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Biology

Collection and Long-Term Maintenance of Leaf-Cutting Ants (Atta) in Laboratory Conditions

Published: August 30, 2022 doi: 10.3791/64154

Summary

Here, a protocol is described to successfully collect and maintain healthy Atta (Hymenoptera: Formicidae) ant colonies in laboratory conditions. Additionally, different nest types and configurations are detailed together with possible experimental procedures.

Abstract

Ants are one of the most biodiverse groups of animals on the planet and inhabit different environments. The maintenance of ant colonies in controlled environments enables an enriched comprehension of their biology that can contribute to applied research. This practice is usually employed in population control studies of species that cause economic loss, such as Atta ants. To cultivate their mutualistic fungus, these leaf-cutting ants collect leaves and for this are considered agricultural pests widely distributed throughout the American continent. They are highly socially organized and inhabit elaborated underground nests composed of a variety of chambers. Their maintenance in a controlled environment depends on a daily routine of several procedures and frequent care that are described here. It starts with the collection of queens during the reproductive season (i.e., nuptial flight), which are then individually transferred to plastic containers. Due to the high mortality rate of queens, a second collection can be carried out about 6 months after the nuptial flight, when incipient nests with developed fungus wad are excavated, hand-picked, and placed in plastic containers. In the laboratory, leaves are daily provided to established colonies, and ant-produced waste is weekly removed along with remaining dry plant material. As the fungus garden keeps growing, colonies are transferred to different types of containers according to the experimental purpose. Leaf-cutting ant colonies are placed in interconnected containers, representing the organizational system with functional chambers built by those insects in nature. This setup is ideal to monitor factors such as waste amount, fungus garden health, and the behavior of workers and queen. Facilitated data collection and more detailed observations are considered the greatest advantage of keeping ant colonies in controlled conditions.

Introduction

Ants compose a diverse group of individuals that exert an influence on most terrestrial environments1. They act as efficient dispersers2,3,4, predators5 and ecosystem engineers6,7,8,9,10, highlighting their importance and ecological success on natural ecosystems. All ant species are classified as eusocial insects; however, their social organization varies greatly among different species groups, i.e., labor division systems, functional groups, communication among individuals, forage organization, colony foundation, and reproduction process11. As a highly diversified group, they resort to several food resources and specialized feeding behaviors. As a matter of fact, agriculture was not only a huge step for human civilization, but also for ant species. Approximately 55 to 65 Ma ago12, attine ants began to culture fungi and incorporate them into an almost exclusive diet. They became so specialized that they developed strict, dependent, and obligatory interactions classified as symbiosis, where one individual does not survive without the other.

Lower fungus-growing ants collect and process dead organic matter, such as fragments of rotting leaf, to grow their mutualistic fungus; while higher fungus-growing ants harvest fresh plant material, composing one of the most successful symbiotic natural systems13. This highly specialized agriculture technique allowed them to seize a new niche. The higher attine ants comprise the leaf-cutting ants, a monophyletic group that arouse between 19 Ma (15-24 Ma) and 18 Ma (14-22 Ma)14,15,16 consisting of four valid genera: Atta Fabricius, Acromyrmex Mayr, Amoimyrmex Cristiano, and Pseudoatta Gallardo. The leaf-cutter agriculture system performed by the leaf-cutting ants, evolved from derived agriculture systems17. Most of these species exclusively exploit the mutualistic fungus species Leucoagaricus gongylophorus Singer18 (also called Leucocoprinus gongylophorus Heim19), marking a significant evolutionary transition11. The fungal cultivars are transmitted vertically, from original nests to offsprings, suggesting that they are clonally propagated20.

Remarkably, Atta societies developed a complex organizational structure of enormous importance in their environment and of great interest to myrmecologists. Their population can be composed of millions of individuals, most of them sterile female workers that display an accentuated polymorphism, i.e., distinct size and anatomical morphology. The population is distinguished by castes according to age, physiological state, morphological type, behaviors, and specialized activities in the colony21. Workers can be discriminated into gardeners and nurses, within-nest generalists, foragers and excavators, and defenders or soldiers21. This organization allows the performance of tasks in cooperation and a self-organizing system that can produce highly structured collective behaviors, allowing them to respond efficiently to environmental disturbances22.

The role of population renewal is played by a single queen (i.e., monogynous), for as long as she lives, constituting the permanent reproductive caste22. Atta queens are known to live for more than 20 years, laying eggs throughout their lifespan23. As the queen is irreplaceable, its endurance is crucial for the survival of the colony13,20,23,24. Yet, thousands of winged reproductive females and males can be found in the nest during breeding seasons, but none stays in the original nest, forming a temporary caste22. In Atta sexdens colonies, nearly 3,000 reproductive females and 14,000 reproductive males are produced25. It occurs when a colony reaches sexual maturity, approximately 38 months from its implementation, and is repeated annually ever since until it is extinguished23,25. New Atta colonies are established through haplometrosis, where one queen commences a new nest.

When environmental conditions are favorable, the reproducers leave the underground nest to begin the nuptial flight. The period of its occurrence differs by region, ranging along the year throughout the brazilian territory depending on the species. However, the event seems to be preceded by rainfalls and humidity elevation26, which can be related to excavation facilitation due to soil moisture22. Frequently, 1-5 weeks before the nuptial flight, nest entrances and channels are widened to facilitate the reproductive individuals to depart. Before leaving their mother colonies, the winged females collect and store, in an infrabuccal cavity, a portion of the mutualistic fungus20,27. Multiple copulations are performed mid-flight, and it is calculated that one queen can be inseminated by three to eight males (i.e., polyandry) in some species28, ensuring genetic variability29. Afterward, the queens proceed to the soil, giving preference to locations with no or few vegetation25, where they remove their wings and excavate their first nest chamber. This is the only period where queens can be seen outside the nest. Although individuals of the temporary caste were seen in artificial nests, it is unknown whether any successful copulation (i.e., nuptial flight) was performed in laboratory conditions24.

The initial nest construction corresponds to the most crucial period of the colony, which can last from 6 h to 8 h23,25. At this moment, the queen cloisters herself in the initial chamber, and in a matter of days, oviposition begins. The first eggs are fed to the mycelial that the queen regurgitates, marking the start of the colony's fungus garden. The first larvae appear in approximately 25 days22, and nearly at the end of the first month, the colony consists of a mat of proliferating fungus, where immatures (eggs, larvae, and pupae) are nested, and the queen, who raises her initial offspring in isolation23. Eggs are also the food resource of the first larvae and highly consumed by the queen13. Additionally, the queen sustains herself with fat-body reserves and catabolizing wing muscles that are no longer of use13. The initial fungus culture is not consumed as the colony survival depends on its development, and during this period, the queen fertilizes it with fecal fluid13. Days after emerging, the first workers open the nest entrance and begin a foraging activity in the immediate area of the nest13. They incorporate the material collected as the substrate of the fungus garden, which is now serving as food for the workers13,22. Before being added to the fungus culture, the plant material carried in by the workers is cut into tiny pieces and moistened with fecal liquid13. The ants manipulate fungus inoculum to increase and control its growth, which will serve for partitioning big soil excavated chambers, specialized in conditioning the garden13,22,25.

About 6 months after the nuptial flight, A. sexdens nests contain a fungus chamber and a few channels. The great specialization in the construction of leaf-cutting ant nests works as a defense mechanism against natural enemies and unfavorable environmental factors22. Leaf-cutting ants are known to fragment the fungus garden and transpose it to chambers with high humidity when chambers start to dry out13. Thus, despite the excavation of the nest having a considerable energy cost, the energy invested is reversed in benefits for the colony itself22. With a few exceptions, Atta species also make specialized chambers for the colony's waste, made mostly of depleted fungus substrate and bodies of dead ants, isolating it from the rest of the nest, and establishing an important social immunity strategy30. In addition, a distinct group of workers manipulate the refuse directly, to avoid contamination of other individuals. Workers constantly forage to nurture the fungus, which is the main nutritional resource of the colony. However, they can feed on plant sap as well while cutting fragments. Plant material is carefully selected for the fungus garden maintenance and influenced by many factors such as leaf traits and properties of the ecosystem13.

The foraging strategy of leaf-cutting ants to obtain fresh material is highly complex, and combined with the high harvest demand of established colonies, result in considerable economic loss to agricultural producers and jeopardizes forest restoration areas22,31. Therefore, these ants can be categorized as pests in most areas where they may be encountered, ranging from southern United States to north-eastern Argentina11,13,22,32. The extinguishing of problematic colonies is challenging due to the series of adaptations inherent in the biology of these insects (i.e., social organization, foraging, fungus-cultivation, hygiene, and complex nest structures)33. Hence, the population control strategies are distinct from those generally applied to other insect pests, and mainly resort to attractive contaminated bait offerings33,34. However, as these ants can reject harmful substances for both the fungus and the colony individuals, and compromise cultivated fields33, new natural compounds and alternatives of control are constantly being tested33,35,36. As experiment results can hardly be monitored on field-tested colonies, preliminary essays are conducted in a controlled environment.

Thus, experimental protocols must be adapted to groups of interest considering the heterogeneous lifestyle of ants, supporting studies on a species level, and accounting for colonies as operational units, where one ant is an element of a complex superorganism11. The reports gathered until now concerning the Atta genus made it attainable to successfully collect and maintain colonies in laboratory conditions and acknowledge their basic needs and general functioning. Based on their natural processes such as reproduction, colony founding, and feeding behaviors, a routine of practices has been developed that permits the long-term establishment of colonies in different types of nests. Here, a procedural protocol to maintain leaf-cutting ants in the laboratory is described and highlights possible general research with distinct experimentation purposes and science outreach.

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Protocol

1. Collection of queens

  1. Search in the literature for the period of Atta reproductive season in the region of interest. The reproductive season occurrence, frequency, and day time of nuptial flights, varies according to regional climate conditions (Table 1). Although it generally takes place in spring, this information must be gathered for the location where the collection is intended to occur37,38,39,40,41,42,43,44,45,46.
  2. Identify and mark locations with Atta nests considered as possible areas for collecting queens and young colonies. During nuptial flights, queens are dispersed around nest locations; therefore, areas with a greater number of colonies have higher chances to have queen-landing spots where they initiate new nest excavations.
  3. Check the areas selected previously for signs of nuptial flight during the reproductive season of Atta ants. Keep track of the environmental conditions of nuptial flight days, such as hot and rainfall weather.
  4. Identify leaf-cutting ant nests in the areas selected previously and look for external features that indicate the upcoming departure of winged-reproductive ants. Nest features include tunnel entrances widened (Figure 1), increased flow of workers showing more aggressive behavior toward possible predators and winged-reproductive ants appearing at tunnel entrances (Figure 1). Beware to high humidity days succeeding rainfall, as they generally precedes nuptial flights.
  5. Prepare plastic lidded containers with a bottom plaster layer to retain the queens individually. Ensure that the container volume is approximately 200 mL, and the plaster layer at the bottom is around 1 cm in height and is highly absorbent for humidity control.
    NOTE: To prepare the plaster base, follow the manufacturer's instructions.
  6. Prepare an environment with a constant temperature of 23 ± 1 °C and approximately 70% ± 10% of relative humidity. Choose a location without intense activities and high flow of people to avoid vibrations and disturbance. Use cleaning products of neutral fragrance to prevent any interference in ant behavior. 
    NOTE: Fluctuations in specified environmental conditions can cause water condensation or moisture loss, and compromise the fungus garden.
  7. After the nuptial flight, collect wingless queens that have initiated nest excavation, and carefully place them in the plastic containers prepared with a plaster layer, individually. Avoid touching the queens with bare hands and use latex gloves or entomological tweezers.
    NOTE: Wing removal and soil excavation behavior indicate reproductive females that have already copulated, and therefore, are able to start a new colony. Queens collection is also treated as the first collection in this work.
  8. Move the containers bearing the queens to the location with a controlled environment previously selected. Perform queens' transportation with utmost caution, avoiding too much disturbance and maintaining a minimum temperature constancy.
  9. Do not manipulate or move the queens for approximately 3 days after collection to avoid stress.

Figure 1
Figure 1. Nest entrance widened with winged ant reproducers and workers. Widened tunnel entrances are one of nests features that indicates Atta nuptial flights occurrence.  Please click here to view a larger version of this figure.

2. Queens' maintenance

  1. Initially, add 2.5 mL of water to the plaster layer of the recipient every 2 days with the help of a needle syringe.
    1. Instead of opening the recipient, carefully puncture the container lids with the needle to avoid disturbance due to manipulation. The same hole can be used during this period. Ensure that the water added does not soak the plaster layer. Avoid directly watering the queen, the initial fungus sponge, and any immatures. For as long as the fungus garden exhibits a dry aspect with water absence, irrigate the plaster layer.
  2. Two weeks after the collection, check whether the fungus has been regurgitated by the queens. If there is no fungus, transfer approximately 2 g of fungus obtained from an established colony. Also, perform this step if the fungus does not develop.
    NOTE: For fungus transfer, it is necessary to collect healthy fungus from an established colony and remove all the ants that might be on it. Use a tablespoon, entomological tweezers, and latex gloves to manipulate the fungus.
  3. After the appearance of the first workers, begin offering fragments of young and thin leaves regularly, according to the cut activity of the colony. Ensure that the offered leaves are healthy, and the plants have not been treated with insecticides or other chemical substances. At early stages, ensure that the leaf fragments are not over 4 cm.
    NOTE: As the first workers initiate leaf foraging, plant material must be offered after their appearance. The offering frequency depends on the agility with which the workers incorporate the plant material on the fungus but can range to 2-3 days a week. Oat flakes and corn flakes can also be offered but should be alternated with leaves to avoid fungus dryness.
  4. When offering new leaves, remove colony waste and dry leaf fragments. Avoid the use of perfumes, moisturizers, creams, or any substance with a strong odor when manipulating the queens. Additionally, use latex gloves during all the processes.
  5. Follow the development of the colony, and when the fungus garden reaches at least half of the container volume, transfer the colony to an artificial perdurable nest.
    NOTE: As the development rate is inherent for each colony, there is not an estimated time for colony transfer. Usually, colonies from the first collection are transfered to nests with fungus garden chamber of 1L volume maximum, due to the small fungus garden.

3. Collection of young colonies

  1. Acquire plastic containers of approximately 500 mL volume.
  2. About 6 months after the nuptial flight, identify indicative tower-shaped mounds with granulated soil particles (Figure 1) of incipient Atta nests (Figure 2) on the locations with leaf-cutting ants occurrence previously marked.
    NOTE: Six months after the nuptial flight, the nests of young colonies are estimated to be up to 1 m deep in the soil. A new collection is indicated at this period to achieve higher chances of successful and enduring colonies in large quantities.
  3. With a garden hoe, excavate the nest entrance until you reach the chamber holding the young colony. Collect the queen, fungus garden, immatures, and young workers, and place them in the plastic container. Perform the collection process as gently as possible.
    NOTE: Naturally, a great amount of soil will be collected too and ought to be removed gradually in future maintenance procedures in the laboratory.
  4. Move the plastic lidded containers holding the colonies to the designated controlled environment. Perform the transportation of young colonies with ultimate caution, avoiding too much disturbance and maintaining a minimum temperature constancy. Refrain manipulating or moving the colonies for approximately 3 days to avoid stress. If the room has an active routine, a dark cloth can be put over the colonies.

Figure 2
Figure 2. Tower-shaped soil mound. The characteristic tower-shaped mound indicates the presence of incipient colonies of Atta sexdens and Atta laevigata. Please click here to view a larger version of this figure.

4. Maintenance of young colonies

  1. Provide thin young leaves 3 times a week.
    1. Ensure that the leaves offered are healthy and the plants have not been treated with insecticides or other chemical substances. At this stage, ensure the leaf fragments are at least 7 cm in length.
      ​NOTE: Oat flakes and corn flakes can also be offered but should be alternated with leaves to avoid fungus dryness.
    2. The offering frequency depends on the agility with which the workers incorporate the plant material on the fungus. With the cut activity being intense, increase the offering two times a day three times a week or 5 days a week.
  2. When offering new leaves, remove colony waste, including soil remnants, with the help of a spoon. Use latex gloves during all the processes. When manipulating the young colonies avoid the use of perfumes, moisturizers, creams, or any substance with a strong odor.
    NOTE: The workers themselves will separate the soil and the waste from the fungus.
  3. Follow the development of the colony, and when the fungus garden reaches at least half of the container volume, transfer the colony to an artificial perdurable nest.
    NOTE: As the development rate is inherent for each colony, there is not an estimated time for colony transfer.

Figure 3
Figure 3: Types of artificial nests to hold Atta sexdens and Atta laevigata colonies. Illustration of perdurable artificial nests of leaf-cutting ants: cloistered vertical nest setup, cloistered horizontal nest setup, and open arena nest setup. Please click here to view a larger version of this figure.

5. Perdurable artificial nests

  1. Prepare a cloistered vertical nest setup as described below (Figure 3 and Figure 4).
    NOTE: Cloistered nest configurations should always have different recipients to separately pose as (1) fungus garden chamber, (2) waste disposal chamber and (3) foraging chamber. Initially, it should start with three containers, but more recipients can be added to increase the fungus garden. The nests may vary in size and material according to their experimental purpose, although here it is described and recommended the use of see-through materials. The containers must be without openings, or else the ants will escape. The type of nest as described below can be used for general research, but it is not well recommended for assertive ant collection due to the disturbance caused when removing the lid, resulting in great agitation among the individuals. Nonetheless, due to the material transparency, it is possible to locate the queen and different ant castes even when the fungus garden has filled the entire container. As it is a tendency, it is always assumed that the immatures are in the center of the fungus garden when it has occupied most of the space available in vertical containers.
    1. Select a transparent lidded container of approximately 1 L and add a 1 cm layer of highly absorbent plaster base. This will be the fungus garden chamber. Select two lidded see-through containers of approximately 500 mL each to be waste disposal and foraging chambers. The ants will choose which chamber will be each, and after that, ensure that they are not interchanged.
      NOTE: To prepare the plaster base, follow the manufacturer's instructions.
    2. Perforate and connect the three containers with a transparent tube or a hose. If necessary, apply masking tape on the tubes border to guarantee a thigh connection with the containers and avoid ants escaping. Place the plaster base container in the middle and the other containers on opposite sides.
    3. Carefully transfer the fungus sponge of selected colonies (see step 2.5 and step 4.3) along with the queen, workers, and immatures to the plaster base container. Before the transference, make sure the plaster base is watered. Use latex gloves.
  2. Prepare a cloistered horizontal nest setup as described below (Figure 3 and Figure 4).
    NOTE: Nests with horizontal configuration allow close observation of the fungus garden and workers' activities toward it. As younger portions of the fungus garden are at the top, it is possible to observe recently offered substrates being incorporated by the workers. New portions of the fungus can be spotted by its color, which will be similar to the color of the last resource offered, whereas the older portions usually bear a beige color. Offsprings and the queen can also be easily located, as in horizontal containers they are usually at the top of the fungus garden, even when it has occupied most of the space. This configuration can be used for behavior-focused research, focal sampling and science outreach purposes, as it gives perception of the organization inside the nest.
    1. Acquire a transparent lidded container with approximately 31 cm x 21 cm x 4.5 cm dimensions, and add a 1 cm layer of highly absorbent plaster base. This will be the fungus garden chamber. Select two lidded see-through containers of approximately 500 mL each to be waste disposal and foraging chambers. The ants will choose which chamber will be each, and after that, ensure that they are not interchanged.
      NOTE: To prepare the plaster base, follow the manufacturer's instructions. If necessary, close the little space between the lid and the container with masking tape to prevent the ants from escaping.
    2. Perforate and connect the containers with a transparent tube or a hose. If necessary, apply masking tape on the tubes border to guarantee a thigh connection with the containers and avoid ants escaping. Place the plaster base container in the middle and the other containers on opposite sides.
    3. Carefully transfer the fungus sponge of selected colonies (see step 2.5 and step 4.3) along with the queen, workers, and immatures to the plaster base container. Before the transference, make sure the plaster base is watered. Use latex gloves.
  3. Prepare an open arena nest setup as described below (Figure 3 and Figure 5).
    NOTE: Open arena nests allow the collection of ants without great disturbance and foraging behavior analysis. They can also provide a reliable representation of a colony found in nature for science outreach purposes.
    1. Select a transparent lidded container of approximately 1 L and add a 1 cm layer of highly absorbent plaster layer base. This will be the fungus garden chamber.
      NOTE: It is recommended to start with 1 L containers and gradually pass on to containers with higher volumes for a bigger fungus garden. However, the containers should not overcome a 5 L volume. Add as many containers as necessary.
    2. Select an open arena. The size of the arena can vary according to study purposes. If a large arena is selected, place the containers holding the fungus garden in its interior (Figure 5). In case of a small arena, connect it to the fungus garden container with a transparent hose or tube (Figure 3). The arena will serve as a foraging and waste disposal chamber, so, ensure that it is not too small.
    3. Apply one layer of polytetrafluoroethylene liquid in a single movement to the arena border to contain the ants. Use a cotton soaked with the liquid and a nitrile glove.
      CAUTION: Avoid inhalation and skin touching while using polytetrafluoroethylene liquid.
    4. Carefully transfer the fungus sponge of selected colonies (see step 2.5 and step 4.3) along with the queen, workers, and immatures to the plaster base container. Before the transference, make sure the plaster base is watered. Use latex gloves.

Figure 4
Figure 4: Artificial cloistered nests of the leaf-cutting ants Atta sexdens and Atta laevigata. Cloistered vertical nest setup top (A) and side view (B); cloistered horizontal nest setup top (C) and side view (D). Please click here to view a larger version of this figure.

Figure 5
Figure 5: Artificial open arena nest of the leaf-cutting ants Atta sexdens and Atta laevigata. Open arena nest setup of Atta sexdens top (A) and side view (B). 1) Fungus garden chambers; 2) Waste; 3) Orange slices; 4) Glass with polytetrafluorethylene (PTFE) layer. Please click here to view a larger version of this figure.

6. Maintenance of developed colonies

  1. Daily offer at least one large leaf into the foraging chamber per colony with 1 L of fungus garden. If the cut activity of the ants is intense, increase the number of leaves. If the fungus is dry, pre-moisten the leaves to provide extra humidity. In cloistered nests, perform the offering quickly to avoid the ants from escaping the foraging chamber.
    NOTE: Here, leaves are collected from plant species such as mulberry (Morus nigra), mango (Mangifera indica), eucalyptus (Eucalyptus sp.), jambolan (Syzygium cumini), hibiscus (Hibiscus sp.), acalypha (Acalypha wilkesiana), and ligustrum (Ligustrum lucidum).
    1. Offer fruits such as orange and apple, and oat and corn flakes to diversify and complement the diet. To big colonies with intense foraging activity, offer oat flakes and corn flakes daily, and fruits once a week. If it is not the case, offer flakes alternately with leaves, but not more than three times a week, and fruits once or twice a month. Adjust the amount and frequency of each food according to the foraging activity of the ants.
    2. If the options described above are not available, identify the ants' foraging preference among leaves, flowers, and fruits of regional trees and shrubs, or even commercialized vegetables, grains, and other flakes. Avoid offering resources with defensive chemical compounds and pesticides.
  2. Remove all the content of the waste chamber every 2 weeks from all colonies. Remove workers as well for population control purposes. If the workers transfer healthy fungus to the waste chamber, ensure that the queen is not on it and remove it. If the amount of waste disposed is high or it is too humid, remove it once per week.
  3. Remove the material not taken by the ants from the foraging chamber whenever offering new ones and make sure it is always clean.
    1. If the workers transfer healthy fungus to the foraging chamber, disturb it, leave the container lid open and apply neutral talcum powder to the chamber margin surface. Perform this procedure only if there is still some space on the fungus chamber, this way the workers will transfer the fungus back to the container without losing it or any immatures.
  4. If more fungus garden is wanted, add another plastered container, and move a portion of the fungus sponge into it. Until the fungus reaches half of the container, add leaves in the fungus chamber. The growth of the fungus garden should happen gradually to not compromise the colony balance. If a bigger container is wanted, make sure to let the fungus occupy the whole space of the smallest containers before transferring it. Colony waste and dry leaves should not be let to accumulate into the fungus garden chamber.
  5. Check the plaster base from the containers, as with time, it may acquire a dark brown color and become ineffective due to ant excretion, waste matter transportation, and high humidity concentration. Also, some colonies may cut the layer and dispose of it. In these cases, transfer the fungus garden to a new plastered container.

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Representative Results

A flowchart depicting the process of ant collection is shown in Figure 6. Here, some results obtained employing the protocol of collection, maintenance, and nest setups described above are shown.

Figure 6
Figure 6: Flowchart for collection of leaf-cutting ants' colonies. Following the protocol, the first collection takes place right after the nuptial flight. The queens that have removed their wings are collected and put in a small container with a plaster base. After 6 months of the nuptial flight, the second collection occurs. Queens that excavated the soil and successfully began colony foundation are collected. After colonies develop, they are transferred to bigger containers, and optionally to different nest setups. Please click here to view a larger version of this figure.

Influence of reproductive status in the immune response of leaf-cutting ant queens
Queens of different species of ants have a long-life span and, therefore, are more likely to be exposed to the same pathogen more than once47. By initiating a new colony, Atta queens are isolated and a trade-off between reproduction and immunity could reduce investment in the immune defenses of these founding ants. Hence, their resistance to pathogens may be related to their reproductive status47,48. By collecting queens in different moments of their early life cycle, and introducing a nylon thread into the gaster of the individuals (Figure 7), it was possible to evaluate individual encapsulation response to pathogens and compare the immune resistance between virgin queens, recently mated queens, and queens that mated 6 months ago into the gaster of the individuals.

Figure 7
Figure 7: Encapsulation response essay. Experiment setup that aimed to evaluate individual immune response of encapsulation variation according to the reproduction status of Atta sexdens and Atta laevigata queens. Virgin queens, newly mated queens and queens that have mated 6 months ago received nylon filament insertions between the 4th and 5th tergite of the gaster to act as an antigen. The nylon filaments were removed after 24h and photographed to color cover quantification. The queens were collected before the nuptial flight, right after the nuptial flight and 6 months after the nuptial flight, following the steps from topics 1, 2, and 3 of the protocol described. Please click here to view a larger version of this figure.

Once the unmated females appear only in nuptial flight events, the study of these individuals at this reproduction status relies on the specific knowledge to predict the occurrence of the particular phenomenon and to know ant collection and maintenance protocols. To guarantee that the reproducers had not yet copulated, the individuals were collected directly from the nest holes that had their entries widened for the departure of those ants (Figure 1). As the identification of reproducers on species level is troublesome, workers from the same colony were collected for validation. In the case of mated individuals, females that had their wings removed and  began to excavate the soil right after the nuptial flight were collected, assuming they had already copulated. For both reproduction stages, a high number of females were collected to assure the quantity of individuals tested, as the mortality rate at this moment is significant. Once the mated ants had dispersed from the original colony, species were identified through cuticular hydrocarbon profile comparison49. Queens mated for 6 months since the nuptial flight have already initiated their colonies, that at this point consist of a small fungus garden, several workers and immatures. These early life colonies were spotted by the distinct tower-shaped soil mound (Figure 2). The collection took place following the steps described previously in section 3 "Collection of young colonies". To study female reproducers at this stage, it was necessary to maintain the young colony, since the queen alone would soon perish. The identification was made using the workers of the colonies. About 200 queens were collected in total using the protocols previously described.

After each collection, the queens were weighed, measured, and soon received the nylon filament insertions, which were removed 24 h later for color cover analysis. For Atta laevigata species, queens that mated 6 months ago showed the darkest nylon filament, thus, the higher encapsulation rate and more efficient immune defense (Figure 8). However, newly mated queens collected just after the nuptial flight, and virgin queens collected before nuptial flight shared approximately the same encapsulation rate (Figure 8). Thus, it was verified that the encapsulation cellular defense in Atta laevigata ants can vary with the reproductive status and the time elapsed after the mating, but it does not alter with weight and head length. Initially, the research aimed to collect and compare Atta sexdens queens as well, but it was not possible to collect virgin queens, since the nests identified did not have any reproducer activities at the holes observed. Despite the difficulty, general studies on the cellular defense mechanisms of leaf-cutting ant reproducers could contribute to the clarification of the immune responses of insects and to the improvement of current control methods.

Leaf-cutting ants' preference to sub products used in baits
Currently, the most applied population control method of leaf-cutting ant colonies is through toxic bait offering. Generally, they are associated with an attractive vegetal origin compound, with citric pulp being the most used. However, it is known that somehow workers can associate the bait toxicity with the attractive compound after prior contact and reject it, making the method inefficient. Therefore, the use of alternative attractive compounds such as soybean (100%), soybean plus citric pulp (50%/50%), and cashew plus citrus pulp (50%/50%) are proposed for a possible rotation between baits. These compounds were selected after preference experiments with Atta sexdens and Atta laevigata colonies. Nests were attached with glass arenas and insecticide-free baits with different compositions were offered (Figure 9).

Figure 8
Figure 8: Encapsulation rate of Atta sexdens and Atta laevigata queens at different reproductive stages. Encapsulation levels were evaluated through the mean gray value from images of the nylon filaments inserted into the gaster of the individuals. The darkest threads were considered to represent an efficient cellular defense because it was assumed that more overlapping hemocytes were lining the target, as shown on the X-axis in the figure. Atta laevigata mated queens showed the highest encapsulation level (ANOVA, Tukey-Kramer, Standard Deviation; p < 0.01; N=29), hence an effective individual immune defense. It was observed that the newly mated queens, and virgin queens of the same species exhibited, similarly, lower rates of encapsulation, and consequently a less effective individual immune defense. The results obtained suggest that mating and nest establishment events affect cellular immune responses of Atta laevigata queens. Atta sexdens queens showed contrary results, and MQ had the lowest encapsulation level (ANOVA, Tukey-Kramer, Standard Deviation, P < 0.01; N = 46). However, as virgin queens of the species could not be collected, the comparison between all groups was not possible. Please click here to view a larger version of this figure.

Figure 9
Figure 9: Leaf-cutting ants' preference to bait subproducts essay. Experiment setup that aimed to evaluate the preference of Atta sexdens and Atta laevigata worker to baits with different attractive subproducts. A glass arena was attached to the fungus chamber to hold the different baits, which were separated by a glass divider. 5 g of each bait were offered in random positions, three at a time. The baits were weighed after the end of the experiment, which occurred either after 1 h of observation or as soon as a bait was completely taken by the workers. Preference was evaluated by the loading activity, which was estimated by the bait load removed from the foraging chamber. Please click here to view a larger version of this figure.

In total, 32 colonies were used with approximately 1 L of fungus garden. The colonies were previously collected and maintained using the protocols described. The most loaded treatments of A. laevigata were citric pulp, soybean plus citric pulp (50%/50%) and cashew plus citric pulp (50%/50%; Figure 10). Similarly, A. sexdens ants' most loaded treatments were citric pulp, soybean (100%), and soybean plus citric pulp (50%/50%; Figure 10). The results obtained for the two species showed that the soy and cashew sub products were the most attractive after citric pulp, suggesting that other bait compositions could be used for chemical population control in leaf-cutting ants.

Figure 10
Figure 10: Attractive baits mean loading percentage by Atta laevigata and Atta sexdens workers. Similarly, the citric pulp (100%), soybean (100%) and soybean plus citric pulp (50%/50%) treatments were the most loaded for both species. However, the least loaded treatment for Atta sexdens (cashew plus citric pulp) was 60% loaded by Atta laevigata workers. Throughout the experiment, five repetitions were performed with five colonies of each species. The baits were differently arranged within the repetitions; however, the citric pulp was always present as the positive control. Please click here to view a larger version of this figure.

Research and science outreach
About 350 queens and 100 colonies are collected annually to be maintained in the laboratory since the year of 1990. At least three interns are designate to daily take care of the ants, and through this time-frame, almost 250 colonies were kept simultaneously for experimental and educational purposes every year. During the years 2016 to 2019, approximately 2,020 students from around 34 schools were able to visit the Laboratory of Leaf-cutting Ants (LAFC) and observe the ant colonies collected and maintained using the protocols described previously. The excursions had different aims, from general biology to social behavior learning. Aside from the known importance of insects such as ants, the complex physical structures built by them in nature, also observed in artificial nests, are an example of collective effort and work organization that have educational interest. To each educational level (Figure 11) a different detailed presentation was given, sometimes following the approach suggested by the teachers responsible for the students. Two colonies with the open arena nest setup are maintained for such purposes, each one with approximately 66 chambers with 177 L of fungus garden (Figure 5). Unfortunately, the excursions were canceled during the pandemic, but it is possible to see a growing interest among educators, as the number of students visiting arose during the last 4 years reported (Figure 12). Nonetheless, to maintain science outreach during the pandemic, virtual visits were made through video sharing platforms. The video50 showing how a nest of leaf-cutting ants is inside, made in partnership with the channel Manual do Mundo, had more than 2.4 million views and more than 3,000 comments in less than a year. There, the biology and creation of leaf-cutting ants in the laboratory were approached.

Figure 11
Figure 11: Students' levels of education on excursions through the years 2016-2019. Number of students that were present in excursions to the Laboratory of Leaf-cutting Ants during the period of 2016-2019, according to the level of education. The excursions aimed at different approaches involving leaf-cutting ant colonies that are maintained in the Laboratory of Leaf-cutting Ants. Please click here to view a larger version of this figure.

Figure 12
Figure 12: Number of students on excursions through the years 2016-2019. Number of students that were present in excursions at the Laboratory of Leaf-cutting Ants during the period of 2016-2019. It is possible to see an increase in the interest of educational organizations for conducting exhibitions of live insects, such as ants, that allow a close observation by the students. Please click here to view a larger version of this figure.

Species Month and Day Year City State Country Continent Latitude Longitude References
Atta bisphaerica November 4 1996 - - Brazil South America - - 37
Atta capiguara October 6 2006 Botucatu São Paulo Brazil South America 22° 50' 60" S 48° 26' 10" W 38
Atta capiguara October 27 2007 Botucatu São Paulo Brazil South America 22° 50' 60" S 48° 26' 10" W 38
Atta capiguara November 2008 Botucatu São Paulo Brazil South America 22° 50' 60" S 48° 26' 10" W 39
Atta capiguara November 2009 Botucatu São Paulo Brazil South America 22° 50' 60" S 48° 26' 10" W 39
Atta cephalotes March to June  - Ilhéus Bahia Brazil South America - - 40
Atta cephalotes September - Ilhéus Bahia Brazil South America - - 40
Atta colombica June 4 1993 - Gamboa Panamá Central America - - 41
Atta colombica June 7 1993 - Gamboa Panamá Central America - - 41
Atta colombica - 1998 - Gamboa Panamá Central America - - 42
Atta laevigata October 6 2006 Botucatu São Paulo Brazil South America 22° 50' 60" S  48° 26' 10" W 39
Atta laevigata October 27 2007 Botucatu São Paulo Brazil South America 22° 50' 60" S  48° 26' 10" W 39
Atta laevigata November 2008 Botucatu São Paulo Brazil South America 22° 50' 60" S  48° 26' 10" W 39
Atta laevigata November 2009 Botucatu São Paulo Brazil South America 22° 50' 60" S  48° 26' 10" W 39
Atta laevigata October 15 2016 Rio Claro São Paulo Brazil South America - - This work
Atta laevigata October 26 2016 Rio Claro São Paulo Brazil South America - - This work
Atta laevigata October 31 2017 Rio Claro São Paulo Brazil South America - - This work
Atta laevigata November 6 2017 Rio Claro São Paulo Brazil South America - - This work
Atta laevigata September 22 2018 Rio Claro São Paulo Brazil South America - - This work
Atta laevigata October 10 2018 Rio Claro São Paulo Brazil South America - - This work
Atta laevigata October 10 2019 Rio Claro São Paulo Brazil South America - - This work
Atta laevigata * October 21 2020 Rio Claro São Paulo Brazil South America - - This work
Atta laevigata November 15 2020 Rio Claro São Paulo Brazil South America - - This work
Atta laevigata November 20 2020 Rio Claro São Paulo Brazil South America - - This work
Atta laevigata October 17 2021 Rio Claro São Paulo Brazil South America - - This work
Atta laevigata October 25 2021 Rio Claro São Paulo Brazil South America - - This work
Atta opaciceps March 8 2022 Macaíba Rio Grande do Norte Brazil South America - - **
Atta sexdens - 1986 to 1991 Viçosa Minas Gerais Brazil South America 20.45° S  42.51° W 43
Atta sexdens October 21 2009 Botucatu São Paulo Brazil South America 22° 49’ 53.25" S 48° 25’ 24.22" W 44
Atta sexdens September 18 2010 Rio Claro São Paulo Brazil South America 22° 23' 70.50" S 47° 32' 54.40" W 45
Atta sexdens October 31 2010 Botucatu São Paulo Brazil South America 22° 49’ 53.25" S 48° 25’ 24.22" W 44
Atta sexdens October 10 2011 Rio Claro São Paulo Brazil South America 22° 23' 70.50" S 47° 32' 54.40" W 45
Atta sexdens October 10 2011 Botucatu São Paulo Brazil South America 22° 23' 70.50" S 47° 32' 54.40" W 45
Atta sexdens October 15 2016 Rio Claro São Paulo Brazil South America - - This work
Atta sexdens October 26 2016 Rio Claro São Paulo Brazil South America - - This work
Atta sexdens October 31 2017 Rio Claro São Paulo Brazil South America - - This work
Atta sexdens November 6 2017 Rio Claro São Paulo Brazil South America - - This work
Atta sexdens September 22 2018 Rio Claro São Paulo Brazil South America - - This work
Atta sexdens October 10 2018 Rio Claro São Paulo Brazil South America - - This work
Atta sexdens October 10 2019 Rio Claro São Paulo Brazil South America - - This work
Atta sexdens* October 21 2020 Rio Claro São Paulo Brazil South America - - This work
Atta sexdens November 15 2020 Rio Claro São Paulo Brazil South America - - This work
Atta sexdens November 20 2020 Rio Claro São Paulo Brazil South America - - This work
Atta sexdens October 17 2021 Rio Claro São Paulo Brazil South America - - This work
Atta sexdens October 25 2021 Rio Claro São Paulo Brazil South America - - This work
Atta sexdens November to March - Ilhéus Bahia Brazil South America - - 40
Atta texana May 1992 Nacogdoche Texas USA North America 32° N  95° W 43
Atta vollenweideri - 2004 to 2010 - Formosa Argentina South America 26° 18' S 58° 49' W 46
Atta vollenweideri October 6 2006 - Formosa Argentina South America 26° 18' S 58° 49' W 46
Atta vollenweideri October 2009 - Formosa Argentina South America 25° 07' S 58° 10' W  46
Atta vollenweideri November 2009 - Formosa Argentina South America 25° 07' S 58° 10' W  46
Atta vollenweideri December 8 2010 - Formosa Argentina South America 26° 18' S 58° 49' W 46
Atta vollenweideri October 24 - - Formosa Argentina South America 26° 18' S 58° 49' W 46
* Only males observed   ** Mendonça, G. A., personal communication (data unpublished)

Table 1: Records of nuptial flight periods of Atta species. Mating flights occur in different periods and frequencies according to regions, however they generally happen during spring, as indicated in reports gathered from Atta ants' territory of occurrence37,38,39,40,41,42,43,44,45,46. Please click here to download this Table.

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Discussion

The protocol described here to maintain leaf-cutting ant colonies has been developed and applied for over three decades in an assertive and replicable way. It allowed the development of research that would be limited by field conditions. Thereby, healthy ants and colonies became available for research in several areas such as comparative morphology, toxicology51,52, histology53, and microbiology54,55,56 at the individual and colony level. Their maintenance in the laboratory includes several procedures described here, which include site preparation under favorable abiotic conditions, collection of queens and young colonies, and continued care. 

Several steps are crucial for the success of leaf-cutting ants maintenance in controlled conditions. Queens and colonies in the early stages need distinguished care and following the initial procedures detailed can guarantee their survival. For example, it is necessary to identify the place where the species are found and the period when the winged reproducers initiate the nuptial flight. In South America, the majority of mating flights occur during the spring (Table 1). Currently, the possibility of captivity breeding is extremely low, as it is to record a nuptial flight in the laboratory24. Additionally, leaf-cutting ant colonies in the fertile phase are large and difficult to excavate, and given the nest architecture, it is a challenge to locate and collect the queen6. For instance, Atta laevigata nests can be up to 67 m2 of nest area, and occupy more than 563 m2 of internal surface area divided into 7,864 chambers in 7 m depth57.

The advantage of collecting queens promptly after the nuptial flight is to guarantee a greater number of reproductive forms in a short period of time and close to the surface. From this first collection, it is possible to develop experiments with reproductive forms (Figure 7), including males. A technique restriction is the need to collect reproductive individuals at an uncertain date. If the queens' survival is low, a second collection of young colonies becomes necessary. Collecting young colonies a few months after the nuptial flight ensures that the queen's chamber is not deep into the surface. In this phase, only the queens with more fitness remain, as they endured the food shortage phase and were able to take care of the fungus and the first workers during the claustral phase58,59,60,61. In laboratory conditions, queens' survival rate can reach 14.5% in the initial months, even with healthy fungus transferred to 90% of queens (data unpublished). Similar survival data of Atta sexdens were recorded by Mota-Filho62. This can be due to the lack of fungus development or natural contamination with entomopathogenic fungi63.

One of the main disadvantages of collecting queens, or colonies at early stages (Figure 6 and Figure 7) is the difficulty in taxonomic identification, as it can more easily be done through major workers12 that only emerge after a few months, when there is more resource for larval feeding. Some critical steps in this technique are associated with promoting the growth of the fungus garden, keeping the nests clean, and avoiding possible escapes. Some actions include the frequent addition of fresh vegetable material, which is constantly being incorporated in the fungus by the workers. The fungus is used as a supply of glucose and starch for the workers, and as protein for the larvae64. Removing old and disposed materials, and maintaining the nests clean, prevents the appearance of possible harmful microorganisms. To avoid escapes, tube connections should always be checked, and in the case of open arenas, upkeep the polytetrafluoroethylene layer. The advantage of maintaining colonies in the nests configurations described to simulate the real structure found in nature, with specific chambers for foraging and waste, and a colony with the social organization complete (i.e. queen, workers, and offsprings). Research conducted with queenless colonies63,65 indicates that workers exhibit distinct behaviors due to the absence of the queen. Behavioral studies concluded that the queen is important for colony cohesion in addition to reproductive function66.

The implementation of the protocols addressed here can be applied to other species of scientific interest, such as other fungus growing ants from the genus Acromyrmex. These leaf-cutting ants are also considered agricultural pests in particular regions of America, and have recently attracted more interest in early-stage colony development67 and toxic bait interaction68 focused research. Although this work is specifically addressed to leaf-cutting ants, the maintenance protocol and the different types of nests described could also be applicable to other groups of ants. Each section of the procedure outlines fundamental ant necessities that must be taken into consideration when preserving them in controlled conditions, such as: identify the most appropriate diet; offer food at regular intervals in a foraging chamber or specific area; provide a high humidity level chamber with plaster base to keep the queen and immatures; avoid ant escape through contingency substances added to the structures of the artificial nests; remove materials disposed by the ants from the nests; and transferring the colonies to another artificial nest when necessary.

Over the past decade, universities and researchers have been dedicated to being more inclusive, inviting the society to be part of the laboratories and their research69. Embracing with great determination the educational purpose, ant laboratories became tools to attract people's curiosity and connect the academic world with common knowledge. The types of nests presented in this work can be used in different activities depending on the objective, such as education and expositions. For example, open nests (Figure 5) are to put on display to visitors at the university, while cloistered nests (Figure 4) are useful for science fair and itinerant exhibitions, due the ease of transport. In all cases, the general public can observe ant colonies, learn about basic biology and fun facts, and be instigated to questioning. On a more expressive level, through extension projects such as "Primeiros Passos na Ciência" (First Steps in Science)69 the students can actually be part of the research teams. Consequently, ant studies with educational purposes benefits student development as it enhances investigation and experiences while encouraging greater interest in scientific research.

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Disclosures

The authors have no conflicts of interest to disclose.

Acknowledgments

Dedicated to Mario Autuori (in memoriam) and Walter Hugo de Andrade Cunha who greatly contributed to the leaf-cutting ant studies. We acknowledge the support of São Paulo State University and the Institute of Biosciences. This study was in part financed by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil (CAPES) - Finance Code 001, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), and Fundação para o Desenvolvimento da UNESP (Fundunesp).

Materials

Name Company Catalog Number Comments
Entomologic forceps N/A N/A N/A
Glass tank N/A N/A Tempered glass, custom made
Hose N/A N/A Transparent, PVC 1/2 Inch x 2,0 mm
Latex gloves Descarpack 550301 N/A
Nitrile gloves Descarpack 433301 N/A
Open arena N/A N/A Polypropylene crate
Plaster pouder N/A N/A Plaster pouder used in construction, must be absorbant
Plastic Containers for collection Prafesta Natural Cód.: 8231/Natural Cód.: 8262 Lidded, transparent , polypropylene
Plastic containers for nests Prafesta Discontinued Polystyrene, hermetic
Teflon Dupont N/A Polytetrafluoroethylene liquid (PTFE Dispertion 30)

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Collection Long-term Maintenance Leaf-cutting Ants Laboratory Conditions Colonies Nests Research Science Outreach Experiment Conditions Controlled Environment Ant Species Acromimix Ants Technique Uncertainty Daily Care Routine Nuptial Flight Queens Excavation Plaster Base Colony Foundation Bigger Containers Nest Setups
Collection and Long-Term Maintenance of Leaf-Cutting Ants (<em>Atta</em>) in Laboratory Conditions
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Nogueira, B. R., de Oliveira, A. A., More

Nogueira, B. R., de Oliveira, A. A., da Silva, J. P., Bueno, O. C. Collection and Long-Term Maintenance of Leaf-Cutting Ants (Atta) in Laboratory Conditions. J. Vis. Exp. (186), e64154, doi:10.3791/64154 (2022).

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