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

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.