Investigating the Biology of Leaf-Cutting Ants to Support the Development of Alternative Methods for the Control and Management of These Agricultural Pests

Abstract

Concerns about the environmental and health risks of synthetic insecticides are driving the search for alternative pest control methods. Leaf-cutting ants (LCAs), one of the most significant pests in the neotropics, cause substantial economic damage to agriculture and present challenges for control due to their complex biology and ecology. While chemical control remains the primary strategy, its intensive use has negative environmental impacts, promotes pest resistance, and endangers non-target species, including plants, animals, and humans. This review describes the biology of LCAs, examines traditional control methods and suggests alternative strategies such as the use of entomopathogenic fungi (EPFs) combined with sublethal doses of insecticides, plant essential oils (EOs), and RNAi techniques. Here, we emphasize the need to address LCA management sustainably by investigating the biology and ecology at both the “colony” and “individual” levels. Colony-level factors include morphology, life cycle, behavior, division of labor, and nest structure, while individual-level mechanisms involve sensory, biochemical, and behavioral adaptations for garden sterilization and decontamination. This review also highlights the potential of sublethal insecticide doses combined with EPFs to induce behavioral changes and worker mortality, and it details the mode of action of EOs and the use of RNAi as promising control strategies. The integration of biological and chemical approaches could offer sustainable alternatives to synthetic insecticides.

1. Introduction

One of the main challenges in agricultural and forestry production is crop destruction caused by pests, primarily insects [1]. Currently, the use of synthetic pesticides is the most commonly used technique for pest control; however, the negative environmental impact of pesticides raises serious concerns [2]. While these compounds can offer broad-spectrum action and rapid effects in treated areas, their effectiveness is often limited in terms of duration and overall impact [3]. This is because these effects cannot be confined solely to crops, leading to their dispersion into surrounding ecosystems, where they cause significant harm to both invertebrates and vertebrates [3]. Additionally, certain synthetic insecticides exhibit a prolonged half-life, persisting in the environment for extended periods, often surpassing several generations of various animal species [4]. They also tend to accumulate in the food chain, amplifying their detrimental effects [5].

Leaf-cutting ants (LCAs) are known to cause billions of dollars in losses in forestry plantations and various agricultural crops [6,7]. Leaf-cutting ants are native to South and Central America, Mexico, and parts of the southern United States [8]. These species exhibit fascinating ecological behavior but have devastating impacts on agricultural productivity. By cutting and transporting leaves to their nests, they can quickly defoliate young plants, weaken trees in reforestation projects, and significantly reduce yields of commercial crops [9]. In farming systems where a single species predominates (monoculture), pest activity becomes even more intense due to the favorable conditions created by such practices, with large expanses of uniformly cultivated land [10,11]. These factors highlight the need for sustainable and effective management strategies that need to take into account the complex biology and adaptive capabilities of LCA.

In this context, innovative approaches for the control of LCAs are necessary, focusing on compounds that target specific physiological processes without affecting other organisms. Promising alternatives include the use of integrated pest management (IPM) strategies such as the application of sublethal doses of insecticides combined with entomopathogenic fungi (EPF), RNA interference (RNAi) technology, and bioinsecticides derived from natural sources, such as essential plant oils (EO) [12,13,14]. These strategies offer significant advantages, including lower toxicity to non-target organisms, effectiveness at low concentrations, and biodegradability. It is very important to develop safe and sustainable solutions for the responsible management of LCAs. This review aims to discuss key information about the biology and ecology of LCAs, highlighting how their complex social and organizational structure influences their behavior, adaptability, and resistance to traditional control methods. Understanding these fundamental aspects is essential for designing effective and sustainable management strategies. Additionally, the review succinctly and objectively describes traditional control methods and explores the development and application of three alternative approaches for managing these pests: (i) the use of sublethal insecticide doses combined with EPF spores, examining their impact on worker behavior and mortality; (ii) the application of RNAi as a novel tool to disrupt specific physiological processes in ants; and (iii) the use of EOs as natural control agents with insecticidal and repellent properties, characterized by their low toxicity to non-target organisms and rapid environmental degradation.

2. Leaf-Cutting Ants

Leaf-cutting ants make up a highly fascinating “autopoietic system” [15] involving a wide variety of ecological interactions. Leaf-cutting ants are eusocial insects that are found in a wide variety of habitats, distributed in regions from North America (~44 °N) to South America (~44 °S) [8,16]. LCAs include 48 species belonging to three genera, Atta (Fabricius 1804), Acromyrmex (Mayr 1865), and Amoimyrmex (Cristiano, Cardoso et al. 2020) [Hymenoptera: Formicidae: Subfamily Myrmicinae: Tribe Attini: Subtribe Attina]. Despite the differences in their biology, ecology, and geographic distribution, all LCA species are characterized by their habit of cutting leaf fragments from living plants, which are used as a substrate for the fungus that they cultivate, which in turn provides the ants with easily assimilable nutrients [17,18]. The mutualistic relationship between LCA and the fungus Leucoagaricus gongylophorus (Möller) Singer (Basidiomycota: Agaricales) is estimated to have evolved over the last 8 to 12 million years [19,20]. These insects play a preponderant role in ecosystems [9,21], increasing the availability and flow of nutrients and energy, increasing seed dispersal, as well as modifying the characteristics of the soil and vegetation [22,23,24].

3. Biology and Ecology

3.1. Ant Morphology

The morphological differences among the three genera of LCA can be observed on the dorsum of the promesonotum, with two pairs of spines in Atta, and three to four pairs of spines in Acromyrmex (the first tergum of the gaster is tuberculated) and Amoimyrmex (the first tergum of the gaster does not have tubercles). LCAs have powerful mandibles [25,26], which they use to cut different types of vegetation; their morphology is linked to the type of plant material they forage [27]. Therefore, species that harvest grasses tend to have large short mandibles, while in species that harvest dicotyledonous plants, the mandibles are longer but smaller [28]. Notable differences are observed in the way grass-cutters and dicot-cutters cut plant material, which appear to be linked to the size of their metathoracic legs. Specifically, grass-cutters have shorter metathoracic legs compared to dicot-cutters [28].

3.2. Colony Structure and Biology

3.2.1. Structure

Leaf-cutting ant colonies present a complex social organization that has a single queen (monogyny) [29], and workers display age polyethism [30], and marked polymorphism according to the genus [31,32,33]. In Atta, worker allometry is diphasic, while in Acromyrmex, it is monophasic [34]. Within the worker caste, distinctions can be made according to the division of labor [35]: with gardeners and generalists spending more time within the nest, whilst foragers, excavators, and soldiers are involved in activities outside of the nest [31,36]. Mature Atta colonies can be very large, with approximately 4–7 million individuals [18], including small “satellite” colonies linked to the primary colony [37,38]. In contrast, Acromyrmex and Amoimyrmex colonies are smaller than those of Atta, with thousands of workers [39]. Although the literature typically describes these colonies as monogynous, it has been exceptionally observed that colonies of certain species can also be polygynous [38,39].

3.2.2. Overview of the Life Cycle of an Atta Colony

An adult or sexually mature colony is considered as such three years after foundation [40,41], because at this stage it can produce winged males and gynes (virgin queens). The nuptial or mating flight is a synchronized event within the same colony as well as in other colonies, which may be of different species and from geographical areas [42,43]. This event takes place over a few days at the beginning of the rainy season when swarming behavior is triggered by weather conditions [44,45]. During the nuptial flight, queens can mate with multiple males whilst in flight [46], and the stored sperm can be used to inseminate eggs during the queen’s lifespan, which is generally more than 10–15 years [47]. Before leaving the nest on the nuptial flight, gynes carry with them in their infrabuccal pocket (a cavity located below the opening of the esophagus, just behind the base of the labium) a small quantity of the mutualistic fungus [8].

Studies have shown that nuptial flights of various Atta species can reach a range of approximately 9–11 km [48] After the nuptial flight, the mated queens land on the ground, shed their wings, and excavate a small underground “nest” consisting of a narrow entrance gallery 12–15 mm in diameter and 20–30 cm deep, which ends in a 6 cm long chamber. Once the chamber is ready, the queen seals the entrance [49]. According to Mariconi [40], on the second day after excavating the small chamber, the queen regurgitates the fungal fragment from her infrabuccal pocket. At this stage, the queen lays three types of eggs: (i) reproductive eggs, (ii) trophic eggs for her own nutrition and for the brood, and (iii) eggs that are used for mycelial growth [8]. The queen carries out the following behaviors in self-confinement: (i) caring for the fungus, (ii) self-grooming, (iii) produces eggs, (iv) feeds herself and her offspring, and (v) general care of the offspring [40,49].

After about 30 days, the brood consists of eggs, larvae, and pupae, inside a small ‘basket’ constructed from mycelium. Studies show that the first foraging adult workers emerge about 70 days after the queen establishes the nest, but this depends on the species in question [40,50]. The average lifetime of the workers varies according to species and the role performed in the nest, with lifespans estimated to be from 2 to 3 weeks and up to 4 months [34].

3.3. Nest Architecture

Leaf-cutting ants build nests to protect the colony and ensure appropriate temperature conditions [51], humidity [52], and air composition [53,54,55] for the fungus that they cultivate. In the case of Atta nests, ants remove a large amount of soil, excavating tunnels, galleries, and underground chambers [56,57]. According to Mariconi [40], it is possible to distinguish four kinds of chambers inside the nests: (i) those containing fungus gardens, (ii) waste deposits (discarded fungal garden material, old plant substrate, ant corpses), (iii) empty chambers, and (iv) chambers containing loose soil. Moser [42] also described so-called “dormancy chambers” containing leaves, workers, and myrmecophilous arthropods. Mature Atta nests can contain between 300 and 7800 chambers when counted from the ground surface down to a depth of 7–8 m [56]. Worker ants are capable of moving between 30 and 40 tons of soil during the construction of their nests, which corresponds to approximately 1.1 tons per hectare [23,58]. Externally, loose soil accumulates, forming one or several mounds with holes for ventilation or foraging [34,59,60]. Acromyrmex nests are smaller than those of Atta, reaching a depth of approximately 5 m, depending on the ant species and the environmental conditions. The number of chambers varies, with a main chamber plus tens or hundreds of smaller chambers, with various dimensions and shapes [61,62,63]. On the surface, they can form a mound of loose earth, covered with sticks, plant waste, or a simple entrance hole [64]. Most species across the three genera translocate their waste deposits to underground chambers [8,65], although there are exceptions where garbage can be discarded outside the nest or at a certain distance from the entrance holes [8,66]. Another characteristic of LCA nests is that the foraging trails are marked with pheromones to recruit workers to collect suitable resources [66,67]. These trails are used to facilitate the transport of the harvested vegetation to the nests [68,69].

3.4. The Silent Collaboration: Fungus Garden and a Small Biocenosis

3.4.1. Fungus Garden

According to the nutritional requirements of the mutualistic fungus, LCAs select plants that are suitable for fungal growth [70,71]. Depending on the ant species, this selection consists of leaves, flowers, fruits, and/or seeds of mono- or dicotyledonous plants that are subsequently cut and transported to the nests [8,70,72]. Inside the nest, the plant material is then processed in preparation for use as a substrate for the fungus, which involves cutting it into smaller pieces, licking, crimping, chewing, sterilizing, and fertilizing the fragments with fecal droplets [73,74,75]. These droplets contain various enzymes such as proteases, amylases, chitinases, cellulases, pectinases, and laccases [75,76,77]. The fungus is inoculated onto this processed material, forming a sponge-shaped structure known as the ‘fungus garden’ [78]. In specific parts of the fungus garden, hyphae present dilatations at their tips called ‘gongilidium’ (plural: gongilidia) [78,79], which form clusters known as ‘staphyla’ [8]. These are mainly consumed by the queen, brood, and some workers [17,80,81], although adult workers can also obtain some of their nutritional requirements by consuming plant sap [17,82]. Gongylidia are structures that concentrate essential amino acids [18,72,83,84], carbohydrates, lipids [85,86], and enzymes, which are produced by the fungus and ingested by the ants, which facilitate the digestion of plant material [86,87,88,89].

3.4.2. Small Biocenosis

The fungus gardens can be considered “small biocenoses” where, in addition to the cultivated fungus, other microorganisms coexist in a state of equilibrium. These microorganisms, isolated from the surface of the ants’ bodies or from the fungus gardens, include bacteria that produce antibiotics (Actinomycetes), along with other bacteria, viruses, yeasts, black yeast-like fungi, and filamentous fungi (

Table 1. Microbial diversity found in the fungus garden (FG) and on/in the bodies (B on the bodies; DS: digestive system) of leaf-cutting ants.

Microbiota		Some Identified Genera	Isolation Source	References
Bacteria	Actinomycetota	Amycolatopsis, Nocardiopsis, Pseudonocardia, Streptomyces, Tsukamurella	FG, B	[90,91]
	Pseudomonadota	Acinetobacter, Burkholderia, Enterobacter, Klebsiella, Pantoea, Pseudomonas	FG, B, DS	[92,93,94,95]
Fungi	Yeasts	Aureobasidium, Candida, Cryptococcus, Rhodosporidiobolus, Sporobolomyces, Starmerella, Tremella, Trichosporon, Wickerhamomyces	FG, B	[96,97,98,99]
	Black yeast-like fungi	Alternaria, Bipolaris, Cladophialophora, Cladosporium, Exophiala, Ochroconis, Paraphaeosphaeria, Phaeococcomyces, Phialophora, Penidiella	B	[100,101,102]
	Filamentous fungi	Aspergillus, Chaetomium, Cunninghamella, Fusarium, Escovopsis, Monilia, Mucor, Penicillium, Phomopsis, Rhizomucor, Rhizopus, Syncephalastrum, Trichoderma	FG, B	[96,103,104,105]
	Entomopathogenic fungi	Aspergillus, Beauveria, Clonostachys, Conidiobolus, Fusarium, Isaria, Metarhizium, Ophiocordyceps, Paecilomyces, Purpureocillium	FG, B	[106,107,108,109]
Virus	Mycovirus	LgTlV1 §, LgMV1 §	FG	[110]

^§ Virus-like particles (+ssRNA): LgTlV1, Leucoagaricus gongylophorus tymo-like virus 1; LgMV1, Leucoagaricus gongylophorus magoulivirus 1.

). While some of these fungi are opportunistic, others exhibit antagonistic interactions with the cultivated fungus, contributing to the complex dynamics of the garden ecosystem.

When considering antagonistic fungi, two genera are regularly associated with LCAs: Trichoderma sp. [111,112] and Escovopsis sp. [113,114,115,116]. Both of these fungi are ascomycetes (Ascomycota: Hypocreales: Hypocreaceae). Entomopathogenic fungi (Ascomycota: Hypocreales e Eurotiales; Entomophthoromycota: Entomophthorales) have also been detected in this system, isolated from the bodies of workers, queens, and from the “fungus garden” (Table 1).

A recent study [110] describes the detection of two virus-like particles isolated from L. gongylophorus, which were visualized using transmission electron microscopy. Through RNA sequencing, two strains of mycoviruses with positive single-stranded RNA genomes (+ssRNA) were identified: Leucoagaricus gongylophorus tymo-like virus 1 (LgTlV1) and Leucoagaricus gongylophorus magoulivirus 1 (LgMV1). This discovery is highly significant, as it suggests that these mycoviruses may play a symbiotic role in the gardens cultivated by leaf-cutting ants. However, further research is needed to clarify whether these strains act as mutualists, commensals, or parasites within this complex biological system [110].

3.4.3. Other Organisms Associated with Nests

Inside the nests, in association with the ants, it is possible to find a variety of myrmecophiles [117]. These organisms can be found in fungal chambers, garbage deposits, galleries, and tunnels. Among the myrmecophilous arthropods are mites [118,119], beetles [8,120,121], moths [122], cockroaches [123], pseudoscorpions [124], spiders [125], flies, and other insects [126,127,128]. Nests can also house vertebrates such as frogs and snakes [129,130].

3.5. Protection of the Microenvironment: A Fundamental Balance

The maintenance of relatively aseptic conditions inside the nests, which helps prevent epizootic and epiphytic diseases, is the result of a set of actions carried out by the ants, the cultivated fungus, and the associated microbiota [131,132,133,134,135,136,137]. Decontamination, disinfection, and sterilization of biosystems (ants–fungus gardens) involve sensory, morphological, biochemical, and behavioral aspects.

3.5.1. Sensory Aspects

Among the actions that LCAs have developed to control biohazards is sensory detection using the antennae. The sensitivity of the antennae can be observed in tests using attractive baits containing spores of pathogenic fungi, which are rejected by the ants [131]. This detection triggers alarm/defense behavior and activates the cleaning of the contaminated parts of the body and the fungus garden by intensive use of the tibio-tarsal comb, as well as allo-grooming [131]. Studies have shown that spores of filamentous fungi such as Aspergillus, Penicillium, Paecilomyces, Scaputariopsis, and Trichoderma produce 2-heptanone [138,139,140], which triggers an alarm reaction similar to the specific alarm pheromone of the ants themselves [141]. Antennal detection of microorganisms is due to neural activity in the antennal lobes, suggesting that chemical compounds are detected by olfactory receptor neurons [141,142]. These neurons are located in specialized olfactory sensilla in the antenna, representing the main detector of pheromones that convert chemical signals into neural activity and relay that information to glomeruli in the antennal lobe [143].

3.5.2. Morphological and Biochemical Aspects

Certain morphological characteristics provide protection against pathogenic organisms. The cuticle, due to its rigidity and hardness, is one of the most important protective barriers [144,145]. Another barrier is the infrabuccal pocket, which acts as a filtration structure, allowing only fluids and small particles to reach the digestive tract [146,147]. Fungal spores or other contaminants that become trapped in the infrabuccal pocket are coated with salivary secretions, which contain heat-resistant chitinolytic enzymes released by the labial glands [85,148,149]. In addition to the salivary glands, labial glands, mandibular glands, and metapleural glands are involved in the prevention of infections by secreting antimicrobial substances [150,151,152,153].

3.5.3. Antibiosis

In addition to the strategies mentioned above, LCA colonies are protected by microbial defenses (beneficial associations with microorganisms) and compounds released by the mutualistic fungus.

• Associated bacteria: Pseudonocardia and Streptomyces actinobacteria coexist in symbiosis on the surface of the exoskeleton of several species of LCAs [154,155]. These actinobacteria produce diffusible and volatile antimicrobial compounds [91,156,157,158]. This microbial complex protects the workers from infection by entomopathogenic fungi [159,160] as well as maintaining homeostasis within the fungal chambers, preventing the development of unwanted microorganisms [161,162]. In addition to actinobacteria, Gram-negative bacteria belonging to the genus Burkholderia (Order: Burkholderiales; Family: Burkholderiaceae) have been isolated from the fungus garden. These bacteria also inhibit entomopathogenic fungi [92]. In addition to bacteria, yeasts isolated from fungal gardens inhibited the growth of entomopathogenic fungi and other L. gongylophorus-antagonistic fungi [163].
• Fungi cultivated by the ants: Although further studies are still needed, mutualistic fungi cultivated by LCAs release substances with antibacterial and antifungal properties [8,131,164,165,166], as well as a complex of volatile organic compounds [167]. However, it is surprising that, despite the potential relevance of these properties, there is a notable lack of recent research exploring in depth the antibiotic, antifungal, or other possible applications of these compounds, leaving a significant gap in the current knowledge of this system.

3.5.4. Behavioral Aspects

Hygienic behavior is one of the most important factors in keeping the colony free of antagonistic organisms [131,135,168,169,170]. These behaviors are as follows:

• Self-grooming involves the meticulous care of personal hygiene, especially the antennae [171,172]. Self-grooming enables the ants to collect debris, spores, hyphae, or dirt particles in their infrabuccal pockets and then discard them in the form of waste pellets. Self-grooming also fulfills the function of spreading antimicrobial substances over the body surface [173,174].
• Mutual grooming or allo-grooming enables workers to keep their bodies clean, especially the parts that are hard to reach by self-grooming [132,175,176,177].
• Weeding behavior is another form of antisepsis, but of the fungus garden by the worker ants, originally proposed by Möller [78] and confirmed by Weber [177] and Currie and Stuart [178]. Weeding involves using the mouthparts to remove regions of the garden contaminated with sporulating foreign invasive fungi [154,179,180]. Currie and Stuart [178] defined another disinfection behavior for fungus gardens, which they called “fungus grooming”. This involves the removal of foreign spores by cleaning the garden with the mouthparts, collecting and accumulating these fragments as pellets in the infrabuccal pocket, and then expelling them in the garbage deposit.

4. Economic Implications and Innovative Management Approaches

Leaf-cutting ants are considered significant pests due to the severe damage they cause to crops and forest plantations (

Table 2. Main damage of economic importance caused by leaf-cutting ants.

Target		Injury Caused	References
Forest plantations	Eucaliptus	The damage caused, up to the third cycle, constitutes up to 30% of plantation management expenses. The total defoliation of the trees causes a reduction of 11 mm in diameter and 0.7 m in height, translating to a 13% loss in wood volume at the end of a seven-year rotation, and also resulting in uneven tree growth. Newly planted seedlings are the most vulnerable, often leading to their death. The characteristic of the plant most affected by defoliation is the diameter, when compared to the height. In plantations with ant nest densities of 2.76 m2 (loose soil per hectare), there was a reduction of 0.87% in wood volume.	[182,183,184]
	Pinus	Attacks on newly planted seedlings can cause different levels of defoliation, which can reach 100%, affecting the apical meristem. This mostly happens in the first month after planting, causing the death of 7.5% of the seedlings. Wood volume loss can reach 43% (compared to controls that did not undergo defoliation), with levels of greater than 75% defoliation 30 days after planting. Acromyrmex species caused losses in seedlings of 20.8% 65 days after planting. A six-year study found significant reductions in total height, diameter, and wood volume, with a mortality rate of 31.2%.	[185,186,187,188,189]
	Salix	The damage produced by Acromyrmex species showed a reduction in wood volume of up to 90%. The weight, diameter, and volume losses of wood in commercial clones of Salix nigra over four years were 70% (wt), 40% (diam), and a 51% to 93% loss of wood volume, depending on the variety of the clone.	[190,191,192,193]
Other plantations and crops	Citrus, vineyards, cocoa, soybean, alfalfa, sunflower, sorghum, alfalfa, flax wheat, maize	Attack in the initial stage of crops, seedlings, caused delays in development and/or losses of seedlings.	[194,195,196,197,198,199,200,201,202,203]
Sugarcane plantations		The losses were calculated at 1.74 tons of sugarcane per ant colony per hectare, in each cycle, with a 30% reduction in the sucrose content of the raw material harvested. A reduction of 3.6 tons of sugarcane per year is equivalent to the loss of 450 Kg of sugar or 300 L of alcohol, as a result of one adult ant colony per hectare.	[43,183,204,205,206]
Pastures		Ten colonies per hectare can consume 52.5 Kg of grass/day, which is equivalent to the daily rations for three oxen. Another type of damage is produced by turning the earth and forming foraging trails.	[28,68,207,208,209,210]
Indirect damage		Environmental pollution caused by the indiscriminate use of pesticides. Structural damage to highways, dams, bridges, mausoleums, or tombs. Accidents with animals and agricultural machinery, loss of land fertility, and negative effects on the grazing behavior of cattle. Problems of water infiltration for irrigation caused by the presence of nests in sugarcane plantations in Colombia.	[23,28,34,184,211,212]

). In agricultural and reforestation areas, their defoliation activity and nest excavation compromise the growth and form of species such as Eucalyptus and Pinus, reducing the wood volume by up to 13% after a single defoliation event and causing more severe losses with successive defoliations [7]. For example, these insect pests can consume up to three tons of sugarcane annually per colony [6]. Additionally, it is estimated that up to 30% of forestry plantation management costs are allocated to control these pests, while indirect impacts, such as soil erosion near the nests and possible damage to machinery or infrastructure due to subsidence, further exacerbate their pest status [6,181].

The control of LCA (summarized in

Table 3. Conventional methods for managing leaf-cutting ants.

Control Method		Description	References
Mechanical		Remove the queen ant by digging up the part of the nest with the queen (for nests >4 months old). Plowing during soil preparation.	[213,214]
Cultural		Crop rotation, destruction of crop residues, pruning, fertilization, and intercropping. Combine crops with alternative plants with repellent effects (castor beans, grasses, sesame).	[7,215,216,217,218]
Physical		Use of fire in forested areas. Flooding in small areas.	[215,219]
Biological	Macro- organisms	Wild and domestic birds. Entomopathogenic nematodes. Arthropods: coleopterans, mites, spiders, parasitoids, predatory ants.	[40,128,220,221,222,223,224]
		Among the parasitoids, phorids (Diptera: Phoridae) have been studied for their possible control. These dipterans lay their eggs in foraging workers when they are transporting leaves along the trail or while cutting leaf fragments.	[225,226]
	Micro- organisms	The use of microorganisms may be a promising biocontrol tactic, by offering baits containing entomopathogenic fungi, antagonistic fungi of L. gongylophorus, and bacteria. More studies are needed following the promising results obtained in the laboratory so that these techniques can be effectively transferred to the field.	[227,228,229,230,231]
		Entomopathogenic fungi (baits): Metarhizium anisopliae, Beauveria bassiana, Aspergillus ochraceus, Conidiobolus lunulus, Purpureocillium lilacinum Antagonistic fungi of L. gongylophorus (baits): Trichoderma spp., Escovopsis weberi	[232,233]
		Bacteria (extracts): Photorhabdus sp., Serratia marcescens, and Xenorhabdus nematophila, on workers of certain species of Acromyrmex, Atta, and the fungus L. gongylophorus	[234,235]
Nano-insecticides		Diatomaceous earth (DE)/nanostructured alumina (NSA). Inert powders with low toxicity to vertebrates and non-target organisms; low impact on the environment. Mode of action on insects: Interfere with the outer waxy protective layer of the cuticle, making the insects vulnerable to water loss and dehydration. DE is a powder composed of fossilized diatoms, which when used alone against Atta colonies, cause very low worker mortality, due to the complex nest architecture.	[236,237]
		NSA is a high-purity homogeneous powder resulting from the combustion synthesis of glycine and aluminum nitrate. NSA shows potential for use as a granular insecticide that can be applied directly to Acromyrmex nests.	[238,239]
	Synthetic/Chemical insecticides	Powders. An active ingredient with contact action, using talc as an inert and application vehicle (spray-dusting). Nests have more than one chamber and therefore the product does not affect the entire ant nest. Soil moisture negatively affects this type of technique.	[7,240,241]
		Liquids. Applied directly to the soil. The ants have to be directly exposed to the liquid, so where the nest is deep, penetration can be poor and there is a loss of the product due to absorption by the soil.	[242,243]
		Nebulization. Equipment: A cylindrical steel tank coupled to a hose, with a rod and nozzle suitable for applying the product (an active ingredient diluted in solvent and mixed with gases, butane, and propane) in the nest through the entrance holes.	[7,242]
		Thermal-fogging. An efficient technique for combating large nests and in areas of reforestation, where the use of bait is economically not feasible. The application method involves heat atomization of an insecticide carried in diesel or mineral oil, introduced through the holes, using a thermo-nebulizer.	[215,244,245,246]
		Toxic bait. Baits contain an attractive substrate and a toxic ingredient (e.g., sulfluramid or fipronil). The toxic compounds are not specific and can cause negative effects on non-target species in addition to water and soil pollution. The workers are contaminated by direct contact during the cultivation of the fungus.	[6,7,181,247,248,249,250]
	Natural control	Pheromones and behavioral control. Many pheromones have attractive properties, which makes them a promising means of improving bait attraction. Some substances, such as β-eudesmol or those contained in jatobá leaves (Hymenaea courbaril), have been shown to produce behavioral changes (e.g., agonistic behavior) in workers from Acromyrmex and Atta colonies.	[215,251,252,253,254]
		Repellents. Plant extracts (PEx): Their low toxicity and persistence make them environmentally safer than pesticides. These extracts can be useful in association with other control tools, such as toxic and attractive types of bait in a “push–pull” strategy.	[255,256,257,258]
		Insecticides/Fungicides. PEx with insecticidal and/or fungicidal properties. Insecticidal activity by contact or ingestion: Citrus seed oils, namely Citrus sinensis, Citrus limon, or Citrus reticulate (Rutaceae); PEx of families such as Amaryllidaceae, Aristolochiaceae, Asteraceae, Euphorbiaceae, Fabaceae, Myrtaceae, Rubiaceae, Rutaceae, Simaroubaceae, and Solanaceae. Certain PEx also affect the mutualistic fungus: Piper piresii (Piperaceae), Simarouba versicolor (Simaroubaceae), Raulinoa echinata (Rutaceae), and Coffea spp. (Rutaceae).	[259,260,261,262,263]

) faces significant challenges, as the available options are limited and mostly ineffective at keeping populations below economically acceptable levels [7]. Furthermore, the use of synthetic compounds often has negative impacts on both human health and the environment [6]. The complexity of control measures arises from the size and structure of nests, their behavior, complex social organization, and the large ant populations, requiring significant efforts and intensive use of resources to achieve successful outcomes. Although chemical options remain the most effective, many insecticides have now been banned, highlighting the urgent need to find more sustainable and effective alternatives [212].

Among the alternative control methods that require further development are (i) the use of sublethal doses of insecticides combined with EPF spores, aimed at enhancing the action of biological agents; (ii) research on RNAi as a novel tool to disrupt key genes involved in ant development or behavior; and (iii) the use of EO, known for their natural repellent and/or insecticidal properties, as ecological, low-impact environmental options.

Exploring these alternatives could lay the foundation for more sustainable and effective strategies, aligned with current demands for environmental conservation and responsible agriculture.

4.1. Combinations of Sublethal Doses of Insecticides and EPF

Neonicotinoids are neuroactive insecticides that act by stimulating nicotinic acetylcholine receptors (nAChRs) in the central nervous system of insects, functioning as agonists of these receptors and mimicking the action of acetylcholine (ACh) [264]. However, unlike ACh, neonicotinoids are not degraded by the enzyme acetylcholinesterase (AChE), resulting in continuous stimulation, which leads to neurotoxicity and cellular death [265,266,267]. This specificity for insect nAChRs, due to electronegative binding sites, gives neonicotinoids high selective toxicity [268]. Although initially considered less harmful to mammals, recent research has highlighted potential impacts on non-target biological systems, such as the immune and reproductive systems, underscoring the importance of assessing their environmental and toxicological risks [269,270]. Within the group of neonicotinoids, imidacloprid is one of the most widely used compounds due to its efficacy and broad-spectrum action [271]. Studies have shown that sublethal doses of this compound affect various aspects of insect behavior [272,273]. In ants, for example, disruptions have been reported in excavation, foraging, brood care, and increased consumption of sugar solutions [274,275]. These findings emphasize the possible influence of imidacloprid on activities essential for colony survival.

Neonicotinoids, including imidacloprid, have been extensively studied for use against invasive ant species such as Linepithema humile [276,277], Solenopsis invicta [274], Lasius niger—Lasius flavus [278], and Lasius neglectus [273]. In Pogonomyrmex occidentalis, low doses (50 ppm) affected foraging capacity by disrupting their orientation system [279]. These sublethal effects, though seemingly minor, can accumulate over time, compromising colony fitness and altering population dynamics [280].

While neonicotinoids have been widely studied in invasive species, their use in leaf-cutting ants remains limited. However, preliminary research suggests they could be integrated into integrated pest management (IPM) strategies when combined with entomopathogenic fungi [281]. This approach takes advantage of the behavioral and physiological disruptions induced by neonicotinoids to enhance the effectiveness of biological agents.

Entomopathogenic fungi belong to different taxonomic groups, such as Oomycota, Microsporidia, Chytridiomycota, Entomophthoromycota, Basidiomycota, and Ascomycota, with Ascomycota and Entomophthoromycota being the most commonly found in natural environments [282,283]. Within Ascomycota, genera such as Beauveria, Metarhizium, Lecanicillium, Paecilomyces, and Isaria have been extensively used in biological applications due to their effectiveness against a wide range of insect pests [284,285,286,287,288,289]. The EPF infection process begins with spore adhesion to the host cuticle, facilitated by electrostatic and hydrophobic interactions and the action of lytic enzymes [283,290]. Following adhesion, spores germinate and produce specialized structures, such as appressoria in many species, allowing them to penetrate the insect cuticle [290,291]. Through this penetration, fungi reach the hemolymph, rich in nutrients such as sugars, proteins, and lipids, where they proliferate and release toxins, disrupting insect physiology and leading to host death [290,292].

However, using EPF as a standalone control tool has limitations. Their action is often slower compared to chemical methods, they exhibit low persistence under adverse environmental conditions, and broad application coverage is required, which can increase costs [282,293,294,295,296]. For these reasons, optimizing their application through technologies such as encapsulation or integrating them into combined strategies with other control methods is essential to overcome their disadvantages [283,297].

Although there are few records of natural infections of LCAs by EPF under field conditions [106,298,299], laboratory studies have demonstrated that LCA are susceptible to fungal infections under controlled conditions [300]. Nevertheless, the failure of many experiments using fungal spores, even at high concentrations [131], is attributed to the ants’ highly efficient prophylactic strategies.

Despite these challenges, recent research has demonstrated the potential for combining fungal spores with sublethal concentrations of neurotoxic insecticides such as imidacloprid [12,244]. The principle behind this combination lies in the ability of neurotoxic insecticides to disrupt the normal hygienic behaviors of ants. This disruption, caused by neurotoxic activity, acts synergistically by allowing fungal spores to germinate and penetrate the host before the insects can remove them [301,302]. This combined strategy represents a promising approach to overcome the natural barriers that hinder efficient pest control [12].

Despite advances in the study of neonicotinoids, research on their impact on leaf-cutting ants remains scarce. Studies evaluating not only sublethal effects under realistic field conditions but also their long-term impact on colony reproductive fitness are necessary. Neonicotinoids offer significant advantages, such as high specificity and efficacy, but also pose environmental and toxicological risks that must be carefully considered. Their integration into more sustainable control strategies, alongside biological and physical approaches, could represent a promising tool for responsible pest management in agricultural systems.

4.2. Applicability of RNAi as a Potential Pest Control Method

RNA interference has emerged as an innovative and promising tool for pest control, offering a safer and more sustainable alternative to conventional chemical pesticides [303,304]. This gene-silencing mechanism interferes with the expression of specific genes, disrupting vital processes in target organisms while reducing the environmental impact [305].

Initially discovered in the model organism Caenorhabditis elegans [306], RNAi is a conserved process across diverse organisms, regulating gene expression at the post-transcriptional level in response to the presence of double-stranded RNA (dsRNA) [307]. Subsequent studies in model organisms like Drosophila melanogaster have clarified the molecular foundations of this process, laying the groundwork for its application in agricultural pest control [303,308]. The RNAi process begins when dsRNA is processed by the endonuclease Dicer, which fragments it into small ~19–25 base pair segments known as small interfering RNA (siRNA) [309,310]. One strand of the siRNA associates with the Argonaute (Ago) protein, forming the RNA-induced silencing complex (RISC). This complex recognizes and degrades complementary mRNA, specifically suppressing gene expression [311,312,313].

Initially identified as an evolutionarily conserved antiviral response [314], RNAi also acts as a gene expression regulator in eukaryotes and has been widely utilized as an experimental tool in biological research [13,304,315,316]. The usefulness of RNAi in pest control depends on an efficient dsRNA delivery system tailored to the species and objectives. The main strategies include (i) microinjection: precise but limited to laboratory experiments; (ii) oral ingestion: ideal for field applications but faces challenges such as enzymatic degradation of dsRNA in the insect digestive tract; and (iii) topical application: useful for insects with permeable cuticles, although less effective for systemic distribution [317,318].

RNAi studies have been conducted on various ant species (

Table 4. Studies on the application of RNAi technology for ant control.

RNAi	Ant Species	Target Gene	Administration Method	Life Stage †	Social Form §	References
dsRNA	Solenopsis invicta	Vitellogenin receptor	Injection	WP	Q	[322]
		Pheromone biosynthesis activating neuropeptide	Injection, Feeding	B, P, A	Q, W	[323]
		Pheromone biosynthesis activating neuropeptide, Pyrokinin-2 receptor	Injection	A	W	[324]
		Short neuropeptide F receptor	Feeding	L, A	Q, W	[325]
		Solenopsis invicta virus 1 capsid	Feeding	Ain	Ec	[326]
		SiOBP1, SiOBP5, SiOBP6, SiOrco	Injection, Feeding	A	W	[327]
		Sifor, 8-Br-cGMP	Feeding	A	Q, M, W	[328]
		Actin, coatomer subunit beta, arginine kinase, V-type proton ATPase catalytic subunit A, V-type proton ATPase subunit B, V-type proton ATPase subunit E	Feeding	A	Q, M	[329]
	Camponotusfloridanus	Peptidoglycan recognition proteins	Feeding	L, A	Ec	[321]
	Diacamma sp.	Yellow	Injection	WP	M, F	[330]
	Polyrhachisvicina	Estrogen-related receptor	Feeding	Ain	Ec	[331,332]
	Pheidolehyatti	Vestigial	Injection	L	W	[320]
	Nylanderiafulva	Actin, coatomer subunit beta, arginine kinase, V-type proton ATPase catalytic subunit A, V-type proton ATPase subunit B, V-type proton ATPase subunit E	Feeding	A	W	[333]
	Linepithemahumile	Spaetzle, Dicer-1	Feeding	A	Q, W	[334]
siRNA	Camponotusfloridanus	cGMP-dependent protein kinase	Injection	A	W	[335]
	Solenopsisinvicta	Chemosensory protein 9, protein kinase	Feeding	L	W	[336]
	Harpegnathossaltator	Corozonin receptor, vitellogenin	Injection	A	Q	[319]

^† Life stage: A, adult; Ain, all individuals; B, brood; L, larva; P, pupa; WP, white pupa. ^§ Social form: Ec, entire colony; F, females; M, males; Q, queens; W, workers.

), including Solenopsis invicta, Camponotus floridanus, Diacamma spp., Pheidole hyatti, Harpegnathos saltator, and Nylanderia fulva (reviewed by Allen [13]). For example, in S. invicta, RNAi is capable of silencing genes related to pheromone synthesis, regulating viruses, and developing oocytes [318]. In H. saltator, interference with the corazonin receptor gene affected reproductive flexibility and hunting behavior [319]. Similarly, genes related to caste development and endosymbiont regulation have been investigated in P. hyatti and C. floridanus [320,321].

However, in the case of LCA, no specific studies have been published to date. These species present unique challenges due to their eusocial colonies and hygienic behaviors, which hinder dsRNA delivery and efficacy [13]. However, RNAi is a promising biotechnological tool for pest control [337]. Its main advantages include high specificity, minimization of impacts on non-target organisms, and the ability to silence essential genes, affecting individuals and colony functioning [13,338]. However, this technology faces several limitations, for example, (i) stability of dsRNA: degrading enzymes in the environment or insect digestive systems [312,339]; (ii) efficient delivery: developing formulations that resist worker manipulation and that could be distributed through trophallaxis within colonies [340,341]; and (iii) specificity and safety: biopesticides must avoid impacts on beneficial species, such as pollinators or predators [338,342].

Incorporating nanomaterials has significantly improved RNAi efficacy. Nanoparticles protect dsRNA molecules from degradation, facilitate their transport to target cells, and increase environmental stability, essential for field applications [318,326]. Additionally, ant genomic sequencing and technologies like CRISPR offer complementary approaches for identifying and manipulating key genes [304,308,332,343].

The design of biopesticides for use against LCAs must consider their unique biology and ecology. It is essential to target genes that affect critical functions such as caste development, reproduction, or social communication while circumnavigating defensive or hygienic behaviors [13]. Advances in formulating dsRNA baits resistant to environmental degradation are promising, although success depends on overcoming current technical barriers and ensuring the safety and specificity of the developed materials [13,341].

Although no specific studies have assessed the impact of RNAi at the colony level in ants, the overall prospects are encouraging [13]. The combination of advances in genomics, biochemistry, and biotechnology paves the way for sustainable pest control, with potential benefits for both agriculture and the ecosystem [342,343].

4.3. Application of Essential Oils as a Low-Impact Alternative to Conventional Control Methods

Plants allocate a significant amount of energy and chemical resources to produce secondary metabolites such as EOs and alkaloids to defend themselves against stress and predators [344,345]. These secondary metabolites, present throughout the plant or in specific localized tissues [346], play a crucial role in insect protection through mechanisms of antixenosis (non-preference) and antibiosis [347]. Additionally, they contribute to indirect defense via volatile compounds released during insect-induced damage, which attract the natural enemies of harmful insect pests [348].

In this context, EOs stand out due to their biodegradability, low environmental persistence, and absence of negative effects on groundwater, positioning them as an eco-friendly and effective solution for pest control [349,350]. EOs are complex mixtures of volatile compounds, including monoterpenes, sesquiterpenes, and phenylpropanoids [351]. These compounds are synthesized as secondary metabolites in the plant’s secretory structures to defend against herbivores and pathogens [352]. Their composition can vary depending on factors such as plant variety, extraction method, and geographical conditions [353,354].

Various studies have confirmed that EOs possess insecticidal, repellent, antifeedant, and growth-regulating properties, demonstrating toxic effects on a wide range of arthropod pests, including Lepidoptera, Coleoptera, mites, Diptera, cockroaches, and termites [355,356,357,358]. These compounds also exhibit antifungal, antibacterial, and antioxidant activities and low toxicity toward non-target organisms, making them valuable tools for sustainable agriculture [359,360].

The toxic mechanisms of EOs against insects involve their ability to interfere with critical physiological processes [361]:

• Acting as repellents [362];
• Inhibiting acetylcholinesterase (AChE) activity, leading to the accumulation of acetylcholine (ACh) in synapses and causing overexcitation of the nervous system and ultimately insect death [363,364];
• Modulating insect GABA receptors, mainly as positive allosteric modulators, which disrupt neuronal function by altering chloride (Cl−) channel activity, resulting in either hyperexcitation or nervous system inhibition, leading to insect death [361,365,366];
• Inhibiting glutamate-activated chloride receptors (GluCls), which are expressed in the head region and neuronal ganglia and also found in legs, intestines, and reproductive systems, affecting olfactory learning, memory, muscles, and antennae [367,368,369];
• Mimicking the effects of octopamine, a biogenic amine essential in the arthropod nervous system, synthesized from tyrosine and playing a key role as a neurotransmitter, neurohormone, and neuromodulator, regulating processes such as memory, learning, fat metabolism, respiration, and muscle function [370,371,372,373,374];
• Acting as insect growth regulators (IGRs), interfering with key developmental processes such as molting and metamorphosis [375,376];
• Altering the cuticle structure, causing dehydration and death. EO components displace cuticle lipids, reducing cuticle hydrophobicity and increasing permeability, facilitating the entry of toxic substances into the insect and weakening its fundamental/primary protective barrier [377,378,379];
• Affecting antioxidant systems by modulating their defense mechanisms against oxidative stress. Some EOs increase reactive oxygen species (ROS) levels and reduce the activity of key antioxidant enzymes, such as superoxide dismutase, catalase, peroxidases, and glutathione-S-transferase, compromising pest defenses and promoting mortality [361,380].

EOs primarily target the insect nervous system, disrupting motor and cognitive functions through interactions with specific receptors, leading to behavioral changes such as intra-colony aggression, reduced foraging, and social disorganization [253,254,381,382]. These effects, caused by compounds such as caryophyllene oxide, β-eudesmol, and thymol not only directly compromise ant survival but can also dismantle the social structure of colonies, hindering their adaptability [14]. However, studies evaluating the use of EOs as bioinsecticides for controlling LCAs are limited, with only one investigation conducted under field conditions (

Table 5. Evaluation of plant essential oils (EOs) for the control of leaf-cutting ants.

Plant Species Source of the EO	Target Species	Application Method	Experimental Context	Effects †	Reference
Aristolochia trilobata (Aristolochiaceae)	A. balzani, A. sexdens	Topical application, fumigation	Lab	In	[383]
	A. balzani/L. gongylophorus	Fumigation	Lab	In/Fc, Fl
Croton tetradenius (Euphorbiaceae)	A. balzani	Volatile exposure	Lab	In, Re, Bm	[384]
Drimys angustifolia (Winteraceae)	A. hispidus, A. crassispinus	Spraying	Field	Bm	[385]
Eplingiella fruticose (Lamiaceae)	A. balzani	Volatile exposure, fumigation	Lab	Re, Bm	[386]
Eucalyptus spp. (Myrtaceae)	A. sexdens	Volatile exposure	Lab	Sar	[387]
Eucaliptus maculate * (Myrtaceae)	A. sexdens	Volatile exposure	Lab	Bm	[252]
	A. sexdens rubropilosa	Volatile exposure	Lab	Bm	[258]
	A. sexdens rubropilosa, A. laevigata, A. bisphaerica	Volatile exposure	Lab	Bm	[253]
Eugenia uniflora (Myrtaceae)	A. laevigatta	Spraying	Lab	In	[388]
Hyptis pectinate (Lamiaceae)	A. balzani, A. sexdens	Direct contact, fumigation	Lab	In	[389]
Lippia spp. (Verbenaceae)	A. balzani	Topical application	Lab	In, Re, Bm	[390]
Melaleuca alternifolia (Myrtaceae)	A. ambiguus, A. lobicornis	Volatile exposure	Lab	Re	[391]
Myrcia lundiana (Myrtaceae)	A. balzani	Fumigation	Lab	In, Bm	[392]
Piper holtonii (Piperaceae)	L. gongylophorus	Direct contact	Lab	Fc	[393]
Pittosporum sp./Pluchea sp. (Pittosporaceae/Asteraceae)	A. ambiguus, A. lobicornis	Direct contact, volatile exposure	Lab, Field	Re	[394,395] **
Pogostemon cablin (Lamiaceae)	A. opaciceps, A. sexdens	Direct contact, fumigation	Lab	Bm	[396]
Brassica juncea (Brassicaceae)/Cinnamomum verum (Lauraceae)/Syzygium aromaticum (Myrtaceae)	A. sexdens, A. subterraneus	Direct contact, ingestion	Lab	In	[397]

^† Effects: Bm, behavior modification; Fc, fungistatic; Fl, fungicidal; In, insecticidal; Re, repellent; Sar, sensitivity of antennal receptors. * Synonym of Corymbia maculata (Hook.) ** ref. [394] Push–pull strategy.

) [14].

In the case of LCAs, EOs have demonstrated significant neurotoxic effects, causing tremors and paralysis in Atta opaciceps and A. sexdens following the application of Pogostemon cablin EO (Lamiaceae) [396], as well as disorientation and migration of entire Acromyrmex crassispinus and Acromyrmex hispidus colonies following the application of Drimys angustifolia EO (Winteraceae) [385].

Additionally, their ability to inhibit the growth of the symbiotic fungus L. gongylophorus is a complementary effect, enhancing their potential as dual management tools [393,394]. EO application methods include direct contact, spraying, fumigation, and exposure to volatiles, although most tests have been conducted in controlled laboratory settings. Expanding research to field scenarios is essential to evaluate the practical feasibility of applying EO in integrated pest management strategies.

5. Conclusions

Leaf-cutting ants have high ecological and economic relevance due to their complex biology, eusocial behavior, and ability to significantly impact agricultural systems. Considered as superorganisms [398,399], their colonies exhibit an advanced level of organization, where the interaction between the colony components and their environment allows them to adapt and thrive. However, this biological success also makes them one of the most prominent pests in tropical and subtropical regions, seriously affecting commercial crops and forest plantations.

The control of these pests has traditionally relied on the use of broad-spectrum chemical insecticides. While effective, these approaches present serious limitations, such as toxicity to non-target organisms, environmental contamination, and the potential for resistance development in colonies. This has driven the search for more sustainable alternatives that include a deeper understanding of the biology and ecology of these ants.

Among alternative methods, the use of sublethal doses of insecticides combined with entomopathogenic fungi has shown promising results by taking advantage of both neurotoxic impacts and fungal infections to overcome the ants’ hygienic defenses. Similarly, the use of RNAi could be a revolutionary tool for targeting key genetic processes, such as caste development and reproduction, though it faces technical challenges in relation to the efficient delivery of dsRNA to colonies. On the other hand, EOs, with their dual action against ants and their symbiotic fungus, represent an eco-friendly option with potential for integrated management, although their application in the field requires further research.

6. Future Directions

• Further studies on sublethal effects of insecticides and EOs: expand research on the sublethal effects on colony behavior and social organization, particularly in natural contexts;
• Integration of biological methods: strengthen the use of EPF in combination with other approaches, assessing their efficacy under different environmental conditions and life stages of the LCAs;
• Optimization of RNAi delivery: develop efficient methods for dsRNA administration, such as baits resistant to environmental degradation and strategies facilitating trophallaxis-based distribution;
• Genomic and transcriptomic analyses: apply ‘omics’ techniques to identify new target genes and crucial metabolic pathways in both ants and their symbiotic fungus, which could be manipulated through biotechnology;
• Comprehensive evaluation of EOs: conduct field-scale studies to validate their efficacy and practical feasibility, including impact analyses on non-target species and an evaluation of application methods;
• Ecological impact of alternative methods: study the long-term consequences of non-chemical approaches on local ecosystems to ensure these solutions are genuinely sustainable.

The management of LCAs requires a multidisciplinary approach that combines biological, chemical, and biotechnological strategies. Only through the effective integration of these methods, supported by robust research, will it be possible to develop sustainable and targeted solutions that minimize the environmental and economic impacts of this pest.