The attini tribe comprises fungusgrowing ants, such as the basal Apterostigma and other more specialized genera, including the higher attine and the ones that cut the fresh plant tissue (Atta and Acromyrmex), maintaining an obligatory mutualistic relation with the fungus Leucoagaricus gongylophorus, which serves as a food source for the ants. Leaf-cutter ants are considered agriculture pests and populate the soil, a rich environment, especially due to the presence of several microorganisms. Some of these microorganisms are natural enemies that may cause epizootics (quickly spreading opportunistic diseases). Such defence strategies include polyethism, that is, division of labor among the individuals. The older ants take on the responsibility of foraging, as their integument is harder and heavily sclerotized, serving as a protective barrier against pathogens (including bacteria and antagonistic fungi). The younger ants, whose metapleural glands synthetize important secretions to eliminate and control microorganisms that could attack the colony fungus garden and the immature (larvae and pupae), remain inside the colony cultivating symbiont fungi. The sum of the survival strategies of ants in general, including social immunity and nest-cleaning behavior, represents a barrier for the application of biological control programs, mainly microbial ones.
Leaf-cutter ants, classified into more than 250 species within 17 genera, are found exclusively in the American continent [1, 2]. The basal genera
Based on the nidification habits of these species, the lowland arid environments with open vegetation in South America are suggested to be the centers of diversification [3, 4]. However, some studies have reported that this diversification occurred in the wet environment of the Amazon Basin [5, 6]. Recent molecular studies on attini ants point to dry environments as a decisive factor in the symbiont fungus domestication .
2. Mutualistic interactions
Attini ants have maintained a mutualistic relationship with basidiomycete fungi, in an obligatory association for around 50 million years . The interaction with the fungus is not restricted to feeding, once symbiotic associations between larvae and fungi in basal attini have been reported in the literature . Based on morphometric analyses,
3. Agronomic importance
4. Biology and behavior
The colonies of
Ants are holometabolous insects, that is, their life cycle includes three stages: larva, pupa and adult. They are organized into castes, which perform different functions, including the maintenance of the colony and brood care and feeding. The males and queens are part of the reproductive castes, ensuring the survival of the species.
Leaf-cutting ants are prevalent herbivores and dominant invertebrates in tropical forests: the volume of the soil occupied by a single 6-year-old nest of
The colonies present a broad behavioral repertoire, varying according to the morphology (polymorphism) and age (polyethism) of the workers [20, 21, 22]. The foraging behavior consists of cutting and transporting the vegetal substrate to the interior of the colony, where it is processed and incorporated in the symbiont fungus garden [5, 17]. It is the most intense and energy-demanding activity performed in the colony, requiring approximately 90% of the workers .
5. Microbial control
5.1. Entomopathogenic fungi
Entomopathogenic fungi (EF) have been proven a promising strategy to control leaf-cutter ants through microorganisms. These fungi present two phases in their biological cycle: anamorphic (vegetative) and reproductive, when they produce conidia, and, according to Sung et al. , they belong to the phylum Ascomicota and class Hypocreales (Table 1).
Considered one of the most efficient pathogens in microbial control, entomopathogenic fungi need a host to spread in the environment, and the literature has reported that they attach to the cuticle of pest insects through physical and chemical processes, producing chitinolytic enzymes and developing a penetration clamp from the apical region of the hyphae [25, 26]. When the host insect is debilitated (low immunity level), some opportunistic fungi can act as saprophytes, accelerating the insect’s death process . Most fungi penetrate the host through the integument [28, 29]. According to Quiroz , who studied
Studies by Kermarrec et al. , which lasted approximately 10 years, used several entomopathogenic fungi, applied on
Machado et al.  reported that ants of the genus
In this sense, it is known that the ant antennae “analyze” different types of material, allowing the recognition of pathogenic elements that could harm the colony . Kermarrec et al.  observed that the sensitivity of the antennae can be demonstrated by the fact that attractive baits containing entomopathogenic fungi and spores are not cut but placed away from the colony area by
6. Other entomopathogenic microorganisms
In addition to entomopathogenic fungi, other organisms have been tested to control pest insects. Entomopathogenic nematodes (
It is important to emphasize that the associations of the bacteria
7. Use of synthetic chemicals
Once toxic substances are capable of overcoming insect immunity barriers (individually and collectively) [47, 48], the development research on strategies to impair the colony organization through immunosuppression, weakening the humoral system of the ants, is fundamental. Such strategies often include the use of chemical substances . In this sense, subdoses of insecticides would aid the biological control; however, this would lead us to the use of granular baits containing a low concentration of the active ingredient. Nevertheless, the bait itself is highly specific to the target insect and, consequently, environmentally friendly, in contrast with other insecticides available in the market, and the demand for efficient and sustainable products is on the increase, considering the stricter requirements of regulatory agencies .
8. Capability to neutralize pathogens through gland secretions
Leaf-cutter microbial control has been subject to the same criticism directed toward plat extracts, that is, considering that the ants evolve in an environment where toxic plants and pathogenic microorganisms abound, probably the concentration is not the only issue to be taken into consideration for the success of these control methods [51, 52, 53]. The virulence of entomopathogenic fungi has been intensely investigated, especially the length of time needed to cause the pest population mortality; however, further investigation is needed on the forms of application and compatibility with adjuvants. Moreover, microbial control faces several natural barriers, including the capability of the ants to inhibit the germination of the conidia through secretions produced by their salivary glands (4-methyl 3-heptanone) and through their fecal fluid (chitinolytic enzymes) as well . According to Pagnocca , in order to prevent the contamination of the symbiont fungus garden, the ants lick the foraged substrate surface, keeping microorganisms in the infrabuccal cavity to dispose them in the waste chamber. Thus, the infrabuccal cavity would function as a filter, preventing the entrance of solid particles into the worker body and in the nest as well [56, 57].
The metapleural glands represent another defense mechanism, secreting several substances, such as phenylacetic acid; 3-hydroxydecanoic acid; indoleacetic acid; and skatole , which produce acid secretion that inhibits the germination of some entomopathogenic fungi, protecting the cuticle against microbial infections . Fernandez-Marin et al.  identified a higher level of cleanliness by the gland secretion in workers inoculated with
Pest control must be thought of from a preventive point of view and, in the case of leaf-cutter ants, in the initial phase of colony establishment. The scientific scenario today allows a more thorough and accurate research .
The use of advanced analysis tools, such as scanning, light and confocal microscopes to investigate what happens in the moment when the insect is infected by the fungus .
Cutting-edge technology software to monitor the agronomic environment and the emergence of entomological radars  can be incorporated as control strategies, indicating the best moment for the technique application (nuptial flight or “revoada”).
Drones have been used in some sampling techniques for leaf-cutter ants .
The use of microbial control agents in different formulations (conidia, blastospores or microscleroids) synergically applied along with adjuvants is a tendency in microbial control .
10. Final consideration
Overall, microbial control has been proven efficient to control some pest insects (
Various isolates of