

Leafcutter ants (Atta sexdens rubropilosa) (Forel 1908) have an elaborate social organization, complete with caste divisions. Activities carried out by specialist groups contribute to the overall success and survival of the colony when it is confronted with environmental challenges such as dehydration. Ants detect variations in humidity inside the nest and react by activating several types of behavior that enhance water uptake and decrease water loss, but it is not clear whether or not a single caste collects water regardless of the cost of bringing this resource back to the colony. Accordingly, we investigated water collection activities in three colonies of Atta sexdens rubropilosa experimentally exposed to water stress. Specifically, we analyzed whether or not the same ant caste foraged for water, regardless of the absolute energetic cost (distance) of transporting this resource back to the colony. Our experimental design offered water sources at 0 m, 1 m and 10 m from the nest. We studied the body size of ants near the water sources from the initial offer of water (time  =  0) to 120 min, and tested for specialization. We observed a reduction in the average size and variance of ants that corroborated the specialization hypothesis. Although the temporal course of specialization changed with distance, the final outcome was similar among distances. Thus, we conclude that, for this species, a specialist (our use of the word "specialist" does not mean exclusive) task force is responsible for collecting water, regardless of the cost of transporting water back to the colony.

Chemical cuticular signature of leafcutter ant Atta sexdens (Hymenoptera, Formicidae) worker subcastes

ABSTRACT In leafcutter ants the division of labour is associated to worker size variation clustered in four subcastes. In this work we used Atta sexdens Forel (1908) as a model to test the hypothesis that each subcaste expresses its own chemical signature comprised of cuticular lipids. To assess it, we extracted epicuticular compounds by using nonpolar solvent hexane and analysed the samples in a combined Gas Chromatography-Mass Spectrometer (GC-MS). We found 24 hydrocarbons with carbon chains ranging from 19 to 39 atoms most of them classified as linear and branched alkanes. No compound occurred in the cuticle of specific worker subcaste, however, the relative proportion pattern varied greatly among them. Our results suggest that although subcastes have similar chemical signatures, significant differences in their relative proportions may play an important role between nestmate and group identification.

Study of anti-microbial and anti-pectinase activity of extracts and compounds isolated from Siphoneugena densiflora (Myrtaceae) and Vitex polygama (Lamiaceae) / Estudo das atividades antimicrobial e inibidora de pectinases dos extratos e compostos isolados de Siphoneugena densiflora (Myrtaceae) e Vitex polygama (Lamiaceae)

Extratos hidroalcoólicos (HA) e doze substâncias naturais isoladas de Siphoneugena densiflora O. Berg and Vitex polygama Cham. foram testadas in vitro como inibidores de crescimento do fungo Leucoagaricus gongylophorus (Singer) Möller, simbionte da formiga cortadeira Atta sexdens, e de suas pectinases. Os efeitos dos extratos e compostos também foram avaliados sobre várias bactérias e leveduras similares às espécies que vivem em simbiose com a formiga. Os extratos HA de S. densiflora exibiram os resultados antimicrobianos mais significativos, principalmente contra a Pseudomonas aeruginosa exibiram os resultados antimicrobianos mais significativos, principalmente contra a Pseudomonas aeruginosa. O 6-O Beta-D-glucopiranosídeo de cafeoila foi o composto mais promissor no controle do crescimento do L. gongylophorus.

Comparison of radial growth rate of the mutualistic fungus of atta sexdens rubropilosa forel in two culture media

In vitro culture of the mutualistic fungus of leaf-cutting ants is troublesome due to its low growth rate, which leads to storage problems and contaminants accumulation. This paper aims at comparing the radial growth rate of the mutualistic fungus of Atta sexdens rubropilosa Forel in two different culture media (Pagnocca B and MEA LP). Although total MEA LP radial growth was greater all along the bioassay, no significant difference was detected between growth efficiencies of the two media. Previous evidences of low growth rate for this fungus were confirmed. Since these data cannot point greater efficiency of one culture medium over the other, MEA LP medium is indicated for in vitro studies with this mutualistic fungus due its simpler composition and translucent color, making the analysis easier.

EFICIÊNCIA DE ISCAS GRANULADAS À BASE DE SULFLURAMIDA E DE CLORPIRIFÓS NO CONTROLE DE Atta sexdens sexdens(HYMENOPTERA: FORMICIDAE), NO TRÓPICO ÚMIDO

This research was developed in hibrid urograndis (Eucalyptus urophylla Eucalyptus grandis)plantation belonging to Jari Celulose S.A., in Pará State, Brazil, from August 1994 to January 1995. The objective was to test one granulated bait with sulfluramid and three others with chlorpirifos in three dosages compared to 10 grams of dodecachlor (0.45%) bait per square meter of ant nest against Atta sexdens sexdens(Hymenoptera: Formicidae). The bait with sulfluramid showed 77.78%; 100.00%; and 88.90% efficiency against A. sexdens sexdensat 6, 8, and 10 grams while the dodecachlor bait showed 100.00% control. The other baits and dosages showed efficiency below 45.00%.

Este trabalho foi realizado em plantios de eucalipto urograndis (Eucalyptus urophylla Eucalyptus grandis)da Jari Celulose S.A., no município de Almeirim, Pará, no período de agosto/94 a janeiro/95. Foi testada a eficiência de uma isca granulada com sulfluramida e de três outras com clorpirifós, em três dosagens cada por metro quadrado de formigueiro, no controle de Atta sexdens sexdens(Hymenoptera: Formicidae). Os resultados foram comparados com os de uma isca à base de dodecacloro (0,45%) a 10 gramas por metro quadrado de formigueiro. A isca granulada com sulfluramida a 6, 8 e 10 gramas por metro quadrado de formigueiro apresentou 77,78%, 100,00% e 88,90% de controle, respectivamente, assemelhando-se à isca a base de dodecacloro que apresentou 100,00% de formigueiros mortos. As outras iscas, em qualquer dosagem, apresentaram eficiência abaixo de 45,00%.

Busca de produtos naturais bioativos em plantas das famílias Myrtaceae (Siphoneugena densiflora Berg) e Verbenaceae (Vitex polygama Cham.) / Search for bioactive natural products in plants of the families Myrtaceae (densiflora Siphoneugena Berg) and Verbenaceae (Vitex polygama Cham.)

BUSCA DE PRODUTOS NATURAIS BIOATIVOS EM PLANTAS DAS FAMÍLIAS MYRTACEAE (Siphoneugena densiflora Berg) E VERBENACEAE (Vitex polygama Cham.). Neste trabalho estão sendo descritas trinta e sete substâncias identificadas de V. polygama Cham. e trinta e duas de S. densiflora Berg, provenientes do estudo fitoquímico de seus respectivos extratos. Destas, quatro substâncias isoladas de S. densiflora são destacadas por serem inéditas na literatura, a saber: 6ßhidroximaslinato de ßD-glucopiranosila; 4-O-α-L-2 -O-acetilramnopiranosídeo do ácido elágico e seu regioisômero com o grupo acetila na posição 3 e a siphoneugenina. São relatadas as atividades inibitórias enzimáticas de várias das substâncias identificadas, e de seus extratos de origem, sobre as enzimas gliceraldeído -fosfatodesidrogenase glicossomal (gGAPDH), de Trypanosoma cruzi; adenina fosforribosiltransferase (APRT), de Leishmania tarentolae; e pectinase, do fungo Leucoagaricus gongylophorus, simbionte da formiga cortadeira Atta sexdens rubropilosa. Dentre todas as substâncias testadas, os taninos castalagina e casuarinina se revelaram os mais promissores inibidores com valores de CI50 de 7,5 e 1,8 µM sobre a gGAPDH e 3,3 e 1,8 µM sobre a APRT, respectivamente. Como os taninos podem precipitar proteínas e causar uma ação inibitória não específica, foram realizados estudos sobre a interação deles com os reagentes dos ensaios enzimáticos. São relatadas as atuações biológicas in vitro de algumas substâncias sobre as formas tripomastigotas de T. cruzi; sobre as lagartas de 1º e 2º instar da mariposa, praga do milho, Spodoptera frugiperda; sobre as bactérias Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, Escherichia coli, Micrococcus roseus e sobre os fungos Leucoagaricus gongylophorus, Candida albicans, Cryptococcus laurentii, Sacharomyces cerevisae e Trichosporon cutaneum. A mistura dos flavonóis glicosilados 3-O + 4 -O-ß-D-glucopiranosil quercetina apresentou a maior atividade tripanocida (1,1 mM; 97 % lise contra 0,6 mM; 100 % lise da violeta genciana), ressaltando-se que sua CI50 sobre a gGAPDH foi de 20 µM. Os extratos metanólicos e hidrometanólicos dos galhos, caule, folhas e cascas da raiz de S. densiflora causaram 100 % de morte das lagartas de S. frugiperda, a uma concentração de 1000 ppm. Do extrato metanólico de folhas de S. densiflora foram isolados dois flavonóides: quercetina e quercitrina que, a uma concentração de 100 ppm, provocaram 78 e 85 % de morte das lagartas. Os taninos casuarinina e 4-O-α-raminopiranosídeo do ácido elágico, na mesma concentração, atuaram principalmente como fagoinibidores. O extrato hidrometanólico de folhas de V. polygama teve uma atividade inseticida de 60 % sobre S. frugiperda a uma concentração de 1000 ppm. Dele foram isoladas as misturas das flavonas di-C-glicosiladas carlinosídeo, schaftosídeo e seus respectivos isômeros, os quais apresentaram atividade inseticida relatada na literatura (SIMMONDS, 2001). O extrato hidrometanólico de folhas de S. densiflora apresentou uma atividade bactericida (halo de inibição de 11 mm) tão intensa quanto a do antibiótico tetraciclina (10 e 12 mm) para as bactérias P. aeruginosa e M. roseus. Das substâncias ensaiadas, apenas a mistura do cafeoil-6-O-a + ß-D-glicopiranosídeo foi relativamente ativa sobre o fungo L. gongylophorus causando 60 % de inibição de seu crescimento, a uma concentração de 50 µg/mL. Também está sendo narrado o desenvolvimento de um método, usando-se CLAE, para a análise e quantificação da 20-hidroxiecdisona presente no extrato metanólico de galhos de V. polygama, em virtude de suas inúmeras atividades biológicas e grande uso e importância na indústria farmacêutica.