Chapter 6

FOOD USE BY LEAF-CUTTER

INTRODUCTION

FEEDING HABITS OF

Most extant belong to Division Endopterygota. Although the origin of holometabolism is not entirely clear, it represents an important adaptive trait that allows adult and juvenile individuals of the same to use different food resources. In addition, approximately 50% of species are phytophagous, feeding directly on plants, and represent the most important primary consumers of terrestrial plants, surpassing vertebrate and directly competing with humans for food. In Hymenoptera, the of feeding habits occurred through two distinct routes: larval herbivory, an ancestral character within the order that is prevalent among species of suborder Symphyta, and larval zoophagy, common in most species of suborder Apocrita. However, the basal species in suborder Apocrita are parasitoids, and how they evolved from phytophagous ancestors has not been properly explained to date; inquilinism has been suggested as an intermediate stage. The adaptations that occurred in the ovipositor enabled parasitoidism, which currently occurs in half of the hymenopteran families. With the appearance of the sting apparatus, the next stage was (Daly et al., 1998). Some Hymenoptera reversed to phytophagy, especially social aculeates. The Vespoidea feed on nectar and pollen, while the Apoidea use nectar, pollen, and oils from plants. In addition to nectar and pollen, the Formicoidea use extrafloral nectaries, seeds, and food bodies, which are common on the plant epidermis and are considered important energy sources for ants (Tobin, 1994) Thus, in addition to the direct use of plants as food, that is, the intake of part or all of a plant, the return to phytophagy also involves the ability to exploit resources that plants already produce in different ways. Thus, not only are all species eusocial, a unique condition among hymenopterans, but they also have a wide range of feeding habits. A classic example are the leaf- cutter ants, which belong to genera and Acromyrmex and use fresh plant material to cultivate their fungal symbiont, considered the main food source for the colony. FOOD TRANSFER AMONG ANTS

In an ant colony, all members of the society have certain skills to perform in the energy cycle for the distribution of food (Hõlldobler & Wilson, 1990). The mechanisms for food transfer are very diverse but may be grouped into three basic categories: 1. Transfer of material stored in the digestive tract: adult workers may transfer food stored in their crops to larvae, to workers (who may specialize on food storage [repletes] or not), and to the queen. In contrast, ant larvae transfer proteins and amino acids to adult workers in different ways, particularly through trophallaxis of glandular secretions and fluids associated with the hemolymph; 2. Transfer through trophic : the source of energy for queens that employ claustral colony founding, such as Atta sexdens rubropilosa, are the food reserve contained in the fat body and the modification of a few organs, such as the degeneration of the flight muscles (Cruz-Landim & Moraes, 1979). In this case, transfer occurs mostly through the laying of trophic eggs; 3. Transfer of glandular secretions: the salivary system of ant workers may contribute for larval feeding, although this is less widespread in ants compared to bees.

THE INFRABUCCAL FILTER—A MAJOR BARRIER

Adult hymenopterans are known to essentially ingest liquid food in the adult stage, but the effectiveness of the mechanism to block solid materials in ants is extraordinary, even preventing the passage of particles less than 1 µm in diameter. Motivated by an interest in clarifying the mechanisms of separation of solid and liquid foods, Bueno (2005) analyzed the foregut of alates of Atta sexdens rubropilosa in detail. The morphology of the digestive tract of these ants varies little. It is organized into three basic regions: foregut, midgut and hindgut. The foregut, or stomodeum, of the adults of Atta sexdens rubropilosa is lined with a distinct cuticle, as in other ants and insects in general. The anteriormost region, located in the head, has several modifications, such as glands and cuticular structures (bristles and hairs). Before the mouth opening there is a preoral cavity and a chamber called the infrabuccal pocket, with the mouth opening in an antero-superior position. Immediately past the mouth opening there is a short rounded elongation of the pharynx, which is dorsoventrally flattened from that point on, that corresponds to the palate. This is followed by the esophagus, a long tube that crosses the rest of the head, the entire mesosoma and the waist to reach the crop, in the anterior part of the gaster, and, eventually, the proventriculus, which marks the boundary between foregut and midgut (Caetano, 1990). Examinations of sagittal sections of the head of the female alate of Atta sexdens rubropilosa, both under stereoscope and using scanning electron microscopy (SEM), allowed the identification of cuticular structures found in the anterior region of the foregut (Figure 1). At the basal portion of the infrabuccal pocket the cuticle has several shallow folds forming coriaceous structures (Figure 1A), while at the posterior region the folds are more pronounced. In the lateral region (at the back of the infrabuccal pocket) there are cuticular projections that resemble short thick bristles (Figure 1L); in the posterior ascending region, the projections are shaped as plates, with longer and thinner bristles (Figure 1K). In the middle and anterior upper regions, these plates have more layers of bristles (Figure 1B), which are longer and spaced more closely together. These layers extend all the way to the opening of the oral cavity (Figure 1C). At the beginning of the pharynx there is a higher density of bristles of different lengths surrounding its entire circumference, arranged in parallel rows inclined at an angle of approximately 45º towards the mouth opening (Figure 1D). After the pharynx bends at a 90º angle towards the mid-region of the palate, the bristle rows on the upper region become more regular, with a uniform size and always bent towards the anterior portion (Figure 1E), while on the lower region they advance a little more (Figure 1F). After that, the upper region becomes more sparsely covered by thinner bristles (Figure 1G), followed by a transition region with bristles and hairs (Figure 1H), while the lower region has structures resembling chemoreceptors (Figure 1J). At the end of the palate, next to the opening of the post-pharyngeal glands, there are deep folds on the pharynx wall with bristles and thin hairs (Figure 1I), followed by the circular opening of the esophagus.

Anterior Dorsal Dilator Muscle of Pharynx Circular Muscle of Postpharyngeal Preoral Pharynx Gland Cavity Pharynx Palate Hypopharynx Glossa Clypeus Head Integument

Posterior Dorsal Dilator Muscle of Pharynx Labrum Brain

Ventral Dilator Muscle of Pharynx

Labial Palps Esophagus Tentorium Base of Maxilla Neck Oral Labium Opening Infrabuccal Subesophageal Muscles Ganglion Pocket

Figure 1. Sagittal section through the head of the female alate of Atta sexdens rubropilosa (Bueno, 2005). A–L: Scanning electron microscopy (SEM) micrographs of the various portions of the anterior region of the foregut, revealing details of the internal cuticular structures. The thin arrows in C, E, and F indicate the direction of food flow; the thick arrow in C indicates the oral opening.

When the workers of a leaf-cutter ant colony manipulate the plant fragment as they tend the garden or the symbiotic fungus itself during larval feeding time, part of the material is ingested and goes directly into the infrabuccal pocket, since the oral opening is closed at that time due to its anterosuperior position. Later, the retraction of mouthparts closes the preoral cavity, and contractions of the muscle bundles of the pharynx creates negative pressure in the palate region, causing the liquid material in the infrabuccal pocket to be suctioned into the mouth and move through the pharynx. Thanks to the action of the circular muscles in the anterior region of the pharynx the liquid does not return. During this process, the food has to pass through the entire cuticular structure apparatus described above, whose function is to retain extremely small solid particles, around 1 µm in the case of Atta sexdens rubropilosa. Images of a pellet inside the infrabuccal pocket of the female alate and another one removed from this cavity revealed the presence of the fungal symbiont and fragments of plant material of various sizes (Figure 2). Minute fragments, less than 1 µm in size, that cross the filtration mechanism at the anterior portion of the foregut are retained in the crop, forming approximately 40 µm lumps surrounded by an acellular membrane, which are not eliminated (Figure 3). This is supported by the observations of Caetano (1998), because the proventriculus, the last portion of the foregut, has the function of filtrating food that moves from the crop to the ventriculus. The remaining food inside the infrabuccal pocket is eliminated as pellets, which result from the intense protraction and retraction movements of the glossa, with the help of maxillae and laciniae. At the nest, these pellets are handled by the smallest workers, who remove the excess of water and transfer them permanently to the waste pile. Measurements of pellets found inside the infrabuccal pocket of female alates of Atta sexdens rubropilosa averaged 1.009 mm in size, with standard deviation of 0.102 mm (N = 57), while in workers of variable sizes the average and standard deviation were 0.145 mm and 0.065 mm, respectively (N = 2194). Analyses of the contents of the infrabuccal pocket under the microscope revealed the presence of particulate material. In medium-sized workers, who are responsible for foraging, there were many silica grains, while medium-small to very small workers had fungal hyphae, small leaf fragments and amorphous wax-like material (Diniz, 2000).

Figure 2. SEM micrographs of the head of the female alate of Atta sexdens rubropilosa in sagittal section, highlighting: A. Empty infrabuccal pocket and B. Pellet removed from the pocket (dashed line); C. Detail of a pellet region rich in hyphae from the fungal symbiont; D. Detail of a pellet region with plant fragments (Bueno, 2005).

Figure 3. A. SEM micrograph of clumps formed inside the crop of Atta sexdens rubropilosa workers, surrounded by an acellular membrane; and B. Detail of a clump, showing it comprises particles less than 1 µm in size (Bueno, 2005).

SEPARATION OF HYDROPHILIC AND LIPOPHILIC COMPOUNDS

Despite the number of published descriptions of the foregut, here we provide the first detailed description of the apparatus responsible for retaining solid particles. This apparatus allows workers, female alates, and queens of Atta sexdens rubropilosa to selectively separate lipid from non-lipid compounds at the final portion of the pharynx. It is also directly related to the capacity of post-pharyngeal glands to retain lipids (Figure 4). If the amount of ingested lipids is large and the system is not big enough to store it, the excess moves into the crop but does not reach the ventriculus. When the stock of the system is reduced the stored lipid returns through the esophagus. In general terms, the ant salivary system has four pairs of glands: 1. Thoracic salivary glands, which open in between the mouthparts (at the base of the labia) and whose excretory portion is located in the mesosoma; 2. Hypopharyngeal glands, which open on both sides of the hypopharyngeal plate; 3. Mandibular glands, connected to the mandibulae; and 4. Post- pharyngeal glands, exclusive to adult Formicidae, which communicate with the end of the pharynx, near the transition to the esophagus. The post-pharyngeal glands originate from two dorsolateral expansions of the posterior region of the pharynx immediately after the body reconstruction during metamorphosis (Gama, 1985). Despite their ectodermal origin, Delage- Darchen (1976) considered their origin distinct from the other glands in the ant salivary system, which derive from the metameres of the mouthparts. In contrast, the post-pharyngeal glands emerge directly from the differentiation of parts of the foregut. The importance of food derived from glandular secretions is very well known in bees, where the workers produce royal jelly to feed larvae and produce new queens. Food of glandular origin may also be important for some ant species, but the nature and function of the glands at the anterior portion of the alimentary canal are not currently well known (Wheeller, 1994). While in bees the mandibular and hypopharyngeal glands produce secretions that are the main components of the larval food (Michener, 1974), in ants the mandibular glands produce multiple compounds that have a role in chemical communication and, although the hypopharyngeal glands have digestive enzymes, they are not well developed (Delage-Darchen, 1976). The thoracic salivary glands seem to act directly in digestion and secretion of substances that are transferred to other members of the society through trophallaxis (Gama, 1985). However, the many functions that have been established for post-pharyngeal glands are, to date, extremely contradictory. It has been suggested that they are a food source for the queen and the brood (Hölldobler & Wilson, 1990), which is consistent with the proposals advanced by Peregrine & Mudd (1974) regarding the transfer of the content of these glands among individuals of a colony and by Ayre (1963), who had already observed secretions from post-pharyngeal glands in larval ventriculi and concluded that they were associated with trophallaxis. The presence of lipids inside post-pharyngeal glands has been observed in many ant species, and their origin is questionable. According to some authors, they derive from secretions of the glandular epithelium itself. For others, they originate from ingested food. Delage-Darchen (1976) put forward three non-exclusive hypotheses regarding their presence: a. transcellular route: the material penetrates the cells that form the glands and is metabolized; b. extraoral route: the material is regurgitated directly from the glands to the other colony members; c. digestive route: the material travels from the interior of the glands towards the crop and the ventriculus. Using molecules labeled with radioactive isotopes, Phillips & Vinson (1980) observed that fatty acids and triglycerides injected into the hemolymph of Solenopsis invicta did not cross into post- pharyngeal glands. Thus, these authors believed that the lipids in the gland lumina must have originated from ingested food. Similarly, based on directly collected data, Vinson et al. (1980) suggested that post-pharyngeal glands act as cephalic caeca in Solenopsis invicta, absorbing fatty acids and triglycerides. Later, Bagnères & Morgan (1991), studying the same ant species, verified that hydrocarbons were the main compounds of the lipids found inside the glands and were similar to those in the cuticle of workers; their function is interspecific and intracolony recognition. As a result, these authors suggested that the nutritional role of the post-pharyngeal glands needed to be reassessed. However, Bueno (2005) directly demonstrated the movement of lipids into gland lumina of workers and queens of Atta sexdens rubropilosa using autoradiography and liquid scintillation with tritiated oleic acid. The lipids are subsequently absorbed by the cellular epithelium, transferred to the hemolymph and distributed throughout the body. The autoradiography technique proved highly effective to reveal the route followed by the oleic acid, from its entrance into gland lumina until it reaches the cytoplasm of the cells, where it can either be stored or transferred to the hemolymph. The liquid scintillation assisted the quantitative analysis, revealing the distribution of the oleic acid to the head and the rest of the ant body after being absorbed by the post-pharyngeal glands. These results support the observations of Bueno et al. (2001) regarding the use of vital stains with solubilities specific for water and lipids as indirect indicators of food flow in workers of Atta sexdens rubropilosa. These authors observed that the lipophilic dye quickly reached the infrabuccal pocket, between 1-2 hours after application, and immediately after the post- pharyngeal glands. In contrast, the amount of this dye in the crop was always very small and could be interpreted as experimental error, or even that after the anterior region of the foregut becomes saturated, the dye may move into the crop. The staining intensity in the infrabuccal pocket and post-pharyngeal glands depended on group size at the various time intervals, that is, as the number of workers in the dish increased, so did the staining intensity per individual. On the other hand, the hydrophilic dye reached the infrabuccal pocket and the crop simultaneously, and there was no relationship between the amount of dye in these structures and the time of exposure. In addition, this dye was never found inside the post-pharyngeal glands. There was no group size effect on staining intensity of the various structures, meaning that dye incorporation was not substantially different as the number of workers in the dish varied (Figure 5).

Figure 4. Micrographs of the post-pharyngeal glands of Atta sexdens rubropilosa, stained blue due to the incorporation of the lipophilic dye sudan black (Bueno, 2005). A. Position of the post-pharyngeal glands inside the dissected head of a soldier; B. Detail of the post-pharyngeal glands removed from the soldier head and their connection to the posterior region of the pharynx; C. Post-pharyngeal glands of a female alate connected at the end of the pharynx; D. Histological section of a gland digit. ct: cuticle; ec: cellular epithelium; lm: lumen; nc: nucleus.

RHODAMINE DYE SUDAN BLACK DYE

A. 1 worker per dish A. 1 worker per dish

Percentage Percentage

Hours Hours

B. 2 workers per dish B. 2 workers per dish

Percentage Percentage

Hours Hours

C. 5 workers per dish C. 5 workers per dish

Percentage Percentage

Hours Hours

Figure 5. Variation in intensity of staining by rhodamine (hydrophilic dye) and sudan black (lipophilic dye) in the various structures of the digestive tract of Atta sexdens rubropilosa workers in different group sizes (Bueno, 2005).

THE ABSENCE OF TROPHALLAXIS

Bueno et al. (2001) also analyzed the behavior of Atta sexdens rubropilosa workers in three nest fragments maintained inside glass dishes. Each contained a small piece of a fungus garden and between 200 and 300 workers. Approximately 30% of them, subdivided into three size classes (small, medium and large), were individually tagged using small numbered disks glued to the mesosoma. The activities inside the dishes were filmed in VHS, generating 10 hours of observation. The analysis of the behavior of the group of workers provided important information for the interpretation of the movement of dyes among individuals. Trophallaxis, an important social behavior, was not observed between adult workers, not even between different castes, confirming the observations of Andrade et al. (2002) and Schneider (2003). Self-grooming was the most frequently observed behavior, 10.6% of the total observation time. In contrast, social grooming was relatively rare, only about 2.5%. Thus, neither trophallaxis nor social grooming can be considered responsible for the movement of dyes among workers of Atta sexdens rubropilosa, suggesting that it occurs instead through physical contact of bodies followed by self-grooming. This sequence of behaviors would also explain the increased amount of lipophilic dyes within the post-pharyngeal glands as the number of workers in the dish increased, because that would make encounters more likely and further promote self-grooming, which is responsible for the transference of oils into the mouth.

FOOD INGESTION BY DIFFERENT CASTES

It is widely accepted that the fungal symbiont is the staple food of leaf-cutter ant colonies, where adults primarily ingest liquids and larvae feed on fungi, which are rich in carbohydrates and proteins and poor in lipids (Martin et al. 1969). It is also accepted that fungi are not the only source of food for the colony, because adult workers directly imbibe plant sap when they are cutting and chopping material (Littledyke & Cherrett, 1976, Quinlan & Cherrett, 1979, Forti & Andrade, 1999). According to Bass & Cherrett (1995), only 9% of the energy needs of adult workers come directly from fungi. Detailed analyses of the anterior region of the foregut, especially the structures of the infrabuccal filter and the apparatus that separates lipid from non-lipid substances, coupled with behavioral observations of the different castes within the colony, allow a new interpretation of how feeding occurs at the individual level. The different adult functional castes in a colony of Atta sexdens rubropilosa use multiple food sources as they perform activities in different areas of the ant nest at certain times. Workers that are active outside of the nest, foraging, may imbibe sap directly while cutting and chopping leaves (Quinlan & Cherrett, 1979), and could potentially also ingest lipids from several other plant structures, such as food bodies and elaiosomes on seeds. When cut plant fragments reach the nest, a complex process of preparation of the plant substrate for incorporation into the fungal garden begins, with the goal of optimizing its use by the fungal symbiont. During initial processing, workers may ingest sap as they chop and squeeze the edges of the plant fragment. At that moment, the workers very frequently lick the edges of the plant fragment, which exudes sap. Another behavior is scraping the surface of the leaf to remove the epicuticular wax layer and facilitate substrate decomposition by the fungus. The workers ingest and store small leaf fragments in their infrabuccal pocket, along with the wax, which may be a potential food source (Table 1) (Diniz, 2000; Andrade et al. 2002). The fungus garden is constantly tended by workers ranging in size from intermediate to very small through their behavior of licking the surface of the garden. Silva et al. (2003) ran a series of laboratory experiments and concluded that glucose was the main source of energy for these ants, responsible for over 50% of their nutritional needs (Figure 6). The glucose in the fungus garden is derived from extracellular digestion by the symbiont.

Figure 6. Median survival (S50, in days) of workers of Atta sexdens rubropilosa maintained on artificial diets with different concentrations of glucose. The filled circle indicates the median survival of workers maintained on fungal garden only (figure from Silva et al, 2003). Table 1. Relative frequency of behavioral acts performed by workers of Atta sexdens rubropilosa to prepare the plant substrate for the cultivation of the fungal symbiont, according to size class (Diniz, 2000).

Behavioral Acts Wk1 Wk2 Wk3 Wk4 Wk5 Total Cutting leaves 0.00 0.00 0.97 0.03 0.00 0.04 Transporting fragments to the nest 0.00 0.00 0.91 0.09 0.00 0.04 Chopping plant fragments 0.10 0.90 0.00 0.00 0.00 0.06 Licking plant fragments 0.39 0.61 0.00 0.00 0.00 0.31 Pressing the edges of the plant fragments 0.00 0.81 0.19 0.00 0.00 0.18 Scraping plant fragments 0.52 0.48 0.00 0.00 0.00 0.21 Depositing fecal fluid on plant fragments 1.00 0.00 0.00 0.00 0.00 0.08 Incorporating plant fragments into the 0.66 0.34 0.00 0.00 0.00 0.05 fungus garden Inoculating the fungal symbiont 0.87 0.13 0.00 0.00 0.00 0.03

Wk1: very small worker; Wk2: small worker; Wk3: medium-small worker; Wk4: medium worker; Wk5: medium-large worker. Total: relative frequency of each behavioral act across all classes.

It is also known that the fungal symbiont produces specialized structures called gongylidia to feed the larvae. These structures are spread throughout the fungus garden, and when observed under the microscope the tips of the hyphae are revealed to be swollen and round shaped; they occur in bundles called staphylae (Figure 7). They are the main food for the larvae, who, despite being in constant contact with the fungus, are unable to feed themselves and are completely dependent on the workers. When a worker feeds larvae, it may do so in two ways: it may collect a staphyla or hyphal bundle and put it directly onto the mouthparts of the , or it may collect a staphyla and manipulate it with its mandibles, labia, and anterior legs, always touching it with its antennae, until the consistency is moist, only then depositing it onto the larval mouthparts. During manipulation, which is very frequent (Table 2), the worker may ingest the exuding liquid and even small portions of the fungal symbiont, which enter its infrabuccal pocket (Schneider, 2003). In addition to sap and other plant components, staphylae, and substances derived from the extracellular digestion promoted by the fungal symbiont, Schneider et al. (2000) reported that leaf-cutter ants may also obtain food from secretions provided by larvae, more specifically from the secretion of fluids from the anal region (Figure 8). Another brood care behavior frequently observed by Schneider (2003) was larval grooming, especially of their oral region, at which time the workers ingest food debris.

Figure 7. Light micrographs of the fungus garden of Atta sexdens rubropilosa (Schneider, 2003).

A. Region rich in staphylae (arrows); B. Detail of the staphyla region (arrows); C. Detail of the hyphal exudate (arrow); D. Detail of a staphyla showing gongylidia (arrow).

Table 2. Relative frequency of the behavioral acts of Atta sexdens rubropilosa workers related to brood care according to size class (Schneider, 2003).

Behavioral Acts Wk1 Wk2 Wk3 Wk4 Wk5 Total Cleaning pupae 0.67 0.30 0.03 0.00 0.00 0.030 Cleaning the bodies of larvae 0.54 0.38 0.05 0.03 0.00 0.150 Cleaning the oral region of larvae 0.41 0.34 0.13 0.06 0.06 0.140 Cleaning the anal region of larvae 0.45 0.28 0.21 0.06 0.00 0.090 Helping larvae/pupae during ecdysis 0.00 0.33 0.67 0.00 0.00 0.007 Transporting eggs 0.88 0.12 0.00 0.00 0.00 0.030 Transporting larvae 0.36 0.44 0.18 0.02 0.00 0.160 Transporting pupae 0.30 0.37 0.23 0.08 0.02 0.070 Offering fungus to larvae 0.47 0.32 0.12 0.07 0.02 0.160 Feeding larvae with intact staphylae 0.33 0.33 0.07 0.27 0.00 0.020 Feeding larvae with manipulated 0.50 0.37 0.09 0.04 0.00 0.140 fungi Feeding larvae with intact hyphae 0.00 1.00 0.00 0.00 0.00 0.001 Feeding larvae with trophic eggs 1.00 0.00 0.00 0.00 0.00 0.002

Wk1: very small worker; Wk2: small worker; Wk3: medium-small worker; Wk4: medium worker; Wk5: medium-large worker. Total: relative frequency of each behavioral act across all classes.

Figure 8. Micrograph of one final-instar (larger) and one intermediate-instar (smaller) larva of Atta sexdens rubropilosa, showing the moment they exude anal secretion (arrows), which is collected by workers (proctodeal trophallaxis) (Schneider, 2003) The usage of the larval anal fluid by workers, proctodeal trophallaxis, is a relatively common event in ants already described by numerous authors (Hölldobler & Wilson, 1990), but it was observed in leaf-cutter ants for the first time by Schneider et al. (2000). These authors reported that, at given moments, the workers would touch the anal region of the larvae, which were variable in size, and the larvae would release a small clear drop that was immediately imbibed by the workers. Chemical analyses of this fluid revealed that it was rich in essential nutrients for the ants, comprising 5–8 mg/mL of protein and 10.98 mg/mL of glucose (Schneider, 2003). The volume of proctodeal fluid released by larvae of Atta sexdens rubropilosa of different sizes was on average 0.055 µL, which corresponds to 0.275–0.440 µg of protein and 0.600 µg of glucose being offered by the larvae each time they were requested to (Schneider, 2003). This fact shows that leaf-cutter ant larvae have a fundamental role in the nutrient flow of the colony. As the adult individuals are unable to feed from the solid parts of the fungi, the larvae do it, digesting the fungal wall and transferring those nutrients to the hemolymph, where they are reabsorbed by the Malpighian tubules, transferred to the hindgut and offered to workers through trophallaxis. This way, nutrients that had been unavailable to the adult individuals of the colony become available. The Atta sexdens rubropilosa queen depends on workers for all of its needs, such as cleaning, protection, -laying, and feeding. In this case, the workers offer food to the queen using the same method used for the larvae, that is, they collect, manipulate, and deposit staphylae directly into the queen’s mouth (Table 3). Thus, it could be said that queens specialize on feeding on the fungal symbiont, since they do not participate in other activities where they would have an opportunity to acquire food. In some species, the staphylae are also the main food source for soldiers and alates, when present. For Atta laevigata, it has been observed that, when the soldiers or alates are inside the nest, on the fungus garden, they remain stationary with extended antennae. When a worker passes by them with a staphyla in its mandibles, they touch its head with the antennae to request food. The worker then deposits the piece of fungus in the mouthparts of the soldier or alate, who quickly consumes it. Not only does the symbiosis between ants and fungi allow for the exploitation of a wider diversity of plants (polyphagia), but it also allows workers to acquire food from multiple sources, increasing adaptive success. This is reflected in their extremely complex and densely populated nests and their great ecological importance, since they are considered dominant herbivores in several , as stressed by Hölldobler & Wilson (1990). Table 3. Relative frequency of behavioral acts of Atta sexdens rubropilosa workers to care for the queen according to size class (Diniz, 2000).

Behavioral Acts Wk1 Wk2 Wk3 Wk4 Wk5 Total Feeding the queen 0.60 0.40 0.00 0.00 0.00 0.18 Cleaning the queen 0.48 0.52 0.00 0.00 0.00 0.30 Standing over the queen 0.36 0.43 0.17 0.04 0.00 0.43 Collecting eggs 0.82 0.18 0.00 0.00 0.00 0.09

Wk1: very small worker; Wk2: small worker; Wk3: medium-small worker; Wk4: medium worker; Wk5: Medium-large worker. Total: relative frequency of each behavioral act across all classes.

FINAL REMARKS

The capacity of insects to act as pests or vectors of plant and diseases stems from their feeding habits. Thus, detailing the structures that form the digestive system and correctly interpreting the functioning of the ingestion mechanism allows us to improve insect management techniques. In addition, the specialized literature on insect feeding has always neglected the use of lipids in this process. With that in mind, could the success of ants be related to the presence of a structure specialized in using energy from lipids available in nature? Taking into account recent discoveries, many paradigms need to be reassessed and new interpretations of the nutritional ecology of ants can be established, especially the following: - The apparatus that forms the infrabuccal filter of the leaf-cutter ant Atta sexdens rubropilosa, locally known as “lemon ants”, blocks the passage of particles greater than 1 µm; - The ants selectively separate lipid and non-lipid components from ingested food, with specific destinations. Lipid compounds reach the post-pharyngeal glands, while the non- lipid compounds move into the crop. This enables adults to obtain energy from lipids available in nature; - The absence of trophallaxis among adult individuals in colonies of Atta sexdens rubropilosa, but its occurrence between larvae and adult workers in the form of proctodeal trophallaxis; - In some species, the feeding of larvae, alates, and soldiers is carried out solely by workers, even if these castes are in the middle of the fungus garden; - The acquisition of food by the adult workers of a colony is diversified, that is, it occurs in the same place where they are performing a basic activity: foraging, preparation of plant material, caring for the fungus garden, and caring for the brood.

Acknowledgements

To the São Paulo Research Foundation (FAPESP) and the National Council for Scientific and Technological Development (CNPq) for their financial support. To the biologists Itamar C. Reiss and Antônio T. Yabuki and the illustrator Cristiane Mileo for technical support.

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