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 S&#227;o Paulo State University and the Institute of Biosciences. This study was in part financed by the Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior-Brasil (CAPES) - Finance Code 001, Conselho Nacional de Desenvolvimento Cient&#237;fico e Tecnol&#243;gico (CNPq), Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado de S&#227;o Paulo (FAPESP), and Funda&#231;&#227;o para o Desenvolvimento da UNESP (Fundunesp).

1. Collection of queens
<ol>
	<li>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,...

The authors have no conflicts of interest to disclose.

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,<sup cl...

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&#39;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&#39;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Entomologic forceps</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Glass tank</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Tempered glass, custom made</td>
		</tr>
		<tr>
			<td>Hose</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Transparent, PVC 1/2 Inch x 2,0 mm</td>
		</tr>
		<tr>
			<td>Latex gloves</td>
			<td>Descarpack</td>
			<td>550301</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Nitrile gloves</td>
			<td>Descarpack</td>
			<td>433301</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Open arena</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Polypropylene crate</td>
		</tr>
		<tr>
			<td>Plaster pouder</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Plaster pouder used in construction, must be absorbant</td>
		</tr>
		<tr>
			<td>Plastic Containers for collection</td>
			<td>Prafesta</td>
			<td>Natural C&#243;d.: 8231/Natural C&#243;d.: 8262</td>
			<td>Lidded, transparent , polypropylene</td>
		</tr>
		<tr>
			<td>Plastic containers for nests</td>
			<td>Prafesta</td>
			<td>Discontinued</td>
			<td>Polystyrene, hermetic</td>
		</tr>
		<tr>
			<td>Teflon</td>
			<td>Dupont</td>
			<td>N/A</td>
			<td>Polytetrafluoroethylene liquid (PTFE Dispertion 30)</td>
		</tr>
	</tbody>
</table>

collection and long-term maintenance of leaf-cutting ants (atta) in laboratory conditions

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.

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.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64154/64154fig6v2.jpg" /> 
Figure 6: Flowchart for collection of leaf-cutting ants&#39; colonies. Following the protoc...

Watch this Scientific Journal Video about Collection and Long-Term Maintenance of Leaf-Cutting Ants Atta in Laboratory Conditions at JoVE.com

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

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.

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

Ants are one of the most biodiverse groups of animals on the planet and inhabit different environments. The maintenance of ant colonies in controlled ...

Successfully collecting and maintaining colonies in different types of nests under laboratory conditions allows the development of general research and can promote science outreach. Since file-tested colonies are difficult to monitor, this protocol allows preliminary experiment conditions in a controlled environment, using whole colonies as operational units. This protocol can be adapted to other groups of interest in considering the heterogeneous lifestyles of ant species, such as the peculiar ants of the genus Acromimix.An individual who has never performed this technique may consider the uncertainty of events and the complex daily care routine as difficulties. A first collection should take place right after the nuptial flight in which queens that have removed their wings and initiated excavation are placed in small containers with a plaster base. A second collection should occur six months after the nuptial flight in which colonies developed by the queens that successfully began colony foundation are collected.After the development of the colonies, they are transferred to bigger containers and optionally to different nest setups. Prepare the plastic-lidded containers with a bottom plaster layer to retain the queens individually. In the reproductive season, identify Atta nests in areas of leaf-cutting ants'occurrence and look for the external features that indicate the upcoming departure of winged reproductive ants.Nest features include tunnel entrances widened, winged reproductive ants appearing at tunnel entrances, and an increased flow of workers showing more aggressive behavior toward possible predators. After the nuptial flight, when the female reproducers return to the soil, remove their wings and start nest excavation. Begin the queens'collection.Carefully place them individually in plastic containers prepared with a plaster layer. Place the queens in a controlled environment, and do not manipulate or move them for approximately three days to avoid stress. Carefully puncture the lids and pour 2.5 milliliters of water with a needled syringe on the plaster layer of the container every two days.For as long as the fungus garden exhibits a dry aspect with water absence, irrigate the plaster layer. Check whether the queens have regurgitated the fungus two weeks after the collection. If there is no fungus, collect two grams of the fungus garden of healthy and established colonies and transfer them to the queens'containers.Perform this step even if the fungus does not develop. After the appearance of the first workers, begin offering fragments of young and thin leaves regularly according to the cut activity of the colony and remove colony waste and dry leaves fragments. 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.Six months after the last nuptial flight, identify indicative tower-shaped mounds with granulated soil particles of incipient attanests on the locations of leaf-cutting ants'occurrence. The nests of young colonies are estimated to be up to one meter deep in the soil after six months of the nuptial flight. At this moment, excavate the nest entrance until you reach the chamber holding the young colony with a garden hoe.Collect the queen, fungus garden, immature, and young workers and place them in a plastic container of approximately 500 milliliters. Move the plastic-lidded containers holding the colonies to the designated controlled environment. Do not manipulate or move the colonies for approximately three days to avoid stress.Start offering leaves. When offering new leaves, remove dry leaves and colony waste. Remove soil remnants from the collection with the help of a spoon.Follow the development of the colonies, and when the fungus garden reaches at least half of the container volume, transfer the colonies to artificial, perdurable nests cloistered vertical nest, cloistered horizontal nest, and open arena nest. Prepare artificial, perdurable nests with the more suitable setup cloistered vertical nest, cloistered horizontal nest, and open arena nest. Prepare a cloistered vertical nest setup with a foraging chamber, waste chamber, and a fungus garden chamber.Then prepare a cloistered horizontal nest setup with a foraging chamber, waste chamber, and a fungus garden chamber. Next, prepare an open arena nest setup with a foraging and waste chamber and a fungus garden chamber connected to it. The arena can also hold the fungus chamber.Apply one layer of polytetrafluoroethylene liquid in a single movement to the arena border to contain the ants. Use cotton soaked with the liquid and nitrile gloves. Offer at least one large leaf daily into the foraging chamber per colony with one liter of the fungus garden volume.The offering frequency depends on the agility with which the workers incorporate the plant material on the fungus. If the cut activity of the ants is intense, increase the number of leaves. In cloistered nests, perform the offering quickly to avoid ants escaping the foraging chamber.Other types of resources can also be offered. In open arena nests, offer leaves according to the number of liters of fungus and away from wastes. Remove all the content of the waste chamber every two weeks from all colonies and per durable nests and the workers as well for population control purposes.If the amount of waste disposed of is high, or it is too humid, remove it once per week. If the workers transfer the healthy fungus to the waste chamber, ensure that the queen is not on it and remove it. Remove the material not taken by ants from the foraging chamber whenever offering new ones, and make sure it is always clean.If the workers transfer the healthy fungus to the foraging chamber, disturb it, leave the container lid open and apply neutral talcum powder to the chamber margin surface. This way, the workers will transfer the fungus back to the container without losing it or any immatures. If more fungus garden is wanted, add another plastered container and move a portion of the fungus sponge into it.The growth of the fungus garden should happen gradually, not to 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 should not accumulate in the fungus garden chamber.By collecting queens in different moments of their early life cycle as described in the protocol and introducing a nylon thread into the gaster of the individuals, it was possible to evaluate and compare individual immune resistance between virgin queens, recently mated queens, and queens mated for six months. 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.Atta laevigata recently mated queens showed the highest encapsulation level, hence an effective individual immune defense. To improve population control methods, colonies collected through the protocol were submitted to Bates attractiveness tests. Throughout the experiment, five repetitions were performed with five colonies of each species, Atta laevigata and Atta Sextens.The non-toxic baits formulated with different subproducts were diversely arranged within the repetitions. The mean loading percentage by Atta laevigata and Atta sexdens workers are shown here. The citric pulp, soybean, and soybean plus citric pulp treatments were the most loaded for both species.However, the last loaded treatment for Atta sexdens was 60%loaded by Atta laevigata workers. During the years 2016 to 2019, approximately 2, 020 students from around 34 schools were able to visit the laboratory of leaf-cutting ants to observe the ant colonies collected and maintained using the protocols described previously. It is possible to see an increase in the interest of educational organizations in conducting exhibitions of live insects such as ants that allow a close observation by the students.The visits had different aims from general biology to social behavior learning according to students'level of education. Some critical steps in this technique are associated with promoting the growth of the fungus garden, keeping the nests clean, and avoiding possible escapes. The protocol can be applied to other species of scientific interest to understand early stage colony development and toxic bait interactions research, for example.This technique has been employed for 22 years by researchers from several areas such as comparative morphology, histology, toxicology, and microbiology, at the individual and colony levels.

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Research

JoVE Journal

Biology

2.3K ビュー.  São Paulo State University. 実験室条件下でさまざまな種類の巣のコロニーをうまく収集して維持することで、一般的な研究の発展が可能になり、科学への働きかけを促進することができます。ファイルテストされたコロニーは監視が難しいため、このプロトコルでは、コロニー全体を操作単位として使用して、制御された環境での予備実験条件が可能になります。このプロトコルは、Acromimix属の?...