Tribe Attini Research Articles

Microbe Profile: Pseudonocardia: antibiotics for every niche.

Pseudonocardia species comprise a genus of filamentous, sporulating bacteria belonging to the phylum Actinomycetota, formerly Actinobacteria. They are found in marine and freshwater sediments and soils and associated with marine animals, insects, and plants. To date, they have mostly been studied because of their mutually beneficial symbiosis with fungus-growing ants in the tribe Attini. They have also attracted interest due to their biosynthetic capabilities, including the production of variably glycosylated polyenes and other novel antifungal compounds, and for their capacity to grow on a variety of hydrocarbons. The majority of clinically used antibiotics are derived from the specialised metabolites of filamentous actinomycete bacteria and most of these come from the genus Streptomyces. However, in the quest for novel chemistry there is increasing interest in studying other filamentous actinomycete genera, including Pseudonocardia. Here we outline the biological properties, genome size and structure and key features of the genus Pseudonocardia, namely their specialised metabolites and ecological roles.

Unearthing a Cryptic Biosynthetic Gene Cluster for the Piperazic Acid-Bearing Depsipeptide Diperamycin in the Ant-Dweller Streptomyces sp. CS113.

Piperazic acid is a cyclic nonproteinogenic amino acid that contains a hydrazine N-N bond formed by a piperazate synthase (KtzT-like). This amino acid, found in bioactive natural products synthesized by non-ribosomal peptide synthetases (NRPSs), confers conformational constraint to peptides, an important feature for their biological activities. Genome mining of Streptomyces strains has been revealed as a strategy to identify biosynthetic gene clusters (BGCs) for potentially active compounds. Moreover, the isolation of new strains from underexplored habitats or associated with other organisms has allowed to uncover new BGCs for unknown compounds. The in-house "Carlos Sialer (CS)" strain collection consists of seventy-one Streptomyces strains isolated from the cuticle of leaf-cutting ants of the tribe Attini. Genomes from twelve of these strains have been sequenced and mined using bioinformatics tools, highlighting their potential to encode secondary metabolites. In this work, we have screened in silico those genomes, using KtzT as a hook to identify BGCs encoding piperazic acid-containing compounds. This resulted in uncovering the new BGC dpn in Streptomyces sp. CS113, which encodes the biosynthesis of the hybrid polyketide-depsipeptide diperamycin. Analysis of the diperamycin polyketide synthase (PKS) and NRPS reveals their functional similarity to those from the aurantimycin A biosynthetic pathway. Experimental proof linking the dpn BGC to its encoded compound was achieved by determining the growth conditions for the expression of the cluster and by inactivating the NRPS encoding gene dpnS2 and the piperazate synthase gene dpnZ. The identity of diperamycin was confirmed by High-Resolution Mass Spectrometry (HRMS) and Nuclear Magnetic Resonance (NMR) and by analysis of the domain composition of modules from the DpnP PKS and DpnS NRPS. The identification of the dpn BGC expands the number of BGCs that have been confirmed to encode the relatively scarcely represented BGCs for depsipeptides of the azinothricin family of compounds and will facilitate the generation of new-to-nature analogues by combinatorial biosynthesis.

Interactions among Escovopsis, Antagonistic Microfungi Associated with the Fungus-Growing Ant Symbiosis

Fungi in the genus Escovopsis (Ascomycota: Hypocreales) are prevalent associates of the complex symbiosis between fungus-growing ants (Tribe Attini), the ants’ cultivated basidiomycete fungi and a consortium of both beneficial and harmful microbes found within the ants’ garden communities. Some Escovopsis spp. have been shown to attack the ants’ cultivated fungi, and co-infections by multiple Escovopsis spp. are common in gardens in nature. Yet, little is known about how Escovopsis strains impact each other. Since microbe–microbe interactions play a central role in microbial ecology and evolution, we conducted experiments to assay the types of interactions that govern Escovopsis–Escovopsis relationships. We isolated Escovopsis strains from the gardens of 10 attine ant genera representing basal (lower) and derived groups in the attine ant phylogeny. We conducted in vitro experiments to determine the outcome of both intraclonal and interclonal Escovopsis confrontations. When paired with self (intraclonal interactions), Escovopsis isolated from lower attine colonies exhibited antagonistic (inhibitory) responses, while strains isolated from derived attine colonies exhibited neutral or mutualistic interactions, leading to a clear phylogenetic pattern of interaction outcome. Interclonal interactions were more varied, exhibiting less phylogenetic signal. These results can serve as the basis for future studies on the costs and benefits of Escovopsis coinfection, and on the genetic and chemical mechanisms that regulate the compatibility and incompatibility observed here.

Searching for Glycosylated Natural Products in Actinomycetes and Identification of Novel Macrolactams and Angucyclines.

Many bioactive natural products are glycosylated compounds in which the sugar components usually participate in interaction and molecular recognition of the cellular target. Therefore, the presence of sugar moieties is important, in some cases essential, for bioactivity. Searching for novel glycosylated bioactive compounds is an important aim in the field of the research for natural products from actinomycetes. A great majority of these sugar moieties belong to the 6-deoxyhexoses and share two common biosynthetic steps catalyzed by a NDP-D-glucose synthase (GS) and a NDP-D-glucose 4,6-dehydratase (DH). Based on this fact, seventy one Streptomyces strains isolated from the integument of ants of the Tribe Attini were screened for the presence of biosynthetic gene clusters (BGCs) for glycosylated compounds. Total DNAs were analyzed by PCR amplification using oligo primers for GSs and DHs and also for a NDP-D-glucose-2,3-dehydratases. Amplicons were used in gene disruption experiments to generate non-producing mutants in the corresponding clusters. Eleven mutants were obtained and comparative dereplication analyses between the wild type strains and the corresponding mutants allowed in some cases the identification of the compound coded by the corresponding cluster (lobophorins, vicenistatin, chromomycins and benzanthrins) and that of two novel macrolactams (sipanmycin A and B). Several strains did not show UPLC differential peaks between the wild type strain and mutant profiles. However, after genome sequencing of these strains, the activation of the expression of two clusters was achieved by using nutritional and genetic approaches leading to the identification of compounds of the cervimycins family and two novel members of the warkmycins family. Our work defines a useful strategy for the identification new glycosylated compounds by a combination of genome mining, gene inactivation experiments and the activation of silent biosynthetic clusters in Streptomyces strains.

Facultative mushroom feeding by common woodland ants (Formicidae, Aphaenogaster spp.)

Despite the remarkable diversity of food resources exploited by ants, fungi are rarely known to be part of their diet. The notable exception is the fungal-feeding specialists in the tribe Attini, which cultivate fungi inside their nests in what is one of the earliest forms of agriculture. Previous accounts of fungivory in ants outside these fungal-feeding specialists have been questioned due to whether or not ants consume fungal tissue or prey on mycophagous insect larvae present in or on mushrooms. Here we show that ants in the widespread genus Aphaenogaster recruit to mushroom baits in the field regardless of whether or not mushrooms contained insects upon which ants might prey. When dye-stained mushrooms were provided to colonies in the lab, ants fed on mushroom tissue and dye was visible throughout their digestive tract. Evidence of mushroom feeding in Aphaenogaster suggests that facultative fungal feeding is more common in ants than previously reported, including within the myrmecine clade that contains the attines. Previous accounts of fungivory in ants have been limited to fungal-feeding specialists, but mushroom feeding by Aphaenogaster shows that fungi can also be part of a generalist ant diet. By feeding on mushrooms and transporting mushroom tissues back to the nest, Aphaenogaster workers may also serve as dispersers of fungal spores.

Springtails (Collembola) Associated with Nests of Fungus-Growing Ants (Formicidae: Myrmicinae: Attini) in Southern Bahia, Brazil

Abstract Four species of Collembola were found in nests of the ant genera Myrmicocrypta, Trachymyrmex and Sericomyrmex of the tribe Attini (Formicidae: Myrmicinae). The panmyrmecophilic collembolan Cyphoderus (Lepidocyrtidae: Cyphoderinae) was the most abundant and was associated with all the ants. The collembolan genus Mucrosomia (Isotomidae: Anurophorinae) was found for the first time in ant nests. This is the first list of springtails found in nests of fungus-growing ants other than those belonging to the genera Atta and Acromyrmex (Formicidae: Myrmicinae).

Escovopsis trichodermoides sp. nov., isolated from a nest of the lower attine ant Mycocepurus goeldii.

Currently, five species are formally described in Escovopsis, a specialized mycoparasitic genus of fungus gardens of attine ants (Hymenoptera: Formicidae: tribe Attini). Four species were isolated from leaf-cutting ants in Brazil, including Escovopsis moelleri and Escovopsis microspora from nests of Acromyrmex subterraneus molestans, Escovopsis weberi from a nest of Atta sp. and Escovopsis lentecrescens from a nest of Acromyrmex subterraneus subterraneus. The fifth species, Escovopsis aspergilloides was isolated from a nest of the higher attine ant Trachymyrmex ruthae from Trinidad. Here, we describe a new species, Escovopsis trichodermoides isolated from a fungus garden of the lower attine ant Mycocepurus goeldii, which differs from the five other species by highly branched, trichoderma-like conidiophores lacking swollen vesicles, with reduced conidiogenous cells and distinctive conidia morphology. Phylogenetic analyses based on partial tef1 gene sequences support the distinctiveness of this species. A portion of the internal transcribed spacers of the nuclear rDNA was sequenced to serve as a DNA barcode. Future molecular and morphological studies in this group of fungi will certainly unravel the taxonomic diversity of Escovopsis associated with fungus-growing ants.

Banding Patterns of Three Leafcutter Ant Species of the GenusAtta(Formicidae: Myrmicinae) and Chromosomal Inferences

Among Neotropical ants, the genera Acromyrmex and Atta are of great importance because they include the major agricultural pest species. Regarding the genus Atta, considered one of the most derived of the tribe Attini, cytogenetic data exists for 5 taxa that present 2n = 22 chromosomes; however, information regarding banding patterns is only available for Atta colombica. Cytogenetics has contributed to several aspects related to the family For- micidae, in particular to the understanding of its evolution. Therefore, the aim of this study was to increase the cytogenetic data on Atta sexdens rubropilosa, Atta laevigata and Atta bisphaerica belonging to populations in the state of Minas Gerais, Brazil. The 3 species were characterized as having 2n = 22 chromosomes with a karyotypic formula of 2n = 18m + 2sm + 2st. Using the C-banding technique, small heterochromatic markings were observed in the centromeres of most of the chromosomes. The presence of GC-rich blocks in the interstitial region on the long arm of the fourth pair of metacentric chromosomes was indicated by CMA3 fluorochrome. The data from this study enabled a comparison with Acromyrmex stria- tus, a phylogenetically close species possessing the same chromosome number as the Atta species studied in this work, although clear differences were evident in the morphologies of 2 chromosome pairs and in the distribution and composition of heterochromatin. These results show the constancy of the chromosome number, morphology and banding pattern for the species studied, highlighting the importance of measuring the chromosomes to enable accurate comparison and using banding techniques to compare species of genera with a conserved chromosome number.

Colony size predicts division of labour in attine ants.

Division of labour is central to the ecological success of eusocial insects, yet the evolutionary factors driving increases in complexity in division of labour are little known. The size–complexity hypothesis proposes that, as larger colonies evolve, both non-reproductive and reproductive division of labour become more complex as workers and queens act to maximize inclusive fitness. Using a statistically robust phylogenetic comparative analysis of social and environmental traits of species within the ant tribe Attini, we show that colony size is positively related to both non-reproductive (worker size variation) and reproductive (queen–worker dimorphism) division of labour. The results also suggested that colony size acts on non-reproductive and reproductive division of labour in different ways. Environmental factors, including measures of variation in temperature and precipitation, had no significant effects on any division of labour measure or colony size. Overall, these results support the size–complexity hypothesis for the evolution of social complexity and division of labour in eusocial insects. Determining the evolutionary drivers of colony size may help contribute to our understanding of the evolution of social complexity.

Novel Phialophora species from leaf-cutting ants (tribe Attini)

Ants in the tribe Attini (Hymenoptera, Formicidae) maintain a 50 million-year-old lifestyle of co-evolution with symbiotic basidiomycetous fungi which they cultivate as essential source of nutrition. However, other microorganisms have been reported from ant habitats indicating a higher diversity of consistently associated species than established to date. Recently, black yeast-like fungi have been reported as a possible symbiont, like a competitor of the actinomycete bacteria that grow in the integument of the insects. During the mating season, gynes of Atta capiguara and A. laevigata were collected from nests located in Botucatu, SP, Brazil and sampled using flotation technique. Phylogenetic analysis based on ITS, partial 28S (LSU) and β-tubulin sequences revealed the occurrence of two novel species of Phialophora among the melanized fungi isolated.

Cytogenetic characterization of the ant Trachymyrmex fuscus Emery, 1934 (Formicidae: Myrmicinae: Attini) with the description of a chromosomal polymorphism

SummaryThe genus Trachymyrmex is a key group in the tribe Attini because of its close phylogenetic relationship to leaf-cutter ants, Acromyrmex and Atta. Cytogenetic data are only available for five taxa of Trachymyrmex, with chromosome numbers of 2n = 12, 18, 20 and 22, and morphology with predominantly metacentric chromosomes. The aim of the present study was to characterize the karyotype of the ant Trachymyrmex fuscus Emery, 1934, by means of the number and morphology of its chromosomes, heterochromatin pattern, CMA3 and DAPI fluorochromes in the population of two nests collected at Paraopeba, state of Minas Gerais, Brazil. Nineteen females presented 2n = 18 chromosomes (16m + 2sm) and a single male presented n = 9 (8m + 1sm). A size chromosomal polymorphism involving the short arm of the submetacentric pair was confirmed by statistical analysis, with three character conditions: heterozygous SB (with a difference in size between the short arms), standard SS (smaller short arms) and homozygote BB (bigger short arms). In the first nest, both SB and SS workers were observed. The other nest contained heterozygous (SB), homozygous (BB), and a male carrying the B chromosome (larger size). The presence of heterochromatin on all centromeric and pericentromeric chromosomes of T. fuscus suggests that the size difference observed in the submetacentric pair in the SB and BB workers is not related to the heterochromatin but to a duplication of euchromatic regions through intra- or inter- chromosomal rearrangements. The fluorochrome CMA3 matched the C-banding markings, indicating that the heterochromatin is rich in GC base pairs. As far as we know, this is the first chromosomal polymorphism reported in the tribe Attini.

Cyatta abscondita: Taxonomy, Evolution, and Natural History of a New Fungus-Farming Ant Genus from Brazil

Cyatta abscondita, a new genus and species of fungus-farming ant from Brazil, is described based on morphological study of more than 20 workers, two dealate gynes, one male, and two larvae. Ecological field data are summarized, including natural history, nest architecture, and foraging behavior. Phylogenetic analyses of DNA sequence data from four nuclear genes indicate that Cyatta abscondita is the distant sister taxon of the genus Kalathomyrmex, and that together they comprise the sister group of the remaining neoattine ants, an informal clade that includes the conspicuous and well-known leaf-cutter ants. Morphologically, Cyatta abscondita shares very few obvious character states with Kalathomyrmex. It does, however, possess a number of striking morphological features unique within the fungus-farming tribe Attini. It also shares morphological character states with taxa that span the ancestral node of the Attini. The morphology, behavior, and other biological characters of Cyatta abscondita are potentially informative about plesiomorphic character states within the fungus-farming ants and about the early evolution of ant agriculture.

Nest architecture, fungus gardens, queen, males and larvae of the fungus-growing ant Mycetagroicus inflatus Brandão & Mayhé-Nunes

All known fungus-growing ants (tribe Attini) are obligately symbiotic with their cultivated fungi. The fungal cultivars of “lower” attine ants are facultative symbionts, capable of living apart from ants, whereas the fungal cultivars of “higher” attine ants, including leaf-cutting genera Atta and Acromyrmex, are highly specialized, obligate symbionts. Since higher attine ants and fungi are derived from lower attine ants and fungi, understanding the evolutionary transition from lower to higher attine agriculture requires understanding the historical sequence of change in both ants and fungi. The biology of the poorly known ant genus Mycetagroicus is of special interest in this regard because it occupies a phylogenetic position intermediate between lower and higher ant agriculture. Here, based on the excavations of four nests in Pará, Brazil, we report the first biological data for the recently described species Mycetagroicus inflatus, including the first descriptions of Mycetagroicus males and larvae. Like M. cerradensis, the only other species in the genus for which nesting biology is known, the garden chambers of M. inflatus are unusually deep and the garden is most likely relocated vertically in rainy and dry seasons. Due to the proximity of nests to the Araguaia River, it is likely that even the uppermost chambers and nest entrances of M. inflatus are submerged during the rainy season. Most remarkably, all three examined colonies of M. inflatus cultivate the same fungal species as their congener, M. cerradensis, over 1,000 km away, raising the possibility of long-term symbiont fidelity spanning speciation events within the genus.

Mandibular and postpharyngeal gland secretions of <i>Acromyrmex landolti</i> (Hymenoptera: Formicidae) as chemical cues for nestmate recognition

Nestmate recognition is fundamental for the cohesiveness of the colony in social insects. Acromyrmex landolti is a higher Attini, a leaf-cutter ant with largest nest and morphological castes. We studied the location of the recognition signal of A. landolti on the whole ant body, the isolation effect on the capacity of nestmate recognition, and the role of the mandibular (MG) and post-pharyngeal glands (PPG) as putative sources for chemical recognition signals in this species. We performed behavioral bioassays and chemical analyses of MG and PPG glands; MG volatiles in different castes were also characterized. In order to determine the occurrence of nestmate recognition signals, behavioral tests were performed for which the whole body, body parts or gland extracts from a nestmate and an alien worker ant, were placed on the central axis of an active field trail. Besides, the isolation effect of the nestmate chemical signal persistence was evaluated by repeating the experiments with whole ant bodies after 12 h, 24 h and 48 h of isolation. The agonistic behavior of the ants on the trail was classified as inspection, threatening and biting. Gland volatiles were obtained by headspace solid phase microextraction, and PPG by solid sample analyses; and chemical analyses were performed using gas chromatography-mass spectrometry. Workers performed agonistic behaviors more frequently towards non-nestmate whole bodies, body parts and gland extracts, primarily those from the post-pharyngeal glands. Nestmate thoraces odorized with alien extracts were also recognized from aliens. Nestmate recognition ability persists for 12 h and 24 h after isolation, but decreases after 48 h, suggesting that the chemical signal changes over time. Inter-colonial chemical analyses showed no differences in the PPG hydrocarbon profiles, while the relative proportions of MG compounds showed significant differences between colonies. These results showed that nestmate recognition was achieved by MG volatiles. Possibly, the PPG secretion works as a matrix that absorbs MG volatiles. We report, for the first time, some mandibular gland compounds for the genus Acromyrmex and for the tribe Attini group.

Cytogenetic and Molecular Analyses Reveal a Divergence between Acromyrmex striatus (Roger, 1863) and Other Congeneric Species: Taxonomic Implications

The leafcutter ants, which consist of Acromyrmex and Atta genera, are restricted to the New World and they are considered the main herbivores in the neotropics. Cytogenetic studies of leafcutter ants are available for five species of Atta and 14 species of Acromyrmex, both including subspecies. These two ant genera have a constant karyotype with a diploid number of 22 and 38 chromosomes, respectively. The most distinct Acromyrmex species from Brazil is A. striatus, which is restricted to the southern states of Santa Catarina and Rio Grande do Sul. Several cytogenetic and phylogenetic studies have been conducted with ants, but the karyotypic characterization and phylogenetic position of this species relative to leafcutter ants remains unknown. In this study, we report a diploid number of 22 chromosomes for A. striatus. The phylogenetic relationship between A. striatus and other leafcutter ants was estimated based on the four nuclear genes. A. striatus shared the same chromosome number as Atta species and the majority of metacentric chromosomes. Nuclear data generated a phylogenetic tree with a well-supported cluster, where A. striatus formed a different clade from other Acromyrmex spp. This combination of cytogenetic and molecular approaches provided interesting insights into the phylogenetic position of A. striatus among the leafcutter ants and the tribe Attini.

Cytogenetic studies of five taxa of the tribe Attini (Formicidae: Myrmicinae)

Cytogenetic studies have been carried out on more than 750 ant taxa and are an important tool in evolutionary, taxonomic and phylogenetic studies. However, less than 10% of the species reported in the tribe Attini have been studied. The aim of the present study was to describe the chromosomes of five attine ants collected in Vicosa, Minas Gerais state, Brazil, at present unknown. The ant karyotypes reported are: Sericomyrmex sp. (2n = 50, 44m + 6sm, n = 25, 22m + 3sm); Trachymyrmex relictus (2n = 20m); Trachymyrmex sp. (2n = 22, 18m + 4sm); Apterostigma madidiense (n = 23, 7m + 10sm + 5st + 1a) and Apterostigma steigeri (2n = 22, 20m + 2sm). C-banding showed that heterochromatin was present in the centromeric region of all chromosomes of T. relictus. Future cytogenetic studies on members of the tribe Attini will provide important information to discuss chromosome evolution in this ant group.

Specialized Fungal Parasites and Opportunistic Fungi in Gardens of Attine Ants

Ants in the tribe Attini (Hymenoptera: Formicidae) comprise about 230 described species that share the same characteristic: all coevolved in an ancient mutualism with basidiomycetous fungi cultivated for food. In this paper we focused on fungi other than the mutualistic cultivar and their roles in the attine ant symbiosis. Specialized fungal parasites in the genusEscovopsisnegatively impact the fungus gardens. Many fungal parasites may have small impacts on the ants' fungal colony when the colony is balanced, but then may opportunistically shift to having large impacts if the ants' colony becomes unbalanced.

First evidence of an intimate symbiotic association between fungi and larvae in basal attine ants

The most conspicuous ants in all of tropical America are those that belong to the tribe Attini which cultivate fungus. The objective of the present study is to verify the alterations that occur in the cuticle of the worker larvae from Myrmicocrypta, Mycetarotes and Trachymyrmex with the purpose of trying to establish the degree of this association. Attine ants from the Atta genus were used as a control group. The analysis of histological sections showed results about the distribution of the cells and tissues on the epidermis of A. sexdens rubropilosa ant larvae and Mycetarotes parallelus, Trachymyrmex fuscus and Myrmicocrypta sp. A cuticle covering the ants was observed in the larvae of all the species, and this is formed by a simple cubic epithelium, whose cells possibly change its shape to prismatic, depending on their secretory activity. Just under this epithelium large adipose cells with reserve granules in their cytoplasm were found. The presence of a space filled by granulose and acellular material was also observed, indicating that the larvae were in a molt period. The presence of fungal hyphae is observed both on the external side of the basal attine larvae as well as emitting projections to the interior of the cuticle reaching the epithelium and the adipocyte cells. Data obtained in the present study demonstrated that the fungus deposited on the surface of immature ants from attine basal species maintain a close relationship with them, once the fungus hyphae have the ability to disorganize the cuticle lamellas, penetrating the interior of the insect cells through the emission of prolongations transporting the cuticle and epithelium barriers and making substances exchanges between larvae and fungus.

Fungus-growing Allomerus ants are associated with antibiotic-producing actinobacteria

Fungus-growing attine ants use natural-product antibiotics produced by mutualist actinobacteria as 'weedkillers' in their fungal gardens. Here we report for the first time that fungus-growing Allomerus ants, which lie outside the tribe Attini, are associated with antifungal-producing actinobacteria, which offer them protection against non-cultivar fungi isolated from their ant-plants.

Expressed sequence tags from Atta laevigata and identification of candidate genes for the control of pest leaf-cutting ants

BackgroundLeafcutters are the highest evolved within Neotropical ants in the tribe Attini and model systems for studying caste formation, labor division and symbiosis with microorganisms. Some species of leafcutters are agricultural pests controlled by chemicals which affect other animals and accumulate in the environment. Aiming to provide genetic basis for the study of leafcutters and for the development of more specific and environmentally friendly methods for the control of pest leafcutters, we generated expressed sequence tag data from Atta laevigata, one of the pest ants with broad geographic distribution in South America.ResultsThe analysis of the expressed sequence tags allowed us to characterize 2,006 unique sequences in Atta laevigata. Sixteen of these genes had a high number of transcripts and are likely positively selected for high level of gene expression, being responsible for three basic biological functions: energy conservation through redox reactions in mitochondria; cytoskeleton and muscle structuring; regulation of gene expression and metabolism. Based on leafcutters lifestyle and reports of genes involved in key processes of other social insects, we identified 146 sequences potential targets for controlling pest leafcutters. The targets are responsible for antixenobiosis, development and longevity, immunity, resistance to pathogens, pheromone function, cell signaling, behavior, polysaccharide metabolism and arginine kynase activity.ConclusionThe generation and analysis of expressed sequence tags from Atta laevigata have provided important genetic basis for future studies on the biology of leaf-cutting ants and may contribute to the development of a more specific and environmentally friendly method for the control of agricultural pest leafcutters.