**2. Leaf–cutting ants of the genera** *Atta* **and** *Acromyrmex* **and their symbiotic fungus,** *Leucoagaricus gongylophorus*

Leaf-cutting ants comprise over 12,000 species and are considered social insects because they participate in parental care, reproductive castes, overlapping generations, and a division of labour [24]. These insects live in permanent colonies and are holometabolous. Taxonomically, they belong to the order Hymenoptera, family Formicidae, subfamily Myrmecinae, and tribe Attini [25]. Leaf-cutting ants belong to the genera *Atta* Fabricius and *Acromyrmex* Mayr as well as the basal genera *Apterostigma* Mayr, *Kalathomyrmex* Klingeberg and Brandão, *Mycetagroi‐ cus* Brandão and Mayhé-Nunes, *Mycetarotes* Emery, *Mycetaphylax* Fr. Smith, *Paramycetophy‐ lax* Kusnezov, *Sericomyrmex* Mayr, *Trachymyrmex* Forel, *Pseudoatta* Gallardo, and *Attaichnus* Lazã. [21] The Attini tribe is found only in the Neotropical region and is distributed between southern South America and the southern United States [26].

The leaf-cutting ants of the genera *Atta* and *Acromyrmex* (known as *saúvas* and *quenquéns* in Brazil, respectively) build nests composed of hundreds of underground chambers that are connected to each other and to the surface through galleries. The exterior of the nest usually exhibits a loose soil mound originating from the chambers formed by the nest [27]. Holes can be found in the loose soil mound or outside of it. According to Mariconi (1970), a large anthill of approximately 200 m2 may contain an estimated population of six million ants [28].

of plant material/day [10]. Thus, these ants cause direct losses, such as the death of seedlings and reduction of tree growth. Indirect losses also occur as a result of the decreased resistance

Leaf-cutting ant control has been performed almost exclusively through the application of conventional insecticides, including cyfluthrin (pyrethroid), imidacloprid (neonicotinoid), furathiocarb (carbamate), sulfluramid (fluoroaliphatic sulfonamide), and fipronil (phenyl pyrazole) [12]. Due to the problems these products may cause to the environment and humans, their use has been restricted by governments and forest product certification bodies, which have demanded and encouraged the development of alternative control strategies to these insecticides, such as the use of plant-derived products, entomopathogenic fungi, and phero‐

Plant-derived products can be used to control ant populations through several mechanisms. Some of these substances can act directly against the ant, leading to its death, such as citrus seed oils obtained from *Citrus sinensis* (L.) Osbeck, *Citrus limon* (L.) Burm. f. or *Citrus reticula‐ ta* Blanco (Rutaceae) [14] and extracts from the castorbean (*Ricinus communis* L.) (Euphorbia‐ ceae) [15], timbo (*(Ateleia glazioviana* Baill.) (Leguminosae) [16] and eucalyptus (*(Eucalyptus*

of trees to other insects and pathogenic agents [11].

258 Insecticides - Development of Safer and More Effective Technologies

**Figure 1.** *Atta sexdens* and its fungal garden: mycelial care. Source: Arnhold, 2012.

mones [13].

Leaf-cutting ants cultivate the symbiotic fungus *L. gongylophorus,* which is used as a source of food and of auxiliary enzymes that function in the ants digestive process [29]. After the origination of the ant-fungus symbiosis, a subsequent evolutionary step involved the acquis‐ ition of staphylae-rich monophyletic cultivars by the highly derived Attini (Figure 2), partic‐ ularly the *Atta* and *Acromyrmex* species, which collect only fresh vegetation as the fungal substrate. The conversion of vegetation into substrate requires several different operations and specialisations. The plants are cut by workers who have a head width of 1.6 mm or greater (other tasks, such as defence, involve larger workers), while care of the fungus requires very small workers, and intermediary steps in the garden are performed by medium-sized workers. Among ant species, this great evolutionary change seems to have placed the derived Attini on the path of producing larger and larger colonies due to the increased differences between the sizes of reproductive females and workers. Relatively larger reproductive ants show an increased ovariole count, enabling rapid colony growth [30], which, due to the increased size variation among nestmate workers, are able to execute all activities involved in fungal cultivation.

excavations, the queen begins oviposition [35]. The queen in a new colony lays both repro‐ ductive and trophic eggs, which are used as a food source for both the queens and their first offspring [36]. Early larvae originating from the reproductive eggs emerge 24 to 25 days after soil excavation by the queen. Ant larvae have been described as the digestive caste of the colony [25] and can actively contribute to the integration of the colony not only as a source of new adults but also by providing nutrients and enzymes for the workers, thus ensuring that the larvae will be cared for, which is essential for their survival. At 62 to 66 days after the nuptial flight, the first adults emerge, which feed on sap or obtain liquid food through trophallaxis or regurgitation [35]. On average, 87 days pass between the initial excavation and the appearance of the first opening created by workers. The second opening is only built 14 months after the first one. Additional openings appear quickly; on average, after 82 days, eight openings are present. The tenth opening is created approximately 20 months after the colony is fertilised. When the nest reaches three years of age (±38 months), it reaches adulthood and produces the

Plant–Derived Products for Leaf–Cutting Ants Control

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The use of plants as a substrate for the growth of the symbiotic fungus has led these ants to become the main pest on Brazilian forest plantations, despite playing a very important ecological role [38,39,40]. Leaf-cutting ants remove and modify the soil during the nest construction process, promoting changes in the chemical-physical proprieties of the soil and nutrient cycling that are favourable to plant growth [34]. However, their position as a key pest is generally cited more often due to the large quantities of vegetation these ants collect for

Leaf-cutting ants show a preference for certain plant species, which are consistently defoliated, while other species are not attacked, despite being abundant and located close to their nests. Some ant species attack only dicotyledonous plants, others target monocotyledons, and some ants collect both plant types. The preference for particular plant species may be related to the nutritional demands of the symbiotic fungus *L. gongylophorus* grown by these ants [41]. Ants can attack plants at any stage of their development, cutting their leaves, flowers, buds, and thin branches, which are then transported to the interior of their underground nests [42,43]. The complete defoliation of one-month-old *Pinus taeda* L. causes 25% mortality of these plants. For the plants that survive, defoliation affects their diameter more than their height [44]. Previous studies have demonstrated that there is a significant reduction in the development of *P. taeda* during the first 24 months of life when attacked by leaf-cutting ants [45]. Total defoliation reduces the growth of *Eucalyptus grandis* W. Hill ex Maiden in both diameter and height as well as the profit of the producer at the end of the rotation, even if the defoliation occurs only once during the beginning of the planting. The decreases in production and profits are enhanced according to the frequency of defoliation, which can make the maintenance of totally defoliated areas economically unviable. The damage caused by ants is more harmful to the plants in their first three years. A single total defoliation event delays the growth of the plant [46] while two or three consecutive defoliation events may lead to plant death [47,48]. Due to the damage they cause, control of these pests is essential to avoid large losses in

first nuptial flight [37].

growing the symbiotic fungus.

agricultural and forest crops [33]

.

**Figure 2.** Microscopic images of *Leucoagaricus gongylophorus*, the symbiotic fungus of *Atta sexdens*. (A) Staphylae (arrows) contrasting with the hyphae (50x magnification). (B) Staphylae viewed under an optical microscope: each sphere corresponds to a gongylidia (1,000x magnification). Source: Schneider and Odair, 2003 [78].

Leaf-cutting ants from the genera *Atta* and *Acromyrmex* are the only ants within the group Attini that exhibit polyandry [31]. The development of this behaviour in the Attini coincided with the practice of cutting the leaves and live parts of plants [30]. The use of leaves, which are a widely available resource, would have allowed the leaf-cutting ants to achieve large colonies, leading to the appearance of long-lived queens and more complex forms of social organisation. Polyandry could favour the development of disease resistance as it increases the genetic diversity of the colony [32].

The consecutive matings between *Atta* and *Acromyrmex* species occur at the regional level throughout Brazil. In the southeast region, the nuptial flight of *Atta* species occurs between October and December; during this period, adult nests (approximately 38 months old) produce alate and fertile ants, and during the nuptial flight, referred to as swarming, the males fertilise the females [33]When leaving the nest for the nuptial flight, the virgin queen carries a small fragment of mycelium in her infrabuccal pocket to start a new fungus garden in a new nest [28]After mating, the reproductive females walk on the soil surface, remove their wings and search for a site to begin the excavation of the new nest. [34] In general, 48 hours after the excavation of the initial chamber [28], the future queen regurgitates the fungal fragment from her infrabuccal pocket. When starting the new colony, this small portion of the fungus is sterilised and fertilised with faecal droplets. Five to six days after the beginning of nest excavations, the queen begins oviposition [35]. The queen in a new colony lays both repro‐ ductive and trophic eggs, which are used as a food source for both the queens and their first offspring [36]. Early larvae originating from the reproductive eggs emerge 24 to 25 days after soil excavation by the queen. Ant larvae have been described as the digestive caste of the colony [25] and can actively contribute to the integration of the colony not only as a source of new adults but also by providing nutrients and enzymes for the workers, thus ensuring that the larvae will be cared for, which is essential for their survival. At 62 to 66 days after the nuptial flight, the first adults emerge, which feed on sap or obtain liquid food through trophallaxis or regurgitation [35]. On average, 87 days pass between the initial excavation and the appearance of the first opening created by workers. The second opening is only built 14 months after the first one. Additional openings appear quickly; on average, after 82 days, eight openings are present. The tenth opening is created approximately 20 months after the colony is fertilised. When the nest reaches three years of age (±38 months), it reaches adulthood and produces the first nuptial flight [37].

sizes of reproductive females and workers. Relatively larger reproductive ants show an increased ovariole count, enabling rapid colony growth [30], which, due to the increased size variation among nestmate workers, are able to execute all activities involved in fungal

**Figure 2.** Microscopic images of *Leucoagaricus gongylophorus*, the symbiotic fungus of *Atta sexdens*. (A) Staphylae (arrows) contrasting with the hyphae (50x magnification). (B) Staphylae viewed under an optical microscope: each

Leaf-cutting ants from the genera *Atta* and *Acromyrmex* are the only ants within the group Attini that exhibit polyandry [31]. The development of this behaviour in the Attini coincided with the practice of cutting the leaves and live parts of plants [30]. The use of leaves, which are a widely available resource, would have allowed the leaf-cutting ants to achieve large colonies, leading to the appearance of long-lived queens and more complex forms of social organisation. Polyandry could favour the development of disease resistance as it increases the

The consecutive matings between *Atta* and *Acromyrmex* species occur at the regional level throughout Brazil. In the southeast region, the nuptial flight of *Atta* species occurs between October and December; during this period, adult nests (approximately 38 months old) produce alate and fertile ants, and during the nuptial flight, referred to as swarming, the males fertilise the females [33]When leaving the nest for the nuptial flight, the virgin queen carries a small fragment of mycelium in her infrabuccal pocket to start a new fungus garden in a new nest [28]After mating, the reproductive females walk on the soil surface, remove their wings and search for a site to begin the excavation of the new nest. [34] In general, 48 hours after the excavation of the initial chamber [28], the future queen regurgitates the fungal fragment from her infrabuccal pocket. When starting the new colony, this small portion of the fungus is sterilised and fertilised with faecal droplets. Five to six days after the beginning of nest

sphere corresponds to a gongylidia (1,000x magnification). Source: Schneider and Odair, 2003 [78].

genetic diversity of the colony [32].

cultivation.

260 Insecticides - Development of Safer and More Effective Technologies

The use of plants as a substrate for the growth of the symbiotic fungus has led these ants to become the main pest on Brazilian forest plantations, despite playing a very important ecological role [38,39,40]. Leaf-cutting ants remove and modify the soil during the nest construction process, promoting changes in the chemical-physical proprieties of the soil and nutrient cycling that are favourable to plant growth [34]. However, their position as a key pest is generally cited more often due to the large quantities of vegetation these ants collect for growing the symbiotic fungus.

Leaf-cutting ants show a preference for certain plant species, which are consistently defoliated, while other species are not attacked, despite being abundant and located close to their nests. Some ant species attack only dicotyledonous plants, others target monocotyledons, and some ants collect both plant types. The preference for particular plant species may be related to the nutritional demands of the symbiotic fungus *L. gongylophorus* grown by these ants [41]. Ants can attack plants at any stage of their development, cutting their leaves, flowers, buds, and thin branches, which are then transported to the interior of their underground nests [42,43].

The complete defoliation of one-month-old *Pinus taeda* L. causes 25% mortality of these plants. For the plants that survive, defoliation affects their diameter more than their height [44]. Previous studies have demonstrated that there is a significant reduction in the development of *P. taeda* during the first 24 months of life when attacked by leaf-cutting ants [45]. Total defoliation reduces the growth of *Eucalyptus grandis* W. Hill ex Maiden in both diameter and height as well as the profit of the producer at the end of the rotation, even if the defoliation occurs only once during the beginning of the planting. The decreases in production and profits are enhanced according to the frequency of defoliation, which can make the maintenance of totally defoliated areas economically unviable. The damage caused by ants is more harmful to the plants in their first three years. A single total defoliation event delays the growth of the plant [46] while two or three consecutive defoliation events may lead to plant death [47,48].

Due to the damage they cause, control of these pests is essential to avoid large losses in agricultural and forest crops [33]

.
