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Recent Res. Devel. Entomol., 5(2006): ISBN: 81-7736-246-1

Studies on Leaf-Cutting ants, Acromyrmex spp. (Formicidae, Attini): Behavior, reproduction and control

Roberto S. Camargo1, Luiz C. Forti1, Juliane F. S. Lopes2

and Nilson S. Nagamoto1 1Laboratório de Insetos Sociais-Praga, Departamento de Produção Vegetal –Setor Defesa Fitossanitária, FCA/UNESP, PO Box 237, Zip Code 18603-970 Botucatu, São Paulo State, Brazil; 2Comportamento e Biologia Animal, Instituto de Ciências Biológicas – ICB, Universidade Federal de Juiz de Fora –UFJF, Campus Universitário Martelos, Zip Code 36036-330 Juiz de Fora, Minas Gerais State, Brazil

Abstract Ants are optimal models for behavioral studies of social groups due to the interactions between individuals of the colony and other organisms. Foraging and reproductive strategies are diverse and highly adapted to the needs of these insects. Leaf-cutting ants, especially the genus Acromyrmexspp., present a complex foraging system, including the

Correspondence/Reprint request: Dr. Roberto S. Camargo, Laboratório de Insetos Sociais-Praga, Departamento de Produção Vegetal - Setor Defesa Fitossanitária, FCA/UNESP, PO Box 237, Zip Code 18603-970, Botucatu São Paulo State, Brazil. E-mail: camargobotucatu@yahoo.com.br

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transfer of information about foraged material and partitioning of food transported among the worker force according to the size of their load and/or trail length. In addition, this genus is characterized by a marked temporal division of labor, with young workers performing tasks inside the nest and older workers being engaged in tasks outside the nest. Another aspect discussed is worker reproduction in orphan colonies of some Acromyrmex spp. where, in the absence of the queen, workers lay haploid eggs that give origin to males. Behavioral studies on these subjects are discussed on the basis of research involving this genus. Finally, considerations about investigations regarding the control of these ants are presented. 1. Introduction Leaf-cutting ants are the dominant herbivores of the Neotropical region which consume more vegetation than any other animal group, including other insects and mammals [1]. These ants, which comprise the genera Atta and Acromyrmex, are considered to be pests of many cultivated plants since they use plant material for the growth of the symbiontic fungus, the primary food source of their larvae [2]. Worker ants use the fluids extravasated from the leaves as an energy source during the leaf-cutting activity [3,4]. In addition, these animals are interesting models for behavioral studies due to their elaborate social organization, as well as their complex system of foraging and reproduction. Species of the genus Acromyrmex are characterized by lower polymorphism of the worker caste and smaller and less populous colonies when compared to the genus Atta spp., although little is known about their biology. Aspects such as the transfer of information from scout workers to other nestmates, as well as the influence of trail length, have been studied in order to understand the organization of labor and the division of tasks inside and outside the nest. Inside the nest, the temporal division of labor has been little investigated in this group, although this division exists and is well pronounced, with the demonstration of temporal in addition to physical subcastes. Another interesting characteristic of this group, in addition to queen reproduction, is worker reproduction in orphan colonies. In some species, in the absence of the queen workers lay haploid eggs that develop into males [5]. We will discuss below various studies on relevant aspects including the behavior, biology and control of the leaf-cutting ant Acromyrmex. 2. The role of scout ants on the decision-making process of recruited workers In leaf-cutting ants, foraging is a complex process during which individual and social elements interact in the determination of substrate carrying to the

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colony in order to permit a maximum productive yield [6]. In addition to the steps of selection, cutting and transport of the plant material, foraging also includes the process of recruitment during which information about the location and, possibly, about the quality of the discovered food resources is conveyed [7]. During mass recruitment, characteristic of leaf-cutting ants, the signal of food source discovery is amplified by recruited workers that participate in the marking and maintenance of chemical trails. Food recruitment is believed to be controlled by the behavior of the scout ant [8]; however, the decision-making process may also be influenced by interactions between workers and by interindividual differences. Recruited workers might learn the odor of the food fragment carried by a scout ant and use it as a memorized signal that influences the decision of which substrate to carry, as demonstrated for A. lundi [9,10]. Furthermore, factors such as the location, quantity and quality of the food resource influence the recruitment intensity [11,12,13,14], especially because workers must combine their individual nutritional needs with those of the symbiontic fungus living in their colonies [15, 16, 17], a fact resulting in a collective foraging pattern [7]. There is evidence indicating that workers use their previous experience as cues [18, 19]. According to [8], foragers inside the nest show differences in the response threshold to recruitment signals, a fact that probably interferes with responses obtained in studies on foraging. Laboratory studies have evaluated how the information conveyed by a scout influences the substrate selection by recruited workers of Acromyrmex balzani, Acromyrmex rugosus and Acromyrmex crassispinus. When the information communicated by a scout ant referred to a preferred substrate, the latter was significantly more carried by recruited workers. On the other hand, when the information received from the scout corresponded to less preferred plant species, different fragments were carried at random. These results reflect the preference of workers for a certain plant type and the fact that the information conveyed by a scout ant influences the decision-making process of recruited workers, considering the random carrying of fragments from different plant species when the information conveyed by the scout ant corresponds to a less preferred substrate [20]. The existence of a communication system during recruitment is highly likely, especially when considering that recruitment is a growing phenomenon, i.e., the number of involved individual increases, with recruited workers becoming able to recruit other individuals. Consequently, the individuals that were not directly recruited by the scout ant receive a chemical information that depends on the decision or preference of another forager. The influence of the information communicated by the scout ant is evident when we consider the random carrying of preferred and non-preferred fragments when the information conveyed by the scout ant corresponds to a less preferred substrate.

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Considering the hypothesis that differences in the response of workers cannot be detected at the beginning of recruitment [8], one may suppose that the signal transmitted by the scout ant is primarily responsible for the decisions of the recruited workers. However, by permitting a recruited worker to come in contact with a substrate that differs from the information received, the ant is able to perform a new assessment which results in a different decision. The unknown option might be better or not than the known option, but the individual can only assess it through direct contact with the substrate [21]. Until they make direct contact with the substrate, the recruited workers base their decision on the signal conveyed by the scout ant, which probably indicates the quality of the food. 3. Influence of trail length during substrate transport One key aspect of foraging is related to the distance between the resource and the nest. Foragers establishing foraging areas at 100 m or more from the nest have been observed [22]. A correlation can be established between the distance from a given plant species and water content [17], transport velocity [22], and size of the cut fragment. Thus, in order to determine the occurrence of task partitioning in three Acromyrmex species and whether this strategy can be correlated with different trail lengths, laboratory studies were performed in which workers had to travel three different distances to arrive at the substrate. Task partitioning, i.e., the transfer of material from one individual to another during leaf transport, was found to be related to an increase in the length of the artificial trails. The results agree with the hypothesis that task partitioning, accompanied by the formation of leaf caches, is an adaptive strategy [23]. In addition, cache formation permits workers on longer trails to more rapidly return to the food source and to maintain the pheromonal trail [24], while, at the same time, they save time and energy. In the field, cache formation may confer an advantage in the prevention against predation [25], and also reduce the loss of leaves, promoting a balance between foraging and processing rates [26]. The benefit of cache formation is that it increases the probability of recovery of fallen leaves, attracting foragers more efficiently than scattered leaves or providing a greater stimulus for collection [26], considering the influence of food volume on carrying rates [27] which may also oscillate according to the distance between the nest and food [28]. Cache formation probably increases the carrying motivation of workers since these caches provide a localized food source. There is also evidence that ants prefer shorter and faster tracks and, in the case of deviations, vertical ones are preferred because they present a reduced risk of navigation errors [29]. In addition, a succession of signals is used by the workers to remain on the trail [30]. However, if the trail is long, the probability

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of a worker to get lost increases and the ant spends time and energy during reorientation. These costs can be avoided with the formation of caches, suggesting that task partitioning during leaf transport is an adaptive strategy that permits leaf-cutting ants to explore food sources distant from the nest.

4. Foraging in grass-cutting versus leaf-cutting species The genus Acromyrmex is polymorphic [31] and presents marked alloethism during growth of the fungus garden [32], a fact suggesting that alloethism may also be observed during foraging. In addition, there are Acromyrmex species that present characteristic food preferences and alternate between the cutting of monocot leaves, dicot leaves or both used as a substrate for the symbiontic fungus [33, 34, 35]. Leaf-cutting ant species that forage on monocotyledon plants to be used as a substrate for the fungus garden differ in their morphology and behavior from those that use dicot leaves. The former have strong and short mandibles, whereas the latter possess longer and less robust mandibles. In the case of grass-cutting ants, the mechanism involved in the determination of the size of the fragment to be cut is unknown. It is only known that workers do not use their legs as pivots [34] and that the length of the transported fragments in the field is larger than the maximum span of the ants [22]. In addition, the conversion of fungal biomass into ant biomass starting from the plant base is probably much higher in leaf-cutting than in grass-cutting ants since the latter process less substrate before incorporation [36]. Furthermore, the size of the cut fragments is influenced by leaf density [37, 38, 10, 39], as well as by the information received from the scout ant [40, 23]. In order to identify the behavioral adaptations exhibited by grass-cutting ants, colonies of A. balzani (grass-cutter) and A. crassispinus and A. rugosus (dicot leaf-cutters) were observed in the laboratory, where Cynodon spp., cultivar Tifton, leaf blades and Acalypha wilckesiana leaves were offered in the foraging arenas to the grass-cutting and leaf-cutting species, respectively. This behavioral study also permitted to compare the performance of different worker size classes of A. balzani, A. crassispinus and A. rugosus during foraging. The subtasks monocot leaf cutting, cutting by couple and measuring leaf length were exclusively observed and described for A. balzani, indicating behavioral differences between grass-cutting and leaf-cutting ants. Monocot leaf cutting: Workers are positioned sidelong in relation to the contact surface. The legs of one side of the body are maintained on the fragment, while the hind and middle legs of the other side are stretched anteroposteriorly. The anterior legs, together with the antennae, hold the fragment and the mandibles open and close in order to cut the fragment. During the process, the worker moves its head from one side to the other of the

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leaf until the transverse cut is concluded. The fragment cut thus possesses a rectangular shape different from the semi-circular shape of fragments cut by leaf-cutting species. Measuring leaf length: This procedure performed prior to cutting consists of the worker moving throughout the leaf area, touching it with the antennas and mandibles that open and close. During this behavior, the leaf-cutting worker approaches the apex of the leaf blade, biting the borders of the leaf surface, and then returns to the lower portion of the blade. Our observations suggest that the “leaf measuring” behavior is related to the determination of the size of the fragment to be cut since it is performed after the transport attempt and before leaf cutting. Supposedly, this mechanism explains that the size of leaf fragments carried by grass-cutting ants is larger than the maximum span of workers, and a correlation between body weight and/or length and weight and/or length of the cut leaf fragment can be expected. Cutting by couple Two workers are positioned at the same point on the fragment and accomplish the cut in a manner similar to that described above, although they do it together. Generally, after cutting, there is a dispute over the transport of the fragment. For the three species, overlapping of the behavioral repertoire was observed between size classes, indicating an interindividual variability between castes and subtask flexibility. This flexibility is directly related to the balance between the entry of food and energy consumption inside the colony through the nutritional status of the worker [41], to an increase in the efficiency of the colony since individuals can rapidly change between tasks [42], or is a consequence of the competition between workers [43]. The species A. crassispinus and A. rugosus presented specialized size classes for the execution of the three subtasks, whereas in the case of A. balzani specialization of MII workers in the subtasks cutting and cutting by couple and an efficient performance of large workers in the transport of leaves were observed. Leaf cutting is a behavior of high energy cost comparable to the cost of flying [44], and the efficient performance of large workers in the transport of leaf fragments without prior cutting therefore suggests the occurrence of an opportunistic behavior in A. balzani. Task and subtask specialization of Acromyrmex workers has been reported [45, 46], promoting an increase in their efficiency and in the efficiency of the colony in general [31, 47]. In addition, individual variability among workers may also act during substrate selection, with an individual preference being observed in A. octospinosus [48].

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The attempted behavioral act of transport is an interesting point to be discussed. This act was observed when workers were unable to transport leaves and immediately gave up or cut the leaf fragment. The results obtained suggest that in A. crassispinus the distribution of subtasks among size classes is more ample during foraging in view of the lack of observation of this behavioral act. In contrast, in A. rugosus and A. balzani this act was observed in excess and was scarce in the MII and large size classes, respectively. In an agonistic manner, the observed frequencies of these behavioral acts permit to hypothesize that in the case of the reported species larger workers are more specialized in the act of transporting and therefore “fail” less. [49] suggested the existence of two worker subcastes (maxima and minima) for A. octospinosus and A. volcanus. However, the data obtained in the present experiment, together with observations made during the process of incorporation of plant substrates into the fungus garden, suggest the existence of at least three worker subcastes. 5. Cultivation of the fungus garden: Monocots versus dicots Cultivation of the fungus garden is an activity that consumes large amounts of worker time and energy since workers have to prevent contamination of the garden [50], increase the production of staphylae through constant pruning, and provide plant substrate for fungal growth [51]. A detailed description of this process has been published by [2] for leaf-cutting ants, especially Acromyrmex colonies, and by [53, 3, 50, 46]. However, little information about the processing of plant substrates is available for grass-cutting ants. In the case of leaf-cutting species, the foraged substrate is chopped into increasingly smaller pieces that are chewed into a pulp and then incorporated [34]. In contrast, grass-cutting species do not process as much the leaf blades brought to the nest [36], suggesting that the fragments are submitted to another type of treatment so that the fungus can consume this substrate. For leaf-cutting ants, the process of licking the leaves is the most time consuming, with medium-sized workers being mainly involved in this activity in the species A. octospinosus [3], as well as in the complex of A. subterraneus [46]. A high frequency of this behavior has also been observed in Atta sexdens rubropilosa, but small (minima) workers were mainly responsible for the licking behavior [52]. Minima and generalist (media) workers predominate in the population of a leaf-cutting ant colony and are responsible for a larger number of tasks related to plant preparation during the process of substrate incorporation [3]. In

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addition, variability is likely to be observed within one case and, consequently, a more refined division of labor [54]. It should be emphasized that the size of workers shows inter- and intraspecies variation [1] and is related to the establishment and separation of niches [55, 56]. Indeed, workers of A. balzani, a grass-cutting ant, present greater head widths than A. crassispinus and A. rugosus, with the head width range of a certain physical subcaste being equivalent to that of another subcaste in some cases (Table 1). Table 1. Head width range according to physical subcaste in three Acromyrmex species.

Head width (mm) Size class

A. balzani A. crassispinus A. rugosus

Large (L) 2.9 – 2.4 2.5 – 1.7 1.6 – 1.3

Medium II (MII) 2.4 – 2.1 1.7 – 1.3 1.3 – 1.0

Medium I (MI) 2.1 – 1.7 1.6 – 1.1 1.0 – 0.9

Small (S) 1.6 – 1.2 1.2 – 1.0 0.9 – 0.75

Very small (VS) 0.99 – 0.76 0.81 – 0.62 0.75 – 0.6

In this respect, an experiment was conducted to investigate behavioral differences between grass-cutting (monocot) and leaf-cutting (dicot) Acromyrmex species, as well as to determine alterations in the division of labor during plant incorporation into the fungus garden between size classes. The experiment consisted of the ad libitum observation [57] of four laboratory colonies of the respective species, with a total of 80 hours for A. rugosus and A. crassispinus, corresponding to 4 continuous hours/5 days/4 colonies, and of 160 hours for A. balzani, corresponding to 4 continuous hours/2 periods/5 days/4 colonies. The periods of continuous observation were determined in order to obtain data referring to all tasks involved in the processing of the plant material. The first difference between species was already observed during the experiment and was related to the velocity of execution of the process of substrate incorporation into the fungus garden, which required a larger sampling effort in the case of A. balzani. This method of observation permitted the elaboration of an ethogram, which consisted of seven behavioral acts exhibited by workers during the processing of plant material for cultivation of the fungus garden. The following behavioral acts were exhibited in an ordered, although overlapping, sequence:

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1) licking leaf fragments; 2) shredding leaf fragments; 3) chewing chopped leaf fragments (except for A. balzani); 4) crimping chopped leaf fragments; 5) incorporating leaf fragment into the surface of the fungus garden; 6) depositing fecal fluid on the freshly incorporated substrate; 7) depositing fungal hyphae on the substrate.

The task of licking leaf fragments, together with the deposition of fungal hyphae on the substrate, was the most frequent and was mainly performed by the MI and MII size classes in all three species. The performance of workers of the MI and MII size classes is unquestionably essential during the incorporation of plant substrates into the fungus garden. In A. rugosus and A. crassispinus, among the six tasks observed only the act of depositing fungal hyphae on the substrate was not preferentially performed by these intermediate size workers, which comprise the generalist caste. These results agree with the findings of [3, 46]. One hypothesis to explain the participation of intermediate size workers in a larger number of tasks is based on the fact that life inside the colony is not always as harmonious as it seems and recent studies have demonstrated that some individuals employ strategies that favor their own interests, thus generating internal conflicts in the society [58]. This non-altruistic behavior might be related to individual variability among workers and act as a balance mechanism between cooperation and conflict in view of the distribution of various tasks within the same size class. According to [59], in Atta sexdens the participation of media workers in a large number of tasks prevents task competition. Sensitivity to changes within a structured working system is important for social organization [47]. In Pogonomyrmex barbatus, signals based on the interaction rate between workers permit ants to respond to changes in the number of workers engaged in a certain task [60]. This may also be true for leaf-cutting ants considering that the tasks observed follow an ordered but, at the same time, overlapping sequence. Thus, workers would be able to obtain information regarding the phase of plant processing for incorporation into the fungus garden and to change tasks according to necessity. On the other hand, in A. balzani the large size class also participated in most tasks together with the MI and MII classes. The different distribution of tasks between size classes in the three species might be related to the fact that A. balzani colonies are less populous, considering that the size of the colony influences the distribution of worker size [61] and, consequently, the distribution of tasks inside the nest. Another hypothesis is related to the fact that grass-cutting ants do not process the plant material as much. Thus, the manipulated leaf fragments are larger and permit the participation of large

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workers. Evidence indicates that workers foraging on larger and heavier substrates are significantly larger [62]. The observation in the three species that the task of depositing fungal hyphae on the substrate was mainly performed by very small workers and the fact that smaller workers are less frequent on the foraging trails [63] provide additional evidence for a close relationship between the type of task and worker size. A. balzani workers are larger than A. crassispinus workers which, in turn, are larger than A. rugosus workers (Table 1). Interspecies size differences are a key aspect in the separation and establishment of niches [55], influencing the determination of the food area that the animal can explore efficiently [64]. In polymorphic species, size differences are considered to be a central point in the economy of the division of labor inside a colony [31]. The tasks of licking and crimping/chewing leaf fragments permit the fungus to rapidly invade the plant tissue [65] and are equally important in grass-cutting and leaf-cutting ants. The licking task is also associated with leaf asepsis, considering the presence of antibiotics in the metapleural gland of workers which inhibit the growth of competing microorganisms [66, 67, 68]. The efficiency of this task is of crucial importance for the development of leaf-cutting ant colonies, especially when considering that endophytic fungi present in most grass species are able to alter the foraging pattern and survival of herbivores, although they do not act as deterrents for leaf-cutting ants [70]. The deposition of fecal fluid might be considered a second step in the auxiliary process of leaf degradation. A large number of nitrogen components that are used for fungal growth [65] and proteases that degrade the substrate [70] have been identified in this fluid. The symbiosis of attine ants with the fungus might be interpreted as a metabolic alliance [71]. The distribution of tasks among size classes was found to differ between the three species. This result might be associated with the relationship existing between the size of the forager and substrate selectivity [72]. The data obtained in this experiment, together with the observations made during foraging, suggest the existence of at least three worker subcastes (large, medium and small), in contrast to [49] who proposed two worker subcastes (maxima and minima) for A. octospinosus and A. volcanus.

6. Age polyethism Almost all adult social insects undergo behavioral changes during aging, a fact that leads to changes in their role in the society. Each species possesses its own distinct pattern of age polyethism, and many of the behavioral changes are accompanied by patterned shifts in the activity of exocrine glands [1]. In this

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respect, younger workers tend to perform tasks inside the nest, whereas older workers are engaged in tasks outside the nest such as foraging [73]. This phenomenon has been well studied in the honeybee Apis mellifera. In the first weeks of life, workers are engaged in licking cells and brood care, in the third week they process nectar and build honeycombs and, finally, workers leave the nest for foraging when they are approximately 25-30 days of age [74]. These behavioral responses are associated with the concentration of juvenile hormone in the individuals, i.e., lower concentrations are associated with behaviors inside the nest during the first 3 weeks of life of the adult bee, and higher concentrations are reached after the third week and are related to foraging [47]. However, honeybees are monomorphic and the division of labor occurs within age groups [74], whereas in highly polymorphic species, in addition to age groups, morphological differentiation occurs among workers, an event that leads to overlapping roles with a mixture of physical and temporal subcastes [75]. In leaf-cutting ants such as Atta and Acromyrmex, worker polymorphism results in a distinct division of labor between physical subcastes, in which larger workers are specialized in the defense of the colony, medium workers forage on plant species, and smaller workers are specialized to work inside the fungus garden [75], observed that in Atta sexdens the tasks are divided among workers into four physical subcastes, three of them being subdivided into three temporal subcastes, for a total of at least seven subcastes. However, little is known about the relationship between physical and age subcastes or about the age polyethism schedule for the genus Acromyrmex. In a recent study, [76] reported that Acromyrmex subterraneus brunneus presents a typical age polyethism pattern, in which immature workers remain inside the nest and older workers perform activities outside the nest. According to these authors, initially all workers were involved in the cultivation of the fungus garden, brood care, and food transport. After the 4th week, workers started to participate in activities outside the nest. A similar age polyethism pattern has been reported by [77] for the desert leaf-cutting ant Acromyrmex versicolor, with most workers caring for the fungus and remaining inside the nest during the first 3 weeks, while the number of foragers started to increase after the 4th and 5th weeks of observation. The authors observed that workers of different matrilines (offspring of different queens) show temporal variations in the transition from tasks inside the nest to tasks outside the nest, for example, foraging. This finding suggests that the division of labor is affected by genetic effects on the choice of age-related tasks. One interesting fact observed by [76] was the presence of a whitish cover, a mutualistic bacterium, on the tegument of A. subterraneus brunneus workers during the initial phase of their lives, a period when they were performing

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tasks inside the nest. These bacteria were no longer visible to the naked eye on the tegument of older workers which performed behavioral acts outside the nest. In A. octospinosus, the mutualistic bacterium (Pseudonocardiaceae) starts its growth on the cuticula of workers a few days after emergence of the adult and reaches its largest body coverage at 13-18 days. This coverage starts to decline 25-30 days after emergence, except for the laterocervical plates [78]. These mutualistic bacteria present on the cuticula of workers specifically act on the control of fungal microparasites of the genus Escovopsis [79]. Interestingly, these facts suggest that these bacteria grow and decline on the cuticula of Acromyrmex spp. workers according to age, guaranteeing the health of the colony and a specific defense against Escovopsis and thus the continuous execution of more than one role (or activity) inside the nest. In summary, the behavioral pattern observed for Acromyrmex spp. demonstrates a clear division of labor between age and physical castes. With respect to the physical castes, the division of labor is based on a bimodal size distribution of the population in A. volcanus and A. octospinosus colonies, with minima workers remaining inside the nest, caring for the fungus garden and brood, and larger workers leaving the nest for foraging [49]. A. subterraneus brunneus probably presents an alloethism similar to that observed for A. volcanus and A. octospinosus but the wide distribution of individuals among polymorphic worker classes in this species should be emphasized [46]. 7. Worker reproduction As a rule, in the sex determination of social Hymenoptera all males are haploid, whereas all females are diploid and can differentiate into reproductive females or workers, a characteristic that depends on environmental and/or genetic factors [80, 81]. One widely known fact is that workers frequently, but not universally, lay non-fertilized eggs that develop into males [1]. This reproductive ability has been reported for many ant species [82, 83, 84, 85], including fungus-growing ants [5, 86, 87]. Worker reproduction is observed in orphan colonies of some Acromyrmex species, with workers laying haploid eggs that give origin to males in the absence of a queen [5, 18]. Although it is known that workers do not lay eggs in queenright colonies, it has been postulated that the self-restriction and sterility of leaf-cutting ant workers are maintained through the queen or through a mechanism of worker policing [88]. In queenless colonies, these workers continue to perform activities for maintenance of the nest such as active foraging, cultivation of the symbiontic fungus and brood care. A decline occurs when the colony suffers loss of the

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worker force and natural death of the subcastes, which results in the lack of replacement of workers and the appearance of males. The behavior of workers in queenless colonies in relation to male larvae is similar to that observed for larvae of workers derived from the queen [92], studied the behavioral responses of workers during parental care to determine whether a differential care exists in A. subterraneus brunneus between male larvae derived from workers and larvae of workers derived from the queen. Although the result was not conclusive, workers presented an elaborate behavioral repertoire during brood care of male and worker larvae (Table 2). Some behavioral acts showed that workers behave differently according to the origin of the larva. The behavioral acts that showed a significant difference were licking the larval body, transporting larvae and depositing hyphae of the symbiontic fungus on the larval body. The differences observed indicate the ability of workers to perceive and discriminate between larvae, probably as a result of differences in their individual needs [92]. Table 2. Description of the behavioral repertoire and probable functions of the behavioral acts performed by Acromyrmex subterraneus brunneus workers during brood care (obtained from [92]).

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Brood care is essential for the success of this group of insects, with workers being engaged in activities ranging from caring for the eggs to help during adult eclosion. When males emerge in queenless colonies, they still require care from the worker caste. According to [92], the behavioral repertoire of winged adults is more limited and includes only the following four behavioral acts: feeding through workers, collecting staphylae and self-feeding, mutual grooming (worker – winged individual), and self-grooming. The authors observed that males are also able to collect food (staphylae or fungal hyphae) from the fungus garden and to feed themselves, thus not requiring workers for this activity. Among basal species of the Attini tribe, Mycetarotes parallelus males do not receive much care from workers, being only groomed, but never fed [93]. [93] also observed that most of the behavioral repertoire was individual, with males grooming and feeding themselves, but never performing worker tasks. Another interesting aspect of these males produced by workers in orphan colonies of A. subterraneus brunneus is their wide morphometric variation in body size [94]. In a previous study [5], comparing head phenotypes of A. rugosus rugosus from queenless and queenright colonies, demonstrated that queenless colonies produced a larger number of phenotypes than queenright colonies. The author still discussed that the queen probably controls the oviposition of workers since a reduced number of male phenotypes was observed in queenright colonies. A recent study using microsatellite genotyping demonstrated that workers from queenless colonies are able to produce males, but no males produced by workers were observed in queenright colonies in eight fungus-growing ant species [87]. These findings indicate that the fertile queen in a colony is able to control and monopolize the production of males and when she dies or is removed from the colony, the workers overtake this reproductive role. Furthermore, it is of fundamental importance to know the ecological role of these males for these species, i.e., whether they are able to copulate or not [94], studied the external genitalia of males in queenless and queenright colonies and observed no morphological differences between males from queenright and queenless colonies. In A. hispidus and A. lobicornis, the male genitalia are robust and consist of curved and wide gonostyli, and the volsellae possess a wide digit and a small tooth in their curvature which differentiates the two species [95, 96]. The same pattern is observed in A. subterraneus brunneus, but the volsella contains few bristles and the base possesses an angular curvature with numerous bristles distributed along the internal margin of the structure, presenting the same specific characteristics of this species. However, the volsella has similar proportions, demonstrating that these males are able to copulate like those produced in queenright colonies [94]. According

Studies on leaf-cutting ants 15

to [97], the male and female copulatory apparatuses fit like a key in a lock and the integrity of the species is preserved due to its morphology. However, the author added that the lock-and-key hypothesis was partially rejected after the observation that the morphology of the male genitalia varies widely in length and shape. The female genitalia do not consist of chitinous rigid parts but of a musculature that promotes contractions which permit greater safety in the connecting of the copulatory organs [97]. Within the class Insecta, males are more variable than females [98]. In Lepidoptera, examples of interspecies, intergenus and even interfamily hybridization exist that demonstrate the inefficacy of the genital anatomy as a mating barrier [99]. Thus, it can be concluded that A. subterraneus brunneus males from queenright and queenless colonies are anatomically able to mate; however, there are some limitations to this hypothesis It is essential to know whether A. subterraneus brunneus males from queenless colonies receive the same care by workers as those reared in queenright colonies, permitting these males to respond to environmental stimuli and to show a good performance during the nuptial flight. The flight and copulation capacity are important parameters for the reproductive success of leaf-cutting ants. In these insects, the nuptial flight basically depends on ideal meteorological conditions and carbohydrate reserves as a flight fuel [100] experiments conducted under laboratory conditions, [100] observed that Atta sexdens males produced in the laboratory flew after a period of 9-10 hours of lights-on even in the absence of environmental cues. Another interesting result was the use of a large amount of carbohydrates as a flight fuel. The existence of worker reproduction in natural situations is still unknown. Hypothetically, if the queen dies in an adult nest in nature, it is possible that workers become able to produce non-fertilized eggs. The production of these males has to be synchronized with other queenright nests that are in the reproductive period, i.e., those producing virgin females for mating and thus perpetuating their descendants. The biological meaning of worker reproduction in leaf-cutting ants is unknown and only permits the conclusion that these males are able to mate, representing an alternative route of reproduction in this species. 8. Current approaches and perspectives for studies on the control of Acromyrmex Leaf-cutting ants (genera Atta and Acromyrmex) use leaves freshly harvested from plants for cultivation of the mutualistic fungus which serves as the main food source [101, 102]. These ants complement their diet with fluids directly ingested from these leaves [51, 52, 103]. Since these ants cut many plant species, including those cultivated by man, they are important pests in

Roberto S. Camargo et al. 16

agriculture, in the cattle-raising industry and in silviculture [34, 104, 105]. Since Acromyrmex colonies are smaller than Atta colonies [2, 106] and the number of colonies per hectare is frequently similar [107], they tend to cause less damage. Therefore, in the daily practice of control of these ants (field conditions), they are usually relegated to a secondary position in most situations. However, Acromyrmex has also been receiving substantial attention in scientific studies regarding methods for the control of leaf-cutting ants, a fact that will be discussed below. Among control methods, the use of toxic baits [104, 108] and resistant plant cultivars [119, 110] are the only methods that can be recommended for the wide and scientifically based application to the control of Acromyrmex. Toxic baits should contain an active ingredient with a delayed action on adult workers (mortality ≤ 15% after 24 hours or ≥ 90% after 21 days) using a vehicle that is highly attractive to workers such as citrus pulp [108, 111]. In contrast to other pest ants such as Solenopsis invicta, no active ingredient is so far available for leaf-cutting ants that does not primarily act on adult workers and that provides efficient control under field conditions such as queen-sterilizing agents, insect growth regulators and inhibitors of the synthesis or deposition of chitin [104, 108, 111, 112, 113]. Additionally, there are no reports of success with the use of baits containing an active ingredient with an effective fungicidal action (against the mutualist fungus) [108, 114]. Although diflubenzuron, an inhibitor of the synthesis or deposition of insect chitin, also inhibits the growth of the mutualist fungus in vitro [115], this active ingredient has no deleterious effect on small colonies maintained in the laboratory [108] or on adult workers [116]. In studies on methods for the control of leaf-cutting ants, including studies directly applicable to control methods or those supporting these applied investigations, a species serving as model for leaf-cutting ants should be selected. Atta sexdens is frequently used as a study model (e.g., [111]) since it is one of the most damaging species, or because of the availability of colonies close to the research centers, with these criteria generally being sufficient and adequate. However, for further improvement we propose here that the choice of the species should preferentially be made so that the specific objective of the study is fulfilled in a more complete manner and that the results have a wider application through an increased interactivity with more basic studies. Thus, in more applied studies in which the size of the colony is an important factor, Acromyrmex should not be used since Atta colonies are much larger. One example is the final phase of the assessment of the efficacy of experimental toxic baits under field conditions, since all evidence indicates that the difficulty is greater exactly for larger colonies [108, 111] and that it would not be useful to control only small colonies that cause less damage.

Studies on leaf-cutting ants 17

Furthermore, it has been suggested that parasitic or antagonistic microorganisms of the ants or of the cultivated fungus exert a synergistic effect with insecticides used for pest control [117, 118]. Since Acromyrmex has been the genus most used as a model in functional studies on the genus Pseudonocardia, a mutualistic bacterium of ants and particularly abundant in this genus, observations are facilitated [67]. Pseudonocardia is an actinomycete (filamentous bacterium) that grows on the body of workers and is used to inhibit the growth of the fungus Escovopsis (asexual stage of Ascomycota), a specific parasite of the mutualistic fungus [119, 120, 121]. Thus, Acromyrmex should be chosen as a model when evaluating the influence of mutualistic bacteria on a given control method. In semi-claustral founding ants such as Acromyrmex, in contrast to the claustral founding in Atta [73, 122], the risk of microbial contamination is higher during the initial phase of colony founding [122]. In addition, mobility of the colonies (change of the nest to another site) is much more frequent than in the genus Atta; thus, the risk of microbial contamination is also higher for already established colonies. We therefore propose the hypothesis that Acromyrmex species and/or their mutualistic microorganisms are inherently more resistant to nonspecialized microbial parasites such as the fungi Metarhizium sp. (entomopathogenic) and Trichoderma sp. (fungal antagonist). We suggest that, in order to support applied studies on control methods, comparative studies including representatives of the two genera (Acromyrmex and Atta) should preferentially be conducted for the investigation of resistance mechanisms (chemical or behavioral) of the ant, fungus or mutualistic bacteria to nonspecialized microorganisms, selecting colonies of similar size (for Atta, it is necessary to use young colonies, less populous). This approach permits to obtain comparative experimental data for one probably more susceptible species and another less susceptible species. Thus, the investigation of the proposed hypothesis is basic research but has importance in applied research. In summary, we suggest that in future studies on the control of leaf-cutting ants the choice of the species used as a model should not only take into account the availability for collection and/or the economic damage caused by the species or genus, but also the bioecology of the ant as well as the microorganisms with which the species co-evolves.

Acknowledgment Since this research has been carried out over several years in the Laboratório de Insetos Sociais Praga, FCA/UNESP, Botucatu, this work was supported by several sources: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico(CNPq). We would like to thank the Journal of Applied Entomology and Blackwell Publishing for permission to use the table.

Roberto S. Camargo et al. 18

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