Sequencing, assembly, and genomic annotation of Leucoagaricus gongylophorus LEU18496, a dikarya mutualistic species.

The basidiomycete fungus Leucoagaricus gongylophorus is able to grow in the fungus garden of leaf-cutter ants. This mutualistic interaction has driven the evolutionary adaptation of L. gongylophorus, shaping its metabolism to produce enzymes adept at lignocellulosic biomass degradation. In this study, we undertook the comprehensive sequencing, assembly, and functional annotation of the genome of L. gongylophorus strain LEU18496, mutualistic fungus of the Atta mexicana. Our genomic analyses revealed a distinctive bimodal nature to the genome: a predominant region characterized by AT enrichment and low genetic density, alongside a smaller region exhibiting higher GC content and higher genetic density. The presence of transposable elements (TEs) within the AT-enriched region suggests genomic compartmentalization, facilitating differential evolutionary rates. With a gene count of 6748, the assembled genome of L. gongylophorus LEU18496 surpasses previous reports for this fungal species. Inspection of genes associated with central metabolism unveiled a remarkable abundance of carbohydrate-active enzymes (CAZymes) and fungal oxidative lignin enzymes (FOLymes), underscoring their pivotal roles in the life cycle of this fungus.

Long-term strict ant-plant mutualism identity characterises growth rate and leaf shearing resistance of an Amazonian myrmecophyte.

Over 125 million years of ant-plant interactions have culminated in one of the most intriguing evolutionary outcomes in life history. The myrmecophyte Duroia hirsuta (Rubiaceae) is known for its mutualistic association with the ant Myrmelachista schumanni and several other species, mainly Azteca, in the north-western Amazon. While both ants provide indirect defences to plants, only M. schumanni nests in plant domatia and has the unique behaviour of clearing the surroundings of its host tree from heterospecific plants, potentially increasing resource availability to its host. Using a 12-year survey, we asked how the continuous presence of either only M. schumanni or only Azteca spp. benefits the growth and defence traits of host trees. We found that the continuous presence of M. schumanni improved relative growth rates and leaf shearing resistance of Duroia better than trees with Azteca. However, leaf herbivory, dry matter content, trichome density, and secondary metabolite production were the same in all trees. Survival depended directly on ant association (> 94% of trees died when ants were absent). This study extends our understanding of the long-term effects of strict ant-plant mutualism on host plant traits in the field and reinforces the use of D. hirsuta-M. schumanni as a model system suitable for eco-co-evolutionary research on plant-animal interactions.

Coastal dunes as drivers of genetic differentiation in the honeypot ant Myrmecocystus baja (Formicidae: Formicinae).

Coastal dunes are unique habitats, threatened by human activities. In biogeographical terms, coastal dunes are habitat islands, being discrete and distinct patches of similar habitat among themselves, separated from each other by a different type of habitat. Furthermore, coastal dunes harbor endemic species, adapted to living solely in the habitats found on specific dune systems. For example, the honeypot ant Myrmecocystus baja is endemic and restricted to coastal dunes of Mexico's Baja California Pacific coast. This ecological and biogeographical scenario led to the questions whether their geographical isolation is reflected in their genetic diversity and structuring, and how their demographic history is related with the formation of the dune system habitats. To answer these questions, population genetic, isolation-with-migration, and phylogeographical analyses were carried out, based on mitochondrial and five nuclear intronic markers. Minimal gene flow was detected only between two of the dune systems sampled; otherwise, the M. baja populations were found to be isolated and genetically structured, and their divergence generally pre-dated the modern-day dune systems. It is therefore highly likely that these ants were already present in paleodunes and that each of the populations was established from founder populations as the dunes formed. These findings highlight the importance of coastal dunes for species such as the honeypot ant from Baja California, in promoting genetic differentiation.

Evolutionary déjà vu? A case of convergent evolution in an ant-plant association.

Obligatory ant-plant symbioses often appear to be single evolutionary shifts within particular ant lineages; however, convergence can be revealed once natural history observations are complemented with molecular phylogenetics. Here, we describe a remarkable example of convergent evolution in an ant-plant symbiotic system. Exclusively arboreal, Myrmelachista species can be generalized opportunists nesting in several plant species or obligately symbiotic, live-stem nesters of a narrow set of plant species. Instances of specialization within Myrmelachista are known from northern South America and throughout Middle America. In Middle America, a diverse radiation of specialists occupies understory treelets of lowland rainforests. The morphological and behavioural uniformity of specialists suggests that they form a monophyletic assemblage, diversifying after a single origin of specialization. Using ultraconserved element phylogenomics and ancestral state reconstructions, we show that shifts from opportunistic to obligately symbiotic evolved independently in South and Middle America. Furthermore, our analyses support a remarkable case of convergence within the Middle American radiation, with two independently evolved specialist clades, arising nearly simultaneously from putative opportunistic ancestors during the late Pliocene. This repeated evolution of a complex phenotype suggests similar mechanisms behind trait shifts from opportunists to specialists, generating further questions about the selective forces driving specialization.

Nasturtium leaf compounds, diphenyl disulfide and lyral, against Atta sexdens (Hymenoptera: Formicidae) and their symbiotic fungi.

Social insect pests, particularly leaf-cutting ants, present a considerable challenge in terms of control. Leaf-cutting ants are significant agricultural, forestry, and pasture pests, and understanding their behavior and defense mechanisms is essential for managing their colonies effectively. While toxic ant baits are a primary control method, the limited availability of effective insecticides and concerns over their hazardous nature has spurred the search for alternative solutions, particularly natural compounds, which aligns with the goals of forest certification groups. In the light of previous evidence demonstrating the efficacy of nasturtium leaves (Tropaeolum majus L. (Brassicales: Tropaeolaceae)) in suppressing leaf-cutting ant colonies, this study investigates 2 active components of nasturtium leaf extracts: diphenyl disulfide and lyral. We tested their impact on Atta sexdens (L.) (Hymenoptera: Formicidae), the most prevalent leaf-cutter ant species in Brazil, and their symbiotic fungus, Leucoagaricus gongylophorus (Möller) Singer (Agaricales: Agaricaceae). We conducted experiments with increasing concentrations of diphenyl disulfide and lyral, assessing their effects on the symbiotic fungus and on forager workers and gardeners of A. sexdens colonies. Our findings revealed no fungicidal activity, and ant mortality was minimal in both topical and ingestion bioassays with the exception of gardeners topically exposed to diphenyl sulfide. Furthermore, the compounds did not affect leaf ingestion, but diphenyl disulfide did increase interactions among foragers. These results suggest that neither diphenyl disulfide nor lyral are the primary contributors to the suppression of leaf-cutting ant colonies by nasturtium leaves. However, they may enhance the formicidal activity of other compounds present in nasturtium leaves.

Antibacterial activity of crude extracts of Camponotus compressus (Fabricius, 1787) (Hymenoptera: Formicidae).

The current study evaluates the antibacterial activity of Camponotus compressus (Hymenoptera: Formicidae) body crude extracts. The increasing antibiotic resistance of bacteria has prompted the world to turn its attention towards insects in the search for new sources of antibacterial compounds. The body crude extract obtained with different solvents were tested against both Gram positive (Staphylococcus aureus, Bacillus subtilis) and Gram negative bacteria (Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae). Standard disc diffusion method was used to perform the activity. The extracts of C. compressus were investigated for their effectiveness against all resistant pathogenic bacteria. Staphylococcus aureus was found to be the most susceptible, exhibiting a high average growth inhibition, while Bacillus subtilis showed a lower average growth inhibition zone. Our findings regarding the inhibitory effect of C. compressus extracts show the presence of a broad-spectrum antibacterial compound. This will be helpful in the search for novel natural antibiotics against robust pathogenic bacterial strains.

With the dead under the mat: the zombie ant extended phenotype under a new perspective.

Some parasitic fungi can increase fitness by modifying the behavior of their hosts. These behaviors are known as extended phenotypes because they favor parasitic gene propagation. Here, we studied three lineages of Ophiocordyceps, a fungus that infects ants, altering their conduct before death. According to fungal strategy, ants may die in leaf litter, with entwined legs in branches, under the moss mat, or biting plant tissue. It is critical for parasites that the corpses stay at these places because Ophiocordyceps exhibit iteroparity, possibly releasing spores in multiple life cycles. Thus, we assumed substrate cadaver permanence as a fungi reproductive proxy and corpse height as a proxy of cadaver removal. We hypothesize that biting vegetation and dying in higher places may increase the permanence of ant corpses while avoiding possible corpse predation on the forest floor. We monitored over a year more than 4000 zombie ants in approximately 15 km2 of undisturbed tropical forest in central Amazonia. Our results show a longer permanence of corpses with increasing ground height, suggesting that the parasites may have better chances of releasing spores and infecting new hosts at these places. We found that the zombie ants that last longer on the substrate die under the moss mat in tree trunks, not necessarily biting vegetation. The biting behavior appears to be the most derived and complex mechanism among Ophiocordyceps syndromes. Our results put these findings under a new perspective, proposing that seemingly less complex behavioral changes are ecologically equivalent and adaptative for other parasite lineages.

Scavenging in two mountain ecosystems: Distinctive contribution of ants in grassland and non-ant invertebrates in forest.

Scavenging is a key process for the cycling of nutrients in ecosystems, yet it is still neglected in the ecological literature. Apart from the importance of specific groups of animals in scavenging, there have been few ecological studies that compare them. Furthermore, the ecological studies on scavenging have mainly focused on vertebrates despite the crucial importance of invertebrates in this process. Here, we performed a large-scale ant suppression and vertebrate exclusion experiment to quantify the relative contribution of ants, non-ant invertebrates and vertebrates in scavenging nitrogen-rich (insect carcasses) and carbon-rich (seeds) baits in two contrasting mountainous habitats in Brazil (grasslands and forests). Overall, bait removal was 23.2% higher in forests than in grasslands. Ants were the primary scavengers in grasslands, responsible for more than 57% of dead insect larvae and seed removal, while, in forests, non-ant invertebrates dominated, removing nearly 65% of all baits. Vertebrates had a minor role in scavenging dead insect larvae and seeds in both habitats, with <4% of removals. Furthermore, our results show that animal-based baits were more consumed in forests than seeds, and both resources were equally consumed in grasslands. Therefore, we demonstrate the superiority of invertebrates in this process, with a particular emphasis on the irreplaceable role of ants, especially in this grassland ecosystem. As such, we further advance our knowledge of a key ecosystem process, showing the relative importance of three major groups in scavenging and the differences in ecosystems functioning between two contrasting tropical habitats.

A Neoponera villosa (Fabricius, 1804) Worker Pupa with too Many Legs (Hymenoptera: Formicidae).

Several somatic and sexual anomalies such as gynandromorphism and other morphological malformations have been described in ants, but cases of supernumerary legs in natural populations are rare, with only six cases reported to date. Moreover, few cases of abnormalities have been reported for the Ponerinae subfamily, with only three cases of genetic chimeras and five teratological cases worldwide. We report here a new case of teratology in this subfamily, with a seven-legged worker pupa found in a Neoponera villosa colony nesting in an epiphytic tank bromeliad. This is the second case for Mexico of a morphological anomaly in a ponerine ant, but the first report of a teratological case in the subfamily for this country.

Taxon-dependent diversity response along a temperate elevation gradient covered by grassland.

Elevational gradients constitute excellent systems for understanding the mechanisms that generate and maintain global biodiversity patterns. Climatic gradients associated with elevation show strong influence on species distribution in mountains. The study of mountains covered by the same habitat type is an ideal scenario to compare alternatives to the energy hypotheses. Our aim was to investigate how changes in climatic conditions along the elevational gradient drive α- and ß-diversity of four taxa in a mountain system located within a grassland biome. We sampled ants, spiders, birds and plants, and measured climatic variables at six elevational bands (with 10 sampling sites each) established between 470 and 1,000 masl on a mountain from the Ventania Mountain System, Argentina. Species richness per site and ß-diversity (turnover and nestedness) between the lowest band and upper sites were estimated. For most taxa, species richness declined at high elevations and energy, through temperature, was the major driver of species richness for ants, plants and birds, prevailing over productivity and water availability. The major ß-diversity component was turnover for plants, spiders and birds, and nestedness for ants. The unique environmental conditions of the upper bands could favour the occurrence of specialist and endemic species.

Considering climate change impact on the global potential geographical distribution of the invasive Argentine ant and little fire ant.

The Argentine ant (Linepithema humile) and the little fire ant (Wasmannia auropunctata) are among the top 100 invasive alien species globally, causing significant ecological and economic harm. Therefore, it is crucial to study their potential geographic distribution worldwide. This study aimed to predict their global distribution under current and future climate conditions. We used distribution data from various sources, including CABI, GBIF, and PIAKey, and key climate variables selected from 19 environmental factors to model their potential geographic distribution using MaxEnt. The AUC values were 0.925 and 0.937 for L. humile and W. auropunctata, respectively, indicating good predictive performance. Suitable areas for L. humile were mainly in southern North America, northern South America, Europe, central Asia, southern Oceania, and parts of Africa, while W. auropunctata suitable areas were mostly in southern North America, most of South America, a small part of Europe, southern Asia, central Africa, and some parts of Oceania. Under climate change scenario, suitable areas for L. humile increased, while highly suitable areas for W. auropunctata decreased. The top four countries with the largest areas of overlapping suitable habitat under current climate were Brazil, China, Australia, and Argentina, while under future SSP585 climate scenario, the top four countries were Brazil, China, Indonesia, and Argentina. Some countries, such as Estonia and Finland, will see an overlapping adaptation area under climate change. In conclusion, this study provides insight into controlling the spread and harm of L. humile and W. auropunctata.

Low Intraspecific Aggression Level, Cuticular Hydrocarbons, and Polydomy in the Bullet Ant.

Ants use chemical cues known as cuticular hydrocarbons (CHCs) for both intraspecific and interspecific recognition. These compounds serve ants in distinguishing between nestmates and non-nestmates, enabling them to coexist in polydomous colonies characterized by socially connected yet spatially separated nests. Hence, the aim of this study was to investigate the intraspecific aggression level between nestmates and non-nestmates of the bullet ant Paraponera clavata (Fabricius, 1775), analyze and compare their CHCs, and evaluate the occurrence of polydomy in this species. We conducted aggression tests between foragers, both in laboratory and field settings. To identify the chemical profiles, we utilized gas chromatography coupled with mass spectrometry (GC-MS). We marked the foragers found at nest entrances and subsequently recaptured these marked ants to validate workers exchange among nests. Across all nests, a low intraspecific aggression level was observed within the same area. However, a significant difference in aggression correlated to distance between nests. Analysis of the cuticular chemical profile of P. clavata unveiled colony-specific CHCs, both qualitatively and quantitatively. Notably, we observed instances of ants from certain nests entering or exiting different nests. This behavior, in conjunction with the observed low intraspecific aggression despite differences in CHCs suggests polydomy for this species. Polydomy can offer several benefits, including risk spreading, efficient exploitation of resources, potential for colony size increasing and reduced costs associated with foraging and competition.

Isocycloseram: A new active ingredient for leaf-cutting ants control.

Leaf-cutting ants are the most important pests in several cropping systems in the Neotropics. Granulated baits containing active ingredients, considered hazardous by the Stockholm Convention, are the usual method to control these ants. Isocycloseram is a new insecticide molecule with high safety margin for mammals, but without registration for the ants in general. Thus, this study investigated the effectiveness of granulated baits with isocycloseram in leaf-cutting ants control under laboratory and field conditions. Initially, the mortality of Atta sexdens workers, fed with dehydrated citrus pulp paste containing different concentrations of isocycloseram was evaluated in the laboratory for 21 days, for toxicological classification. Subsequently, the loading, devolution, and incorporation of baits with different concentrations of isocycloseram and the mortality of A. sexdens colonies were evaluated in the laboratory. After that, the percentages of loading and devolution of baits, foraging activity, and colony mortality treated with 0.05, 0.1, 0.2, and 0.3% of isocycloseram were evaluated for the species A. sexdens, A. laevigata, and Acromyrmex lundii in field conditions. All concentrations of isocycloseram killed more than 15% of ants in 24 h and more than 90% in 21 days in the laboratory, being classified as a fast-acting and highly effective active ingredient. Baits with 0.001 to 0.03% of isocycloseram were highly loaded and exhibited low rate of devolution. The mortality of A. sexdens colony was higher at concentrations between 0.075 and 0.3%, in the laboratory. Baits containing isocycloseram at concentrations of 0.2 and 0.3% were highly loaded, presented low devolution rates, and were highly efficient in controlling A. sexdens, A. laevigata, and A. lundii in the field, at dosages of 6, 10, and 12 g/m² of nest. This is the first report of the use of isocycloseram against leaf-cutting ants, contributing to the development of efficient and toxicologically safer ant baits.

Leafcutter ants enhance microbial drought resilience in tropical forest soil.

We conducted a research campaign in a neotropical rainforest in Costa Rica throughout the drought phase of an El-Nino Southern Oscillation event to determine microbial community dynamics and soil C fluxes. Our study included nests of the leafcutter ant Atta cephalotes, as soil disturbances made by these ecosystem engineers may influence microbial drought response. Drought decreased the diversity of microbes and the abundance of core microbiome taxa, including Verrucomicrobial bacteria and Sordariomycete fungi. Despite initial responses of decreasing diversity and altered composition, 6 months post-drought the microbiomes were similar to pre-drought conditions, demonstrating the resilience of soil microbial communities to drought events. A. cephalotes nests altered fungal composition in the surrounding soil, and reduced both fungal mortality and growth of Acidobacteria post-drought. Drought increased CH4 consumption in soils due to lower soil moisture, and A. cephalotes nests decrease the variability of CH4 emissions in some soil types. CH4 emissions were tracked by the abundance of methanotrophic bacteria and fungal composition. These results characterize the microbiome of tropical soils across both time and space during drought and provide evidence for the importance of leafcutter ant nests in shaping soil microbiomes and enhancing microbial resilience during climatic perturbations.

Influence of Host Plants and Tending Ants on the Cuticular Hydrocarbon Profile of a Generalist Myrmecophilous Caterpillar.

In myrmecophilous organisms, which live in symbiosis with ants, cuticular hydrocarbons (CHCs) play a pivotal role in interspecific communication and defense against chemical-oriented predators. Although these interactions form complex information webs, little is known about the influence of biotic environmental factors on the CHC profiles of myrmecophiles. Here, we analyzed the effect of different host plants and tending ants on the larval CHC profile of Synargis calyce (Lepidoptera: Riodinidae), a polyphagous species with facultative myrmecophily. Groups of caterpillars were fed individually with three host plant species (without tending ants), and with two tending ant species. Through gas chromatography analysis, we compared the cuticular profiles of treatments and found a high similarity between plants and caterpillars (65-82%), but a low similarity between caterpillars and their tending ants (30 - 25%). Cluster analysis showed that caterpillars, ants, and plants form distinct groups, indicating that S. calyce caterpillars have their own chemical profile. These results are similar to those observed for Lycaenidae caterpillars indicating that there is functional convergence in the chemical strategies used by myrmecophilous caterpillar species with similar ecology. Also, the results suggest that the cuticular compounds of S. calyce are primarily influenced by their host plants rather than their tending ants. Thus, we propose that these caterpillars present a trade-off between camouflage and directly informing their presence to ants, maintaining their unique chemical profile, though slightly affected by biotic environmental factors.

LncRNA gene ANTSR coordinates complementary sex determination in the Argentine ant.

Animals have evolved various sex determination systems. Here, we describe a newly found mechanism. A long noncoding RNA (lncRNA) transduces complementary sex determination (CSD) signal in the invasive Argentine ant. In this haplodiploid species, we identified a 5-kilobase hyper-polymorphic region underlying CSD: Heterozygous embryos become females, while homozygous and hemizygous embryos become males. Heterozygosity at the CSD locus correlates with higher expression of ANTSR, a gene that overlaps with the CSD locus and specifies an lncRNA transcript. ANTSR knockdown in CSD heterozygotes leads to male development. Comparative analyses indicated that, in Hymenoptera, ANTSR is an ancient yet rapidly evolving gene. This study reveals an lncRNA involved in genetic sex determination, alongside a previously unknown regulatory mechanism underlying sex determination based on complementarity among noncoding alleles.

Semi-arid's Unsung Heroes: Hymenoptera and the Vital Ecosystem Services Enabled by Encholirium spectabile, a Rupicolous Bromeliad in the Brazilian Semi-arid Region.

The concept of Ecosystem Services (ES) recognizes the importance of natural ecosystems in supporting human well-being. Hymenoptera, a diverse group of insects including ants, bees, and wasps, play crucial roles in providing ESs. Despite their significance, the provision of ESs by Hymenoptera is often undervalued, leading to ecosystem degradation and loss of important services. This study focuses on the association between Hymenoptera and a rupicolous bromeliad species (Encholirium spectabile) and explores the ESs promoted directly and indirectly by these insects. The study area is located in the Caatinga region of Brazil, characterized by irregular rainfall and a dry season. The results show that Hymenoptera, particularly bees, ants, and wasps, provide a range of ESs including pollination, honey production, pest control, cultural symbolism, and educational value. These services are vital for plant reproduction, food production, and ecosystem functioning in both seasons; there are no differences in species richness between seasons, but rather in species composition. Understanding the importance of Hymenoptera for ESs is crucial for informing conservation and management practices to ensure the sustainability of natural ecosystems. The study highlights the need for conservation actions to protect the intricate ecological relationships between Hymenoptera and bromeliads, which indirectly support ESs by providing habitat and resources, especially during droughts when resources are scarce in the region. By recognizing the importance of bromeliads in supporting Hymenopteran communities, conservation efforts can focus on preserving these critical ecological interactions and maintaining ES provision.

Hydrocarbons in Formicidae: influence of chemical footprints on ant behavioral strategies.

When an insect walks, it leaves chemical cues that derive from the arolium, a tarsal structure. These cues may contain important information about other species that occur in their community and can then mediate interactions of competition, predation, and information about resources with ants from their own colony. The compounds of these cues are released into the substrate in the form of chemical footprints. There are still few species studied, and little is known about the behavior of ants regarding these signals and how they use them in their interactions. Therefore, the aim of this study was to assess the behavioral strategy of different ant species when confronted with chemical footprints left by other ants, as well as identify their compounds and their relationship with the cuticular hydrocarbon profile. The experiments were performed using a Y-maze, where in one of the arms, there were chemical footprints of their own species or of other species, and the other Y arm was footprint-free. The chemical compounds of footprints and cuticle were analyzed by gas chromatography-mass spectrometry. The results show that foragers of all species detect and respond to the presence of chemical cues in the form of footprints left by other ants. Foragers of all species followed footprints of individuals of the same species both nestmates and non-nestmates; however, Neoponera villosa avoided the footprints of Cephalotes borgmeieri, and C. borgmeieri avoided the footprints of the other two species. The chemical compositions of the cuticle and footprints are related to each other and are specific to each species.

Interspecific insect relationships on Terminalia argentea (Myrtales: Combretaceae) trees in the Cerrado biome.

Terminalia argentea Mart. (Combretaceae), native to Brazil, is used in habitat restoration programs. Arthropods are bioindicators because their populations reflect changes in the environment. We evaluated the recovery of a degraded area by using ecological indices and analyzing arthropod interactions on T. argentea plants. The richness and diversity of sap-sucking Hemiptera and the abundance of tending ants and Sternorrhyncha predators increased with the number of T. argentea leaves. The correlation of the abundance of tending ants and Sternorrhyncha predators was positive with that of the sap-sucking Hemiptera, and the abundance of Sternorrhyncha predators was negative with that of tending ants and sap-sucking Hemiptera. The positive correlation between the abundance, richness, and diversity of insect groups and numbers of T. argentea leaves is an example of the bottom-up regulation mechanism, with the population dynamics of the lower trophic levels dictating those of higher trophic levels. The contribution of T. argentea, a host plant of many arthropods, to the recovery of ecological relationships between organisms in degraded ecosystems is important.

Imprints of indirect interactions on a resource-mediated ant-plant network across different levels of network organization.

Indirect interactions are pivotal in the evolution of interacting species and the assembly of populations and communities. Nevertheless, despite recently being investigated in plant-animal mutualism at the community level, indirect interactions have not been studied in resource-mediated mutualisms involving plant individuals that share different animal species as partners within a population (i.e., individual-based networks). Here, we analyzed an individual-based ant-plant network to evaluate how resource properties affect indirect interaction patterns and how changes in indirect links leave imprints in the network across multiple levels of network organization. Using complementary analytical approaches, we described the patterns of indirect interactions at the micro-, meso-, and macro-scale. We predicted that plants offering intermediate levels of nectar quantity and quality interact with more diverse ant assemblages. The increased number of ant species would cause a higher potential for indirect interactions in all scales evaluated. We found that nectar properties modified patterns of indirect interactions of plant individuals that share mutualistic partners, leaving imprints across different network scales. To our knowledge, this is the first study tracking indirect interactions in multiple scales within an individual-based network. We show that functional traits of interacting species, such as nectar properties, may lead to changes in indirect interactions, which could be tracked across different levels of the network organization evaluated.